Regulation appears to rely on a variable and dynamic interaction between mRNA target and ARE-specific binding proteins (AUBPs). The AUBP-ARE mRNA recognition is directed by multiple intracellular signals that are predominantly targeted at the AUBPs. These include (but are unlikely limited to) methylation, acetylation, phosphorylation, ubiquitination and isomerization. These regulatory events ultimately affect ARE mRNA location, abundance, translation and stability. In this review, we describe recent advances in our understanding of phosphorylation and its impact on conformation of the AUBPs, interaction with ARE mRNAs and highlight the role of Pin1 mediated prolyl  The cytoplasmic level of all mRNAs is dictated by a complex interplay between the rates of gene transcription and mRNA decay. mRNA decay allows cells to eliminate aberrant/mutant messages (nonsense-mediated) as well as modulate mRNA pools and abundance to rapidly adapt to an ever-changing environment ,2. The dIn steady state mammalian cells, mRNAs show a wide variety of decay rates. Protein with critical cellular functions such as cytokines, oncogenes, kinases, phosphatases and cell-surface receptors are often, but not exclusively coded by short-lived mRNAs that are unstable in resting cells ,2. Alongin vitro transcribed, radiolabeled ARE RNA fragments derived from GM-CSF mRNA [Trans-acting factors , that bind directly or indirectly to the AREs  are esseCSF mRNA . AUF1 waCSF mRNA ,9,10 havAUBPs bind to AREs via a variety of domains including the so-called RNA-recognition motif (RRM), CCCH tandem zinc finger, and the K-homology domain (KH) . A singl−/− MEFs [The breadth of AUBP-mediated regulation is substantial. For example, a genome-wide analysis identified ~250 mRNAs that were stabilized in TTP−/− MEFs . These r−/− MEFs . While 2−/− MEFs .The expression of different AUBPs varies depending on cell/tissue types and external stimuli. However, it is clear that multiple AUBPs coexist in cells and that numerous target mRNAs can interact with and presumably be regulated by multiple AUBPs. Thus, a critical question remains as to what determines the recognition between an AUBP and specific target ARE mRNA. As the interaction appears to be extremely plastic and dynamic, signal transduction events that trigger AUBP post-translational modification(s) seem likely to alter the affinity or localization (among other options) of preexisting AUBPs ,15. Indecis-trans prolyl isomerase (PPIase), was cloned from a human cDNA library and found to be essential for cell-cycle progression [cis-trans isomerases of the cyclophilin and FKBP families. While the latter proteins can isomerize X-Pro peptide bonds (where X is any amino acid), Pin1 is the only known mammalian isomerase with rigid specificity for Ser-Pro or Thr-Pro peptide bonds. Isomerization is bidirectional with cis to trans or trans to cis conversions but occurs approximately 1000 fold faster when the N-terminal Ser or Thr has been phosphorylated [trans pSer/pThr-Pro bonds often show enhanced phosphatase accessibility, this is likely a common event.Pin1, a gression . Pin1 isorylated ,22,23. Sorylated ,25. The cis-trans isomerization has profound effects on target protein folding, altering subsequent protein-protein and protein-nucleic acid interactions, protein stability and subcellular localization thereby altering a variety of cellular processes including cell cycle progression, apoptosis, innate and acquired immunity, and gene regulation [Pin1 mediated prolyl gulation ,22,26. RIn this review, we will discuss the post-translational modification of AUBPs by phosphorylation and potential role of Pin1 isomerization in the regulation of AUBPs conformational remodeling, AUBPs-RNA interaction and ARE mRNA turnover in mammals. Finally, we will address several physiological cytokines and immune disorders associated with the function of Pin1.Kinases involved in AUBP phosphorylation and ARE mRNA stability include the proline directed kinases  and others  . The MAP−/− knockout mice show low TNF-α levels while double MK2−/−:TTP−/− mice express high levels of TNF, reminiscent of TTP−/− mice [TTP is a destabilizing AUBP that can be phosphorylated in murine cells by MK2 (substrate of p38 MAPK) at two serine residues . Phospho−/− mice . These d−/− mice , it is l−/− mice . Dephosp−/− mice ,12 (TablAUF1 (hnRNP D) appears to be unique as an AUBP with both stabilizing and destabilizing activity. It can destabilize  or stabilize  ARE mRNAs . Alternain vitro on Ser87 and Ser83 by protein kinase A (PKA) and glycogen synthase kinase 3 beta (GSK3β), respectively [in vivo action of these kinases on AUF1 has not been confirmed et but Ser83 phosphorylation may require prior Ser87 phosphorylation. Phospho-Base predicted seven additional phosphorylation sites, but to date, only CK1 site at Thr91 has been investigated [The first phosphorelationsites in p40 AUF1 were identified on Ser83 and Ser87 in polysome-associated protein ,28. Thesectively  . The resulting phenotype was enhanced GM-CSF mRNA stability and cytokine release. As GM-CSF blocks the default apoptosis of eosinophils, the excess release of prosurvival cytokine contributed to the accumulation of pathologic cells in the airways of active asthmatics. Conversely, Pin1 blockade prevented GM-CSF mRNA stabilization or cytokine secretion, and attenuated allergic inflammation and airway fibrosis in the rodent models of asthma [In the context of AUBPs, Pin1 constitutively interacted with the AUF1 complex in eosinophil cytoplasm  Figure . Cell acf asthma . Pin1 haf asthma ,159. In f asthma  whereas f asthma , suggestIn vitro FRET experiments revealed that non-phosphorylated p40 AUF1 promotes formation of a condensed, less flexible structure within the TNF-α ARE [cis-trans isomerization is not required for the effect of AUBPs on target mRNA turnover. Moreover, a role for phospho-HuR in modulating miRNA access to mRNA has been proposed by several independent studies, and may involve similar mechanisms as discussed above [cis-trans isomerization and phosphorylation-dependent changes in the architecture of AUBPs and local RNA/DNA structure provides a diverse population of molecular determinants to direct downstream macromolecular events.Post-translational modifications have impact on protein conformation and thus can alter the ability of AUBPs to bind ARE-containing transcripts. Depending on which AUBP occupies an ARE, the decay rate is increased or decreased. NF-α ARE . While dNF-α ARE ,38. In rNF-α ARE , leadingNF-α ARE  (Figure NF-α ARE . Pin1-meNF-α ARE . Pin1 reNF-α ARE ,160, altNF-α ARE ,162. In NF-α ARE ,164,165.ed above ,167,168.ed above ,170. As ed above , Pin1 meIn vivo treatment with juglone not only suppressed cytokine production but also preserved transplanted organ architecture and function [ARE-mediated post-transcriptional regulation is particularly important for a rapid cellular response. This is observed during defense against pathogens, cardiovascular toning, organ rejection, and allergic reactions ,171,172.function ,158. Thifunction  also demfunction ,174. As function ,176,177,cis-trans conversion alters AUBP binding to targets or cofactors and how drugs can be developed that can affect Pin1 or its interaction with protein ligands.The regulation of mRNA stability through the ARE allows a fine-tuning of responses to extra- and intra-cellular signals. The process requires the regulated association of one or more AUBPs with target mRNAs in a sequential manner. Proper regulation of AUBPs is thus essential for normal cellular, tissue, and organ homeostasis. While kinase-mediated AUBP phosphorylation is critical, downstream events including Pin1 mediated binding and isomerization are also important. As seen in a variety of pathologies including cancer, allergy and infection, Pin1 dysregulation can have profound consequences. Critical, unanswered questions include the identification of additional Pin1 AUBP targets, characterization of how the


To evaluate the functional role of this phosphorylation site in vivo, we generated transgenic flies that can express identical amount of either wild-type or phosphorylation-resistant mutant Drosophila TSC2 in a tissue-specific manner. In response to transgenic Sestrin induction, which causes ectopic AMPK activation and subsequent mTORC1 inhibition, wild-type Drosophila TSC2 synergistically reduced tissue growth in the dorsal epithelium of Drosophila wings. However, phosphorylation-resistant mutant Drosophila TSC2 was unable to show such a growth-inhibiting effect, suggesting that this phosphorylation is important for AMPK-dependent regulation of cell growth.AMP-activated protein kinase (AMPK) is an important metabolic regulator that mediates cellular adaptation to diverse stresses. One of the AMPK substrates, tuberous sclerosis complex 2 (TSC2), was suggested to mediate AMPK-induced silencing of mTOR complex 1 (mTORC1) signaling that is critical for cell growth. However, it is not known whether the AMPK-dependent TSC2 phosphorylation, originally observed in mammalian cells, is conserved in invertebrates. Here we show that energy depletion inhibits mTORC1 signaling through the AMPK-TSC2 axis in Drosophila S2 cells. We have discovered an AMPK phosphorylation site in TSC2-like genes from many different invertebrate species including Drosophila. The site (Ser1338 in Drosophila TSC2) is specifically and efficiently phosphorylated by AMPK  AMP-activated protein kinase (AMPK) is a cellular energy sensor whose protein kinase activity is upregulated upon binding to AMP, which is accumulated when energy source has been depleted . Once acde novo pyrimidine synthesis. On the other hand, mTORC1 suppresses autophagic catabolism by inhibiting an autophagy-initiating protein kinase ULK1/2 through direct phosphorylation [Mechanistic target of rapamycin complex 1 (mTORC1) is another important nutrient-sensing protein kinase, which can respond to the level of various nutrients, including glucose and amino acids . Unlike rylation  and by irylation . Thus, iAlthough it has been repeatedly observed that mTORC1 is activated by amino acids, it is still poorly understood how mTORC1 biochemically senses amino acids . Severalin vitro. Mutation of this phosphorylation site also diminished the function of TSC2 in suppressing mTORC1-dependent cell growth in Drosophila wing epithelium. Thus, our study shows that the signal transduction pathway composed of AMPK, TSC2 and mTORC1 is present in invertebrates including Drosophila.Here, by using cluster analysis for performing multiple sequence alignment between TSC2-like genes from various vertebrate and invertebrate species, we were able to identify an AMPK phosphorylation motif that is conserved throughout all the examined metazoan species. The target serine in this motif (Ser1338 in Drosophila TSC2) is corresponding to Ser1387 in human TSC2. Using Drosophila TSC2, which is also known as Gigas or dTSC2, we showed that AMPK can efficiently phosphorylate invertebrate homolog of TSC2 Although it has been known that energy-dependent AMPK signaling can induce cell structure changes in both insect and mammalian cells , it is nWe tested whether AMPK and TSC2, two known mediators of energy-induced mTORC1 silencing in mammalian cells , are alsAlthough human and Drosophila TSC2 show significant homology to each other, pairwise sequence alignment between them shows that the region around Ser1387 in human TSC2 (the AMPK phosphorylation site) is very poorly conserved in Drosophila TSC2, as formerly reported . Thus, iFor this, we conducted two independent approaches. In the first approach, we have performed multiple sequence alignment of TSC2-like proteins from various vertebrate and invertebrate animals of distinct taxa. The rationale for this approach is that we would be able to minimize alignment errors or noises by comparing multiple sequences and identifying more functionally important residues that are widely and strictly conserved in different organisms. Interestingly, when we analyzed 10 different animal species for the multiple sequence alignment analysis, we were able to observe that the region surrounding Ser1387 is significantly conserved among the TSC2 proteins . In thisin vitro kinase assay using a recombinant Drosophila TSC2 protein purified from E. coli and an active AMPK holoenzyme purified from rat liver. Using this assay, we have found that Drosophila TSC2 is efficiently phosphorylated by AMPK, which was further activated by addition of AMP, an AMPK activator (in vitro phosphorylation event mediated by AMPK (We were curious whether this putative phosphorylation site (Ser1338 in Drosophila TSC2) can be indeed phosphorylated by AMPK. Thus, we have set up an ctivator c. In con by AMPK c, indicaTSC2 is an important tumor suppressor whose loss can induce hyperplastic cell growth due to mTORC1 overactivation . OverexpIt has been recently shown that Sestrin-family proteins can inhibit mTORC1-dependent cell growth through the AMPK-TSC2 axis ,22,23 aswww.flyrnai.org). Oligomycin and insulin were purchased from Sigma . dsRNAs against white (control), TSC2 and AMPK were generated and treated as recommended by DRSC, and corresponds to DRSC30065, DRSC29781 and DRSC28055 amplicons.Drosophila S2 cells are from the Drosophila Genomics Resource Center (DGRC) and cultured in Schneider’s medium plus 10% FBS and Peni-strep, according to instructions from the Drosophila RNAi Screening Center  containing protease inhibitor cocktail , and processed as formerly described [Cells and tissues were lysed in cell lysis buffer  were formerly shown to cross-react with phospho-Thr184 Drosophila AMPK and phospho-Thr37/46 Drosophila 4EBP ,28. Phos32P-ATP for radioactive assay), and 1 mg of protein substrate at 30 °C for 20 min, with or without 0.3 mM AMP. Active AMPK holoenzyme was purchased from Upstate (14-305). GST-tagged recombinant dTSC2 proteins, purified from BL21 E. coli transformed with pGEX 4T-1 vector expressing dTSC2 (a.a. 1278-1371), were used as substrates. The assay samples were subjected to SDS–PAGE, coomassie staining and autoradiography.As formerly described , the proWT and pUAST-attB-dTSC2S1338A were microinjected into 1 M{vas-int.Dm}ZH-2A w*y; M{3xP3-RFP.attP'}ZH-22A flies and stable transformants were isolated by presence of +mini-white marker. hs-Gal4, ap-Gal4 and XP4dSesn were formerly described [Wild-type dTSC2-coding sequence was derived from LD36178 cDNA (Berkeley Drosophila Genome Project). Ser1338Ala mutation was introduced to dTSC2 through a PCR-based site-directed mutagenesis. Both forms of dTSC2 was cloned into a pUAST-attB vector  and fullescribed . We have shown that energy deprivation in Drosophila cells can provoke mTORC1 inhibition in an AMPK- and TSC2-dependent manner. In addition, we have revealed that Drosophila TSC2 has an authentic AMPK phosphorylation site, which plays a key role for tissue growth-regulatory function of the protein. These data indicate that the AMPK-TSC2 signaling axis, originally discovered in mammalian cells, is highly conserved in invetebrate systems such as in Drosophila, and is critical for energy- and stress-dependent modulation of cell and tissue growth.


Vmax represents the maximal velocity. The reaction rate constant of enzyme–substrate–inhibitor complex E. coli. The present method provides a simple way for determining kinetic parameters of the substrate inhibition irrespective of complete and partial types.A simple graphical method was described for determining the kinetic parameters of substrate inhibition of complete and partial types. The method consists of plotting experimental data as  E. coli phosphofructokinase II.Enzyme inhibition by its substrate in excess, substrate inhibition, is one of the common deviations from Michaelis–Menten kinetics, and means that the velocity curve of a reaction rises to a maximum as substrate concentration increases and then descends either to zero (complete inhibition) or to a non-zero asymptote . Substrate inhibition is an extremely widespread phenomenon in enzyme kinetics, and plays critical regulatory roles in a number of metabolic pathways Kaiser . The simE, has two binding sites for its substrate S, a catalytic site for binding that can produce the product, P, and a non-catalytic or allosteric site that generates the product at a reduced rate. We may write the following reaction scheme:We now derive an equation for substrate inhibition. The enzyme, ES1 and ES2, the substrates bound to the catalytic site and to the non-catalytic site, respectively, and further by ES1S2 two substrate molecules bound to both the catalytic and the non-catalytic sites. It is formally convenient to regard Vmax, that is to say the ES1S2 complex is assumed to break down at a different velocity from the ES1 complex, and the rate constants k and k′ apply to the breakdown of the ES1 complex and ES1S2 complex, respectively. A rate equation for the reaction in Eq. /v can be approximated as follows:The second term of Eq.  is negliThen,Vmax − v)/v and the substrate concentration gives a hyperbolic curve under the conditions of higher substrate concentrations /v and [S] of complete inhibition gives straight lines under the higher concentrations of substrate  by ATP with the present method.Phosphofructokinase is a key enzyme in glycolysis, and is controlled by various allosteric effectors Ramaiah . Of reguE. coli phosphofructokinase II showed non-allosteric properties, and was subject to typical substrate inhibition with respect to ATP . v/(Vmax − v) and the reciprocal of ATP concentrations gave straight lines converging on the origin, indicating the inhibition to be of complete type. We calculated the apparent P Figure a. Vmax ventclass1pt{minimaentclass1pt{minimaES1 can generate a reaction product; however, ES1S2 complex, the enzyme that binds two substrate molecules at the active site and the inhibitory site, breaks down at a reduced velocity than ES1 complex. This inhibition is classified as being of partial type (k′/k < 1). On the other hand, when ES1S2 complex cannot produce the product, that is, k′ = 0, the inhibition is called complete substrate inhibition.Substrate inhibition is explained by the binding of two substrate molecules at the active sites and the inhibitory sites. Enzyme-substrate complex, v/(Vmax − v) versus 1/[S] at higher and inhibitory concentrations of the substrate: this plot can decide whether the substrate inhibition is of the complete or the partial type, and further can determine dissociation constant of the ES1S2 complex.The purpose of the study on enzyme inhibition is determination of inhibition constants and inhibition types. Several graphical methods have been used for analyzing kinetic parameters Dixon ; howeverVmax values for calculation. This plot can contribute to analysis of the inhibition mechanism and metabolic control. Recent kinetic studies use nonlinear regression analysis with computer simulation methods; thus, present plot and various linearization methods may be useful for initial orientation regarding the action of two substrate molecules.The present study serves as a useful graphical tool for determining substrate inhibition types and inhibition parameters such as v/(Vmax − v) versus 1/[S] gave a linear relationship under the conditions of higher and inhibitory concentrations of substrate. The rate constant of the enzyme–substrate–inhibitor complex (k′/k) and A simple graphical method for determining the inhibition constant of substrate inhibition was presented. Plot of pfkB) was purified from the archived clone of the pfkII-overexpressed E. coli mutant, and the determination of the activity was described previously (Ogawa et al. 2, 0.1 mM NADH, l mM dithiothreitol, l unit of aldolase, 3 units of triose phosphate isomerase, and 8 units of glycerol 3-phosphate dehydrogenase in the presence of 0.1, 0.5, and 5 mM fructose 6-phosphate. The reaction was started by the addition of phosphofructokinase, and the decrease in absorbance at 340 nm was recorded.The source of materials used in this work as follows: NADH, glucose 6-phosphate, aldolase, triose phosphate isomerase, glycerol 3-phosphate dehydrogenase. Phosphofructokinase II (encoded by


In addition, phosphopeptide mapping analysis determined that the S373 residue of mSK1a was the only site phosphorylated by cPKC. Interestingly, alanine substitution of S373 made mSK1a refractory to the inhibitory effect of phorbol esters, whereas glutamate substitution of the same residue resulted in a significant reduction in mSK1a activity, suggesting the significant role of this phosphorylation event. Taken together, we propose that mSK1a is negatively regulated through cPKC-dependent phosphorylation at S373 residue.Sphingosine kinase is a lipid kinase that converts sphingosine into sphingosine-1-phosphate, an important signaling molecule with intracellular and extracellular functions. Although diverse extracellular stimuli influence cellular sphingosine kinase activity, the molecular mechanisms underlying its regulation remain to be clarified. In this study, we investigated the phosphorylation-dependent regulation of mouse sphingosine kinase (mSK) isoforms 1 and 2. mSK1a was robustly phosphorylated in response to extracellular stimuli such as phorbol ester, whereas mSK2 exhibited a high basal level of phosphorylation in quiescent cells regardless of agonist stimulation. Interestingly, phorbol ester-induced phosphorylation of mSK1a correlated with suppression of its activity. Chemical inhibition of conventional PKCs (cPKCs) abolished mSK1a phosphorylation, while overexpression of PKCα, a cPKC isoform, potentiated the phosphorylation, in response to phorbol ester. Furthermore, an  Phorbol 12-myristate 13-acetate (PMA), 4-deoxy pyridoxine, GF109204X, Go6976, and RO-31-8220 were from Merck Millipore . Silica TLC G60 plate was from Merck Millipore. Serum and medium were obtained from HyClone , and other chemicals were from Sigma-Aldrich .S1P and SPH were from Biomol .  ATP for 20 min at 37°C. The labeled lipids were extracted and resolved by TLC in the solvent of 1-butanol/ethanol/acetic acid/water (8:2:1:2). The formation of S1P was visualized and quantitated on a PhosphoImager system .As described previously with minor modifications , the cel3H]SPH  preloaded onto 1 mg/ml fatty acid-free BSA, together with PMA (100 nM) or vehicle. After incubation for 5 min, the assay medium was replaced with 1 ml of pre-chilled methanol. The lipids were extracted, dried, and resolved by TLC in 1-butanol:acetic acid:water (3:1:1) as described previously  inorganic phosphate (0.4 mCi) in 2 ml of phosphate-free DMEM for 3 hrs. After PMA (100 nM) treatment for 10 min, cells were washed twice with ice-cold PBS, lysed in 500 μl of lysis buffer  containing protease inhibitors, and centrifuged at 15000×g for 5 min at 4°C. mSK1a was isolated with α-FLAG Affi-Gel, separated with SDS-PAGE, electro-blotted onto NC membrane, and analyzed by autoradiography.For In vitro phosphorylation by PKC was carried out under standard kinase assay conditions. Briefly, recombinant mSK1a (50 ng) was incubated with purified PKCα (10 ng) in the presence or absence of 1 μM PMA in a total volume of 50 μl of PKC assay buffer  containing 2.5 μCi of [32P]-ATP and 10 μM ATP for the times specified at 30°C. Reactions were terminated by adding 12.5 μl of 5×SDS sample buffer and boiling for 5 min. Proteins were separated by SDS-PAGE and electro-blotted onto NC membrane. The phosphorylated protein bands were localized by autoradiography to PhosphoImager or photographic film.In vivo immunoprecipitated or in vitro32P-labeled mSK1a was resolved by 10% SDS-PAGE and electro-blotted onto NC membrane. Two-dimensional phosphopeptide mapping of phosphorylated mSK1a and phosphoamino acid analysis were carried out on a Hunter thin layer electrophoresis system  as described previously -[_/F/K/V/L/Q/V]-[K/R]-[K/R/G]- - , further[K/R/G]- . Thus, a[K/R/G]- . PKCα ar2+-calmodulin complexes was not affected by phosphorylation or mutation at S373, suggesting that the altered activity with phosphorylation or mutation of S373 is unlikely due to interference of the Ca2+-calmodulin-dependent regulation of mSK1a. Also, notwithstanding the phosphorylation- or S373E substitution-dependent transition to the inactive form, mSK1a exhibited no visible change in protein–protein interaction(s)  . These ovo assay . Collectin vivo.We found that mSK1a is phosphorylated in two distinct sites, S225 and S373. Our results identify S373 of mSK1a as the cPKC-dependent phosphorylation site, as well as S225 as the PKC-independent phosphorylation site. A previous report showed that S225 phosphorylation of hSK1a in response to PMA treatment is mediated by ERK1/2, which plays a key role in PMA-induced activation of mSK1a by facilitating both membrane translocation and enzymatic activation of hSK1a . PresumaIn summary, our results demonstrate that mSK1a is inhibited by cPKC-dependent phosphorylation, and that mSK1a phosphorylation is mediated specifically and directly by cPKC. We also demonstrated that phosphorylation at S373, located in the C-terminal regulatory region, plays a pivotal role in the negative regulation of mSK1a. Nonetheless, we have to admit that our study was conduced mostly with cell stimulation with PMA, a type of phorbol esters which does not exist endogenously in mammals, but mimic the action of diacyl glycerol (DAG), potent activator of PKC . In this2 generates IP3 and diacyl glycerol (DAG), leading to elevation of intracellular Ca2+ and cPKC activation. The increase in cellular Ca2+ may result in Ca2+-calmodulin-dependent activation of mSK1a via direct interaction, whereas cPKC activation may lead to S373 phosphorylation, which results in subsequent inactivation of mSK1a , or its S373E mutant (S373E). After serum deprivation, the cells were treated for 10 min with PMA (100 nM). The cell lysates were incubated with α-FLAG Affi-Gel. The protein complexes co-immunoprecipitated with mSK1a were analyzed with SDS-PAGE followed by Coomassie Brilliant Blue (CBB) staining. Abbreviations: IgGh; Immunoblobulin G heavy chain, IgGl; immunoglobulin G light chain.(TIF)Click here for additional data file.


HIV-1 gene expression is driven by the long terminal repeat (LTR), which contains many binding sites shown to interact with an array of host and viral factors. Selective pressures within the host as well as the low fidelity of reverse transcriptase lead to changes in the relative prevalence of genetic variants within the HIV-1 genome, including the LTR, resulting in viral quasispecies that can be differentially regulated and can potentially establish niches within specific cell types and tissues.Utilizing flow cytometry and electromobility shift assays, specific single-nucleotide sequence polymorphisms (SNPs) were shown to alter both the phenotype of LTR-driven transcription and reactivation. Additional studies also demonstrated differential loading of transcription factors to probes derived from the double-variant LTR as compared to probes from the wild type.This study has identified specific SNPs within CCAAT/enhancer binding protein (C/EBP) site I and Sp site III  that alter LTR-driven gene transcription and may alter the course of viral latency and reactivation. The HIV-1 LAI LTRs containing the SNPs of interest were coupled to a plasmid encoding green fluorescent protein (GFP), and polyclonal HIV-1 LTR-GFP stable cell lines utilizing bone marrow progenitor, T, and monocytic cell lines were constructed and utilized to explore the LTR phenotype associated with these genotypic changes.Although the 3 T and 5 T SNPs have been shown to be low-affinity binding sites, the fact that they can still result in effective HIV-1 LTR-driven gene expression, particularly within the TF-1 cell line, has suggested that the low binding site affinities associated with the 3 T C/EBP site I and 5 T Sp site III are potentially compensated for by the interaction of nuclear factor-κB with its corresponding binding sites under selected physiological and cellular conditions. Additionally, tumor necrosis factor-α and Tat can enhance basal transcription of each SNP-specific HIV-1 LTR; however, differential regulation of the LTR is both SNP- and cell type-specific. Flow cytometry analysis was performed utilizing a Calibur Flow Cytometer  and analyzed the results using Flowjo version 8.8.7 software .TF-1, U-937, and Jurkat cells were washed with flow cytometry buffer [Hanks balanced saline solution (Mediatech), FBS (3%), and NaN32P-ATP and T4 polynucleotide kinase as described . The sequences of the probes used in this study are as follows: LAI LTR WT: TCG AGC TTG CTA CAA GGG ACT TTC CGC TGG GGA CTT TCC AGG GAG GCG TGG CC; LAI LTR 3 T: TCG AGT TTG CTA CAA GGG ACT TTC CGC TGG GGA CTT TCC AGG GAG GCG TGG CC; LAI LTR 5 T: TCG AGC TTG CTA CAA GGG ACT TTC CGC TGG GGA CTT TCC AGG GAG GTG TGG CC; LAI LTR 3T5T: TCG AGT TTG CTA CAA GGG ACT TTC CGC TGG GGA CTT TCC AGG GAG GTG TGG CC. The SNPs are indicated as underlined nucleotides.Complementary single-stranded oligonucleotides corresponding to the LAI LTR with and without the 3 T SNP in C/EBP site I and/or the 5 T SNP in Sp site III were synthesized (Integrated DNA Technologies) and annealed by brief heating at 100°C followed by slow cooling to room temperature as previously described . Blunt-eElectrophoretic mobility shift assay binding reactions were performed as previously described . The rad7 cells were harvested for each nuclear extraction and washed once with 1× phosphate-buffered saline. The cells were incubated for 15 minutes with lysis buffer on ice and then 2 μL 10% NP-40 detergent was added and cells were inverted several times. Cells were centrifuged at 1100 RPM for 5 minutes and then resuspended, through inverting, in lysis buffer and then centrifuged again. The nuclei pellets were then resuspended in nuclear extract buffer and then incubated for at 4°C for 30 minutes on a vortexer set to a speed of 3. The nuclear lysate was centrifuged at 14,000 RPM for 10 minutes at 4°C, after which time the supernatant was removed and quantitated with respect to protein concentration as previously described [Nuclear protein extraction was performed as previously described . Brieflyescribed . NuclearBBB: Blood–brain barrier; BSA: Bovine serum albumin; C/EBP: CCAAT/enhancer binding protein; CNS: Central nervous system; FBS: Fetal bovine serum; GFP: Green fluorescent protein; HAART: Highly active antiretroviral therapy; HIVD: HIV-associated dementia; HPC: Hematopoietic progenitor cell; LTR: Long terminal repeat; MFI: Mean fluorescent intensity; NF-κB: Nuclear factor-κB; RASF: Rheumatoid arthritis synovial fibroblast; SNP: Single-nucleotide polymorphism; TNF-α: Tumor necrosis factor-α; WT: Wild type.The authors declare that they have no competing interests.SS contributed to the development of stably transfected cell lines, developed the stably transfected cell clones, completed all flow cytometric acquisition and analysis, contributed to study conception and design, drafted figures, and drafted the manuscript. KA constructed the LTR-plasmid constructs and contributed to the development of stably transfected populations. SD contributed to the design and completion of the electrophoretic mobility shift assays. VP helped draft the manuscript. SS, MN and BW conceived of the study and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


Inhibition of CDK9 kinase activity did not interfere with in vitro fertilization and pronuclear formation. However, when in vitro produced zygotes were treated with CDK9 inhibitors, their development beyond the 4-cell stage was impaired. In these embryos, inhibition of CDK9 abrogated global transcriptional activity and rRNA production. Collectively, our data suggested that P-TEFb kinase activity is crucial for oocyte maturation, embryo development and regulation of RNA transcription in pig.Positive transcription elongation factor b (P-TEFb) is a RNA polymerase II carboxyl-terminal domain (Pol II CTD) kinase that phosphorylates Ser2 of the CTD and promotes the elongation phase of transcription. Despite the fact that P-TEFb has role in many cellular processes, the role of this kinase complex remains to be understood in mammalian early developmental events. In this study, using immunocytochemical analyses, we found that the P-TEFb components, CDK9, Cyclin T1 and Cyclin T2 were localized to nuclear speckles, as well as in nucleolar-like bodies in pig germinal vesicle oocytes. Using nascent RNA labeling and small molecule inhibitors, we showed that inhibition of CDK9 activity abolished the transcription of GV oocytes globally. Moreover, using fluorescence in situ hybridization, in absence of CDK9 kinase activity the production of ribosomal RNAs was impaired. We also presented the evidences indicating that P-TEFb kinase activity is essential for resumption of oocyte meiosis and embryo development. Treatment with CDK9 inhibitors resulted in germinal vesicle arrest in maturing oocytes  Embryonic genome activation is a highly regulated process by which an embryo begins to produce its own gene products from its newly formed genome. Before the embryonic genome is activated, the embryo is transcriptionally inactive and is dependent on the factors already provided by the oocyte. These factors (mainly proteins and mRNAs) are produced during the course of oocyte growth until the oocyte becomes competent for resumption of meiosis. Before the oocytes become competent to maturation process, the oocyte genome undergoes changes in genome architecture and function which prepare an epigenetic context for the developmental regulation of the global gene expression . Along wPol I mainly synthesizes ribosomal RNAs, while Pol II is responsible for mRNAs and snRNAs production. Although the regulation of rRNA synthesis is well studied in GV oocytes, the mechanism(s) regulating Pol II-dependent transcription is less understood in mammalian oocytes. Pol I and its related transcription factors such as UBF and SL1, are located specifically in the nucleolus. The nucleolus is a prominent sub-nuclear structure that is responsible for the biogenesis of ribosome subunits, 18S, 5.8S and 28S rRNAs. Electron microscopy has permitted researchers to discern three main nucleolar compartments: the fibrillar centers (FCs), the dense fibrillar component (DFC), and the granular component (GC) . Pol I iIn few studies, the presence and the phosphorylation status of Pol II in mammalian GV oocytes have been investigated –19. Pol Gene expression is highly regulated at transcription phase. Pol II function is controlled in multiple steps mainly via phosphorylation and dephosphorylation of its CTD –22. The P-TEFb contributes to multiple cellular functions. For instance, multiple steps of gene expression are influenced by P-TEFb. Beside its crucial role in elongation phase of transcription, P-TEFb function regulates co-transcriptional control of mRNA processing, export as well as mRNA translation in the cytoplasm . ExpressC. elegans cyclin T genes by RNAi had no dramatic change in its development. But when both cyclins were genetically inactivated simultaneously, embryos died at ~100-cell stage. Same phenotype was observed when CDK9 was knocked down by RNAi, indicating the crucial role of P-TEFb in early embryonic development . Incubations with primary antibodies proceeded in PBG for 16 h at 4°C. Cells were subsequently washed four times, for 5 min. each time, in PBG and incubated with the appropriate secondary antibodies for 1 h in PBG at room temperature. Next, the cells were washed twice, for 5 minutes each time, in PBG and twice for 5 minutes each time in PBS. For microscopic observation, embryos were deposited on slides and mounted under coverslips using Vectashield mounting medium containing DAPI (Vector Laboratories). In situ run-on transcription was performed as described earlier , with soOocytes or embryos were fixed in 2% formaldehyde for 15 min and then washed twice in PBS/PVA for 5 min. at room temperature. Cells were then permeabilized with 1% Triton X-100 in PBS for 30 min. Then, cells were washed in 2X saline-sodium citrate (SSC) plus 50% formamide for 5 min. at room temperature. Hybridizations occurred over night at 38.5°C in hybridization buffer consisting of: 10% dextran sulfate, 2mM vanadyl ribonucleoside complex, 0.02% BSA, 40 μg tRNA, and 10 ng of RNA probe in 2X SSC plus 50% formamide. Then, cells were washed with pre-warmed 2X SSC/50% formamide at 37°C for 30 min. Finally, cells were washed twice in PBS/PVA and mounted in Vectashield mounting medium containing DAPI. The sequences of the probes corresponding to pig 5’ETS rRNA (Accession no: L31782) and 28S rRNA (Accession no: 4W24_5) were:5’ETS antisense: Cy3-gccagagaccggaggagacaccgcagcacgggcc; 5’ETS sense: Cy3-ggcccgtgctgcggtgtctcctccggtctctggc. 28S antisense: FAM-ccccacccgtttacctcttaacggtttcacgccc; 28S sense: FAM-gggcgtgaaaccgttaagaggtaaacgggtgggg. The probes were produced by Bioneer, Inc. Korea.Images were captured using a Zeiss scanning laser confocal microscope running Zeiss LSM Image Browser software. Serial optical sections (the Z-series) were collected at 0.5 μm intervals. The Z-stacks were captured and images depicting staining patterns and intensities of all nuclear areas were generated. All oocyte and embryo samples were prepared and processed simultaneously prior to fluorescence intensity measurements. The laser power was adjusted to ensure that signal intensity was below saturation for the specimen that displayed the highest intensity and all images were next scanned at that laser power.The presence and localization of P-TEFb components have never before been addressed in porcine oocytes. Initially, using specific antibodies, we observed that CDK9, Cyclin T1 and Cyclin T2 are present and dominate the GVs of oocytes obtained from pig antral follicles . SimilarTo our knowledge, the nucleolar association of P-TEFb components had not been addressed previously in oocytes or embryos and we were curious to see the extent, if any, of CDK9 or Cyclin T1's spatial connection to the nucleolus in pig GV oocytes. Co-immunostaining of CDK9 with nucleolar markers, fibrillarin revealed that in NSN oocytes, fibrillarin was strongly accumulated inside the NLB and some of the CDK9 accumulations closed to NLB were colocalized with marginal nucleolar fibrillarin . In pNSNWe and others have previously shown that NSN oocytes are mostly found in COCs obtained from small follicles. Also, the population of SN oocytes is higher when COCs are extracted from large follicles , 6. We wThe association of CDK9 to the nucleolar structures prompted us to consider the existence of a functional relation between CDK9 and the nucleolus. To study this, we selected GV oocytes in their active transcription state, NSN configuration. Regarding the sensitivity of pig oocytes to permeabilization and microinjection, we employed a cell-permeable modified RNA precursor, 5-fluorouridine (FU), to identify transcription sites and changes occurring during pig oocyte growth. Our previous experiments showed that FU can be successfully incorporated into both nascent RNAs by simple incubation of oocytes in FU-containing culture medium followed by immunostaining. Moreover, FU incorporation appears sensitive to both RNA synthesis inhibitors and activators . First, To obtain a better understanding about the CDK9’s role in oocyte transcription, we treated GV oocytes in their transcriptionally active state (NSN) with CDK9 specific inhibitors Flavopiridol and CDK9 Inhibitor II (CAN508). When COCs were treated with either compound for 1 hour and labeled with FU, no transcriptional activity was observed in NSN or in pNSN oocytes. When analyzed carefully under a confocal microscope, we observed that inhibition of CDK9 activity resulted in decline in transcription almost globally which differed with the situation in α-amanitin-treated oocytes reported previously . To obtaBecause FU labeling of nascent RNAs does not distinguish between RNA polymerases’ products, we set out to revisit the effect of CDK9 inhibition on oocyte nucleolar transcription by applying rRNA-FISH. To this end, fluorescently labeled DNA probes were designed to detect two distant parts of pre-rRNA; one probe complementary to 34 nucleotides of the 5’ external transcribed spacer (5’ETS), and another probe complementary to 34 nucleotides of 28S rRNA. Since we found no report of similar study in pig oocytes, before the analysis of CDK9 inhibition, we first collected all the oocytes of different follicular size to analyze the situation of pre-RNA transcription and processing in these cells. We found that in NSN oocytes, a strong central signal emitted from 5’ETS surrounded by mutually exclusive localization of 28S rRNA probe. In pNSN oocytes the state of 5’ETS remained almost unchanged but 28S RNA became weaker in its intensity and marginal in its localization . As expein vitro up to 44 hours. First, we treated COCs with 50, 100 or 300 nM Flavopiridol for 22 hours and monitored the expansion of cumulus cells. Measurements under a light microscope equipped with a graded optical lens revealed that by 100 nM or higher concentrations, the expansion of cumulus cells was dramatically declined compared to non-treated control group  activity in GV oocytes. To address this question, we set to analyze the effect of CDK9 inhibition on both CDK1 kinase activity and inhibitory phosphorylation of CDK1 on Thr14/Tyr15 residues by immunocytochemistry. Low activity of CDK1is required for meiotic prophase I arrest, which resembles the G2 phase of mitosis in somatic cells . IncreasThen, we repeated the experiment to compare the effects of these two CDK inhibitors with that of Flavopiridol. COCs were cultured in a complete maturation medium in absence or presence of compounds for 6 hours and then subjected to immunostaining against MPM-2 antigens. Analysis of the specimens by confocal microscopy revealed that in untreated oocytes, the fluorescence intensity of MPM-2 was only slightly higher than that of the oocytes in which CDK9 activity was inhibited by Flavopiridol . In Dinain vitro by in vitro fertilization or parthenogenetic activation, were cultured and, at different stages, subjected to immunocytochemical analyses. Initially we observed that in embryos produced by IVF, CDK9 is present in all the stages of preimplantation development and is well colocalized with Pol II  with different mechanism of action revealed that both Pol I- and Pol II-dependent transcriptions are sensitive to CDK9 activity. To understand the potential role of CDK9 in Pol I-dependent transcription, we inhibited the enzyme’s kinase activity using a CDK9 specific inhibitors, Flavopiridol and CAN508 . Flavopi in vivo , 43, 44. in vivo , 45. In  studies . When thLabeling of nascent RNAs with a cell permeable halogenated nucleotide, FU, overcomes the difficulty of BrUTP application that should mostly be injected into oocytes or embryos. However, these nucleotide analogues do not distinguish between the transcripts of different RNA polymerases. To address the potential role of CDK9 specifically in Pol I transcription, we applied fluorescently labeled probes to detect pre-rRNA transcripts. We could not find any similar report in the literature addressing the situation of rRNA transcription in pig oocytes or embryos, so we applied a standard RNA-FISH procedure with some modifications . The proAlso we showed that CDK9 function is crucial for resumption of meiosis in pig oocytes . It is win vitro. Our analyses showed that Flavopiridol did not inhibit CDK1 kinase activity, but both Dinaciclib and JNJ-7706621 negatively affected the level of phosphorylation of CDK1 targets. Also, treatment with JNJ-7706621 confirmed that CDK1 inhibition blocked GVBD in a transcription-independent manner.The second most sensitive CDK to Flavopiridol is CDK1 , 72. CDKin vitro [C. elegans [We previously have shown that CDK9 inhibition blocks mouse embryos at 2-cell stage in vitro . We alsoin vitro , 76. The elegans  and Dros elegans . In both elegans  and the  elegans , 79. In In conclusion, our data show the importance of CDK9 kinase activity in both resumption of meiosis and embryonic genome activation in pig. Moreover, we find that CDK9 is involved in both Pol I- and Pol II-dependent transcription in pig early development.S1 FigA, B and C) treated COCs and A’, B’ and C’) untreated COCs assessed after 0, 22 and 44 hours, respectively. D) Mean diameters of COCs in both group at indicated times of culture.COCs were collected from large antral follicles and were cultured in complete maturation medium with gonadotropins in presence or absence of 100 nM Flavopiridol. After 22 hours, the expansion of cumulus cells was assessed under a graded light microscope and then, COCs were transferred to the same medium without gonadotropins and analyzed 22 hours later. The figure shows that Flavopiridol strictly inhibited the cumulus cell expansion in pig COCs. (DOCX)Click here for additional data file.


Resveratrol (RSV) is a natural polyphenol that has a beneficial effect on health, and resveratrol-induced autophagy has been suggested to be a key process in mediating many beneficial effects of resveratrol, such as reduction of inflammation and induction of cancer cell death. Although various resveratrol targets have been suggested, the molecule that mediates resveratrol-induced autophagy remains unknown. Here, we demonstrate that resveratrol induces autophagy by directly inhibiting the mTOR-ULK1 pathway. We found that inhibition of mTOR activity and presence of ULK1 are required for autophagy induction by resveratrol. In line with this mTOR dependency, we found that resveratrol suppresses the viability of MCF7 cells but not of SW620 cells, which are mTOR inhibitor sensitive and insensitive cancer cells, respectively. We also found that resveratrol-induced cancer cell suppression occurred ULK1 dependently. For the mechanism of action of resveratrol on mTOR inhibition, we demonstrate that resveratrol directly inhibits mTOR. We found that resveratrol inhibits mTOR by docking onto the ATP-binding pocket of mTOR . We propose mTOR as a novel direct target of resveratrol, and inhibition of mTOR is necessary for autophagy induction. Resveratrol  is a natural polyphenolic compound found in the roots of plants and in edible fruits including berries and grapes. Administration of resveratrol is thought to elicit beneficial effects, including alleviation of inflammation and tumor cell death in applied cells and organisms256mTOR , a Ser/Thr kinase present in cells, functions within two distinct complexes: mTORC1 (mTOR complex 1) and mTORC2 (mTOR complex 2). These complexes share mTOR as a kinase subunit, but contain different adaptors and scaffolds for distinct functions and regulatory mechanisms. mTORC1 controls cell growth and proliferation through the regulation of various processes and promotes translation through the phosphorylation of S6K and 4E-BP1. Lipid synthesis is also enhanced by active mTOR through the regulation of PPAR-γ, SREBP, and Lipin18111213515Here, we demonstrate that resveratrol induces autophagy through the mTOR-ULK1 pathway. Additionally, we found that resveratrol induces death of the cancer cells known to be sensitive to mTOR inhibition in ULK1 dependent manner. With respect to the mechanism by which resveratrol reduces mTOR activity, we demonstrated that resveratrol directly inhibits mTOR in an ATP-competitive manner. Through this study we suggest that mTOR is a distinct and direct target of resveratrol, and inhibition of mTOR is necessary for resveratrol-induced autophagy.In order to understand resveratrol-induced autophagy, we examined the effect of resveratrol on autophagy in GFP-LC3 expressing HeLa cells. Resveratrol treatment induces autophagy, as evidenced by the accumulation of LC3B-II and LC3 puncta formationmTOR regulates autophagy through inhibitory phosphorylation of ULK1 and inhibition of mTOR decreases the inhibitory phosphorylation level of ULK1 and increases autophagy13in vitro and in vivo46As previous studies showed, resveratrol-induced autophagy suppresses cancer et al. showed that resveratrol increased the interaction affinity between mTOR and DEPTOR and suggested that DEPTOR is required for the suppression of mTOR activity upon resveratrol treatmentDEPTOR is a negative regulator of both mTORC1 and mTORC2. Liu in vitro kinase assay. We observed that resveratrol dose-dependently decreased the phosphorylation level of 4E-BP1  mTOR but not D2195A mTOR. The basal activity level of the D2195A mutant was lower than that of WT mTOR, possibly due to the fact that the mutated residue resides in the kinase domain of the protein. We observed that PP242 also inhibited DA mTOR, albeit the inhibitory potency was less than WT mTOR , were used as substrates. These results suggested that SIRT1 may not be the direct target of resveratrol, although resveratrol clearly activates SIRT1 in vivo25262729303133et al. suggested PDEs (phosphodiesterases) as a direct target of resveratrol. In their report, they found that resveratrol inhibits PDEs through competition with cAMP (cyclic AMP), a substrate of PDE. They also observed that inhibition of PDE is enough to mimic the effects of resveratrol administration in mice, such as AMPK activation, SIRT1 inhibition, and enhancement of metabolic featuresBecause of its beneficial effects, many researchers have attempted to identify the molecular target of resveratrol. SIRT1, a NADResveratrol was initially identified as a SIRT1 activator because of its structural similarity to quercetinin vitro activity assays. Among these enzymes, PI3K and PKC showed ATP-dependent inhibition by resveratrol3750 value for resveratrol compared to PI3K, PKC, and PDE. Resveratrol, ATP, and cAMP are similar in structure, which suggested that resveratrol could compete with cAMP or ATP for single binding sites. Additionally, PP242 and quercetin, an mTOR kinase inhibitor and a natural polyphenol, respectively, are similar in structure.Resveratrol was reported to directly inhibit various enzymes as demonstrated by et al. suggested acquired resistance to resveratrol in SW620 cells, which is dependent on the DNA-damage responseWe found that resveratrol showed a cancer-cell-type-selective effect on viability in that it suppressed only the viability of MC7 cells, but not that of SW620 cells . Howeveret al. showed that resveratrol suppresses mTOR activity by enhancing the affinity between mTOR and DEPTOR, a negative regulator of mTOR. However, we observed no changes in the level of mTOR-interacting DEPTOR , 200 ng of 4E-BP1 (Stratagene) and the indicated amount of resveratrol for 10 min on ice. The reaction was started with the addition of 50 or 100 μM ATP and was conducted at 30 °C for 10 min. The reaction was stopped by the addition of 5x sample buffer. For a kinase assay using recombinant mTOR, GST-mTOR (Life Technologies) was used and GST-S6K, which is generated by PCR amplification of kinase dead fragment S6K (322 to C-term) and subcloning into pGEX-4T1 vector was purified from E.coli and used as a substrate. An innhibitory curve was drawn using Sigma Plot.mTORC1 kinase assay was performed as described previously, with some modificationsHA-mTOR cDNA, HA-Raptor cDNA , FLAG-mTOR cDNA (insertion of PCR product from HA-mTOR cDNA into pCMV vector) and FLAG-D2195A mTOR (generated by site directed mutagensis) were transfected with Lipofectamine (Invitrogen) according to the manufacturer’s instructions. Cells were lysed in buffer containing 40 mM HEPES, 120 mM NaCl, 50 mM NaF, 10 mM β-glycerophosphate, 1 mM EDTA, 10 mM sodium pyrophosphate, 1 mM PMSF, 1 μg/μl aprotinin, 2 μg/μl leupeptin and 0.3% CHAPS. Except for the IP samples, 1% TX-100 was used as a detergent. Then 700–1000 μg of protein was incubated with the respective antibody for 4 h followed by incubation with protein A-conjugated agarose beads. Antibody immobilized beads (Sigma-Aldrich) were used for FLAG-mTOR. Beads were washed three times with the lysis buffer and resuspended with the sample buffer. Prepared samples were subjected to SDS-PAGE (6–16% gradient) and transferred to NC membranes (Amersham Biosciences). The membranes were blocked with TTBS buffer containing 5% skim milk and incubated overnight at 4 °C with antibodies against ULK1 (Santa Cruz Biotechnology), Rictor (Bethyl Laboratories), actin  and other proteins . The membranes were next washed with TTBS, incubated with HRP-conjugated secondary antibodies at room temperature for 1 h, and washed again with TTBS. Antibody-bound membranes were developed using ECL (Thermo Fisher).On day 1, HeLa cells stably expressing GFP_LC3  were plated onto poly-lysine coated glass coverslips. On day 2, medium was changed with fresh medium containing DMSO or resveratrol at the indicated concentration for 2 h. The medium was then aspirated and cells were washed twice with PBS. Cells were fixed with 4% formaldehyde for 10 min at RT and stained with DAPI for 5 min at RT. Washing three times with PBS was conducted between each step. The coverslips were mounted on a slide glass and visualized using confocal microscopy (Carl Zeiss), and randomly captured images from various fields were used for autophagy formation analysis. GFP punctate positive cells were quantified as autophagy induced cells. The total number of cells was determined by measuring DAPI-positive cells.Lentiviral shRNAs were prepared and transduced according to the Addgene protocol. Briefly, the pLKO empty vector or pLKO shULK1_8 (purchased from Addgene) was transfected with pMD2.G and psPAX2 (purchased from Addgene) using Lipofectamine according to the manufacturer’s instructions(Invitrogen). Media were collected at 24 h and 48 h after transfection. Collected media were centrifuged at 1200 rpm for 5 min and filtered with 0.45-μm filter. For virus infection, virus-containing media were diluted with growth medium and added to plated cells. Twenty-four hours after infection, the medium was replaced with fresh medium. Twenty-four hours after changing the medium, infected cells were used for the various experiments based on experimental purpose.Three dimensional 3D) atomic coordinates were prepared from the X-ray crystal structure of mTOR in complex with the potent inhibitor, Torin2 (PDB code: 4JSX)D atomic Microsoft Excel was used for statistical analysis. Data are presented as means ± SEM. Comparisons between two groups were made by unpaired two-tailed Student’s t-tests. p values < 0.05 were considered statistically significant.How to cite this article: Park, D. et al. Resveratrol induces autophagy by directly inhibiting mTOR through ATP competition. Sci. Rep.6, 21772; doi: 10.1038/srep21772 (2016).


Interestingly, status epilepticus induced by systemic kainate injection in mice led to specific upregulation of the CPEB3 isoforms containing exon seven. Extensive analysis of CPEB3 phosphorylation in vitro revealed two other phosphorylation sites. In addition, we found plethora of potential kinases that might be targeting the alternatively spliced kinase consensus site of CPEB3. As this site is highly conserved between the CPEB family members, we suggest the existence of a splicing-based regulatory mechanism of CPEB function, and describe a robust phosphospecific antibody to study it in future.Cytoplasmic Polyadenylation Element Binding proteins (CPEBs) are a family of polyadenylation factors interacting with 3’UTRs of mRNA and thereby regulating gene expression. Various functions of CPEBs in development, synaptic plasticity, and cellular senescence have been reported. Four CPEB family members of partially overlapping functions have been described to date, each containing a distinct alternatively spliced region. This region is highly conserved between CPEBs-2-4 and contains a putative phosphorylation consensus, overlapping with the exon seven of CPEB3. We previously found CPEBs-2-4 splice isoforms containing exon seven to be predominantly present in neurons, and the isoform expression pattern to be cell type-specific. Here, focusing on the alternatively spliced region of CPEB3, we determined that putative neuronal isoforms of CPEB3 are phosphorylated. Using a new phosphospecific antibody directed to the phosphorylation consensus we found Protein Kinase A and Calcium/Calmodulin-dependent Protein Kinase II to robustly phosphorylate CPEB3  Xenopus laeavis as translational activators of otherwise dormant mRNA during oocyte maturation  was pre-activated (15 min at 30°C) in 1x kinase buffer in the presence of 2 mM CaPhosphorylated peptides (PSL) were desalted on the C18 ZipTips (Millipore). C18-bound peptides were eluted with 5 μl of the matrix (α-HCCA saturated in 33.3% acetonitrile/0.1%TFA), and applied on a MALDI target plate. Samples were dried at room temperature and analyzed on the Biflex III MALDI-TOF MS spectrometer , using the following parameters: IS1: 19 kV, IS2: 17.2 kV, lens: 8.8 kV, PIE: 200 ns, gating: maximum. Spectra were recorded in the linear positive mode at a laser frequency of 20 Hz, within a mass range from 1,000 to 6,000 Da. For each spectrum, at least 300 laser shots in 30 shot steps were collected. Each sample was analyzed at least in duplicate. Visual estimation of the mass spectra, raw data smoothing and baseline subtraction was performed using the FlexAnalysis 1.0 software. Obtained data was analyzed using the GraphPad Prism 5.0 software.HEK-293 cells were grown in DMEM medium supplemented with 10% FCS, 25 mM glucose, 6 mM L-glutamine, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate . CPEB3a-EGFP CMV expression plasmid was generated by inserting mouse CPEB3a ORF into EGFP-N1 vector backbone (Clontech). CPEB3a-FLAG and EGFP-FLAG were generated by inserting respectively CPEB3a and EGFP ORF into 3xFLAG-CMV-7.1 vector backbone (Sigma). mRuby-CaMKII was generated by replacing mGFP by mRuby (gift from J. Wiedenmann) in the mGFP-CaMKII plasmid ) . All tra2 [HEK-293 cells were transfected with either the CPEB3a-EGFP vector only or both the CPEB3a-EGFP and mRuby-CaMKII vectors. 16 h post-transfection, the cells were transferred for 5 min in 1x Hank’s Balanced Salt Solution  only or stimulated in HBSS with 10 μM ionomycin and 5 mM CaCl2 . Immedia2 . The mea2 and 5 mM MgCl2 supplemented with the same drugs used for pre-incubation. After 5 min, cells were either transferred for 90 s in depolarizing solution consisting of HBSS supplemented with 40 mM KCl, 2.5 mM CaCl2, and 1 mM MgCl2 plus either 10 μM KN93, 10 μM H89, 100 μM RP-cAMPS or no drugs, or for 5 min in a standard solution consisting of HBSS supplemented with 2 mM glucose, 1.2 mM CaCl2 and 1 mM MgCl2 and containing the drugs used in pre-incubation plus 50 μM FS  for the PKA activation experiments. Thereafter the cells were immediately fixed in 4% PFA. The immunofluorescence, image acquisition and analysis were performed as described for the HEK-293 cells.Dissociated hippocampal neurons were prepared as previously described , 34. Neustatus epilepticus were killed by cervical dislocation, decapitated and the brain was quickly removed, followed by dissection of the hippocampus. Total hippocampal RNA was extracted using RNAeasy Lipid Tissue Mini kit (Qiagen) with on-column gDNA digestion. Reverse transcription was done with SuperScript III RT (Invitrogen), using equal amount (1–2 μg) of RNA template, and random hexamers for priming. 0.5 μl of obtained cDNA was used for Taq-Man RT-PCR, carried out using Gene Expression Master Mix (Applied Biosystems), in Optical 384-Well Reaction Plate PCR plates (Applied Biosystems) with a 7900HT Fast Real-Time PCR System (Applied Biosystems), according to product recommendations. For each reaction, critical threshold cycle (Ct) value was determined using SDS 5.0 Software (Applied Biosystems). Efficiency of the primer/probes sets was calculated according to the serial dilution method [C57BL/6N mice (Charles River) at 3 months of age were injected intraperitoneally with kainite (2 mg/kg body weight) or sham injected with PBS. 30 min post injection, mice experiencing consistent n method , 37. Dat3, 270 mM sucrose, 1% Triton X-100, 1% NP-40, supplemented with Complete Mini Protease Inhibitors and PhosStop Phosphatase Inhibitor Cocktail (both from Roche). Lysates were sonicated for 10 min and spun down (10 min / 10.000 g / 4°C) to pellet cell debris. Total protein content was assayed with a BCA (Pierce). Lysates were mixed with denaturing sample buffer and heated for 5 min at 95°C. Proteins  were separated by discontinuous SDS-PAGE in denaturing conditions [HEK-293 cells were transfected with CPEB3a-EGFP vector and 24 h post transfection medium was supplemented with 50 μM or 200 μM FS (New England Biolabs) or DMSO (as diluent control) for 1 h. After a brief rinse with ice-cold PBS, cells were harvested by scraping in a buffer containing 50 mM Tris-Cl, pH 7.5, 50 mM NaCl, 50 mM NaF, 2 mM EDTA, 2 mM NaVOnditions  and elecSSLFPFED peptide coupled to KLH were used for immunization. Obtained serum was purified positively against modified (phosphorylated) peptides, following depletion of the non-phosphospecific fraction against the unmodified peptide. Assessing specificity: biotynylated peptide substrate corresponding to CPEB3a/c was phosphorylated in vitro with recombinant PKA-C (New England Biolabs), immunoblotted, and detected with phospho-CPEB3a/c antibody following goat-anti-rabbit HRP-coupled secondary antibody . Biotin-HRP was used to control for equal loading. Assessing crossreactivity with CPEB2 and CPEB4: recombinant vectors encoding CPEB2a-EGFP and CPEB3a-EGFP  were transfected into HEK-293 cells. Cells were harvested by scraping in ice-cold kinase lysis buffer (1x kinase buffer (NEB), 1% Tx-100) supplemented with Complete Mini Protease Inhibitors and PhosStop Phosphatase Inhibitor Cocktail (Roche). Lysates were spun down (15.000 g / 10 min) to pellet the cell debris, incubated with PKA-C  and analyzed by Western blot.Rabbit polyclonal antibody was generated at Eurogentec GmbH. Bi-phosphorylated (pS419 and pS420) and monophosphorylated (pS419 or pS420) forms of the RRGR2. Beads were mixed with kinase buffer containing 1 mM ATP and PKA-Cα , pre-activated CaMKII , or the corresponding buffer without enzyme. Sample was incubated for 30 min at 30°C with rotation and then mixed with 50 μl of NuPage reducing sample buffer and separated by SDS PAGE.HEK-293 cells expressing recombinant FLAG-CPEB3a protein were harvested 36 h post-transfection by scraping in ice-cold lysis buffer containing 10 mM HEPES pH 7.4, 200 mM NaCl, 2 mM EDTA, 1% Tx-100, Complete Mini protease inhibitors (1 tablet/10 ml) and Phos-Stop phosphatase inhibitors (1 tablet/10 ml). Lysates were sonicated for 10 min in ice-cold sonication bath and spun down (15 min/17.000 rcf). Supernatants were mixed with 50 μl of FLAG M2 agarose beads (Sigma), previously blocked with 3% BSA, and incubated for 2 h at 4°C on a rotator, followed by 5x washing with lysis buffer. CaMKII was preactivated at 30°C for 30 min in kinase reaction buffer (New England Biolabs) supplemented with 1.2 μM calmodulin and 2 mM CaCl6). The eight most intense precursor ions (threshold intensity 5000) were subjected to collision induced dissociation with multiple stage activation  and fragment spectra recorded in the linear ion trap. After acquisition of eight MSA-spectra, the same eight precursor ions were subjected to higher energy collisional dissociation  and fragment spectra recorded in the Orbitrap detector. Fragmented peptide ions were excluded from repeat analysis for 20 s. Raw data processing and analysis of database searches were performed with Proteome Discoverer software 1.3 (Thermo Fisher Scientific). Peptide identification was done with an in house Mascot server version 2.3 (Matrix Science Ltd) and the Sequest search node in Proteome Discoverer. MS2 data were searched against mouse sequences from SwissProt (release 2012_07). Precursor ion m/z tolerance was 8 ppm, fragment ion tolerance 0.6 Da (CID) or 0.02Da (HCD). b- and y-ion series were included. Semitryptic peptides with up to two missed cleavages were searched. The following dynamic modifications were tested: phosphorylation of serine, threonine, and tyrosine, oxidation of methionine, propionamide or carbamidomethyl on cysteine, and acetylation of the protein N-terminus. Mascot and Sequest results from searches against SwissProt were sent to the percolator algorithm version [Coomassie-stained proteins were excised and subjected to tryptic in gel digestion , 40. In  version  1.17 as in silico predictions [in vitro, followed by Matrix-assisted laser desorption/ionization–time of flight (MALDI-TOF) mass spectrometry. PKA activity led to a shift of the a/c-peptide mass peak, as expected from addition of one (80 Da) or two (160 Da) phosphate (Pi) groups  groups , while t) groups . S420 wa) groups . Interes2+/ionomycin stimulation compared with CaMKII-free controls . The immunization strategy was designed so that the antibody would recognize the monophosphorylated forms of the protein (pS419 or pS420), as well as the bi-phosphorylated species (pS419 and pS420). The specificity of the antibody was confirmed on synthetic peptide substrate corresponding the CPEB3a/c isoform, and on the recombinant FLAG-tagged CPEB3a protein. Antibody pCPEB3-S419/S420 detected the synthetic peptide and the overexpressed FLAG-CPEB3a with high affinity and phosphospecificity . We obsecontrols . Forskolcontrols , p<0.05.controls .Primary hippocampal neurons expressing CPEB3a-mGFP and depolarized with 40 mM KCl for 90 s showed significantly higher phosphorylation of CPEB3a compared with unstimulated controls , p<0.05.in-situ hybridization (ISH) that hyperactivity leads to upregulation of CPEB3 in principal neurons of the hippocampus and cortex [We previously showed by d cortex . Since nin vitro by CaMKII, but the number of PSMs associated with this site was low. Localization of phosphorylation to S439, S443, and S444 was complicated by the ambiguity of spectra produced by mono- and bisphosphorylated peptides containing these amino acids. Many longer monophosporylated peptides containing S419 to T446 were identified after PKA treatment. Exact localization of the phosphosites was not possible but fragment ion spectra indicated a probable modification on (S419 or S420) or  rather than S432, S439, S440, or T446. Evidence for double phosphorylation on (S419 or S420) and on (S443 or S444) was found after CaMKII treatment. In PKA treated samples three spectra of triply phosphorylated peptide species appeared indicating phosphorylation on S419 and/or S420 along with S444 and possibly S432. Triply phosphorylated species were not found in other samples. Results are summarized in To look for other phosphorylation sites on CPEB3a, a FLAG-tagged CPEB3a was transiently expressed in HEK-293 cells and then immunoprecipitated . Part ofin vitro phosphorylation of this region, which was not observed for the corresponding peptide lacking the B-region (data not shown). The analysis revealed CPEB3a/c to be a likely substrate for such kinases as the tumor suppressor WNKII, Ribosomal Protein S6 Kinase (RPS6K) and Calcium/Calmodulin-dependent Protein Kinase IV (CaMKIV)  . The comXenopus CPEB is phosphorylated by Aurora kinase, leading to increased polyadenylation of maternal mRNA [Xenopus CPEB) is phosphorylated by Aurora and upon NMDA receptor activation, via Aurora and CaMKII kinase [Phosphorylation was previously shown to regulate CPEB activity. nal mRNA , 45. In I kinase , 8, 46. I kinase –49, overI kinase . In contin silico [The CPEB3 protein in mouse brain occurs in four splice variants: a, b, c, and d, differing by the presence (or absence) of the B- and C-regions . The B-rn silico , 21, 25.n silico , 25, conin vivo leads to changes in ratios of CPEB3 isoforms. To this end we have designed a quantitative multiplex RT-PCR assay, to discriminate between splice variants containing- and lacking the B-region. We then performed systemic injections of kainate (an agonist of non-NMDA type glutamate receptors) in mice to induce epileptic seizures. Such treatment led to a shift in mRNA levels of CPEB3 splice variants containing the B-region, but not the ones lacking it, consistent with our previous observations [As CPEB3 is implicated in synaptic plasticity , 27, 51,rvations . Certainin silico predictions [2+/ionomycin stimulation in HEK-293 cells. KCl depolarization of primary neurons transfected with CPEB3a showed CaMKII- and PKA-dependent S419/S420 phosphorylation. Addition of OA led to increased phospho-CPEB3a, suggesting a rapid dephosphorylation under basal conditions. We speculate that depolarization might lead to activation of CaMKIIα and PKA, but also to activation of phosphatases (e.g. PP1), as suggested by the additive effect of co-application of FS and OA. In a parallel study we showed that interfering with CPEBs-1-4 function by overexpressing an N-terminally truncated CPEB-1  leads to deficits in LTP and spatial reference memory . Considering the overlap mRNA target specificity between CPEBs, such a transgene likely interferes with the function of CPEBs2-4. Indeed, a similar memory impairment phenotype was observed in CPEB3 KO mice, due to upregulation of AMPA receptors in postsynaptic density (PSD) [in vivo in purified post PSD fractions [Aplysia CPEB (ApCPEB) is involved in long-term facilitation (LTF) by forming a prion-like multimers upon neuronal (serotonin) stimulation [To explore this further, and to facilitate future studies, we have raised an antibody detecting phosphorylation of CPEB3a at serine residues S419/S420. With this antibody we showed for the first time in cells, that CPEB3 splice variants containing the B-region are targets of PKA and CaMKII. We tested CPEB3a phosphorylation in HEK-293 cells and in cultured hippocampal neurons. In each experimental paradigm, FS (adenylyl cyclase activator) led to a robust increase in phospho-CPEB3 levels, which was reversed by the PKA inhibitor H89. As FS also indirectly activates CaMKII by triggering intracellular calcium release, we cannot exclude its contribution to CPEB3 phosphorylation in neurons , which wdictions , and woudictions , 53. CaMty (PSD) . In addity (PSD) . CPEB3 Kty (PSD) , and wasractions . Of notemulation , 56. ApCmulation  and to mmulation . To datein vitro analysis of CPEB3 phosphorylation. By LC/MS we confirmed that S419 and S420 are the primary targets of phosphorylation, at least by PKA and CaMKII. In addition, the analysis revealed two other sites undergoing phosphorylation in HEK-293 cells, which might have a regulatory role as well. Altogether, this detailed analysis of CPEB3 phosphorylation in vitro and in cells expands our understanding of the CPEB family, while the pCPEB3-S419/S420 antibody may be a useful tool to study the regulation of CPEB3 in physiological and pathological conditions.In addition to the four isoforms we studied , additional CPEB3 isoforms have been found in mouse retina . We founS1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S1 Text(PDF)Click here for additional data file.


The transition metal copper enhances amyloid β aggregation and neurotoxicity, and in models of concomitant amyloid and tau pathology, copper also promotes tau aggregation. Since it is not clear if the effects of environmental copper upon tau pathology are dependent on the presence of pathological amyloid β, we tested the effects of copper overload and complexing in disease models which lack pathological amyloid β.We used cell culture and transgenic murine models to test the effects of environmental copper on tau phosphorylation. We used oral zinc acetate as a copper lowering agent in mice and examined changes in blood and brain metals through inductively coupled plasma mass spectroscopy. Behavioral effects of copper lowering were assessed with Morris water maze and novel object recognition tasks. Changes in tau phosphorylation were examined by phosphorylation specific antibodies on Western blots.In human neuroblastoma cells, excess copper promoted tau phosphorylation and a copper complexing agent, tetrathiomolybdate, attenuated tau phosphorylation. In a transgenic mouse model expressing wild type human tau, copper-lowering by oral zinc suppressed plasma and brain levels of copper, and resulted in a marked attenuation of tau phosphorylation. No significant changes in behavior were observed with copper lowering, but a trend to improved recognition of the novel object was observed in zinc acetate treated mice.We propose that reduction of brain copper by blocking uptake of copper from the diet may be a viable strategy for modulating tau pathology in Alzheimer’s disease. The potential benefits of this approach are tempered by the absence of a behavioral benefit and by the health risks of excessive lowering of copper.The online version of this article (doi:10.1186/2047-9158-3-24) contains supplementary material, which is available to authorized users. Alzheimer’s disease (AD) is a progressive neurodegenerative disease that manifests symptoms years after initial abnormal processes begin. It is characterized pathologically by the presence of extracellular amyloid β (Aβ) plaques and intracellular neurofibrillary tau tangles (NFTs). The largest risk factor for AD is age, however increasing evidence points to other environmental factors as playing a role in AD. In particular, the transition metals zinc and copper have been shown to be involved in the progression of AD. Studies examining metal levels in humans indicate an increase of copper during aging, and further increased concentration of copper within AD brains . Aβ has Previous studies examining various copper lowering therapies have attempted to chelate copper from the brain. Clioquinol and its derivative PBT2 were developed as blood brain barrier permeable chelators and had promising results in clinical trials at improving cognition and Aβ in the CSF , 14, 15.We tested the hypothesis that copper modulates tau phosphorylation in the absence of pathological Aβ in a human neuroblastoma cell line (SH-SY5Y) and in a transgenic mouse expressing wild type human tau (hTau). The SH-SY5Y cell line is a widely used model, including studies of tau biology. The hTau mouse model was selected due to the expression of full length wild-type human tau, which more closely resembles idiopathic AD. This mouse model contains the entire wild-type human tau gene with the endogenous mouse tau gene disrupted, resulting in hyperphosphorylated tau and accumulation in an age dependent manner.The human neuroblastoma SH-SY5Y cells were obtained from ATCC CRL-2266) and grown in DMEM/F12 Media (11320–082 Gibco) and supplemented with 10% FBS (16000–044 Gibco), non-essential amino acids (NEAA) (11140–050 Gibco), and penicillin/streptomycin (15140–122 Gibco). For experiments, cells were plated in growth media with 10 μM retinoic acid (R2625 Sigma) (in DMSO) to induce outgrowth of projections and upregulation of tau protein  (Thermo) 1:500 mouse IgG, AT180 [phosphorylated T231/S235 tau] (Thermo) 1:500 mouse IgG, PHF-1 [phosphorylated S396/S404 tau] (Peter Davies) 1:500 mouse IgG, pT231 [phosphorylated T231 tau] (Thermo) 1:1000 rabbit IgG, Tau12 (Skip Binder) 1:5000 mouse IgG, GSK-3β total  1:1000 mouse IgG, Phosphorylated-GSK-3β S9  1:1000 rabbit IgG, CDK5  1:1000 rabbit IgG, p35/p25  1:500 rabbit IgG, and GAPDH (Thermo) 1:5000 mouse IgG. Blots were imaged using a Bio-Rad Gel Doc XR Imaging platform with Image Lab software. Images were exported as *.tiff, quantified using Image J, and assembled using Adobe Illustrator.For the MWM and NORT, data was plotted using GraphPad Prism 5 software (GraphPad software Inc.) and analyzed using both GraphPad Prism and R 3.0  as previously described . Data foBased upon previously published results , 17, we The major copper carrying enzyme in the blood is ceruloplasmin (Cp). Cp expression is regulated by systemic copper, therefore, plasma Cp can be used as a surrogate marker of copper status. We found a marked reduction in plasma Cp activity in zinc treated male and female mice as compared to untreated mice. We further analyzed the brain metal composition of both copper and zinc using ICP-MS to determine if systemic copper lowering produced lower brain copper levels and unchanged zinc levels, as we have previously shown in other murine models . We founOur proposed hypothesis was that a reduction in brain copper levels would affect tau phosphorylation, as was shown in previous studies and in our cell culture model. We analyzed tau phosphorylation through Western blots using site specific anti-phosphorylated tau antibodies Figure . We founThere are many kinases known to phosphorylate tau at the sites reported here, however CDK5 and GSK-3β have been previously examined in relation to chronic copper exposure . We examIn our previous study, we observed that despite a reduction in insoluble Aβ, there was no appreciable difference in the learning and memory between control and zinc treated mice. However, Aβ load correlates less well to cognition in humans, as compared to tau aggregation. We first analyzed our hTau mice utilizing the Morris Water Maze where we found no differences in swim speed between genders or treatment groups (data not shown). No significant difference was observed in learning by either visual platform or hidden platform in the hTau mice, regardless of gender Figure A, B. WheThese studies demonstrate that copper modulates tau phosphorylation in the absence of pathological Aβ. This is distinct from previous studies showing effects of copper upon Aβ pathology or upon concomitant Aβ and tau pathology , 19, 27,At the same time, we recognize that these model systems are not ideal in other respects. Differentiated neuroblastoma cells are not fully differentiated neurons, and there is no functional change in these cells to indicate any consequences of tau phosphorylation. Additional experiments with primary neurons will permit the assessment of copper manipulations upon relevant markers of neuronal health, such as dendritic complexity and spine number.The concentration of copper required to induce tau phosphorylation in vitro is also a relative weakness, as this relatively high concentration may not be physiologically relevant and may represent a non-specific stressor , there was a trend toward treatment efficacy upon frontal cortex-dependent memory (Novel Object). We used an hTau-/- and mouse tau (mTau) -/- strain to compare behavioral changes of the mTau-/-, hTau+/+ strain. A more appropriate control for behavioral comparisons may have been the mTau+/+, hTau -/- strain. However, the mice used in this study were still able to learn the tasks, suggesting that they are suitable for detecting symptomatic effects. Additional experiments in mice with more robust tau pathology, neuronal damage, and behavioral effects are under way and will help clarify whether the observed effect on tau phosphorylation is functionally relevant.Another limitation of this strategy is the health risks of lowering systemic copper levels. There are both hematologic and neurologic complications of copper deficiency which will require close monitoring if this copper lowering approach is applied clinically. The dosage used in mice in these experiments is based on previous dose finding work to achieve copper lowering of 30-50%, which was well tolerated by the mice in terms of body weight and survival. Similarly, human subjects with Wilson’s disease tolerate oral zinc therapy and achieve a negative copper balance , so clinFinally, the mechanism by which tau phosphorylation is modulated is not entirely clear, as the in vitro studies implicate one kinase involved in tau phosphorylation (CDK5) while the in vivo data implicate another (GSK-3β). Resolution of this point will require additional experimentation, but may not be necessary for decisions about translating this strategy to clinical situations in which modification of tau phosphorylation is desirable, regardless of the kinase involved.In summary, we present evidence that human wild type tau phosphorylation can be modulated by copper, and a copper-modulating strategy suitable for human use is effective in an animal model of tau expression. Translation to human subjects is consequently possible, but justification for such a trial depends on the demonstration that this intervention is functionally important for brain neurons, and will require careful monitoring to avoid adverse effects from copper deficiency.Quantification of p35 and p25 bands from Figure S1. Western blot bands for p35 and p25 were quantified. The mean ± the SEM of the band intensity is represented. Brackets indicate p < 0.05 between the two treatments. (JPEG 830 KB)Additional file 1:Iron levels in mouse brain and plasma after zinc treatment. ICP-MS was used to analyze levels of iron in the brain (A) and plasma (B). Metal levels are plotted as parts per billion (ppb) for plasma and μg of metal per gram of homogenized brain tissue. Data is represented as the mean ± SEM of all mice in the treatment group. (JPEG 1 MB)Additional file 2:Plot of pTau to total tau for soluble mouse brain fractions following zinc acetate treatment. Bands from Figure S3 for AT8, AT180, PHF-1 and Tau12 were plotted as a ratio of the phospho-tau species/total tau for each mouse and represented as mean ± SEM of all mice in each treatment group. (JPEG 723 KB)Additional file 3:Low concentrations of copper do not affect tau phosphorylation. Varied copper concentrations were added to the media of SH-SY5Y cells in an effort to clarify whether copper at concentrations below 400 uM affect tau phosphorylation. Western blot bands for phosphorylated tau at T231 show no effect of exogenous copper at concentrations from 5 to 100 uM. (TIFF 745 KB)Additional file 4:


Several splice variants of IgE exist in human plasma, including a variant called IgE-tailpiece (IgE-tp) that differs from classical IgE by the replacement of two carboxy-terminal amino acids with eight novel residues that include an ultimate cysteine. To date, the role of the secreted IgE-tp isoform in human immunity is unknown. We show that levels of IgE-tp are raised in helminth-infected donors, and that both the classical form of IgE (IgE-c) and IgE-tp interact with polymers of the serine protease inhibitor alpha-1-antitrypsin (A1AT). The association of IgE-tp with A1AT polymers in plasma protects the antibody from serine protease-mediated degradation, without affecting the functional interaction of IgE-tp with important receptors, including FcεR1. That polymers of A1AT protect IgE from degradation by helminth proteases may explain why these common and normally non-disease causing polymorphic variants of A1AT have been retained by natural selection. The observation that IgE can be complexed with polymeric forms of A1AT may therefore have important consequences for our understanding of the pathophysiology of pulmonary diseases that arise either as a consequence of A1AT-deficiency or through IgE-mediated type 1 hypersensitivity responses. Immunoglobulin E (IgE) functions by binding to IgE-receptors (FcεRI and FcεRII) found on the surfaces of immune cells, including basophils and mast cells, which when activated in the lung induce the release of toxic mediators responsible for the symptoms of asthma12567Only one species of secreted IgE was thought to exist until the discovery of several isoforms generated by alternative splicing of the human Cε gene91011121314101110911in vivo this variant may interact with plasma proteins that may bestow unique immunological functions on this IgE variantLevels of IgE-tp are unchanged in atopy and may not be a major determinant of allergic inflammation9Parasitic helminth infections, including those responsible for the disease schistosomiasis, are also associated with high titers of specific and non-specific IgE antibody and many reports have shown an important role for human IgE in parasite killing19202222ex-vivo by helminth and human serine proteases that cleave IgE in the Fc, resulting in IgE molecules that are unable to interact with Fcε-receptors25in vivo could not be found, and we therefore speculated that human IgE associates with another plasma protein to protect it from serine-protease mediated degradation. Here we show that IgE and IgE-tp interact with plasma alpha1-antitrypsin (A1AT), encoded by the serpin peptidase inhibitor, clade A gene (SERPINA1). The interaction with A1AT protects IgE from cleavage by serine proteases and enhances interactions with FcεR1 expressed by fibroblasts. Although the mechanism(s) for the increased interactions with FcεR1 observed here have not been elucidated, they nonetheless point to a potentially important role for IgE in modifying A1AT mediated clearance of pathogenic A1AT polymers and serine proteases.Human IgE and IgE-tp are efficiently degraded ex-vivo by proteases released from S. mansoni25in situ, we screened numerous plasmas derived from helminth-infected donors for the presence of degraded IgE and IgE-tp  detected IgE at varying high molecular weights (HMW) of approximately 250–400 kDa, as determined by their relative electrophoretic mobility compared with the monomeric recombinant IgE-c and IgE-tp controls which run at the expected molecular weight for IgE of 190 kDa  or an anti-IgE-Fc confirmed that: i) IgE was also present in the complex, and ii) that the interaction with A1AT caused no intrinsic degradation to either IgE or IgE-tp. The formation of the tetramer and >250 kDa species of A1AT seen with IgE-tp was reliant on the 8 amino acid C-terminal tailpiece of this IgE isoform, since prior incubation with the tailpiece-specific monoclonal antibody 367 blocked their formation , whilst PiZ  plasma that was recognized by the anti-IgE Cε2 monoclonal 4.15 or anti-A1AT antibodies . Although raised levels of IgE and IgE-tp were observed in both donor cohorts compared with healthy controls 9proteins . This inRGDC) and is already known to form complexes with A1ATThe forms of A1AT giving rise to dimers most likely bind IgE-tp and IgE-c on the kappa-light chain, which also ends in a cysteine residue  did not pull down either IgE or A1AT . The IgERRGGC) may be involved in forming hydrogen bonds with A1AT that also encourage polymer formation. Arginine glycine repeats are already known to facilitate protein-protein interactions that include self-associationsAlthough dimer and tetramer forms of A1AT clearly bind at different positions on IgE, the mechanism of binding of either form is presently unknown. Low concentrations of guanidine hydrochloride are known to induce A1AT polymer formation3949The high population frequency of the Z allele of A1AT suggests that it confers a selective advantage in affected individualsThere is evidence that PiZ heterozygotes without demonstrable lung disease display an airway IL-8 related neutrophilic inflammation60Cleavage of the reactive centre loop (RCL) of A1AT by a target enzyme, typically elastases, results in a striking conformational transition that ‘flips’ the enzyme from the upper pole of A1AT to the lower pole, essentially trapping the enzyme in a molecular ‘mouse-trap’ for clearance from the site of inflammation49To test this hypothesis, we allowed recombinant IgE-tp to bind A1AT in plasma, prior to incubating the complex with schistosome proteases that we had previously shown cleave IgE-tp at the solvent exposed Cε3/Cε4 inter-domain region of IgE-Fc42http://www.chogenome.org). Therefore the increased binding seen with IgE-opsonized beads complexed with A1AT may arise from additional cross-linking of IgE-A1AT to LRP1 and/or SRB1 in addition to FcεRI  and Immunet (http://immunet.princeton.edu) databases that help identify functional relationships between genes and proteins. A search of both databases with SERPINA1 revealed such a potential association with human FcεR1. As polymers of A1AT are believed to be pro-inflammatory52To explore if there might be a relationship between A1AT and FcεR1 we interrogated the UCSC Gene Sorter . Cell lines were maintained and IgE antibodies purified from culture supernatants as previously described915oC before using. Plasma samples from Piida, Uganda have been characterized in a previous studyFresh heparinized blood (20 ml) was collected from healthy human volunteers by venepunture in vacutainers containing lithium-heparin . Blood was transferred to Universal tubes containing a 5 ml solution of 6% dextran T70  in 0.9% saline and red cells allowed to sediment for 30 mins at 37 °C. The resultant buffy coat was centrifuged at 1500 g for 5 min to remove leucocytes and remaining plasma stored at −80 3, pH8.5. 0.25 g of dansyl chloride (Sigma) was dissolved in di-methyl formamide (DMF), and both solutions were mixed together at room temperature under gentle rotation for 2 h before dialysis against five liters of phosphate buffered saline (PBS) pH 7.4.Briefly, 1.2 g of bovine serum albumin (BSA) was dissolved in 35 ml of 0.2M NaHCO3, 0.5 M NaCl, pH 8.3) and gently agitated overnight together with the sepharose, and then washed with excess coupling buffer to remove unconjugated hapten, followed by blocking of reactive groups with 0.1 M Tris-HCl buffer, pH 8.0 for 2 h. The sepharose was then washed with alternating high  and low  buffers to remove non-covalently bound material before storage in 20% ethanol. Recombinant IgEs were affinity purified from large volumes (~3 litres) of tissue culture supernatant using dansyl-BSA-sepharose packed columns using an AKTA FPLC frac-950 system (Amersham). Bound IgE was eluted off the column with 0.2 M sodium phosphate pH2 into 100μl of 0.7 M sodium phosphate pH11 to neutralize and eluted IgE containing fractions pooled, concentrated and dialyzed against PBS. Concentrations of purified IgE were determined by bicinchoninic acid (BCA) protein assay kit (Pierce), and purity determined by SDS-PAGE on 4–15% gradient gels.Cyanogen bromide activated sepharose (Amersham) was swollen with 1 mM HCl. 10 mg/ml of dansyl-BSA was diluted in coupling buffer (0.1 M NaHCOoC for 5 min prior to loading at 20μl per lane on 4–15% gradient gels. Proteins were transferred to nitrocellulose membranes (Schleicher & Schuell) and blocked with PBS/0.05% Tween-20 (PBST) containing 5% non-fat milk powder for 1 h under gentle agitation at room temperature before washing 3 times each for 5 minutes with PBST. Blots were cut and incubated in PBST containing a 1:500 dilution of peroxidase-conjugated anti-human A1AT (Serotec or Dako) or alkaline phosphatase-conjugated anti-human IgE (Sigma) for 1 h in PBST. Monoclonal antibodies 367, 4.15, 8E/4F4 or ATZ11 were incubated with blots at 1:500 in PBST prior to washing and detection with peroxidase-conjugated or alkaline phosphatase-conjugated anti-mouse IgG (Pierce). After washing blots were developed in DAB reagent or BCIP/NBT solution according to manufacturers instructions. For experiments investigating the role of the RCL, plasma was incubated with varying concentrations of human neutrophil elastase (Sigma) prior to precipitation of A1AT with recombinant IgE as described above. Resistance of IgE-tp A1AT complexes to degradation by S. mansoni cercarial elastases (CE) was determined by Western blotting with monoclonal 367 as described previouslyFor precipitation studies, typically 50μl of human plasma was incubated with 50μg of antibody for two hours prior to precipitation with 50μl dansyl-BSA-sepharose slurry (in the case of recombinant IgEs) or sheep anti-mouse IgG coated Dynabeads M-280  in a final volume of 0.5 ml of PBS for 1 h. Beads were pelleted by gentle centrifugation and washed five times in 1.5 ml PBS. Up to 80μl of non-reducing or reducing sample buffer was then added to the pelleted beads and heated at 100 Fifty microliter of dansyl-sepharose beads were opsonized with up to 500μg/ml of recombinant IgE-c or IgE-tp antibodies making use of their specificity for the hapten dansyl. After three washes in PBS, IgE-opsonized beads were incubated in the presence or absence of varying dilutions of plasma for 1 h prior to five further washes in PBS. IgE-opsonized beads were then incubated with the FcεRI expressing CHK1E1 transfectants grown as described previously to confluence in twenty-four well platesFull informed consent was obtained for all procedures, and ethical approval for use of anonymized plasma samples was obtained from the Department of Genetics, University of Nottingham ethics review board or the respective institutions at the time of sampling.How to cite this article: Quinn, P. M. et al. IgE-tailpiece associates with α-1-antitrypsin (A1AT) to protect IgE from proteolysis without compromising its ability to interact with FcεRI. Sci. Rep. 6, 20509; doi: 10.1038/srep20509 (2016).


This is in contrast to findings in patients with allergy to classical type 1 allergens, e.g. grass or peanuts. Besides that, our investigations indicates that histamine is only released in the minority of positive intracutaneous tests. The reason for this is not known. A hypothesis could be that the specific IgE against penicillin measured in serum is not present in the skin. The aim of this project is to investigate, if you by priming the skin with autologous serum in patients with specific IgE to penicillin subsequently are able to develop a positive ICT upon testing.In patients allergic to penicillin only a minority of those who have specific IgE against penicillin also have a positive intracutaneous test (ICT) to penicillin and The patients are intradermally injected with autologous serum, 0,1 ml, in 3 different places on the volar forearm on day one.On day two, cutaneous microdialysis is performed at the 3 primed sites and on two additional sites. Forwith microdialysis, semipermeable fibers with an inner diameter of 0,2 mm are inserted into the dermis and subsequently perfused with saline at a rate of 3 µl/min. During the experiment, ICT with penicillin is performed 1 mm away from the fiber both in the primed and not-primed areas. Histamine concentrations in the eluate are subsequently measured by fluorescence spectroscopy. ICT with saline and codeine served as negative and positive control in both primed and non-primed skin.So far 8 patients have been investigated. All have measurable levels of IgE at the time of microdialysis. 6/8 were primed with serum obtained earlier where IgE levels were higher than the actual. On the day of the experiment 2/8 had a positive ICT in not-primed skin, the rest were negative. ICT performed in the primed areas did not result in any further positive ICTs. In the positive ICTs, no histamine was detected. All patients released significant amounts of histamine upon ICT with codeine.Our data do not support the hypothesis that the presence of IgE against penicillin and lack of a positive ICT is due to lack of IgE in the skin. Possibly, there may be both functional and non-functional IgE, or a positive ICT is not IgE mediated.