Dicer in the forebrain displayed progressive neurodegeneration accompanied by disease-like changes in tau phosphorylation and splicing. Dicer is a key enzyme in the biogenesis of microRNAs (miRNAs), small noncoding RNAs that function as part of the RNA-induced silencing complex (RISC) to repress gene expression at the posttranscriptional level. We identified miR-16 and miR-132 as putative endogenous modulators of neuronal tau phosphorylation and tau exon 10 splicing, respectively. Interestingly, these miRNAs have been implicated in cell survival and function, whereas changes in miR-16/132 levels correlate with tau pathology in human neurodegenerative disorders. Thus, understanding how miRNA networks influence tau metabolism and possibly other biological systems might provide important clues into the molecular causes of tauopathies, particularly the more common but less understood sporadic forms.Abnormal regulation of tau phosphorylation and/or alternative splicing is associated with the development of a large (>20) group of neurodegenerative disorders collectively known as tauopathies, the most common being Alzheimer's disease. Despite intensive research, little is known about the molecular mechanisms that participate in the transcriptional and posttranscriptional regulation of endogenous tau, especially in neurons. Recently, we showed that mice lacking  Since their discovery almost 20 years ago , 2, reseet genes , 14, thuAbnormal phosphorylation and insoluble deposition of tau are observed in more than 20 neurodegenerative disorders, collectively known as tauopathies (reviewed in ). In AlzAlthough the exact physiological role of tau remains under scrutiny, it is proposed to function to promote microtubule assembly, stabilization, spacing, and parallel-ordered organization, which are necessary for axonal transport and neurite outgrowth (reviewed in ). Tau biTo date, several groups have identified factors involved in the regulation of tau splicing. These include regulatory sequences (cis-elements) within or around tau exon 10 as well as specific regulatory proteins (trans-acting factors) (reviewed in ). Similain vivo was enhanced by loss of R3D1/loquacious, a double-stranded RNA binding protein that is required for the activity of Dcr-1 (Dicer homologue) in miRNA processing. Interestingly, upregulation of bantam suppressed tau-induced degeneration, suggesting that this miRNA could mitigate tau-induced neurotoxicity. However, as bantam is not conserved in humans, it is tempting to speculate that other miRNAs play a similar role in the mammalian brain. More recently, a neuronal miRNA, miR-128, was shown to modulate the expression of BAG2, a cochaperone potentially involved in tau degradation and aggregation in cultured COS-7 cells and in primary neurons [Dicer  in the adult mouse brain results in disease-like hyperphosphorylation of endogenous tau [Dicer mutant mice display changes in tau exon 10 splicing [In 2006, Bilen et al.  showed a neurons . Our recnous tau . Moreovesplicing , as seenin vivo is of particular importance. It is noteworthy that some of those central key kinases also regulate indirectly tau splicing through phosphorylation of splicing factors [Tau is a phosphoprotein that contains more than 80 potential phosphorylation sites (reviewed in ). It is  factors –36. TogeDicer loss in the brain, we observed significant changes in endogenous tau phosphorylation and splicing [in vivo, as most biological models express exogenous and/or mutated human tau. Remarkably, several phosphoepitopes related to disease, including serine 422, were increased in the Dicer mutant mice when compared with controls [Dicer loss (approximately 4 weeks), we could not determine whether tau hyperphosphorylation concurred before or after neurodegeneration. Nevertheless, these results provide a proof of concept that miRNA haploinsufficiency causes abnormal tau hyperphosphorylation and missplicing.As general posttranslational regulation of gene expression, miRNAs are potential modulators of kinase, phosphatase, and/or splicing factor expression. While studying the effects of splicing . This wacontrols . Unfortuin vivo. In attempt to identify such enzymes in the Dicer cKO mice, we performed whole-genome microarrays and western blot analyses. These experiments led to the identification of ERK1/MAPK3 (and possibly ERK2/MAPK1) as a major regulator of neuronal tau phosphorylation in vivo, at least in this model. In line with this observation, several ERK1-dependent epitopes were hyperphosphorylated in the Dicer mutant mice. Of course, several other enzymes can potentially contribute to tau phosphorylation misregulation in this and other models, and in particular disease conditions in humans. In line with this hypothesis, a number of proposed tau kinases, including GSK3β and JNK/MAPK8, are prone to miRNA regulation in various cell types [in vivo.As stated above, several tau kinases and phosphatases have been identified, some of which are believed to contribute significantly to tau hyperphosphorylation ll types , 38. Nevll types –41. In all types , 43. SpeDicer cKO mice provide a unique and unbiased model to study global miRNA function in the brain. To identify miRNAs involved in the regulation of ERK1 (and consequently tau phosphorylation), we used several prediction programs that are available online, including TargetScan (http://targetscan.org/). This program identified a number of potential miRNA-binding sites in the 3′UTR of ERK1. Our functional assays provided the validation that several members of the miR-16 family  could directly modulate endogenous ERK1 and tau phosphorylation in neuronal cells in vitro, including rat primary neurons. Of mention, both endogenous miR-16 and tau are enriched in distal axons of sympathetic neurons [in vivo in the mouse brain [in vivo recapitulates, at least in past, the observed effects on ERK and, most importantly, tau phosphorylation in vivo.Although it is a conceptually crude experimental approach, the  neurons . Intrigu neurons , 45, 46, neurons –49. In ase brain . Taken tse brain . It willDicer deficiency in the adult brain is also associated with changes in tau splicing [in vitro [As discussed above, abnormal regulation of tau exon 10 splicing can cause disease. It is interesting to note that splicing . Using ain vitro .β in mouse primary neurons [β peptides, tau missplicing and hyperphosphorylation remain an exciting possibility. Interestingly, differential splicing of the tau transcript has also been reported in AD [While not discussed in detail in our study, other miRNAs, including miR-124 and miR-9, could also regulate endogenous tau exon 10 splicing in neuronal cells by targeting specific regulatory and/or splicing factors . Both mi neurons . Whethered in AD , 61.PSP is a cause of late-onset atypical parkinsonism described by Steele et al. . DementiAlthough the above-mentioned studies are interesting, many questions remain unanswered. For instance, what are the biological and clinical significance of these findings? Are other miRNAs involved in tau metabolism regulation? Can miRNAs be used as diagnostic and possibly therapeutic agents for sporadic tauopathies? Without a doubt, these and other questions will require extensive followup studies in various models, from cells to animals to humans.


As an upstream signature component of FcεRI signaling, inhibition of spleen tyrosine kinase (Syk) abrogated the IgE-induced HASM proliferation. Further analysis of IgE-induced signaling depicted an IgE-mediated activation of Erk 1/2, p38, JNK MAPK, and Akt kinases. Lastly, lentiviral-shRNA-mediated STAT3 silencing completely abolished the IgE-mediated HASM cell proliferation. Collectively, our data provide mechanisms of a novel function of IgE which may contribute, at least in part, to airway remodeling observed in allergic asthma by directly inducing HASM cell proliferation.Airway remodeling is not specifically targeted by current asthma medications, partly owing to the lack of understanding of remodeling mechanisms, altogether posing great challenges in asthma treatment. Increased airway smooth muscle (ASM) mass due to hyperplasia/hypertrophy contributes significantly to overall airway remodeling and correlates with decline in lung function. Recent evidence suggests that IgE sensitization can enhance the survival and mediator release in inflammatory cells. Human ASM (HASM) cells express both low affinity (FcεRII/CD23) and high affinity IgE Fc receptors (FcεRI), and IgE can modulate the contractile and synthetic function of HASM cells. IgE was recently shown to induce HASM cell proliferation but the detailed mechanisms remain unknown. We report here that IgE sensitization induces HASM cell proliferation, as measured by  Nearly 80% of children and more than 50% of adult asthma is thought to be allergic/immunoglobulin E (IgE)-dependent. ClassicAirway smooth muscle (ASM) cells are structural entities of airways which are believed to confer an abnormally exaggerated bronchoconstriction in asthma, the phenomenon commonly known as airway hyperresponsiveness (AHR). Clinically, majority of asthma patients show a significant increase in ASM bundles, likely due to increase in cell number, collectively contributing to airway remodeling. Tissue 2+]i) mobilizationi) mobilization, altogether suggesting a critical role of this pathway in airway inflammation and hyperresponsiveness[In vivo, anti-IgE treatment decreased the thickness of ASM layer compared with the ovalbumin (OVA)-challenged mice[in vivo. Although the low affinity (FcεRII/CD23) receptor has also been described in ASM cells with enhanced signal in ASM tissue from asthma[Serum IgE levels were shown to affect ASM cell function and tend to correlate with AHR. Cumulatsiveness,12,14,15siveness. Moreovesiveness. Althougsiveness, possiblsiveness. Furthersiveness. Finallysiveness. In vivoged mice, suggestm asthma, and Rotm asthma; presentm asthma, abolishm asthma,31. Furtm asthma and in vm asthma. TherefoSTAT3 has been shown earlier to regulate allergic response in asthma. Particularly, epithelial STAT3 was identified as a critical regulator of allergen-induced inflammation and AHR in a murine model of asthma, IL-17A-Although IgE-induced signaling pathways are well characterized in inflammatory cells, there is limited information on this area in HASM cells. MAPK family is fundamental in regulating multiple cell functions such as cytokine expression, proliferation, and apoptosis. Although Erk1/2 and p38 MAPK were shown to mediate IgE-induced proinflammatory gene expression in HASM recently, Akt wasin vivo observations where IgE can promote immune sensitization to hapten in the skin, without the need of antigens[Mechanistically, there is sufficient evidence from past decade to convince that the 'IgE sensitization/monomeric IgE exposure’ of FcεRI on inflammatory cells itself can activate multiple signaling pathways; induce a plethora of proinflammatory mediators release and cell survival factors, and subsequent repression of apoptosis. Interesantigens. Not onlantigens. Of noteantigens,15,17 anantigens,27 modelin vivo. In conclusion, blocking the IgE-FcεR interaction, not only on inflammatory cells but also on the airway structural (HASM) cells should be considered as a novel tool to inhibit allergic sensitization-mediated airway remodeling in asthma.Omalizumab, the clinically approved anti-IgE antibody blocks the interaction of IgE with FcεRI and has shown clinical benefits in controlling allergic inflammation, and offered improvement in asthma symptoms, reduced frequency of asthma exacerbations, and significantly lowered the use of inhaled corticosteroids,47. FinaThe authors declare that they have no competing interests.NSR and ASG conceived the study, NSR performed the experiments, analyzed the data, and wrote the manuscript; LS, DA, HLA, HM performed the experiments; BL provided critical scientific input; ASG provided the resources, and provided editorial and scientific input on manuscript. All authors read and approved the final manuscript.


Immunoglobulin E (IgE) binds to high affinity receptor FcεRI numerously expressed on mast cells. Recent findings have revealed that IgE by itself may regulate various aspects of mast cell biology, however, detailed data is still limited.in vivo differentiated mature tissue mast cells isolated from rat peritoneal cavity. Mast cells were exposed to IgE alone and then the release of preformed and de novo-synthesized mediators, surface FcεRI expression and mast cell migratory response were assessed. IgE by itself was found to up-regulate FcεRI expression and activate mast cells to degranulation, as well as de novo synthesis and release of cysteinyl leukotrienes and TNF. We have provided evidence that IgE alone also amplified spontaneous and CCL5- or TNF-induced migration of mast cells. Importantly, IgE was effective only at concentrations ≥ 3 µg/mL. A molecular basis investigation using an array of specific inhibitors showed that Src kinases, PLC/PLA2, MAP kinases (ERK and p38) and PI3K were entirely or partially involved in IgE-induced mast cell response. Furthermore, IgE alone stimulated the phosphorylation of MAP kinases and PI3K in rat mast cells.Here, we have examined the influence of IgE alone, used at different concentrations, on mast cell activity and releasability. For the study we have employed in vivo IgE is one of the important factors modulating mast cell biology within tissues.Our results clearly demonstrated that IgE by itself, at higher concentrations, influences mast cell activity and releasability. As there are different conditions when the IgE level is raised it might be supposed that  Under physiological conditions, immunoglobulin E (IgE) synthesis and, hence its concentration in the blood or within tissues is constantly low via their involvement in angiogenesis, tissue remodeling and repair, but also are key players in inflammatory processes and the host response to pathogens. Moreover, mast cells play an important role in the course of many diseases, other than IgE-dependent hypersensitivity reactions, for example, atherosclerosis, rheumatoid arthritis, congestive heart failure, malignancies, Crohn's disease and pulmonary fibrosis 2+. Of note, the majority of studies were carried out using cell lines differentiated in vitro , which differ with respect to phenotype and activity from mast cells, which develop and mature under influence of microenvironmental factors. It should be also emphasized that the available data concerning the influence of IgE exposure on mast cell response and activity still remains limited.IgE binds with high affinity to the FcεRI abundantly expressed on mast cells. Mast cells are widely distributed throughout the body and are the source of large numbers of biologically active mediators. Mast cell-derived mediators exert diverse proinflammatory, anti-inflammatory, and/or immunoregulatory effects and modulate the activity of many cell populations. Thus, mast cells participate not only in maintaining homeostasis mainly de novo synthesis. Another important question to clarify was whether IgE alone affects spontaneous migratory response of mast cells, as well as migration induced by strong mast cell chemotactic factors such as CCL5 and TNF via FcεRI, i.e. mitogen-activated protein (MAP) kinases  and p38 kinase), Src kinases, phosphatidylinositol 3-kinase (PI3K) and phospholipase (PL)C/PLA2.In the present study we scheduled to examine the direct impact of IgE alone, used at different concentrations, on various effector functions of fully mature rat peritoneal connective tissue mast cells. To this end, we examined the IgE-induced mast cell preformed mediators release, arachidonic acid metabolite generation and tumor necrosis factor (TNF) 2, CaCl2, 2-hydroxyethylpiperazine-N'-ethanesulphonic acid (HEPES), NaOH, glucose, HCl, o-phthaldehyde (OPT), Percoll, hematoxylin, toluidine blue, trypan blue, bovine serum albumin (BSA), PLC/PLA2 inhibitor (U73122), PI3K inhibitor (LY294002), Src kinase inhibitor , RIPA buffer and laminin from human placenta were obtained from Sigma-Aldrich . ERK inhibitor (PD98059) and p38 kinase inhibitor (SB203580) were purchased from InvivoGen . Recombinant rat (rr) tumor necrosis factor (TNF) was obtained from R&D Systems , rrCCL5, and mouse anti-rat IgE monoclonal IgG1 antibodies (anti-IgE) were purchased from AbD Serotec . Mouse monoclonal IgG1 anti-FcεRI and anti-PI3K antibodies were obtained from Abcam Inc.  and purified rat myeloma IgE was obtained from Invitrogen, Life Technologies . Fluorescein isothiocyanate (FITC)-labeled goat anti-mouse polyclonal antibodies, purified mouse monoclonal IgG1 antibodies (isotype control), and BD CellFIX were purchased from BD Biosciences . The 48-well Boyden microchamber as well as the 8-μm-pore-size polycarbonate filters were purchased from Neuro Probe . Rat TNF and cysLT specific immunoassay kits were purchased from Gen-Probe Inc.  and Cayman Chemical , respectively. The RNeasy Mini Kit was obtained from Qiagen . The High Capacity cDNA Reverse Transcription Kit, TaqMan® probes dyed 6-carboxyfluorescein (6-FAM) (rat TNF Rn01525859 and rat Actb Rn00667869) and TaqMan® Gene Expression Master Mix were purchased from Applied Biosystems  and bicinchoninic acid (BCA) Protein Assay Kit was obtained from Pierce . The Western Lightning luminol-based enhanced chemiluminescence (ECL) Pro system was purchased from Perkin-Elmer . Anti-phospho-ERK1/2 (Thr-202/Tyr-204) and anti-ERK1/2, anti-phospho-p38 (Tyr-182) and anti-p38 as well as horseradish peroxidase (HRP)-labeled donkey anti-goat IgG, goat anti-mouse IgG-HRP and goat anti-rabbit IgG-HRP all were obtained from Santa Cruz Biotechnology Inc. . Anti-phospho-PI3K (p85 Tyr458/p55 Tyr199) antibodies were obtained from Thermo Scientific .Dulbecco's Modified Eagle's Medium (DMEM), Hank's balanced salt solution (HBSS), sodium bicarbonate, fetal calf serum (FCS), gentamicin and glutamine were purchased from GIBCO, Life Technologies . NaCl, KCl, MgClad libitum. Isoflurane-induced anesthesia was carried out prior to animal decapitation. All efforts were made to minimize animal suffering. Animal experiments were approved by the Local Ethics Committee for Experiments on Animals of the Medical University of Łódź . All manipulations performed on animals were in accordance with university guidelines.Mast cells were collected from peritoneal cavities of female Wistar rats weighing ∼250 g, aged 3–4 months, bred in the animal quarters of the Medical Faculty of the Medical University of Łódź. Standard storage conditions for animals were provided, i.e. room temperature (20±2°C), artificial lighting for 12 h and 12 h of darkness, in metal cages, 5 rats in each. The animals were fed with LSM Murigran granulated fodder for rodents and water 2, 1 mM CaCl2, 10 mM HEPES, 5.6 mM glucose, and 1 mg/mL BSA (for histamine release assay and cysLT synthesis measurement), to obtain mast cell concentration of 1.5×106 cells/mL. Mast cell were prepared with purity > 98%, as determined by toluidine blue staining.Peritoneal cell suspensions were obtained from peritoneal cavities by lavage with 50 mL of 1% HBSS supplemented with 0.015% sodium bicarbonate. Peritoneal cell suspension was washed twice  in complete (c)DMEM containing DMEM supplemented with 10% FCS, 10 µg/mL gentamicin, and 2 mM glutamine. In order to collect purified mast cells, peritoneal cells were resuspended in 72.5% isotonic Percoll solution and centrifuged . After being washed twice , mast cells were counted and resuspended in an appropriate volume of cDMEM  or in medium for rat mast cells, containing 137 mM NaCl, 2.7 mM KCl, 1 mM MgClPurified mast cells were incubated with IgE at final concentrations of 0.1, 0.5, 1, 1.5, 3, or 5 µg/mL, or without IgE (negative control) in a water bath for 1 h at 37°C with constant stirring. In every experiment a positive control for the determination of anti-IgE-induced histamine release from native mast cells  was included. For time-course experiments mast cells were incubated with IgE at final concentrations of 5 µg/mL for 0, 5, 10, 30, or 60 min. After incubation the reaction was stopped by adding 1.9 mL of cold medium for rat mast cells and the release of histamine was measured using spectrofluorometric method, as described previously Purified mast cells were incubated with IgE at final concentrations of 0.1, 0.5, 1, 1.5, 3, or 5 µg/mL, or without IgE (negative control) in a water bath for 1 h at 37°C with constant stirring. In every experiment a positive control for the determination of native mast cell cysLT synthesis in response to anti-IgE stimulation  was included. The supernatants were collected by centrifugation  and analyzed for cysLT by an ELISA commercial kit according to the manufacturer's instructions. The sensitivity of this assay was < 13 pg/mL.2. Native mast cells were treated with anti-IgE at concentration of 5 µg/mL under the same experimental conditions (positive control). The supernatants were collected by centrifugation  and analyzed for TNF by an ELISA commercial kit according to the manufacturer's instructions. The sensitivity of this assay was < 20 pg/mL.Purified mast cells were incubated with IgE at final concentrations of 0.1, 0.5, 1, 1.5, 3, or 5 µg/mL, or without IgE (negative control) for 6 h at 37°C in a humidified atmosphere with CO2. Total RNA was isolated from mast cells using an RNeasy Mini Kit. Synthesis of cDNA was performed using High Capacity cDNA Reverse Transcription Kit. For qRT-PCR, TaqMan® probes and TaqMan® Gene Expression Master Mix were used. All reactions were performed with the use of 7900 HT Fast Real-Time PCR System (Applied Biosystems). The expression of TNF mRNA was corrected by normalization based on the transcript level of the housekeeping gene rat Actb. All qRT-PCRs were conducted in triplicate, and the changes in gene expression are presented as fold-increases relative to non-stimulated mast cells.Purified peritoneal rat mast cells were stimulated with IgE or anti-IgE (positive control) both at 5 µg/mL for 0, 15, 30, 60, 180, and 360 min at 37°C in a humidified atmosphere with 5% CO2. After incubation, the non-migrating cells were removed from the upper surface of the filter by scraping, and migrating cells adherent to the lower surface of the membrane were fixed in 99.8% ethanol, washed in distilled water, stained for 10 min with hematoxylin, cleared in distilled water, and mounted on microscope slides. Mast cell migration was quantified by counting the total number of cells migrating through the filter in 10 high power fields (HPF).Mast cell migration was analyzed in a 48-well Boyden microchamber using 8-μm-pore-size polycarbonate filters. The filters were coated overnight at room temperature with laminin at a concentration of 100 µg/mL and air dried for at least 1 h before use. Purified mast cells were preincubated with IgE at 1 µg/mL and 5 µg/mL or medium alone (native mast cells) for 1 h at 37°C with constant stirring. After preincubation, mast cells were washed in cDMEM . Next, 30 µL of rrTNF at 10 ng/mL, rrCCL5 at 100 ng/mL or medium alone (spontaneous migration) was placed into the lower compartment of a microchamber, and 50 µL of native or IgE-coated mast cells was pipetted into the upper compartments. The mast cells were incubated in the chemotaxis chamber for 3 h at 37°C in a humidified incubator with 5% CO2 inhibitor (U73122) was used at a concentration of 0.1 µM, ERK kinase inhibitor (PD98059) was used at a concentration of 100 µM, PI3K inhibitor (LY294002) was used at a concentration of 50 µM, and p38 inhibitor (SB203580) was used at a concentration of 100 µM. It should be underlined that the concentrations of all applied inhibitors were chosen in the preliminary experiments, and neither of inhibitors affected mast cell viability, as examined by staining with trypan blue.In independent experiments, mast cells were pretreated with various signaling pathway inhibitors for 1 h at 37°C in a water bath with constant stirring, before main procedure accomplishments . Src kinase inhibitor (Src I-1) was used at a concentration of 0.01 µM, PLC/PLAAfter incubation with 5 µg/mL of IgE alone for either 0, 5, 15, 30, 60 or 120 min, purified mast cells were washed with PBS and lysed in RIPA buffer on ice for 20 min, sonicated and centrifuged . Total protein of the cell lysate was assessed by BCA reagent. Cell lysates containing 50 µg of crude cell lysate protein were analyzed by 10% SDS-PAGE followed by immunoblotting. Protein expression was detected using anti-phospho-ERK1/2 (Thr-202/Tyr-204), anti-phospho-p38 (Tyr-182) or anti-phospho-PI3K (p85 Tyr458/p55 Tyr199) antibodies, and appropriate antibodies that recognize these MAP kinases or PI3K, irrespective of their phosphorylation states. HRP-conjugated anti-mouse, anti-goat or anti-rabbit IgGs were used as secondary antibodies and then visualized using chemiluminescence reagents.6 cells/mL, were incubated with IgE at 1 µg/mL or 5 µg/mL or without IgE (native mast cells) for 1, 6 or 24 h at 37°C in a humidified atmosphere with 5% CO2. After incubation, cells were washed with cDMEM in order to remove unbound IgE, fixed using 1% CellFIX solution for 15 min at 4°C, washed twice, and finally resuspended in 1% PBS. For the measurement of FcεRI expression, mast cells were initially incubated with mouse anti-rat FcεRI IgG1 antibodies or mouse IgG1 isotype control with irrelevant specificity  at 4°C for 45 min. After washing with 1% PBS, cells were stained with FITC-conjugated goat anti-mouse IgG1 antibodies at a final concentration of 5 µg/mL at 4°C for 45 min in the dark. Following this, the cells were washed twice and finally resuspended in 300 µL of 1% PBS prior to fluorescence assessment. Ten thousand events in each sample were analyzed using a FACSCalibur flow cytometer with CellQuest software (BD Biosciences). IgE-dependent mast cell FcεRI expression was presented as a percentage of FcεRI MFI (mean fluorescence intensity) measured in native mast cells (referred to as 100%). After each period of incubation mast cell viability was examined using the trypan blue exclusion test.Purified mast cells, at a concentration of 2×10Results are expressed as mean value ± standard error of the mean (SEM) and tested statistically by Student's t-test for “small groups”. Results were considered statistically significant when p<0.05.The effects of various concentrations of IgE, from 0.1 µg/mL to 5 µg/mL, on mast cell degranulation and preformed mediator release were evaluated first. We found that IgE alone, used at concentrations of 0.1, 0.5, 1, 1.5, and 3 µg/mL, failed to induce detectable mast cell degranulation and histamine release. However, it was noted that when stimulated with 5 µg/mL of IgE for 1 h, mast cells were activated to secrete histamine. The level of 5 µg/mL IgE-induced histamine release was significantly (p<0.01) higher than in control but statistically (p<0.05) lower than anti-IgE-challenged histamine secretion . Time-code novo TNF production. As shown in de novo TNF synthesis, the expression of TNF mRNA was analyzed. qRT-PCR was carried out and the relative expression of TNF mRNA in IgE-stimulated mast cells (5 µg/mL) compared to non-stimulated cells was assessed. It was revealed that IgE alone was capable of elevating TNF mRNA expression in mast cells in a time-dependent manner . ERK inhibitor and Src kinase inhibitor almost completely abolished, and PLC/PLA2 inhibitor significantly decreased, cysLT generation mediated by IgE alone. Mast cell pretreatment with Src I-1 or SB203580 resulted in a statistically significant (p<0.001) inhibition of IgE-induced TNF production and release. By contrast, ERK inhibitor was found to slightly attenuate IgE-mediated TNF synthesis . The same inhibitor did not affect spontaneous mast cell migration. Src inhibitor and PI3K inhibitor both caused significant  reduction in CCL5-induced, TNF-induced as well as in spontaneous migration of IgE-coated mast cells  and PI3K upon stimulation with IgE alone. As can be observed in In the current study, the FcεRI expression was evaluated on native mast cells as well as on mast cells exposed to IgE used at concentrations of 1 µg/mL and 5 µg/mL for 1, 6 and 24 h. Flow cytometry analysis revealed that native mast cells expressed surface FcεRI receptor. We found that the baseline level of FcεRI expression was significantly up-regulated (p<0.05) upon 1 h incubation with IgE at 5 µg/mL, and reached 163.6±44.3% of control FcεRI expression on native mast cells. The treatment of mast cells with 5 µg/mL of IgE following a 6 h incubation resulted in a statistically significant (p<0.05) increase in FcεRI level, compared with the control unstimulated mast cells. Interestingly, the FcεRI expression on mast cells after prolonged exposure (24 h) to IgE at 5 µg/mL was comparable with control expression. The exposure of mast cells to 1 µg/mL of IgE did not cause alteration in surface FcεRI level, at any time point examined , Fig. 7Bde novo synthesis and release of eicosanoids, cytokines and chemokines The central dogma of mast cell biology holds that FcεRI cross-linking by specific IgE and antigen initiates signaling cascade activation, leading not only to immediate preformed mediator secretion, but also to de novo TNF synthesis and release de novo TNF generation than anti-IgE stimulation at both TNF mRNA and TNF released levels.In our study we also stated that IgE alone directly triggered mature rat mast cells to preformed mediator secretion, as assessed by histamine release, and cysLT production. Importantly, the release of mediators was substantial only when IgE at 5 µg/mL was employed. So far, only a handful of studies have examined the direct effect of IgE on mast cell degranulation and eicosanoid generation, and the results remain ambiguous. Some earlier studies have shown that IgE by itself induced BMMCs and human lung mast cells (HLMCs) degranulation, however only when mast cells were stimulated with at least 3 µg/mL of IgE Under physiological conditions, the mast cell number in tissues is high and relatively constant de novo-synthesized TNF. The Src kinase-initiated signal also appeared to be vital for IgE-affected rat peritoneal mast cell increased migration towards CCL5 and TNF. Interestingly, spontaneous migratory response of IgE-coated mast cells was only partially abrogated through the withdrawal of Src kinases signal.The precise signaling mechanisms underlying mast cell response mediated by IgE alone have yet to be clarified. It is highly likely, however, that IgE-dependent signaling events are similar, if not the same, to the classical pathway cascade elicited by FcεRI aggregation 2+ in a PLC-dependent fashion 2 and ERK i.e. signaling molecules deeply involved in arachidonic acid metabolism de novo TNF generation are consistent with these observations. We also documented that IgE-affected spontaneous migratory response was independent of PLC/PLA2, and was only partially generated through the PI3K-involved pathway. Alternatively, the effect of IgE alone on the CCL5- and TNF-induced migration of mast cells was mediated by PI3K and to some extent by PLC/PLA2. These discrepancies in the mechanistic basis between spontaneous and induced migratory responses of IgE-coated mast cells may be a result of additional/common pathways triggered via chemoattractant receptors.It was established that IgE alone triggered the phosphorylation of PKB  and several MAP family kinases, i.e. ERK, p38, JUN N-terminal kinase (JNK) in vivo, and so mast cell FcεRI occupation with IgE can be relatively high. Under these conditions, IgE per se may act as a mast cell activating factor. Moreover, since it is well-known that FcεRI is present not only on mast cells but also on basophils, eosinophils, neutrophils, monocytes, and on different subsets of dendritic cells In conclusion, our results, taken in concert with previous data, clearly indicated that IgE by itself influences mast cell activity and releasability. It should be emphasized, however, that IgE alone effectively influences mast cell biology only when used at higher concentrations. Of note, there are some circumstances when IgE level is elevated


In addition to AChE, O. latipes has a ChE that is different from either vertebrate AChE or BChE, which we are classifying as an atypical BChE, and which may resemble a transitional form between the two. Of the fourteen aromatic amino acids in the catalytic gorge of vertebrate AChE, ten are conserved in the atypical BChE of O. latipes; by contrast, only eight are conserved in vertebrate BChE. Notably, the atypical BChE has one phenylalanine in its acyl pocket, while AChE has two and BChE none. These substitutions could account for the intermediate nature of this atypical BChE. Molecular modeling supports this proposal. The atypical BChE hydrolyzes acetylthiocholine (ATCh) and propionylthiocholine (PTCh) preferentially but butyrylthiocholine (BTCh) to a considerable extent, which is different from the substrate specificity of AChE or BChE. The enzyme shows substrate inhibition with the two smaller substrates but not with the larger substrate BTCh. In comparison, AChE exhibits substrate inhibition, while BChE does not, but may instead show substrate activation. The atypical BChE from O. latipes also shows a mixed pattern of inhibition. It is effectively inhibited by physostigmine, typical of all ChEs. However, although the atypical BChE is efficiently inhibited by the BChE-specific inhibitor ethopropazine, it is not by another BChE inhibitor, iso-OMPA, nor by the AChE-specific inhibitor BW284c51. The atypical BChE is found as a glycophosphatidylinositol-anchored (GPI-anchored) amphiphilic dimer (G2a), which is unusual for any BChE. We classify the enzyme as an atypical BChE and discuss its implications for the evolution of AChE and BChE and for ecotoxicology.Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are thought to be the result of a gene duplication event early in vertebrate evolution. To learn more about the evolution of these enzymes, we expressed  Acetylcholinesterase  hydrolyzes acetylcholine at the neuromuscular junction of vertebrates. Higher vertebrates also contain an evolutionarily related cholinesterase (ChE), butyrylcholinesterase . The function of BChE is unknown but is suggested to play a role in growth and development and to act as a scavenger of cholinergic toxins as well as having an auxiliary role in synaptic transmission Torpedo marmorataPleuronectes platessaPlatichthys flesusAcanthuras dussumieriPetromyzon marinusMyxine glutinosaWithin species, AChE and BChE have ∼50% amino acid identity, and the overall tertiary structures of the two enzymes are similar R, AChEH, or AChET) subunits 1naH has a hydrophobic C-terminus, which is replaced by a glycophosphatidyl-inositol phospholipid (GPI) anchor and produces amphiphilic dimers, G2aT is capable of forming G1a, G2a, and G4na4a (or G4P). ColQ anchors AChE to the junctional basal lamina of the neuromuscular junction, producing A4, A8, and A12, which represent one, two or three tetramers attached to the ColQ triple helix. While AChET is found in all classes of vertebrates, AChEH exists in cartilaginous fish (Torpedo spp.) Xenopus laevis)  AChE and BChE also exist in a variety of homomeric and heteromeric molecular forms. The catalytic subunit of AChE is found in different variants as a result of alternative splicing of the C-terminus, producing R, H, and T (or AChET) Xenopus tropicalis genome project Xenopus species. The atypical BChEs of T. marmorata and A. dussumieri are T variants (BChET), assembling a collection of globular and asymmetric forms P. flesus is BChEH, assembling only into GPI-anchored G2a forms  BChE does not exhibit alternative splicing and is considered to be found solely as a T variant  and an enzyme that we are classifying as an atypical BChE O. latipes contains 564 amino acids , while the atypical BChE resembles both AChE and BChE more or less equally (46/68% for AChE and 49/67% for BChE). A phylogenetic tree of vertebrate and deuterostome invertebrate ChEs is shown in O. latipes is found in the AChE clade, while the atypical BChE of O. latipes is found in the BChE clade.Two expressed sequences for ChEs are present in the no acids . Pair-wiO. latipes AChE, and ten are conserved in the atypical BChE; in contrast, eight are conserved in vertebrate BChE , near the carboxyl terminal that normally mediates inter-chain disulfide bonding, is also conserved in the atypical BChE , propionylthiocholine (PTCh), and butyrylthiocholine (BTCh) by the recombinant enzyme  is seen with PTCh. Additionally, ATCh and PTCh produce substrate inhibition , although the inhibition by PTCh is weak  values of the enzyme for the inhibitors physostigmine, which inhibits all ChEs; BW284c51, which inhibits AChE preferentially; and iso-OMPA and ethopropazine, which inhibit BChE preferentially. Physostigmine and ethopropazine inhibit the enzyme at sub-µM concentrations; by contrast, much higher concentrations of BW284c51 and Iso-OMPA are required for inhibition under the conditions tested  and its shift to higher values in the absence of detergent due to aggregation of the enzyme  (2a produced is a glycophosphatidylinositol-anchored (GPI-anchored) form. It is unusual for any BChE to be found as a GPI-anchored form ChEs exist in various homomeric and heteromeric molecular forms depending, in part, on the nature of their carboxyl termini. Since the amino acid sequence of the atypical BChE indicates an H-type C-terminus, we performed velocity sedimentation on sucrose gradients in the presence and absence of the non-ionic detergent Triton X-100 to determine the molecular forms of the recombinant enzyme produced , N = 6) . Digesti, N = 6) . These dO. latipes based on the X-ray structures of H. sapiens BChE, and T. californica and Drosophila melanogaster AChEs, in order to get a structural understanding of the special pharmacological and enzymatic properties of the enzyme  is much closer to that of H. sapiens BChE (690 Å3) than that of T. californica AChE (410 Å3). The difference in volume is related to the lack of aromatic residues in three gorge subsites: the peripheral site, the acyl binding pocket, and the choline binding pocket. T. californica AChE possesses three conserved residues  that form an aromatic peripheral binding site and restrict access to the gorge. None of these aromatic residues is conserved in H. sapiens BChE and only one aromatic residue is present in the O. latipes atypical BChE (Tyr282), thus enlarging the gorge entrance  is substituted by a non-aromatic residue in both BChEs . Phe330 can adopt different conformations in the presence of different ligands thus providing an efficient way to modulate the shape and size of the choline binding pocket, even acting as a molecular lid. The absence of a gating aromatic residue at this position in BChE leaves the gorge wide open at all times. Notably, just next to this position, the substitution of Met442 in H. sapiens BChE by Ala440 in O. latipes BChE results in a significantly widened choline binding pocket.Another major difference between the O. latipes shares more structural features with that of H. sapiens BChE than that of T. californica AChE, and it appears legitimate to consider this enzyme as an atypical BChE from a structural point of view.All things considered, the active site gorge of the atypical BChE from O. latipes has an atypical BChE that is in many respects different from either vertebrate AChE or BChE  H. sapiens BChE) O. latipes has one of the two Phe residues, Phe291 (Phe290 in Torpedo spp. AChE) but not the other, present as Val288 (Phe290), seems sufficient to explain the intermediate substrate specificity of the enzyme. Our molecular modeling is consistent with this explanation.The atypical BChE from Mus musculus AChE, the F295L (F288L) mutation has little effect on the mK for ATCh but lowers the mK for BTCh 30-fold H. sapiens AChE, F295L has little effect on the mK for ATCh or PTCh but decreases mK for BTCh 10-fold mK for ATCh, but decreases mK for PTCh and BTCh 4-fold and 33-fold, respectively. With respect to catk, the F295L M. musculus AChE mutant decreases catk 4-fold for ATCh and increases it 14-fold for BTCh. In H. sapiens AChE, the F295L, A mutants do not affect appreciably the catk for ATCh but increase mK 400-fold. For wild type H. sapiens and M. musculus AChE, the highest catalytic efficiency (cat/Kmk) by far is seen for ATCh; whereas, for the F295L, A mutants, the highest efficiency is found for BTCh, with the efficiency for all three substrates within a factor of three. These data are consistent with, if not identical to, the results that we have obtained for the O. latipes atypical BChE, which lacks the homologous Phe: all three substrates are hydrolyzed appreciably, the mK is lowest for BTCh but lower for PTCh compared to ATCh, and the catalytic efficiency for all the substrates are within a factor of two. Thus, it appears that the residue corresponding to Phe288 affects both the binding and hydrolysis of substrate.In the mouse H. sapiens BChE, and Phe288 is not conserved in the atypical BChE from O. latipes (Leu291). Thus, this stabilizing network is absent in BChE. However, the catalytic histidine of BChE is well stabilized by interactions with an adjacent phenylalanine, Phe398 in H. sapiens BChE or Phe403 in O. latipes atypical BChE, which is absent in AChE . As a matter of fact, the catalytic histidine of H. sapiens BChE has never been observed in an alternate conformation, whereas such a conformation is not unusual in liganded AChE. (See X-ray structure of VX-AChE (pdb entry 2VXR), tabun-AChE (pdb entry 3DL4) and the NMR study of Masiah et al. O. latipes atypical BChE, there is an aromatic residue facing it, Phe401. Reciprocally, when there is an aromatic residue at this position, as with Phe288 in T. californica AChE, there is an aliphatic residue facing it, Val400  that shape the acyl binding pocket of AChE, along with the aromatic peripheral site residues, form an aromatic continuum with Phe330 and Phe331 of the hydrophobic patch. This aromatic network in AChE has been suggested to play an important role in the stabilization of the catalytic histidine , Val400 . This syO. latipes is efficiently inhibited by physostigmine and ethopropazine but not BW284c51 or iso-OMPA, a pattern of inhibition different from AChE or BChE. However, we think that the pattern more closely resembles BChE rather than AChE with the inhibition by iso-OMPA being exceptional. All ChEs are inhibited by physostigmine, so its effective inhibition simply confirms that the enzyme is a ChE M. musculus AChE, the peripheral site double mutant Y72N/Y124Q (Y70N/Y121Q) increases iK for BW284c51 69-fold H. sapiens AChE the same double mutation increases iK for the inhibitor 35-fold. Moreover, the choline-binding site mutation Y337A (Phe330A) increases the iK 5-fold iK by over two orders of magnitude. Thus in the O. latipes atypical BChE, which has the aliphatic substitutions Met69, Val124, and Cys333 at the homologous sites, preventing the necessary cation-π and π-π interactions via the aromatic residues, one might expect inefficient inhibition by BW284c51, which is exactly what is observed. Notably, the structure of the acyl pocket does not appear to influence the binding of BW284c51 O. latipes.The atypical BChE of O. latipes is inhibited by a low concentration of the tricyclic amine-containing phenothiazine, ethopropazine, which contains a bulky diethylamino-2-isopropyl exocyclic group – inhibition typical of vertebrate BChE. Inhibition of AChE and BChE by ethopropazine appears to be sensitive to the nature of the residue corresponding to Phe330 of the aromatic patch in Torpedo spp. AChE and to the volume of the active center in BChE M. musculus AChE, the Y337A (F330A) mutation decreased iK almost 2700-fold, making it comparable to BChE H. sapiens BChE do not substantially affect the iK for the inhibitor Torpedo spp. AChE was reported to be 302 Å3, and in a modeled H. sapiens BChE it was measured as 502 Å3. The van der Waals volume of ethopropazine is 318 Å3, explaining why the drug does not bind well to AChE. The F330A mutation of Torpedo spp. AChE increases the lower gorge volume to 338 Å3 and allows ethopropazine to bind. In contrast, the A328Y and A328F mutations in the H. sapiens BChE model only decrease the volume of the lower gorge to 410 and 406 Å3, respectively, which allow the gorge to still be large enough to bind ethopropazine easily 3 for H. sapiens BChE, 630 Å3 for O. latipes atypical BChE and 410 Å3 for Torpedo spp. AChE, in good agreement with the observed trend for ethopropazine inhibition.The atypical BChE of M. musculus AChE, the acyl pocket mutation F295L (F288L) increases ik 90-fold, F297I (F290I) 200-fold, and the double mutant, over 500-fold for iso-OMPA H. sapiens AChE, similar, although more complex, results were seen for the less bulky organophosphates, diisopropyl phosphorofluoridate (DFP), diethyl phosphorofluoridate (DEFP), and paraoxon ik up to 130-fold, with substitutions at Phe295 (Phe288) having the greater effect. The differences in ik were primarily due to decreases in dK with 2k relatively unaffected, suggesting that the substitutions relieved steric interference in the binding of the inhibitors and enhanced enzyme affinity, but did not alter the rate of phosphorylation. Unlike ethopropazine, converse site-directed mutagenesis of the acyl pocket has not been reported for BChE and iso-OMPA. The atypical BChE from O. latipes is relatively insensitive to, but is inhibited by iso-OMPA. This result, as well as the substrate specificity of the enzyme, is in good agreement with the reduction in size of the acyl pocket, compared to BChE, associated with the presence of only one of the two aromatic residues in the subsite in our molecular modeling. This explanation, as well as the others offered on the molecular basis of substrate and inhibitor specificity could be tested by site-directed mutagenesis. In any event, it should be kept in mind that vertebrate ChEs cannot always be characterized as AChE or BChE simply on the basis of diagnostic inhibitor specificities T. marmorata, P. flesus, P. platessa, and A. dussumieri, we cannot compare at the molecular level the atypical BChE from O. latipes with these enzymes. Iso-OMPA is an effective organophosphate inhibitor of BChE but not AChE O. latipes is a GPI-anchored G2a membrane-bound dimer and thus resembles a BChEH, possessing an H-type C-terminus, which is removed upon the addition of the GPI anchor. The atypical BChE of P. flesus is also a GPI-anchored G2aT. marmorata and A. dussumieri are BChET types since they are capable of assembling into G1, G2, and G4 molecular forms, and are able to interact with the collagenic tail protein ColQ P. platessa enzyme are not known. While many vertebrate AChEs, including Torpedo spp. and mammalian AChE are found as AChET and AChEH as the result of alternative splicing, all vertebrate BChEs are considered to be of the T-type O. latipes and P. flesus are the only certain exceptions to this rule. The nature of the ChE activity in Xenopus spp. is perplexing. A PIPLC-sensitive G2 AChEH has been reported in X. laevis muscle X. laevis tadpoles was found to be resistant to various carbamate and organophosphate inhibitors and to not show inhibition by excess substrate X. tropicalis indicates that only AChET sequences and not AChEH sequences are present. In striking contrast though, a BChEH sequence is found. The H-peptide is widespread in the AChEs of invertebrates, although these sequences are not homologous among the invertebrates nor with the vertebrates, containing only a few functional similarities: a cysteine near the carboxyl terminal that mediates inter-chain disulfide bonding in the dimer, and a GPI anchor signal consisting of an ω cleavage/attachment site followed by a non-conserved stretch of hydrophobic amino acids The atypical BChE from O. latipes and the other atypical BChEs discussed represent a transitional form of ChE as BChE gradually evolved from an ancestral AChE in the vertebrates subsequent to a gene duplication event early in vertebrate evolution. Assuming Darwinian gradualism, one would expect that if AChE were the ancestral vertebrate ChE, with two Phe residues in its acyl pocket, that upon gene duplication the two residues would be replaced sequentially with an intermediate enzyme having one of the Phe residues and decreased substrate specificity, a property that is consistent with the putative role of BChE in detoxification mechanisms and our data In terms of substrate and inhibitor specificity and inhibition, it is tempting to speculate that the atypical BChE of P. marinus and the hagfish M. glutinosa, appear to have only one ChE, AChE T. marmorata, AChE and an atypical BChE O. latipes can serve as a model for the evolutionarily intermediate ChE between AChE and BChE.Because of the possibility of three extensive (even genome-wide) gene duplication events early in vertebrate evolution at (1) the origin of the vertebrates, (2) the emergence of the jawed fish, and (3) the appearance of the ray-finned fishes Torpedo spp. and mammals, and apparently in a BChE in the amphibian Xenopus spp., but not reported in reptiles or birds, Massoulié et al. P. flesus and O. latipes. Combes et al.  Given that the poorly conserved H-transcripts are widespread in the AChEs of invertebrates, while in vertebrates they are present only in the AChE of the elasmobranch Pesticide use is a major concern in aquatic environments, where runoff from agricultural and urban ecosystems impacts their ecology. The major pesticides in use today are organophosphate and carbamate acetylcholinesterase inhibitors, and pyrethroid ion-channel agents All animal procedures were conducted in strict adherence to the European Council Directive of November 24, 1986 (86-609). Approval for this study was provided by Comité Régional d'Ethique Languedoc Roussillon C34-172-10.Dulbecco's modified Eagle medium, fetal bovine serum, OptiMEM medium, and phosphatidylinositol-specific phospholipase C (PIPLC) were purchased from Invitrogen, Carlsbad, California. FuGene was obtained from Roche, Indianapolis, Indiana. Acetylthiocholine (ATCh), butyrylthiocholine (BTCh), propionylthiocholine (PTCh), [4-[5-[4-(dimethyl-prop-2-enyl-ammonio)phenyl]-3-oxo-pentyl]phenyl]-dimethyl-prop-2-enyl-azanium dibromide (BW284c51), 5-(3-carboxy-4nitro-phenyl)disulfanyl-2-nitro-benzoic acid (DTNB), N-[bis(propan-2-ylamino)phosphoryloxy-(propan-2-ylamino)phosphoryl]propan-2-amine (iso-OMPA), 10-(2-diethylaminopropyl) phenothiazine hydrochloride (ethopropazine), and (3aS-cis)-1,2,3,3a,8,8a-hexahydro-1,3a,8-trimethylpyrroloindol-5-ol methylcarbamate (physostigmine) were purchased from Sigma, St. Louis, Missouri. The high-affinity ligand 7-[(diethoxyphosphoryl)oxy]-1-methylquinolinium iodide (DEPQ) was a gift from Yacov Ashani.O. latipes adults and embryos were from facilities at INRA. Fish were maintained at 28°C on a 13-h light/11-h dark cycle.O. latipes library was obtained from UniGene. The clone was extended by PCR on the basis of the gene structure on scaffold2582 in UCSC genome project, and the full cDNA sequence (Genbank GU797251) was cloned into the expression vector pCMV SPORT 6.1 (Invitrogen).The cDNA clone Ola.23452 (Genbank AV668390) from an Sequences were aligned with Clustal W or Clustal X for molecular modeling or phylogenetic analysis by the neighbor-joining method 6 cells/75 cm2 culture flask, incubated overnight, and transferred to OptiMEM medium. FuGene was then used to transfect the cells with 7.8 µg of DNA. The cells were then incubated for 48 h at 37oC before the medium was removed and the cells extracted in high ionic strength (HIS) buffer: 10 mM NaHPO4, pH 7, 1 M NaCl, 1% Triton X-100, 1 mM EDTA. Extracts were centrifuged at 20,000g for 20 min, and the supernatants were assayed for ChE activity.COS-7 monkey cells (American Type Culture Collection) were grown in Dulbecco's modified Eagle medium containing 10% fetal calf serum. Cells were plated at a density of 2.5×10et al.et al.4, pH 7, 0.3 mM DTNB, 167 mM NaCl, and 258 µM Triton X-100; some BChEs are inhibited by Triton X-100 O. latipes is not at the concentrations used in this study (data not shown). ATCh, BTCh, and PTCh were used as substrates at various concentrations; for pharmacological analyses and assays of sucrose gradients, the concentration of ATCh was 1 mM. The kinetic parameters mK, ssK, b, and maxV, were determined by using SigmaPlot to fit the data to the equation below as described by Radić et al. ssK is the dissociation constant for the binding of substrate to a second site on the enzyme, and the parameter b indicates the relative catalytic efficiency of the SES complex compared to SE. If b <1, the enzyme shows substrate inhibition; if b>1, the enzyme shows substrate activation, and if b = 1, Michaelis-Menten kinetics is observed.AChE activity was measured according to the method of Ellman catk (maxV/[Enzyme]) was determined by enzyme titration with DEPQ 50 for the inhibitors used were determined by incubating enzymes with various concentrations of drug for 20 min and then assaying for enzyme activity in the presence of ATCh. SigmaPlot was then used to fit the data to a three-parameter logistic function, yielding IC50. Biphasic inhibition curves were fit with GOSAfit. Since we were just looking for classical diagnostic differential inhibition, it was not necessary to determine ik or IK values for the inhibitors The turnover number 2×t (h)]  =  2.5×1010 as described previously The molecular forms of ChE were analyzed by velocity sedimentation in 5–25% isokinetic sucrose gradients prepared in HIS buffer (with or without Triton X-100) containing 1 mg/ml bovine serum albumin. Sedimentation was in an SW 41 rotor at 30,000–37,000 rpm for times satisfying the equation [(rpm)H. sapiens BChE (pdb code 1p0i), T. californica AChE (pdb code 1ea5) and D. melanogaster AChE (pdb code 1dx4) as structural templates. Structural water molecules that are conserved among the three templates were also retained in the modeling procedure. A series of 200 runs of MODELLER were carried out using standard parameters, and the outcomes were ranked on the basis of the internal DOPE scoring function. The model with the highest score was chosen as the candidate model. Then, energy minimization was performed using GROMACS 4.05 according to the software protocol Sequence alignment was performed with ClustalX and homology modeling was carried out using MODELLER 9v8 program Figure S1O. latipes BChE.The pdb dataset for the model of (DOC)Click here for additional data file.


Ser410 and p-PDK1Thr513 are modulated by small-molecule PDK1 inhibitors, and their degree of dephosphorylation correlates with inhibitor potency. We note that classical, ATP-competitive PDK1 inhibitors do not modulate PDK1 T-loop phosphorylation (p-PDK1Ser241), highlighting the value of an unbiased approach to identify drug target-regulated phosphorylation sites as these are complementary to pathway PD biomarkers. Finally, we extend our analysis to another protein Ser/Thr kinase, highlighting a broader utility of our approach for identification of kinase drug-target engagement biomarkers.Pharmacodynamic (PD) biomarkers are an increasingly valuable tool for decision-making and prioritization of lead compounds during preclinical and clinical studies as they link drug-target inhibition in cells with biological activity. They are of particular importance for novel, first-in-class mechanisms, where the ability of a targeted therapeutic to impact disease outcome is often unknown. By definition, proximal PD biomarkers aim to measure the interaction of a drug with its biological target. For kinase drug discovery, protein substrate phosphorylation sites represent candidate PD biomarkers. However, substrate phosphorylation is often controlled by input from multiple converging pathways complicating assessment of how potently a small molecule drug hits its target based on substrate phoshorylation measurements alone. Here, we report the use of quantitative, differential mass-spectrometry to identify and monitor novel drug-regulated phosphorylation sites on target kinases. Autophosphorylation sites constitute clinically validated biomarkers for select protein tyrosine kinase inhibitors. The present study extends this principle to phosphorylation sites in serine/threonine kinases looking beyond the T-loop autophosphorylation site. Specifically, for the 3′-phosphoinositide-dependent protein kinase 1 (PDK1), two phospho-residues p-PDK1 Drug development remains an expensive, uncertain and challenging endeavor. The cost from discovery to regulatory approval of a novel drug averages about $900 million out of pocket, with a capitalized cost of ∼$1.78 billion, and the process now typically takes up towards 13 years Are there active concentrations of drug in plasma, blood and tissue? Is the target engaged? Is there downstream pathway inhibition? Is there a biological effect or clinical response?Biomarkers are defined as molecular entities that can be measured and evaluated objectively as indicators of normal biological processes, pathogenic processes or pharmacological response to therapeutics and can be categorized as either proximal (target-related) or distal (disease-related) For kinase drug discovery, phosphorylated protein substrates of a kinase drug target represent attractive PD biomarker candidates. However, an inverse linear relationship between the stoicheometry of substrate phosphorylation and the degree of kinase inhibition seldom exists as the phosphorylation state of a given protein usually depends on convergent upstream input from multiple signaling pathways as well as negative and positive feedback loop regulation. Moreover, the phosphorylation of a particular substrate site is subject to variable rates of turnover depending on the dedicated phosphatase activity against said phosphorylation site. Despite these limitations, if a drug with a novel unproven mechanism fails to achieve the desired clinical activity, evaluation of PD biomarkers, when available, is an important means to assess the degree of target engagement by the drug. If the PD biomarkers indicate strong target engagement, the lack of clinical activity is likely due to the biology/mechanism of the target. Conversely, minimal target engagement due to non-favorable properties of the molecule or insufficient dosing will dictate additional pharmacological activities to address these issues.cis or trans- activation For protein kinases, amino acid sequence alignments combined with site-directed mutagenesis and enzymatic assay have revealed conserved regulatory phosphorylation sites In order to assess kinase drug-target engagement independent of prior knowledge of kinase (auto)phosphorylation sites, such as the conserved T-loop phosphorylation site, we here report a broadly applicable, quantitative, differential mass spectrometric (MS) approach for identification and quantification of all direct and indirect drug-regulated phosphorylation sites in any kinase drug target.Thr308 and p90RSKSer221 in multiple cell lines Ser241) as a candidate target engagement biomarker for PDK1 inhibitor development. While it is informative to monitor the inhibition of proximal PD biomarkers (i.e. p-AKTThr308 and p-p90RSKSer221) in response to PDK1 inhibitor treatment, it is well established that the phosphorylation state of AKT is regulated indirectly by many other protein kinases including a majority of receptor protein tyrosine kinases, as well as lipid kinases, most notably phosphoinositide 3′-kinase (PI3′K). Accordingly, the observation that a PDK1-targeted small molecule inhibitor modulates p-AKTThr308 does not guarantee that this is due to direct inhibition of PDK1, despite PDK1 is the upstream activating kinase of AKT that directly phosphorylates the T-loop phosphorylation site Thr308 in AKT. Based on these facts, we set out to identify drug-regulated phosphorylation sites on PDK1 that could be used to link direct PDK1 drug-target engagement in cells to biological responses. Specifically, we mapped and quantified 12 serine/threonine phosphorylation sites on PDK1, including novel phosphorylation sites, and found that two of these sites, p-PDK1Ser410 and p-PDK1Thr513, were modulated by pharmacological inhibitors of PDK1. Moreover, the degree of dephosphorylation of these sites correlated with the inhibitor potency (IC50) in PDK1 enzymatic assays.We recently characterized a panel of structurally diverse PDK1-targeted lead compounds that inhibit the phosphorylation of PDK1 substrates such as AKTcis inhibition/trans activation mechanism and phosphorylation changes exerted by upstream kinases or phosphatases regulate kinase catalytic activity through allosteric conformational changes The catalytic activity of protein kinases is tightly controlled by reversible protein phosphorylation bona fide PDK1 substrates such as p-AKTThr308 and p-RSKSer221Thr308 inhibition in cells in response to inhibitor treatment (Ser221 in PC3 cells (50) and the cellular inhibition of p-RSKSer221 in PC3 cells (Thr308 (Thr308 is effectively inhibited in PC3 cells using a highly selective (>1000-fold) PDK1 inhibitor Thr308 in mouse embryonic fibroblasts (MEFs) Thr308 correlated with lack of kinome selectivity  and/or lipid kinases such as PI3′K. Indeed, side-by-side comparison of PDK1-inhibitor-treated PC3 and LNCap cells confirmed that p-AKTThr308 is not a universal PD biomarker for PDK1 kinase inhibitor development whereas p-RSKSer211 appears to be consistently inhibited across cell lines  that have been reported for the PI3K-pathway  in PC3 cells, yet inhibit reatment . The PTEC3 cells . In totaC3 cells  using thC3 cells , when sm  phosphorylation site analysis on immunoprecipitated PDK1 from cells treated with 4 structurally diverse PDK1 inhibitors for 24 hrs according to the workflow shown in hibitors . The corThe human genome encodes a little more than 500 protein kinases and an estimated twenty percent of drugs currently in development target this enzyme class 90) in vivo and hence inform about true drug target exposure. Such combined information has far-reaching potential, and can provide evidence whether a molecule fails clinical proof-of-concept (POC) for efficacy in clinical trials due to the biological mechanism or simply due to lack of target modulation. It is important to emphasize that target engagement biomarkers, while defining the molecular effects of the drug on the purported target, may not necessarily have any correlation with the desired clinical effects  in high glucose, L-glutamine, 110 mg/L sodium pyruvate and 10% FBS. Myc-tag PDK1 (pCMV5-PDK1-wt) was transiently transfected into 293FT cells (5×106 cells per 10 cm petri dish) using standard 293FT LV packaging transfection protocols (i.e. lipofectamine in OPTI-MEM). Kinase inhibitors or control (DMSO) were added 18 hours prior to cell lysis using immunoprecipitation buffer , 1 mM b-Glycerolphosphate, 1 mM Na3VO4, 2.5 mM Sodium pyrophosphate). ‘Heavy’ and ‘light’ isotope-labeled samples were mixed 1∶1 based on the protein concentration measurement using standard BCA protocol. 250 µl lysate was pre-cleared by adding 30 µl of a 50% Protein-A agarose slurry for ∼2 hours and PDK1 was immunoprecipitated via tag-specific pull down (anti-c-MYC antibody) and subjected to SDS-PAGE and stained with Coomassie blue. The identical procedure was also carried out for a 1;1 mix of cells grown in ‘heavy’ and ‘light’ isotope without compound treatment as an internal control.T293 cells were grown for seven passages in DMEM media containing either normal-isotope lysine, arginine (‘light medium’) or Western blotting was performed for 2 h using a BioRad SemiDry apparatus with Immobilon-P PVDF membrane (Millipore), at a constant voltage of 25 V and 10A/10 cm2. Chemiluminescent detection was carried out using CDP Star™  per recommendations by the manufacturer.Ser221 and phospho-AKTThr308 were previously reported and performed as described Development of biochemical assays to report on phospho-p90RSKExcised protein bands were reduced, alkylated and digested with Trypsin (Promega), GluC (Roche) and AspN (Roche) overnight at the room temperature. The resulting peptides were analyzed by a reverse-phase nano-liquid chromatography system  online-coupled with a LTQ Orbitrap hybrid mass spectrometer with electron transfer dissociation (LTQ Orbitrap XL ETD) (ThermoFisher Scientific). Briefly, an aliquot of the digested sample was loaded onto a micro-capillary liquid chromatography column (50 µm i.d.×25 cm packed with Magic C18). Bound peptides were eluted from the column via a 75 min solvent gradient program. Eluted peptides were continuously converted to multiply charged peptide ions by electrospray ionization and introduced into the vacuum system of a LTQ Orbitrap XL ETD. High resolution mass spectra were acquired at a rate of 1 spectrum per second. These spectra recorded the intensity, retention time, and accurate mass-to-charge ratio for the discrete peptide ions in each sample. MS/MS spectra that were simultaneously acquired were used to provide amino acid sequence information for selected peptide ions.http://pubchem.ncbi.nlm.nih.gov/ using SEQUEST. Parameters for the searches included the following: Trypsin, AspN, GluC for enzyme specificity respectively; static modifications of 57.02 Da on Cys (Carbamidomethylation); differential modification of 79.9 Da on Ser, Thr, and Tyr (phosphorylation) and 15.99 Da on Met (oxidation). The precursor ion mass tolerance was set as 50 ppm and the fragment-ion mass tolerance was set as +/−0.8 Da Mono/Mono. Each MS/MS spectra exhibiting possible phosphorylation were manually validated. ‘Light’ and ‘heavy’ SILAC labeled peptide pairs are identified based on m/z and the retention time of the features contained in the data set. The ratio of the identified peptide pairs were then derived based on the ratio of their corresponding areas under the ion chromatographic curves.Acquired MS data were uploaded to the Elucidator data analysis system  as previously described Figure S1Relative quantification of MARK3 phosphorylation by SILAC-IAP-MS.A) MARK3 targeting inhibitors. B) Relative quantification by SILAC-IAP-MS showed the signal intensity of three phosphorylation sites  were dose-dependently reduced in response to both inhibitor 5 and 6.(TIF)Click here for additional data file.Figure S2Relative quantification of c-Met phosphorylation by SILAC-IAP-MS.A) Fold changes for c-Met phosphopeptides treated with MK-2461 treatment vs. DMSO (red columns) were obtained as described in materials and methods. B) Reconstructed ion chromatogram and C) high resolution full MS spectra of both light (black) and heavy (red) peptides ISAIFSTFIGEHpYVHVNATpYVNVK (Y1349/Y1356) respectively.(TIF)Click here for additional data file.Table S1Relative quantification of c-Met phosphorylation by SILAC-IAP-MS. DMSO and IC90 refer to treating cells either using DMSO or MK-2461 at IC90 concentration, (L) and (H) refer to light and heavy isotope media.(DOC)Click here for additional data file.


The HIV-1 protease is initially synthesized as part of the Gag-Pol polyprotein in the infected cell. Protease autoprocessing, by which the protease domain embedded in the precursor catalyzes essential cleavage reactions, leads to liberation of the free mature protease at the late stage of the replication cycle. To examine autoprocessing reactions in transfected mammalian cells, we previously described an assay using a fusion precursor consisting of the mature protease (PR) along with its upstream transframe region (p6*) sandwiched between GST and a small peptide epitope.In this report, we studied two autoprocessing cleavage reactions, one between p6* and PR  and the other in the N-terminal region of p6*  catalyzed by the embedded protease, using our cell-based assay. A fusion precursor carrying the NL4-3 derived protease cleaved both sites, whereas a precursor with a pseudo wild type protease preferentially autoprocessed the proximal site. Mutagenesis analysis demonstrated that several residues outside the active site  contributed to the differential substrate specificity. Furthermore, the cleavage reaction at the proximal site mediated by the embedded protease in precursors carrying different protease sequences or C-terminal fusion peptides displayed varied sensitivity to inhibition by darunavir, a catalytic site inhibitor. On the other hand, polypeptides such as a GCN4 motif, GFP, or hsp70 fused to the N-terminus of p6* had a minimal effect on darunavir inhibition of either cleavage reaction.Taken together, our data suggest that several non-active site residues and the C-terminal flanking peptides regulate embedded protease activity through modulation of the catalytic site conformation. The cell-based assay provides a sensitive tool to study protease autoprocessing reactions in mammalian cells. HIV-1 protease (PR) is one of three virus-encoded enzymes essential for virus propagation and infectivity. The catalytic site of protease has been mapped to residue D25. Alteration of D25 to A, Y, H, or N completely abolishes enzymatic activity . In the The released mature HIV-1 PR forms stable dimers and recognizes at least 10 different cleavage sites in the Gag and Gag-Pol polyproteins. Accurate and precise protease processing of these sites is absolutely required for the production of infectious progeny virions -13. Therin vitro [E. coli [In distinct contrast to the extensive studies on the mature protease, the molecular and cellular mechanisms of HIV-1 protease autoprocessing are largely undefined. It is known that the protease domain embedded in the precursors is essential and sufficient to mediate autoprocessing because various precursors containing an active PR domain are able to release the mature protease when expressed in vitro ,21, in E[E. coli ,22-24, o[E. coli ,25. Of t[E. coli . A trans[E. coli . In addi[E. coli . In cont[E. coli ,30. Remo[E. coli . These s[E. coli ,24,31,323KMN). Because a protease antibody with high sensitivity is not available, a C-terminal peptide epitope was included to facilitate detection of the precursor substrate and processing products. The resulting fusion precursor effectively autoprocessed in E. coli and in transfected mammalian cells, and faithfully reproduced autoprocessing phenotypes observed in other systems [Structural information on the embedded protease is currently unavailable in spite of more than 500 reported structures for the mature protease. Therefore, the mechanism by which the embedded protease mediates the autoprocessing cleavage reactions remains obscure. To facilitate examination of the cleavage reactions involved in protease autoprocessing, we previously engineered a fusion precursor consisting of a miniprecursor (p6*-PR) sandwiched between GST and a small peptide epitope Figure . GST was systems ,25. ThisIn this report, we examined two cleavage reactions involved in protease autoprocessing using protease inhibitors as a structural probe to gain insights into the catalytic site conformation of the protease under different contexts. Our data demonstrated that different protease constructs displayed varying sensitivities to inhibition by the currently available protease inhibitors, suggesting the existence of more than one catalytic site conformation. Interestingly, several surface residues far from the PR catalytic site, and residues adjacent to the PR C-terminus, also regulated the activity of the embedded protease involved in the autoprocessing cleavage reactions. Our data highlights a different catalytic mechanism driving liberation of the mature protease and provides a glimpse of the embedded protease as it functions during autoprocessing.NL hereafter; the other was a pseudo wild type protease, PRpse, which was engineered to reduce protease self degradation  and protein aggregation mediated by thiol oxidation (C67A and C95A) for structural analysis of the mature protease in vitro [pse is also self degraded [NL and PRpse; all others are identical in these precursors  cleavage site) and the other at the N-terminal region of p6*  cleavage site) Figure . We testin vitro ,31. Whendegraded . There as Figure . Interese Figure . Becausepse precursors containing individual or combinatorial amino acid substitutions reflecting those present in PRNL . Therefore, we are inclined to suggest that different embedded proteases display different substrate preferences.It is very intriguing that different proteases display different preferences to the D and P cleavage sites. Since the cleavage sequences are identical in these fusion constructs, we suggest that different proteases have different catalytic sites that determine different substrate preferences. One could argue that different substrate accessibility might also be attributed to the observed difference. Although it is possible that the P site accessibility is altered by the adjacent PR, it is difficult to explain how the PRWe next utilized darunavir, the most potent HIV-1 protease inhibitor, as a structural probe to examine the catalytic site conformation of various proteases. Darunavir binds to the catalytic site of the mature protease with low nanomolar affinity ,41. A prNL fusion precursor carries two native cleavage sites, D and P, respectively. To examine whether the cleavage reactions at these two sites interfere with each other, we engineered two fusion precursors to examine the individual reaction. The P site was mutated in the MG precursor, and the D site was deleted in the M1 precursor [The wild type p6*-PRrecursor  .We next examined effects of darunavir on the released proteases. In the absence of darunavir, the two PR-containing products, PRradation , while te Figure , middle.d Figure , right. pse-HA was less sensitive than PRNL-HA to darunavir inhibition of self degradation  approximately 6-fold higher than that for PRNL-HA (~10 nM), suggesting that they are not enzymatically identical. This is consistent with a previous report demonstrating that p6*-PR is incapable of processing many of the cleavage sites in the Gag polyprotein normally processed by the mature protease [pse vs. PRNL) and also by the p6* peptide fused to the N-terminus of the mature protease (PRNL vs. p6*-PRNL).The released PRn Figure &E, suggein vitro . In addiprotease . Taken t50 determined in our system is very similar to the IC50 identified for the mature protease activity in HIV-1 infected cells. Darunavir has an IC50 of ~5 nM to inhibit the production of p24 [50 for PRNL-HA was ~10 nM. The slight difference might be attributed to varied protein concentrations. At least two factors might be attributed to this slight difference.  One factor is different readouts; the ~5 nM reflects the darunavir concentration required to achieve 50% inhibition of p24 production whereas the ~10 nM represents the concentration required to suppress the mature protease from self degradation by 50%.  The other factor is varied protein concentrations. Subsequently, more darunavir is required to suppress PR-HA self degradation in our cell-based assay. Nevertheless, our result is in agreement with the established darunavir IC50, further validating the utility of our assay for protease activity and autoprocessing analyses.It should be noted that the self degradation ICn of p24 ,41, wher50) of the D site reaction to be ~7500 nM darunavir, as indicated by open triangles . The same trend was observed for the P site cleavage reaction, which had an apparent IC50 of ~1500 nM, i.e., ~150-fold higher than the self degradation IC50 of PRNL-HA (~10 nM). Additionally, the cleavage reaction IC50 of the embedded p6*-PRpse was ~7500 nM, whereas the self degradation IC50 for the released PRpse was ~40 nM  than the HA and Myc epitopes, although there is no obvious correlation between the lengths or charge properties of the tags with this observed difference. Our data suggested that the embedded protease activity was modulated by different C-terminal tags, but self degradation of the mature protease after it was released from the precursor was not significantly affected by these tags. One possible cause for the increased sensitivity to darunavir could be that the C-terminal tags increased difficulty in precursor dimerization, and thus less active site were formed and less darunavir were required to suppress their catalytic activity. Alternatively, the C-terminal tag could directly modulate the enzymatic activity of the embedded protease by influencing the catalytic site conformation. Biophysical and structural analyses of these proteases are essential to definitely distinguish the possible causes.To examine whether different amino acid sequences fused to the C-terminus of PR influence protease autoprocessing, we engineered a panel of fusion precursors carrying different C-terminal epitopes Figure . A trunc50 ~20 nM darunavir, whereas the IC50 for the cleavage reaction was greater than 1500 nM. This was similar to the HA-tagged fusion precursor. We also detected additional fragments likely produced as a result of cleavage reactions at alternative sites at high darunavir concentrations, suggesting the existence of a catalytic conformation(s) induced by darunavir binding that process amino acid sequences not recognized at low darunavir concentrations. These data further confirmed that the embedded protease is much less sensitive to darunavir inhibition and its activity could be influenced by different C-terminal peptides that are adjacent to it.To gain further insight into the effect of C-terminal flanking sequences on protease autoprocessing, we constructed more fusion precursors containing longer C-terminal fusions. The GCN4 dimerization motif derived from a yeast transcription factor -52 was d50 of the released PR-RT-Flag was at the low nanomolar range (2-4 nM), and the cleavage reaction mediated by the embedded protease displayed an apparent IC50 of ~60 nM darunavir , and there are reports suggesting a possible contribution of RT to regulation of protease activity ,54. We gr Figure . We werer Figure , while t50s that were very similar to the corresponding values observed for the fragments released from the GST fusion precursors  affect the catalytic site conformation, which in turn alter the response sensitivity to inhibition by catalytic site inhibitors. Several non-active site residues as well as residues flanking the protease also contributed to modulation of catalytic site conformation. Our results are consistent with extensive structural studies that demonstrate multiple slightly different catalytic site conformations in mature protease as a molecular basis for the evolution of drug resistant strains. The advantage of our assay is simplicity without a compromise of sensitivity, allowing for broad application in the examination of the protease autoprocessing mechanism and/or in the identification and characterization of novel anti-HIV drugs. Our results also imply that novel inhibitors targeting important surface residues may be developed as alternative therapeutic agents. An encouraging report along this vein identified a novel inhibitor from combinatorial libraries that suppresses HIV-1 protease activity likely through binding to a groove outside of the catalytic site . IdentifNL-HA, GST-p6*-PRpse-HA, GST-p6*-PRpse D25N-HA, GST-p6*-PRNLH69D-HA, GST-M1-PRNL-HA, and GST-M2-PRNL-HA was previously described [pse-HA expression plasmid. The GST-p6*-MG-PRNL-HA construct was generated by mutating the amino acids at the P site (from TVSFSF⇓PQIT to TVMG⇓PQIT) to block proteolytic cleavage. The indinavir resistant mutation V77V82 was cloned into the plasmid encoding GST-M1-PRNL-HA. To generate fusion precursors with various C-terminal sequences, the Flag tag coding sequence in GST-M2- M2-PRNL-Flag was replaced by sequences encoding the indicated epitopes, motif or proteins. Similarly, the GST coding sequence in the GST-Flag-PRNL-HA expression plasmid was replaced with GCN4, GFP, or Hsp70 [Construction of plasmids encoding GST-p6*-PRor Hsp70  encoding2O was mixed with 9.3 μl of 2 M CaCl2. Then 75 μl of 2xHBS  was added to the DNA-Ca mixture followed by gentle agitation. The resulting mixture was directly added drop-wise to each well. Protease inhibitors darunavir  and indinavir  were dissolved in autoclaved H2O to generate 250 μM darunavir and 10 mM indinavir stock solutions, respectively; these were stored at -20°C. darunavir or indinavir were then diluted and added to cells directly after transfection at the indicated working concentration. After 7-8 h incubation at 37°C, 5% CO2, the transfection medium was replaced with fresh chloroquine-free medium containing the corresponding protease inhibitors. At 30 h post-transfection, cells were washed once with PBS and lysed with 100 μl lysis buffer A . Cell lysates were clarified by brief centrifugation at 20800 × g for 2 minutes and stored at -20°C for further western blot analysis.HEK293T cells  were maintained in DMEM containing 10% fetal bovine serum and penicillin/streptomycin . Plasmid DNA was transfected into HEK293T cells using calcium phosphate as previously described ,46. BriePrimary antibodies used in this study include polyclonal rabbit anti-GST , anti-V5 , anti-HIV1 protease ; mouse monoclonal anti-HA , anti-flag , and anti-myc . Secondary antibodies included IR700 goat anti-rabbit  and IR800 goat anti-mouse . For protein detection, about 1/6 of the cell lysate from one well of a 12 well plate was subjected to SDS-PAGE followed by western blot analysis. The blots were probed simultaneously or separately with the corresponding primary and secondary antibodies, and visualized with an Odyssey infrared dual laser scanning unit . To reduce background noise, the rabbit anti-HIV protease antibody was first incubated with cell lysates from untransfected 293T cells that had been resolved by SDS-PAGE and transferred onto a PVDF membrane.For quantification of protein intensity, Western blot images captured by an Odyssey infrared dual laser scanning unit were analyzed using TotalLab software (Nonlinear Dynamics Inc.). The total pixel volume of each band was quantified and normalized; the highest value was arbitrarily set at 1000.Colorado State University has filed for patent protection of this work.CC designed the experiments and wrote the manuscript. LH constructed most of the plasmids used in this study and performed 293T transfections and Western blot analyses. YL made the plasmids used in Figures


The c-Jun-N-terminal kinase (JNK), glycogen synthase kinase-3β (GSK-3β) and cyclin-dependent kinase 5 (Cdk5) can all be responsible for this phosphorylation. These kinases are activated by excitotoxic stimuli fundamental hallmarks of AD. The exposure of cortical neurons to a high dose of NMDA (100 μM) for 30’-45’ led to an increase of P-APP Thr668. During NMDA stimulation APP hyperphosphorylation has to be assigned to GSK-3β activity, since addition of L803-mts, a substrate competitive inhibitor of GSK-3β reduced APP phosphorylation induced by NMDA. On the contrary, inhibition of JNK and Cdk5 with D-JNKI1 and Roscovitine respectively did not prevent NMDA-induced P-APP increase. These data show a tight connection, in excitotoxic conditions, between APP metabolism and the GSK-3β signaling pathway.The phosphorylation of Amyloid Precursor Protein (APP) at Thr N- and O-glycosylations, and phosphorylations that can induce a preferential cleavage of APP in the amyloidogenic pathway eventually leading to Aβ production [668) in the cytoplasmic domain plays a pivotal role in APP processing and is highly relevant to AD [Alzheimer’s disease (AD) is the most common form of dementia in the elderly. One of the pathological hallmarks of AD is the abnormal accumulation of amyloid-β (Aβ) produced by proteolytic cleavage of the Amyloid Precursor Protein (APP). APP processing is influenced by post-translational modifications, oduction ,2. In pant to AD . 668phosphorylation, are three: Cyclin-dependent kinase 5 (Cdk5), glycogen synthase kinase-3β (GSK-3β) and c-jun-N-terminal kinase (JNK) [The candidate kinases, responsible for Thrse (JNK) ,5,6,7, bse (JNK) ,10 and ase (JNK) ,12.Amongst them, JNK is a key pathway in excitotoxicity ,14,15, A668, as well as Tau and Presenilin1 [in vitro [Finally, GSK-3β is of particular relevance to neurological disorders since it phosphorylates APP at Thrsenilin1 ,23,24, isenilin1 . An abnosenilin1  and inhisenilin1 . Moreovein vitro .668 in cortical neurons stimulated with a high dose of NMDA (100 μM). This dose of NMDA correlated with an increase in APP phosphorylation and amyloidogenic processing (β-APPs) and led to activation of all three kinases without causing neuronal death in this short temporal window. Because glutamate playsa key role in AD pathology and NMDA stimulation for prolonged periods leads to increased production and secretionof Aβ fragments in primary neuronal cultures  we invesN-methyl-D-aspartate (NMDA) 100 μM for 30’-45’. As reported previously by Borsello et al., this dose of NMDA induces 90% neuronal death after 24 h [Cortical neurons were exposed to ter 24 h . InsteadThe first clear signs of neuronal death became apparent after 1 h of NMDA stimulation, with (a) a significant increase in LDH release in comparison to control and NMDA treated cultures for 30’-45’ (b) nuclear fragmentation  and at 45’ rose further   b. Total = 0.003) , d. The = 0.003) , c. Nota= 0.003) , e. More= 0.003) , f.Moreover NMDA treatment induced a significant increase in the amyloidogenic processing of APP as demonstrated by the increase of βAPPs/APP ratio after 45’ (p = 0.02) and reduction of αAPPs/APP ratio at 30’-45’ (p = 0.037 and p = 0.039) see , a-b-c. 668 we studied their activation following NMDA stimulation. Because JNK, Cdk5 and GSK-3β kinases can all contribute to APP phosphorylation at Thr(1) JNK. In agreement with our previous reports ,15, we c(2) Cdk5. The effect of NMDA on Cdk5 activity was examined with Cdk5 immunoprecipitation kinase assays using histone H1 as protein kinase substrate. NMDA application for 45’ induced a significant increase  in Cdk5 activity , c-d. (3) GSK-3β. Concomitantly, NMDA application led to a powerful activation of GSK-3β (here expressed as a decrease of the inhibitory phosphorylation in Ser-9). GSK-3β phosphorylation decreased to 0.5 (p = 0.002) by 30’, to reach a 0.7 value (p = 0.0002) by 45’ , e-f. These results demonstrate that NMDA treatment leads to the activation of all three kinases, indicating the involvement of complex signaling mechanisms.668 in cortical neurons [As previously shown, in control conditions, D-JNKI1 treatment for 24h prevented APP phosphorylation on Thr neurons . To inveWe can conclude that NMDA-induced hyperphosphorylation of APP is not mediated by JNK. We then investigated the role of Cdk5 in our model. To prevent Cdk5 action we used the ATP competitive inhibitor Roscovitine, a well-characterized inhibitor of cdc-2 like kinases and the most common inhibitor to block Cdk5 activity . Neurons were pre-treated with Roscovitine (10 μM) for 30’ before NMDA administration and the P-APP/APP ratio was compared to untreated neurons. Roscovitine effectively blocked NMDA induced activation of Cdk5 but did not reduce APP phosphorylation levels. Quantification of P-APP/APP ratio confirmed that there was no significant reduction in the Roscovitine/NMDA samples compared to NMDA alone , c-d. Thus Cdk5 does not play a pivotal role in NMDA mediated hyperphosphorylation of APP. To examine if GSK-3β plays a role in NMDA induced hyperphosphorylation we used L803-mts, a small cell permeable peptide, which competes for the substrate binding site of GSK-3β . ApplicaTo detect the effect of L803-mts during NMDA treatment on APP amyloidogenic processing we analysed the level of βAPPs (amyloidogenic pathway) and αAPPs fragments (non-amyloidogenic pathway) , a. As dThese results indicate that L803-mts modulates the APP amyloidogenic processing induced by NMDA.668, in the C-terminal cytoplasmic domain of APP (AICD). Such phosphorylation facilitates β-secretase cleavage and can lead to an increase in Aβ production [Amyloid Precursor Protein metabolism has a fundamental role in AD pathogenesis. Very important in the processing of APP is the role of the phosphorylation at Throduction . It is tWe investigated the impact of an excitotoxic stimulus such as NMDA on the phosphorylation of APP and on the activation of the three kinases that are involved in this phosphorylation, namely: JNK ,17, Cdk52+ homeostasis, oxidative stress and NMDA responses [2+ homeostasis and oxidative stress through the NMDA receptors (NMDA-R) [The neurotoxic effect of extracellular aggregates of Aβ peptide, derived from APP processing, is mediated by excitotoxic events such as dysregulation of Caesponses ,33,34,35(NMDA-R) ,37. Conc(NMDA-R)  and dere(NMDA-R) ,40. in vitro model. In order to investigate the direct effect of NMDA on APP, we stimulated 12 DIV fully differentiated cortical neurons with NMDA 100 μM for 30’-45’, a combination of concentration and duration that is not toxic for our neuronal 668. Similar data were described by Hoey et al., [668 for the APP amyloidogenic processing. Stimulation of cortical neuronal cultures with NMDA for 30’-45’ induces APP hyperphosphorylation at Thr et al., . AdditioAt the same time, NMDA stimulation for 30’-45’ led to an increase in Cdk5 activity and, in agreement with others, induced an increase of GSK-3β  as well In the second part of this study we investigated the role of Cdk5, GSK-3β and JNK in APP phosphorylation using specific kinase inhibitors. Although several studies have investigated the involvement of these kinases in APP phosphorylation their exact roles in models of excitotoxicity are still very unclear. We could show that inhibition of GSK-3β with a substrate competitive inhibitor (L803-mts) abolished NMDA induced APP phosphorylation after 45’ stimulation. Moreover we observed a positive effect of L803-mts on the APP amyloidogenic processing induced by NMDA treatment. In this short time window, the GSK-3β substrate competitive inhibitor led to a decrease of βAPPs production and an increase of αAPPs levels, even if it did not completely restore to control levels. This could be explained by the short-term treatment that is sufficient to significantly reduce P-APP levels but not all the APP processing. et al. described a cross-talk between GSK3 and Cdk5, where Cdk5 over-activation leads to GSK3 inhibition [et al. [On the other hand, neither inhibition of Cdk5 by roscovitine, nor inhibition of JNK by D-JNKI reduced NMDA-induced APP hyperphosphorylation. Notably, inhibition of Cdk5 and/or JNK in NMDA stimulated neurons led to a further increase of APP hyperphosphorylation without affecting neuronal survival. Further studies are needed to elucidate the mechanisms behind such regulation. However we think that this data are not so surprising since several studies have reported cross-talks among JNK, GSK-3β and Cdk5 pathways ,43,44. Phibition . Similar [et al. . We coulUnfortunately dissecting further these signaling pathways is difficult: a) due to the rapidity of their response and b) because a combinational treatment with these inhibitors was toxic in our model.5 cells/dish) pre-coated with 25 μg/mL poly-D-lysine (Sigma Aldrich). Plating medium was B27/neurobasal supplemented with 0.5 mM glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin. The experiments were performed 12 days from plating date, at which time neurons are considered differentiated. Neurons were treated with NMDA , for 30’-45’-1 h. The inhibitors L803-mts , roscovitine , and D-JNKI1  were administered to neurons 30’ before NMDA treatment. L803-mts and Roscovitine were diluted in DMSO. Treatment of neurons with DMSO only did not affect the results (data not shown).Primary neuronal cultures were obtained from the cerebral cortex of two days post-natal rats, incubated with 200U of papain (Sigma Aldrich) (30’-34 °C), then with trypsin inhibitor (Sigma Aldrich) (45’-34 °C) and subsequently mechanically dissociated. All experimental procedures on animals were performed in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and all efforts were made to minimise animal suffering. Neurons were plated in 35 mm dishes , . LDH assays were performed in triplicates.Total protein extracts were obtained by washing cells twice in ice-cold PBS and lysed (20’-4 °C) in 1% Triton x-100 lysis buffer supplemented with proteases  and phosphatases  inhibitors .Four hundred μL of medium were incubated with 100 μL of TCA 50% . After centrifugation , pellets were washed twice with 500 μL acetone and reconstituted in 50 μL Urea 4 M.Protein concentrations were quantified using Bradford Assay (Bio-Rad Protein Assay 500-0006) and 20 μg of whole cell proteins were separated by 8–10% SDS polyacrylamide gel. PVDF membranes were blocked in Tris-buffered saline  . Primary antibodies were diluted in the same buffer  using: 1:2000 anti APP clone 22C11 (APPs) , 1:500 anti P-APP , 1:250 anti βAPPs , 1:1,000 anti αAPPs clone 6E10 , 1:2,000 anti P-GSK3β (Ser9) , 1:2000 anti GSK-3β (27C10) , 1:1,000 anti P-JNK  1:1000 anti JNK . All blots were normalized to α-tubulin  and at least three independent experiments were performed. Western blots were quantified by densitometry using Quantity One software (Biorad).32P-ATP in kinase buffer  . Quantification of kinase assays was done using Quantity One software (Biorad) and based on at least three independent experiments.Cells were washed in ice-cold PBS and lysed (10 h - 4 °C) in RIPA buffer with proteases  and phosphatases  inhibitors. Three hundred μg of whole cell protein were incubated with 3 μg of anti Cdk5 (C-8)  (2 h - 4 °C) and precipitated with Protein A Sepharose CL-4B  (1 h - 4 °C). Immunoprecipitated complexes were incubated with 6 μg/μL of Histone H1 and 1 μCi/μL of γ-p value of < 0.05 was considered significant.All experiments were repeated using at least three independent culture preparations. Quantitative data were statistically analyzed by paired T-test with two-tailed distribution. A Altogether, these findings suggest that in adult differentiated stressed neurons GSK-3β is the kinase responsible for APP phosphorylation in excitotoxic conditions and an appropriate regulation of GSK-3β activity can be useful for modulation of APP processing.