Here, we have tested if this is due to the loss of nucleotide binding to the noncatalytic site that is required for the activation. Measurements with a tryptophan mutant of BF1 indicated that the noncatalytic sites could bind ATP normally. Furthermore, the mutant BF1 that cannot bind ATP to the noncatalytic sites showed much lower ATPase activity. It was concluded that the cause of strong MgADP inhibition of BF1 is not the weak nucleotide binding to the noncatalytic sites but the other steps required for the activation.F FoF1 consists of a water-soluble F1 part (F1-ATPase) connected to a membrane-embedded H+ channel, Fo 1-ATPase consists of α3, β3, γ, δ and ε subunits and its hydrolysis of one ATP molecule at a catalytic site on the β subunit drives a discrete 120° rotation of the γε subunits relative to the α3β3δ 1, rotation of the rotor subunits of F1 (γ and ε) is transferred to the c subunit ring of Fo to couple ATP synthesis/hydrolysis and H+ flow.FoF1, which prevent ATP hydrolysis; The chloroplast ATP synthase has a pair of cystein residues in the γ subunit and the formation of the disulfide between them inhibits the activity; The ε subunit of bacterial and chloroplast ATP synthase inhibits ATP hydrolysis: and so on. Among them, the most prominent is MgADP inhibition 1-ATPase is stalled. It is a common mechanism among all ATP synthases examined so far. Several factors are known to affect MgADP inhibition; Sodium azide stabilizes MgADP inhibition N-oxide (LDAO) releases MgADP inhibition 1 reaches equilibrium between active and MgADP inhibited states, resulting in lower steady-state activity compared to the initial one The catalytic mechanism of ATP synthase has been extensively studied by structural studies and single-molecular experiments and the mechanism of the regulation of ATP synthase becomes attracting more interests. Several regulatory mechanisms are known: The mitochondrial ATP synthase has specific regulatory protein called IF1-ATPase from Bacillus subtilis (BF1) is highly suppressed by the MgADP inhibition 1-ATPases because they only fall into half, one third or so 1 more than a hundredfold 1-ATPases  1 has a strange ATP concentration dependency of steady-state ATPase activity, the ATPase activity at 20∼100 µM ATP is lower than those at 1∼10 µM or 200∼5000 µM 1 by releasing MgADP inhibition 1 will help us to understand how the regulation of ATP synthase varies depending on the environment where the source organisms live.Our recent study revealed that the ATPase activity of F1-ATPases from other species showed that the ATP binding to the noncatalytic site promotes release of inhibitory MgADP from catalytic sites and results in the substantial activation 1-ATPase from thermophilic Bacillus PS3 (TF1) that cannot bind nucleotide to the noncatalytic site showed large initial inactivation that reached to essentially no steady-state activity 1-ATPases, the noncatalytic B subunit does not bind nucleotide and V1-ATPase from Thermus thermophilus HB8 showed strong MgADP inhibition and no steady-state activity 1 is due to the inability of noncatalytic sites to bind nucleotide. To examine this hypothesis, we prepared a mutant α3β3γ complex of BF1 in which nucleotide binding to the noncatalytic nucleotide binding sites can be monitored by the changes in the fluorescence from the tryptophan residues introduced near the noncatalytic sites. The result indicated that the noncatalytic sites of BF1 could bind ATP. Thus, the cause of strong MgADP inhibition of BF1 is not the weak binding ability of the noncatalytic sites but other steps required for the recovery from the MgADP inhibition. However, the mutant α3β3γ complex of BF1 that cannot bind nucleotide to the noncatalytic sites showed lowered ATPase activity, indicating that the nucleotide binding to the noncatalytic sites has a substantial role for recovery from MgADP inhibition in BF1.Studies with FEscherichia coli F1-ATPase (EF1) 3β3γ complex of BF1, pET21-BF1 5′-CTCAGGCGTATGGCCAGCGATCAATGCCGG-3′ and 5′-TTGATCGCTGGCCATACGCCTGAGAAGAAC-3′ and the franking primers were 5′-GCCGTATTGTAAACCCGCTAGGCCAG-3′ and 5′-TCTTGTGTGATGGCTGCTTGGCGAG-3′. The resulting 2.2 kbp DNA fragment was introduced into the EcoRV site of pZero2.1 vector (Novagen). Then the 0.8 kbp DNA fragment containing mutation was excised out by cutting this plasmid with NotI and NcoI. The fragment was put back to the original site of WT pET21-BF1 by ligating with NcoI/BamHI fragment (1.2 kbp) and NotI/BamHI fragment (7.3 kbp) of WT pET21-BF1. The resulting plasmid, pET21-BF1(αR354W) was used for protein expression. The mutations (αK175A/T176A), which is known to suppress nucleotide binding to the noncatalytic site 5′-CCGTCAAACAGGTGCAGCATCTGTTG-3′ and 5′- ATCGCAACAGATGCTGCACCTGTTTG-3′ and the franking primers were 5′-GAAATTAATACGACTCACTATAGG-3′ and 5′- GATAAGCACTCCGTAAAACCGAACTG-3′. The resulting 2.0 kbp DNA fragment was introduced into the EcoRV site of pZero2.1 vector (Novagen). Then the 1.6 kbp DNA fragment containing mutations was excised out by cutting this plasmid with XbaI and NcoI. The fragment was put back to the original site of pET21-BF1(αR354W) by ligating with NcoI/BamHI fragment (1.3 kbp) and XbaI/BamHI fragment (6.4 kbp) of pET21-BF1(αR354W). The resulting plasmid, pET21-BF1(αK175A/T176A/R354W) was used for protein expression. Mutations were confirmed by DNA sequencing. WT (α3β3γWT), αR354W mutant (α3β3γRW), and αK175A/T176A/R354W mutant (α3β3γΔNC) α3β3γ complexes of BF1 were prepared as described previously The mutation (αR354W), which corresponded to the same mutant of 2SO4 (pH 7.5), 50 mM K2SO4 and 2 mM MgSO4 was transferred to a quartz cuvette (1.5 ml). The cuvette was placed in a fluorescence spectrophotometer, FP-6500  and the temperature was controlled to 25°C. The α3β3γ complex of BF1 was added to 100 nM. The concentrated ATP-Mg solution  was injected into the cuvette at the time indicated and the changes in the fluorescence were measured every 0.5 s or 1 s until the fluorescence reached a plateau. Excitation and emission wavelengths were set at 300 nm and 350 nm, respectively. Excitation and emission slit widths were 5 and 10 nm, respectively. The solution was stirred continuously during the measurement. Emission spectra were measured before and after the time-course measurement at a rate 50 nm/min.The assay mixture consisted of 50 mM Tris-HThe time course of the fluorescence was corrected for baseline with buffer. The fluorescence change at a plateau was plotted against the ATP concentration and fitted with the simple binding equation or the Hill equation by the computer software . The sum of two simple binding equations did not improve fitting (data not shown).ATPase activity was measured by NADH-coupled ATP-regenerating system at 25°C as described previously Protein concentration was determined by the method of Bradford 3β3γRW showed large fluorescence compared to the WT  The mutant αo the WT . Additiouenching . There wequation . These v3β3γRW showed similar ATPase properties to the α3β3γWT  to test if the nucleotide binding to the noncatalytic sites of BF1 promotes recovery from MgADP inhibition, even if weak. With the α3β3γΔNC, no fluorescence quenching upon addition of ATP was observed Click here for additional data file.


The low‐affinity IgE receptor FcϵRII (CD23) is part of the regulatory system controlling IgE synthesis in human B cells and exists in membrane and soluble forms. Binding of IgE to CD23 has been described to have stabilizing effects and to prevent cleavage of CD23. Previous experiments using anti‐CD23 antibodies reduced IgE synthesis but were difficult to interpret as the antibody Fc part might also mediate feedback mechanisms. The purpose of this study was to investigate the regulatory role of CD23, by using designed ankyrin repeat proteins (DARPins) that specifically recognize CD23. Anti‐CD23 DARPins were isolated by ribosome display and were produced as monovalent and bivalent constructs. Affinities to CD23 were measured by surface plasmon resonance. IgE synthesis and up‐regulation of CD23 in human peripheral B cells were induced by IL‐4 and anti‐CD40 antibody. We assessed CD23 expression and its stabilization by FACS and used an ELISA for detecting soluble CD23. IgE synthesis was measured by ELISA and real‐time PCR. Surface plasmon resonance revealed affinities of the DARPins to CD23 in the pico‐molar range. Anti‐CD23 DARPins strongly inhibited binding of IgE to CD23 and share thus a similar binding epitope as IgE. The DARPins stabilized membrane CD23 and reduced IgE synthesis in an isotype specific manner. Furthermore, the anti‐CD23 DARPins decreased IgE transcript through inhibition of mature Cϵ RNA synthesis suggesting a posttranscriptional control mechanism. This study demonstrates that targeting CD23 alone is sufficient to inhibit IgE synthesis and suggests that a negative signaling occurs directly through the CD23 molecule. C‐type lectin‐domain at the C‐terminus, where binding of IgE occurs KD 3.7 × 10−8 M) through an avidity effect FcϵRII CD23) is the low affinity receptor for IgE. It is a type II transmembrane glycoprotein consisting of a short cytoplasmic N‐terminus followed by an extracellular part with a stalk region and a 3 is the In B cells, alternative splicing results in the expression of CD23 in two isoforms (CD23a and CD23b) differing in six or seven amino acids at the N‐terminus The monoclonal chimeric anti‐CD23 antibody, lumiliximab  was shown to reduce IL‐4‐induced IgE synthesis in vitro Escherichia coli; moreover they can be easily fused to each other To investigate the effects of targeting CD23 in more detail we have used non‐immunoglobulin‐like binding scaffolds termed designed ankyrin repeat proteins (DARPin) lacking an Fc‐domain. DARPin binders with high specificities and high affinities have been already isolated against various targets n = 5) as determined by flow cytometry .Buffy coats were purchased from the blood donation center . The study was approved by the local ethics committee. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation on Ficoll Paque . B cells were isolated by negative selection using magnetic beads . The average purity of B cells from five independent donors was 94% ± 0.8 . Expression and purification of monovalent, bivalent, and bispecific DARPins was performed as published The principle of DARPin selection is published elsewhere 6 cells/mL in RPMI 1640  containing 10% FBS , Transferrin 40 µg/mL , mercaptoethanol 50 µM , Sodium pyruvate 1 mM (Sigma–Aldrich), bovine insulin 4 µg/ml (Sigma–Aldrich), l‐glutamine 2 mM (Gibco), and MEM Non‐essential amino acids 1:100 (Gibco). Cells were activated with IL‐4 20 ng/mL  and anti‐CD40 antibody 1 μg/mL  for up to 13 days. RPMI 8866 cell line was cultured in RPMI 1640 medium  supplemented with 10% FBS. Anti‐CD23 DARPins were added to the cell culture at day 0 at a concentration of 300 nM if not otherwise stated.B cells were cultured in 48‐well plates at 2 × 10Affinities of DARPins were measured on a Biacore X100 instrument  with HBS‐EP+ running buffer . Recombinant CD23  was immobilized on a CM5 biosensor chip using a standard amine coupling kit  to an immobilization level of 2400 response units (RU). Binding of anti‐CD23 DARPins was assessed in a twofold serial dilution with at least ten concentrations. Samples were injected for 2 min, followed by 10 min dissociation. Kinetic parameters were calculated with BIAevaluation software  using global fitting of the binding data with a 1:1 Langmuir binding.To visualize CD23, CD20, and CD45 on B cells, cells were stained using Phycoerythrin(PE)‐conjugated mouse anti‐human CD23 IgG1 1:500 (R&D) or with PerCP‐conjugated mouse anti‐human CD20 IgG2b 1:500  or with FITC‐conjugated mouse anti‐human CD45 1:500  for 30 min at 4°C, washed and analyzed on guava easyCyte™ Flow cytometer . In the inhibition assays, the chimeric IgE‐JW8 Ninety six‐well microtiter plates  were coated with monoclonal mouse anti‐human IgE HB235 at 5 µg/mL , polyclonal sheep anti‐human IgG at 5 µg/mL , with sheep anti‐human IgG1 1:1000 (Binding Site), with sheep anti‐human IgG4 1:1000 (Binding Site), with sheep anti‐human IgM 1:1000 (Binding Site), or with monoclonal mouse anti‐human CD23 at 2 µg/mL (R&D) overnight in PBS. Unspecific binding sites were blocked with PBS‐casein (0.15%) for 2 h at 37°C. Cell culture supernatants were incubated for 3 h at 37°C. Detection antibodies were the following: horseradish peroxidase HRP‐conjugated sheep anti‐human IgE 1:1000 (Binding Site), HRP‐conjugated sheep anti‐human IgG 1:2000 (Binding Site), HRP‐conjugated sheep anti‐human IgM 1:1000 (Binding Site) and HRP‐conjugated goat anti‐human CD23 1:500 (R&D). TMB  was used as substrate and was stopped with 1 M sulphuric acid. Monoclonal human IgE (Sus11) β2‐microglobulin and relative expression was determined on 7500 system software (Applied Biosystems). Following primers and probes were used: β2‐microglobulin, Hs00187842_m1; IgG1, Hs00378340_m1 (Applied Biosystems). IgG4 forward: 5′‐CTGGTCACCGTCTCCTCA‐3′, reverse: 5′‐AGTAGTCCTTGACCAGGCA‐3′, probe: 5′‐FAM‐TTCAAGGGCCCATCGGTCTTCC‐tamra‐3′; IgE forward (mature): 5′‐ACCCTGGTCACCGTCTCC‐3′, forward (germline): 5′‐ACCATCCACAGGCACCAA‐3′, reverse: 5′‐GAGTCACGGAGGTGGCATT‐3′, probe: 5′‐FAM‐CCCTTGACCCGCTGCTGCAAA‐tamra‐3′; CD23a forward: 5′‐TGGAGGAAGGTCAATATTCAGAG‐3′, CD23b forward: 5′‐ATGAATCCTCCAAGCCAGGA‐3′, reverse: 5′‐GCCACAGGAGAAGCAGAGTC‐3′, probe: 5′‐FAM‐GGTGTTGCAGGCGTGGGACT‐tamra‐3′ were ordered at Microsynth .Total mRNA was isolated using Guanidinum thiocyanate‐phenol‐chloroform extraction . Isolated mRNA was reverse transcribed using random hexamer primers  and Superscript II . qPCR was performed using TaqMan Universal Master Mix (Applied Biosystems) on a 7500 Real Time System (Applied Biosystems). Expression was normalized to the endogenous control E. coli as monovalent  and bivalent  proteins.Binders recognizing recombinant CD23 were isolated from two DARPin libraries coding for either two (N2C) or three (N3C) randomized ankyrin repeat modules. Binders were expressed and crude DARPin extracts were screened for specificity in an ELISA. Two N3C DARPins termed D86 and D89, displaying no reactivity to control proteins, were chosen for further investigation and expressed in In order to investigate whether the two DARPin molecules D86 and D89 recognize different epitopes on CD23, we performed an assay on surface plasmon resonance. The chip surface was coated with bivalent DARPin D86‐86  and subsequently incubated with recombinant CD23 until the sensogram reached a stable response. DARPin D89 was then injected and was still able to bind to CD23 indicating that the two DARPins, D86 and D89, recognize different epitopes on CD23 which is a prerequisite to generate bispecific DARPins. Monovalent DARPins D89 and D86 were then fused and expressed as a bispecific DARPin (D89‐86). SDS‐PAGE showed that DARPins D86, D86‐86 and D89‐86 run as monomeric molecules, while DARPins D89 and D89‐89 tend to form dimers .KD values of nearly four orders of magnitude. These improved affinities might be mainly due to increased avidity. Interestingly, bivalent DARPin D89‐89 has a faster off‐rate, which results in an affinity ten times less compared with the other bivalent and bispecific molecules.Kinetic parameters of anti‐CD23 DARPins were assessed by surface plasmon resonance on immobilized CD23  had the highest affinities. Therefore, the experiment was performed by mixing monovalent , bivalent (D86‐86), and bispecific (D89‐86) DARPins at different concentrations with a saturating concentration (25 nM) of FITC‐labeled IgE before adding to the cells. As shown in Figure As targeting CD23 by IgE has been shown to stabilize surface CD23 As expected, supernatants of IL‐4/anti‐CD40‐stimulated B cells showed an increase of soluble CD23 in comparison with unstimulated samples. Monovalent DARPins D89 and D86 as well as bispecific DARPin D89‐86 significantly decreased the amount of soluble CD23 after 6  and 13 . Incubation of B cells with bispecific DARPin D89‐86 did not significantly influence expression of CD23 mRNA, indicating a post‐transcriptional mechanism of membrane CD23 stabilization , who own the intellectual property on the DARPin technology used in this study. All the authors declare no competing financial interests.Additional supporting information may be found in the online version of this article at the publisher's web‐site.Supporting DataClick here for additional data file.Figure S1. Epitope specificities of anti‐CD23 DARPins. The chip was saturated with bivalent DARPin D86‐86, which has a low off‐rate dissociation constant.Figure S2. Protein analysis of anti‐CD23 DARPins. Anti‐CD23 DARPins (1.5 μg) were visualized on a 15% SDS‐PAGE stained with Coomassie.Figure S3. SPR sensorgrams for the assessment of binding kinetics of anti‐CD23 DARPins to CD23.Figure S4. Expression of CD20 and CD23 on freshly isolated B cells.Figure S5. Effects of anti‐CD23 DARPins on surface CD23 after 6 days of culture.Figure S6. Effects of anti‐CD23 DARPins on different immunoglobulin isotypes.Click here for additional data file.


In erythrocytes, the regulation of the redox sensitive Tyr phosphorylation of band 3 and its functions are still partially defined. A role of band 3 oxidation in regulating its own phosphorylation has been previously suggested. The current study provides evidences to support this hypothesis: (i) in intact erythrocytes, at 2 mM concentration of GSH, band 3 oxidation, and phosphorylation, Syk translocation to the membrane and Syk phosphorylation responded to the same micromolar concentrations of oxidants showing identical temporal variations; (ii) the Cys residues located in the band 3 cytoplasmic domain are 20-fold more reactive than GSH; (iii) disulfide linked band 3 cytoplasmic domain docks Syk kinase; (iv) protein Tyr phosphatases are poorly inhibited at oxidant concentrations leading to massive band 3 oxidation and phosphorylation. We also observed that hemichromes binding to band 3 determined its irreversible oxidation and phosphorylation, progressive hemolysis, and serine hyperphosphorylation of different cytoskeleton proteins. Syk inhibitor suppressed the phosphorylation of band 3 also preventing serine phosphorylation changes and hemolysis. Our data suggest that band 3 acts as redox sensor regulating its own phosphorylation and that hemichromes leading to the protracted phosphorylation of band 3 may trigger a cascade of events finally leading to hemolysis. Due to their function in carrying oxygen and their high iron content, red blood cells (RBCs) are constantly exposed to oxidative stress . In addiIt is noteworthy that approximately 7% of world population is affected by those mutations which have been selected by malaria.It is well known that RBCs respond to oxidative stress with a metabolic response finalized to maximize the production of NADPH and to regenerate the stores of GSH and thioredoxin. In parallel, RBCs also respond by activating tyrosine kinases determining the tyrosine (Tyr) phosphorylation of band 3, the most abundant RBC membrane protein and the major linkage between the cytoskeleton and the lipid bilayer –12. In RThe redox regulation of band 3 Tyr phosphorylation apparently involves different components. In a previous report, it has been demonstrated that oxidized band 3 is selectively phosphorylated . Lyn is 2O2 of the inhibitory Cys is 0.005-fold lower than GSH, indicating that, at its normal concentrations, GSH should very effectively protect PTPs from oxidative inhibition [Phosphatases (PTPs) have also been implicated in the phosphorylation of band 3 that follows oxidative stress –29 and ihibition , 31. Addhibition ; Lyn acthibition , 26. Mor2O2 and by hemichromes which cause irreversible oxidation.In the present report we performed a series of experiments to gain more information on the mechanisms that are involved in the Tyr phosphorylation of band 3 following a reversible membrane protein oxidation triggered by diamide and H2HPO4, 1.5 mM KH2PO4, 20 mM HEPES, 1 mM MgCl2, and pH 7.4) in 5 mM glucose (PBS glucose) to obtain packed cells. RBCs were suspended at 20% hematocrit in PBS glucose and incubated at 37°C in 0.5 mM diamide at different incubation times  and then in the presence of different diamide concentrations. Separate experiments were also performed in 5 mM H2O2 or 1 mM phenylhydrazine (PHZ). pH was measured after 180 minutes and adjusted to 7.4 with NaOH. When necessary to avoid tyrosine phosphorylation, RBCs were pretreated with 10 μM of Syk inhibitor II , for 1 hour at 37°C in the dark, before oxidant treatment. For all protocols described, untreated controls were processed identically except that the inhibitor was omitted from the incubation. To prevent further phosphorylation of band 3, after incubation we washed the cells with cold buffer and membranes were immediately prepared.Venous blood was drawn from healthy volunteers following informed consent and pelleted at 1000 g for 10 minutes at room temperature. After removal of the buffy coat, RBCs were again pelleted and washed 3 times with phosphate buffered saline   containing a protease and a phosphatase inhibitor cocktail and then washed up to 4 more times in the same buffer (until membranes became white) in a refrigerated Eppendorf microfuge at 25000 g. The preparations were stored frozen at −80°C until use. Membrane protein content was quantified using the CD Protein Assay (Bio-Rad).Membrane proteins were prepared at 4°C on ice as previously described . Brieflyµg of proteins for analytical gels, 1 µg of proteins for anti-band 3, and 30 µg of proteins for anti-phosphotyrosine, anti-phosphoserine, and anti-Syk antibodies were loaded for western blot analysis and separated on 8% of polyacrylamide gel under reducing and nonreducing conditions. SDS-PAGE analysis was conducted by heating the sample for 5 minutes at 95°C and was run on the Bio-Rad mini-protean 3 setup.To perform one-dimensional electrophoresis, membrane proteins were solubilized in Laemmli Buffer  in a volProteins separated by SDS-PAGE were transferred to nitrocellulose membranes as previously described  and thenµM band 3 concentration. Band 3 concentration was estimated measuring total membrane proteins in packed membranes (approximately 4 mg/mL) considering that band 3 represents approximately 25% of total membrane proteins and a band 3 M.W. of 95.000 Da. Resuspended membranes were incubated for 10 minutes on ice, with 0.1 mM diamide in the presence of increasing concentrations of GSH. The reaction was stopped by washing the solution 3 times with HB. The percentage of oxidized band 3 was evaluated by western blot following nonreducing 8% SDS-PAGE and expressed as percentage of the maximal oxidation measured in the absence of GSH. In the absence of GSH an average of 95.2 ± 4.5% of the band 3 was found present in reducible aggregates with a M.W. >200.000 KDa.RBC membranes were diluted in HB to obtain a 5 μM Syk inhibitor II  was added to prevent further phosphorylation of band 3. The rate of band 3 dephosphorylation was expressed as PTP activity and as a percentage of maximal activity in untreated RBCs.Erythrocyte PTP activity was measured using phosphorylated band 3 as substrate. Phosphorylated band 3 was obtained treating RBCs with 1 mM diamide. Membranes were prepared and incubated for 10 min at 37°C with the cytoplasmic fraction of RBCs treated with different concentrations of diamide. 10 We used a simplified method to measure the relative changes of Hb in RBC cultures supernatant; after discarding RBC membranes by centrifugation, lysis was quantified by measuring hemoglobin absorbance at 405 nm in RBC supernatant and expressed in nmoles/mL .RBCs were solubilized in HB containing 1% Triton X-100, centrifuged at 15.000 g at 4°C. High molecular weight hemichrome aggregates were separated from the supernatant on a Sepharose CL-6B microcolumn. The hemichrome fraction was then diluted and quantified measuring heme absorbance at 560, 577, and 630 nm  and exprRBC membrane proteins were treated in the presence or the absence of 2 mM diamide, solubilized for 10 minutes on ice with 3 volumes of 1% Triton X-100 in HB. After centrifugation in a refrigerated Eppendorf microfuge at 15000 g, supernatants were collected and incubated with anti-mouse anti-band 3 cross-linked to Protein A-Sepharose (1 : 10) via bifunctional coupling reagent dimethyl pimelimidate for 2 hours at 4°C under gentle mixing. Beads were washed three times with 1% Triton X-100 in HB . LaemmliTo obtain the oxidized and nonoxidized cdbd3 fragment, RBCs were incubated with or without diamide (2 mM). Membranes were prepared as described above and cytoskeletal proteins were eliminated incubating the membranes with 0.1 M NaOH at 4°C (stripped membranes). Cdbd3 was then purified from RBC membranes as previously described . The pur4HCO3/ACN (acetonitrile) (50/50 v/v) and successively dried with pure ACN. The gel slices were rehydrated for 45 minutes at 4°C in 20 μL of 5 mM NH4HCO3 digestion buffer containing 10 ng/μL of trypsin. Excess protease solution was removed and the volume was adjusted with 5 mM NH4HCO3 to cover the gel slices. Digestion was allowed to proceed overnight at 37°C.Bands were excised from electrophoresis gels and were destained by doing several washes in 5 mM NHμL of the tryptic digests mixed 1 : 1 with a solution of CHCA  . MS analysis of peptides from 1-DE gel bands was performed with a MALDI-TOF micro MX  according to the tuning procedures suggested by the manufacturer. Peak lists were generated with Proteinlynx Data Preparation using the following parameters: external calibration with lock mass using mass 2465.1989 Da of ACTH fragment 18-39 background was subtracted using the adaptive mode, performing deisotoping with a threshold of 3%. The MS spectra were converted into pkl files using Mass Lynx 4.0. Peak lists containing the 20 most intense peaks of the spectrum were sent to MASCOT PMF search (http://www.matrixscience.com/) using a Swiss-Prot database . Search settings allowed one missed cleavage with the trypsin enzyme to be selected, carboxymethylated cysteine as fixed modification and oxidation of methionine as potential variable modification and a peptide tolerance of 50 ppm. Only protein identifications with significant Mascot scores (p < 0.05) were taken in consideration.Samples were loaded onto MALDI target using 1 2O2) that is physiologically generated from superoxide anion during methaemoglobin formation and by denatured hemoglobin products [Time dependent phosphorylative changes of the RBC membrane proteins have been measured comparing the effects of (i) diamide, a single electron oxidant that induces disulfide formation , 26, . This is plausibly due to the potent scavenging activity of catalase and glutathione peroxidase in RBCs on H2O2.Diamide caused an intense and transient Tyr phosphorylation of band 3 and of proteins 4.1 and 4.2 though to a lesser extent and Ser phosphorylation changes in additional membrane protein Figures . H2O2 inλ-phosphatase, which was used to remove phosphate groups from blotted proteins (data not shown). Control experiments to exclude a direct oxidant effect of PHZ (2 mM) on isolated membranes revealed no effect on band 3 sulfhydryl groups (data not shown). The lack of short term effects of PHZ on membrane protein phosphorylation is coherent with its specific action on hemoglobin and the slow formation of hemichromes [Conversely PHZ caused a slow phosphorylation response measurable only after 60 minutes of incubation Figures . Tyr phoichromes , 39. A lichromes , 18.μM) that, due to the buffering effect of cellular GSH, are not expected to exert an effect on protein thiols. We observed a dose-dependent increase of the phosphorylation signal (p < 0.01) while no significant difference has been observed between the amount of cdbd3 immunoprecipitated from oxidized and nonoxidized samples  in forming band 3 (5 μM) intermolecular disulfide bonds on increasing concentrations of GSH. This experiment showed that at 0.1 mM GSH concentration (20-fold higher than band 3) approximately 40% of band 3 was still oxidized by diamide; at 1 mM GSH concentration (200-fold higher than band 3) approximately 20% of band 3 was still oxidized (μM) no oxidation of GSH was detectable (data not shown).The characteristic accessibility of the two cysteins 201 and 317 located in the cytoplasmic domain of band 3 has been already demonstrated . To obtaoxidized , indicatErythrocyte PTPs have been implicated in promoting the Tyr phosphorylation of band 3 due to an inhibitory Cys residue located in their catalytic domain , 40.To rule out the possibility that diamide treatment, at concentrations that induce band 3 phosphorylation, may also determine a substantial inhibition of PTPs, we compared the levels of band 3 phosphorylation and PTP inhibition at different diamide concentrations. Differently from the effect of diamide that caused reversible changes, after phenylhydrazine treatment, band 3 oxidation, and its phosphorylation, Syk translocation to the membrane and its phosphorylation increased progressively, paralleling the generation of hemichromes . Also inInterestingly, RBCs rapidly react to oxidative stress through very intense Tyr phosphorylation of band 3, their major integral membrane protein. We previously found that the phosphorylation of band 3 affects its interactions with the cytoskeleton inducing membrane destabilization . This phAnyway, the involvement of Syk kinase of band 3 oxidation and of all the major steps of the pathway such as the mechanism of redox sensing, its transduction, the regulation of Syk activation, and docking to band 3 need to be clarified to envisage the physiological role of this intense redox response characteristic of erythrocytes. To address those issues, the present work has been performed to obtain a series of quantitative data to study (i) the temporal and dose effects of different physiological and nonphysiological oxidants in eliciting the minimal band 3 and Syk modifications, (ii) the role of disulfide cross-linked band 3 in docking Syk, (iii) the buffering effect of GSH on the oxidation of band 3 Cys residues to rule out if band 3 could display activity as redox sensor in intact erythrocytes, and (iv) the relative roles of Syk activation and docking versus PTPs inhibition in the phosphorylation of band 3.2O2. The comparative measurement of band 3 phosphorylation and of PTPs inhibition at different concentrations of diamide revealed that intense phosphorylation can occur at concentrations that minimally inhibit erythrocytes PTPs acting on phospho-band 3. This finding is in accordance with the relatively low reactivity of the Cys residue located in the catalytic site of PTPs [The presented results indicate that band 3 possess highly reactive Cys residues capable of being easily oxidized in the presence of physiological concentration of GSH and that disulfide cross-linked band 3 docks Syk and acts as competitive inhibitor of band 3 phosphorylation. Those results support the observed changes in whole RBCs with very low concentration of a specific sulfhydryl reagent (diamide) or following the formation of minute amounts of hemichromes. In both models band 3 phosphorylation exactly parallels its oxidation. On the other hand erythrocytes seem to be fairly protected by H of PTPs  and with2O2 which induces a transient phosphorylation of band 3 does not cause hemolysis, a persistent phosphorylation of band 3 induced by irreversible hemichromes apparently leads to a severe membrane destabilization. It should be anyway noticed that hemichromes formation was also accompanied by serine phosphorylative changes involving some membrane protein; those phosphorylation changes have been usually considered to cause a decrease of the affinity between some components of the RBC membrane junctional complexes [In the present report, we observed that Syk inhibitors are potent inhibitors of the hemolysis that follows to the generation of hemichromes. Considering that treatment with diamide or Homplexes –50 and mConsidering that band 3 phosphorylation may have a function in remodeling the RBC membrane to remove noxious hemichromes , the pre


This shows that tau oligomers are not necessarily always toxic.Furthermore, their formation correlates with increased tau levels, causedintriguingly, by either pharmacological or genetic inhibition of tau kinaseglycogen-synthase-kinase-3beta (GSK-3β). Moreover, contrary to commonbelief, these tau oligomers were neither highly phosphorylated, and nor did theycontain beta-pleated sheet structure. This may explain their lack of toxicity. Ourstudy makes the novel observation that tau also forms non-toxic insoluble oligomersin vivo in addition to toxic oligomers, which have been reported byothers. Whether these are inert or actively protective remains to be established.Nevertheless, this has wide implications for emerging therapeutic strategies such asthose that target dissolution of tau oligomers as they may be ineffective or evencounterproductive unless they act on the relevant toxic oligomeric tau species.Aggregation of highly phosphorylated tau is a hallmark of Alzheimer’sdisease and other tauopathies. Nevertheless, animal models demonstrate thattau-mediated dysfunction/toxicity may not require large tau aggregates but insteadmay be caused by soluble hyper-phosphorylated tau or by small tau oligomers.Challenging this widely held view, we use multiple techniques to show that insolubletau oligomers form in conditions where tau-mediated dysfunction is rescued  All tauopathies, including Alzheimer’s disease (AD), are characterized by theaccumulation of insoluble, hyper-phosphorylated aggregates of the microtubule-associatedprotein tau. Both tau aggregation and hyper-phosphorylation are implicated intau-mediated dysfunction and toxicity1Tau can be phosphorylated at a large number of sites, and many of these sites areabnormally hyper-phosphorylated in AD45781011131415In contrast, the case for tau aggregates as a primary toxic species is less clear. Indeedthe toxicity of aggregates has been challenged for other aggregating proteins in otherproteinopathies as well1920212526303234in vivo in situations where tau-mediated neuronaldysfunction is rescued. Thus tau oligomers are not necessarily made up ofhyper-phosphorylated tau and they are not necessarily associated with tau toxicity.Though the ever-increasing studies on tau oligomers clearly describe a variety ofoligomers that differ in size (and number of tau protein constituents), shape andsolubility, these differences are rarely acknowledged or discussed. Consequently theircontribution to the pathogenic potential of oligomeric tau species is not fullyappreciated0N3R) inDrosophila. These phenotypes include locomotor impairment anddisrupted axonal transport789370N3R-expressing neurons in these animals,we made an unexpected observation: treatment with either drug led to theformation of 20–50 nm electron-dense granules in axonalEM sections  rescues phenotypes induced by human tau , the Drosophila homolog ofGSK-3β, similarly reduced tau phosphorylation .0N3R fliessuggested that insoluble tau species were present but were unable to enter theSDS-resolving gel , a procedure routinely used to visualise tauoligomersR brains , but werR brains . The dimin situ, weperformed in situ immuno-gold labeling for hTau on sections of peripheralnerve. In conditions in which electron-dense granules were observed, such asLi-treated hTau flies, we found granules decorated with gold particles withinaxons .However, unlike the soluble tau fraction, the hTau in the insoluble (GTO)fraction was largely un-phosphorylated in all animals in which GTOs wereproduced  and greater intensity than thosefrom untreated hTau flies  so the only signal possible is from the tau oligomers .−146Phosphorylation has previously been measured by Raman spectroscopy, and it hasbeen clearly demonstrated that, under basic conditions, a strong peak is evidentat around 980 cm−1 (amide I)and 1220–1300 cm−1 (amideIII). These are typically used for determining the secondary structure ofproteins, although the former is more reliable as it is free from contributionsfrom side-chain vibrations4245−1 (arrowhead– −1 (and more soin the untreated hTau spectrum) suggests a helical arrangement of the proteinbackbone. This is also consistent with the slight increase in intensity of thepeak at ~1250 cm−1 observed,which indicates an increase in the non-β-sheet structures (such asα-helices or random secondary structures), in the oligomeric proteincompared to the monomerTo probe the secondary structure of our tau oligomers, we studied thespectroscopic traces within regions1625–1700 cmWe present here novel data that genetic or pharmacological inhibition ofGSK-3β-mediated tau phosphorylation rescues phospho-tau phenotypes, andunexpectedly increases total tau levels and produces insoluble tau oligomersresembling granular tau oligomers (GTO)s. These insoluble tau oligomers are notphosphorylated, and do not contain β-pleated sheet structure. Contraryto prevailing opinion, this demonstrates that oligomeric tau is not necessarilytoxic.invivo. Though our findings do not necessarily imply that tau oligomers aredirectly neuroprotective, they clearly shows that they are certainly not alwaystoxic when formed in vivo.In all proteinopathies there are a variety of soluble and insoluble misfoldedproteins. It is now generally accepted that the largest insoluble structures inthese diseases (such as neurofibrillary tangles in tauopathies) are not the mosttoxic species. In the case of tau protein, suspicion has now fallen on smallsoluble and insoluble oligomers251920Drosophila924As we, and others have previously shown, tau phosphorylated at GSK-3βsites is associated with toxicity in both increasingGSK-3β activity promotes tau aggregation61451et al.Drosophila modelsof tauopathy, we do not believe this to be the case. Instead we speculate thatthe discrepancy between our results and those of others is due to two reasons.Firstly, in studies in which the effect of GSK-3β inhibitors on tauaggregation was analysed51loose small insoluble tau oligomers. As discussed inin vitro) is critically dependent upon tau concentrationIt is intriguing that reduction of tau phosphorylation in this study promotes tauaggregation, because conventional opinion dictates that hyper-phosphorylation oftau precedes and promotes aggregation. Although one other study is consistentwith ours in showing that decreasing GSK-3β activity leads toincreased levels of insoluble tau oligomersIt will be interesting to investigate the mechanism by which GSK-3βinhibition increases total tau. Like us, others have also shownGSK-3β mediated regulation of tau turnover in both invertebrate andvertebrate models33555655Though phosphorylation of tau is believed to precede tangle formation, it isintriguing that not all tau proteins derived from paired helical filaments in ADare phosphorylatedThe failure of amyloid-based therapies in clinical trials has stimulated researchinto tau-based targets for the treatment of tauopathies likeAlzheimer’s disease. Two major tau-based strategies are directed atinhibiting GSK-3β-mediated tau phosphorylation, and reducing tauaggregation/oligomerisation by various means34Our finding that inhibition of tau phosphorylation leads to production ofinsoluble tau oligomers raises a cautionary note regarding the therapeuticstrategies aimed at reducing tau phosphorylation. Our results suggest theoutcome of this strategy may conflict with the aim of the other key therapy if,as we show, inhibition of tau phosphorylation leads to formation of tauoligomers.Additionally, our finding that tau oligomers are not always toxic hasimplications for the effectiveness of strategies aimed at reducing tauaggregation. Such strategies would be ineffective if the tau oligomers that theyare clearing are not toxic. Furthermore, these approaches might even becounter-productive if solubilising aggregated tau releases the more toxicspecies. Our results underscore the importance of establishing which species ofoligomeric tau are toxic, neutral or protective to determine the mostappropriate target for effective tau-based therapies.in vivo. This has profound implications for therapeuticapproaches aimed both at inhibiting tau phosphorylation and reducing tauaggregation.The mechanism(s) by which abnormal tau causes toxicity in tauopathies is not clear.Although it is established that abnormal tau phosphorylation plays a critical role,the role of tau aggregation, and in particular oligomerisation, is less wellunderstood. We have found that reduction of tau phosphorylation promotes tauaggregation, and that this is, surprisingly, associated with rescue of neuronaldysfunction Drosophila melanogaster expressing the GAL4 driverselavC155-Gal4 or D42-Gal4were crossed with either wild-type Oregon-R flies (wt) as a control, or withflies transgenic for human 3-repeat tau(UAS-hTau0N3Rfrom Bloomington Stock centre),with or without UAS-sgg* (UAS-sggS9A fromBloomington Stock centre) or UAS-sggDN. Flies were raised at 25 °C on standard flyfood with or without 20 mM LiCl or 20 μmAR-A01448 (Sigma-Aldrich). For all experiments, equivalent numbers of males andfemales were utilized for each condition compared.10 fly heads (0-3d flies) were pooled and homogenized in40 μl aqueous buffer  at4 °C, and centrifuged at 1,000 g for2 minutes to remove unhomogenized debris. The supernatant was thencentrifuged for 2 h at 200,000 g(4 °C). The resulting supernatant (S1) is theaqueous-soluble fraction. Pellet (P1) was resuspended in SDS buffer andcentrifuged for 2 h at 200,000 g(25 °C). Supernatant (S2) is the detergent-solublefraction. P2 was washed by repeating this spin and, after discarding thesupernatant, P2 was resuspended in buffer containing 8 M urea with agitation for 18 h at room temperature.S3 is the detergent-insoluble fraction.Samples of pooled fly heads (0-3d flies) were either prepared for solubilityfractionation, as above, or homogenized in buffer containing protease, kinaseand phosphatase inhibitors . Samples were heated for5 minutes at 95 °C in Laemmli buffer,separated by 10% PAGE, and transferred to PVDF membrane (Amersham). Blots wereprobed with the following primary antibodies: anti-human-tau antibody , anti-human-tau N-terminal , tau-1 , PHF-1 , AT8 , AT180, MC1 , or anti-pS262, followed by HRP-conjugated anti-rabbit secondary antibody and Chemiluminescent substrate (Amersham). Band densities weremeasured using Image J.Filleted L3 larvae were fixed  for 1 hour. Specimens wererinsed in 0.1 M PIPES buffer, post-fixed in 1% buffered osmiumtetroxide (1 hour), block-stained in 2% aqueous uranyl acetate(20 min), dehydrated in ethanol and embedded in Spurr resin (EMScience). Ultra-thin sections were cut through the nerves at the base of theventral-cord on a Leica OMU 3 ultramicrotome. The sections were stained withReynolds lead stain and viewed on a Hitachi H7000 transmission electronmicroscope with SIS Megaview-III digital camera. The number of microtubuleprofiles per axon was counted in cross sections of peripheral nerves. Comparableregions of peripheral nerves were studied and all animals were processed inparallel. All visible axons were analysed in 5 animals per condition, with the experimenter blinded tocondition.Filleted CNS preparations from L3 larvae were fixed in 4% PFA(15 min), then the anterior half of the fillet was cut away andpost-fixed overnight in fresh 4% PFA. Samples were cryoprotected by incubationin a glycerol series up to 30%. Samples were briefly plunge-frozen in liquidethane (−170 °C). Frozen samples wereincubated in 1.5% uranyl acetate in methanol at-90 °Cfor 30 h, then at −45 °Cfor11 h. This was followed by infiltration with a series ofresin:methanol mixtures at −45 °C,culminating in pure Lowicryl HM-20 resin (EM Science) overnight. Specimens werepolymerized by UV light , then the temperature increased to0 °C over 9 h. Gold/silver sections were cutthrough the proximal peripheral nerves on a Leica ultramicrotome, andtransferred to formvar-coated Ni grids (EM Science). Immunochemistry was carriedout as describedThe insoluble protein fraction (P2 from the solubility fractionation method) wasresuspended in 20 μl distilled water, and5 μl spotted onto carbon-formvar copper grids (EMScience) for 2 min. Grids were blocked in 1% milk in PBS; incubatedin anti-human-tau (Sigma) or anti-v-glut at 1:200 in 1% milk in PBS(60 min room temperature); washed in PBS; incubated ingold-conjugated anti-rabbit secondary antibody (EM Sciences - 1:30) in 1% milkin PBS (60 min room temperature); washed in PBS then water; negativestained with 2% uranyl acetate (10 s). Grids were viewed asabove.20 brains of L3 larvae per sample were dissected on ice and homogenized in500 μl buffer , centrifuged  and unhomogenized material discarded. Samples wereimmuno-precipitated as follows: preclear in 20 μlprotein A/G beads (Sigma) at 4 °C 1 hour;incubate with 1 μl anti human-tau (Dako) at4 °C 1 hour; add60 μl beads and incubate at 4 °C1 hour; wash beads 3 times in buffer; reduce precipitated sample in20 μl Laemlli buffer for 5 min90 °C. Precipitates were spotted onto freshly cleavedmica discs (Agar Scientific), incubated for 2 minutes, rinsed with200 μl ultrapure water and dried with compressed air.Samples were imaged in air with a digital multimode Nanoscope III AFM(www.veeco.com) operating in tapping mode with an uncoated silicon cantilevers with set points of 0.6–0.8 at a scan frequencyof 3 Hz. Oligomer sizes were determined by cross-sectional heightanalysis of individual aggregates.Insoluble protein fractions (P2 from solubility fractionation protocol above),from hTau and hTau-Li brains were placed on slides. An InVia Renishaw Ramanmicroscope system was used for obtaining spectra. A 633 nm laser wasused for excitation with an acquisition consisting of 3 exposures of10 s each. Spectra were processed with Wire 3.1 software:fluorescence background was subtracted and spectra were smoothed.0N3R expression was driven by the motor neuron specific driveD42-GAL4. Third instar L3 larvae were allowed to crawl freely on anagarose plate under standardized lighting conditions. Their crawling behaviorwas recorded using standard video recording equipment and body wall contractionsundertaken in one min were counted.HtauHow to cite this article: Cowan, C. M. et al. Rescue from tau-inducedneuronal dysfunction produces insoluble tau oligomers. Sci. Rep.5, 17191; doi: 10.1038/srep17191 (2015).


Protein misfolding and aggregation is a common hallmark in neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), and fronto-temporal dementia (FTD). In these disorders, the misfolding and aggregation of specific proteins occurs alongside neuronal degeneration in somewhat specific brain areas, depending on the disorder and the stage of the disease. However, we still do not fully understand the mechanisms governing protein aggregation, and whether this constitutes a protective or detrimental process. In PD, alpha-synuclein (aSyn) forms protein aggregates, known as Lewy bodies, and is phosphorylated at serine 129. Other residues have also been shown to be phosphorylated, but the significance of phosphorylation in the biology and pathophysiology of the protein is still controversial. In AD and in FTD, hyperphosphorylation of tau protein causes its misfolding and aggregation. Again, our understanding of the precise consequences of tau phosphorylation in the biology and pathophysiology of the protein is still limited. Through the use of a variety of model organisms and technical approaches, we are now gaining stronger insight into the effects of phosphorylation in the behavior of these proteins. In this review, we cover recent findings in the field and discuss how targeting phosphorylation events might be used for therapeutic intervention in these devastating diseases of the nervous system. Neurodegenerative disorders, such as Parkinson's disease (PD), Alzheimer's disease (AD) and frontotemporal dementia (FTD), result from the progressive loss of specific neuronal populations leading to the progressive appearance of the clinical symptoms that are characteristic of each disorder. Current treatments for these disorders are palliative rather than curative and their effectiveness is still far from satisfactory. Thus, tremendous efforts are underway to elucidate the causes underlying these disorders and to find a cure. From a molecular perspective, the common hallmark of neurodegenerative disorders is the misfolding and aberrant aggregation of proteins in amyloid-like beta-sheet filaments. This feature is not only characteristic of classic neurodegenerative disorders but also of prion disorders and other amyloidosis inside and outside the central nervous system, suggesting that neurodegenerative disorders are part of a much greater superfamily of protein misfolding disorders, or aggregopathies , such as phosphorylation, ubiquitination or sumoylation, which alter the conformation and/or biological function of proteins, can also affect protein folding and aggregation, and thereby play a critical role in neurodegenerative disorders. For example, ubiquitination can direct proteins for either degradation by the proteasome or to certain subcellular compartments, and glycosylation is related to the secretion of proteins to the extracellular medium. Both processes could therefore influence protein concentration, folding, localization and, ultimately, aggregation. Phosphorylation can also affect protein conformation, function and fate in many different ways: it may be required for proper protein folding; it may induce conformational changes that can result in lower or higher catalytic activity; it may precede or function as a recognition signal for further modifications, such as ubiquitination; it may alter the subcellular localization of the protein; and it may modify protein-protein interactions  is the main protein component of Lewy bodies (LBs), the typical pathological hallmarks of PD and other disorders collectively known as synucleinopathies. The vast majority of PD cases are sporadic, multiplications and missense mutations in the gene encoding for aSyn have been associated with familial forms of PD  single cell models ; and (iii) animal models of PD (mice or rat models). In these studies the relation between toxicity and aggregation was not always explored  aSyn was initially associated with pathology in a transgenic Similarly, while some reports suggest that pS129 promotes inclusion formation 2 was found to form fibrils more readily than unphosphorylated aSyn in vitro  in vitro  were used in several Initial studies suggested aSyn might be predominantly unphosphorylated under physiological conditions  region, in which this residue is located. As an example, it is possible that S87 phosphorylation alters the interaction with phospholipase D (PLD) 2, an enzyme involved in lipid-mediated signaling cascades and vesicle trafficking , GRKs may also phosphorylate non-receptor substrates, including the four members of the synuclein family   were found to phosphorylate aSyn at S87, and this was based on Several tyrosine kinases phosphorylate aSyn. The Y125 residue is target of phosphorylation by Fyn  mutation was reported in a family with AD-typical histology and proposes that accumulation of an APP cleavage product, beta amyloid (Aβ), induces the biochemical, histologic, and clinical changes AD patients manifest  assembly in a more unphosphorylated state  at S400 was inversely correlated with tau phosphorylation at S396  tau oligomers was detected at human AD synapses concomitant with dysfunction of the UPS  function and to induce excitotoxicity  to proteolytically degrade excess tau  rescued from tau-induced toxicity. Interestingly, Par-1 phosphorylation of tau was a prerequisite for downstream phosphorylation through GSK-3β and Cdk5, and the generation of disease-associated phosphorylation epitopes . Moreover, three sites, S113, S238, and S433, were phosphorylated only by the action of CK1δ suggesting a relevant role of this kinase in tau pathology  by eicosanoyl-5-hydroxytryptamide resulted in dephosphorylation of aSyn at S129, inhibition of aSyn aggregation with concomitant improved neuronal integrity, reduction in inflammation and amelioration of behavioral deficits in an aSyn transgenic mouse model, (Lee et al., Dysregulated phosphatase activity also seems to be partially responsible for tau pathology. A number of pharmacological agents, such as the FDA-approved drug memantine, prevented okadaic acid or calyculin-induced PP2A inhibition and tau phosphorylation (Li et al., The induction of aSyn degradation through clearance pathways is also seen as an attractive therapeutic strategy. aSyn can be degraded by direct proteolysis, by the UPS, by chaperone-mediated autophagy, or by general autophagy (Lashuel et al., Several studies indicate that the reduction of tau phosphorylation was paralleled by an overall decrease in protein levels. Moreover, targeting unphosphorylated tau protein had deleterious effects since tau is a normal component of the cytoskeleton (Rosenmann et al., in vitro tested substances showed severe side effects on the normal biology of tau and its MT stabilization in cell culture and brain slices (Duff et al., Positive lysosomal modulation was described for several factors and may be an attempt of cells to clear amyloidogenic species such as tau and aSyn oligomers (Lee et al., Recent studies strongly suggest that disease progression in AD, PD, and other neurodegenerative disorders may, at least in part, be due to cell-to-cell transmission of amyloidogenic species. Thus, the elimination of these toxic species may help to slow or contain neurodegeneration.The mechanisms behind the spreading of aSyn are not fully understood. However, aSyn secreted by neurons could strongly contribute to cell-to-cell propagation (Marques and Outeiro, Immunization with cell-penetrating phospho-tau antibodies was already discussed as a therapeutic approach to clear from toxic tau species. Similarly, antibodies that remain in the extracellular space may inhibit spreading and this includes both, anti-tau and anti-Aβ antibodies (Giacobini and Gold, While it is clear that phosphorylation of aSyn and tau is relevant in the context of their aggregation and toxicity, there is still no final consensus on the precise contribution this type of PTM has toward the disease process. For example, there is still no consensus on whether aSyn phosphorylation is a cause or a consequence of aggregation, or whether phosphorylation is neurotoxic or neuroprotective. Furthermore, only a few phosphorylation sites have been confirmed in human tissue so far, so there may be other sites relevant to human pathology that remain to be identified. Tau phosphorylation regulates tau's function in many ways while abnormal phosphorylation of tau is neurotoxic. The relationship between tau phosphorylation and aggregation is clearly complex with evidence that tau phosphorylation precedes, prevents or is irrelevant to its aggregation. It will also be important to explore the cross-talk between different PTMs, as this will likely have a strong impact on our understanding of the biology/pathobiology of different proteins associated with neurodegeneration. In addition, the identification of the kinases and phosphatases involved in the phosphorylation/dephosphorylation of aSyn and tau will certainly open novel possibilities for pharmacological intervention. Ultimately, solving the problems and inconsistencies surrounding the phosphorylation of these important players in AD and PD will be essential for advancing the development of novel therapeutic strategies.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Abnormal phosphorylation and aggregation of tau is a key hallmark of Alzheimer's disease (AD). AD is a multifactorial neurodegenerative disorder for which Diabetes Mellitus (DM) is a risk factor. In animal models for DM, the phosphorylation and aggregation of tau is induced or exacerbated, however the underlying mechanism is unknown. In addition to the metabolic dysfunction, DM is characterized by chronic low-grade inflammation. This was reported to be associated with a neuroinflammatory response in the hypothalamus of DM animal models. Neuroinflammation is also implicated in the development and progression of AD. It is unknown whether DM also induces neuroinflammation in brain areas affected in AD, the cortex and hippocampus. Here we investigated whether neuroinflammation could be the mechanistic trigger to induce tau phosphorylation in the brain of DM animals. Two distinct diabetic animal models were used; rats on free-choice high-fat high-sugar (fcHFHS) diet that are insulin resistant and streptozotocin-treated rats that are insulin deficient. The streptozotocin-treated animals demonstrated increased tau phosphorylation in the brain as expected, whereas the fcHFHS diet fed animals did not. Remarkably, neither of the diabetic animal models showed reactive microglia or increased GFAP and COX-2 levels in the cortex or hippocampus. From this, we conclude: 1. DM does not induce neuroinflammation in brain regions affected in AD, and 2. Neuroinflammation is not a prerequisite for tau phosphorylation. Neuroinflammation is therefore not the mechanism that explains the close connection between DM and AD. CR1, CD33, and TREM2, which are all expressed in microglia  is a multifactorial neurodegenerative disorder characterized by aggregation of Aβ and abnormal phosphorylation and aggregation of tau  is a risk factor for AD and that the incidence of AD is higher in people with DM  and tap water. Two distinct diabetic animal models were used: an insulin resistance diet model and insulin deficient model. Rats on fcHFHS diet received in addition to the control groups a dish of saturated fat [beef tallow ] and a bottle of 30% sugar water  in the cage for 1 week (n = 6), 4 weeks (n = 6), or 10 weeks (n = 9). For the second diabetic model, T1DM, rats received under anesthesia of isoflurane subcutaneous an injection of STZ   or vehicle (citrate buffer). Animals were sacrificed 20 days after injection . For western blot analysis, animals were decapitated under CO2/O2 and brains were quickly removed and collected on dry ice before frozen in −80°C. Subsequently, hippocampus and cortex were dissected for protein lysates. For immunohistological analysis, animals were transcardially perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde under Nembutal anesthetics (120 mg/kg). Brains were embedded in paraffin. During the experiment body weight, food and water intake was measured 3 times a week. Glucose and insulin levels were not determined since the therefore required clamp studies or fasting of the animals both have a profound effect on the brain and could interfere with our results . Rats were individually housed under a 12:12 h light/dark cycle (lights on 07.00 h) at 21–23°C with Brain lysates were homogenized with a seirin needle and incubated for 30 min on ice in RIPA buffer  supplemented with protease and phosphatase inhibitors. Subsequently, brain lysates were centrifuged for 15 min at 20.000 × g at 4°C. Supernatant protein content was determined by BCA protein assay kit . Equal amounts of protein were loaded on 10% polyacrylamide gels and blotted onto nitrocellulose membrane . Blots were pre-incubated with 5% bovine serum albumin  in TBS-T [0.05% Tween-20 in Tris buffered saline (TBS)] for 60 min at room temperature and subsequently incubated at 4°C overnight with primary antibodies. Membranes were washed 3 × 10 min in TBS-T and subsequently incubated with species-specific secondary antibodies conjugated to horseradish peroxidase . Reactive protein bands were visualized using LumiLightPLUS Western blotting substrate (Roche Applied Science). Results were analyzed using Advanced Image Data Analyzer software  version 3.44.035 and using Image Studio Version 2.0 software . Brain samples from the experimental and the control group were all loaded on the same SDS-PAGE gel. The primary antibodies and their dilution factors are listed in Table 2O2 in TBS for 30 min to quench endogenous peroxidase activity. Sections were treated with 10 mmol/l, pH 6.0, sodium citrate buffer (AT8) or with 10 mM Tris and 1 mM EDTA buffer, pH 9.0 (IBA-1) for 10 min at 99°C for antigen retrieval and subsequently incubated with primary antibodies at 4°C overnight. Antibodies (Table Brains were cut into 5 μm sagittal sections. The sections were immersed in 0.3% Ht-test was used for single statistical comparison. No significance difference was defined as P > 0.05.GraphPad Prism software was used for graphs and statistical analysis. All data are compared to the control group. Two-sided unpaired Student's To investigate whether there is neuroinflammation in AD-affected brain areas as a complication of diabetes we used a STZ model and fcHFHS diet model. In both diabetic models body weight gain was measured at the end of the experiment to ensure effectiveness of the treatment. As expected, STZ treatment reduced body weight Figure . In lineThe differential effects of the STZ treatment and the fcHFHS diet on tau phosphorylation provides the opportunity to study whether neuroinflammation is inextricably linked to increased tau phosphorylation. Inflammation has been associated with the progression and peripheral complications of both T1DM and T2DM King, . It is nThe present study demonstrates absence of reactive microglia and increased levels of GFAP and COX-2 in the hippocampus and the cortex of two distinct diabetic animal models. This indicates that DM does not directly lead to neuroinflammation in AD-affected brain areas. An increased level of endogenous tau phosphorylation was observed in STZ-treated rats demonstrating that neuroinflammation is not a prerequisite for diabetes-induced tau phosphorylation. The absence of increased tau phosphorylation in our fcHFHS diet model is in accordance with the literature Table . Other sInflammation was reported in the hypothalamus of STZ-treated animals and animals on a high-caloric diet (Luo et al., Although we demonstrate that neuroinflammation is not a prerequisite for increased tau phosphorylation, our data do not exclude that peripheral inflammation could have an effect on the brain resulting in increased tau phosphorylation independent of neuroinflammation. In addition, DM may alter the neuroinflammatory response or even trigger neuroinflammation after a priming stimulus and thus facilitate AD pathology. As mentioned above there is a body of evidence to suggest that neuroinflammation plays a role in the progression of AD (Hoozemans and O'Banion, in vivo indicating that also metabolic stress in DM could be the trigger to induce tau phosphorylation (van der Harg et al., As neuroinflammation is not the trigger for increased tau phosphorylation the question remains which mechanism induces endogenous tau phosphorylation in the diabetic brain. Interestingly, dysfunction in insulin signaling has been reported in post-mortem AD brain material and in animal models of AD (Steen et al., In conclusion, our data demonstrate that increased tau phosphorylation occurs independently of neuroinflammation. Eliminating neuroinflammation as causal factor facilitates further elucidation of the complex connection between DM and AD and helps to put the multiple underlying mechanisms contributing to AD in an integrated framework (van Dijk et al., All authors substantial contributed to the conception and design of the work and the critically drafting of the manuscript. All authors approved the manuscript to be published and agreed to be accountable for the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: p47phox is a regulatory subunit of NADPH oxidase, which produces superoxide.Results: We propose a molecular dynamics model of p47phox phosphorylation and assembly with p22phox in NADPH oxidase activation supported by biochemical experiments.Conclusion: Ser-379 phosphorylation in the C-terminal tail is a molecular switch in p47phox activation.Significance: This report provides novel structural and mechanistic information of p47phox activation. phox is a key regulatory subunit of NADPH oxidase; however, due to the lack of full protein structural information, the mechanistic insight of p47phox phosphorylation in NADPH oxidase activation remains incomplete. Based on crystal structures of three functional domains, we generated a computational structural model of the full p47phox protein. Using a combination of in silico phosphorylation, molecular dynamics simulation and protein/protein docking, we discovered that the C-terminal tail of p47phox is critical for stabilizing its autoinhibited structure. Ser-379 phosphorylation disrupts H-bonds that link the C-terminal tail to the autoinhibitory region (AIR) and the tandem Src homology 3 (SH3) domains, allowing the AIR to undergo phosphorylation to expose the SH3 pocket for p22phox binding. These findings were confirmed by site-directed mutagenesis and gene transfection of p47phox−/− coronary microvascular cells. Compared with wild-type p47phox cDNA transfected cells, the single mutation of S379A completely blocked p47phox membrane translocation, binding to p22phox and endothelial O2⨪ production in response to acute stimulation of PKC. p47phox C-terminal tail plays a key role in stabilizing intramolecular interactions at rest. Ser-379 phosphorylation is a molecular switch which initiates p47phox conformational changes and NADPH oxidase-dependent superoxide production by cells.Phagocyte superoxide production by a multicomponent NADPH oxidase is important in host defense against microbial invasion. However inappropriate NADPH oxidase activation causes inflammation. Endothelial cells express NADPH oxidase and endothelial oxidative stress due to prolonged NADPH oxidase activation predisposes many diseases. Discovering the mechanism of NADPH oxidase activation is essential for developing novel treatment of these diseases. The p47 Brie: 1K4U) – and PHDs: 1K4U) –. The geneviously .The secondary structure, residue contacts and water affinities of the energy minimized models were analyzed using the protein geometry function in MOE. The stereochemical qualities of the models were assessed by using Ramachandran plot analysis and structural analysis using the protein report function in MOE. This searches for disallowed bond angles, bond lengths and side chain rotamers. There were no unacceptable deviations in the models with less than 2 outliers in the Ramachandran plot.phox conformational changes was investigated in silico by adding a phosphate group (PO43−) to the side-chain of each serine residue of Ser-303, Ser-304, Ser-310, Ser-315, Ser-320, Ser-328, Ser-345, Ser-348, Ser-359, Ser-370, and Ser-379 using the molecular builder function of MOE. The phosphorylated p47phox model then underwent energy minimization for structural refinement to correct any potential disallowed bond angles, bond lengths and unfavorable torsion angles following PO43− addition.The effect of phosphorylation-induced p47phox conformational changes associated with serine phosphorylation were studied by molecular dynamics (MD). Prior to MD simulation the protein was prepared by addition of missing hydrogen atoms which were unable to be located in low resolution protein x-ray structures. The hydrogen atoms were added at positions calculated using the Protonate 3-dimensional module of MOE. Next, the protein was solvated in a square-box of water, which was positioned randomly to occupy the volume using the solvate module in MOE. Finally, sodium ions were added to the simulation box to balance charges, and followed by energy minimization to remove any high energy interactions or van der Waals violations. The MD simulation was recorded every 0.5 picoseconds (ps) at a constant temperature of 310K for 500 ps, with a time step of 0.002 ps. The algorithm employed was the Nosé-Poincaré-Anderson with an NPT ensemble. The force field used was AMBER99 with a distance-dependent dielectric of 4 for the protein and 80 for the solvent. This resulted in a stable simulation after initial equilibration of the structure under calculated conditions. The simulation was considered to have been equilibrated after the first 100 ps.The p47g is the number of degrees of freedom in the atomic system plus one if V is not fixed. The s coordinate is a time scaling coordinate used to enforce constant temperature. The quantity H is conserved, and the equations of motion can be derived by differentiating H with respect to the conjugate coordinates and momenta to obtain the equations of motion.H  in which q are the coordinates and p the conjugate momenta at time t can be integrated through a time step h with Equations 4The NPA Hamiltonian used in MOE is shown in phox sSH3 domain and the p22phox PRR domain was examined by molecular docking using MOE. A 1.80Å resolution x-ray structure of the p22phox (phox peptide would migrate into the p47phox sSH3 binding groove. This dynamics simulation was followed by energy minimization to convergence. The resulting root mean squared deviation (RMSD) following MD when compared with the orientation of the crystallized p22phox peptide was 1.676Å, and the peptide interacted successfully with the p47phox in the sSH3 binding groove.Protein docking is a well-established technique for probing the proteome or interactome . Protein p22phox  PRR was phox cDNA (GenBankTM: AF330627.1) was performed exactly as described previously using the QuikChange Multi Site-directed Mutagenesis kit (Agilent Technologies) (phox cDNA was cloned into pcDNA3.1/Zeovector (Invitrogen) and used for the mutagenesis. Mutated p47phox cDNAs were then sequenced and cloned into Escherichia coli, DH5α (Invitrogen) (The substitution of serine (S) by alanine (A) in human p47ologies) . Brieflyitrogen) . The plaphox knock-out (KO) mice on a 129sv background were obtained from the European Mouse Mutant Archive, and backcrossed to C57BL/6J for 10 generations. All studies were performed in accordance with protocols approved by the Home Office under the Animals (Scientific Procedures) Act 1986 UK. The p47phox−/− coronary microvascular endothelial cells (CMEC) were isolated from the hearts of 10–12 week-old p47phox KO mice as described previously  containing PMSF (1 mmol/liter), EDTA (0.1 mmol/liter), sodium fluoride (50 mmol/liter), sodium vanadate (2 mmol/liter), leupeptin (2 μmol/liter), and pepstatin (2 μmol/liter). The fractions of cell nuclei, mitochondria, submitochondria, and small organelles were eliminated by sequential ultracentrifugation as described previously , and thephox was performed as described previously -chemiluminescence as described previously (m) was added into the cell homogenates as substrate for the detection of NADPH-dependent O2⨪ production. The specificity of O2⨪ detection was confirmed by tiron . Potential enzymatic sources of O2⨪ production were verified using inhibitors: rotenone (100 μmol/liter), oxypurinol (100 μmol/liter), or diphenyleneiodonium . As an independent approach, the intracellular ROS production by adherent cells was examined by 5-(and 6)-chloromethyl-2′,7′-dichlorodrofluorescein diacetate   . BrieflyImmunofluorescence microscopy was performed as described previously . Antibodin vitro studies, data were presented as means±S.D. of results taken from at least 3 independent cell cultures/per condition. In the case of CMEC, each isolation used 6 mice/per group, and the data presented were the mean from at least 3 isolations. Comparisons were made by one-way ANOVA with Bonferroni test analysis, and p < 0.05 was considered statistically significant.For the phox protein, we initially generated computational models from three available crystal structures of the p47phox functional domains: the PX-domain , the sSH3/AIR-domain , and the PRR domain  (a and b). Two missing linking peptides (142-MKDGKSTATDITGPII-156 and 340-PGPQSPGSPLEEERQTQRSK-360) between these structures were generated using the homology modeling server Phyre2 with a 50 and 90% structural confidence, respectively. A full p47phox structure of the auto-inhibited form was generated using the in silico homology modeling technique (c). The structure has no distorted torsion angles or residues in disallowed regions as analyzed using a Ramachandran plot (d), and all internal hydrogen-bonds were optimally occupied as an independent measure of structural reliability. Furthermore, the generated p47phox structure is extended rather than globular, with a radius of gyration in the same order of magnitude that was published previously in a study employing small angle x-ray scattering  , a and bechnique c. The stran plot d, and alattering .phox structure toward the C terminus, the PX domain was omitted, and the rest of the structure (a.a. 156–390) was subjected to MD simulation  . Importantly, there were no noticeable core structural changes between the pre-MD and after MD with a RMSD of the α-carbon backbone being 1.493 Å . The lack of gross structural changes throughout the MD simulation confirms the stability of the generated p47phox model used in this study.To examine the conformational changes in the p47mulation . We foun43−) group to the side-chains of Ser-303, Ser-304, Ser-310, Ser-315, Ser-320, Ser-328, Ser-345, Ser-348, Ser-359, Ser-370, and Ser-379 . The addition of phosphate groups before MD had no significant effect on the core structural stability of the p47phox because the radius of convergence of molecular mechanics used in this way was quite small and the local structural energy was accordingly minimized to accommodate the changes to keep the structural stability. The sSH3 remained completely masked by the AIR . However, when the structure was subjected to MD simulation, the interaction between the C-terminal tail, AIR, and sSH3 underwent rapid dissociation that released AIR in less than 100 ps of MD. There was a time-dependent sequential structural change of the AIR during the course of MD simulation with the movement of the phosphorylated double serines (Ser-303/4) as the final step to fully open the sSH3 groove . Compared with the un-phosphorylated structure, the p47phox α-carbon backbone RMSD kinetics in the phosphorylated form increased rapidly during the equilibration period (<100 ps) and plateaued afterward during the MD time course . The phosphorylated p47phox structure after MD had no distorted torsion angles or residues in disallowed regions as analyzed using a Ramachandran plot . There was no significant difference in the experimental settings of unphosphorylated and phosphorylated forms in terms of MD simulation parameters, i.e. temperature and volume .We then added a phosphate (POa) revealed an unexpected discovery that the movement of the phosphorylated C-terminal tail occurred before AIR destabilization. There are at least three known phosphorylation sites of PKC  located in p47phox C-terminal tail. The phosphorylation of Ser-359 and Ser-370 had been found to have no significant effect on p47phox interaction with other phox subunits , and formed a H-bond with Ser-215 located in the hinge region of sSH3 and another H-bond between Ser-379 side chain and the main chain of Ser-315 located in the central part of the AIR, which tied AIR into the tandem SH3 groove , MD simulation (500 ps) of un-phosphorylated Ser-379 had no significant effect on the autoinhibited structure of p47phox . When Ser-379 was phosphorylated and subjected to MD simulation, the negative charge generated broke the previous links immediately during the equilibration period and caused the C-terminal tail to move . After 500 ps of MD simulation, new H-bonds were formed between phosphorylated Ser-379 and Thr-382 located in the C-terminal tail and between the phosphate group of Ser-379 and the Asp-217 as a way to neutralize the local environment charges . The AIR was free, and the sSH3 groove was partially exposed .Our MD data a revealesubunits . Howeversubunits . To find the AIR a, and fophox activation, we need to confirm if it is also linked with any other single serine phosphorylation. The p47phox has a double serine phosphorylation site (Ser-303/4) located within the AIR, therefore, we checked firstly the Ser-303/4 phosphorylation. There was no significant structural change associated with Ser-303/4 phosphorylation at post-equilibration  and at 500 ps of MD . Overlapping images of the unphosphorylated and phosphorylated structures overlaid further confirmed no significant structural change . We then examined one by one the single phosphorylation plus 500 ps of MD of Ser-303, Ser-304, Ser-310, Ser-315, Ser-328, Ser-345, Ser-348, Ser-359, and Ser-370, and confirmed that there was no significant structural change associated with these single serine phosphorylations. The local residue interaction maps taken after phosphorylation and 500ps of MD  remained the same as the maps taken before phosphorylation.Although our data strongly indicate that Ser-379 phosphorylation is an early key event in p47phox sSH3 groove and p22phox PRR  (a and b). We found a time-dependent decrease in the intramolecular interactions between the p47phox AIR and sSH3 (r2 = 0.954), and this was accompanied with a time-dependent increase in the interaction between the p22phox PRR peptide and the p47phox sSH3 (r2 = 0.927) (a). At 500 ps of MD simulation, the p22phox peptide migrated completely into the p47phox sSH3 binding groove in contact with the key p47phox residues as shown in the crystallized structure (1OV3) (B). We also examined the protein-protein interaction between our phosphorylated p47phox model and the consensus p22phox model published previously . We found that both the p22phox and p47phox models remained stable throughout the MD simulations and bound together successfully through the interaction between p47phox sSH3 and p22phox PRR to form a p47phox/p22phox protein complex, which for the first time provides mechanistic insight into the global association of these two proteins.It is well documented that the cytosolic subunits of the NADPH oxidase anchor at membranes through an interaction between the p47phox PRR . HoweverNPPPRPP) , a and b= 0.927) a. At 500e (1OV3)  except fe (1OV3) B. We alseviously   was used as a negative control and the wild-type p47phox cDNA was used as a positive control. Mutated and wild-type p47phox cDNA constructs were used to transfect the primary CMEC isolated from the p47phox−/− mice. The cells were then stimulated with either vehicle or a potent protein kinase C activator, PMA  to induce p47phox phosphorylation, and then examined for NADPH oxidase activity by detecting the levels of NADPH-dependent O2⨪ production (a). Tiron (an O2⨪ scavenger) was used to confirm the assay specificity. We found that there was no significant difference in the basal (without PMA) levels of O2⨪ production by p47phox−/− cells after gene transfection with different constructs. However, when the cells were stimulated with PMA and compared with cells transfected with an empty vector, cells transfected with any constructs i.e. wild-type (WT) p47phox cDNA, S303–4A, S310A, S315A, S320A, S328A, S345A, S349A, S359A, and S370A increased significantly the levels of O2⨪ production, except the cells transfected with S379A. It was obvious that Ser-379 phosphorylation had a key role in initiating p47phox phosphorylation-dependent O2⨪ production by the NADPH oxidase.To confirm the oduction a. Tiron 2⨪ production by endothelial cells was further investigated in detail (b). Without PMA stimulation, there was no significant difference in the basal levels of O2⨪ production between cells transfected with an empty vector and cells transfected with wild-type p47phox cDNA or with S379A mutated construct . However, when the cells were stimulated with PMA, WT p47phox cDNA transfected cells significantly increased O2⨪ production, which was completely inhibited back to the vector control levels in S379A-transfected cells (p < 0.05) . The enzymatic source of PMA-induced O2⨪ production by WT p47phox cDNA transfected cells was examined using different enzyme inhibitors. It was abolished by tiron, an O2⨪ scavenger, significantly inhibited by DPI (a flavo-protein inhibitor) and apocynin (a NADPH oxidase inhibitor), but not by inhibitors to the mitochondrial complex-1 enzymes (rotenone) or to xanthine oxidase (oxypurinol) . As an independent approach, the effect of S379A on PMA-induced O2⨪ production by intact adherent CMEC was double confirmed by tiron-inhibitable DCF fluorescence (a).The effect of S379A on acute PMA-induced On detail b. Withourescence a.phox phosphorylation, membrane translocation, and complex formation with p22phox, we immune-pulled-down (IP) total p47phox protein and detected for the levels of serine phosphorylation and the levels of p22phox pulled-down together with p47phox (b). In vehicle-stimulated cells, the levels of p47phox phosphorylation and binding to p22phox were not detected in vector-transfected cells, and were just detectable in cells transfected with wild-type p47phox or S379A. However, when the cells were stimulated with PMA, WT p47phox cDNA-transfected cells significantly increased the levels of p47phox phosphorylation and the association with p22phox, and these were inhibited back to vector-transfected control levels in S379A construct transfected cells.To confirm if Ser-379 phosphorylation was indeed required for the chain reaction of PMA-induced p47 p47phox b. In vehphox membrane translocation was examined in membrane fractions isolated from p47phox−/− CMEC after gene transfection (c). The p47phox was absent in the membrane fraction of vector transfected cells. After PMA stimulation, p47phox was highly detected in the membrane fraction of WT p47phox cDNA transfected cells, and this was significantly reduced in S379A-transfected cells (c). The inhibitory effect of S379A on p47phox membrane translocation and association with p22phox was further confirmed by immunofluorescence (d). The p47phox was labeled by a goat polyclonal antibody and detected by Cy3 (red color), and p22phox was labeled by a rabbit polyclonal antibody and detected by FITC (green color). In vector-transfected cells, p22phox was detected across the entire cells. PMA stimulation induced p22phox clustering and membrane localization. The p47phox was detected in wild-type p47phox cDNA and S379A construct transfected cells. PMA stimulation induced p47phox plasma membrane translocation and association with p22phox as indicated by the yellow fluorescence seen only in WT p47phox cDNA-transfected cells, but not in S379A mutation transfected cells.The p47sfection c. The p4ed cells c. The inrescence d. The p4phox phosphorylation has been recognized as a prerequisite for NADPH oxidase assembly and activation  between the C-terminal tail and the AIR and the sSH3, and the movement of the phosphorylated C-terminal tail occurred before the AIR phosphorylation. The entire AIR phosphorylation process was a well organized chain reaction with the movement of phosphorylated double serine, Ser-303/4 as the final step to fully open the sSH3 binding site. This is important novel information on p47phox regulation, and may have a broad application to other SH3 domain-containing proteins.Although crystal structures of several functional domains of p47ously 14–, 36, 37,rylation . The novphox has multiple serine phosphorylation sites within and outside the AIR toward its C terminus. Previously we only knew that extensive p47phox phosphorylation of AIR is required for p47phox activation, but we did not know the molecular switch of the AIR phosphorylation cascade. An important contribution of the current study is that we have discovered Ser-379 phosphorylation as a molecular switch to initiate the AIR phosphorylation cascade. At rest, unphosphorylated Ser-379 forms H-bonds with the hinge of sSH3 and the AIR, which connects the sSH3 and AIR together and stabilizes p47phox in its autoinhibited form. When the Ser-379 is phosphorylated, it disrupts the links with the sSH3 and AIR, and in turn frees AIR for subsequent phosphorylation. The unique role of Ser-379 phosphorylation as a molecular switch in p47phox activation has been further confirmed by extensive experimental approaches of single serine phosphorylation and MD simulation one by one of eleven potential phosphorylation sites. It is clear that none of the other single serine phosphorylation sites or even the double serine phosphorylation sites (Ser-303/4) can cause significant conformational change of AIR.The p47phox was reported to interact with the p47phox N-terminal SH3a domain rather than the C-terminal SH3b domain  p47phox binding to p22phox, as evidenced by immuno-pull-down assay and Western blot; (ii) p47phox membrane translocation, as evidenced by cell membrane fractionation followed by Western blot and immunofluorescence; and (iii) O2⨪ production by NADPH oxidase, as examined by both lucigenin-chemiluminescence in cell homogenates and DCF fluorescence in intact cells. These in vitro data provide strong evidence in support of our in silico models that Ser-379 phosphorylation is a molecular switch of the p47phox phosphorylation cascade in response to agonist stimulation.The in silico three-dimensional model of the entire p47phox protein and revealed an important mechanism of p47phox activation. The molecular insight of how Ser-379 phosphorylation in the C-terminal tail initiates p47phox activation and how phosphorylation dynamically controls partial or full opening of the p47phox binding site can be further exploited for developing specific NADPH oxidase inhibitors to treat oxidative stress-related diseases.In summary, we have presented for the first time an