Stimulation with ephrinA1 produced rapid increases of EphA2 phosphorylation that were inhibited by dasatinib, although the effects on activation of downstream signalling differed among the pancreatic cancer cell lines. Dasatinib also inhibited ligand-induced binding of EphA2 to the ubiquitin ligase Cbl, and the internalisation and degradation of EphA2, suggesting that these processes are dependent on kinase activity. Treatment with dasatinib decreased EphA2 phosphorylation in BxPC-3 xenografts, suggesting that dasatinib might have activity in pancreatic cancer due to EphA2 inhibition, besides its effects on Src.Eph receptors constitute the largest family of receptor tyrosine kinases in the human genome. EphA2 is one prominent member that is overexpressed and functionally altered in many invasive cancers, including pancreatic cancer. Dasatinib, which is a multi-targeted kinase inhibitor mainly developed for Bcr-Abl and Src family kinases, has recently been shown to have significant activity against EphA2. As selective small molecule EphA2 inhibitors are not currently available, we investigated the therapeutic potential to target EphA2 by dasatinib in pancreatic cancer cell lines. Using  The Eph family of receptor tyrosine kinases (RTKs), with at least 14 distinct members in mammals, constitutes the largest subfamily of RTKs . Eph RTKEphA2 critically controls many aspects of cell behaviour . EpithelHowever, despite the strong correlation of EphA2 receptor expression with malignant phenotypes, the mechanisms by which EphA2 contributes to tumour cell malignancy are far from clear  293 cell line were obtained from American Type Culture Collection . BxPC-3 was grown in RPMI-1640 with 10% fetal bovine serum (FBS). PANC-1 was grown in Dulbecco's modified Eagle's medium (DMEM)-H21 with 10% FBS. MIA PaCa-2 was grown in DMEM-H21 with 10% FBS and 2.5% horse serum. HEK-293 was grown in DMEM with 10% FBS. Cells were maintained at 37°C under standard cell culture conditions.γ fragment-specific was from Jackson ImmunoResearch laboratories Inc. . [γ-32P]ATP was from New England Nuclear .Mouse monoclonal antibodies against phosphotyrosine (P-Tyr-100), phospho-STAT3 (Tyr705) as well as rabbit polyclonal antibodies against phospho-Src (Tyr416), phospho-FAK (Tyr576/577), phospho-FAK (Tyr925), phospho-Akt (Ser473), phospho-Paxillin (Tyr118), phospho-p44/42 MAPK (Thr202/Tyr204) and antibodies directed against their nonphosphorylated counterparts, were purchased from Cell Signaling Technology . Mouse monoclonal antibody against phospho-STAT3 (Ser727) was from BD Biosciences . Rabbit polyclonal antibodies directed against EphA2 and Cbl were obtained from Santa Cruz Biotechnology Inc. . A mouse monoclonal antibody against EphA2 and recombinant mouse ephrin-A1/Fc chimera were from R&D Systems Inc. . The secondary antibodies for western blots (anti-mouse and anti-rabbit IgG antibodies) were from Amersham Biosciences . Protein G-Sepharose was from Sigma-Aldrich Canada Ltd. . AffiniPure goat anti-human IgG, Fc−1 suspension in 80 mM sodium citrate/citric acid buffer, pH 3.0 for oral gavage in vivo.Dasatinib was purchased from Toronto Research Chemicals . PP2 was purchased from Calbiochem . Dasatinib and PP2 used for tissue culture were both dissolved in DMSO, aliquoted, and stored at appropriate temperature. Dasatinib was prepared freshly as a 12.5 mg mlIn vitro autophosphorylation assays were essentially performed as described earlier (−1 HEPES (pH 7.5), 10% glycerol, 1% Triton X-100, 150 mmol l−1 NaCl, 1 mmol l−1 EGTA, 1.5 mmol l−1 MgCl2, 100 mmol l−1 NaF, 10 mmol l−1 Na4P2O7·10H2O, 1 mmol l−1 Na3VO4] and kinase reaction buffer, incubated with dasatinib  at room temperature for 10 min and then incubated with 10 μCi of [γ-32P]ATP at room temperature for 30 min. The reactions were terminated by the addition of 6 × Laemmli sample buffer and boiling. After resolving samples by 10% SDS–PAGE, the gel was stained with Coomassie blue to check for equal loading, then dried and exposed.μl complete medium and then pretreated with 0, 25, 50, 100 and 200 nM dasatinib for 48 h. According to the manufacturer's instructions, viable cell number was determined using a colorimetric method based on the cellular reduction of the tetrazolium compound 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) into a soluble coloured formazan product .BxPC-3, PANC-1 and MIA PaCa-2 cells were cultured in 96-well plates at 5000 cells per well in 100 M dasatinib for 24 h, and then permeabilised with Triton X-100, treated with ribonuclease, and stained with 50 μg ml−1 propidium iodide. DNA histograms were analysed using ModFit LT™ .Subconfluent BxPC-3, PANC-1 and MIA PaCa-2 cells were treated with 0, 25, 50, 100 and 200 nμg ml−1 ephrinA1-Fc for the indicated times and then processed for immunoblotting and immunoprecipitation. Briefly, cells were washed with ice-cold PBS and lysed in lysis buffer containing protease inhibitor cocktail tablets  for 1 h on ice. Equivalent amounts of protein  were separated on 10% SDS–PAGE gels. Proteins were transferred to polyvinylidene difluoride membranes  and probed with the appropriate antibodies according to the manufacturer's instructions. For immunoprecipitation, EphA2 or Cbl was immunoprecipitated from 200–500 μg cleared lysates using anti-EphA2 or Cbl antibody plus protein G-Sepharose. Products were fractionated as above and blots probed with anti-phosphotyrosine or anti-EphA2 antibody. Blots were stripped and re-probed with anti-EphA2 or anti-Cbl antibody. Detection was conducted using SuperSignal West Pico chemiluminescent substrate kits  or enhanced chemoluminescence Plus detection reagents .Cells in logarithmic growth phase were serum-starved (0.5% serum) overnight, stimulated with preclustered 2 μg ml−1, BD Biosciences, Mississauga, Ontario, Canada). For staining, cells were fixed with PBS containing 4% paraformaldehyde for 15 min at room temperature, permeabilised with 0.2% Triton X-100, then blocked with PBS containing 10% FBS. After blocking, cells were incubated with diluted monoclonal antibody overnight at 4°C followed by Cy3-conjugated goat anti-mouse IgG  at room temperature in the dark for 1 h. Control experiments with secondary antibody alone were performed to confirm the absence of background staining. Between each step, cells were washed with PBS. After final washes, slides were coverslipped with Vectashield Mounting Medium with DAPI and examined by confocal microscopy.Cells were grown on glass chamber slides coated with collagen (125 6 cells) was injected subcutaneously into the left flank of 6-week-old male severe combined immunodeficient mice. After 4 weeks, the tumours became obviously visible. Five groups of three tumour-bearing mice were treated with a single dose of dasatinib (50 mg kg−1) or vehicle control  by oral gavage, and killed at 2, 4, 8, and 24 h. Each tumour was rapidly dissected from the surrounding tissues and cut into pieces that were snap frozen in liquid nitrogen, fixed in formalin for 24 h then paraffin-embedded, or homogenised in 1 ml lysis buffer for western blot and immunoprecipitation.Animal experiments were done in the animal facility of the Princess Margaret Hospital, operated under the guidelines approved by the Canadian Council for Animal Care. BxPC-3 cell suspension   .We next examined the effects of dasatinib on the activation of downstream signalling in response to ephrinA1-Fc stimulation. In the absence of ligand, treatment with dasatinib partially inhibited Akt phosphorylation at Ser 473, ERK phosphorylation at Thr 202/Tyr 204, and STAT3 phosphorylation at Ser 727 but not Tyr 705 in all three cell lines . UnexpecM dasatinib for 2 h, then stimulated with 2 μg ml−1 ephrinA1-Fc for 30 min. In the absence of dasatinib, a large increase in EphA2 association with Cbl was observed upon ligand stimulation, concordant with the increased level of EphA2 autophosphorylation. As shown in Previous work has shown that ligand-binding results in the internalisation and proteasomal degradation of EphA2, which is Cbl-dependent  pathway , STAT3 pα-MOTIF (SAM) domain or the PDZ recognition motif might regulate ligand-stimulated signalling events (Dasatinib strongly inhibited EphA2 tyrosine phosphorylation of EphA2 at dose levels similar to those that inhibited Src in all three cell lines. However, as seen in g events  independCell–cell contacts promote ligand binding, and ligand binding induces so-called ‘forward signalling’ mostly through phosphotyrosine-mediated pathways . Upon liin vivo. Dasatinib might therefore have activity in pancreatic cancer because of EphA2 inhibition, in addition to its effects on Src.Preliminary experiments showed that treatment with dasatinib results in a transient decrease of EphA2 phosphorylation in BxPC-3 xenografts, indicating that this compound can modulate EphA2 at doses that are achievable EphA2 is overexpressed in a number of human cancers, including pancreatic cancer, and this is associated with poor prognosis. Various strategies have been proposed to target EphA2 in cancer, including the use of monoclonal antibodies directed at EphA2 surface antigens and RNAi to EphA2 (


The changes in the actomyosin crossbridge cycle underlying altered contractility of the heart are not well described, despite their importance to devising rational treatment approaches.2+ sensitivity of fibers from ischemic hearts were both reduced. Muscle activation by photolytic release of Ca2+ and ATP suggested that the altered contractility was best described as a reduction in the rate of activation of noncycling actomyosin crossbridges to activated, cycling states. More specifically, the apparent forward rate constant of the transition between the nonforce bearing A-M.ADP.Pi state and the bound, force bearing AM*.ADP.Pi state was reduced in ischemic fibers, suggesting that this transition is commensurate with initial crossbridge activation. These results suggested an alteration in the relationship between the activation of thin filament regulatory units and initial crossbridge attachment, prompting an examination of the post-translational state of troponin (Tn) T and I. These analyses indicated a reduction in the diphosphorylated form of TnT during ischemia, along with lower Ser23/24 phosphorylation of TnI. Treatment of perfused fibers by 8-Br-cAMP increased Ser23/24 phosphorylation of TnI, altering the reverse rate constant of the Pi isomerization in a manner consistent with the lusitropic effect of β-adrenergic stimulation. However, similar treatment of ischemic fibers did not change TnI phosphorylation or the kinetics of the Pi isomerization.A rat ischemia–reperfusion model was used to determine the transitions of the crossbridge cycle impacted during ischemia. Compared to perfused hearts, the maximum force per cross-sectional area and CaIschemia reduces the isomerization from A-M.ADP.Pi to AM*.ADP.Pi, altering the kinetics of crossbridge activation through a mechanism that may be mediated by altered TnT and TnI phosphorylation. The fibers were typically <250 µm in diameter and <1.5 mm in length. Prepared fibers were permeabilized for 2 h at 4°C with pCa8.0 relaxing solution containing 1% Triton X-100. To test the impact of cAMP-dependent protein kinase (PKA) activation on perfused and ischemic anterolateral papillary muscle fibers, the cell permeable analog 8-Br-cAMP  was included during the permeabilization step.This model was described in detail previously 2+, 10 mM total EGTA, 25 mM total BES, pH 7, and when appropriate, 25 mM creatine phosphate and 15 units/mL creatine kinase. Ionic strength was maintained at 200 mM using potassium methanesulphonate. Activating solutions of varying free Ca2+ were prepared using an iterative program The workstation to test permeabilized fiber contraction has been described previously 2+ relationship, the fibers were activated with solutions of varying free [Ca2+] between pCa8.0 and 4.0. For the measurement of the influence of ADP or Pi on the force-Ca2+ relationship, a separate batch of free [Ca2+] solutions were mixed including either 1 mM added ADP or 5 mM added Pi. All forces were normalized to cross-sectional area with the assumption that fibers were elliptical in cross-section. Data were fit using the Hill equation to determine the concentration of free Ca2+ required for half-maximal force (EC50). Fiber stiffness was measured using four consecutive fiber length releases of 0.05, 0.1, 0.15 and 0.2% F) was measured by a single exponential fit of the force recovery profile following a 5% decrease in fiber length within 3 ms t-test using a Bonferroni adjustment when multiple comparisons were necessary between three surgery groups.To test fiber mechanics, an initial control contraction was recorded from each fiber by changing the bathing solution from pCa8.0 to pCa4.0. To measure the force-CaAct) was determined for perfused, ischemic and ischemia-reperfused fibers. The fiber was placed in a pCa8.0 relaxing solution that was prepared as before except for the substitution of 20 mM PIPES for BES and inclusion of 10 mM dithiothreitol 2+-rigor (kATP) was measured by flash photolysis of NPE-ATP. A fiber in pCa8.0 relaxing solution was transferred to pCa8.0 rigor solution (0 mM ATP) including 1 unit/mL apyrase Act and kATP, were determined from the force transients by single exponential fits.The rate constant of force development upon flash photolysis of NP-EGTA -1 and -2 phosphoforms essentially as described To measure the extent of Ser23/24 phosphorylation of TnI, a multiplex Western blotting strategy was used. Samples resolved by SDS-PAGE were transferred to PVDF membrane and incubated simultaneously with a mouse anti-cardiac muscle TnI monoclonal antibody  and a phospho-Ser23/24 TnI rabbit polyclonal antibody . Following washing, the blots were incubated simultaneously with Cy3 labeled anti-mouse IgG and Cy5 labeled anti-rabbit IgG (GE Lifesciences). Blots were scanned on a Typhoon 9410 imager at the appropriate channels, and the scanned images were analyzed using ImageQuant TL software. The relative extent of Ser23/24 phosphorylation of TnI was determined to be the Ser23/24 phosphospecific antibody signal divided by the total TnI mAb signal for each sample. To determine the extent of Ser23/24 phosphorylation of TnI in perfused versus ischemic hearts, homogenates of the anterolateral papillary muscle from five perfused hearts and five ischemic hearts were resolved on a single gel and blotted. The extent of Ser23/24 phosphorylation in the ischemic samples was reported relative to the perfused samples. To determine the effect of 1 mM 8-Br-cAMP treatment on Ser23/24 phosphorylation of TnI in perfused and ischemic rat hearts, ten fibers from the anterolateral papillary muscle were dispersed into two groups, with one group receiving 8-Br-cAMP treatment during permeabilization and the other group remaining untreated. All ten samples from each individual rat were resolved on a single gel and Western blotted to measure the extent of Ser23/24 phosphorylation of TnI. The extent of phosphorylation in the five treated samples was reported relative to the untreated samples. The experiments were repeated with an independent pair of rats, resulting in measurements from two perfused rat hearts and two ischemic rat hearts.The overall phosphorylation state of the thick filament associated protein myosin binding protein-C (MYBP-C) was examined by sequential Pro-Q Diamond phosphoprotein and Deep Purple total protein staining as previously described in vivo rat model was used to determine the effect of myocardial ischemia-reperfusion on the maximum force per cross-sectional area (Fmax), maximum fiber stiffness or the force – Ca2+ relationship of permeabilized small fiber bundles isolated from the anterolateral papillary muscle. Following thirty minutes of ischemia, Fmax fell from 50.1±2.5 mN/mm2 (n = 12) to 36.1±2.4 mN/mm2 , which increased significantly in ischemic fibers to 3.61±0.11 µM (n = 8) compared to 3.32±0.13 µM in perfused fibers (n = 7). This loss of force during ischemia was partially recovered by reperfusion to 41.3±5.2 mN/mm2 (n = 5) whereas the EC50 of reperfused fibers was 3.57±0.12 µM (n = 5). The Hill coefficients were not different for any of the conditions tested (data not shown). To determine if the change in Fmax correlated with a change in crossbridge attachment, we measured maximum stiffness per cross-sectional area based on the assumption that measured changes in stiffness would be proportional to the number of attached crossbridges 2/mm (n = 7) to 350.2±40.5 mN/mm2/mm (n = 8) with ischemia (P<0.016), and there was not full recovery with reperfusion .An  n = 12; . A subse2+ relationship of perfused, ischemic and ischemia - reperfused fibers in the presence of 1 mM added ADP. In agreement with previous studies, the addition of 1 mM ADP decreased the EC50 of fibers from the three surgery groups We probed the ADP-dependent transition of the actomyosin crossbridge cycle by steady state measurements of the force - Ca2 to 35.9±3.0 mN/mm22.The phosphate-bound transitions of the crossbridge cycle were examined at steady state by similar experiments. As expected, the addition of 5 mM Pi decreased the Fmax of perfused fibers from 50.1±2.5 mN/mmP<0.016). These data were complemented by measurements of the rate constant of force redevelopment, kF, following a length release F was significantly reduced in ischemic (19.3±0.8 s−1) and ischemia – reperfused fibers (20.7±0.6 s−1) as compared to perfused fibers (25.9±1.1 s−1). For all fibers, increasing [Pi] increased the rate of force redevelopment, but ischemic fibers had significantly reduced rates at all Pi concentrations tested, whereas ischemia – reperfused fibers were intermediate (F = k1+(k−1 [Pi])/(K2+[Pi]) according to Scheme 1 1/k−1) whereas K2 is the equilibrium constant for the Pi release step:We measured the effect of Pi on the Fmax of perfused, ischemic and ischemia-reperfused fibers to further explore the steady state response to exogenous Pi. We hypothesized that if the relative insensitivity of ischemic fibers to 5 mM Pi is reflective of a change in the rate of the transitions between Pi-bound states, then the effect should manifest at other Pi concentrations. Fibers from a new cohort of rats were activated at pCa4.5 and then moved to pCa4.5 solutions containing 20 and 30 mM Pi. When compared to zero added Pi, relative force declined to 61.3±1.7% (n = 14) of maximum in ischemic fibers as compared to 54.2±0.9% (n = 17) and 50.0±1.9% (n = 12) in perfused and reperfused fibers. This relationship was similar at 30 mM Pi, wherein the relative force maintained was 51.3±1.7% (n = 14) in ischemic fibers as compared to 41.9±0.9% (n = 17) and 40.0±1.5% (n = 12) in perfused and reperfused fibers, respectively. Under all cases, the maintained relative force in ischemic fibers was significantly higher than for perfused and reperfused fibers (rmediate . We fit 1 and k−1 were reduced (18.9±0.8 s−1 and 30.7±1.7 s−1 respectively) versus perfused fibers . Values were intermediate for ischemia – reperfused fibers where k1 was 20.4±0.6 s−1 and k−1 was 37.4±2.9 s−1. The value of K2 determined from the fits was 39.4±4.6 mM for perfused fibers, which was significantly higher than in ischemic fibers  but not different from ischemia-reperfused fibers (29.4±3.3 mM).In this scheme, the necessary transition that activates crossbridges from non-cycling to cycling states 2+ release following flash photolysis of NP-EGTA resulted in a rapid rise in force, with a maximum value of 58.0±4.2 mN/mm2 and a rate constant of 22.0±1.2 s−1 (n = 11). Both the force produced and kAct were significantly reduced in ischemic fibers to 33.6±2.9 mN/mm2 and 15.4±1.1 s−1, respectively . For reperfused fibers, the force produced and rate constant of activation were intermediate at 42.1±1.6 mN/mm2 and 19.3±1.6 s−1 (n = 9). As the major difference in rate and force was observed between perfused and ischemic fibers, the NP-EGTA activation data were complemented with NPE-ATP activation data for perfused and ischemic fibers in Ca2+-rigor. Force produced by transferring perfused and ischemic fibers from relaxing to Ca2+-rigor solution was 18.4±2.2 mN/mm2 and 11.9±1.5 mN/mm2, respectively (P<0.05). Following photolysis of NPE-ATP, there was a small, brief drop in force prior to force activation . Total force produced (Ca2+-rigor + active) in these experiments was 55.6±5.8 mN/mm2 (n = 13) for perfused fibers and 35.3±3.5 mN/mm2 (n = 12) for ischemic fibers. However the rate constant of force activation from Ca2+-rigor, kATP, was not different between perfused and ischemic fibers, measuring 58.5±3.0 s−1 (n = 13) and 56.3±2.9 s−1 (n = 12) respectively  and the percentage of diphosphorylated TnT decreased to 60.3±2.9% (The levels of phosphorylation of MLC-1 and MLC-2 were determined in perfused and ischemic fibers. Both MLC-1 and MLC-2 were present largely as nonphosphorylated and singly phosphorylated forms 0.3±2.9% .P<0.05). These data were complemented by an examination of the phosphorylation state of MYBP-C as determined by phosphoprotein and total protein staining (P<0.05). As both TnI and MYBP-C are downstream targets of β-adrenergic signaling P<0.05). However, 8-Br-cAMP treatment of anterolateral papillary muscle fibers from ischemic rat hearts did not result in an increase in Ser23/24 phosphorylation of TnI. In untreated ischemic fibers, the relative level of phosphorylation was 100±5.9% (n = 10 fibers from two rats) whereas treatment with 8-Br-cAMP resulted in a relative level of 95.2±5.5%  whereas k−1 was significantly increased from 36.7±3.0 s−1 to 49.7±3.9 s−1 (P<0.05). In contrast, neither k1  nor k−1 (23.3±1.5 s−1 vs 25.7±2.5 s−1) was significantly influenced by 8-Br-cAMP treatment of ischemic fibers. Compared to untreated perfused fibers, 8-Br-cAMP treated perfused fibers trended towards higher Fmax when activated by pCa4.5 in the presence of 0, 5, 10, 20 and 30 mM Pi. In fibers from ischemic rat, the Fmax at all [Pi] increased significantly following 8-Br-cAMP treatment (P<0.05).To determine the impact of 8-Br-cAMP treatment on the Fmax and force redevelopment in the presence of Pi, 8-Br-cAMP treated and untreated fibers from perfused and ischemic rat hearts were examined . Treatme±3.9 s−1 ; P<0.05.2+ availability 2+] relationships were determined in the presence and absence of exogenous Pi and ADP. These products of ATP hydrolysis influenced the force – [Ca2+] relationship of fibers generally as expected 2+-sensitization of muscle fibers from all surgery groups, although the measured effects were not significantly different between groups. By contrast, Pi decreased the Ca2+-sensitivity of force production in all surgery groups. However, ischemic fibers better retained Ca2+ sensitivity and force in the presence of 5, 20 and 30 mM Pi when compared to perfused and reperfused fibers. This distinct response suggested that the cellular events associated with ischemia impacted the Pi-bound states of the actomyosin crossbridge cycle. In the presence of Pi, the higher Ca2+-sensitivity of ischemic fibers may signal retention of attached crossbridges through near-neighbor thin filament regulatory unit interactions that better preserve the level of activation Deciphering the mechanisms contributing to the decline in contractility of the heart is fundamental to developing new treatment modalities. We have previously demonstrated that permeabilized fibers from ischemic hearts produce submaximal force regardless of maximum ATP or Ca2+ and ATP. Both the maximum force and the rate constant of force activation with Ca2+, kAct, were lower for ischemic fibers as compared to perfused fibers , crossbridges readily cycle between force bearing and non-force bearing states with a higher apparent rate for k1 (Scheme 1), allowing kATP to be faster than both kF and kAct. Therefore, at high Ca2+, thin filaments freely permit re-attachment of detached crossbridges, allowing them to readily transition between force bearing and non-force bearing states 2+ release is best described as a change in the rate of crossbridge activation into cycling states, which itself is a function of the molecular events that facilitate thin filament activation through an inter-related mechanism mediated by Ca2+ and strong crossbridge attachment Act of ischemic fibers is slowed as a result of their reduced Ca2+ sensitivity, which may alter the cooperative recruitment of crossbridges into activated, cycling states.These fiber mechanics measurements therefore allow us to speculate about the steps of muscle activation that limit the magnitude and rate of force production during ischemia. Since koduction . In the han kAct  but unaleriments  is due tvivoin vivo sites of cardiac muscle TnT phosphorylation. A recent study suggested an in vivo site of phosphorylation within the first 29 amino terminal residues in vitro, with Thr206 phosphorylation having a dominant, depressive effect on fiber contraction in vitro work using in vivo phosphorylated or dephosphorylated TnT will be required to address this mechanism.To begin to decipher the underlying molecular basis for reduced contractility during ischemia, we concentrated on the regulatory troponin complex of the thin filament. Both TnT and TnI of the troponin complex are phosphorylated in 2+ sensitivity under β-adrenergic stimulation 2+ sensitivity with reduced Ser23/24 phosphorylation in this and a previous model −1 without a change in k1 (To examine the possible impact of TnI phosphorylation, we focused on the adjacent residues Ser23 and 24, two sites that are especially relevant for β-adrenergic signaling. This signaling pathway may be altered in heart failure through a reduction in the content of PKA regulatory subunits, reduction in β-adrenergic receptor density, or desensitization of β-adrenergic receptors ge in k1 , which wge in k1 . Nonethege in k1 , 2.et al−1 and force following 8-Br-cAMP treatment of perfused fibers (32P into MYBP-C was significantly less in magnitude than other prominent targets such as TnI and phospholamban 32P incorporation over baseline At this point, it is difficult to determine how the change in MYBP-C phosphorylation contributed to the accumulated results. Previous studies have reported reduced MYBP-C phosphorylation in models of altered myocardial blood flow, suggesting that this is a conserved target d fibers  occurredd fibers . Althoug2+ but not ATP is reflective of altered thin filament regulatory function and crossbridge recruitment during ischemia that modulates the apparent rates of activation of the actomyosin crossbridge cycle without impacting the intrinsic activity of the myosin enzyme. The in vivo model demonstrated reduced phosphorylation of TnT and TnI, suggesting that the integrated effect of altered troponin phosphorylation may be involved in the observed contractile response. Future studies will benefit from a focus on the thin filament and the post-translational state of regulatory proteins during ischemia in order to understand their individual and combined contributions to pathophysiological states marked by reduced contractility.In conclusion, the present study demonstrates that the actomyosin crossbridge cycle during ischemia is unique for a reduction in the forward rate constant of the isomerization of the Pi-bound actomyosin transition, which may be closely linked to the transition of non-cycling crossbridges to activated, cycling crossbridges. Additionally, the decrease in the rate of force activation with Ca


Many hypotheses attempting to explain the speed and sensitivity with which a T-cell discriminates the antigens it encounters include a notion of relative spatial and temporal control of particular biochemical steps involved in the process. An essential step in T-cell receptor (TCR) mediated signalling is the activation of the protein tyrosine kinase ZAP-70. ZAP-70 is recruited to the TCR upon receptor engagement and, once activated, is responsible for the phosphorylation of the protein adaptor, Linker for Activation of T-cells, or LAT. LAT phosphorylation results in the recruitment of a signalosome including PLCγ1, Grb2/SOS, GADS and SLP-76. In order to examine the real time spatial and temporal evolution of ZAP-70 activity following TCR engagement in the immune synapse, we have developed ROZA, a novel FRET-based biosensor whose function is dependent upon ZAP-70 activity. This new probe not only provides a measurement of the kinetics of ZAP-70 activity, but also reveals the subcellular localization of the activity as well. Unexpectedly, ZAP-70 dependent FRET was observed not only at the T-cell -APC interface, but also at the opposite pole of the cell or “antisynapse”. The question of how the signalling machinery downstream of the T-cell Receptor (TCR) rapidly integrates the binding information resulting from the encounter of a T-lymphocyte with an antigen presenting cell (APC) is central to our understanding of the adaptive immune response. Many hypotheses attempting to explain the speed and sensitivity with which a T-cell discriminates the antigens it encounters, include a notion of relative spatial and temporal control of particular biochemical steps involved in the process  which is comprised of elements of mouse Grb2, mouse LAT and the CFP-YFP pair of fluorescent donor and acceptor. Here we report the use of ROZA expressing cells to directly visualize ZAP-70 dependent phosphorylation in T-cell lines and primary human lymphocytes with subcellular resolution during the formation of an immunological synapse. Furthermore, we observe that ZAP-70 dependent activity revealed by ROZA displays a bipolar distribution, appearing at times at the pole opposite that of the lymphocyte-APC contact. To our knowledge, ROZA is the first biosensor engineered for a TCR dependent tyrosine kinase activity and as such represents a key step forward towards bringing the wealth of biochemical information available about TCR mediated signal transduction into the realm of live cell microscopy.Numerous studies employing ZAP-70 GFP fusion proteins have demonstrated that ZAP-70 is rapidly recruited to the cell membrane upon TCR engagement N-terminal residues of mouse Lck. As shown in N-terminal 35 residues of LAT, encompassing the transmembrane domain and palmitoylation sites, however this anchor led to a largely vesicular probe expression (data not shown).As previously described Stimulation of ROZA-expressing Jurkat T-cells with pervanadate (PV), a phosphatase inhibitor that unveils constitutive tyrosine kinase activity, triggered a simultaneous increase of the 436→470 nm signal and a decrease of the 436→535 nm signal . The stiIn order to establish that the observed FRET changes were due to a ZAP-70-dependent phosphorylation of the probe, Jurkat T-cells transiently expressing ROZA were stimulated, lysed, and subsequently subjected to anti-phosphotyrosine immunoprecipitation and anti-GFP immunoblotting . The resYVNV tyrosine phosphorylation motif present in the probe cannot be phosphorylated by Src kinases in the absence of ZAP-70.Additional specificity tests were performed by imaging. As expected, the FRET changes triggered by anti-CD3 stimulation were strongly inhibited in cells pretreated with PP2 for 30 minutes before stimulation (data not shown). The strong fluorescence of piceatannol precluded its use in FRET experiments. Cells expressing the YF175 ROZA mutant also displayed no significant change in the FRET signal upon PV stimulation confirming that phosphorylation of the LAT sequence found within ROZA was necessary for the FRET ratio change. Finally, in ZAP-70-deficient Jurkat T-cells, P116, transfected with ROZA, no significant change in the FRET ratio was observed upon stimulation . This clIn order to validate the utility of ROZA for examining the evolution and subcellular distribution of ZAP-70 dependent phosphorylation during a cell-cell activation event, we followed the formation of conjugates between Jurkat clones stably expressing ROZA and Raji B cells loaded with superantigen under 40X magnification. Rapidly after conjugate formation, a synaptic clustering of the probe was observed. Such a recruitment could be due to the fact that the probe is anchored to the membrane through a palmitoylated and myristylated N-terminal sequence derived from the Src kinase Lck, that presumably targets ROZA into lipid rafts The appearance of a double signalosome could be considered as a simple case of biological pattern formation. In the theory of biological pattern formation initially proposed by Turing and later revisited by others, a pattern-forming reaction uses a combination of self-enhancing local activator and a long-range inhibitor triggered by the activator, and may create a dead zone for activator accumulation around the initial accumulation JTAG, J77 clone 20 and Raji B cells were kinds gifts of Georges Bismuth ; ZAP-70 deficient Jurkat T-cells P116 were a kind gift of Claire Hivroz . Human T lymphocytes (PBT) were isolated from blood donors by Ficoll density gradient centrifugation, followed by negative depletion on magnetic beads . Cells were cultivated in RPMI 1640 supplemented with 10% FCS. Superantigen was a mix of recombinant staphylococcal enterotoxin E, staphylococcal enterotoxin A, staphylococcal enterotoxin B, and staphylococcal enterotoxin C3 (Toxin Technology). Raji B cells were loaded for 30 min at 37°C in RPMI with superantigen at a final concentration of 200 ng/ml. All PCR amplifications were performed using Taq polymerase (Invitrogen). Sequencing was performed by MWG-Biotech AG . DNA modifying enzymes were obtained from Invitrogen. Synthetic nucleotides were obtained from Sigma-Genosys, Ltd. . Plasmids encoding mCFP and mYFP were kindly provided by R.Y Tsien . Kinase inhibitors PP2 and piceatannol were from Calbiochem. Mouse anti-human anti-CD3 (UCHT1) was from BD Pharmingen. HRP coupled goat anti-mouse antibody was from Immunotech SA (Marseille France). Anti-phospho-tyrosine antibody 4G10 ascites was a kind gift of Hai-Tao He, . Anti-GFP antibody was from Abgene (U.K.) Anti phospho-ZAP70 319 and anti-phospho LAT171 were from Cell Signalling (USA).HindIII site, a kozak sequence, and the 13 N-terminal residues of mouse Lck and a non-coding primer incorporating a SphI restriction site. Mouse Grb2 (residues 56-152) cDNA was amplified using a coding primer incorporating a SphI site and a non-coding primer which incorporated the linker sequence, mouse LAT residues 171-178 and a SacI site. The specific amino acids created were as shown in SacI site and a non-coding primer incorporating a stop codon and an EcoRI site. The three fragments were ligated simultaneously into the HindIII/EcoRI sites of pCDNA3.1. The Tyr to Phe mutation of the LAT tyrosine 175 present in central fragment of the ROZA sequence was created using nested amplifications of the Grb2-linker LAT sequence. The complete nucleotide sequence encoding this structure has been deposited in Genebank (accession number EU035753).The sequence encoding ROZA was constructed from three fragments. A CFP fragment was created by PCR amplification of mCFP residues 1-228 using a coding primer incorporating a 6 cells were transfected with 5 µgs plasmid DNA. Cells were used 24–48 hours after nucleofection. Stable clones of the Jurkat J77 cl20 expressing ROZA were established first sorting the YFP positive cells with a Becton Dickenson FACSAria cell sorter followed by cloning by limiting dilution in the presence of 1.5 mg/ml G418 (Invitrogen).Jurkat T-cells and PBT were transfected by Amaxa nucleofection with solution V/ Program G-10 or S-18 and human T solution / Program U-14, respectively. Typically 5×10Fluorescence acquisition was performed with a Nikon TE2000 equipped with cooled CCD camera . Three images were acquired every 10s: visible, excitation at 435 nm and emission successively at 530 nm (FRET) and 470 nm (CFP). The ratio R = FRET/CFP that gives an estimation of the inverse of ZAP-70 activity, was calculated with MetaFluor (Roper Scientific) after background subtraction. The following filters were used, all from Chroma , except when otherwise mentioned. For CFP excitation and emission: 436±5 nm→470±15 nm. For YFP: 500±10 nm→535±20 nm.6 cells per ml, recovered, rinsed once in serum-free medium and suspended at 5×106 cells per point in RPMI supplemented with 10 mM HEPES with or without kinase inhibitors. Cells were stimulated with the pervanadate or antibody for the indicated time, briefly spun, and resuspended in ice cold NP-40 lysis buffer  as per manufacturers instructions) at 4°C for 20 minutes. In the indicated experiment, post nuclear supernatants were subjected to immunoprecipitation with an anti-GFP antibody to pull down the probe. Immunoprecipitates were subjected to SDS-PAGE transferred to PVDF membrane. Membranes were probed with indicated antibodies as per manufacturers recommandations for each specific antibody followed by HRP conjugated secondary antibody . Images were revealed using ECL plus (Amersham).ROZA expressing cells were cultured at densities less than 0.5×10


PARP activation is also relevant to the development of complications of diabetes. Hence, agents capable of inhibiting PARP may be useful in preventing the development of diabetes and in slowing down complications of diabetes.Poly(ADP-ribose) is a NADPARP inhibition was assessed with a colorimetric assay kit. Molecular docking studies on the active site of PARP were conducted using the crystalline structure of the enzyme available as Protein Data Bank Identification No. 1UK1. Type 2 diabetes was induced in male Sprague-Dawley rats with streptozotocin . The test compounds  were given by the i.p. route 45 min before STZ at 2.4 mM/kg  or 1.2 and 3.6 mM/kg (only NIC and TAU). Blood samples were collected at 24 hr after STZ and processed for their plasma. The plasma samples were used to measure glucose, insulin, cholesterol, triglycerides, malondialdehyde, nitric oxide, and glutathione levels using reported methods.+ and TAU to interact with the binding site for the adenine moiety of NAD+. While STZ-induced diabetes elevated all the experimental parameters examined and lowered the insulin output, a pretreatment with 3-AB, NIC or TAU reversed these trends to a significant extent. At a dose of 2.4 mm/kg, the protective effect decreased in the approximate order 3-AB>NIC≥TAU. The attenuating actions of both NIC and TAU were dose-related except for the plasma lipids since NIC was without a significant effect at all doses tested.3-AB, NIC and TAU were able to inhibit PARP, with the inhibitory potency order being 3-AB>NIC>>TAU. Molecular docking studies at the active site of PARP showed 3-AB and NIC to interact with the binding site for the nicotinamide moiety of NADAt equal molar doses, 3-AB was generally more potent than either TAU or NIC as an antidiabetogenic agent, but the differences were not as dramatic as would have been predicted from their differences in PARP inhibitory potencies. NIC and TAU demonstrated dose-related effects, which in the case of TAU were only evident at doses ≥2.4 mM/kg. The present results also suggest that in the case of NIC and TAU an increase in dose will enhance the magnitude of their attenuating actions on diabetes-related biochemical alterations to that achieved with a stronger PARP inhibitor such as 3-AB. Hence, dosing will play a critical role in clinical studies assessing the merits of NIC and TAU as diabetes-preventing agents. Contill death -8. Overaycolysis ,5,9. Wheycolysis ,10.+ consumption and decreases of the ATP pool, and to protect pancreatic β-cells from chemically induced necrosis but not from cytokine-mediated apoptosis, a major cause of autoimmune diabetes [+ and NADPH caused by diabetes [+, has been the subject of intensive clinical trials around the world as a means of preventing or delaying the clinical onset of diabetes in humans [3 possesses other properties that may be of benefit in diabetes such as antioxidant action, modulatory role on neuronal Ca++ influx, inhibitory action on apoptosis [PARP inhibitors have the ability to prevent intracellular NADdiabetes . Furtherdiabetes . Becausediabetes -13. Howediabetes , studiesdiabetes . In contn humans ,16-18. Ipoptosis , and abiAmong natural compounds, taurine (TAU) is among those most extensively investigated for its attenuating effects on diabetes-related alterations such as decreased insulin secretion ,20, hypeIn view of the similarity of protective actions manifested by TAU, 3-AB and NIC against diabetes it appeared of interest to determine whether or not TAU is endowed with PARP-inhibiting action and, if this is the case, to what extent differences in PARP-inhibiting potency among TAU, 3-AB and NIC impact on the extent of their antidiabetic effects. To attain these goals, TAU, 3-AB and NIC were tested both in vitro for PARP-1 inhibiting power and for mode of interaction with the active site of PARP-1 in rats and in vivo, using rats made diabetic with streptozotocin, (STZ), a known generator of oxygen and nitrogen reactive species, for their counteracting actions on hypoinsulinemia, hyperglycemia, hyperlipidemia and oxidative stress when administered in equimolar doses as a pretreatment to STZ. To our knowledge these comparisons have not been previously reported.All the experiments were carried on male Sprague-Dawley rats, 200-225 g in weight, purchased from Taconic Farms, Germantown, New York, USA, and housed in a temperature controlled room (21±1°C) with a 12 hr light-12 hr dark cycle. The animals were allowed free access to a standard rat chow and filtered tap water for at least 5 days. The solid food, but not the water, was removed 12 hr prior to an experiment. The experimental groups consisted of 6 animals each. The study received the approval of the Institutional Animal Care and Use Committee of St. John’s University, and the animals were cared in accordance with the guidelines established by the United States Department of Agriculture.Solutions of STZ, 3-AB, NIC and TAU were made in citrate buffer pH 4.6, and they were administered by the intraperitoneal (i.p.) route in a volume not exceeding 2 ml. The doses used were: STZ 60 mg/kg, 3-AB 2.4 mM/kg, TAU 1.2-3.6 mM/kg, NIC 1.2-3.6 mM/kg. 3-AB and NIC were administered 45 min before STZ. TAU was administered in divided doses, one-half at 75 min before STZ and one-half at 45 min before STZ. Control animals received only citrate buffer pH 4.6 in a volume equal to that of a treatment solution. All the animals were decapitated at 24 hr after a treatment with STZ, and their blood samples, collected into heparinized tubes, were processed for their plasma fractions.2HPO4, NaH2PO4, and NaCl) and 0.1% Triton X-100. Then, 50 µL of diluted Strep-HRP (blocking solution) was added to each well, and the strips were further incubated at room temperature for 60 min. After washing the wells 4 times each with PBS and with 0.1% Triton X-100, they were mixed with 50 µL of TACS-Sapphire™ colorimetric substrate, and allowed to stand in the dark for 10-15 min. The intensity of the blue color that developed in each well was read on a microplate reader set at 630 nm. After stopping the reaction by adding 50 µL of 5% phosphoric acid to each well, the absorbance of the yellow color was measured at 450 nm. Parallel experiments were conducted by substituting the test solution with an equivalent volume of dimethyl sulfoxide or distilled water to verify the effect of the vehicles on the enzyme activity. All the samples were tested in triplicate.The inhibitory action of the test compounds towards PARP-1 was determined using a commercially available microplate assay kit  and in accordance with the instructions provided by the manufacturer. Stock solutions of the various test compounds were made in dimethyl sufoxide, which were then serially diluted to the required concentration (100 µM) with distilled water. For the assay, each strip well was filled with 10 µL of the inhibitor solution, 15 μL of diluted PARP-1 enzyme (providing 0.5 Unit/well), and 25 µL of PARP Cocktail . The strip wells were incubated at room temperature for 60 min, and then washed 4 times with phosphate buffered saline  and the IC50 value for each plot was obtained using Regression Wizard from Sigma Plot version 10.0 . All the assays were conducted in two separate occasions, each time in triplicate. The results of these studies are presented as mean ± standard deviation (SD) and are reported in μM.To determine the ICMolecular docking computations were carried out on a Dell Precision 470n workstation with the RHEL 4.0 operating system using Glide 5.0. 3D Structures of the various test compounds were constructed using the fragment dictionary of Maestro 9.0. The geometry of the ligands was optimized by Macromodel program v9.5 using the Optimized Potentials for Liquid Simulations-all atom (OPLS-AA) force field  with theortho-dianisidine, and the reaction is allowed to proceed at 37°C for 30 min. The absorbance of the colored solution is read on a spectrophotometer at 450 nm. The concentration of glucose in the sample was calculated by reference to a glucose standard solution provided by the manufacturer and treated in identical manner as the plasma sample. The result was expressed in mg/dl of plasma.The content of glucose in a plasma sample was measured using a commercially available colorimetric assay kit  which is based on the method of Rabbo and Terkildsen . In the The quantity of insulin released into the plasma was measured using a commercially available solid phase two-site Insulin ELISA immunoassay kit and strips coated with insulin MAb . This assay is based on a direct sandwich technique in which two monoclonal antibodies (enzyme (HRP)-conjugated anti-insulin and anti-insulin antibody bound to a microtitration well) are directed against separate antigenic determinants on the insulin molecule. Following a washing step to remove any unbound enzyme labeled antibody, the bound HRP complex is detected by reaction with TMB to yield a colored product that can be read on an ELISA plate reader. The results were expressed as μIU/ml.® Cholesterol, Beacon Diagnostics Pvt. Ltd., Navsari, India). In the assay, 10 μl of plasma sample was mixed with 1.0 ml of enzyme-color reagent solution , incubated at 37°C for 5 min, and the absorbance of the solution was read on a spectrophotometer at 505 nm against a blank preparation (the enzyme-color solution). A standard preparation of cholesterol, treated in identical manner as the plasma sample, was analyzed alongside and used as a reference for calculating the plasma cholesterol content, in mg/dl.The concentration of total cholesterol in a plasma sample was measured using a commercially available enzymatic-colorimetric assay kit . In the assay, 10 μl of plasma sample was mixed with 1.0 ml of enzyme-color reagent solution , incubated at 37°C for 5 min, and the absorbance of the solution was read on a spectrophotometer at 500 nm against a blank preparation (the enzyme-color solution). A standard preparation of triglyceride, treated in identical manner as the plasma sample, was analyzed alongside and used as a reference for calculating the plasma triglyceride content, in mg/dl.The concentration of triglycerides in a plasma sample was measured using a commercially available enzymatic-colorimetric assay kit  in the plasma was measured as TBARS by the method of Buege and Aust . In the The content of GSH in a plasma sample was measured using the method of Ellman  and in wEvidence of the formation of NO in the plasma was obtained indirectly by measuring the content of nitrite by the method of Kauser et al.  which isResults are expressed as the mean ± SEM for n = 6. Intergroup comparisons were carried out by Student’s t-test, one-way ANOVA, and Neuman-Keuls post hoc test. Differences were considered to be significant when p was <0.05.50 values, 3-AB (33 ± 1.8 μM) was about 6.4-fold more potent than NIC (210 ± 2.9 μM) and about 9.7-fold more potent than TAU (320 ± 3.4 μM).3-AB, NIC and TAU were tested for their ability to inhibit PARP-1 using a colorimetric assay kit. The results of this test indicated that a wide difference in inhibitory potency existed among these compounds. In terms of their mean ± SD IC+. In the donor site, three subsites are described: a NIC-ribose (NI) binding site, a phosphate (PH) binding site and an adenine-ribose (AD) site [904 and amino and carbonyl groups of the backbone of Gly863 at the NI binding site of PARP-1, the highly polar TAU entered into hydrogen bonding at the AD site with the carbonyl group on the backbone of Asp766 and the carboxyl group of Asp770 via its β-amino group and to the carbonyl and amino groups on the backbone of Arg878 via its sulfonic acid group. Assessment of ligand binding efficiency based on docking scores indicated a good correlation with the PARP-1 inhibitory actions of the test compounds .PARP-1 is a polypeptide whose activation requires binding as a homodimeric protein to a nicked DNA. This polypeptide possesses a highly conserved organization consisting of three main domains: a N-terminal DNA-binding domain which acts a DNA nick sensor, a central portion designated as the automodification domain (AMD) and which contain regions for dimerization and for modulating interactions with DNA and with proteins, and a C-terminal region, representing the most conserved part of the enzyme, and capable of catalyzing poly(ADP-ribose) synthesis and of binding to target proteins . In turnAD) site . To be aFrom the data presented in Figure While a 60 mg/kg i.p. dose of STZ induced type 2 diabetes in the rat and suppressed the insulin output by about 82% of the control value (p<0.001), a pretreatment with any of the test compounds was able to preserve the ability of pancreas to secrete insulin following a treatment with STZ Figure . In paraAs shown in Figure From the results shown in Figure The occurrence of oxidative and nitrosative stress as a result of STZ-induced diabetes was assessed on the basis of the plasma MDA Figure , NO Fig and GSH NO is one of the metabolic products of STZ responsible for DNA strand breakage in pancreatic cells and for the formation of the highly toxic peroxynitrite anion following interaction with reactive oxygen species (ROS) . In thisGSH is a biomolecule that functions as an antioxidant by participating in the detoxification of electrophilic/oxidizing xenobiotics, free radicals and peroxides . During + and ATP as a result of several possible mechanisms. First, metabolism of its nitrosoureido moiety can yield chemical species  for alkylating DNA, inhibiting the incorporation of precursors into DNA, and causing DNA strand breaks [O-GlcNAc residues from modified nucleocytoplasmic proteins acting as a glucose sensor [In the present study, diabetes was induced by the intraperitoneal administration of STZ. This methylnitrosoureido derivative of D-glucose is well suited as pharmacological tool for studying the effects of compounds with PARP-1 inhibitory action and with potential for preventing the onset of type 1 diabetes in experimental animals. Following its entry into pancreatic β-cells, STZ will be toxic to cells because it depresses the levels of NADd breaks ,35. Therd breaks ,36-38. Hd breaks ,37. Addid breaks , and thee sensor ,42.The three compounds evaluated in the present study are endowed with biological properties that are of benefit in preventing or delaying the onset of type 1 diabetes and to minimize the development of complications of diabetes. In this context, while 3-AB and NIC have been extensively studied for their PARP-1 inhibitory actions, in the case of TAU such link appears lacking. Although both 3-AB and NIC are examples of PARP inhibitors that are presently considered to be of low potency when compared to compounds designated as second- and third-generation inhibitors, they are still used experimentally to explore the function of the PARP family of enzymes . Among P50 values for both 3-AB and NIC fall within the range of values reported for these compounds in the scientific literature [904 and Gly863 at the NI binding site of the active site, the much weaker inhibitor, TAU, was forming hydrogen bonds with Asp766, Asp770 and Arg878 at the AD site of the active site. Calculation of the corresponding docking scores for each compound, which are inversely related to the binding affinities, suggested 3-AB to be more tightly bound than NIC to the active site of the enzyme than NIC, and TAU to be weakly bound to PARP-1. In common with most PARP inhibitors, the three compounds under evaluation are inhibiting the enzyme in a competitive manner since they block the binding of NAD+ to the catalytic domain of PARP-1 [In vitro testing of 3-AB, NIC and TAU for their PARP-1 inhibitory activity indicated that 3-AB was about 6.4-fold more potent than NIC and about 9.7-fold more potent than TAU. The experimental ICterature ,44. WithIn addition to differences in inhibitory activity imposed by their structural features , the actA role of PARP activation in pancreatic islet cell damage by STZ can be ascertained from the circulating levels of both insulin and glucose, with the type of diabetes being a determined by the dose of STZ administered . In the In rodents, the induction of diabetes with a STZ is known to lead to a hyperlipidemia characterized by elevations in the circulating levels of total cholesterol, triglycerides, very low density lipoprotein and low density lipoprotein-cholesterol and by a decrease of high density lipoprotein-cholesterol ,39,47. IOxidative stress is a hallmark of STZ-induced diabetes since this diabetogen can promote the formation of ROS and NO in the pancreas ,38,48 anThe present study finds that all the test compounds were able to lower the plasma levels of NO and MDA, a secondary product of LPO, and to increase the plasma GSH content relative to plasma levels from animals on STZ alone. In all likelihood, these changes reflect the intracellular status of those organs and tissues, particularly erythrocytes, being affected by STZ-induced diabetes along with the activities of those intracellular systems responsible for their formation , Further2-induced LPO [+. In turn, NAD+ undergoes metabolic conversion to NADP, which in its reduced form is an obligatory cofactor for the redox cycling of GSH with its disulfide form GSSG under the mediation of glutathione reductase [++ influx, apoptosis and cell injury in a dose-related manner [The reduction in LPO by these compounds is not unexpected since they have all shown antioxidant properties in vivo, in vitro and ex vivo. Thus, 3-AB was found to ameliorate peroxynitrite-induced cytotoxicity , to effeuced LPO . NIC caneductase . Furthereductase ,57. In reductase . The samd manner .2O2 [In addition to its protective actions against STZ-induced hyperglycemia and hypoinsulinemia , TAU is 2O2 ,66, has 2O2 . In brie2O2 , and, moThe results of the present work indicate that TAU possesses PARP-1 inhibitory activity and that this activity is rather weak when compared to classical PARP-1 inhibitors such as 3-AB and NIC. Through molecular docking analysis, TAU is found to bind to subsite of the active site of PARP-1 that is different from that shared by 3-AB and NIC. When administered to rats at a 2.4 mM/kg dose 45 min before a 60 mg/kg dose of STZ, 3-AB, NIC and TAU demonstrated common attenuating actions on the hyperglycemia, hypoinsulinemia and oxidative stress induced by STZ, with 3-AB exhibiting the greatest potency and TAU the least. However, at a dose of 3.6 mM/kg the effects of TAU and NIC against STZ-related alterations became either equal or greater than those by a 2.4 mM/kg dose of 3-AB. Irrespective of the dose used, TAU was the most protective among the compounds tested against oxidative stress by STZ. On the other hand, the lack of effect of NIC, a more potent PARP-1 inhibitor than TAU on hyperlipidemia by STZ suggests that PARP-1 inhibitory potency is not a reliable predictor of the type and extent of the effect that a PARP inhibitor may have on the plasma lipids associated with diabetic hyperlipidemia.TMB: 3,3´,5,5´-tetramethylbenzidine; TBARS: thiobarbituric acid reactive substances; TCA: trichloroacetic acid; TBA: thiobarbituric acid; HCl: hydrochloric acid; TEP: 1,1,3,3-tetraethoxypropane; GSH: reduced glutathione; GSSG: oxidized glutathinoe; DTNB: 5,5’-dithiobis(2-nitrobenzoic acid); NED 2HCl: N-1-naphthylethylenediamine hydrochloride.The authors declare that they have no competing interests.KGP carried out all experimental work on live animals, performed all the biochemical assays and statistical analyses, helped with the collection of literature information, prepared the figures, and made editorial comments to the article. MRP performed the in vitro tests for PARP-1 inhibition, carried out the molecular modeling studies, prepared the figures for the molecular modeling results, and made editorial comments to the article. CLP conceived the project and guided its development, assembled, organized and interpreted the experimental data, and reviewed the pertinent scientific literature.


The secreted enzyme autotaxin (ATX) stimulates tumor cell migration, tumorigenesis, angiogenesis, and metastasis. ATX hydrolyzes nucleotides, but its hydrolysis of lysophospholipids to produce lysophosphatidic acid (LPA) accounts for its biological activities. ATX has been identified only as a constitutively active enzyme, and regulation of its activity is largely unexplored. In spite of its presence in plasma along with abundant putative substrate LPC, the product LPA is found in plasma at unexpectedly low concentrations. It is plausible that the LPA-producing activity of ATX is regulated by its expression and by access to substrate(s). For this reason studying the interaction of enzyme with substrate is paramount to understanding the regulation of LPA production.In this study we determine ATX hydrolytic activities toward several artificial and natural substrates. Two novel point mutations near the enzyme active site (H226Q and H434Q) confer attenuated activity toward all substrates tested. The Vmax for LPC compounds depends upon chain length and saturation; but this order does not differ among wild type and mutants. However the mutant forms show disproportionately low activity toward two artificial substrates, pNpTMP and FS-3. The mutant forms did not significantly stimulate migration responses at concentrations that produced a maximum response for WT-ATX, but this defect could be rescued by inclusion of exogenous LPC.H226Q-ATX and H434Q-ATX are the first point mutations of ATX/NPP2 demonstrated to differentially impair substrate hydrolysis, with hydrolysis of artificial substrates being disproportionately lower than that of LPC. This implies that H226 and H434 are important for substrate interaction. Assays that rely on hydrolyses of artificial substrates (FS-3 and pNpTMP), or that rely on hydrolysis of cell-derived substrate, might fail to detect certain mutated forms of ATX that are nonetheless capable of producing LPA in the presence of sufficient exogenous substrate. H420Q-ATX could not be differentiated from WT-ATX, indicating that histidine at position 420 is not required for any of the activities of ATX tested in this study. Autotaxin ATX, NPP2), a secreted enzyme originally purified as a tumor cell motility-stimulating factor , enhance, a secreAlthough products of the ATX nucleotide phosphodiesterase activity, such as adenosine nucleotides , have beThe biological activities of ATX are now understood to arise from its lysophospholipase D (LPLD) activity  that catThe LPLD, nucleotide PDE, and motility-stimulating activities of ATX all require the presence of threonine at (human sequence) position 210 (T210) as well as three metal-binding histidines  . The catWe have identified two novel point mutations in the vicinity of the active site of ATX that disproportionately discriminate against the hydrolysis of two artificial substrates (FS-3 and PpNpTMP), have attenuated activity toward LPC, and are defective in the stimulation of migration. In this manuscript, we define the Km and Vmax of native ATX for its various substrates, and characterize the altered kinetic and biological properties of ATX-H226Q and ATX-H434Q.Nucleotide pyrophosphatase and phosphodiesterases (NPPs) are metallo-enzymes that contain a number of histidine and aspartate residues in the presumed metal-binding site. Several of these residues have been shown to be required for their enzymatic activities ,17,19. WThe enzymatic activities of the wild type vs. mutant forms of ATX were tested for three structurally distinct types of ATX substrates. Fig. To better understand the altered hydrolytic efficiency and substrate selectivity of H226Q-ATX and H434Q-ATX, we determined the effects of substrate concentrations on the reaction rates and calculated the kinetic parameters Km and Vmax for each substrate. Fig. Km and Vmax values from these data, normalized for ATX protein concentration, are included in Table We compared the kinetic parameters of the mutant forms toward LPC 16:0, LPC 18:0, and LPC 18:1; these results are also included in Table ® Red, the fluorescent detector for choline oxidase activity, may be susceptible to direct hydrolysis, which we occasionally observed in partially purified ATX preparations from transfected insect (HiFive) cell conditioned medium (data not shown). Notably, none of the ATX preparations used in this study (from transfected COS1 cell conditioned media) hydrolyzed Amplex® Red directly (data not shown).The kinetic data demonstrate the strengths and weaknesses of each enzymatic assay for the detection of ATX activity. Product formation kinetics were linear at 30 – 100 ng WT-ATX protein for FS-3 and LPC, and at 30 – 60 ng WT-ATX protein for pNpTMP (data not shown). LPC has the advantage of being ATX-specific. Although FS-3 hydrolysis by NPP1 and NPP3 is considered unlikely because they lack activity toward lysophospholipids, direct evidence that FS-3 itself is resistant to hydrolysis by NPP1 or NPP3 has not been reported and this limits the interpretation of ATX specificity . In contMigration responses of A2058 cells, with or without inclusion of exogenous LPC (18:1), to WT-, H226Q-, and H434Q-ATXs are shown in Fig. Migration stimulation by H420Q-ATX was indistinguishable from WT-ATX (data not shown). At tested concentrations, migration stimulation by H226Q-ATX or by H434Q-ATX, without exogenous LPC, was not significantly different from background. Inclusion of sufficient exogenous LPC with either mutant resulted in a significant migration response. Together these results suggest that, compared to WT-ATX, H226Q-ATX and H434Q-ATX are not only less efficient in hydrolyzing exogenously-provided LPC but are also less efficient in hydrolyzing and/or retrieving cell-derived substrate. LPC alone failed to stimulate migration responses in the absence of exogenous ATX. Therefore the migration response data reflect properties intrinsic to the various ATX constructs.The product of extra-cellular ATX activity is LPA, a powerful stimulatory lipid which acts via cell surface receptors and whose concentration must be tightly regulated in space and time. This suggests that ATX itself is localized near its products' site of action. If both WT-ATX and the attenuated mutants, e.g. H226Q-ATX, have a common target, competition could occur between the two forms. To detect a dominant negative effect of H226Q-ATX on WT-ATX activity, we tested the migration response of WT-ATX in the presence and absence of excessive amounts of H226Q-ATX, and the results are shown in Fig. At higher concentrations of H226Q-ATX  activity of WT-ATX, and the results are shown in Fig. The prevailing context of this study was cancer theory and therapy, but as noted in the Background section, the findings have implications for development and for other pathologies. ATX is extra-cellular and a metallo-enzyme; therefore it is an attractive pharmacological target. Knowledge of the details of the lysophospholipase D enzyme mechanism can inform efforts at drug design. For example, ATX and the other NPPs use a "ping-pong" type mechanism that involves two hydrolytic steps. The second step is an attack by water on the covalent enzyme intermediate, and this releases the final product, LPA. Enzymes using this type of mechanism are often sensitive to product inhibition, and it is now apparent that the proposed natural inhibitor, S1P, and the recently reported synthetic inhibitors, are either a product or product analog.The role of particular amino acids within ATX in this enzyme mechanism is incompletely understood. Studies using chimeric constructs with regions from other NPPs indicate that regions throughout the entire length of ATX are irreplaceable for LPC hydrolysis. It is known that threonine at position 210, the site of intermediate formation, is absolutely required; and certain histidine residues have been implicated as coordinators of required metals. Histidine residues within enzymes can also participate directly in other aspects of catalysis. For example, in histidine phosphatase, histidine plays a crucial role in the phospho-transfer reaction . MeasuriTwo mutants, H226Q-ATX and H434Q-ATX showed anomalous results: they could hydrolyze LPC, albeit at a reduced rate, but did not stimulate cell migration. The initial study, identifying plasma LPLD as ATX  reportedH226Q-ATX and H434Q-ATX are the first point mutations of ATX/NPP2 demonstrated to differentially impair substrate hydrolysis, including that of endogenous substrate(s) in the migration assay. These two histidine residues appear to be important in ATX-substrate interaction. H420Q-ATX could not be differentiated from WT-ATX, indicating that histidine at position 420 is not required for any of the activities of ATX tested in this study. There was no apparent cross-inhibition of either migration stimulation or hydrolytic activity between WT-ATX and the attenuated mutant H226Q-ATX.Except as noted, reagents were from Sigma-Aldrich . FS-3 was from Echelon Biosciences , and Amplex UltraRed was from Invitrogen Molecular Probes .The HA-tagged ATX was obtained by amplification utilizing the Platinum Pfx DNA polymerase . An antisense oligonucleotide primer was used to introduce the HA epitope tag (YPYDVPDYA) and an XbaI site into the C terminus of ATX:(5'ATGCATGCTCTAGAATAGTCAGGAACATCGTATGGGTACTCGAGAATCTCGCTCTCATATGT3'). The sense primer that we utilized to introduce a HindIII site was (5'ATCTATCTAAGCTTATGGCAAGGAGGAGCTCGTT3'). ATX/NPP2 cDNA, derived from MDA 435 cells cloned into pcDNA3.1 vector (pcDNA3.1wATX/NPP2) was used as a template [All point mutants of ATX were constructed utilizing the QuikChange XL Site-Directed Mutagenesis Kit from Stratagene per the manufacturer's protocol . Each mutant plasmid was sequenced to confirm the presence of the mutation and the fidelity of the PCR amplification. COS-1 cells were transiently transfected with pcDNA3.1-wtATX or with each mutant plasmid using Lipofectamine 2000 reagent (Invitrogen Life Technologies) per the manufacturer's protocol. A non-vector control was produced by adding appropriate medium and identical treatments but no vector to COS-1 cells. Following transfection, medium was switched to DMEM and cells were incubated for 48 hr. The conditioned media were collected, concentrated, and analyzed by immunoblot using 4–12% polyacrylamide gels, anti-ATX-peptide rabbit polyclonal affinity-purified primary antibody , goat-anti-rabbit HRP-conjugated secondary antibody, ECL-plus (Amersham), and quantification by direct scanning . Relative ATX protein concentrations were determined and normalized prior to the assays by comparing immunoblot signal intensities.Cell migration was measured using A2058 human melanoma cells as described earlier  with min2, 1 mg/ml BSA (fatty acid free); substrates and their concentrations were included as indicated. LPC hydrolysis assays were based on detection of released choline. The reactions contained choline oxidase (0.1 U/ml), peroxidase (1.0 U/ml), and Amplex UltraRed (10 μM). Product formation kinetics (peroxide-driven Amplex UltraRed hydrolysis to yield fluorescent resorufin) was monitored by fluorimetry (Ex560 nm/Em590 nm). The assays for hydrolysis of pNpTMP [Reaction buffer (TCB) was 50 mM Tris pH 7.4, 5 mM CaClf pNpTMP  and FS-3f pNpTMP  were adaATX: (autotaxin); DB: (DMEM supplemented with 0.1% FAF-BSA); FAF-BSA: ; LPA: (lysophosphatidic acid); LPC: (lysophosphatidylcholine); LPLD: (lysophospholipase D); NPP: (nucleotide pyrophosphatase and phosphodiesterase); PDE: (phosphodiesterase); pNpTMP: (p-nitrophenyl-TMP).The authors declare that they have no competing interests.EK performed the mutagenesis, construction of vectors, and ATX expression. RWB developed the enzyme assay methodology. DDR analyzed and interpreted data. TC performed the enzyme activity and migration assays. MLS and TC designed the study, performed statistical analysis, and wrote the manuscript. All authors read and approved the final manuscript.


GBA) cause Gaucher disease (GD), an autosomal recessive lysosomal storage disorder. Individuals with homozygous or heterozygous (carrier) mutations of GBA have a significantly increased risk for the development of Parkinson’s disease (PD), with clinical and pathological features that mirror the sporadic disease. The mechanisms whereby GBA mutations induce dopaminergic cell death and Lewy body formation are unknown. There is evidence of mitochondrial dysfunction and oxidative stress in PD and so we have investigated the impact of glucocerebrosidase (GCase) inhibition on these parameters to determine if there may be a relationship of GBA loss-of-function mutations to the known pathogenetic pathways in PD. We have used exposure to a specific inhibitor  of GCase activity in a human dopaminergic cell line to identify the biochemical abnormalities that follow GCase inhibition. We show that GCase inhibition leads to decreased ADP phosphorylation, reduced mitochondrial membrane potential and increased free radical formation and damage, together with accumulation of alpha-synuclein. Taken together, inhibition of GCase by CβE induces abnormalities in mitochondrial function and oxidative stress in our cell culture model. We suggest that GBA mutations and reduced GCase activity may increase the risk for PD by inducing these same abnormalities in PD brain.Mutations of the gene for glucocerebrosidase 1 ( GD patients have reduced GCase activity while heterozygote carriers generally have an intermediate level  is a ubiquitous lysosomal enzyme responsible for the breakdown of glucocerebroside to glucose and ceramide. Diverse mutations within the gene  on mitochondrial function and free radical generation.The mechanism(s) whereby e stress . Given t22.1Reagents were supplied by Sigma–Aldrich  and Merck  unless otherwise stated.2.2SHSY-5Y cells were maintained as described . During 2.3d-glucopyranoside as substrate in a plate reader . The increase in fluorescence of released 4-Methylumbelliferone at 460 nm following excitation at 360 nm was followed over 1 h. Protein levels were estimated using a bicinchoninic acid (BCA) kit  with reference to the protein standard supplied with the kit.Respiratory chain , proteas2.4Cells were harvested, washed with PBS and processed as described . 25–40 μ2.5Ψm) was quantified by steady-state fluorescence (excited using the 543 nm laser line and measured using a 560 nm longpass filter) of mitochondrial patterns produced by 25 nM tetramethyl rhodamine methyl ester  stained at room temperature for 45 min. Free radical generation was measured by the rate of ratiometric change of reduced and oxidised dihydroethidium  fluorescence. 10 μM of DHE was loaded at room temperature, and measurements were made typically over 120 s. Oxidised DHE was measured with the 543 nm laser line and 560 nm long-pass filter, while for reduced DHE measurement it was excited at 351 and measured at 435–485 nm. Cells were treated accordingly and measured at the same time to minimise variability of fluorescence measurements. Individual cells were marked and mean fluorescence of individual cells measured by the ImageJ software ; where the image was captured with Z-stack, z-projection was performed using max intensity and net fluorescence was obtained by subtracting the background fluorescence. Mitochondrial morphology was measured on the circularity and aspect ratio from the TMRM images by ImageJ as described . Stable clones were isolated following selection with puromycin  at 4 μg/ml and characterised by analysis of GCase activity, actin-normalised GBA mRNA by a ‘StepOne’ QPCR machine  using SyBr Green  and appropriate primers for GBA and β-actin  and GCase protein levels (by Western blotting). Clones were assessed after several passages (in the presence of a maintenance dose of 2 μg/ml puromycin) to check for the continuation of any knockdown effect.SHSY-5Y cells were transfected with a ‘Hush’ GBA knockdown plasmid , empty plasmid and scrambled control  and by 33% at 30 days (p < 0.05), compared to control. Complex II/III-linked ADP phosphorylation using succinate as substrate was significantly reduced by 30% at 20 days (p < 0.05) and 26% at 30 days (p < 0.05). There was no significant change in complex IV-linked ADP phosphorylation with ascorbate/TMPD as substrate. Basal levels of ATP (determined from time zero acid-treated samples) were 616 ± 149 and 643 ± 270 pmol/million cells, for control and 30 day treated cells respectively. These values represented approximately 1.5–4.5% of the values determined for ADP phosphorylation with glutamate/malate over the incubation period.3.2.2There was no statistically significant effect of CβE on the function of individual isolated respiratory chain complex enzymatic activities 3.2.3Ψm and TMRM fluorescence, while the ATPase inhibitor oligomycin increased Ψm and TMRM fluorescence , 23% at 20 days (p < 0.01) and 28% at 30 days (p < 0.01). We also observed fragmentation of the mitochondrial network at 30 days , normalised to actin as a loading control, showed no significant change with CβE. This indicates that CβE treatment for up to 30 days had no significant effect on mitochondrial content E and F.3.3p < 0.01) and by 71% at 30 days (p < 0.01). Aconitase activity, a measure of free radical mediated damage, was reduced by 60% (p < 0.001) after 30 days incubation with CβE  in alpha synuclein levels in the treated cells, as determined by Western blotting . As shown in GBA caused a significant fall in TMRM fluorescence (p < 0.05) compared to the controls i.e. parental SHSY-5Y and cells stably expressing the scrambled shRNA. There was a 38% reduction (p = 0.003) in aconitase activity (p < 0.067); To confirm the effects of GCase inhibition by CβE, we generated a stable shRNA-mediated knockdown model of activity . An incr3.6Inhibition of lysosomal function by bafilomycin or ammonium chloride produced a different pattern of impaired ADP phosphorylation (bafilomycin) or no effect (ammonium chloride) . These i4GBA mutations have now been reproducibly associated with a substantially increased risk for PD estimated variously as 5 to 20-fold  in PD. Abnormalities of the chaperone-mediated autophagy (CMA) pathway have been demonstrated in PD brain in anatomic regions that map to neuronal degeneration and Lewy body deposition . GBA mutescribed . It is n pathway . Alpha s pathway . It is o pathway . We haveutations . In addiGBA mutations and decreased GCase activity increase the risk for PD by inducing or exacerbating these same abnormalities of mitochondrial function, oxidative stress and alpha-synuclein accumulation to cause or accelerate the development of PD.The results from the present study indicate that inhibition of GCase induces defects in mitochondrial function, increases oxidative stress and confirms previous reports that inhibition of this enzyme increases alpha-synuclein levels. These abnormalities are also found in the PD brain, particularly in the substantia nigra, the main site of neuronal degeneration. We suggest that


The CN class of peptides, derived from the inhibitory region of CaM-KIIN, provides powerful new tools to study CaMKII functions. The goal of this study was to identify the residues required for CaMKII inhibition, and to assess if artificial mutations could further improve the potency achieved during evolution.CaM-KIIN has evolved to inhibit stimulated and autonomous activity of the CaFirst, the minimal region with full inhibitory potency was identified (CN19) by determining the effect of truncated peptides on CaMKII activity in biochemical assays. Then, individual residues of CN19 were mutated. Most individual Ala substitutions decreased potency of CaMKII inhibition, however, P3A, K13A, and R14A increased potency. Importantly, this initial Ala scan suggested a specific interaction of the region around R11 with the CaMKII substrate binding site, which was exploited for further rational mutagenesis to generate an optimized pseudo-substrate sequence. Indeed, the potency of the optimized peptide CN19o was >250fold improved (IC50 <0.4 nM), and CN19o has characteristics of a tight-binding inhibitor. The selectivity for CaMKII versus CaMKI was similarly improved . A phospho-mimetic S12D mutation decreased potency, indicating potential for regulation by cellular signaling. Consistent with importance of this residue in inhibition, most other S12 mutations also significantly decreased potency, however, mutation to V or Q did not.These results provide improved research tools for studying CaMKII function, and indicate that evolution fine-tuned CaM-KIIN not for maximal potency of CaMKII inhibition, but for lower potency that may be optimal for dynamic regulation of signal transduction. CN19 was identified as the minimal region that contains the full inhibitory potency. Mutational analysis showed that the region around R11 of CN19 is of special importance, and that potency of CN19 can be >250fold further increased. Additionally, the results indicated a potential for regulation of CaM-KIINα by phosphorylation (at S12 of CN19).CaM and CaMKII was purified after bacterial or baculovirus/Sf9 cell expression as described 2, 10 mM MgCl2, 100 µM [γ-32P]ATP (∼1 Ci/mmole), 75 µM substrate peptide , and inhibitor peptide as indicated. Reactions with 0.1 nM CaMKII were done for 10 min.Standard reactions (1 min at 30°C) were started by adding CaMKIIα  to a mix of 50 mM PIPES pH 7.2, 0.1% BSA, 1 µM CaM, 1 mM CaClFor all kinase activity assays, reactions were stopped by spotting onto P81 cation exchange chromatography paper (Whatman) squares. After extensive washes in water, phosphorylation of the substrate peptide bound to the P81 paper was measured by the Cherenkov method.2 and CaM. Prior to activity assays, CaMKI (12.5 nM) and CaMKIV (80 nM) were phosphorylated by CaMKKα (1.25–4 nM) for 5 min at 30°C in Ca2+/CaM containing buffer. Additionally, kinase amount, reaction time, and the substrate (obtained from different companies as indicated) was varied as follows: 2.5 nM CaMKIα, 3 min, syntide 2 (Genescript); 5 nM CaMKIV, 10 min, CaMK2g 345–358 peptide (Anaspec); 2.5 nM DAPK1 1–363, 3 min, MLC-derived peptide (Anaspec); 5 nM AMPK-α2β1γ1, 10 min, SAMStide ; 5 nM PKA, 10 min, Kemptide (Genescript); 2 nM PKC, 10 min, MARCKS peptide (Anaspec). PKA and PKC were purified from bovine heart or rat brain, respectively. PKA was catalytic subunits only (without regulatory R subunits) and PKC was the constitutively active tryptic PKM fragment; thus, both kinases did not require stimulation for activity. Other kinases were recombinant protein from mouse (CaMKI) or human , of the isoform and/or subunit composition indicated above.Reactions were performed essentially as described for CaMKII. However, for AMPK, 100 µM AMP was added to the reaction. For AMPK, PKC, and PKA, the reaction mix contained 1 mM EGTA instead of CaClWas done in the UC Denver Biophysics Core using a Jasco J815 Circular Dichroism spectropolarimeter. Peptide concentration was 0.25 mg/ml.All pair-wise comparisons were done using a two-tailed t-test, using Excel software (Microsoft). Comparisons of multiple conditions were done by one-way ANOVA with Newman-Keuls multiple comparison post hoc analysis, using Prism software (GraphPad).Previous studies showed that the 21 mer CN21  containsCN peptides and the helical CaMKII auto-inhibitory region interact with the same site on the CaMKII kinase domain, the T-site Truncation to a 19 mer enabled efficient small-scale peptide synthesis in a 96-well format, and thus efficient generation of CN19 mutation series. In an initial screen, we substituted each CN19 residue individually for Ala, and tested the effect on CaMKII inhibition . As expeThe CN19 region most sensitive to Ala substitutions (surrounding R11) contains a Ser (S12) that could be subject to phosphorylation. A phospho-mimetic S12D mutation of CN19 significantly decreased potency of CaMKII inhibition , indicatAs a next step towards improved CN peptides, we compared combinations of the three mutations that individually increased potency of CaMKII inhibition. Indeed, the triple mutant P3A, K13A, R14A (CN19a3) did significantly further increase potency compared to single mutant with the highest potency, R14A . HoweverImportantly, CN19a2 could be combined with S12V  without loss of its potency . Again, Several additional rounds of mutations  did not yield any further improvements over CN19a2v. However, several residues that could be substituted without reduction in potency were identified . This inCN inhibitors are highly selective for CaMKII The initial Ala scan revealed that R11 is the single most important charged residue for potency of CaMKII inhibition. As the minimal consensus sequence for CaMKII substrates is RxxS/T, we reasoned that R11 might provide the R at the −3 position for a pseudo-substrate sequence within CN19 that directly blocks the substrate binding site . Thus, wCN19o inhibited CaMKII with an IC50 of <0.4 nM , and thuAs CN19a2v increased potency not only for CaMKII but also for CaMKI inhibition, we tested if the optimized CN19o also shows such undesired correlation. On the contrary, potency of CaMKI inhibition by CN19o (∼38 µM IC50) was reduced compared to CN19a2v (∼18 µM IC50), and instead similar to CN19 (∼36 µM IC50). Thus, tAdditionally, CN19o was tested for effects on a panel of other kinases . This paFine tuning of CaMKII activity and localization by a complex set of regulatory mechanisms is required for neuronal plasticity underlying higher brain functions. Here, we identified and characterized the minimal inhibitory region (CN19) of the neuronal CaMKII-regulatory protein CaM-KIINα. The region around R11 of CN19 was especially important for potency of CaMKII inhibition. S12 was sensitive to substitutions with most other residues, including phosphomimetic S12D mutation, indicating a possible mechanism for dynamic regulation by phosphorylation in response to neuronal stimulation. Remarkably, by combining random and rational mutation strategies, it was possible to increase CN19 potency >250fold, thereby generating a much improved tool (CN19o) for studying CaMKII functions. With an IC50 of <0.4 nM, the dose required for efficient inhibition is no longer limited by the concentration of CN19o, but by the amount of CaMKII.CN19 is the minimal inhibitory region of CaM-KIINα with full potency, as CaMKII inhibition was significantly reduced only by further truncation. CN19 consists of over 40% charged residues, which appeared to indicate that inhibition involves strong electrostatic interaction. However, only substitution of R11 decreased potency by >3fold, while substitution of K13 and R14 even increased potency. By contrast, substituting any of the three long hydrophobic residues decreased potency, two of them (L6 and I9) by >3fold. Overall, the region around R11 (i.e. I9-S12) contributed most to CaMKII inhibition, indicating that R11 may constitute the −3 position R in a pseudo-substrate interaction. Indeed, by far the greatest increase in CN19 potency was achieved by engineering an optimized CaMKII pseudo-substrate sequence around R11: The optimized CN19o had >250fold increased potency. Selectivity of CaMKII vs CaMKI inhibition was similarly increased, and is almost 100,000fold for CN19o. High selectivity for CaMKII was further corroborated by lack of CN19o effects on a panel of other related kinases , even at 5 µM .A recent crystal structure of CaMKII-bound CN21 2+/CaM stimulus, dissociation of CaM reverses only binding to regular substrates but not to CaM-KIINα If CaMKII inhibition by CN peptides involves a pseudo-substrate interaction, why is the inhibitory mechanism non-competitive with regular substrates A database search (protein blast) revealed that CaM-KIIN homologues are found in mammals, birds, frogs, and fish. At first glance, it seems unlikely that one could significantly improve on >300 M years of evolution in the laboratory. Upon more careful consideration, this is much dependent on how one defines “improvement”. Obviously, it was possible to dramatically enhance potency of CN19. Thus, evolution has fine tuned CaM-KIIN not for maximal potency of CaMKII inhibition, but for a lower potency that may be sufficient for effective CaMKII inhibition and may additionally allow better dynamic control of CaMKII activity. Indeed, the inhibitory region of CaM-KIINβ is identical from zebra fish to humans, indicating evolutionary pressure also against mutations that further increase potency of CaMKII inhibition. The inhibitory region of CaM-KIINα (CN19) may have appeared later in evolution, and is identical in mammals and birds. The only difference to the inhibitory regions of CaM-KIINβ is a single Ala to Ser substitution (S12 in CN19). This generates the potential for dynamic control directly regulated by cellular signaling, as our results indicate that S12 phosphorylation would interfere with CaMKII inhibition. The only other known mechanism to regulate CaM-KIIN is control of its expression, which indeed occurs in response to learning Clearly, CaM-KIIN can participate in the fine tuning of the CaMKII activity required for regulation of synaptic plasticity that underlies higher brain function. Additionally, CaM-KIIN may curb over-activation of CaMKII in pathological glutamate signaling that causes neuronal damage after stroke. Indeed, CN peptides were neuroprotective even when applied after glutamate insults in culture Figure S1CN19 is the minimal CaMKII inhibitory region of CaM-KIINa with full potency. CN21 and CN19 (ΔC2) showed the same IC50 (∼1 mM) for inhibition of CaMKII activity towards the T-site binding substrate AC2 (40 µM); further truncation at the C-terminus or any truncation at the N-terminus significantly reduced CaMKII inhibition (p<0.001).(TIF)Click here for additional data file.Figure S2CN19 peptides are largely unordered.A, Circular dichroism indicated that CN19 (0.25 mg/ml) is largely unordered; no significant content of α-helix of β-sheet structure was detected Click here for additional data file.Figure S3Effects of S12 mutations in CN19a3 and a2 on CaMKII inhibition.A, The CN19a3 S12R mutation retained potency of CaMKII inhibition, but the S12A, G, P, or N mutations did not. B, The CN19a2 S12V mutation retained potency of CaMKII inhibition, but the S12F, L, or K mutations did not. Error bars indicate s.e.m. in all panels.(TIF)Click here for additional data file.Figure S4Effects of further CN19a3 and a2 mutations on CaMKII inhibition.A, The CN19a3 mutations K5R, G7R and I9L retained potency of CaMKII inhibition, but the mutations K1R, L6R, Q8R or I9R did not. The A3R mutation also lowered CaMKII inhibition of CN19a3, however, this R mutation still increased potency compared to the original P3 in CN19. B, The CN19a2 mutations G7K and Q8R retained potency of CaMKII inhibition. So did the combination mutant 3,5,7,12R, but subsequent additional mutations D19A, I17R, and D19R reduced CaMKII inhibition. Error bars indicate s.e.m. in all panels.(TIF)Click here for additional data file.Figure S5CN19, CN19a2v, and CN19o inhibit CaMKI, but with 360–100,000fold less potency compared to CaMKII. The IC50 for CaMKI inhibition of CN19 (36 µM), CN19a2v (18 µM) and CN19o (38 µM) 360–100,000fold greater than their IC50 for CaMKII inhibition Click here for additional data file.Figure S65 nM CN19o blocked CaMKII activity almost completely, while the same concentration of CN19a2v only mildly reduced CaMKII acitiviy (to ∼75%) and CN19a2v V15I left some residual CaMKII activity (∼10%). Error bars indicate s.e.m.(TIF)Click here for additional data file.