The expression of c-Fos defines brain regions activated by the stressors hypotension and glucoprivation however, whether this identifies all brain sites involved is unknown. Furthermore, the neurochemicals that delineate these regions, or are utilized in them when responding to these stressors remain undefined. Conscious rats were subjected to hypotension, glucoprivation or vehicle for 30, 60 or 120 min and changes in the phosphorylation of serine residues 19, 31 and 40 in the biosynthetic enzyme, tyrosine hydroxylase (TH), the activity of TH and/or, the expression of c-Fos were determined, in up to ten brain regions simultaneously that contain catecholaminergic cell bodies and/or terminals: A1, A2, caudal C1, rostral C1, A6, A8/9, A10, nucleus accumbens, dorsal striatum and medial prefrontal cortex. Glucoprivation evoked phosphorylation changes in A1, caudal C1, rostral C1 and nucleus accumbens whereas hypotension evoked changes A1, caudal C1, rostral C1, A6, A8/9, A10 and medial prefrontal cortex 30 min post stimulus whereas few changes were evident at 60 min. Although increases in pSer19, indicative of depolarization, were seen in sites where c-Fos was evoked, phosphorylation changes were a sensitive measure of activation in A8/9 and A10 regions that did not express c-Fos and in the prefrontal cortex that contains only catecholaminergic terminals. Specific patterns of serine residue phosphorylation were detected, dependent upon the stimulus and brain region, suggesting activation of distinct signaling cascades. Hypotension evoked a reduction in phosphorylation in A1 suggestive of reduced kinase activity. TH activity was increased, indicating synthesis of TH, in regions where pSer31 alone was increased  or in conjunction with pSer40 . Thus, changes in phosphorylation of serine residues in TH provide a highly sensitive measure of activity, cellular signaling and catecholamine utilization in catecholaminergic brain regions, in the short term, in response to hypotension and glucoprivation. Hypotension and glucoprivation are two physical stressors. Hypotension unloads baroreceptors, which leads to the activation or inhibition of a number of brain sites in an attempt to restore arterial pressure (AP). Similarly, glucoprivation, which results in low effective plasma glucose concentration, leads to the activation of centrally mediated counter regulatory events in order to restore plasma glucose levels. The brain sites activated by these stimuli have been demonstrated using the expression of c-Fos to mark activated neurons. With respect to hypotension limited brain sites including the nucleus of the solitary tract (NTS), ventrolateral medulla (VLM), A5 cell group, A6 cell group (not in rat), periaquaductal grey (PAG), central amygdaloid nucleus (CeA), supraoptic nucleus (SON), lateral hypothalamic area (LHA), paraventricular nucleus (PVN), and bed nucleus of the stria terminalis (BNST) are activated in vitro conditions 32P incorporation in vivo or in situ conditions The approach taken here was to determine the involvement of the catecholamines  within the brain, in response to hypotensive and glucoprivic stress. Multiple catecholamine cell groups are found within the brain, each capable of synthesizing one of the three catecholamines. The end product in each cell group depends upon the synthetic enzymes present. Tyrosine hydroxylase (TH) is the initial and rate limiting enzyme for all catecholamine biosynthesis. We will take advantage of the short term regulation of TH via phosphorylation of three serine residues at the N-terminal domain that can directly or indirectly result in increased activity of the enzyme Specific kinases are responsible for the phosphorylation of the three serine residues on the N terminus of TH In the adrenal medulla, another site of catecholamine synthesis, catecholamine release induces TH phosphorylation and enzyme activity increases in order to maintain the catecholamine stores in vivo dialysis, and this powerful technique further provides evidence of the time course of response although only single or at most two central sites can be explored simultaneously. Catecholamines are released in prefrontal cortex and A6 following hypotension Catecholamines could be utilized in the central response evoked by glucoprivation or hypotension. Following hypotension or glucoprivation the medullary C1 cell group expresses c-Fos Thus, the major aims of this study were toDetermine, at ten brain sites simultaneously, the response to acute hypotension or glucoprivation by identifying the changes in phosphorylation at Ser19, Ser31 and Ser40 in TH in order to determine the involvement of catecholamines and catecholaminergic structures in mediating the counter regulatory responses.Compare and contrast the brain sites and phosphorylation patterns activated/inhibited by hypotension or glucoprivation at two time points 30 and 60 min post stimulus.Using the data generated in combination with previous literature we can also deduce information related to:The intracellular signaling processes that result in phosphorylation of TH and/or release of catecholamine.The neural substrate  responsible for the changes in TH phosphorylation evoked.We have demonstrated that changes in phosphorylation of TH were evoked in multiple and distinct brain regions dependent upon the stimulus. The approach used here is more sensitive than other methods for detecting activation as sites not previously described are activated, the effects can be detected earlier, a time course of response can be determined, the effect is neurochemically selective, activation of terminal fields alone can be identified and bidirectional changes in phosphorylation can be detected. In addition, the utilization of catecholamines in some brain regions mediating baroreceptor reflex function and the response to glucoprivation is indicated.The administration of hydralazine , a potent vasodilator, caused a persistent hypotension, which evoked a baroreflex mediated increase in HR  as descrUsing phospho-specific antibodies, the level of TH phosphorylation at specific serine residues was determined in 10 brain regions, 30 min after HDZ (n = 5–6), 2-DG (n = 5–6) or saline (n = 5–6) administration using western blot analysis. Recombinant rat TH (rTH) specifically phosphorylated to full stoichiometry at Ser40, or Ser31, or Ser19 (see methods) and also non-phosphorylated recombinant rat TH were run on each blot. In the A2 cell region no changes in phosphorylation at any serine sites were seen 30 min after either stimulus .The C1 cell group was analyzed in two parts, the caudal C1 and rostral C1 cell regions and effects were determined at both 30 and 60 min following administration of HDZ, 2DG or saline . RepreseIn the caudal C1 region, 30 min following HDZ administration, increases in TH phosphorylation were evoked at Ser40 , 30 min following HDZ administration TH phosphorylation was not changed at any serine residue , whereasIn the medial prefrontal cortex, 30 min after HDZ treatment TH phosphorylation was increased at Ser31  TH phosphorylation changes were identified in discrete brain regions following hypotensive or glucoprivic stimuli, with some regions activated by both, some by only one and some by neither stimulus, 2) hypotension evoked phosphorylation changes were seen in multiple brain regions, whereas glucoprivic evoked effects were more restricted, 3) TH phosphorylation changes were identified in brain regions containing catecholaminergic cell bodies  or terminals alone, 4) increases in TH phosphorylation reflected increases in TH activity in sites containing terminal fields, with or without cell bodies, 5) increases in neuronal activation as measured by c-Fos expression reflected changes in TH phosphorylation in some brain regions, however changes in TH phosphorylation were also a sensitive measure of activation/inhibition in some sites such as the midbrain dopaminergic cell regions that showed no c-Fos expression, 6) different patterns of serine residue phosphorylation were often evoked by the two stimuli in the same brain region, suggesting different levels of neuronal activation and/or cellular signaling, 7) TH phosphorylation at individual serine residues was seen to increase, decrease or not change, 8) the TH phosphorylation changes were, in general, transient with larger changes evident at 30 min compared to 60 min after the stimulus.These findings indicate that changes in site specific TH phosphorylation can be used as a short term marker of neuronal activation at brain sites containing either catecholaminergic cell bodies and/or terminals. Furthermore, select catecholaminergic regions are activated by each acute stimulus and the catecholamine mediated consequences of hypotension are more common than those of glucoprivation. Catecholamines are most likely released or metabolized at some brain sites which contribute to counter regulatory responses evoked by each stimulus. Different TH phosphorylation patterns reflect intracellular signaling cascades evoked by diverse receptor mechanisms in different brain regions.The dose and route of administration of each drug (stimulus) used was based on previous studies where maximal effects were evoked The time points chosen for analysis (20–30 and 50–60 min) were based on our previous studies The phospho-specific TH antibodies used here have been generated, well characterized and tested against specific TH serine residues in vivo. Phosphorylation at Ser19 depends primarily upon the activation of CaMKII in response to increases in levels of Ca2+ which enters the cell after depolarization Ser19 phosphorylation was increased in the caudal C1, rostral C1, A8/9, A10 and medial prefrontal cortex following one or both stimuli. In adrenal cell cultures Ser19 is phosphorylated rapidly and then is dephosphorylated sometimes to below basal levels in vivoin vivo. Salvatore et al (2009) Changes in pSer31TH were seen in the caudal C1, rostral C1, A6, the nucleus accumbens and the medial prefrontal cortex following one or both stimuli. In adrenal cell cultures Ser31 is slow to be phosphorylated and very slow to be dephosphorylated Phosphorylation at Ser40 was only found in caudal C1 and the A10 region. In adrenal cell cultures Ser40 was phosphorylated at times following the phosphorylation of Ser19 and prior to the phosphorylation of Ser31 and was not rapidly dephosphorylated In the present study phosphorylation at Ser31 was often evoked, as were changes in Ser19, with changes in Ser40 least commonly elicited. The ease with which Ser31 is activated in the brain does not appear to depend upon the stimulus used, the brain site activated 2DG evoked changes in TH phosphorylation in a restricted population of brain regions (4 of 10 examined) compared to HDZ (7 of 10 examined). On the other hand c-Fos-ir revealed neuronal activation following 2DG in only 3 regions  and HDZ in only 2 brain regions  when data from the present study and the data of others pertaining to the A1 and A2 regions is considered 2DG evoked dephosphorylation in the A1 region and increased phosphorylation in the caudal C1, rostral C1 regions and the nucleus accumbens.In the A1 region pSer31 was decreased in response to 2DG. This was surprising as c-Fos was detected in some A1 cells Increased pSer19TH was evident in caudal and rostral C1 regions in response to 2DG. This is in keeping with c-Fos activation of C1 cells seen previously 2DG evoked phosphorylation of Ser31 in the nucleus accumbens which must arise from the TH containing terminals. This is in keeping with previous findings where hypoglycemia increased dopamine release in the region Although the increases pSer31TH in A6 evoked by 2DG did not reach significance about 60% of A6 neurons did contain c-Fos-ir. There have been inconsistent reports of c-Fos-ir in A6 following 2DG or hypoglycemia with only a proportion of the TH-ir cells activated, if at all In contrast to that seen following 2DG, a wide range of brain sites showed altered phosphorylation of TH in response to hypotension.Decreases in pSer19, pSer31 and pSer40 TH were seen in the A1 cell group in response to hypotension. This may reflect a reduced activation as c-Fos levels were similar to control when a similar stimulus was used in vivo. The potential sources of adrenaline release are local C1 cells or contralateral C1 Caudal C1 shows the most robust pattern of increased phosphorylation seen in this study with all three serine residues phosphorylated in response to hypotension. Rostral C1 is also phosphorylated at both Ser19 and Ser31. Hypotension evoked c-Fos-ir in caudal and rostral C1 as demonstrated here and elsewhere The noradrenergic A6 region shows an increase in pSer31 but a decrease in pSer19 that could indicate kinase inhibition/phosphatase excitation supporting the finding of little to no c-Fos activation. Previous studies show mixed levels of c-Fos activation following hypotension An increase in TH phosphorylation at Ser19 in A8/9 and at Ser19 in A10 indicates these regions are activated and the increases in Ser 40 in A10 suggest that TH activity is increased, in response to hypotension. It is possible that the increases in pSer19 seen in the A8/9 region may result from contamination by the closely adjacent A10 cells. As chemical activation of the A10 region evokes cardiovascular responses and decreases in baroreflex sensitivity In contrast to other sites in the present study changes in pSer19 did not correlate with c-Fos induction in the A8/9 or A10 regions nor do previous studies indicate that c-Fos is activated in these regions following hypotension. It has been suggested that c-Fos may not be easily induced in midbrain dopaminergic neurons in vivo and reflects catecholamine release. Baroreceptor denervation reduces c-Fos activation in higher brain regions Hypotension evoked increases in both pSer31TH and TH activity in the mPFC suggesting catecholamine release. This must arise from terminal fields of the A6 and/or A10 cell groups Thus, this study demonstrates that the TH phosphorylation profile is a powerful and reliable marker for acute activation in catecholamine containing brain regions including dopaminergic cell groups and regions containing only terminal fields. This has advantages over other methodologies, such as c-Fos, as changes can be detected within 20–30 min of a stimulus, time course data can be determined, both increases and decreases in phosphorylation can be detected and phosphorylation in nerve terminals can be identified. Most importantly, the action measured is of a single neurochemical entity providing chemical specificity. Some evidence for anatomical resolution is provided by immunohistochemical appraisal of the pSerTH as demonstrated here however difficulties associated with quantitative analysis of such data together with small ranges of minimal to maximal stoichiometry may reduce the impact of using immunohistochemical data in isolation. Although other phosphorylated markers including pMAPK and pCREB can be very useful The identification of changes at three serine residues on TH provides information not only regarding neuronal activation (Ser19) but also has the potential to indicate signaling pathways activated (e.g. MAPK or CDK for Ser31) and the probability of catecholamine synthesis/metabolism being activated (if Ser31 and particularly Ser40 are phosphorylated). Ser 19 and Ser31 are the sites most sensitive to phosphorylation in the brain regions examined and although phosphorylation of Ser31 may indicate dopamine turnover at higher brain regions Different patterns of phosphorylation were detected in the same cell group by different stimuli indicating that different levels of activation or perhaps different cell signaling mechanisms are recruited. However some changes detected included directionally opposite effects at different serine residues in the same cell group. This complex pattern raises a challenge to determine what is happening at the cellular level. This not only involves monitoring the three serine residues that demonstrate changes in the activity of the enzyme but also indicates the importance of monitoring the time course of these changes. In the present study only two time points were measured based on previous findings in the adrenal medulla utilizing similar stimuli. Although significant changes were detected at 20–30 min, the earliest time point investigated here it remains a possibility that some phosphorylation changes may be detected earlier.Our findings demonstrate that central responses to glucoprivation and hypotension rely not just on neural pathways containing catecholamine but on the involvement of catecholamines themselves, at least in some brain regions. In future studies it will be important to determine the prevalence of somatodendritic release of catecholamines in the brainstem in response to these stimuli and also to determine in higher brain regions particularly A6, A10 and medial prefrontal cortex that hypotension evoked changes are baroreceptor mediated.. . Phospho-Ser40TH, phospho-Ser31TH, and phospho-Ser19TH specific antibodies were generated and tested for specificity as described by Hydralazine (HDZ), 2-Deoxy-D-glucose (2DG), ethylene glycol tetraacetic acid (EGTA), ethylene diamine tetraacetic acid (EDTA), Tween-20, reduced glutathione and mouse anti-tyrosine hydroxylase antibody  were purchased from Sigma Chemical Co. . Criterion Tris-HCl precast gels (10%) and molecular-weight PAGE standards were from Bio-Rad Laboratories . Nitrocellulose membrane (Hybond ECL) and ECL Advanced kit were from GE Health Care . Anti-rabbit immunoglobulin , anti-mouse immunoglobulin , Cy3 donkey anti rabbit IgG  and DyLight 488 AffiniPure Donkey Anti-Mouse IgG  were from Jackson Immunoresearch Laboratories Inc. . Anti-sheep immunoglobulin  was from Pierce Biotechnology . Rabbit anti-c c-Fos antibody was from Santa Cruz Biotechnology IncAll experiments were carried out with the approval of the Animal Ethics Committees of Macquarie University, Sydney and conducted in accordance with the Australian Code of Practice for the Care and Use of animals for scientific purposes.Animals  rats ) were purchased from the Animal Resource Centre . One day prior to experimentation rats were housed singly in a temperature controlled room (21±1°C) with ad libitum access to food and water. Multiple cages were housed in a single room for a minimum of 12 hours and rats were always within sight, sound and smell of other rats in order to reduce the stress of isolation.−1 i.p.) and ventilated with oxygen during the procedure. The left femoral vein and artery were cannulated for drug administration and AP recording. Supplemental doses of urethane were administered i.v. if required. Core temperature was measured with a rectal probe and maintained at 37°C with a homeothermic blanket and an infra-red lamp. After a stable recording of AP and heart rate was achieved, 2DG (400 mg/kg i.v. n = 3) or HDZ (10 mg/kg i.v. n = 3) was administered.Rats that were anesthetized with 10% urethane , 2DG  or saline , was administered intraperitoneally (0.4 ml) and food and water were removed. Animals were sacrificed 20–30 min  or 50–60 min  following treatment by an anaesthetic overdose, sodium pentobarbital . When rats no longer responded to painful stimuli they were decapitated by guillotine (<5 min) and blood and tissue samples were collected.125I Corticosterone RIA kit  according to the manufacturer’s instructions. Blood glucose was measured immediately using Accu-check performa glucometer from Roche .Whole trunk blood (5 ml) was collected into tubes containing 4 mM EGTA and 4 mM reduced glutathione as described previously . Blood samples were centrifuged for 10 min at 1800 rpm (4°C). Resulting plasma was then spun for 10 min at 2700 rpm (4°C) and then stored at −80°C until analysis. Plasma corticosterone level was measured using an 3. All sites except the VTA and SN were taken from sections 1 mm thick which were taken from the same 3 mm thick section.The brain was rapidly removed from the skull and placed into ice-cold buffer before being placed into a plastic brain matrix . One to 3 mm thick coronal sections were isolated containing the 10 areas of interest based on the rat brain atlas Ten brain regions containing catecholaminergic cell bodies and/or terminals were isolated by microdissection and their dorsoventral and mediolateral dimensions are illustrated in Brain regions were weighed and homogenised, in 20–40 volumes of homogenization buffer ), by sonication, on ice , then boiled for 5 min and centrifuged for 20 min. Supernatants were carefully removed. 100 µl of supernatant was mixed with 5 µl of 10% dithiothreitol (DTT) and 35 µl of sample buffer . 25 µL of each sample was run on Criterion Tris-HCl precast gels (10%) and then transferred to nitrocellulose as described previously 3H] tyrosine and 100 µM tetrahydrobiopterin, final volume 50 µl. Assays were performed for 20 min at 30°C and were stopped by addition of 700 µl charcoal slurry . Unbound 3H2O was analysed by scintillation spectrometry  for 20 min per sample. The changes in TH activities were expressed as a fold increase of the mean of the basal samples.The caudal C1  and mPFC  were homogenised in homogenization buffer . Samples were sonicated 3 times×30 seconds at 4°C, using a micro-sonicator, and then centrifuged for 20 min at 16,000 rpm. Supernatants were collected and protein levels were determined using a BCA protein assay kit  according to the manufacturer’s instructions. TH activity was measured using a method based on the tritiated water release assay with minor modifications To detect c-Fos protein animals were administered HDZ , 2DG  or saline (n = 3) and left for 2 hours in a quiet environment and deprived of food and water. To detect phospho-TH animals were administered HDZ  or saline (n = 2) and left for 30 min. All animals were anesthetized with sodium pentobarbital  and perfused trans-cardially with 300 ml ice-cold saline followed by 400 ml ice cold fixative solution . The brain was removed rapidly and stored in the same fixative . The following day, the brain was blocked and serial sections  were cut on a vibrating microtome  into ice-cold 0.1 M PBS. After three 30 min washes in 0.01 M tris phosphate buffered saline (TPBS), sections were incubated in a solution containing 1∶4000 mouse anti-tyrosine hydroxylase antibody and 1∶1000 rabbit anti-c-Fos antibody with 5% normal horse serum in TPBSm or sections were incubated in a solution containing 1∶1000 of rabbit anti- pSer19TH or 1∶2000 rabbit anti-pSer31TH antibody, for 48 hrs at 4°C. After washing three times for 30 min each in TPBS, sections were then incubated overnight at 4°C in a solution containing 1∶500 Cy3 donkey anti rabbit IgG or together with 1∶500 DyLight 488 Donkey Anti-Mouse IgG with 2% NHS in TPBSm. The next day sections were washed 3 times for 30 min, and mounted onto glass slides and coverslipped with ProLong Gold .For analysis of c-Fos and TH immunoreactivity anatomically matched sections for each brain region were analyzed for all animals in each treatment group . Optical sections of each region were taken using a Zeiss Z2 microscope  at 2 µm intervals (in the z direction) using filters selective for each fluorophore. Images were then analyzed and the number of cells showing immunoreactivity for TH were counted and assessed for immunoreactivity for c-Fos. For phospho-specific TH immunoreactivity, anatomically matched sections were photographed. Further analysis was not attempted as immunohistochemistry cannot be reliably used for quantitative assessment.Statistical analyses were performed using Student’s unpaired t-tests for single comparisons. Differences were considered to be significant at the p<0.05 level.


Phosphorylated forms of microtubule-associated protein tau accumulate in neurofibrillary tangles in Alzheimer's disease. To investigate the effects of specific phosphorylated tau residues on its function, wild type or phosphomutant tau was expressed in cells. Elevated tau phosphorylation decreased its microtubule binding and bundling, and increased the number of motile tau particles, without affecting axonal transport kinetics. In contrast, reducing tau phosphorylation enhanced the amount of tau bound to microtubules and inhibited axonal transport of tau. To determine whether differential tau clearance is responsible for the increase in phosphomimic tau, we inhibited autophagy in neurons which resulted in a 3-fold accumulation of phosphomimic tau compared with wild type tau, and endogenous tau was unaffected. In autophagy-deficient mouse embryonic fibroblasts, but not in neurons, proteasomal degradation of phosphomutant tau was also reduced compared with wild type tau. Therefore, autophagic and proteasomal pathways are involved in tau degradation, with autophagy appearing to be the primary route for clearing phosphorylated tau in neurons. Defective autophagy might contribute to the accumulaton of tau in neurodegenerative diseases. Several neurodegenerative diseases, collectively termed tauopathies, are characterized by intraneuronal inclusions of straight or paired helical filaments of insoluble, highly phosphorylated tau . InteracIn Alzheimer's disease, tau is highly phosphorylated, and many of the tau phosphorylation sites targeted by glycogen synthase kinase-3 (GSK-3), the primary candidate kinase for tau phosphorylation, are also phosphorylated in insoluble tau isolated from Alzheimer brain , 2009. CBecause, in addition to affecting its transport, tau phosphorylation affects its ability to be degraded , we examIn this study, we have used mutant forms of tau, which mimic permanent states of phosphorylation and dephosphorylation, to investigate tau function. In particular, we have examined the effect of tau phosphorylation on its rate of axonal transport and its degradation by the UPS and autophagy. We show here that phosphomimic tau binds less to microtubules and exhibits an increased number of motile tau particles in axons compared with wild type tau. We also show that, in primary cortical neurons, exogenously expressed phosphomimic and wild type tau are targets for autophagy, and endogenous tau is not. In the absence of autophagy, degradation of tau by the UPS in neurons is minimal.22.1Plasmids expressing the longest human central nervous system tau isoform  and 2 tau phosphomimic mutants, E18tau and E27tau, with 18 or 27 serine/threonine residues mutated to glutamate, respectively, were gifts from M. Goedert . The tau sequences were cloned into the enhanced green fluorescent protein (EGFP)-C1 vector  to generate a construct in which EGFP was fused to the amino terminus of tau. Site-directed mutagenesis  was used to create an additional EGFP-tagged tau mutant construct encoding A18tau in which the same 18 serine/threonine residues that were mutated in E18tau were substituted by alanine . Thus, A2.2Embryonic day 18 (E18) rat cortical neurons were cultured in Neurobasal medium , supplemented with B27 (Invitrogen), as described previously . Chinese2.32 perfusion on the stage of an Axiovert 200M Zeiss microscope. Epifluorescence illumination was attenuated to 8% using neutral density filters. Images were collected at 1–2-second intervals for 2–4 minutes using a Plan-ApoChromat 100× 1.4 NA oil immersion objective (1 pixel = 0.063 μm), a green fluorescent protein filter set and an AxioCam MRm camera. Movement of fluorescent particles was analyzed using AxioVision software 4.6.3 (tracking module). Fluorescent particles that moved <0.3 μm between consecutive frames were considered to be pausing . Statistical analyses were performed using a 2-tailed Student t test.E18 rat cortical neurons (5 days in vitro [DIV]) were transfected with plasmids expressing WTtau, E18tau, E27tau or A18tau, and time-lapse images were recorded after 48 hours. Coverslips were placed in a sealed chamber with Neurobasal medium containing B27 and maintained at 37 °C with 5% CO2.4Transfected CHO and MEF cells were fixed in 4% (wt/vol) paraformaldehyde 24 hours after transfection. All incubations were carried out at room temperature. Cells were blocked using 1% (wt/vol) Triton X-100, 10% (vt/vol) fetal calf serum in phosphate buffered saline (PBS) for 20 minutes, and then stained for α-tubulin  for 1 hour. The anti-mouse Texas Red-coupled secondary antibody was added for 1 hour in the dark. Nuclear staining was performed using Hoescht 33342 (5 μg/mL bisbenzimide in PBS). Fluorescence microscopy was performed using an Axioskop microscope (Zeiss), equipped with a camera  and with Plan-NeoFluor 20× 0.50 NA, 40× 0.75 NA, and 100× 1.30 NA objectives.2.56 neurons per well, were rinsed with PBS at 4 °C and cells were scraped into hot (2×) Laemmli sample buffer. Proteins were separated on 10% (wt/vol) sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were probed with antibodies to tau , β-actin , S6  or P-S6 . For LC3 analysis, proteins were separated on 15% (wt/vol) gels and blots were probed with LC3 antibody . Antigens were visualized using secondary antibodies coupled to infra-red dyes and an Odyssey scanner (Li-Cor Biosciences).For protein analysis, 6-well plates containing 1 × 102.62, 1 mM ethylene glycol-bis(2-aminoethyl)-N,N,N′,N′-tetraacetic acid, 0.5% (wt/vol) Triton X-100 and 30% (vol/vol) glycerol), containing 1 mM phenylmethylsulfonylfluoride, Complete protease inhibitor (Roche), 0.5 μM okadaic acid , and 10 μM taxol (Sigma). Cell suspensions were centrifuged at 5000g for 10 minutes at room temperature and an aliquot of the supernatant was retained as the postnuclear lysate (input). The remaining postnuclear lysate was centrifuged at 100,000g for 1 hour at room temperature. The supernatant (unbound) was retained and the pellet (microtubule-bound) was rinsed twice and resuspended in PIPES buffer. The proteins in each fraction were separated on 10% (wt/vol) SDS-polyacrylamide gels and blots were probed with the tau polyclonal antibody (DAKO).Assays for microtubule binding of tau were performed as described previously , with so2.7Rat cortical neurons (5 DIV) were transfected with constructs expressing EGFP-tagged WTtau or phosphomutant tau. After 48 hours, neurons were lysed and EGFP-tau was immunoprecipitated using an agarose-conjugated polyclonal antibody to GFP , as described previously . Immunop2.8g for 1 minute at 4 °C, washed 3 times with modified RIPA buffer, and resuspended in Laemmli sample buffer containing 40 mM dithiothreitol. GST-bound proteins were analyzed on Western blots probed with an antibody to total tau (TP70) (Purified recombinant WTtau and E27tau proteins were prepared as described . Glutathu (TP70) .33.1We first analyzed the interaction of wild type and phosphomutant tau with microtubules. Tau constructs included: (1) the longest human central nervous system tau isoform ; (2) mutant E18tau, in which serine or threonine residues are mutated to glutamate at 18 sites to mimic a permanent state of phosphorylation; (3) mutant E27tau, with 27 serine and threonine sites mutated to glutamate; or (4) A18tau, in which alanine replaces serine or threonine to preclude phosphorylation at these sites  Fig. 1)Fig. 1).t test, p < 0.01). Low expression of A18tau resulted in bundled microtubules in 84% of the cells, compared with only 26% of the cells containing bundles with WTtau. Low expression of either of the 2 phosphomimic tau mutants resulted in negligible microtubule bundling , in agreement with earlier in vitro findings . The results support previous findings that binding of tau to microtubules is dependent on the phosphorylation state of tau  WTtau particles per axon (n = 106) and E27tau  were detected, both of which were statistically significantly different from WTtau . These results demonstrate that the frequency of motile particles of phosphomimic tau was approximately 3–5-fold greater than that of WTtau .Examination of individual axons harboring motile tau particles showed that fewer motile particles of WTtau were present in the same axon, compared with each of the tau phosphomimics . Thus, tThese results indicate that, based on the increased number of motile tau puncta, phosphorylation of tau significantly increases its probability of being transported in neurons and that tau axonal transport is hindered by dephosphorylation. However, the likelihood of movement of phosphomimic tau puncta did not appear to influence their kinetic parameters. Furthermore, this mechanism could help to explain in part the reduced overall rate of slow axonal transport of tau observed after tau dephosphorylation in the presence of lithium, that we observed previously with these phosphomutant forms of tau .3.4p = 0.077) and the phosphomimic tau mutants bound a similar amount of kinesin-1 as WTtau.We have previously shown that in vitro phosphorylation of recombinant tau by GSK-3 increases the ability of tau to bind to the anterograde motor protein, kinesin-1 . To deteBecause kinesin and tau bind microtubules, it is possible that tau could coprecipitate with kinesin through its binding to microtubules, rather than through a direct interaction with tau. Therefore, we investigated this interaction in vitro using beads containing GST alone, or fused to kinesin light chain (KLC) 1 or KLC2, to pull down recombinant WTtau or E27tau in the absence of microtubules. Beads linked to GST-KLC1 or GST-KLC2, but not GST only, pulled down substantial amounts of WTtau C, howeve3.5Autophagy has been implicated as a potentially important process in tau degradation, with some reports showing an influence of tau phosphorylation on autophagic clearance . To detep < 0.05, 1-way analysis of variance). E27tau and A18tau also accumulated with 3-MA treatment, but these increases were similar to that of WTtau , which blocks autophagy by targeting phosphoinositide 3-kinase (PI3K) . Neuronaof WTtau B. These 3.6+/+ and autophagy-deficient Atg5-/- cells were probed on blots with an antibody to LC3. As expected, LC3-I was present in both cell lines, but LC3-II was undetectable in Atg5-/- MEFs because of the absence of autophagy . The estimated half-lives for WTtau, A18tau, E18tau, and E27tau in MEFs were approximately 16, 18, 21, and >24 hours, respectively. These results are in line with those obtained from 3-MA-treated neurons expressing E18tau . However, phosphomimic tau particles are indistinguishable from WTtau with respect to their velocity and the number and duration of pauses. This finding suggests that, when the molecular motor complex binds to tau, its phosphorylation state does not influence transport kinetics. We did not detect any movement of the nonphosphorylatable A18tau, possibly because of its increased microtubule binding. Notably, we found that coexpression of WTtau and E27tau in neurons resulted in their codistribution, although a proportion of E27tau puncta did not overlap with the generally more diffuse axonal WTtau. A plausible explanation for this finding is that phosphorylated tau has a reduced ability to bind to microtubules, which could result in accumulation of E27tau.We examined the axonal transport kinetics of tau because regulation of microtubule binding and tubulin stabilization are important for the axonal transport of multiple cargoes . InteresThe increased number of motile phosphomimic tau particles observed in the axonal transport analysis led us to investigate changes in tau degradation that could explain the increase of pseudophosphorylated tau protein. The autophagic and proteasome systems have been linked to tau degradation and we decided to study the contribution of both pathways to tau accumulation. Autophagosome dynamics in primary neurons has been described by The role of autophagy in neurodegeneration is controversial, with some reports concluding that inhibiting autophagy is neuroprotective , and othIn contrast to the lack of effect of autophagic inhibition on endogenous tau, exposure of neurons to 3-MA resulted in the accumulation of exogenously expressed wild type and phosphomutant tau. 3-MA induced the accumulation of all forms of exogenously expressed tau, with E18tau increasing 3-fold relative to WTtau. This finding implies that there might be conformational differences between the 2 tau phosphomimics, which could result in differential recognition of phosphorylated forms of tau as a substrate for degradation. Our findings support and extend reports of the effects of 3-MA on wild type and phosphomutant tau in cells  and suggTo determine the effect of the proteasome on tau in neurons, in conditions in which autophagy is inhibited, we treated cultured neurons with cycloheximide to prevent synthesis of proteins involved in early autophagosome formation and hence measure primarily proteasomal degradation. In conditions when autophagy is inhibited, tau degradation was minimal after 24 hours, in line with its reported half-life of approximately 60 hours in HT22 cells . All forInterestingly, the effect of autophagy inhibition using 3-MA is not recapitulated in neurons expressing the phosphomutants and treated with cycloheximide, which also inhibits autophagy. A possible explanation for this is that cycloheximide inhibition of autophagy might be secondary to blockage of protein synthesis . Treatme-/- MEFs are dependent for proteolysis on cellular mechanisms other than autophagy, and proteins are therefore eliminated by the proteasome and/or by calpain-mediated degradation. Even when highly expressed, WTtau, E18tau, E27tau, and A18tau did not form aggregates in Atg5-/- MEFs, suggesting that inhibition of autophagy might not have a marked effect on tau accumulation in these cells. However, there was a significant reduction in the rate of degradation of the tau phosphomutants compared with WTtau, indicating that increased phosphorylation might reduce tau degradation by the UPS and/or calpain, possibly directing tau toward an autophagic route.The effect of proteasome inhibition on tau in cultured embryonic neurons is also somewhat controversial. Inhibiting the proteasome has been reported not to affect the amount of tau in neuronal cells . In contExtrapolation of these results to neurons undergoing neurodegeneration, in which tau phosphorylation plays a key role, suggests a mechanism whereby highly phosphorylated tau could result in abnormal accumulation of cytoplasmic tau and a cascade of secondary events that might contribute to neurotoxicity and cell death. For example, increased steady-state amounts of phosphorylated cytoplasmic tau could overload kinesin-based molecular motor transport mechanisms, thereby blocking or reducing the movement of other cargoes, including mitochondria and vesicles. Thus, it is plausible that increased axonal transport could contribute to the mislocalization and accumulation of tau, and thereby participate in the spreading of tau pathology in neurodegenerative disease. Secondly, increased phosphorylation could lead to the removal of tau from the plasma membrane, and potentially to disruption of intracellular signaling pathways . FinallyWe conclude that in autophagy-deficient mouse embryonic fibroblasts, but not in neurons, proteasomal degradation of phosphomutant tau is reduced compared with wild type tau. Autophagic and proteasomal pathways are involved in tau degradation, with autophagy appearing to be the primary route for clearing phosphorylated tau in neurons. Enhancing the autophagic pathway might have potential as a novel therapeutic approach for the removal of damaging, highly phosphorylated and aggregated proteins in Alzheimer's disease and other neurodegenerative disorders.The authors declare no conflicts of interest.All animal work in this study was performed following UK Home Office guidelines.


Scaffold proteins modulate cellular signaling by facilitating assembly of specific signaling pathways. However, there is at present little information if and how scaffold proteins functionally interact with each other.Here, we show that two scaffold proteins, caveolin-1 and IQGAP1, are required for phosphorylation of the actin associated pool of extracellular signal regulated kinase 1 and 2 (ERK1/2) in response to protein kinase C activation. We show by immunofluorescence and proximity ligation assays, that IQGAP1 tethers ERK1/2 to actin filaments. Moreover, siRNA experiments demonstrate that IQGAP1 is required for activation of actin-bound ERK1/2. Caveolin-1 is also necessary for phosphorylation of actin-bound ERK1/2 in response to protein kinase C, but is dispensible for ERK1/2 association with actin. Simultaneous knock down of caveolin-1 and IQGAP1 decreases total phorbol ester-induced ERK1/2 phosphorylation to the same degree as single knock down of either caveolin-1 or IQGAP1, indicating that caveolin-1 and IQGAP1 operate in the same ERK activation pathway. We further show that caveolin-1 knock down, but not IQGAP1 knock down, reduces C-Raf phosphorylation in response to phorbol ester stimulation.Based on our data, we suggest that caveolin-1 and IQGAP1 assemble distinct signaling modules, which are then linked in a hierarchical arrangement to generate a functional ERK1/2 activation pathway. Scaffold proteins facilitate the assembly of signaling cascades by simultaneous binding of several consecutive components of a signaling pathway. By doing so, they regulate speed, specificity, intracellular localization and amplification of signal propagation . ScaffoThe canonical ERK pathway consists of three kinase tiers: Raf, MEK (MAPK and ERK kinase) and ERK. ERK/MAPK scaffolds, in the narrow sense, assemble two or all three tiers of the canonical ERK pathway , thus - when expressed at optimal stoichiometry - facilitating and accelerating ERK activation, but at the same time restricting signal amplification. KSR1, which binds to Raf, MEK and ERK, belongs to this category. Scaffold proteins in the broader sense associate with one or more members of the MAPK pathway within a larger complex or protein platform, such as paxillin, which interacts with Raf, MEK and ERK within the focal adhesion complex . AnotherAlthough much is known about interaction, function and regulation of the various scaffolds, there is at present little information if and how MAPK scaffold proteins functionally interact with each other. Since most studies focus on only one scaffold protein, the available literature concerning scaffold proteins appears to give the impression that most scaffolds function autarkically, i.e. independently of other scaffolds.In smooth muscle, ERK1/2 activation can lead to different signaling outcomes ranging from proliferation to contraction, depending on the stimulus. In an effort to unravel stimulus-specific ERK1/2 signaling, we have recently shown that ERK1/2 is divided into subfractions in aortic smooth muscle cells, and that these subfractions respond differently to distinct signaling cues . In partHere, we hypothesized that in addition to caveolin-1, a second scaffold protein is necessary to maintain ERK1/2-actin association during PKC stimulation. In the present study, we identify the actin-binding IQGAP1 as the ERK1/2 scaffold that targets ERK1/2 to the actin cytoskeleton. Our data show that for phosphorylation of actin-associated ERK1/2 in response to PKC activation, both caveolin-1 and IQGAP1, in a serial arrangement, are required. Thus, our results demonstrate that the hierarchical nature of scaffolding is an important concept to consider in understanding signaling pathways.We have recently shown that an actin-associated fraction of ERK1/2 is activated in a stimulus-specific manner in vascular smooth muscle cells . MoreoveWe next hypothesized that stimulus-specific activation of ERK1/2 is mediated by scaffold proteins. To identify possible changes in ERK1/2 interaction with its scaffolds during stimulation, we carried out endogenous ERK1/2 co-immunoprecipitation experiments with unstimulated, DPBA- or FCS-stimulated cells, and looked for changes in co-precipitation of several known ERK1/2 scaffolds including paxillin, KSR1, IQGAP1 and basic calponin. In the cases of IQGAP1 and KSR1, we found changes in ERK1/2 interaction upon stimulation, and added co-immunoprecipitation experiments in the reverse direction (anti-IQGAP1 and anti-KSR) as control  in the immunoprecipitates. In IQGAP1 immunoprecipitates, the pERK:tERK ratio after phorbol ester stimulation reflects the pERK:tERK ratio found in input samples . Four days after transfection, cells were either left unstimulated, or stimulated with DPBA or FCS. Whole cell lysates were analyzed for expression of IQGAP1, KSR1, ERK1/2, phospho-ERK1/2, and GAPDH. Figure We found no effect of KSR1 knock down on phorbol ester or serum induced ERK1/2 phosphorylation Figure B,C. ThisAs an actin binding protein , IQGAP1 To quantify these results, we employed in situ proximity ligation assays (PLA) in siRNA-transfected cells. PLA produces fluorescent dots if pairs of proteins are within 30 nm of each other [Figure We have shown previously shown that a Triton X-insoluble fraction of ERK1/2 moves into the Triton X-soluble fraction after serum stimulation, but not after phorbol ester stimulation . These fAn analogous set of PLA experiments was performed to test for proximity between phosphorylated ERK1/2 and actin. As shown in Figure In the PLA experiments, both IQGAP1 and caveolin-1 are required for ERK1/2 activation in response to DPBA. That IQGAP1 was also required for actin association, suggests that IQGAP1 interacts with ERK1/2 and actin directly and simultaneously, which eliminates the need for any further downstream scaffold protein. Since caveolin-1 knock down, like IQGAP1 knock down, also reduced ERK1/2 activation in response to DPBA, it is therefore most likely positioned upstream of IQGAP1 in this pathway. To further elucidate the hierarchical arrangement of caveolin-1 and IQGAP1, we assayed for activation of Raf. Since C-Raf associates with caveolin-1  and is kKnock down of caveolin-1, IQGAP1, B-Raf and C-Raf reduces activation of cytoskeletal ERK1/2, but not soluble ERK1/2, after DPBA stimulationTo find out to what extent caveolin-1 and IQGAP1 scaffolding activites as well as B-Raf and C-Raf signaling are required for activation of cytoskeletal ERK1/2, we enriched actin-associated ERK1/2 by subcellular fractionation. In these experiments, cells were transfected with siRNA directed against caveolin-1, IQGAP1, B-Raf and C-Raf. Non-targeting control siRNA (Dharmacon) was used as control. Four days after transfection, cells were stimulated with DPBA and then subjected to Triton X-100 (TX) extraction before preparation of soluble and insoluble lysates. Target protein knock down was confirmed by western blots Figure C. ERK1/2IQGAP1 has been demonstrated to bind to B-Raf , whereasBased on our results, we present a model in which caveolin-1 and IQGAP1 act as scaffolds in the same PKC-induced ERK1/2 activation pathway, with caveolin-1 facilitating signaling along the upstream part of the pathway, and IQGAP1 tethering the downstream part of the signaling cascade to the actin cytoskeleton Figure A. After We do not know yet the precise spatiotemporal arrangement of this signaling complex. The possibilities include (1) direct interaction of caveolin-1 and IQGAP1, (2) sequential or (3) simultaneous interaction with the Raf-MEK-ERK cassette, possibly through dimerization of Raf, MEK and/or ERK . Of noteIn the few currently known examples of functional interaction between scaffolds, scaffolds cooperate in a complementary manner, rather than in a hierarchical arrangement as shown here. In the drosophila Hippo pathway for example, activation of the transcription factor Warts (Wts) requires the concerted action of the scaffold proteins Sav and Mats; as another example, the scaffold proteins BNIP-2 and JLP are required simultaneously for activation of p38 by cdc42 in myogenic and neuronal differentiation .Scaffold proteins group signaling proteins into signaling modules. Here, we show how, on a higher level, two scaffold proteins functionally interact to link two signaling modules. Our data show that the phorbol ester stimulated pathway PKC-Ras-Raf-MEK-ERK is broken down into two modules, of which one, PKC-Ras-(C-)Raf, is assembled at the caveolae with the help of caveolin-1, and the other one, (B-)Raf-MEK-ERK, is tethered to the actin cytoskeleton with the help of IQGAP1. Enhanced proliferative activation of ERK signaling is held responsible for the oncogenic effect of Ras or B-Raf mutations, which are found at a high incidence in many types of human cancers -50. As sGeneral laboratory reagents were of analytical grade or better and were purchased from Sigma  and Bio-Rad . For stimulation, fetal calf serum  was used at 10% and 12-deoxyphorbol 13-isobutylate 20-acetate  was used at 3 μmol/L. Duration of stimulation was 5 minutes in all experiments. Since DPBA was dissolved in dimethylsulfoxide (DMSO), which may affect ERK1/2 activation -53, unstA7r5 rat aorta cells  were cultured in DMEM high glucose (Invitrogen) with 10% FCS, 1% glutamine, 50 units/ml penicillin and 50 μg/ml streptomycin. Cells were grown to confluency and incubated in medium containing 0% serum for 24 h prior to all experiments to ensure differentiation of the cells to the smooth muscle-like phenotype ,55. SiRN2 (PEM) buffer . Cells were examined with an Eclipse TE2000-E fluorescence microscope  equipped with a CCD camera and using filters optimized for double-label experiments. Images were optimized for display with Photoshop CS3 software . For proximity ligation assays (PLA), fixed cells were stained with primary antibodies as indicated and subsequently stained essentially according to the manufacturer’s instructions [Cells were fixed and stained as previously described . For imaructions . For eac2, 1 dithiothreitol and 0.5% Nonidet-P40 in a 20 mmol/L sodium phosphate buffer, pH 8.0) or IP lysis buffer  supplemented with protease inhibitor cocktail . Cells were lysed on ice for 30 min. Lysates were cleared by centrifugation .Prior to preparation of cell lysates, cells were either stimulated with DPBA (3 mmol/L) or with FCS (10%) for five minutes, or left unstimulated. To prepare whole cell extracts, plates were washed with ice-cold phosphate buffered saline (pH 7.2) and then scraped off in lysis buffer  were incubated with anti-IQGAP1, anti-KSR1, anti-B-Raf or anti-C-Raf primary antibodies (see “Reagents and antibodies”) cross-linked to Protein G-dynabeads® (Invitrogen) or with anti-ERK1/2 cross-linked to protein A agarose beads (Millipore) at 4°C over night. The immobilized antigen-antibody complexes were washed three times with IP lysis buffer and eluted in sample buffer. Proteins in the samples were separated on 12.5% SDS polyacrylamide gels according to standard procedures. For western blot analysis, proteins on SDS gels were transferred onto nitrocellulose membranes . Bound proteins were detected with specific primary antibodies and appropriate secondary antibodies. Bands were visualized on an Odyssey® infrared imaging system (LI-COR). Densitometry analysis was perfomed on raw data with the Odyssey 2.1 software. For analysis of protein expression, bands of interest were normalized to GAPDH on the same membrane. For analysis of protein phosphorylation, phospho-ERK1/2 and total ERK1/2 were analyzed in parallel on the same membrane, and phospho-C-Raf and total C-Raf were analyzed on duplicate membranes after normalization to GAPDH. For statistical analysis of immunoprecipitation experiments, background signal as detected in control immunoprecipitations was either subtracted from immunoprecipitated protein bands  supplemented with 50 mmol/L NaCl. Cell homogenates were centrifuged at 100,000 g for 1 hour and the supernatant collected as the cytosolic fraction. The pellet was resuspended in buffer A supplemented with 0.5% Triton X-100, extracted at 4°C for 1 hour and centrifuged at 100,000 g for 1 hour. This supernatant was collected as the Triton X-soluble or membrane fraction. The pellet was resuspended in buffer A with 0.5% Triton X-100 and 1.2% SDS, extracted at 4°C for 1 hour and briefly centrifuged. This final supernatant was collected as the Triton X-insoluble or cytoskeletal fraction. Equal volumes of buffers I-III were used. All buffers were supplemented with protease inhibitor cocktail  and homogenates were kept on ice or at 4°C between centrifugations to prevent proteolysis. Triton X soluble and -insoluble fractions were prepared as described previously [2, 4% PEG). Cells were scraped off in prewarmed PEM buffer with 0.25% Triton X-100 and incubated at 37°C for 3 minutes with gentle agitation. After centrifugation (400 rcf at room temperature) for 2 minutes, the supernatant was collected and the pellet was resuspended in sample buffer (= Triton X-100 insoluble fraction). Proteins in the supernatant (=Triton-X soluble fraction) were precipitated over night after adding 2.5 volumes of ethanol. Precipitated protein was pelleted by centrifugation  and resuspended in sample buffer.Subcellular fractionation for differential ultracentrifugation was performed as described previously . Brieflyeviously . BrieflyAll values given in the text and displayed in the graphs are mean ± standard error. Differences between means were evaluated using two-tailed Student’s t-tests. Western blots were analyzed by densitometry using an Odyssey infrared scanner (LiCor). Data from at least four independent experiments were used for statistical analyses. For proximity ligation assay (PLA) experiments, because of the high number of analyzed cells (n=60) significance was taken at the p<0.001 level to minimize type I errors. In all other analyses, differences were considered significant at the p<0.05 level.DPBA: 12-deoxyphorbol 13-isobutylate 20-acetate; ERK1/2: Extracellular signal regulated kinase 1 and 2; FCS: Fetal calf serum; IQGAP1: IQ domain containing GTPase activating protein 1; KSR1: Kinase suppressor of ras 1; MAPK: Mitogen activated protein kinase; PKC: Protein kinase C.The authors declare that they have no competing interests.SV and KGM designed the project, analyzed the data and wrote the manuscript. SV designed the experiments. SV, RP and QQL performed the experiments. All authors read and approved the final manuscript.


The nature of “toxic” tau in Alzheimer’s disease (AD) has been unclear. During pathogenesis, the importance of tau oligomerization vs. tau phosphorylation is controversial and the investigation of both remains critical toward defining the “toxicity” of tau. The phosphorylation of tau on serines and/or threonines occurs early in the disease course and altering phosphorylation has been shown to disrupt neuropathogenesis. We have recently reported that in PC12-derived cells, tau had a role in signal transduction processes activated by NGF. By depleting tau, NGF-induced MAPK activation was attenuated and by restoring tau, MAPK activation was restored. Furthermore, the phosphorylation of tau on Thr231 was required for tau to potentiate MAPK activation. Here we report the effects of additional disease-related tau phosphorylation sites and tau isoform on the ability of tau to potentiate MAPK activation. Our findings, which tested three other sites of phosphorylation, showed that phosphorylation at these other sites mainly lessened MAPK activation; none potentiated MAPK activation. In comparing 0N3R tau to the other five brain tau isoforms, most showed a trend toward less MAPK activation, with only 2N4R tau showing significantly less activation. Since MAPK activation has been reported in AD brain and is characteristic of cell proliferation mechanisms, tau phosphorylation that promotes MAPK activation could promote cell cycle activation mechanisms. In neurons, the activation of the cell cycle leads to cell death, suggesting that abnormally phosphorylated tau can be a toxic species. The relationship between tau oligomerization and its ability to potentiate MAPK activation needs to be determined. Drosophila and C. elegans, tau-induced neurodegeneration occurred in the absence of neurofibrillary tangles  is the most prevalent and it has been suggested that the presence of tau is critical for disease progression , 2. Neur tangles , 9. Most tangles –12 and n tangles –16. HoweTau phosphorylation is required for its neurotoxic effects , 18 and PC6-3 cells  were culMAPK activation was measured by a luciferase reporter assay as described by Leugers and Lee . The abiD5 cells were grown with or without NGF for 30 min and then harvested in RIPA buffer with protease and phosphatase inhibitors . After rTo examine MAPK activation, PC6-3 cells, a PC12-derived cell line , were trTo select tau phosphorylation sites to test, we sought sites that were modified in both AD and in the PC6-3 cells. We chose to examine Ser214, Ser396/Ser404, and Ser199/Ser202, all of which are known to be phosphorylated in AD. While tau phosphorylation in NGF-treated PC12 cells has been examined, NGF treatments greater than 24 h were often used and we were interested in earlier time points as our focus was on signal transduction rather than neurite outgrowth. To assess the phosphorylation of both endogenous rat tau and exogenously expressed human tau (0N3R), we utilized the previously described PC6-3-derived cell line D5, that stably expresses human tau (To examine MAPK activation, we tested a panel of phospho-mimetic mutations at these tau phosphorylation sites. In advance of measuring MAPK activation, protein produced by each plasmid was visualized by western blotting in order to confirm that equivalent amounts of each protein was being expressed in each experiment Figure A. In thiTau mutations that affect the alternative splicing of tau mRNA can result in increased levels of 4R tau and cause neurodegenerative disease . ToLastly, we compared the effects of the phospho-mimicking substitutions on 0N3R and 0N4R tau. Comparing the effects of S202D on 0N3R and 0N4R, we found that the mutation inhibited MAPK signaling to a larger extent in 0N4R, where a significant decrease occurred (compare Figures Our data shows that phosphorylation differentially affected the function of 3R and 4R tau isoforms. This result resembles previous data reported for the interaction between tau and the SH3 domain of Fyn, where we found that phosphorylation differentially affected the equilibrium binding constant of 0N3R and 0N4R tau for the SH3 domain of Fyn . For theDuring development, tau phosphorylation changes, with phosphorylation at Ser199, Ser202, Ser214, and Ser404 first increasing, then decreasing while phosphorylation at Thr231 and Ser396 remained unchanged . In our Our tests have investigated the ability of disease-related tau phosphorylation to affect the ability of tau to upregulate MAPK signaling. Among the sites we have investigated, Thr231 was the site whose phosphorylation occurred earliest during neurodegeneration , 34. PhoThe activation of MAPK can occur in signal transduction pathways where cell proliferation is upregulated . ThIt is not known whether tau participates in signal transduction in adult neurons. Gene expression in the tau-depleted mouse was compared to that of WT mouse, using microarray analysis of 8-week-old mice . In the While it is clear that phosphorylated tau can form oligomers, we have not determined if the tau that upregulates MAPK activation is a monomer, dimer, trimer, or oligomer. Moreover, the mechanism by which tau affects NGF signaling is under investigation. The ability of proteins to form dimers during signal transduction processes is not unusual and in some cases, dimer formation is linked to phosphorylation. It would be important to determine if tau dimerization or oligomerization occurred in the same manner as disease-related phosphorylation, where dimerization or oligomerization would occur normally during development, then become down-regulated in the adult. If tau oligomers are found during normal development, the toxicity of tau oligomers during neurodegeneration may be related to specific tau oligomer functions that were inappropriate or abnormal for adult neurons.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.


BCR-ABL. We hypothesize that treatment with small molecule inhibitors of JAK2 will similarly generate inhibitor-resistant mutants in JAK2.The non-receptor tyrosine kinase JAK2 is implicated in a group of myeloproliferative neoplasms including polycythemia vera, essential thrombocythemia, and primary myelofibrosis. JAK2-selective inhibitors are currently being evaluated in clinical trials. Data from drug-resistant chronic myeloid leukemia patients demonstrate that treatment with a small-molecule inhibitor generates resistance via mutation or amplification of a priori, we utilized TEL-JAK2 to conduct an in vitro random mutagenesis screen for JAK2 alleles resistant to JAK Inhibitor-I. Isolated mutations were evaluated for their ability to sustain cellular growth, stimulate downstream signaling pathways, and phosphorylate a novel JAK2 substrate in the presence of inhibitor.In order to identify inhibitor-resistant JAK2 mutations Jak2 V617F allele, we observed that a subset of mutations conferred resistance to inhibitor, validating the use of TEL-JAK2 in the initial screen. These results demonstrate that small-molecule inhibitors select for JAK2 inhibitor-resistant alleles, and the design of next-generation JAK2 inhibitors should consider the location of mutations arising in inhibitor-resistant screens.Mutations were found exclusively in the kinase domain of JAK2. The panel of mutations conferred resistance to high concentrations of inhibitor accompanied by sustained activation of the Stat5, Erk1/2, and Akt pathways. Using a JAK2 substrate, enhanced catalytic activity of the mutant JAK2 kinase was observed in inhibitor concentrations 200-fold higher than is inhibitory to the wild-type protein. When testing the panel of mutations in the context of the  Myeloproliferative neoplasms (MPNs) are diseases characterized by an excess production of one or more fully differentiated blood cell types, and can be precursors to more severe disorders including myelodysplastic syndrome and acute leukemia The identification of a somatic valine to phenylalanine mutation at residue 617 of JAK2 was made in 90% of PV, 50% of ET, and 50% of PMF patients BCR-ABL fusion gene or a mutation in the kinase domain that prevent small-molecule inhibitor binding in vitro system was developed to identify IM-resistant mutations Chronic myeloid leukemia (CML) is a Philadelphia chromosome-positive MPN characterized by the presence of the t chromosomal translocation in vitroin vitro.Discovery of JAK2 V617F and its role in PV, ET, and PMF started the search for a small-molecule inhibitor for JAK2. More than a dozen inhibitors have since been identified to reduce JAK2 V617F kinase activity The anti-phosphotyrosine antibody 4G10, anti-ERK1/2, and anti-STAT5a/b antibodies were purchased from Upstate Biotechnology . The anti-phospho-ERK1/2 (pY204) and anti-GST antibodies were purchased from Santa Cruz Biotechnology . The anti-phospho-STAT5 (pY694) antibody was purchased from Zymed . The anti-JAK2, anti-phospho-S6 (pS235/236), anti-S6, anti-phospho-Akt (pS473), anti-Akt antibodies were purchased from Cell Signaling . The horseradish peroxidase (HRP)-conjugated protein A, and donkey anti-rabbit-HRP IgG, sheep anti-mouse-HRP IgG antibodies were purchased from GE Healthcare UK .KEYYKVKE; referred to as KEYY) and cloned into pEBG-GST in order to express a GST fusion protein. KEYY, KEYF, KEFY and KEFF were used in substrate optimization experiments with TEL-JAK2. KEYF was used to test the phosphorylation ability of TEL-JAK2 and its associated mutations, whereas KEYY was utilized to test the kinase activity of Jak2 V617F and its associated mutations. KEFF was used as a negative control.Human TEL-JAK2(5-12) and full-length murine Jak2 were cloned into the retroviral expression vector pMPG2. TEL-JAK2(5-12) contains the PNT dimerization domain of TEL fused to the kinase and pseudokinase domains of JAK2. The indicated point mutations in TEL-JAK2 and Jak2 were induced using the QuikChange site-directed mutagenesis kit . The JAK2 substrate was modeled after the activation loop of JAK2 . CEP-701 (Lestaurtinib) was purchased from LC Laboratories . TG101348 was kindly donated by Ross Levine, Memorial Sloan-Kettering Cancer Center, New York, NY.2.BaF3 cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 50 nM β-mercaptoethanol , and 10% WEHI-conditioned medium. BaF3-EPO-R cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 50 nM β-mercaptoethanol, and 0.5 units/mL of human recombinant erythropoietin (EPO). HEK-293T and the HEK-293T-based Phoenix cells were cultured in Dulbecco’s Modified Eagle’s Medium H21 supplemented with 10% heat-inactivated fetal calf serum. All cells were incubated at 37°C with 5% COCells were transfected with Lipofectamine 2000 , according to the manufacturer’s instructions, and 0.1 µg of pEBG and/or 1.0 µg of pMPG2, unless otherwise indicated.BaF3 cells were transduced using the Phoenix cell system as previously described E. coli (Stratagene). A large volume of mutagenized plasmid was isolated from the XL1-Blue strain using a Maxiprep kit . BaF3 cells were cultured and transduced with the mutagenized pMPG2-TEL-JAK2 library (as above). Transduced BaF3 cells were selected in cytokine-free RPMI medium for three days. Cells were then plated at a low concentration in soft agar containing cytokine-free medium plus 1.93 µM JAK Inhibitor-I. Colonies were then isolated and grown in cytokine-free RPMI containing 2.5 µM JAK Inhibitor-I. DNA was isolated using a mammalian genomic DNA extraction protocol. The TEL-JAK2 kinase and pseudokinase domains were sequenced to identify mutations.pMPG2-TEL-JAK2(5-12) was used to transform XL1-Red Competent 2 -CaCl2 PBS). Cells were washed and resuspended in 200 µL lysis buffer ). BaF3 cells were washed once with Hank’s balanced salt solution buffered with 10 mM HEPES and resuspended in 200 µL lysis buffer (as above). Cell lysates were incubated on ice and cell debris was pelleted.HEK-293T cells were gently washed with magnesium and calcium-free phosphate-buffered saline (-MgClHEK-293T cells expressing both pMPG2 and pEBG (Addgene plasmid 2227) were lysed (as above). Glutathione Sepharose 4B beads  were added to the cell lysis solution and incubated overnight with agitation at 4°C. The beads were then washed three times with PBS, and 50 µL of 1×sample buffer  was added prior to SDS-PAGE.Cell lysis and GST pull-down samples were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane. Western blotting was performed as previously described 3 cells/well. BaF3 and BaF3-EPO-R cells expressing TEL-JAK2 or Jak2 V617F, respectively, bearing the indicated mutation were diluted into medium (as above) containing the drug to a total volume of 100 µL/well. Each cell line and mutation was represented in triplicate, with the values averaged for plotting and statistical analysis. Cells were incubated in drug for 48 hours at 37°C. Post-48 hour incubation, 25 µL of pre-warmed XTT solution (diluted in medium: 125 nM Phenazine methosulfate (Sigma); 930 µM XTT ) was added to each well, and cells were incubated at 37°C for an additional eight hours. Absorbance at 450 nm was determined using a 96-well plate spectrophotometer (Molecular Devices Optimax Turntable Microplate Reader).In order to quantify resistance conferred by specific TEL-JAK2 and Jak2 V617F mutations, an XTT assay was performed. All XTT experiments were performed in 96-well plates (Nunc) at an initial concentration of 2×10http://www.rcsb.org/, PDB ID: 2B7A). Structural analysis and image rendering was performed with PyMOL (http://www.pymol.org/).The JAK2 kinase domain structure complexed with JAK Inhibitor-I was obtained through the protein data bank , producing a mutagenized library. TEL-JAK2(5-12), referred to as TEL-JAK2, contains the PNT oligimerization domain of TEL and the kinase and pseudokinase domains of JAK2. BaF3 cells were transduced with the mutagenized TEL-JAK2 library and incubated in soft agar containing 1.93 µM JAK Inhibitor-I. Colonies presumably expressing mutagenized TEL-JAK2 were observed on the plates. One hundred colonies were selected, expanded, and both the kinase domain and pseudo-kinase domain were sequenced. Nine kinase domain mutations . Three additional constructs were generated as controls: PQDKEYFKVKE (GST-J2s-KEYF), PQDKEFYKVKE (GST-J2s-KEFY), and PQDKEFFKVKE (GST-J2s-KEFF). 293T cells were transfected with pMPG2-TEL-JAK2 and one of the four JAK2 substrate variants in order to assess the ability of TEL-JAK2 to phosphorylate the tyrosines within these substrate fusion proteins . TEL-JAK2 stimulates tyrosine phosphorylation of a doublet in GST-KEYY  GST fusion construct to examine Jak2 kinase activity in the presence of JAK Inhibitor-I. 293T cells were co-transfected with a vector expressing Jak2 V617F wild type, G935R, or R975G, and the GST-J2s fusion vector. Cells were treated with JAK Inhibitor-I for four hours and lysed. The JAK2 substrate protein was isolated with glutathione sepharose beads, and probed for phosphorylation . Jak2 V6in vitro prediction of BCR-ABL mutations against multiple inhibitors was robust and provided the field with significant data to aid in the design of second and third generation kinase inhibitors Inhibitor resistance is currently one of the biggest challenges facing effective treatment of CML. Evidence suggests that BCR-ABL mutations are present at the commencement of treatment, and the inhibitor provides strong selective pressure for affected clone outgrowth and consequent patient relapse Identification of a single point mutation, JAK2 V617F, thought to play an important role in MPN development and progression, initiated the search for small-molecule inhibitors of the JAK2 tyrosine kinase. We hypothesized that inhibitor-resistant JAK2 alleles may become apparent as large cohorts of MPN patients progress through clinical trials testing JAK2-selective drug therapies. The objective of our study was to identify JAK2 mutations that provide resistance to small molecule inhibitors before patient relapse is observed in the clinic.TEL-JAK2 is a fusion gene created by the t translocation in vitro kinase activity. For example, TEL-JAK2 V881A had high Erk2 phosphorylation in the absence of JAK Inhibitor-I, but weak kinase activity upon drug addition. We also examined growth ability in the presence of two clinically relevant inhibitors, TG101348 and CEP-701 . We observed only JAK2 V617F G935R to display a striking difference in survival, downstream signaling, and substrate phosphorylation in comparison to the wild-type protein and other mutants. There are at least two possible explanations for this finding. First, the difference may be due to the relative kinase strength of TEL-JAK2 compared to Jak2 V617F. The Jak2 V617F allele is not transforming unless it has a functional FERM domain and is provided with a cytokine scaffold In order to understand how the panel of identified mutations contributes to inhibitor resistance, mutations were modeled using the previously published JAK2 kinase domain crystal structure complexed with JAK Inhibitor-I A recent publication has identified activating JAK1 mutations selected for by cytokine deprivation et al. demonstrated that G935R displayed broad inhibitor-resistance using a wide panel of JAK2-selective inhibitors. Similarly, Y931C  293T cells were transfected with TEL-JAK2 or empty vector and various GST-JAK2 substrate constructs, as indicated. Forty-eight hours post-transfection, cells were lysed, GST fusions were captured on glutathione-sepharose beads, and immunoblotting was performed with anti-phosphotyrosine or GST antibodies. KEXX denotes either KEYY, KEYF, KEFY, or KEFF GST-JAK2 substrate fusion constructs (GST-J2s). The phosphorylation of TEL-JAK2 was confirmed by immunoblotting with an anti-phosphotyrosine antibody. (B) 293T cells were transfected with wild-type Jak2, Jak2 V617F or TEL-JAK2 expression vectors. G935R or R975G mutations were introduced on to each Jak2 backbone. JAK2 expression vectors were co-transfected with GST-J2s vectors as shown. Post-lysis, GST fusion proteins were isolated by glutathione-sepharose and immunoblotting was performed with anti-phosphotyrosine or anti-GST antibodies. Expression of Jak2 constructs was confirmed by performing an immunoblot with an anti-Jak2 antibody.(TIF)Click here for additional data file.Figure S2Human JAK2 and murine Jak1 domain alignment demonstrate clustering of activating/inhibitor-resistant mutations discovered in separate screens. Results from Hornakova et al.et al.et al. (TIF)Click here for additional data file.Table S1Isolated TEL-JAK2 mutations identified in a soft agar screen, with both TEL-JAK2 and Jak2 amino acid numbering. Mutations are indicated on the full-length Jak2 backbone. The precise location of each mutation in TEL-JAK2(5-12) is indicated for reference. The asterisk denotes an engineered mutation (M929 homologous to T315 in BCR-ABL).(DOC)Click here for additional data file.


Although all the enzymatic reactions follow michaelian kinetics, vmax decreased significantly (P < 0.05) and KM increased  with the increase in t. The conformational changes produced in the starch chains as a consequence of the ageing seemed to affect negatively the diffusivity of the starch to the active site of the enzymes and the release of the reaction products to the medium. A similar effect was observed when the enzymatic reactions were carried out with unaged starches supplemented with different concentrations of gelatine [G]. The inhibition in the amylolytic activities was best mathematically described by using three modified forms of the Michaelis-Menten model, which included a term to consider, respectively, the linear, exponential, and hyperbolic inhibitory effects of t and [G].The effect of increasing ageing time ( This inhibition can be overcome with the increase in substrate concentration. The pure noncompetitive inhibitor binds to the enzyme, but at a site away from the active site, producing a reduction in the value of vmax but the KM value is unchanged. In this case, the inhibitor has an identical affinity for the enzyme and the enzyme-substrate complex. Its action changes the shape of the enzyme and thus the active site, limiting the interaction between the enzyme and the substrate. In contrast, a mixed noncompetitive inhibitor has a different affinity for both the free enzyme and the enzyme-substrate complex. The uncompetitive inhibition takes places when the inhibitor binds to the enzyme-substrate complex but not to the enzyme. As a consequence, the values of vmax and KM decreased ),v=vmax·[Kt·[t]),v=vmax· and low values of gelatine concentration [G].The response surfaces  generateconstant . From thThe main contribution of this paper is the description of the kinetics of the amylolytic enzymes San Super, Termamyl, and BAN in starches aged for different times. The results showed that the three enzymes follow a Michelian behaviour with independence of the ageing time of the starch, which acted as an inhibitor of the activity enzymatic. The addition of increasing gelatine concentrations to the reaction mixture inhibited the activity of the amylolytic enzymes in a similar way as that produced by the ageing time. The effect of both inhibitors was successfully modelled by using three modified forms of the Michaelis-Menten model.To our knowledge, it is the first time that the amylolytic activities of these three enzymes are modelled as a function of the increase in substrate concentration and the ageing time or gelatine concentration. The above-described mathematical models could be applied to operate and control the enzymatic hydrolysis of starch by the three amylolytic enzymes in an industrial bioreactor .


Mytilus coruscus, were investigated and the variable factors, protease concentration, hydrolysis time, pH, and temperature, were optimized using Uniform Design, a new statistical experimental method. The results proved that the hydrolysate of alkali proteases had high ACE-inhibitory activity, especially the alkali protease E1. Optimization by Uniform Design showed that the best hydrolysis conditions for preparation of ACE-inhibitory peptides from Mytilus coruscus were protease concentration of 36.0 U/mL, hydrolysis time of 2.7 hours, pH 8.2, and Temperature at 59.5°C, respectively. The verification experiments under optimum conditions showed that the ACE-inhibitory activity (91.3%) were agreed closely with the predicted activity of 90.7%. The amino acid composition analysis of Mytilus coruscus ACE-inhibitory peptides proved that it had high percent of lysine, leucine, glycine, aspartic acid, and glutamic acid.The angiotensin-I-converting enzyme (ACE) inhibitory peptides from mussel,  Hyperte as milk , seed pr as milk , blue mu as milk , bovine  as milk , casein  as milk –11, zein as milk , sardine as milk , and tun as milk . ACE inh as milk , soy sau as milk , and soy as milk . Mytilus coruscus is one of the most important bivalves in both Chinese aquaculture and Chinese market [Mytilus mussel protein, such as Mytilus inhibitory peptides [Mytilus coruscus mussel protein hydrolysate.e market . Like otpeptides , antimicpeptides , and antpeptides . In addipeptides . HoweverUniform Design method, a new experimental technique, is established together by Fang . One of Mytilus coruscus muscle protein. Uniform Design method was applied to investigate the effects of protease concentration, hydrolysis time, hydrolysis temperature, and hydrolysis pH for the ACE inhibitory activity of hydrolysates from Mytilus coruscus.In the present study, we want to optimize the hydrolysis conditions for achieving ACE inhibitory peptides from Mytilus coruscus, was obtained from local aquatic product market . Hippuryl-histidyl-leucine (HHL) was used as substrate of ACE. The HHL and ACE were purchased from local chemical company . Five kinds of proteases (E1 to E5) were purchased from local food additives market . The labeled optimum hydrolysis temperature and pH were shown in Mussel,  Mytilus coruscus, were washed with water to remove salt and other materials. The mussels were filleted and defatted with petroleum ether at 50°C by reflux extraction. Then the mussels were minced and mixed with distilled water (ratio of 1 : 10). The mixture was homogenate and then was boiled for 10 minutes to inactive the inner protease. Then the mixture was digested by five proteases at designed conditions, respectively. The pH of the reaction mixture was maintained stably by addition of either 1 N NaOH or HCl. Then, the mixture was incubated at 90°C for 10 min to terminate the reaction. After centrifugation  for 10 min, the supernatant of the hydrolysate was collected for test the ACE inhibitory activity.Mussels,μL) and ACE solution  were mixed together. After the mixture being preincubated at 37°C for 5 min, 50 μL 7.6 mmol/L HHL substrate solution, which was solved in 50 mM sodium borate buffer and 6.8 mM NaCl at pH 8.3, was added. The mixture was incubated at 37°C for 25 min. The reaction was terminated after addition of 10 μL of 20% trifluoroacetic acid (TFA). The solution was filtrated through 0.22 μm membrane. The hippuric acid liberated by ACE was analyzed by reversed-phase high performance liquid chromatography (RP-HPLC) on an Inertsil ODS C18   column. The mobile phase was 30% methanol, which contained 0.1% TFA and 0.05% acetic acid. The flow rate was 1.0 mL/min. The UV detection wavelength was 228 nm. The ACE inhibitory activity was obtained from peak area and expressed as percent. The ACE inhibitory activity was determined by Wang et al. method  with sliFive kinds of proteases were used for hydrolysis at their labeled optimum temperature and pH shown in . And the7(74) was applied to determine the optimum hydrolysis conditions for obtaining ACE inhibitory activity peptides from Mytilus coruscus. The combination effects of independent variables X1 , X2 , X3 (hydrolysis pH), and X4  at 7 variation levels in the hydrolysis process were shown in Y) were ACE inhibitory activity. These values were related to the variables by a second-order polynomial . Therefore, alkali protease E1 was chosen to next experiments to optimize hydrolysis conditions for producing ACE inhibitory peptides from Mytilus coruscus.ACE inhibitory peptides generally were short peptides and enzymatic hydrolysis of food derived protein was one of important measures to obtain ACE inhibitory peptides. A lot of ACE inhibitory peptides had been reported from food derived proteins hydrolysates. In this investigation, five kinds of commercial proteases, including three alkali proteases, one neutral protease, and one acid protease, were chosen to obtain ACE inhibitory peptides from A regression analysis was conducted to fit a mathematical model to the experimental data. The results of regression analysis were summarized , and a rP  value (P = 0.047 < 0.05) and satisfactory values of the regression coefficient R2  (R2 = 0.9712) for the response. The high regression coefficient make clear that the experimental values of the ACE inhibitory activity agreed with predicted values, which meant that the predicted model seemed to reasonably represent the observed values. The largest relative error of predicted value was less than 5% shown in t-test and P value in The statistical analysis indicated the predicted model was adequate, possessing significant X1): 36.0 U/mL; hydrolysis time (X2): 2.7 h; hydrolysis pH (X3): 8.2; hydrolysis temperature (X4): 59.5°C. The predicted ACE inhibitory activity was 90.7% at optimum hydrolysis conditions. Under this optimum hydrolysis conditions, other three verification experiments were conducted and the average actual ACE inhibitory activity was 91.3%, which was in agreement with the predicted values of 90.7%. Then the optimum hydrolysis conditions of protease E1 and the prediction ACE inhibitory activity were obtained by . The optMytilus coruscus were determined and the results were shown in  The compositions of free amino acid and amino acid in peptides of the ACE inhibitory peptides from Mytilus coruscus protein for producing ACE inhibitory peptides. The factors, including protease concentration, hydrolysis time, hydrolysis pH, and temperature, affected the ACE inhibitory peptides of hydrolysates. Uniform Design was chosen to investigate the effects of preceding variables on ACE inhibitory activity. And the best hydrolysis conditions of alkali protease E1 optimized by Uniform Design were protease concentration of 36.0 U/mL, hydrolysis time of 2.7 hours, pH 8.2, temperature at 59.5°C. The optimal predicted ACE inhibitory activity of 90.7% was obtained at the optimum conditions. The experimental activity (91.3%) under optimized conditions was agreed closely with the predicted activity. The amino acid composition analysis of the ACE inhibitory peptides proved that it had high percent of lysine, leucine, glycine, aspartic acid, and glutamic acid. It was suggested that the ACE inhibitory peptides derived from Mytilus coruscus could be utilized to develop nutraceuticals and pharmaceuticals.Alkali protease was a good choice for hydrolyzing


R = 0.928, and the model was applied in ACE-inhibitory peptides preparation from defatted wheat germ protein (DWGP). According to the QSAR model, the C-terminal of the peptide was found to have principal importance on ACE-inhibitory activity, that is, if the C-terminal is hydrophobic amino acid, the peptide's ACE-inhibitory activity will be high, and proteins which contain abundant hydrophobic amino acids are suitable to produce ACE-inhibitory peptides. According to the model, DWGP is a good protein material to produce ACE-inhibitory peptides because it contains 42.84% of hydrophobic amino acids, and structural information analysis from the QSAR model showed that proteases of Alcalase and Neutrase were suitable candidates for ACE-inhibitory peptides preparation from DWGP. Considering higher DH and similar ACE-inhibitory activity of hydrolysate compared with Neutrase, Alcalase was finally selected through experimental study.A quantitative structure-activity relationship (QSAR) model of angiotensin-converting enzyme- (ACE-) inhibitory peptides was built with an artificial neural network (ANN) approach based on structural or activity data of 58 dipeptides , the overall correlation coefficient of the predicted versus actual data points is  In recent years, some progress have been made in bioinformatics study of functional peptide preparation, such as comparing active peptide sequences in database, hydrolysis enzyme choosing, simulated hydrolysis, activity prediction of hydrolysate, and so forth –6. HowevBesides comparing characterized peptide sequences in databases, peptide quantitative structure-activity relationship (QSAR) models could also be used in peptide bioinformatics study. QSAR models are mathematical functions that describe the relationship between activity and chemical structure expressed by variables. Such models are applied both to predict activity of untested chemical structures and to predict the chemical structure of compounds with specific activity . SeveralAn artificial neural network (ANN) is an interdisciplinary technique, involving biology, mathematics, physics, electronics, and computer technology. It is a kind of information processing system based on imitation of the structure and function of brain networks. It is the theoretical model of the human neural network. ANN technique can simulate any nonlinear process; therefore, it can avoid the linear deficiencies , 16, 18.In this study, illustrated by preparation of ACE-inhibitory peptides from defatted wheat germ protein, a QSAR model was built with ANN. The structural characteristics of the ACE-inhibitory peptides were investigated according to the model. Based on the structural characteristics analysis and experimental result of DWGP digestion, appropriate protease was selected to produce high-activity ACE-inhibitory peptides from DWGP isolates.Defatted wheat germ protein was purchased from Man Tian Xue Flour Industry . Alcalase 2.4 LFG (2.670 AU/g) and medium temperature amylase 480 L (527.50 KNU/g) were purchased from Novo Co. . Angiotensin I-converting enzyme  was purchased from Sigma Chemical Co. . N-(3[2-Furyl]Acryloyl)-Phe-Gly-Gly (FAPGG) was purchased from Fluka Chemical Corp. . All the other reagents were in analytical purity grade.The instruments used were as follows: thermostat-controlled water-bath (model HH), Jintan Zhongda Instruments Co., Ltd. ; pH meter (model PHS-3C), Shanghai Precision & Scientific instrument Co., Ltd. ; electrothermal blast drying oven, Shanghai Laboratory Instrument Works Co., Ltd. ; Agilent 1100 HPLC, Agilent Technologies Inc. ; SPX-250B biochemistry incubator, Changzhou Guohua Electric Co., Ltd. ; Multiskan Spectrum Microplate Reader, Thermo Scientific Inc. .α-amylase was added in. After stirring for 180 min at 70°C, it was adjusted to pH 4.0 with 1.0 mol/L HCl to precipitate the protein, and the solution was centrifuged at 8000 r/m for 20 min. The precipitate was washed several times with distilled water (pH 4.0), and was then dispersed in a small amount of distilled water, then it was adjusted to pH 7.0 with 0.1 mol/L NaOH. The dispersed precipitate was dried by spraying dryer  to get DWGP isolates.DWGP isolates were prepared according to the method described by XIN Zhi-hong  with minTen grams of DWGP was dispersed in 1 L distilled water and was digested in batch by Alcalase at pH 9.0, 50°C or by Neutrase at pH 7.0 at 50°C, both at the enzyme/substrate mass ratio of 8% ([E]/[S]). Samples were collected at 0.5, 1, 1.5, 2, 3, 4, and 5 h and were immediately heated in a boiling water bath for 10 min. After cooling, the samples were centrifuged at 10,000 r/m for 15 min, and the supernatants were diluted with distilled water to determine their ACE-inhibitory activities.Z descriptor was used to predict the ACE-inhibitory activity of peptides, amino acids descriptor selected Z-scales, Z1, Z2, and Z3 means the hydrophilic amino acids, three-dimensional shape, size, and electrical parameters, respectively  were used in the text and were shown in 50]) and six independent variables (Z parameters).Fifty-eight kinds of ACE-inhibitory peptides (dipeptides) samples and their activity data  the number of training steps is controlled in 6000. The number of hidden layer neurons was determined through repeated verification.A three-level BP neural network model was built using MATLAB neural network tool (from Matrix Laboratory). Transfer functions of neurons in hidden layer and output layer were Tansig function and Purelin function, respectively. Because the BP neural network is not easily converged or easily falls into local minimum, the following steps were applied to avoid it: (1) network training algorithm using gradient descent momentum Traingdm, (2) network training objectives (mean square error) is set to 10a1) and sample (b1), and the final absorbance  were recorded. The absorbance decrease of blank and sample are A ( = a1 − a2) and B ( = b1 − b2), respectively. Then, ACE-inhibitory activity (%) was expressed as I = (A − B)/A.N-(3-[2-Furyl]Acryloyl)-Phe-Gly-Gly  was used as substrate in ACE-inhibition assay. The reagents were sequentially added in for test reaction according to The degree of hydrolysis (DH) was measured by pH-stat method. The release of amino acids in protein digestion makes pH of the hydrolysate decrease significantly, the alkali solution was added into hydrolysates to maintain pH value. By recording the amount of alkali consumed, the degree hydrolysis of protein and the amount of the rupture protein bonds can be figured out according to the following formula:VNaOH is consumption volume of alkali (mL) in titration; NNaOH is the concentration of alkali (mol/L) in titration; Mp is total protein (g) used; hhot is the total number of peptide bonds per gram of protein ; α is a-amino acid dissociation degree, it can be calculated according to formula  precolumn derivatization RP-HPLC determination .R of study samples  were shown in R reaches to 0.928, the training set mean square error is 0.0188, and the prediction set mean square error is 0.2091. The predicting results of BP network model to the set of prediction were shown in In this study, 4–10 hidden layer neurons were selected to build QASR model, each hidden layer neuron was modeled five times in order to identify the optimal number of hidden layer neurons. Network convergence speed rises when the number of neurons increases, but too many or too few of hidden layer neurons will decrease the generalization performance of model. Under the premise of guaranteed network convergence, a fewer number of neurons are preferred. The correlation coefficients Z operator) has impact on the found hidden layer neurons. The back stepping method was used to find out the operator which has the greatest impact on the activity. The steps are as follows: (1) find out which hidden layer neuron has the greatest impact on output (activity), (2) find out which input neuron (specific Z operator) which affected the 6 and 7 hidden layer neurons were investigated.In After searching the hidden layer neurons, the input layer neurons with the greatest impact on the hidden layer neurons were subsequently searched. In Z operators on 6 and 7 hidden layer neurons, we found that the 21Z parameters  have the greatest impact on the activity, followed by the Z22 . As we have defined that the Z1 operator represents the hydrophobicity of amino acids [Z descriptors to investigate quantitative structure-activity relationship of ACE-inhibitory dipeptides, and they found that ACE-inhibitory activity was greatly affected by the three-dimensional chemical properties and hydrophobicity of C-terminal amino acids, that is, the higher the volume and the greater hydrophobicity of amino acids were, the nicer the ACE-inhibitory activity was; so some dipeptides with hydrophobic amino acids at the C-terminal, such as phenylalanine, tryptophan, and tyrosine, will have high ACE-inhibitory activity. Cheung et al. [no acids , we coulno acids  used Z dg et al.  have alsg et al. . As for g et al.  found thg et al.  also reaThe DWGP contains 42.84% hydrophobic amino acids , it is sNeutrase  tends to hydrolyze protein to produce peptides whose C-terminals are hydrophobic amino acids, such as Tyr, Try, or Phe. Alcalase  tends to hydrolyze protein to obtain peptides whose C-terminals are amino acids with large side-chain and no charge , such as Ile, Leu, Val, Met, Phe, Tyr, or Trp. Moreover, the hydrolysis process will be accelerated when N-terminals of peptides have hydrophobic amino acids , 29. ProFrom Based on data of activity, hydrophilic amino acids, three-dimensional shape, size, and electrical parameters of 58 dipeptides, a quantitative structure-activity relationship (QSAR) of amino acids ACE-inhibitory peptides was built with ANN, the related coefficient is 0.928, and by analyzing the ANN model, it was found that (1) C-terminal is primarily important to ACE-inhibitory activity; (2) proteins containing abundant hydrophobic amino acids are potential good source to produce ACE-inhibitory peptides; (3) as for DWGP, Alcalase was a proper protease for ACE-inhibitory peptides preparation.