Optogenetic mapping of connectivity in the primary somatosensory cortex shows that mature cortical circuits adapt to sensory deprivation by selectively altering specific network motifs. Although neocortical connectivity is remarkably stereotyped, the abundance of some wiring motifs varies greatly between cortical areas. To examine if regional wiring differences represent functional adaptations, we have used optogenetic raster stimulation to map the laminar distribution of GABAergic interneurons providing inhibition to pyramidal cells in layer 2/3 (L2/3) of adult mouse barrel cortex during sensory deprivation and recovery. Whisker trimming caused large, motif-specific changes in inhibitory synaptic connectivity: ascending inhibition from deep layers 4 and 5 was attenuated to 20%–45% of baseline, whereas inhibition from superficial layers remained stable (L2/3) or increased moderately (L1). The principal mechanism of deprivation-induced plasticity was motif-specific changes in inhibitory-to-excitatory connection probabilities; the strengths of extant connections were left unaltered. Whisker regrowth restored the original balance of inhibition from deep and superficial layers. Targeted, reversible modifications of specific inhibitory wiring motifs thus contribute to the adaptive remodeling of cortical circuits. Many natural and engineered networks contain recurring patterns of local connectivity. Although these so-called network motifs are thought to have functional significance, direct tests of the idea that network topology reflects function remain scarce. We have performed such a test in the area of mammalian neocortex that is devoted to the sensory representation of touch. To this end, we equipped inhibitory interneurons in the mouse with light-activated ion channels that allowed us to stimulate interneuron activity optically and record light-evoked inhibitory currents in their postsynaptic partners, thereby revealing maps of connectivity. We find that excitatory pyramidal cells in layer 2/3 of primary somatosensory cortex receive inhibition from GABAergic interneurons located in different cortical layers, with a characteristic balance of inhibitory connections from deep and superficial layers. Trimming the whiskers to remove sensory input in adult animals alters this balance—inhibitory connections from deep cortical layers are depleted, while inhibitory connections from superficial layers are augmented. These changes revert when the whiskers regrow, restoring the original balance between wiring motifs. This see-saw relationship between deep and superficial inhibition demonstrates that mature cortical circuits adapt to functional change by selectively altering specific network motifs. Neocortex has a similar multilayered histology throughout The organization of inhibitory neocortical circuits also obeys principles of some generality. A recent survey of inhibitory-to-excitatory wiring patterns in primary motor (M1), somatosensory (S1), and visual cortex (V1) of the mouse uncovered 25 interlaminar connection motifs common to all three regions This interpretation raises several questions. Is the presence of a specific wiring motif linked to the particular type of input a cortical area receives? In other words, does the motif change when the type of input changes? If so, is plasticity limited to a critical developmental period, or does the capacity to adapt persist into adulthood? And how motif-specific is the change? Are variable wiring motifs inserted or removed on demand, akin to plug-in devices that add new functionalities, or are circuits reconfigured more broadly?To answer these questions, we have analyzed and compared the laminar organization of inhibitory inputs to pyramidal neurons in L2/3 of adult mouse barrel cortex (S1) under physiological conditions, during sensory deprivation (whisker trimming), and after recovery (whisker regrowth). In agreement with recent observations in visual cortex Experiments were performed on acute neocortical slices of a mouse knock-in line expressing the optogenetic actuator To determine the density and strength of inhibitory inputs from different cortical layers, pyramidal cells in L2/3 were voltage-clamped at 0 mV, while a focused laser beam scanned a pseudorandom pattern of 14×20 locations arranged in a 60-µm grid . When a −13 of cortical tissue. This volume is at least an order of magnitude larger than what could realistically be sampled by two-photon random-access glutamate uncaging With a lateral optical resolution of ∼60 µm FWHM  [5], a rBarrel cortex of adult mice was deprived of sensory input by trimming whisker rows A, B, D, and E every other day for 2–3 wk. Animals were aged 8–11 wk at the beginning of the manipulation and 10–14 wk at the time of analysis. To exclude potential confounds due to deprivation-induced changes in the optical excitability of ChR2-expressing interneurons, we compared the light responses of ChR2-positive cells in deprived barrel-related columns and nondeprived cortex. Our analysis concentrated on L5B, which exhibited the largest functional changes associated with deprivation (see below). We distinguished between fast-spiking and the two principal types of non-fast-spiking interneurons in layers 1 and 5, termed accommodating and nonaccommodating (or regular-spiking) cells , and exaIn barrel-related columns representing trimmed whisker rows A, B, D, and E, sensory deprivation was linked to a conspicuous loss of ascending inhibitory inputs to L2/3 pyramidal cells from deeper layers . The norAfter whiskers had been allowed to regrow for 3 mo, the inhibitory charge flow from all cortical layers returned to baseline values . Full whSparing a single central whisker from deprivation can cause excitatory circuits to expand, so that signals from the spared whisker now activate surrounding deprived barrel-related columns We also analyzed the horizontal inhibitory input distribution of L2/3 pyramidal neurons residing in the spared barrel-related columns of row C. Trimming the principal whiskers associated with adjacent barrel-related columns altered neither the horizontal spread  nor the Remarkably, deprivation-induced changes in vertical inhibitory connectivity also affected the spared barrel-related columns of row C. As in deprived columns, the number of home column inputs decreased significantly , but theSeveral mechanisms could generate these adaptations, singly or in combination. Elaboration or retraction of inhibitory terminals could alter the number of pyramidal cells contacted by one interneuron (a change in connection probability) or the number of synapses between one interneuron and one pyramidal cell (a change in connection strength). Differences in connection strength could also arise if synaptic release probability or quantal size were modulated. A formal, though remote, possibility is that the number of interneurons themselves might change during deprivation.p = 0.002; ANOVA). Most of the few surviving sources of inhibition attributed to L5B arose from stimulation sites that straddled the border to L5A .In adult mice with intact or fully regrown whiskers, identically sized majorities of pyramidal cells in L2/3 were targeted by L5B interneurons . By contrast, in deprived barrel-related columns approximately one half (11/23) of L2/3 pyramidal cells lacked any detectable input from layer 5B . Where cr to L5A , raisingr to L5A . IPSCs cp<0.001; ANOVA; Inhibition from layers 4 and 5A underwent qualitatively similar but less extensive changes. Absolute input numbers, as well as the fractions of connected locations, fell during sensory deprivation but rebounded fully when sensory input was restored . Connectp<0.05; t test; p<0.05, t test). Even in L1 the number of connected locations fell slightly  or regular-spiking (nonaccommodating) type (n = 5), consistent with the notion that non-fast-spiking Martinotti cells are the principal conduits of L5-to-L2/3 inhibition To search for structural correlates of these functional changes, we analyzed the wiring motif undergoing the largest deprivation-induced change: ascending inhibition from L5B . Forty-nThe fractional loss of inhibitory varicosities after whisker trimming was, however, smaller than the fractional reduction in inhibitory charge flow from L5B compare . This apInterneurons are thought to innervate each of their postsynaptic targets via multiple boutons  Of course, optical stimulus-locked IPSCs may represent compound events if multiple presynaptically connected interneurons are activated simultaneously. Because the mean integrated current depends on the number and the individual strengths of all contributing synapses, it cannot be equated with the strength of a monosynaptic connection. In several instances, however, the two variables of synapse number and average synapse strength could be disambiguated by considering changes in charge transfer in the context of simultaneously occurring changes in the number of connected locations. For example, sensory deprivation greatly reduced the number of connections from layers 4 and 5 but left the charge transfer per remaining connection unchanged. If it is reasonable to assume, in light of this general trend toward synapse elimination, that persisting connections will be made through a constant or smaller rather than a larger number of synapses, then the average strength of these synapses must remain level or increase after deprivation. Our morphometric finding that L5B-derived axonal varicosities in deafferented whisker columns retained their pre-deprivation size or even expanded slightly  reinforcAn analogous argument applies to L1 of the spared whisker-related row C. The charge transfer per IPSC in these barrel-related columns increased significantly after whisker trimming, while the number of connected locations fell marginally . This coA history of deprivation had profound aftereffects on the strengths of inhibitory inputs originating in superficial layers 1 and 2/3. The inhibitory charge flow per IPSC from these layers increased marginally upon sensory deprivation, echoing similar changes during the critical period The adaptations documented here lay bare four remarkable features of experience-dependent plasticity of inhibitory connections. First, extensive changes take place in adult neocortex, long after the critical period for refining neuronal connections has closed p<0.001, t test; Although the experimental settings and analytical approaches differ, it is instructive to compare our present findings with those of previous reports of experience-dependent inhibitory plasticity in adult neocortex From hindsight, hints of motif-specific plasticity are already evident in some earlier studies of excitatory The adaptive changes displayed by different inhibitory circuit motifs offer some clues to the possible roles of these motifs in normal cortical function. Interneurons in L2/3 are thought to be driven by L2/3 excitatory neurons and the thalamocortical input layers 4 and 5A The inhibitory L5B-to-L2/3 motif, in contrast, is all but deleted from deprived whisker columns . This stQualitatively similar considerations may apply to inhibition originating in layers 4 and 5A. L5A and septal regions of L4 form part of a cortico-thalamo-cortical loop involving the posterior medial nucleus (POM) Inhibitory interneurons in L1, in contrast, are likely mediators of top-down control of cortical areas by hierarchically higher regions Plasticity that is wiring motif–, cell type– All procedures complied with the UK Animals (Scientific Procedures) Act 1986.R26::CAG-lox-STOP-lox-ChR2-EGFP responder and T2Gad2::CreER driver transgenes at both targeted loci Experimental animals were knock-in mice homozygous for Slices of acutely deprived somatosensory cortex were harvested no later than 36 h after the last whisker trimming session. To examine the effects of recovery from deprivation, whiskers were allowed to regrow for 4–5 wk (1-mo regrowth) or 12–14 wk (3-mo regrowth). Starting at 6–8 d before slices were cut, mice were injected intraperitoneally (i.p.) on 5 consecutive days with 0.3–0.5 mg 4-OH-tamoxifen (Sigma-Aldrich), which was dissolved in sterile sunflower oil at 5 mg/ml.2PO4, 25 NaHCO3, 10 glucose, 240 sucrose, 0.5 CaCl2, 7 MgCl2, pH 7.4, 320 mOsm. The brain was recovered into perfusion solution, and 310-µm slices of the left primary somatosensory cortex (S1) were cut on a Leica VT1200S vibratome. The cutting plane was oriented across whisker barrel rows, maintaining angles of 45° with the midline and nearly 90° with the cortical surface 2PO4, 25 NaHCO3, 25 glucose, 1.25 CaCl2, 2 MgCl2, pH 7.4, 315 mOsm. Recordings were performed at room temperature in aCSF containing (in mM): 125 NaCl, 3.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 25 glucose, 1.25 CaCl2, 1 MgCl2, pH 7.4, 310 mOsm. All extracellular solutions were bubbled with 95% O2/5% CO2.Experiments were performed on mice 2–4 d after the last 4-OH-tamoxifen injection. Animals were anesthetized by i.p. injection of 150 µl of a 3∶5 mixture of ketamine  and medetomidin  and perfused cardially with ice-cold solution containing (in mM): 2.5 KCl, 1.25 NaH2GTP, 4 NaCl, 5 QX-314-Br, 0.2% neurobiotin, pH 7.25, 274 mOsm. The internal solution for current-clamp recordings from interneurons contained (in mM): 120 K-gluconate, 10 KCl, 10 Hepes, 4 MgATP, 0.3 Na2GTP, 10 phosphocreatine, 0.2% neurobiotin. Signals were amplified and low-pass-filtered at 2 kHz by a Multiclamp 700a amplifier (Molecular Devices) and digitized at 5–10 kHz .Whole-cell recordings were obtained from cells in barrel-related columns corresponding to whisker rows A through E. Patch pipettes had tip resistances of 4–6 MΩ. The internal solution for voltage-clamp recordings from pyramidal cells contained (in mM): 110 CsOH, 110 gluconic acid, 0.2 EGTA, 30 Hepes, 2 MgATP, 0.3 NaThree criteria were used to distinguish fast-spiking (fs) from non-fast-spiking (non-fs) interneurons. Fs neurons (i) attained firing rates >90 Hz during a 1,000-ms depolarizing current step, (ii) exhibited a ratio of >0.7 of the average interspike interval (ISI) at the beginning and end of the depolarizing current step (averages of 3 ISIs each), and (iii) displayed a spike width of ≤1 ms at half-maximal amplitude. Cells that met all three criteria were classified as fs and cells that failed all three criteria as non-fs.Optical stimulation experiments were performed on a Zeiss Axioskop 2FS microscope. A 40×, 0.8 NA water immersion objective with DIC optics was used for electrode placement and a 10×, 0.3 NA water immersion objective, without DIC optics, for optical stimulation. The output of a continuous-wave solid-state laser with a maximum power of 325 mW at 473 nm  was digitally switched and intensity-modulated by an acousto-optic deflector (IntraAction model ASN-802832 with ME-802 driver), positioned by a pair of galvanometric mirrors (GSI Lumonics VM500 with MiniSAX servo controllers), and merged with the epi-illumination path of the microscope via custom-built optics. Light pulses carried 0.5–1.8 mW of optical power at the exit pupil of the objective. To generate maps of inhibitory inputs, a virtual instrument written in LabVIEW 8.5 delivered focused stimulation light pulses  at intervals of 680 ms to 60-µm grids encompassing 14×20 locations in pseudorandomized order.n = 2 in each condition) were perfused with phosphate-buffered saline  containing 4% (w/v) paraformaldehyde (PFA) and 0.2% (v/v) picric acid under general ketamine-medetomidine anesthesia. The brain was removed, incubated for 24 h in perfusion solution, and infiltrated with 30% (w/v) sucrose in PBS for at least 24 h. Coronal sections of 50 µm were cut on a Leica SM 2000R sliding microtome. The sections were rinsed three times in Tris-buffered saline , three times in TBS containing 3% (w/v) Triton X-100 (TBS-T), and once for 1 h in TBS-T containing 20% (v/v) horse serum (Vector Labs) and then incubated for 48 h at 4°C in TBS-T containing 1% horse serum and combinations of the following primary antibodies: anti-GFP , anti-parvalbumin , or anti-somatostatin . The sections were rinsed 4 times in TBS and stained in TBS-T containing 1% horse serum and Alexa488- and Alexa546-labeled secondary antibodies (Invitrogen). After four rinses in TBS, the slices were mounted in VectaShield (Vector Labs) and imaged on a Leica TCS SP5 confocal microscope.Animals  PFA and 0.2% (v/v) picric acid. The slices were rinsed in TBS and stained in TBS-T containing 1% (v/v) horse serum, 4 µg/ml Alexa546-labeled streptavidin (Invitrogen), and 0.0001% DAPI (Sigma) for 12–24 h. After four rinses in TBS, the slices were mounted in VectaShield (Vector Labs) and imaged on a Leica TCS SP5 confocal microscope.Interneurons were identified from their spiking responses to step-current pulses during the neurobiotin infusion as well as on the basis of morphological criteria after filling . The axonal arbors of 11 out of 49 neurobiotin-filled neurons  showed high-contrast axonal labeling in L2/3 that could be traced back to the filled soma in L5B; these arbors were chosen for morphometric analysis. Ten L2/3 axon segments in each condition, ranging in length from 125 to 2,063 µm, were reconstructed manually using the freeware Neuromantic Data were analyzed as described t test. Pairwise hypotheses were evaluated by t test.The presynaptic sources of IPSCs were allocated to individual cortical layers, which were identified by differences in shading and cell density To visualize inhibitory input maps and their rearrangements following whisker trimming and regrowth, input strengths  were coded in normalized gray scale .Figure S1Passive membrane properties of layer 2/3 pyramidal cells. Membrane resistance (left) and membrane capacitance (right) of L2/3 pyramidal cells in barrel-related columns of mice with intact  or trimmed  whiskers. Measurements used the membrane test routine built into pClamp10 (Axon Instruments) immediately after establishment of the whole-cell patch-configuration.(TIF)Click here for additional data file.Figure S2Individual input maps. Maps of inhibitory inputs to individual L2/3 pyramidal neurons in barrel-related columns representing intact (top), trimmed (second from top), or previously deprived whiskers after regrowth for 1 mo (second from bottom) or 3 mo (bottom). The amplitude of the IPSC evoked at each location is represented in gray scale, according to the look-up table at the bottom. The selected neurons represent deciles in the frequency distribution of the number of L5B-derived inputs to L2/3 pyramidal cells. Maps are scaled to the size of a standard whisker-related barrel (yellow outline).(TIF)Click here for additional data file.Figure S3Variability of IPSC amplitudes. Coefficients of variation of the individual IPSC amplitudes shown in (TIF)Click here for additional data file.Figure S4Distribution of IPSC amplitudes. Histograms display the number of verified input sources (x-axis) of a given amount of charge transfer (y-axis) grouped in bins of 1 pC (layer 1–4) or 0.5 pC (layer 5). Data correspond to IPSC traces in (TIF)Click here for additional data file.Table S1Estimate of the number of interneurons activated per stimulation site. The number of ChR2-expressing interneurons activated by a single optical pulse was calculated using two estimates of interneuron densities in specific layers of primary somatosensory cortex (S1) of the mouse −1(DOCX)Click here for additional data file.


Brazil is a very rich country in terms of biodiversity offering broad spectra of possibilities to study new allergens, particularly from foods. In poor and developing countries manioc is a major source of carbohydrates in the diet of millions of people. In Brazil is the ninth cultivated food and the country takes the second place in the world as manioc cultivator. Allergic reactions to manioc were previously reported, but the allergen molecules involved and the cross-reactivity with other plant food sources or latex remain unknown. The aim of present work is to identify new manioc allergens, clone and express them. Based on clinical history, nine patients allergic to manioc were submitted to prick-to-prick test and basophil activation tests and sera were used for immunoblot analysis and ISAC. All patients demonstrated positive IgE reactivity to manioc proteins of approximately 30 and 40 kDa in 1D gel electrophoresis. Using 2D analysis, 6 immunoreactive spots were identified as allergenic-related protein Pt2L4, GADPH and fructose-bisphosphate-aldolase. Three out of five tested sera showed IgE reactivity to Hev b 5 in ISAC. The rich glutamic acid protein, Pt2L4 shows around 40% sequence identity with Hev b5 and 2 of 5 linear IgE epitopes identified for Hev b 5 are partially conserved in Pt2L4. Therefore, the mature sequence was amplified from manioc cDNA using specific primers and cloned into a pET-based expression vector. Clones from two different cultivars were checked and they are all the same sequence but deviate 3 amino acids from the three previously published sequences. Recombinant allergenic-related protein was expressed in E. coli BL21 Star and purified by ion exchange and size exclusion chromatography. Protein will be characterized regarding its physicochemical properties using aminoacid analysis, mass spectrometry, circular dichroism and simulated duodenal digestion. The IgE-binding activity of the recombinant molecule is assessed by IgE immunoblot and ELISA. In addition, IgE cross-reactivity with purified natural Hev b 5 is evaluated using sera of manioc and latex allergic patients.FAPESP, INCT-iii


Based on these data, we suggest that the attenuation of the melanopsin-based light response by dopamine is mediated by direct PKA phosphorylation of melanopsin, rather than phosphorylation of a downstream component of the signaling cascade.The visual pigment melanopsin is expressed in intrinsically photosensitive retinal ganglion cells (ipRGCs) in the mammalian retina, where it is involved in non-image forming light responses including circadian photoentrainment, pupil constriction, suppression of pineal melatonin synthesis, and direct photic regulation of sleep. It has recently been shown that the melanopsin-based light response in ipRGCs is attenuated by the neurotransmitter dopamine. Here, we use a heterologous expression system to demonstrate that mouse melanopsin can be phosphorylated by protein kinase A, and that phosphorylation can inhibit melanopsin signaling in HEK cells. Site-directed mutagenesis experiments revealed that this inhibitory effect is primarily mediated by phosphorylation of sites T186 and S287 located in the second and third intracellular loops of melanopsin, respectively. Furthermore, we show that this phosphorylation can occur  This third class of mammalian photoreceptors differs from the classical photoreceptors, the rods and cones, in both function and physiology. The ipRGCs primarily contribute to non-image forming light responses including circadian photoentrainment, pupil constriction, suppression of pineal melatonin synthesis, and direct photic regulation of sleep. The melanopsin-based signaling cascade causes the ipRGCs to depolarize in response to light, in contrast to the light-induced hyperpolarization seen in rod and cone photoreceptors In the work reported here, we demonstrate that melanopsin’s activity can also be modulated by protein kinase A (PKA). PKA is a serine-threonine kinase that is activated by increased levels of cyclic adenine monophosphate (cAMP). In many regions of the nervous system, the neurotransmitter dopamine is known to increase intracellular levels of cAMP via activation of adenylyl cyclase by D1-like dopamine receptors in vivo using an in situ proximity-dependent ligation assay (PLA).One class of cells in the retina that produces and releases dopamine is the dopamanergic amacrine cells, whose dendrites co-stratify with those of one class of melanopsin-expressing RGCs Predicted PKA phosphorylation sites within the melanopsin sequence were identified using the Group-based Prediction System (GPS) 2.0 A proximity-dependent ligation assay (PLA) (Olink Biosciences) was used to determine if any of these predicted sites were actually phosphorylated in a cellular environment. The PLA assay is designed to determine the proximity of two epitopes to one another using antibodies specific for each epitope. In this case, antibodies against the carboxy tail of melanopsin and against phosphoserine were used in conjunction with secondary antibodies conjugated with oligonucleotide linkers. If the primary antibodies are bound within ∼ 40 nm of each other, the oligonucleotides will anneal to form a piece of circular DNA, which can then be amplified by a polymerase using rolling circle amplification to produce concatamers of single-stranded copies of the DNA sequence. Addition of fluorescently-labeled oligonucleotide probes specific for the amplified DNA results in a single fluorescent spot for each pair of interacting antibody molecules −6). The PLA signal remained slightly elevated even in the S287G mutant, and only returned to untreated levels of fluorescence in the triple mutant . Although S182 may not be the primary target of PKA phosphylation in HEK cells, these results suggest that this residue may still be phosphorylated in the absence of other available sites.In order to confirm the importance of these putative PKA phosphorylation sites, each was individually mutated to non-phosphorylatable residues ; a double mutant  and a triple mutant containing all mutations  were also constructed. These mutant constructs were also examined by PLA in the dark in the presence of 200 µM 8-Br-cAMP . The quaHaving shown that the activation of PKA leads to phosphorylation of melanopsin in the dark, we next examined the effect of this phosphorylation on melanopsin function. This was tested using a kinetic calcium-signaling assay as previously described In order to confirm the importance of the previously identified PKA phosphorylation sites , mutant melanopsin proteins with were screened using the calcium kinetic assay . As predin vivo as it does in transfected cells, we performed a PLA assay using murine retinal sections. We have previously demonstrated light-dependent phosphorylation of the C-terminal tail of melanopsin in the retina, similar to the phosphylation observed in HEK cells, and that it remains largely unphosphorylated in the dark To demonstrate if PKA-dependent phosphorylation of mouse melanopsin occurs In order to investigate the broader implications of the identified PKA sites on melanopsin, these sites were examined across the twenty-five mammalian melanopsin sequences previously analyzed by Porter and colleagues  [26]. SePresented here is the novel finding that melanopsin signaling can be regulated by phosphorylation serine and threonine residues found in intracellular loops two and three by protein kinase A. Phosphorylation occurs at three locations, S182 and T186 of loop two and site S287 of loop three. Of these sites, T186 and S287 appear to be the most functionally important for regulating melanopsin signaling in rodents, and mediate an inhibition of the total amount of calcium mobilized by melanopsin in response to light in transfected HEK cells. These findings identify a potential mechanism for the dopamine modulation of the photoresponse which has recently been reported in rat ipRGCs The conservation of these sites across the 25 species examined here implies that it is advantageous to have at least one of the two sites  found to be functionally important. In the non-mammalian vertebrate species, there is a division between the OPN4m sequences and OPN4x, with OPN4x containing these three phosphorylatable sites, while the OPN4m does not. This may reflect a division of labor between the two genes, with OPN4x being more sensitive to regulation by dopamine than OPN4m. From this current analysis it is unclear if the common ancestor of melanopsin shares these PKA phosphorylation sites. In some species, however, the melanopsin sequence contains none of the identified regulatory phosphorylation sites. Although the third site, S182, is the most conserved residue, its mutation had no effect on the PKA-dependent suppression of melanopsin signaling. Further analysis of this site shows that it is a better match as a protein kinase C (PKC) phosphorylation site, which may provide an explanation for its high degree of conservation.This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Washington State University (Permit Number: 3766). To sacrifice animals we anesthetized mice with isofluorane inhalation, followed by cervical dislocation, and all efforts were made to minimize suffering.mouse melanopsin (GenBank accession no. 6693702).Mouse melanopsin construct is as previously described 2+-imaging assay as previously described 2+-imaging at a density of 8×104 cells per well in a 96 well plate with a clear bottom and black walls (Becton-Dickinson). Twenty-four hours after re-plating, cells were washed two times with Hank’s Balanced Salt Solution (HBSS) plus 20 mM HEPES, pH 7.4, and subsequently incubated in HBSS/HEPES, supplemented with 4 µM Fluo-4 AM (Invitrogen), 0.02% pluronic acid (Invitrogen), and 20 µM 11-cis retinal. Fluorescent measurements were performed on a Tecan Infinite M200 microplate reader (Tecan Group Ltd.) , sampling at a rate of 1 Hz for 60 seconds. Background fluorescence was subtracted to account for variation in dye loading and cell density.Melanopsin activity was determined using a fluorescent Ca8-Br-cAMP (Tocris Biosciences) was added to a final concentration of 200 µM (except where noted) 30 minutes prior to reading. To inhibit PKA activity, KT5720 (Tocris Biosciences) was added 10 minutes prior to addition of 8-Br-cAMP.Site directed mutagenesis was performed to remove predicted phosphorylation sites from the intracellular loops of melanopsin. Mutations were introduced by PCR using primers containing one base mismatches corresponding to the desired change in the coding sequence as previously described in vivocis-retinal for 1 hr, and fixed in ice-cold 4% PFA in the dark, before or after a 30 min incubation in 200 µM 8-Br cAMP. For experiments with endogenous melanopsin, C57/B6 mice were dark adapted overnight (>16 hrs) and sacrificed under dim red light. Animals were enucleated, and eyes were pierced through the cornea with a scalpel to create a large hole to allow solutions access to the retina. Untreated retinas were placed directly into ice-cold 4% PFA for 1-hour, while treated retinas were first soaked in 50 nM of the dopamine D1 receptor agonist A68930 (Tocris Biosciences) for 30 minutes before fixation. Cornea and lens were removed and the remaining eyecup was cryoprotected in 30% sucrose, embedded in OCT, and sectioned at 16 µm on a cryostat. Melanopsin-expressing cells or retinal sections were permeabilized in PBS containing 0.3% Triton-X, and blocked in PBS containing 0.3% Triton-X and 10% normal goat serum. Cells were probed with rabbit anti-melanopsin C-terminal antibody  and mouse anti-phosphoserine . Specificity of the anti-melanopsin C-terminal antibody has been demonstrated previously in situ proximity ligation assay (PLA) was performed according to the manufacturer’s protocol. PLA probes were diluted in 0.1% Triton X-100/PBS/1% fetal calf serum and incubated in a pre-heated humidity chamber for 1 h at 37°C, followed by hybridization, ligation, amplification, and detection. Fluorescence was visualized by confocal microscopy (TCS SP5 Lecia Microsystems). Fluorescent spots were counted and error bars represent standard deviation of 50 cells counted for each condition pooled from two separate transfections.We have previously used this assay to demonstrate that mouse melanopsin’s carboxy tail is phosphorylated in a light-dependent manner both in tissue culture cells and


N-acetyltransferases (NATs) are xenobiotic-metabolizing enzymes that play a key role in the biotransformation of AA carcinogens. Changes in NAT activity have long been associated with variations in susceptibility to different cancers in relation with exposure to certain AAs.Cadmium (Cd) is a carcinogenic heavy metal of environmental concern. Exposure to both Cd and carcinogenic organic compounds, such as polycyclic aromatic hydrocarbons or aromatic amines (AAs), is a common environmental problem. Human arylamine We explored the possible interactions between Cd and the NAT-dependent biotransformation of carcinogenic AAs.We exposed purified enzymes, lung epithelial cells, and mouse models to Cd and subsequently analyzed NAT-dependent metabolism of AAs.in vivo toxicokinetics with a significant decrease (~ 50%) in acetylated 2-AF in plasma. We found that human NAT1 was rapidly and irreversibly inhibited by Cd , with results of acetyl coenzyme A (acetyl-CoA) protection assays indicating that Cd-mediated inhibition was due to the reaction of metal with the active-site cysteine residue of the enzyme. We found similar results for human NAT2, although this isoform was less sensitive to inactivation .We found that Cd, at biologically relevant concentrations, impairs the NAT-dependent acetylation of carcinogenic AAs such as 2-aminofluorene (2-AF) in lung epithelial cells. NAT activity was strongly impaired in the tissues of mice exposed to Cd. Accordingly, mice exposed to Cd and 2-AF displayed altered Our data suggest that Cd can alter the metabolism of carcinogenic AAs through the impairment of the NAT-dependent pathway, which may have important toxicological consequences. Cadmium (Cd), a ubiquitous toxic element and widespread pollutant, is introduced to the environment mainly through anthropogenic activities, such as lead mining, fossil fuel combustion, and the manufacturing of Cd-containing products. Another major source for human exposure to Cd is cigarette smoke . The chiBecause of its stability in the environment and long retention time in the human body , Cd can accumulate and cause a variety of adverse effects . The tarAromatic amines (AAs) represent one of the most important classes of occupational or environmental pollutants . AAs accN-acetyltransferases (NATs) are xenobiotic-metabolizing enzymes (XMEs) that play a major role in the biotransformation of AA carcinogens  or CYP1A2 promotes their metabolic activation into DNA-binding electrophiles , 2-aminofluorene (2-AF), 4-ABP, acetyl coenzyme A (acetyl-CoA), coenzyme A (CoA), and protease inhibitors were obtained from Sigma . Cell culture reagents were from Invitrogen . All other reagents were purchased from Euromedex  unless otherwise stated.Cadmium chloride (CdClEscherichia coli strain BL21 (DE3) as previously described (Hexa-histidine (6xHis)-tagged human NAT1 was produced and purified from escribed . We used2 in 25 mM Tris-HCl (pH 7.5) for 10 min at 37°C. Recombinant NAT1 or NAT2 enzymatic activities were determined spectrophotometrically using p-nitrophenylacetate (PNPA) as the acetyl donor and 2-AF as arylamine substrate, as reported previously  with increasing concentrations of CdCleviously . In all We assessed the reversibility of the reaction of Cd with NAT1 and NAT2 by incubating recombinant enzymes with Cd  for 10 min at 37°C. Mixtures were then dialyzed overnight at 4°C against 25 mM Tris-HCl, pH 7.5. For controls, enzymes not treated with Cd were dialyzed overnight. After dialysis, residual enzyme activities were assayed.We tested whether reducing agents [reduced glutathione (GSH) and dithiothreitol (DTT)] or the chelating agent EDTA was able to restore the activity of Cd-inhibited NAT1 and NAT2. To this end, recombinant enzymes were preincubated with Cd  for 10 min at 37°C. Mixtures were then incubated with different concentrations of DTT or GSH  or EDTA  for 10 min at 37°C. Residual enzyme activities were then assessed. Control assays were carried out as described above in the absence of Cd but with GSH, DTT, or EDTA. We also tested whether these reducing or chelating agents were able to protect NAT1 and NAT2 enzymes from the inhibitory effects of Cd by carrying out Cd treatments  in the presence of high concentrations of DTT, GSH, or EDTA  and then determining residual enzyme activities.We investigated the extent to which acetyl-CoA and CoA protected NAT1 and NAT2 from Cd-dependent inhibition. To this end, recombinant enzymes were preincubated with different concentrations of acetyl-CoA or CoA in 25 mM Tris-HCl, pH 7.5, for 5 min at 37°C. Mixtures were then incubated with Cd  for 10 min at 37°C . Samples were then assayed. Control assays carried out in the absence of Cd treatment gave 100% enzyme activity.kinact were obtained by fitting residual enzyme activity to the equationNAT1 or NAT2 was incubated under second-order conditions as described previously by E is the enzyme concentration, E0 is the initial enzyme concentration, and t is time. We used KaleidaGraph, version 3.5  for mathematical analyses of the data.where The murine mtCC1-2 Clara lung epithelial cell line  was prov2 in 10 mL PBS or DMEM for 2 hr at 37°C in a cell incubator. Control cells were incubated with PBS or DMEM only. After incubations, monolayers were washed with PBS and scraped into 0.5–1 mL lysis buffer  and protease inhibitors. Extracts were sonicated and centrifuged for 15 min at 13,000 × g. Supernatants (whole-cell extracts) were removed, and their protein concentration was determined using Bradford reagent with bovine serum albumin as a standard. All cell extracts were adjusted to the same protein concentration by adding lysis buffer and were used for enzyme assays. We determined NAT activity in cells or mouse tissue extracts by measuring the formation of N-acetylated metabolites using reverse-phase high-performance liquid chromatography (HPLC) as described previously (Cell monolayers (~ 80% confluence) were washed with phosphate-buffered saline (PBS) and exposed to different concentrations of CdCleviously . All ass2 cytotoxicity using a 3--2,5-diphenyltetrazolium bromide (MTT)-based assay  and to 750 μM 2-AF (or 4-ABP) in culture medium. At different time points, aliquots were analyzed as described above.All procedures involving animals were carried out in accordance with the French Agriculture Ministry’s internal guidelines for animal handling. The number of mice and suffering were minimized whenever possible.in vivo by treating 12-week-old female C57BL/6J mice with a sublethal dose of CdCl2 (2 mg/kg in PBS), as reported previously (n = 7) were sacrificed by cervical dislocation, and endogenous NAT activity was measured in protein lysates of tissues known to be targeted by Cd, such as blood, liver, kidney, and lung. Tissue extracts were prepared as described previously (We investigated the ability of Cd to inhibit NAT functions eviously . Two houeviously .in vivo pharmacokinetics of acetylated 2-AF in mice (n = 7) with or without CdCl2 treatment (2 mg/kg in PBS). Two hours after Cd treatment, 2-AF (50 mg/kg) dissolved in dimethyl sulfoxide was administered by IP injection. Animals were then anesthetized with 10 mg/kg ketamine and 1 mg/kg xylazine, and blood samples were drawn from retro-orbital venous plexus at five sequential time points . Samples were diluted 1:50 in HPLC mobile phase and analyzed for parent and acetylated metabolites by HPLC as described above. Area under the curve (AUC) was determined by the trapezoidal rule using Microsoft Excel 2007 .In a second set of experiments, we analyzed the t-test between two groups using StatView 5.0 .Data are presented as mean ± SD of three independent experiments performed in triplicate, unless otherwise stated. One-way analysis of variance was performed, followed by Student’s 50) for Cd was approximately 0.055 μM (50 (~ 1 μM). These data indicate that both human NAT isoforms are readily inhibited in vitro by low, biologically relevant concentrations of Cd.To test whether Cd can inhibit human NAT1 and NAT2 activity, we assessed its effect on purified recombinant enzymes. As shown in 0.055 μM , inset. We tested whether the inhibition of NAT1 by Cd could be reversed by physiological (GSH) and nonphysiological (DTT) reducing agents that are known to react with Cd. DTT and GSH  did not significantly reverse Cd-dependent inhibition of NAT1 . We obseWe also tested whether EDTA, a known Cd-chelating agent, was able to reverse Cd-dependent inhibition of NAT1 and NAT2. Incubation of inhibited enzymes with different concentrations of EDTA for 30 min at 37°C did not reverse the inhibitory effect of Cd . To furtIn a second set of experiments, we analyzed the ability of DTT and GSH to prevent Cd-dependent inhibition of NAT1. Incubation of the enzyme with Cd in the presence of these compounds at high concentrations  protected NAT1 only partially against inhibition . At highkinact). The kinact constant for Cd-dependent inhibition of NAT1 was 5.2 × 104 M−1 · s−1 (kinact for NAT2 was slightly lower (1 × 104 M−1 · s−1). These results confirm the high reactivity of Cd toward NAT1 and NAT2 enzymes in vitro.To further characterize the reaction of Cd with NAT enzymes, we performed kinetic analyses. Pseudo-first-order conditions  could not be used because the enzymes were almost instantly inhibited by Cd under these conditions. Therefore, we used second-order conditions  to determine the second-order rate constant of inhibition  treated with a sublethal dose of CdCl2 (2 mg/kg) as described previously  afforded only partial protection  against Cd-dependent inhibition . These data suggest that Cd reacts more rapidly with NAT1 (and NAT2) than with GSH or DTT. Accordingly, the kinetics of the Cd reaction with GSH is at least two orders of magnitude lower (kassoc < 102 M−1 · s−1) than the inactivation rates found for NAT1 and NAT2 (kinact > 104 M−1 · s−1). Conversely, we found that EDTA afforded significant protection against Cd-dependent inhibition of human NAT enzymes. These results are similar to data obtained with the DNA repair enzyme Ogg1 and indicate the dependence of inhibition on the presence of free metal ions in solution  endogenous NAT activity in lung, liver, kidney, and blood, tissues known to accumulate Cd , from mia]pyrene (a]pyrene metabolites (Increasing evidence suggests that genotoxicity induced by Cd also depends on the synergic interactions of Cd with genotoxic chemicals . Cd inhia]pyrene . Interesabolites .Cd toxicity is caused by both acute and chronic exposure. Although the toxicity of Cd is considered mostly chronic, several studies using acute conditions have yielded a substantial amount of information pertinent to Cd toxicity, including mechanistic information . MoreoveHumans are exposed to AA carcinogens mainly through cigarette smoke or occupational/industrial pollutants. Several studies have suggested that NAT-dependent acetylation is a susceptibility factor for cancers associated with AA exposures , possibl


We identify phosphorylation of the membrane-intercalated proteoglycan syndecan-4 as an essential switch controlling integrin recycling. Src phosphorylates syndecan-4 and, by driving syntenin binding, leads to suppression of Arf6 activity and recycling of αVβ3 to the plasma membrane at the expense of α5β1. The resultant elevation in αVβ3 engagement promotes stabilization of focal adhesions. Conversely, abrogation of syndecan-4 phosphorylation drives surface expression of α5β1, destabilizes adhesion complexes, and disrupts cell migration. These data identify the dynamic spatiotemporal regulation of Src-mediated syndecan-4 phosphorylation as an essential switch controlling integrin trafficking and adhesion dynamics to promote efficient cell migration.Precise spatiotemporal coordination of integrin adhesion complex dynamics is essential for efficient cell migration. For cells adherent to fibronectin, differential engagement of α ► c-Src phosphorylates syndecan-4 in response to extracellular stimuli ► Syndecan-4 phosphorylation and engagement regulate Arf6 activity ► Syndecan-4-mediated Arf6 activity regulates differential integrin recycling ► Syndecan-4-mediated integrin recycling controls FA dynamics and cell migration 5β1 or αVβ3 integrin recycling in the control of adhesion dynamics.Efficient cell migration requires dynamic coordination of cell-matrix interactions, in part through precise spatiotemporal control of integrin adhesion dynamics. Morgan et al. show that phosphorylation of the extracellular matrix receptor syndecan-4 acts as a molecular switch, differentially directing either α Haptotactic migration, in which cells are guided by direct interactions of adhesion receptors with extracellular matrix (ECM) fibers, is fundamental to tissue morphogenesis, homeostasis, and repair and for the pathogenesis of inflammatory and neoplastic diseases. Focal adhesions (FAs) are sites of cell-ECM integration where topological features of the ECM are interpreted. FAs contain clusters of integrin receptors and hundreds of cytoskeletal and signaling molecules. These complexes function as both physical links to the contractile cytoskeletal machinery and dynamic signaling nexuses. Crucially, efficient cell migration requires the precise spatial and temporal regulation of FA turnover and stabilization .5β1 and αVβ3 exhibit distinct biomechanical, mechanoresponsive, and signaling properties that directly influence the dynamic interaction with the ECM and cell migration  or syndecan-4 with a mutated Y180 residue (HA-Syn4Y180L) was expressed in 293T cells and levels of tyrosine phosphorylation were assessed. Disruption of the Syn4Y180 residue substantially reduced syndecan-4 tyrosine phosphorylation, as determined by both solid-phase immobilization and immunoprecipitation B. SyndecTo test whether c-Src phosphorylates syndecan-4 directly, we assessed c-Src-mediated phosphorylation of isolated syndecan-4 cytoplasmic domains. Recombinant active c-Src phosphorylated GST-Syn4WT, but not GST alone, in a time-dependent manner E. SimilaTo investigate the contribution of c-Src-mediated syndecan-4 phosphorylation and signaling to cell migration, we expressed wild-type syndecan-4 or cytoplasmic domain mutants in syndecan-4-null fibroblasts. We generated cells expressing Syn4Y180L, which could not be phosphorylated on Y180, and Syn4Y180E, in an attempt to generate a phosphomimetic syndecan-4 that emulates c-Src-mediated phosphorylation of the Y180 residue A. ModulaCell movement has an essential reliance on precise coordination of dynamic cell-ECM interactions. Overall, FA dynamics are a function of adhesion contact formation, maturation, stabilization, translocation, dissociation, and the diffusion and mobility of individual FA components. In view of the effects of Syn4Y180 phosphorylation on migration, we sought to determine whether modification of this residue was involved in the regulation of FA dynamics. As the FA adaptor protein vinculin provides a physical link between ECM-bound integrins and the actin cytoskeleton, we initially used live-cell imaging of GFP-vinculin to monitor FA dynamics in Syn4WT-, Syn4Y180L-, and Syn4Y180E-expressing cells A and S2BTo assess the dynamics of FA components, we next used fluorescence recovery after photobleaching (FRAP). Expression of mutant syndecan-4 constructs had no significant effect on the GFP-vinculin mobile fraction D, but siThese data suggest that the phosphorylation status of Syn4Y180 dictates both the migratory characteristics of cells and the dynamics of their FAs. Indeed, Syn4Y180 appears to function as a switch between two distinct modes of behavior characterized either by accelerated turnover of FAs and a complete inability to migrate (Syn4Y180L) or by increased FA stabilization resulting in directionally persistent migration (Syn4Y180E).−/−, Syn4Y180L, or Syn4Y180E cells, which are all unable to be modified on Y180 by c-Src  were prespread on a fragment of fibronectin containing the integrin-binding central cell-binding domain (50K/Fn6-10) and stimulated with a soluble fragment of fibronectin containing the syndecan-binding domain (H0), as described previously . StimulaAs c-Src phosphorylates syndecan-4 on Y180, we tested whether c-Src activity could influence Arf6 activation. Steady-state Arf6 activity was assessed in cells expressing either wild-type or constitutively active c-Src . Consistent with the reduced levels of Arf6 activity observed in cells expressing phosphomimetic Syn4Y180E, expression of constitutively active c-Src significantly reduced Arf6 activity I. Import5β1 and αVβ3 integrins  substantially reduced migration of cells expressing endogenous syndecan-4, Syn4WT, and Syn4Y180E to levels similar to cells expressing nonphosphorylatable Syn4Y180L c-Src phosphorylates syndecan-4 directly on Y180, and this modification leads to a stabilization of FAs.(2)ECM engagement of syndecan-4 activates the small GTPase Arf6, and c-Src-mediated syndecan-4 phosphorylation suppresses Arf6 activity.(3)5β1 integrin to the plasma membrane, and suppresses trafficking of αVβ3, to promote FA turnover.Syndecan-4-mediated Arf6 activation drives recycling of α(4)5β1, increases levels of αVβ3 at the cell-ECM interface, and stabilizes FAs.Syndecan-4-dependent inactivation of Arf6 suppresses recycling of αIn this study, we have identified syndecan-4 phosphorylation as a key control point driving membrane delivery of specific integrin heterodimers to control FA dynamics and coordinate cell migration. Specifically, we show the following:5β1 or αVβ3 integrins are recycled to the membrane to coordinate FA dynamics. During cell migration, FA turnover and stabilization need to be controlled precisely to orchestrate protrusion, tail retraction, and the application of locomotive cytoskeletal forces  detected and developed with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) substrate (ABTS). Syndecan-4 tyrosine phosphorylation was calculated relative to total syndecan-4. Further details are available in Proteins were immunoprecipitated from cell lysate using 1–3 μg antibody and 45–80 μl protein G Sepharose beads for 1.5 hr at 4°C. Immune complex-bound beads were washed with lysis buffer and, where appropriate, heparitinase treated, and proteins were eluted with reducing sample buffer. Immunoprecipitated proteins and total cell lysates were resolved by SDS-PAGE or Phos-tag gel electrophoresis and detected by western blotting. Further details are available in Proteins were solubilized with SDS sample buffer, resolved by SDS-PAGE, and transferred to nitrocellulose. Proteins were detected using the Odyssey western blotting fluorescence detection system (LI-COR Biosciences), as described previously .33P]ATP (excluded for mass spectrometry samples) at 30°C for the appropriate time. Reactions were stopped with 3% phosphoric acid, and proteins were analyzed by mass spectrometry or resolved by SDS-PAGE and analyzed on a phosphoimager. Further details are available in Purified GST-tagged or recombinant custom-synthesized syndecan-4 cytoplasmic domain peptides were incubated with 43 ng active recombinant Src, 50 μM ATP, and 5 μCi [γ-3 cells at 37°C and 5% CO2 in MEF medium for 4 hr prior to filming. For scratch wound assays, 5 × 104 cells were plated on fibronectin-coated 24-well plates for 20 hr in MEF medium. Cell monolayers were wounded with sterile pipette tips. Time-lapse bright-field images were acquired on an AS-MDW live-cell imaging system (Leica) using a 5×/NA 0.15 Plan Fluotar objective and 1.5× magnification. Point visiting was used to allow multiple positions to be imaged within the same time course, and cells were maintained at 37°C and 5% CO2. Images were collected every 10 min over 14 hr (sparsely plated cells) or every 7.5 min over 15 hr and 20 min (scratch wound assays) using a Coolsnap HQ (Photometrics). Cell migration was tracked manually using the MTrackJ plug-in for ImageJ. Migration tracks were plotted using the Chemotaxis Tool and Manual Tracking ImageJ plug-ins.We coated 24-well plates with plasma fibronectin from solution (10 μg/ml) and plated 5 × 10Cells expressing GFP-vinculin were plated on fibronectin 24–48 hr posttransfection in the presence of 2% FBS. Time-lapse epifluorescence images were acquired on an Olympus inverted microscope (IX71) using DeltaVisionRT software (Applied Precision) (1 image every 7.5 min over 8 hr). Live-cell TIRF images were acquired on a TE2000 microscope (Nikon) using a 488 nm laser (1 image every 1.5 min over 2 hr) using point visiting and a Cascade 512B EM CCD camera (Photometrics). Details of FA translocation and lifetime analysis and data presentation are available in 1/2) and mobile fraction were calculated using softWoRx FRAP photokinetic analysis software (Applied Precision). The mobile fraction indicates the amount of a molecule that is freely mobile, whereas changes in the t1/2 signify alterations in diffusion or binding to, or release from, an immobile substrate within a complex .Cells expressing GFP-vinculin were plated on fibronectin 24–48 hr posttransfection and imaged at 37°C in Ham’s F12 medium supplemented with 2% FBS. FRAP was performed on an Olympus inverted microscope (IX71) equipped with a 488 nm FRAP laser under the control of DeltaVisionRT software (Applied Precision) using a 100×/NA 1.40 Plan Apo objective. Images were acquired every 3 s for 180 s postphotobleaching. One micrometer-diameter regions of interest were selected, and half-time of recovery (tCells were detached using trypsin/EDTA or enzyme-free Hanks’-based cell-dissociation buffer (Invitrogen), washed, immunolabeled, and analyzed on a Beckman Coulter Cyan ADP. Further details are available in For standard immunofluorescence, cells were plated on fibronectin-coated coverslips in the presence of 2% FBS for 3 hr, fixed, and immunostained. Immunofluorescent images were acquired on an Olympus IX71 using DeltaVisionRT software and analyzed using ImageJ. Further details are available in Recycling of surface-biotinylated receptors from the perinuclear recycling compartment was assessed as previously described  with theArf6 activity was assessed by GST-GGA3 pull-down assay, as described previously . Arf6 acData were analyzed using Student’s unpaired t tests or Z tests. p < 0.05 was considered statistically significant. p values and statistical tests are quoted in the figure legends.


OCTN2 - the Organic Cation Transporter Novel family member 2 (SLC22A5) is known to be a xenobiotic/drug transporter. It transports as well carnitine - a compound necessary for oxidation of fatty acids and mutations of its gene cause primary carnitine deficiency. Octn2 regulation by protein kinase C (PKC) was studied in rat astrocytes - cells in which β-oxidation takes place in the brain. Activation of PKC with phorbol ester stimulated L-carnitine transport and increased cell surface presence of the transporter, although no PKC-specific phosphorylation of Octn2 could be detected. PKC activation resulted in an augmented Octn2 presence in cholesterol/sphingolipid-rich microdomains of plasma membrane (rafts) and increased co-precipitation of Octn2 with raft-proteins, caveolin-1 and flotillin-1. Deletion of potential caveolin-1 binding motifs pointed to amino acids 14–22 and 447–454 as the caveolin-1 binding sites within Octn2 sequence. A direct interaction of Octn2 with caveolin-1 in astrocytes upon PKC activation was detected by proximity ligation assay, while such an interaction was excluded in case of flotillin-1. Functioning of a multi-protein complex regulated by PKC has been postulated in rOctn2 trafficking to the cell surface, a process which could be important both under physiological conditions, when carnitine facilitates fatty acids catabolism and controls free Coenzyme A pool as well as in pathology, when transport of several drugs can induce secondary carnitine deficiency. SLC22A5 gene, belongs to a superfamily of organic ion transporters, specific towards organic anions (OATs), urate (URAT) and organic cations (OCTs and OCTNs) for review see, +-independent way, transports a broad spectrum of organic cations, including xenobiotics/drugs as substrates +-dependent way SLC22A5 gene can cause systemic carnitine deficiency, classified as an inherited disease OMIM212149 Several solute transporters are important for proper functioning of astrocytes, moreover, their activity is a necessary prerequisite of a close cooperation in the brain between astrocytes and neurons, just to mention maintenance of neurotransmitters pool. Organic cation/carnitine transporter OCTN2 octn2 was shown to be up-regulated in peripheral tissues by peroxisome proliferators activator receptor α (PPARα), what correlated with an increased expression of genes coding several enzymes involved in β-oxidation of fatty acids octn2 confirms an important physiological role of carnitine in transfer of acyl moieties in form of carnitine esters through the inner mitochondrial membrane. However, such an up-regulation of octn2 by PPARα was not observed in astrocytes The OCTN2 can be also regulated post-transcriptionally, its mRNA was shown to be stabilized in endoplasmic reticulum by cartregulin +/Cl–dependent neurotransmitter transporters were reported to undergo internalization upon PKC stimulation Many plasma membrane transporters are regulated post-translationally by phosphorylation. Analysis of OCTN2 sequence reveals presence of 6 potential protein kinase C (PKC) phosphorylation sites 0,+Several transporting proteins have been reported to be localized at least partially in cholesterol/sphingolipid-rich microdomains - so-called rafts SLC22 gene superfamily, the present work was focused on the effect of this kinase on Octn2 in astrocytes, in particular on the phosphorylation status of the transporter, its activity, localization in membrane microdomains, as well as a possible interaction with raft proteins. Apart from lack of a direct Octn2 phosphorylation by PKC in rat astrocytes, activation of this kinase was correlated with increased carnitine transport and augmented Octn2 presence in plasma membrane, in particular in rafts. A direct interaction with caveolin-1 and amino acid sequence fragments responsible for this binding have been established.Since there is no information on trafficking regulation by PKC of transporters coded by 3H]carnitine and inulin[14C]carboxylic acid were from Amersham . Polyclonal antibody against rOctn2 peptide 537–553 0,+L--carnitine (600 Ci/mol), with inulin[14C]carboxylic acid as a control, was measured, as described in 2CO3, 1% SDS and samples were taken for radioactivity counting and protein estimation.Astrocytes were washed and incubated in either 137 mM NaCl, 10 mM HEPES, pH 7.4 or in 137 mM sodium gluconate, 10 mM HEPES, pH 7.4 for experiments without chloride. The cells were covered with 3 ml of the corresponding buffer and preincubated with either PMA alone or PMA with Bis II, as indicated in the Figure legend. Transport of 5 M L-[methyl-The mean ± SEM was calculated for each set of experiments and statistical analysis  was performed using one-way ANOVA with subsequent Bonferroni’s multiple comparison test. For Western blot analyses the representative blots have been shown and the number of independent experiments (n) has been indicated in Figure legends.+/Cl–dependent amino acid/carnitine transporter ATB0,+0,+ activity, results in a 25% decrease of carnitine transport, pointing to a substantial (75%) contribution of Octn2 in the process of carnitine accumulation in astrocytes. In the absence of Cl− ions, PMA stimulated carnitine transport by 25% . We further verified the phosphorylation status of proteins co-precipitating with Octn2 using two different clones of anti-phosphoserine antibodies recognizing phosphoserine in a vicinity of positively charged amino acids, a consensus site recognized by PKC r of 130,000 and 46,000, as recognized by PSR-45. It should be emphasized that clone 4A9 was previously shown by our group to detect an increased phosphorylation of a low affinity carnitine transporter ATB0,+, both in astrocytes and HEK293 cells treated with PMA 0,+ revealed a shift of the band recognized by anti-ATB0,+ antibody towards more acidic pH, indicating lower pI of the protein, what could result from phosphorylation  after PMA . Since ar about 250,000 , while x can be any amino acid, can bind to caveolin scaffolding domain Since the double deletion did not eliminate completely co-precipitation of mutated Octn2 with caveolin-1 and many transporters are known to form homooligomers, we wanted to verify if over-expressed Octn2 can form oligomers, in particular with the endogenous hOCTN2. Analysis with use of TEM confirmed Octn2 localization both in the plasma membrane and in the cytoplasm , as deteCo-precipitation of two proteins does not prove their direct interaction, therefore we used a proximity ligation assay which allows to detect interaction at the distance not exceeding 40 nm. As presented in SLC superfamily of genes, as reported for hetero-trimers of glutamate transporters SLC6 family +-dependent transporters with 12 transmembrane domains Octn2 present in rat astrocytes in majority is localized intracellulary . Although flotillin-1 can be phosphorylated by PKC, what correlated with internalization of dopamine transporter in vivo an important role in regulation of brain osmolarity. Octn2 regulation can be also important in understanding of secondary carnitine deficiency, known to be induced by several drugs, since OCTN2 is widely recognized as a drug transporter and among its substrates are β-lactam antibiotics, as well as oxaliplatin, varapamil, mildronate, valproate, imatinib and quinidine Oxidative metabolism of fatty acids in the brain was detected in astrocytes Our observations indicate that Octn2 can be regulated post-translationally by trafficking, a process dependent on formation of a multiprotein complex (containing caveolin-1 and flotillin-1) and regulated by PKC-dependent phosphorylation of other than Octn2 protein(s). Amino acids 14–22 and 447–454 of Octn2 sequence are involved in a direct interaction with caveolin-1. PKC activation directs this complex to rafts, leading in consequence to an increased transporting activity.


An exception was the case of the most kinase-proximal P-site-992, the catalytic site binding of which occurred exclusively in cis via an intramolecular reaction. We discovered that the in cis interaction of P-site-992 with the catalytic site was facilitated by a cleft between the N-terminal and C-terminal lobes of the PTK domain that allows the short CT domain sequence tethering P-site-992 to the PTK core to reach the catalytic site. Our work provides several new mechanistic insights into the EGFR self-phosphorylation reaction, and demonstrates the potential of coarse-grained molecular simulation approaches for investigating the complexities of self-phosphorylation in molecules such as EGFR (HER/ErbB) family receptors and growth factor receptor PTKs in general.Upon the ligand-dependent dimerization of the epidermal growth factor receptor (EGFR), the intrinsic protein tyrosine kinase (PTK) activity of one receptor monomer is activated, and the dimeric receptor undergoes self-phosphorylation at any of eight candidate phosphorylation sites (P-sites) in either of the two C-terminal (CT) domains. While the structures of the extracellular ligand binding and intracellular PTK domains are known, that of the ∼225-amino acid CT domain is not, presumably because it is disordered. Receptor phosphorylation on CT domain P-sites is critical in signaling because of the binding of specific signaling effector molecules to individual phosphorylated P-sites. To investigate how the combination of conventional substrate recognition and the unique topological factors involved in the CT domain self-phosphorylation reaction lead to selectivity in P-site phosphorylation, we performed coarse-grained molecular simulations of the P-site/catalytic site binding reactions that precede EGFR self-phosphorylation events. Our results indicate that self-phosphorylation of the dimeric EGFR, although generally believed to occur  The epidermal growth factor receptor (EGFR) is one of a large group of cell surface receptors that allow cells to respond to growth-stimulating signals in their environment. Upon sensing of growth factor, the EGFR is activated, which triggers a signaling cascade leading to the cell nucleus and ultimately initiating cell division. The first event following receptor activation is an intramolecular kinase reaction that results in the introduction of phosphate groups onto several specific amino acids (phosphorylation sites or P-sites) in the tail of the EGFR protein. Thus, the tail of the receptor undergoes self-phosphorylation, which involves conformational motions enabling the various P-sites to access the catalytic site. The structure of the tail of the receptor is unknown, and hence the mechanism of the self-phosphorylation reaction is not well understood. To investigate this mechanism, we generated a structural model of the EGFR protein and performed computer simulations of EGFR P-site/catalytic site binding reactions. These simulations indicated how the distribution of P-sites along the tail of the receptor and restrictions in molecular movements of the tail lead to selectivity in the phosphorylation of the different P-sites. Our simulations yielded unique insights into the mechanism of EGFR self-phosphorylation that have important biological implications. Classical polypeptide growth factor receptors are intrinsic membrane proteins, each possessing a ligand binding domain in the N-terminal extracellular portion of the molecule, a protein tyrosine kinase (PTK) domain in the C-terminal intracellular portion, and a single transmembrane (TM) domain in the intervening sequence cis mechanism) in the case of the receiver molecule or an intermolecular reaction (in trans) in the case of the activator molecule. As the roles of the activator and receiver PTK domains might be exchanged by a dissociation and ensuant reassociation of the kinases in the opposite orientation cis and trans mechanisms.A case in point is that of the prototypical epidermal growth factor receptor  PTK, about which there exists extensive structural information in vitro phosphorylation studies (employing purified EGFR protein) and live cell phosphorylation analyses have identified the various major and minor sites of phosphorylation  in growth factor receptor PTKs can alter the downstream signaling effectors activated and the physiologic response (e.g. induction of proliferation or cellular transformation in the case of oncogenic growth factor receptors) cis or trans mechanisms, is effectively intramolecular nature. We reasoned that the determinants of P-site usage might, in addition to the usual elements of enzyme-substrate recognition, also include topologic/configurational factors that influence the access of individual P-sites to the active site. Thus, because multiple P-sites within the lengthy CT domain of the EGFR can be phosphorylated and one CT domain can indeed be multiply phosphorylated When considering the dimeric EGFR as a molecular unit, the self-phosphorylation reaction, whether occurring by 2+ cations (i.e. an AMPPNP.2Mg2+ substrate complex)  in the active dimeric form see . This stlex) see .α atoms and physical interactions between pseudo-atoms were treated using the approach of Gō cis or trans interaction of each P-site with the active site of either the receiver molecule  or the activator molecule  was higher (p<0.0001) than that for P-sites of the activator molecule . Interestingly, the frequency of binding for individual P-sites of the receiver molecule (cis binding events) was highest (p<0.05) for the site most proximal in sequence to the PTK domain  and lower for more distal sites (e.g. P-site-1114 and -1148 each had 23 cis binding events each). With two exceptions, the frequencies of binding for differing P-sites of the activator molecule (trans binding events) mirrored those of the receiver molecule (cis binding events), ranging from 17 to 32 events. The exceptions here were that P-site-1086 of the activator molecule had a lower number of binding encounters (n = 17) than that of the receiver molecule (n = 25)  and that P-site-992 of the activator molecule had no encounters during the simulations. The discrepancy between the frequencies of cis versus trans binding for P-site-992 was responsible for the overall bias in favor of cis binding events, as when P-site-992 was ignored in the analysis, the numbers for cis (n = 184) versus trans (n = 167) binding events were not statistically different. Also, because the unusually high number of P-site-992 cis binding events was negated by a lack of P-site-992 trans binding events, the summed frequencies of cis and trans binding events for each P-site  than was P-site-992 of the activator molecule (a trans interaction). However, the relative frequencies of catalytic site interactions of individual P-sites did not seem to be entirely determined by topologic factors. Thus, P-site-1086 had the lowest frequency of catalytic site binding events . Thus, some P-sites in trajectories of EGFR molecular motion might make more frequent excursions near the catalytic site, independent of conventional enzyme/substrate recognition phenomena, and for this reason be more efficiently phosphorylated. To assess how topologic factors might alter the frequency of catalytic site interactions of individual P-sites, we examined the set of five hundred EGFR structures taken from five simulated trajectories of EGFR motion  to ascertain the frequency with which individual P-sites were in proximity of the catalytic site see  and 6, ients see , althougents see .in trans interaction with the catalytic site. This issue was explored by examining the conformations of the two CT domains in each of 650 structures sampled from trajectories of EGFR motion to identify those structures in which P-site Tyr-992 of either the receiver , it seemed that the lack of occurrences of in trans P-site-992/catalytic site binding events in our simulations  versus activator  molecule, we considered that it might be physically impossible for P-site 992 to make an receiver  or activreceiver  moleculeions see  was due in trans with the catalytic site, P-site-992 of the receiver molecule had the most catalytic site binding events of any P-site. In our randomization of the EGFR CT domain structure, P-site-992 of the receiver molecule (P-site-992A) made very frequent excursions near the catalytic site  with the active site than did other more distal P-sites, and this site interacted almost exclusively in cis. This indicated that the high frequency of P-site-992 interactions observed in our first simulations was largely attributed to topological factors that much facilitated the in cis interaction of P-site-992 with the active site, including the short length of the sequence between the catalytic domain and P-site-992 and the substrate binding cleft we identified above (see in cis (see p<0.05) in favor of trans (n = 32) versus cis (n = 8) binding events for P-site-1148 in simulations with no CT domain electrostatic interactions that was not evident in our initial simulations.To examine the role that electrostatic interactions might play in facilitating or inhibiting the interaction of individual P-sites with the catalytic site, we performed further simulations using an EGFR model in which the charges on all pseudo-atoms representing CT domain residues  were set to zero see . This EGbove see . Hence, bove see  and 9 ifbove see  with thein vivo, we considered that the pattern of P-site phosphorylation subsequent to an initial phosphorylation event might differ significantly from that of the first. Hence, we simulated P-site binding events subsequent to a first event that resulted in the phosphorylation of a single P-site, using the initial phosphorylation of P-site-992A of the receiver monomer (the most frequently bound P-site) as an example. The simulation method applied above was modified so that iterative simulations were initiated with structures randomly chosen from a set representing 59 different P-site-992A binding events that occurred in our initial P-site binding simulations  might occur ions see . Also, icis  versus trans  binding events (p<0.05). This is noteworthy in that in our earlier simulations  and was most obvious in the case of P-site-1045, with the binding in cis of P-site-1045 being the most frequent binding event overall (n = 57) and its binding in trans occurring much less frequently (n = 4). Our simulation results, appeared to reflect a potential processivity in EGFR phosphorylation, i.e. the phosphorylation of one P-site (here P-site-992 of the receiver) greatly enhanced the likelihood that a neighboring P-site (P-site-1045 of the receiver) would be bound to the catalytic site and in turn phosphorylated , which is presumably dependent upon the identity of the bound P-site. How the rates of phosphorylation of different P-sites sequences when bound in the catalytic site vary should be reflected in the steady state kinetics parameters reported by Fan et al.KM and kcat for several 17-amino acid P-site-derived peptide substrates of the EGFR kinase (see vphos) as would be determined biochemically can be predicted from the results of our P-site binding simulations by use of the relationk′intra is the relative frequency of binding site interactions for a given P-site as determined in our simulations performed without CT domain electrostatic interactions and kcat and KM are the catalytic constants for the phosphorylation of the corresponding P-site-derived peptide Supporting Information, vphos for those P-sites for which the steady state kinetic parameters of the corresponding peptides are available are given in vphos values, and thus would be predicted to be efficiently phosphorylated, in agreement with the latter three being identified as major sites of phosphorylation. The low value of vphos for P-site-1086, in part a reflection of its very low kcat/KM ratio, is not consistent with its prior identification as a major site of phosphorylation. Conversely, while this analysis predicts that P-site-992 would be the most efficiently phosphorylated, it has not been considered a major site of EGFR phosphorylation. It should be recognized that our characterization of major and minor sites of EGFR phosphorylation was based upon the mainly qualitative biochemical analyses summarized in in vitro EGFR phosphorylation under controlled conditions, experimentation that we are now initiating.The numbers of catalytic site encounters for the various P-sites of the EGFR CT domain occurring in our simulations should be related to the relative levels of P-site phosphorylation seen in biochemical experiments. We recognize that the frequencies of P-site binding seen in our simulations would not be exact predictors of the relative rates of P-site self-phosphorylation, given that they relate only to the substrate binding event and do not take into account the rate of the subsequent catalytic phospho-transfer reaction  with the active site of the activator (see Supporting Information). Thus, if a P-site of either CT domain interacted with the active site of the activator , it tended to preclude the interaction of other P-sites in the same CT domain with the true catalytic site. In some cases, a productive P-site/catalytic site binding event was preceded by several nonproductive P-site binding events involving the active site of the activator molecule  and with or without classical electrostatic interactions in the case of formally charged residues. Obviously, this model might not accurately recapitulate the atomistic steric interactions involved in active site substrate recognition and only approximates the sampling of conformational states of the CT domain polypeptide that occurs in the course of its movement. In terms of the former issue, we attempted to reintroduce those elements of substrate recognition lost in our modeling and the P-site-dependence of the rate of the catalytic phospho-transfer reaction by a simple theoretical treatment that takes advantage of published steady state catalytic constants for P-site phosphorylation. Regarding the latter issue, we must concede that the CT domain conformational sampling occurring in our simulations is an approximation of that occurring in reality, and the conclusions we make concerning the relative propensities of individual P-sites for active site binding are therefore tentative. We do consider that our simulations have identified possible new paradigms with regard to EGFR/HER family receptor signaling that might not otherwise have been discovered. These include the possibilities that cis and trans self-phosphorylation within dimeric receptors might occur with similar efficiencies and that the in cis interaction of P-site-992 with the catalytic site might be facilitated by a substrate binding cleft and could represent a unique evolutionarily conserved mechanism for the self-phosphorylation of this crucial regulatory P-site.Although our coarse-grained modeling of the EGFR self-phosphorylation reaction was necessitated by the large numbers of atoms involved and the limitations of the computational machinery available, we must recognize some limitations of our simulations. In the model employed, amino acid residues were represented by pseudo-atoms centered on the CIn summary, the investigation by simulation of the first step of the EGFR self-phosphorylation reaction we present here provides significant new insights into the mechanism of self-phosphorylation, and raises several intriguing questions about this mechanism in the contexts of the larger HER/ErbB receptor family and growth factor receptor PTKs in general. Issues that remain to be explored include the selectivity of P-site phosphorylation in heterodimeric receptors, such as in the highly transforming HER2/HER3 coreceptor combination that drives breast cancer progression A structure of the full-length active dimeric EGFR was created from known structures of the dimeric extracellular domain (3NJP) Because we wished to have a complete EGFR dimer model with structurally identical extracellular ligand binding domains, versus the nearly identical extracellular domains seen in the crystal structure 3NJP, we superposed a copy of molecule A of the 3NJP dimer structure upon molecule B in the original structure by aligning selected residues in the nearly symmetric contact interface between molecules A and B (residues 191 to 308 and 573 to 614). Thus, we constructed a nearly symmetrical EGFR extracellular domain dimer composed of structurally identical extracellular domains (residues 1 to 614) each with a bound EGF molecule, specifically residues 5 to 51 of the 53-residue EGF sequence that are ordered in the structure 3NJP. Subsequently, two transmembrane domain structures (residues 622–644) were modeled as α-helices by use of Swiss PDB, and remaining structural elements of the dimeric EGFR holoreceptor were modeled as follows.The several structural elements described above  were manually reoriented with respect to each other in the Swiss PDB viewer such that they might be connected by modeling the missing structural elements, specifically residues 615 to 621 of the two extracellular JM domains, residues 645 to 654 of the intracellular JM sequence proximal to the active conformation kinase domain, residues 645 to 654 and 665 to 678 of the intracellular JM sequence that connect the TM domain, JM domain helical segment, and inactive conformation kinase structures, and lastly, the two CT domains of the active (residues 968 to 1186) and inactive (960 to 1186) conformation kinases.  The several segments connecting known structures were structurally modeled with the Loopy algorithm 2+ cations, as would be presumed to be the relevant substrate for the self-phosphorylation reaction, we docked the nucleotide substrate complex (AMPPNP with two chelated Mg2+ cations) from a structure of the active insulin receptor PTK domain with bound nucleotide and peptide substrates (1IR3) .2Mg2+ complex in 1IR3 with the AMPPNP in the 2GS7-derived structure, which again appropriately placed the substrate complex in the active site.As no available active conformation EGFR kinase structure contained a nucleotide substrate complex of ATP  and two bound MgN-4 to N+4, where residue N is the P-site tyrosine, see Each CT domain in the active receptor dimer contains eight candidate P-sites that potentially interact with the active site of the receiver molecule  and become phosphorylated. Our simulation of the self-phosphorylation reaction therefore required the generation of structural models descriptive of the interaction of each P-site with the EGFR catalytic site. Because in most cases the primary determinants of protein kinase substrate specificity are located within four residues on either side of the target residue α atom with bonds formed between pseudo-atoms of adjacent residues. Models of the bound AMPPNP.2Mg2+ complexes comprised pseudo-atoms centered on atoms C2, C6 and N9 of the adenine moiety, C4′ of the deoxyribose, P1, P2 and P3 of the α-, β-, and γ-phosphates, and each of the two Mg atoms, with bonds connecting every pseudo-atom pair. The energetic model was the summation of (1) standard molecular mechanics potential functions to treat interactions between bonded pseudo-atoms, (2) Gō-inspired potential functions to model short-range attractive interactions between those non-bonded pseudo-atoms representing native contacts, (3) short-range repulsive potentials to treat interactions between all other non-bonded pseudo-atoms, and (4) electrostatic potential terms relating to all charged residue/atom pairs  The force constants kbond and kangle were set to 20 kcal/mol/Å2 and 10 kcal/mol/rad2, respectively, and the energy maxima V1 and V3 for the dihedral terms were set to 0.5 and 0.25 kcal/mol, respectively.In the energetic model, the interactions between bonded pseudo-atoms were described byi and j that represented native contacts. A residue was considered to form a native contact if any of its atoms were within a specified distance cut-off (here 5.5 Å) of any of another residue in the native structure, with exceptions as described below. Here the energy well depth ε was assigned a value of 0.6 kcal/mol, and ijσ and ijr are the distances separating the pseudo-atoms in the native and evolving structures, respectively. Non-bonded pseudo-atoms not representing native contacts were modeled with strictly repulsive terms:σvdw set here at 4 Å and ε at 0.6 kcal/mol. In addition to these bonded and non-bonded energy terms, any pseudo-atom representing a residue (or atom in the AMPPNP.2Mg2+ substrate complex) with a significant partial charge at the simulation pH (here 7.6) was assigned that partial charge and allowed to interact with other such pseudo-atoms via additional Debye-Hückel electrostatic potential termsiq and jq are the charges on pseudo-atoms i and j, κ is the ionic strength (set at 0.15 M), rij is again the distance between the pseudoatoms.The interactions between non-bonded pseudo-atoms (those separated by three or more residues) were modeled with additional short-range attractive potential functionsz-axis  and separated by 33 Å, and additional terms in the energetic model that held the TM domain residues between the membrane planes and kept extracellular and intracellular EGFR structural elements from penetrating the membrane. Specifically, each pseudo-atom representing one of the TM residues 622 to 644 was subject to a restraining potentialz0 and z are the initial and evolving, respectively, displacements of the TM pseudo-atom along the z-axis and krestraint is a force constant set at 5.0 kcal/mol/Å2. Also, extracellular pseudo-atoms (those of the receptor extracellular domains and bound EGF molecules) were subject to half-harmonic repulsive potential termsz is the evolving z-coordinate of the extracellular pseudo-atom, zwall indicates the position (16.5 Å) of the extracellular membrane, and kwall is a force constant set again at 5.0 kcal/mol/Å2. Analogous repulsive potential terms applied to intracellular pseudo-atoms (those of the intracellular domains and the nucleotide substrate complexes).A membrane in which the dimeric receptor was constrained to diffuse laterally was modeled with two planar barriers normal to the .2Mg2+ complexes representing atoms within 5.5 Å of any atom of an active site residue in the all-atom model were considered to form native contacts and assigned short-range attractive potential terms corresponding to these interactions. Secondly, those residues of the eight different nine-amino P-site sequences that formed contacts with active site residues (or atoms of the AMPPNP.2Mg2+ substrate complex) when docked in the kinase active site were also considered to make native contacts (again a 5.5 Å cutoff was applied). In the case of each P-site peptide, the P-site tyrosine in the docked structure formed nine total native contacts with active site residues (in addition to three involving the γ-phosphate of the bound AMPPNP substrate), and the simultaneous formation of these nine contacts in the course of a simulation was used to define a stable P-site/catalytic site interaction. This was analogous to evaluating the extent of protein folding using a reaction coordinate or order parameter (e.g. Q) defined as the fraction of native contacts formed in the evolving system The all-atom structural model of the dimeric EGFR from which our coarse-grained model was generated contained elements of known structure derived from published crystal structures, as well as some modeled structural elements (e.g. sequences in the extracellular and intracellular JM domains and the entirety of the two CT domains). Only those pseudo-atoms representing residues in elements of known structure were considered able to form native contacts and thus interact with other such non-bonded pseudo-atoms via short-range attractive versus purely repulsive potentials, which ensured that the folding of the structured domains remained intact during dynamic simulations. There were two exceptions. Firstly, those pseudo-atoms of the active site-bound AMPPNP.2Mg2+ substrate complex model set at 3.5 Å. In the course of P-site binding simulations, the number of P-site tyrosine/catalytic site native contacts formed was periodically determined, and the simulation terminated if all of the nine possible contacts were simultaneously formed in the case of any P-site. Simulations were performed in sets of five on separate Dell PowerEdge R815 computers, each with four AMD Opteron 6168 12-core processors. Snapshots of simulations were saved every 100 psec for analysis.Given the above coarse-grained structural and energetic model, the motion of the dimeric EGFR structure was propagated using a Langevin dynamics algorithm developed by Winter and Geyer A multinomial test was used to determine if differences in the frequencies of P-site binding were statistically significant.In structural modeling, homologous structural elements were aligned using the superpositioning functions of the Swiss PDB program Figure S1Influence of initiating structures on the relative frequency of simulated P-site binding events. P-site binding simulations were performed iteratively on five computers with initial structures randomly chosen from five different sets of one hundred structural models derived from five trajectories of 10 µsec randomizations of independent EGFR structures (see t) separating the two initial structures used in the simulations, and made a histogram of the number of same-site binding events versus Δt. Because the relative likelihood (p) that two times chosen randomly from a total time interval T (here 10 µsec) are different by a time Δt decreases linearly with Δt according to p = 2.(1−Δt/T), we evaluated for comparison the frequency with which differing Δt values would arise via a random selection of the same total number of pairs of time values. If simulations initiated with structures more closely spaced in trajectory time had an increased tendency to result in the binding of the same P-site, the observed frequency of same-site binding events (Simulated Events) with smaller Δt values would be greater than the frequency with which such Δt values would arise from randomly selecting pairs of times from within the interval T (Uncorrelated Events). The analysis showed that only for pairs of initial structures most closely spaced in trajectory time (<500 nsec) was there a significantly higher propensity for same-site binding events. This indicated that although the propensity for binding of a given P-site in a simulation was somewhat dependent upon the initiating structure, the diversity of the set of initiating structures sampled from the 10 µsec trajectories of the five independent structural randomizations would largely negate any dependence of the observed frequencies of interaction of differing P-sites upon the initiating structures.(TIF)Click here for additional data file.Figure S2Potential competition of the non-catalytic active site with the true catalytic site in P-site binding. In some simulations, the binding of a P-site to the catalytic site in receiver molecule appeared to be delayed by the binding of a P-site(s) to the alternative active site in the activator molecule, which restricted the access of the CT domain containing that P-site to the catalytic site . When P-site binding was especially delayed, there were often a number of nonproductive interactions of a P-site with the activator active site that preceded the binding of a P-site to the true catalytic site. Shown here are plots of P-site/activator active site distances for P-sites interacting nonproductively with the active site of the activator molecule over the course of one such simulation taken from those described in (TIF)Click here for additional data file.Video S1Representative P-site binding simulation showing competition between active sites. Animation showing a trajectory from a representative P-site binding simulation in the first of three views . Pseudo-atoms of the receiver and activator receptor monomers are represented as beads colored in shades of pink and blue, respectively, with bound growth factor molecules in dark blue. In both monomers the catalytic Asp-813 residue is colored cyan and the tyrosines of all P-sites are colored green. The 1840 trajectory images represent 1 nsec intervals during the complete 1.8 µsec simulation. This first animation shows the motion of the dimeric EGFR from a view through the plane of the membrane. The simulation begins with P-site-1148A of the receiver monomer binding to the active site of activator (see close-up in (MPG)Click here for additional data file.Video S2Representative P-site binding simulation showing competition between active sites: view of receiver catalytic site. Close-up animation of the trajectory shown in (MPG)Click here for additional data file.Video S3Representative P-site binding simulation showing competition between active sites: view of activator active site. Close-up animation of the trajectory shown in (MPG)Click here for additional data file.Text S1Interpretation of simulated P-site binding event frequencies. A theoretical digression on the prediction of P-site phosphorylation rates by use of the frequencies of P-site binding observed in simulations and the steady state catalytic parameters for phosphorylation of P-site-derived peptides (citing references (PDF)Click here for additional data file.


Glutathione transferases (GSTs) are dimeric enzymes containing one active-site per monomer. The omega-class GSTs (hGSTO1-1 and hGSTO2-2 in humans) are homodimeric and carry out a range of reactions including the glutathione-dependant reduction of a range of compounds and the reduction of S-(phenacyl)glutathiones to acetophenones. Both types of reaction result in the formation of a mixed-disulfide of the enzyme with glutathione through the catalytic cysteine (C32). Recycling of the enzyme utilizes a second glutathione molecule and results in oxidized glutathione (GSSG) release. The crystal structure of an active-site mutant (C32A) of the hGSTO1-1 isozyme in complex with GSSG provides a snapshot of the enzyme in the process of regeneration. GSSG occupies both the G (GSH-binding) and H (hydrophobic-binding) sites and causes re-arrangement of some H-site residues. In the same structure we demonstrate the existence of a novel “ligandin” binding site deep within in the dimer interface of this enzyme, containing S-(4-nitrophenacyl)glutathione, an isozyme-specific substrate for hGSTO1-1. The ligandin site, conserved in Omega class GSTs from a range of species, is hydrophobic in nature and may represent the binding location for tocopherol esters that are uncompetitive hGSTO1-1 inhibitors. The inducible phase II enzymes known as glutathione transferases  conjugate endogenous and xenobiotic toxins with electrophilic centers to glutathione . Several classes function as glutathione peroxidases or as reductases Like all cystosolic GSTs, the omega-class isozymes have an N-terminal thioredoxin-like domain and a unique helical C-terminal domain via several pathways. Some α-haloketones have been identified as metabolites of insecticides kcat) by a factor of five Recently the role of omega-class GSTs in the disposition of α-haloketones has been investigated. The α-haloketones are a class of biologically active compounds that can enter the human body In addition to activities involving GSH and its conjugates, several classes of GST have been shown to exhibit “ligandin” activity, i.e., non-catalytic ligand binding. In the case of a squid sigma- and a blood fluke mu-class GST, this has been demonstrated to occur in the dimer interface In this report, we have describe a crystal structure in which GSSG is observed in the active site of an inactive hGSTO1-1 mutant (C32A), giving us a snapshot of enzyme regeneration occurring. The same structure reveals the binding of 4NPG in the dimer interface, revealing a non-catalytic ligandin binding site.Escherichia coli BL21 (DE3) cells as an N-terminal poly-His-tagged ubiquitin fusion protein from the pHUE plasmid Protein was purified as described previously FO-DFC or mFO-DFC electron density maps. POINTLESS was therefore used to combine the two datasets and ascertain their Laue symmetry before scaling with SCALA. The starting model for refinement was again the previously published structure, (PDB code: 1EEM) FO-DFC density was performed with COOT Data was subsequently collected remotely at the SSRL using an X-ray wavelength of 1.034375 Å (12 keV). X-ray data was processed using software within the CCP4 suite The final structure contains one protomer (residues 4 to 241), two sulfate molecules, one each of 4NPG, GSSG and DTT molecules. A total of 140 water molecules were built into the model. The asymmetric unit contains a single monomer: the physiologically relevant dimer is produced by two-fold crystallographic symmetry. The crystals of hGSTO1-1 C32A mutant are similar to that reported for the wild-type enzyme Schistosoma japonica4NPG binds in the dimer interface, deep within the cleft formed between monomers. Because it sits on a crystallographic two-fold axis, the electron density corresponds to two overlapping 4NPG molecules at half-occupancy . The gluArabidopsis thalianaSchistosoma japonicum50 of 4 µM Some members of the GST family of enzymes were originally identified as “ligandins”, due to their apparent capacity to bind a wide variety of large (>400 Da) lipophilic compounds such as bile acids, fatty acids and certain drugs. This function was thought to play a role in storage and transport of these compounds in the aqueous phase of the cell Bombyx mori GSTO3-3 Sphingobium sp. SYK-6 LigG Phanerochaete chrysosporium GSTO3-3 Phanerochaete chrysosporium GSTFuA Bombyx mori GSTO3-3 and Sphingobium sp. SYK-6 LigG equivalent to the hGSTO1-1 L-site appear more occluded (Phanerochaete chrysosporium GSTO3-3 and GSTFuA is significantly altered due to the fundamentally different nature of dimerization interactions in these GSTs: the putative L-site regions are no longer on the dimer interfaces and are more solvent exposed (It is instructive to compare the newly identified L-site with features in other omega-class and related GSTs. Residues in hGSTO1-1 binding the 4-Nitrophenacyl moiety are conserved or conservatively substituted in homologues from other species, and in hGSTO2 occluded , ligandi exposed . It is nProteus mirabilis enzyme Ochrobactrum anthropi beta-class GST has been observed Ochrobactrum anthropi beta-class GST and GSH has not yet been observed Like the Omega-class GSTs, the beta-class GSTs from bacteria feature an active-site cysteine that forms a mixed disulfide with GSH, as demonstrated in the crystal structure of the A snapshot of hGSTO1-1 in the process of being regenerated has been observed by crystallography. We show that a GSSG molecule can bind in the active site, with one half of the molecule in the canonical G-site, and the other half in the H-site. There are few specific interactions of the glutathionyl moiety bound in the H-site. This apparent lack of specificity gives a possible explanation as to why other sulhydyl containing compounds can substitute for GSH in the recycling of oxidized hGSTO1-1. We have further identified a potential non-catalytic ligand-binding site in the dimer interface that may be the binding location of uncompetitive inhibitors such as tocopherol.