N-methyl-d-aspartate receptors (NMDARs) are ligand-gated ion channels (‘ionotropic’ receptors) activated by the major excitatory neurotransmitter, l-glutamate. While the term ‘the NMDAR’ is often used it obscures the fact that this class of receptor contains within it members whose properties are as different as they are similar. This heterogeneity was evident from early electrophysiological, pharmacological and biochemical assessments of the functional properties of NMDARs and while the molecular basis of this heterogeneity has taken many years to elucidate, it indicated from the outset that the diversity of NMDAR phenotypes could allow this receptor family to subserve a variety of functions in the mammalian central nervous system. In this review we highlight some recent studies that have identified structural elements within GluN2 subunits that contribute to the heterogeneous biophysical properties of NMDARs, consider why some recently described novel pharmacological tools may permit better identification of native NMDAR subtypes, examine the evidence that NMDAR subtypes differentially contribute to the induction of long-term potentiation and long-term depression and discuss how through the use of chimeric proteins additional insights have been obtained that account for NMDAR subtype-dependency of physiological and pathophysiological signalling.This article is part of the Special Issue entitled ‘Glutamate Receptor-Dependent Synaptic Plasticity’. ► Reviews the control of NMDAR functional properties by GluN2 subunits. ► Assesses structural elements of GluN2 subunits that determine NMDAR heterogeneity. ► Considers the utility of orthosteric and allosteric ligands acting at NMDARs. ► Potential of the NMDAR C-terminal domain as a novel therapeutic target is discussed. The obligate GluN1 subunit harbours the co-agonist glycine (d-serine) binding site and, although being encoded by a single gene, can exist in eight, spatially and temporally regulated, isoforms  which arise from the alternative RNA splicing of exons 5, 21 and 22  represents the most highly conserved portion of NMDAR subunits and consists of three transmembrane helical segments  with an additional a short re-entrant ‘P’-loop (M2) between the M1 and M3 segments. The crystal structure of the closed AMPAR pore demonstrates that the M3 helices interact close to the iGluR-conserved ‘SYTANLAAF’ motif at the extracellular side of the membrane to form a constriction that forms the channel gate . For the2+ block .The large (>600 amino acids in the GluN2A and GluN2B) intracellularly located CTD is least conserved between GluN2 . The CTD32+, their permeability to Ca2+ and their single-channel conductance  each NMDAR subtype performs. The repertoire of NMDAR subtype-selective antagonists and negative and positive allosteric modulators has been extensively reviewed recently  and whilR--α-(4-hydroxyphenyl)-β-methyl-4-(phenylmethyl)-1-piperidine propranol -1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol -[(S)-1-(4-bromo-phenyl)-ethylamino]--methyl]-phosphonic acid (NVP-AAM077)   showed 1fference . Residuefference . Indeed fference  where NVfference . In otheN-[4-[[2-(phenylcarbonyl)hydrazino]carbonyl]benzyl]benzenesulphonamide) and TCN 213 (N-(cyclohexylmethyl)-2-([4-thiadiazol-2-yl]thio)acetamide) each antagonise GluN1/GluN2A NMDAR-mediated currents in a non-competitive but glycine-dependent manner (d-serine) is used at a concentration of 30 μM (to ensure saturation at the GluN1 co-agonist binding site) complete inhibition of a GluN1/GluN2A NMDAR-mediated cannot be achieved (d-serine) in experimental preparations so that the expected level of inhibition of GluN1/GluN2A NMDAR-mediated responses can be predicted – this is not always possible in, for example, in vitro slice preparations. In addition it should be remembered that many formulations of culture media contain high concentrations of glycine that are considered to be saturating with respect to the NMDAR glycine binding site and therefore the TCN compounds are rendered ineffective under such conditions. Despite these caveats, non-competitive glycine-site negative allosteric modulators such as TCN 201 do not suffer from the same equilibrium/non-equilibrium issues that are highlighted above when using potent competitive glutamate-site antagonists if it is assumed that steady-state conditions are achieved prior to the synaptic glutamate release. The discovery of a novel allosteric binding site in GluN1/GluN2A NMDARs raises the potential for future development of more potent ligands that possess better solubility to allow for the selective and complete block of GluN2A-containing NMDARs.Recently, however, novel GluN2A-selective inhibitors have been identified  which sht manner . In addit manner  Fig. 6)N-[4-[[2-t manner  and it iachieved . FurtherR*,3S*)-1-(phenanthrene-3-carbonyl)piperazine-2,3-dicarboxylic acid -4-(6-methoxy-2-(3-nitrostyryl)-4-oxoquinazolin-3(4H)-yl)-benzoic acid  that are responsible, along with the CTD of GluN1, for linking the NMDAR to an array of signalling and scaffolding proteins that together form the NMDAR signalling complex . The amivice-versa), the effects of the CTD subtype can be investigated in isolation. These approaches are valid because there is no evidence that swapping the GluN2 CTD subtype would influence the gating properties of the NMDAR. Indeed, this possibility has now been directly tested and ruled out (An important requisite of such studies is to uncouple any influence of the channel/ligand-binding portion of the GluN2 subunit from that of the C-terminus. An approach taken by several labs has been to investigate the functional consequences of the expression of both wild-type GluN2A and GluN2B compared to expression of chimeric GluN2 subunits in which the CTD of one subunit has been switched with that of the other. By studying, for example, the consequence of GluN2B expression compared with that of expressing GluN2B with its CTD replaced with that of GluN2A (and uled out .Using this approach Sheng and co-workers demonstrated an important role for the GluN2B CTD in hippocampal LTP: knockdown of GluN2B inhibited LTP and could be rescued by expression of a RNAi-resistant form of GluN2B but not by GluN2A or by GluNB with its CTD replaced by that of GluN2A . ConceptGrant, Komiyama and co-workers recently used an alternative, and elegant, approach which enabled them to study the role of GluN2 CTD subtype in the whole animal . Rather 2+ influx to neuronal death. Indeed, this was observed regardless of whether the GluN2B was attached to the rest of the GluN2B subunit, or as part of a chimeric subunit linked to GluN2A. In collaboration with Grant and Komiyama we studied excitotoxicity in vitro and in vivo utilizing their mouse expressing GluN2B with its CTD replaced with that of GluN2A. In exact agreement with our ectopic expression studies, replacing GluN2B's CTD with that of GluN2A reduced neuronal sensitivity to NMDAR-dependent Ca2+ influx both in vitro culture and in vivo following an excitotoxic insult (We recently addressed the role of GluN2 CTD subtype in determining NMDAR excitotoxicity . Ectopicc insult . The molc insult . The toxc insult . Under tc insult  and tranc insult  with delc insult .It will also be important to determine whether GluN2 CTD subtype determines the progression or severity of neurological conditions associated with abnormal or inappropriate NMDAR function, such as ischaemia, traumatic brain injury or chronic progressive disorders such as Huntington's disease. Neuroprotective peptide mimetics of the GluNB CTD and downstream interacting proteins are an emerging area of interest, with several showing promise in rodent stroke models . ParticuMoreover NA-1/Tat-NR2B9c was shown to be effective post-treatment in reducing lesion size and improving outcome following stroke in the non-human primate brain . NA-1 ha8In this review we have focussed on the role played by the GluN2 subunit in controlling NMDAR function. These subunits are critical determinants of NMDAR heterogeneity and provide the major influence in determining NMDAR subtype biophysical, pharmacological and signalling properties. In recent years our rapidly advancing understanding of NMDARs has allowed for the identification of structural elements and, at times, single amino acid residues, which determine GluN2-specific control of function. This understanding provides us with opportunities to develop novel pharmacological agents with which we can probe NMDAR subtype-specific physiological and pathophysiological functions, the ultimate aim of which is the treatment of neurological dysfunction. Indeed, as indicated in the final section of the review selective targeting of GluN2-dependent function shows such strategies possess considerable clinical promise.


Drosophila, Hh dependent phosphorylation activates Smo. Whether this is also the case in vertebrates is unclear, owing to the marked sequence divergence between vertebrate and Drosophila Smo (dSmo) and the involvement of primary cilia in vertebrate Hh signaling. Here we demonstrate that mammalian Smo (mSmo) is activated through multi-site phosphorylation of its carboxyl-terminal tail by CK1α and GRK2. Phosphorylation of mSmo induces its active conformation and simultaneously promotes its ciliary accumulation. We demonstrate that graded Hh signals induce increasing levels of mSmo phosphorylation that fine-tune its ciliary localization, conformation, and activity. We show that mSmo phosphorylation is induced by its agonists and oncogenic mutations but is blocked by its antagonist cyclopamine, and efficient mSmo phosphorylation depends on the kinesin-II ciliary motor. Furthermore, we provide evidence that Hh signaling recruits CK1α to initiate mSmo phosphorylation, and phosphorylation further increases the binding of CK1α and GRK2 to mSmo, forming a positive feedback loop that amplifies and/or sustains mSmo phosphorylation. Hence, despite divergence in their primary sequences and their subcellular trafficking, mSmo and dSmo employ analogous mechanisms for their activation.Hedgehog (Hh) signaling regulates embryonic development and adult tissue homeostasis through the GPCR-like protein Smoothened (Smo), but how vertebrate Smo is activated remains poorly understood. In  Drosophila to human, and its malfunction has been implicated in a wide range of human disorders. Hh signal is received by the twelve-transmembrane receptor Patched and transmitted intracellularly by the seven-transmembrane protein Smoothened (Smo). How vertebrate Smo is activated in order to transmit the Hh signal remains poorly understood. Here we investigate the molecular mechanism of mammalian Smo (mSmo) activation and find it is similar to that described for Drosophila Smo despite the marked sequence divergence between them. We show that mSmo is activated via phosphorylation at multiple sites by the serine/threonine kinases CK1α and GRK2. We provide evidence that Sonic hedgehog  can regulate the accessibility of mSmo to these kinases and that phosphorylation promotes the ciliary accumulation of this transmembrane protein in its active conformation. Moreover, increasing concentrations of Shh induce a progressive increase in mSmo phosphorylation that fine-tunes mSmo activity. Thus, our results provide novel insights into the biochemical mechanism of vertebrate Hh signal transduction and reveal a conserved mode of Smo activation.Hedgehog (Hh) signaling governs embryonic development and adult homeostasis in species ranging from  The Hh family of secreted proteins plays pivotal roles during embryonic development and adult tissue homeostasis R). Hh signaling blocks Gli processing and converts full-length Gli2/3 into activator forms (GliA). The reception system for the Hh signal consists of a twelve-transmembrane protein Patched (Ptc) as the Hh receptor and a seven-transmembrane protein Smoothened (Smo) as the obligatory Hh signal transducer R and GliA are still poorly understood.Hh exerts its biological function through a signaling cascade that ultimately controls a balance between activator and repressor forms of the Gli family of transcription factors Drosophila and mammalian Smo proteins exist as constitutive dimers/oligomers, but in the absence of Hh, Smo C-tails adopt a closed conformation that prevents their association. Hh induces a conformational switch in Smo, leading to dimerization/oligomerization of the C-tails In mammals, Hh signaling depends on the primary cilium, a microtubule-based membrane protrusion found in almost all mammalian cells Drosophila, Hh and Ptc reciprocally control Smo cell surface accumulation and conformation through regulating Smo phosphorylation Drosophila Smo and do not contain the PKA/CK1 phosphorylation clusters found in Drosophila Smo C-tail Drosophila Smo are regulated by fundamentally distinct mechanisms In Several studies suggested that G protein coupled receptor kinase 2 (GRK2) positively regulates Shh signaling In this study, we investigate the activation mechanism of mammalian Smo (henceforth referred to simply as Smo). We demonstrate that Smo is activated via multiple phosphorylation events mediated by CK1α and GRK2 that induce its ciliary accumulation and active conformation. We provide evidence that graded Shh signals induce increasing levels of Smo phosphorylation that fine-tune Smo ciliary localization, conformation, and activity. In addition, we provide evidence that Shh promotes Smo phosphorylation by regulating the accessibility of Smo to its kinases.Gli-luciferase (Gli-luc) reporter gene, although the fold of activation was less dramatic compared with Shh stimulation ; however, Smo C-tail folds back and is in close proximity to the intracellular loops, resulting in high intramolecular FRET between CFP inserted in the second intracellular loop (L2) and YFP fused to the C-terminus (FRETL2C) and low intermolecular FRET between CFP and YFP fused to the C-termini of two Smo molecules (FRETC)   [22]. Shng FRETN  [22], suFRETN  608–670 and aa 770–793, were phosphorylated by both CK1 and GRK  and aa 770–793 contains one sequence (DADS791DF) that match GRK consensus sites , S642 (SA2), or S666 (SA3) reduced and their combined mutations (SA123) abolished phosphorylation of aa 608–670  or GRK  in the presence of γus sites . Indeed,3 by GRK , lane 141–3S/T 615, S619, and S620 mediated CK1 phosphorylation of aa 608–670  abolished CK1 phosphorylation of aa 581–612 (CK1 phosphorylation sites conform to the consensus: D/E/S/T(P)Xd by CK1 , lane 8, 581–612 , lane 19592, S594, T597, and S599 collectively as S0; S615, S619, and S620 as S1; S642 as S2; S666 as S3; S774 and S777 as S4; and S791 as S5  that specifically recognizes phosphorylated S1  promote whereas cyclopamine blocks Smo activation Gli-luc reporter assay in smo−/− MEFs. Mutating S0 (SA0) or S1 (SA1) slightly reduced whereas their combined mutations (S01) markedly inhibited Shh-induced Smo activity  to mimic different levels of phosphorylation . SD mutaTo determine whether the oncogenic mutation activates Smo through its phosphorylation, we mutated several CK1/GRK sites to Ala in SmoA1. Mutating S2/3  had little if any effect on SmoA1 activity . In contWe also found that mutating SA0–5 abrogated SAG-induced Smo activation, whereas SD0–5 exhibited resistance to cyclopamine inhibition , suggestΔloop2 (PtcΔ2), as well as prevented the derepression of the dorsal marker Pax7  or mutant forms of Smo were electroporated into one side of the neural tube, leaving the other side as an internal control, followed by immunostaining to visualize the expression of various Hh responsive genes. Electroporation of SmoWT or Smo variants that mimic low-level phosphorylation  did not significantly alter the expression of the marker genes ; howeverker Pax7  [42]. ThIn line with tissue culture experiments, SmoA1 is more potent than SmoSD variants in inducing ectopic expression of ventral marker genes in chick neural tubes . MutatinShh induces ciliary accumulation of Smo that correlates with pathway activation, but the underlying mechanism is poorly understood A recent study suggested that β-arrestins mediate Smo ciliary localization by binding to Smo and facilitating its interaction with the kinesin-II motor We also confirmed that phosphorylation regulates Smo/β-arr2 association using FRET assay. We found that Shh and A1 increased the FRET between Smo-CFP and β-arr2-YFP, and this increase was abolished by the SA0–5 mutation . ConversC, whereas individual mutations at other sites  had no effect . NIH3T3Smo-CFP did not exhibit significant basal ciliary localization of Smo-CFP or ciliary PS1 signal but accumulated both signals in the cilia upon stimulation with Shh, SAG, or 20-OHC . In line with a previous report To examine the dynamics of Smo phosphorylation, we treated NIH3T3We also monitored Smo phosphorylation in whole cells by western blot using the PS1 and GFP antibodies. We found that the ratio of PS1 versus Smo-CFP signal intensity was lower at early time points and gradually increased over time . Thus, STo investigate whether primary cilia regulate Smo phosphorylation, we disrupted the cilia using a dominant negative form of Kif3b (DN-Kif3b), a subunit of the kinesin-II motor required for cilia formation C in the wild type or Kif3a−/− MEFs transfected with wild type or mutant forms of Smo-CFPC/YFPC. We found that Shh or A1-induced FRETC was dramatically reduced in Kif3a−/− MEFs compared with WT MEFs  and three loss-of-function mutations in or near the CK1α binding pocket identified by previous studies  [28], ×100.For protein expression, cells were transfected with FuGENE 6 transfection reagent (Roche) according to the manufacturer's instructions, harvested and lysed in RIPA buffer , 1% NP-40 supplemented with protease inhibitors (Roche), and lysates were frozen and thaw 2∼3 times. Immunoprecipitation experiments were performed as previously described 5 cells/ml in 24-well plates, and cells were transfected with 8XGliBS-luciferase and pRL-TK at 4∶1 ratio, and 5% w/w of pGE-Smo constructs with Fugene 6 (Roche) according to the manufacturer's instructions. After 2 d of transfection, cells were changed to low serum medium  with or without Shh-conditioned medium combined with additional treatments as indicated, and cells were harvested and luciferase activities were determined using the Dual Luciferase Reporter Assay System (Promega) and FLUOstar OPTIMA (BMGLABTCH). Each sample was performed in triplicate and the assays were repeated for at least 3 times.The day before transfection, different cell lines were seeded at a density of 1∼2×10All constructs were electroporated into the neural tube of HH st11–12 chick embryos Stable NIH 3T3/shRNA cell lines against kinase CK1α, GRK2, or GRK5 were generated by retroviral infection and selected with 3 µg/ml of puromycin.HEK 293T cells were transfected with XZ201 retrovirus vectors encoding variant Smo cDNAs and pCL-Eco packaging vector, and supernatants were collected 72 h post-transfection, filtered through a 0.45 µM syringe filter, and added to 50∼70% confluent wild type MEFs with 8 µg/ml polybrene (Sigma) overnight.Figure S18XGliBS-luc reporter and control pRL-TK construct, and treated with or without Shh-conditioned medium, followed by dual Luciferase assay. (F) Cell extracts from stable NIH 3T3/shRNA cell lines or control NIH 3T3 cells transfected with Smo-Myc and treated with or without Shh-conditioned medium were separated on Phos tag-conjugated SDS-PAGE gel and probed with Myc antibody. (G) Gli-luciferase assay in NIH 3T3 cells in response to Shh stimulation or kinase overexpression. (H) Gli-luciferase assay in control or CK1α/GRK2 shRNA expressing NIH 3T3 cells treated with or without Shh-conditioned medium.CK1α and GRK2 regulate Smo phosphorylation and Shh signaling activity. (A) Diagrams showing the sequences of the corresponding shRNAs targeting CK1α, GRK2, or GRK5. (B) Knockdown efficiency by the indicated shRNAs. (Top) Cell extracts were prepared from NIH3T3 cells with integrated LMP control vector (CT) or vectors expressing shRNA against different regions of CK1α, GRK2, or GRK5 and immunoblotted with CK1α, GRK2, GRK5, and GAPDH antibodies. Representative western blots were repeated 3 to 5 times. The intensity of each band was analyzed using the ImageJ software. The numbers indicated percentage of knockdown. (Bottom) Knockdown efficiency of individual stable NIH 3T3/shRNA lines measured by real-time PCR. (C–E) Stable NIH 3T3/shRNA lines were transfected with Smo and WT or dominant-negative (DN) bovine CK1α (bCK1α) or GRK2 (bGRK2) together with the (TIF)Click here for additional data file.Figure S2Gli-luc assay in NIH 3T3 cells transfected with Smo or SmoSA0–5 with or without the indicated kinase expressing constructs and treated with or without Shh-conditioned medium. (E) FRET analysis in NIH 3T3 cells transfected with Smo-CFPC/YFPC or SmoSA0–5-CFPC/YFPC with or without the indicated kinase expressing constructs and treated with or without Shh-conditioned medium . (F) Evaluation of the specificity of the PS1 antibody. (Top) A schematic drawing of full-length Smo with the antigen peptide sequence for generating the PS1 antibody indicated. (Bottom) Western blot analysis using the PS1 antibody or antibodies against the non-phosphorylated peptide (S1). The PS1 antibody recognized GST-Smo608–670 but not GST-Smo608–670SA phosphorylated by GRK or CK1. In addition, the PS1 antibody did not recognize the unphosphorylated GST-Smo608–670. Equal amounts of GST fusion proteins were loaded as indicated by western blot with the S1 antibody. (G) Knockdown of CK1α or GRK2 affected Shh-induced Smo phosphorylation. Cell extracts from indicated stable NIH 3T3/shRNA lines transfected with Smo-Myc and treated with or without Shh-conditioned medium were separated on SDS-PAGE gel and immunoblotted with PS1 and Myc antibodies. Knockdown of CK1α or GRK2 but not GRK5 reduced Shh-induced Smo phosphorylation at S1 site. (H–I) The effect of mutating CK1/GRK sites on A1-induced Smo phosphorylation and Smo activity in response to Shh and small molecules. (H) Cell extracts from NIH 3T3 cells transfected with Smo-Myc, SmoA1-Myc, or SmoA1SA1–5-Myc and treated with or without Shh-conditioned medium were separated on Phos tag-conjugated SDS-PAGE gel and immunoblotted with a Myc antibody. Mutating multiple CK1/GRK sites abolished Shh- or A1-induced mobility shift of the Myc-tagged Smo. (I) Gli-luc assay in NIH 3T3 cells transfected with Smo, SmoSA0–5, or SmoSD0–5 and treated with or without the indicated reagents. The activity of SmoSA0–5 was no longer induced by Shh or SAG, whereas SmoSD0–5 exhibited elevated basal activity and was more resistant to cyclopamine (CYC) inhibition.CK1 and GRK phosphorylate multiple sites in Smo. (A–C) CK1 and GRK phosphorylate individual serine in the S1 site. (A) A schematic drawing full-length Smo with the sequences for S1, S2, and S3 indicated underneath. Amino acid substitutions for individual constructs are indicated. (B–C) In vitro kinase assay using recombinant CK1δ (B) or GRK5 (C) and purified GST-Smo608–670 fusion proteins with wild type (WT) sequence or indicated substitutions. (D–E) CK1/GRK sites in Smo C-tail mediate Smo activation by Shh, CKIα, GRK2, and GRK5. (D) (TIF)Click here for additional data file.Figure S3Mutating CK1/GRK sites affect Smo activity in chick neural tube. (A) Activity of Smo SD variants in chick neural tube. SmoWT, SmoSD1, SmoSD12, SmoSD123, SmoSD1–5, or SmoSD0–5 were transfected by in ovo electroporation into the thoracic region of HH st11–12 chick neural tube and the expression patterns of the indicated markers analyzed 48 h later. In embryos transfected with SmoSD123, SmoSD1–5, or SmoSD0–5, the expression of Pax7 was repressed and expression of Isl1, Olig2, and Nkx2.2 expanded dorsally (arrows). By contrast, the expression patterns of the neural tube markers in SmoSD1 or SmoSD12 electroporated embryos were similar to those in embryos transfected with SmoWT. (B) Mutating S1 affects SmoA1 activity in chick neural tube. SmoA1 or SmoA1 with different combination of SA mutations  were transfected by in ovo electroporation into the thoracic region of the neural tube of HH st11–12 chick embryos and the expression patterns of the indicated markers analyzed 48 h later. SmoA1 exhibited constitutive signaling activity, resulting in the dorsal expansion of ventral markers, including Islet1, Nkx6.1, Olig2, and Nkx2.2 and the repression of Pax7 (Brackets). Mutating S1 alone (A1SA1) or in combination with other sites  markedly reduced the signaling activity of SmoA1 and these constructs only induced mild ectopic expression of ventral markers (arrows). By contrast, mutating S2 and S3 (A1SA23) did not significantly affect SmoA1 activity.(TIF)Click here for additional data file.Figure S4Smo-CFP treated with different reagents. NIH 3T3Smo-CFP cells were either untreated or treated with Shh-conditioned medium (Shh), SAG (200 nM), 20-OHC (10 µM), CYC (10 µM), or a combination of Shh and CYC (10 µM), SAG (200 nM) and CYC (10 µM), or 20-OHC (10 µM) and CYC (10 µM). The histogram indicates the percentage of Smo-CFP or PS1 positive cilia. Over 100 ciliated cells were counted for each time point (n = 3). (B) FRET analysis in wild type or Kif3a−/− MEFs transfected with Smo-CFPC/YFPC, SmoA1-CFPC/YFPC, or SmoSD0–5-CFPC/YFPC and treated with or without Shh-conditioned medium . (C) Gli-luc assay in Kif3a−/− MEFs transfected with the indicated constructs and treated with or without Shh-conditioned medium.Primary cilium and Smo phosphorylation. (A) Quantification of Smo-CFP or PS1 positive cilia in NIH 3T3(TIF)Click here for additional data file.Figure S5p<0.05, **p<0.01, ***p<0.005. The signal intensity for each band was quantified by ImageJ software followed by Prism analysis, n = 3.Quantification of CK1α binding to different forms of Smo. Histograms for the western blot analyses shown in (TIF)Click here for additional data file.


Climate warming has been related to glacial retreat along the Western Antarctic Peninsula. Over the last years, a visible melting of Fourcade Glacier  has exposed newly ice-free hard bottom areas available for benthic colonization. However, ice melting produces a reduction of light penetration due to an increase of sediment input and higher ice impact. Seventeen years ago, the coastal sites close to the glacier cliffs were devoid of macroalgae. Are the newly ice-free areas suitable for macroalgal colonization? To tackle this question, underwater video transects were performed at six newly ice-free areas with different degree of glacial influence. Macroalgae were found in all sites, even in close proximity to the retreating glacier. We can show that: 1. The complexity of the macroalgal community is positively correlated to the elapsed time from the ice retreat, 2. Algae development depends on the optical conditions and the sediment input in the water column; some species are limited by light availability, 3. Macroalgal colonization is negatively affected by the ice disturbance, 4. The colonization is determined by the size and type of substrate and by the slope of the bottom. As macroalgae are probably one of the main energy sources for the benthos, an expansion of the macroalgal distribution can be expected to affect the matter and energy fluxes in Potter Cove ecosystem. Antarctica is one of the regions most seriously affected by climate change; particularly, the Western Antarctic Peninsula is exhibiting a rapid regional warming The term “newly ice-free areas” refers to those places available for colonization and biological succession, due to glacier retreat According to the recent observations of the glacier retreat in Potter Cove, these questions arise: Is macroalgal distribution expanding into the newly ice-free areas? Which species are colonizing these areas? Is the structure of the communities related to the prevailing physical conditions in these areas?To tackle these questions, underwater video transects were performed at six newly ice-free areas to assess the spatial macroalgal distribution and to compare the present situation with a study performed seventeen years ago The study was carried out at Potter Cove , from March to August 2008. The Cove is divided into an outer and an inner part. The bottom of the outer Cove consists of hard substrate whereas the inner part is a soft one. The southern shore of the inner Cove is a sandy beach, where creeks discharge. Glacier cliffs reach the Cove in the north and east Previous studies have shown that the rocky shores of the outer side of Potter Cove are colonized by a high biomass of macroalgae −2 s−1) was obtained at 0, 5, 10, and 20 m depth in summer 2009–2010 (December-March), data was measured weekly around noon. Kd, the light attenuation coefficient, was calculated according to Kirk 0/Ez) where E0 is the surface incident irradiance and Ez is the irradiance at depth z. Low Kd values describe transparent water with little attenuation of radiation, whereas high Kd values mean high suspended particles in the water column producing high radiation extinction.Underwater photosynthetically active radiation  was considered a proxy for glacial sediment input. Photosynthetically active radiation (PAR) was measured in the six different selected newly ice-free areas (Sites 1 to 6) using a Licor LI 1400 datalogger. Instantaneous PAR data , the Environmental and Tourism Antarctic Management Program of the National Direction of the Antarctic  in the Republic of Argentina, has issued the appropriate permissions to all the stages of this research:To the Specially Protected Area N°132 “Peninsula Potter” Taking and harmful interference and introduction of species Both of these permissions properly followed the regulations in force.i log2 pi where pi is the relative abundance (estimated by cover data) of taxon i in the range of depth and evenness was calculated using Pileoús Index as J =  H/log2 S. Richness (S) was the total number of species. Abundance was estimated as percent cover data.The newly ice-free areas were characterized by ecological indexes: diversity, richness and evenness were calculated for each range of depth. Diversity was determined by Shannon index: H = ∑ pThe macroalgal taxa were classified according to their life history  in annuaOne way ANOVA were performed to test the effects of the site on the analyzed variables. Post-hoc multiple means comparisons were analyzed using Tukey Test. Homogeneity of variances was checked using Cochran’s Test. Tests were performed with Statistica TM 6.0 software package.In summer, significant differences of Kd were found between the studied sites . Kd for Macroalgae colonized all the studied sites. A total of 18 species of macroalgae and two genera of encrusting red algae  were identified .Diversity and evenness were lower in S5 compared to the rest of the studied sites  and S1 The 20 taxa recorded in this study constitute a low number compared to the registered macroflora of Potter Cove, which includes 42 species S1, S2 and S3 were the first to be available for benthic colonization and it is plausible that macroalgal communities have been developing there for a longer time period. Hence, a higher macroalgal cover and more mature communities are found there. Indeed, a high contribution of large brown species of order Desmarestiales  was registered in these three sites. This macroalgal composition was similar to the sites situated on the outer Cove On the other hand, S5 and S6 were ice-released later in time and a less mature macroalgal community would be expected there.However, an older age of the site is not always related to the maturity of the community. Indeed, S5 is older than S6 and shows a significantly lower diversity. Other factors should also be considered to explain the observed patterns (see below).Light availability is a major factor determining the vertical distribution limits of subtidal macroalgae in Antarctica In addition, glacial run-off not only reduces light penetration but also causes accumulation of sediment on the sea floor which may limit the attachment of benthic algae Palmaria decipiens as by far, the dominant species, immersed in a high sediment load. This species seemed to be able to cope with both low light penetration and high sediment accumulation, probably due to its physiological plasticity At S5, the video images showed Even though S5 and S6 had similar optical conditions, S5 showed significantly lower diversity than S6. Thus, other factors should also be considered, as ice disturbance (see below) to better explain the observed patterns.Ice scouring can be a strong driver of the distribution and abundance of macroalgae, affecting zonation and diversity patterns in Polar Regions Substrate and slope play a key role in determining algal settlement Substrate and slope could also explain the differences between S5 and S6.These sites undergo high sediment input conditions but the vertical wall of the island prevents sediment accumulation. Vertical substrate was shown to favor the establishment of sessile macrofaunal communities et al.A special consideration is necessary for the encrusting red algae . In many quantitative studies of subtidal communities, this group is mentioned as “encrusting coralline algae” on hard substrate, without a complete taxonomic species identification. Some authors emphasize the importance of their presence, primarily because they will provide future answers of the evolution of the acidification of the ocean To explain the pattern of spatial distribution of macroalgal species, a conceptual model was developed . The iceet al.Macroalgae are an important food source for fish and benthic invertebrates in Potter Cove Smale and Barnes


Inteins are intervening protein domains with self-splicing ability that can be used as molecular switches to control activity of their host protein. Successfully engineering an intein into a host protein requires identifying an insertion site that permits intein insertion and splicing while allowing for proper folding of the mature protein post-splicing. By analyzing sequence and structure based properties of native intein insertion sites we have identified four features that showed significant correlation with the location of the intein insertion sites, and therefore may be useful in predicting insertion sites in other proteins that provide native-like intein function. Three of these properties, the distance to the active site and dimer interface site, the SVM score of the splice site cassette, and the sequence conservation of the site showed statistically significant correlation and strong predictive power, with area under the curve (AUC) values of 0.79, 0.76, and 0.73 respectively, while the distance to secondary structure/loop junction showed significance but with less predictive power (AUC of 0.54). In a case study of 20 insertion sites in the XynB xylanase, two features of native insertion sites showed correlation with the splice sites and demonstrated predictive value in selecting non-native splice sites. Structural modeling of intein insertions at two sites highlighted the role that the insertion site location could play on the ability of the intein to modulate activity of the host protein. These findings can be used to enrich the selection of insertion sites capable of supporting intein splicing and hosting an intein switch. Native intein coding sequences are elements found inserted in-frame into their host gene. Post-translationally, the intein domain is capable of splicing out of the host, in either cis- or trans-splicing reactions, connecting the amino(N-)- and carboxy(C-)-terminal residues of the host exteins resulting in an active mature protein Inteins are not universally transferable between proteins. There are specific characteristics for intein insertion sites that allow functional splicing The specificity of sites for both intein splicing and host activation is understandable given the number of interconnected events that must occur in this process. First, the intein must be located in a site that allows adequate protein expression, stability, and folding, preventing intracellular degradation. Second, if the intein regulates the host protein’s activity, the intein insertion must reversibly block the activity of its protein host. This could in principle occur in a number of different ways including blocking the access of the substrate to the active site, altering the local structure of the active site, or interfering with dimerization required to form the active complex. Finally, the local extein environment must allow the intein to efficiently splice out of the host protein In this work we examined features of native inteins to determine which features are highly correlated with the location of the intein insertion site. These include sequence and structure based properties of the intein’s flanking extein, such as location of insertion sites in conserved sequence region of the host, sequence similarities of the splice site cassette, local secondary structure, flexibility of the intein insertion site, burial of the insertion site and distance of the insertion site to the active site and the dimer interface. Of these, conservation of position, local sequence, local secondary structure and distance to both the active site and dimer interface were seen to be statistically significant predictors of the insertion site, while the flexibility of the insertion site and the degree of burial were not significant. Additionally, the conservation of the site, local sequence and distance to the active site/dimer interface were shown to have strong predictive power in identifying native splice sites, while the predictive strength of the local secondary structure was somewhat weaker. Finally, to see if this method could be used to insert inteins into a non-native host protein, we looked at a case study of 20 insertion sites in the XynB xylanase.http://www.neb.com/neb/inteins.html); To analyze the properties of native inteins, flanking extein sequences were extracted  from a pUsing this set of exteins and cassettes, sequence and structural properties were evaluated to determine which had a strong correlation with the location of the intein insertion site. These properties were analyzed by evaluating the characteristics of native insertion sites in relation to the characteristics of all sites that contain a cysteine, serine or threonine (C/S/T sites). The properties evaluated were the conservation of the position of intein insertion site, the sequence similarity of native cassettes, the local secondary structure, the flexibility of the site, the degree of burial and the distance of the insertion site to the active site and dimer interface.−34. This clearly indicates that the insertion sites occur in more conserved positions that the average C/S/T site.Conservation among intein insertion regions within the exteins has been previously reported through the analysis of small numbers of inteins −25.We used a support vector machine (SVM) to evaluate the similarity of all C/S/T sites to native insertion sites. The support vectors describe the intein insertion site cassettes in vector form. The positive training set was selected from the set of native intein insertion site cassettes. These were filtered for redundancy. Additionally, three random, non-native, “decoy” cassettes were also selected from each extein with a cassette in the positive test set. Each set of three cassettes was selected so as to not include duplicates. The SVM was trained using SVMlight In order to determine structural information about the intein insertion site environment, homology models of the exteins were generated. Extein sequences were modeled onto structures in the Protein Data Bank −9). Further analysis showed that native intein insertion sites are more likely to occur close to a loop/β-sheet or loop/α-helix boundary (54% of sites), than to occur in the middle of a β-sheet or an α-helix (31% of sites). For all C/S/T sites these values are 38% and 43% respectively  using a chi-squared test.Using the homology models, the local secondary structure of all residues was evaluated using the program Stride ectively . This diα position of the native intein insertion site to the closest identified active site or dimer interface residue. Measurements were also carried out for all C/S/T sites. These distances were binned into groups of 5 Å and shown in −13). Similarly for the dimer interface site, the native insertion sites are on average closer to the dimer interface sites than the average C/S/T sites. These were shown to have a mean distance of 16.6 to 24.1 Å respectively and have a significance value of 0.0045.The proximity of the intein insertion site to two different types of functionally important residues, active site residues and dimer interface residues of split inteins was measured in our analysis. Of the homology models described above, 43 models were identified with annotated active site residues and 24 with annotated dimer interface residues. These annotations were derived from homologous sequences in the NCBI genpept database. We then measured the distance of the Cβ-density. This method has been used to describe the burial environment of sites in different applications β-density was calculated as the number of Cβ atoms within 8 Å of a particular site. For each amino acid type of cysteine, serine and threonine, the native insertion sites were compared to the remainder of the extein sites. For each amino acid type the distribution of the Cβ-densities for native intein insertion sites is not significantly different from the random sites. These distributions are shown in The degree of burial for each intein insertion site was examined and compared with the degree of burial for random C/S/T sites using a method of Cα-atom position of all C/S/T sites were calculated.The relative flexibility of the insertion site may be an important issue when determining sites that are amenable to intein insertion. Here we investigated relative flexibility at the insertion site by performing molecular dynamic equilibration simulations on our set of homology models as described in the methods. Briefly, each model was solvated into a TIP3 water sphere that had a radius that was 5 Å larger than the protein in each dimension. These were minimized for 1000 steps and run with equilibrium molecular dynamics for 5 ns in steps of 2 fs. Snapshots of this trajectory were taken every 50 ps. For the last 50 (2.5 ns) snapshots the time-averaged root mean square deviation of the CThe distribution of native sites is compared to the distribution of all other C/S/T sites in this data set and presented in Of the features tested, four showed significant correlation with the location of the intein insertion site. To determine the predictive quality of these features, first the true positive rate (TPR) (sensitivity) and the false positive rate (FPR) (1-specificity) were calculated for each feature as shown in the following equation. The cutoff point above which a target would be predicted as a positive and below which it would be predicted as a negative was optimized. For each cutoff, the true positives (TP), true negatives (TN), false positives (FP) and false negatives (FN) were counted and used to calculate the TPR, and the FPR.These values are plotted in Dictyoglomus thermophilum (P77853) to facilitate splicing of the Thermus thermophilus DnaE-1 intein (Tth) based on these predictors. The tested sites included 20 different cysteine, serine, and threonine (C/S/T) sites between amino acid residues 64 to 206 of the catalytic domain. To evaluate the ability of insertion sites to support splicing we have Western blotted lysates of E.coli cells expressing XynB:Tth . The distance to the active site may be functionally important to the intein. By blocking activity in the precursor state, the host protein requires the intein to be functional for the host to retain its activity. This would create selective pressure on the intein to splice or the host protein itself will lose functionality. If the intein does not block the activity of the protein, then there is not the same pressure on the intein to be functional. The intein could be inactivated and the host protein could still function.The proximity of the insertion site to active site or dimer interface strongly correlates with the location of the insertion site. Previously, it had been suggested that active sites and dimer interface sites could be ideal locations for inteins because conserved sites that are not in the core tend to be active sites, binding and docking sites, and dimer interfaces with surface loop regions between α-helices and β-sheets Our goal was to predict native like intein insertion sites in enzymes that do not natively host inteins and have these insertions permit intein splicing. Insertion site selection may be done by linker scanning or similar methods that can identify insertion tolerant sites The total computational time required for these predictive models is dominated by the generation of the homology model and the psi-blast search used to predict conservation rank. Each of these can takes up to 30 minutes on a single cpu depending on the size of the protein. After that the remaining calculations are done in seconds. This compares favorably to the 2 weeks required to perform the experimental validations of the 20 insertion sites of XynB. The predictive power we have seen from these methods can be used to prioritize the screening of a given protein, as well as to increase confidence in the selection of follow-up mutagenesis. As is the case for the T134 and S158 sites, mutagenesis to improve the splicing of an intein could take several weeks to months to complete. Any upfront pruning of possible sites could then save a lot of downstream experimental work by prioritizing the sites to focus on.We have shown the predictive methods to be useful, but there is still room for improvement. This becomes apparent when comparing the sites predicted by to all the methods to be splicers . The resulting set comprised of 412 inteins . From this set of inteins, the corresponding precursor proteins were extracted from the NCBI genpept library. This was done using the GI accession number from the Inbase library as well as using a Blast search to identify cases where the accession number was missing or not up-to-date. The intein insertion site was then confirmed, the intein was removed from this precursor protein and the native extein was defined as the remainder of the sequence.Inteins for this set were selected from the NEB Inbase Intein Library −15, and the expectation value set to 10−15.Conservation of the intein insertion site was determined using an entropy based position-specific scoring matrix (PSSM) based method. The PSSM was calculated using the NCBI PsiBlast program i) for each site i was calculated using equation 3, where pij is the probability of amino acid type j at site i.From the PSSM for each sequence, the sequence entropy  was generated, which describes the cassette surrounding the site. This vector is a concatenation of six 20-dimensional vectors (sp), one for each of position of the cassette (−3 to −1 and +2 to +4). These are represented in equation 4.To generate a SVM of the intein insertion site cassettes, a subset of inteins was chosen to minimize the bias of sequence homology. This was done by removing sequences from the dataset that had identical insertion site cassettes (defined as the −3 to −1 and the +2 to +4 positions). When two sequences had identical cassettes, the sequence with the larger extein was kept. This reduced the total to 188 extein cassettes. Using this set of 188 native exteins, a SVM model was generated to predict the intein insertion site. The training set was determined by using the native intein insertion site as a true positive, and 3 other random C, S or T cassettes as true negative sites. For each insertion site sp, all 20 components are 0 except for the component corresponding to the amino acid present at that site. This has a value of 1. To train the SVM model the positive set were given a target value of +1. In addition, three true negatives were chosen from each extein as a random T, S or C site in the extein . These were given a target value of −1. The SVM model was then trained using SVMlight version 6.02 Here for each vector This method was tested using a leave-one-out cross validation. For each target extein, the remaining 187 exteins were selected as the training set using the same training method as for the whole set. All C, S, and T sites were then scored for the target extein using the SVM and the rank of the true positive was determined as the fraction of decoy insertion site cassettes with a score lower than the true positive insertion site. This was repeated 25 times for each extein using a different random training set.−10 or lower was included in this set to generate homology models. This included 300 extein sequences. These sequences and the alignments to the blast hits were then used to generate homology models. The top blast hit was downloaded from the PDB and all non-aligning structure was removed. The sequence of the extein was then repacked onto the template structure using Rosetta v3.1 fixed backbone repacking. All non-identical residues were repacked and residues that were identical in the alignment were held fixed. Ten different repacks were performed and the lowest energy one was selected. Parts of the alignments with gaps were then predicted using the loop modeling module of Rosetta v3.1 loop model with the ccd_closure method. This set of structures was then filtered to limit it to non-identical sequences. When structures had identical sequences, the structure with the longer sequence was chosen.All 412 exteins were blasted against the sequences contained in the PDB (4/20/2010). Any extein sequence that had a hit with an E-value of 10Using the homology models constructed for all of the exteins, the local secondary structure for all possible insertion sites were determined and compared to the native secondary structures. Their Dictionary of Protein Secondary Structure (dssp) secondary structure assignments were made using the program Stride with default parameters. i the rmsd was defined as in equation 5, where xi,s is the position of the Cα-atom of residue i at in snapshot s.The flexibility of insertion sites were determined using molecular dynamics equilibration simulations. The initial structures used were from the set of homology models described above. These structures were solvated into a solvent sphere of TIP3 waters using the VMD solvate package. β density from the homology model described above. This calculation was performed by counting that number of Cβ atoms that are within 8 Å of the target site Cβ atom. The ranks for the cysteine, serine and threonine residues were compared separately for the native and non-native intein insertion sites.The degree of burial of the C/S/T sites was determined by calculating the Ctype = “active site” described in their “Features” section of their genpept file. All active site residues identified whose alignment was to part of the extein sequence in the homology model and had identical residue types were set as the active site residues. The sequence alignment that produced active site residues with the lowest E-value was used. All PsiBlast alignments gave E-values of 10−100 or lower. For all homology models in which there were both an intein insertion site and an active site residue contained in the protein, the distance between the Cα atoms of these positions was calculated. The closest distance for any active site to the insertion site was set as the distance for that insertion site position. A similar method was used to find the dimer interface positions. The only difference was that the list of dimer interface sites was determined by identifying sites that had type = “other” and note = “dimer interface”. The distances were calculated as above described above.To determine the distance of the intein splice site to the active site of the protein or dimer interface, information for the exteins was extracted from the NCBI genpept database. To find the active site positions the sequences of the extein were blasted against the NCBI nr sequence database using PsiBlast D. thermophilum (Uniprot accession # P77853) T. thermophilus DnaE-1 intein (Tth) was corn codon optimized, inserted 5′to 20 C/S/T sites of XynB  using overlapping PCR and cloned into the EcoRI and XhoI site of the precut λ Uni-ZAP XR expression vector (Agilent Technologies). Packaging, phage infection and plating was done as described in the lambda ZAP-II manual (Stratagene), except that the top agar of the diagnostic plate was supplemented with IPTG (2.5 mM) and 0.2% AZCL-xylane oat (Megazyme) to detect xylanase activity.XynB xylanase from To score xylanase activity of the intein modified XynB:Tth, plates were incubated at 37°C overnight till confluent lysis then incubated at 60°C for 3 hrs. Blue color development indicated xylanase activity.XynB:Tth and the control XynB were phagemid rescued from λ UniZapII to SOLR according to the manufacturer’s protocol (Stratagene) and were grown in 1 ml auto induction medium (Novagen) supplemented with Carbenicillin (100 mg/l) at 37°C overnight. Cells were pelleted, resuspended in 100 µl of polybuffer pH = 6.5 containing 1×FastBreak Lysis Buffer™ (Promega) and 1/5000 dilution of Benzonase (Novagen) and lysed at 30°C for 1 hr. Lysate was diluted into 400 µl Polybuffer pH = 6.5 and an aliquot was heated at 60°C for 3 hrs. 2.5 ul of lysate was separated on a 12% SDS/PAGE (BioRad), transferred onto PVDF membrane and the Western blot was developed using the “THE mouse anti-6×His”  primary antibody at 0.25 µg/ml, and the secondary antibodies peroxidase-goat anti-mouse IgG  and the HRP-anti-biotin  to detect the biotinylated mol. weight markers .To test the intein mutagenized XynB:Tth at the T134 and S158 insertion site, Tth intein was random mutagenized using Mutazyme (Stratagene), inserted into XynB and expressed from the lambda ZAP-II onto xylanase diagnostic plates as above, but the phage titer for plating was reduced to grow individual plaques. Plaques that developed distinctively strong blue color indicative of high xylanase activity were phagemid rescued to SOLR and lysates of bacteria expressing the intein mutagenized XynB::Tth were Western blotted as above.Dictyoglomus thermophilum (XynB) were generated by inserting the homology model of the Tth intein into the S158 and T134 sites of a X-ray crystal structure of the XynB catalytic domain (pdbID 1f5j). Chain A of 1f5j was used and renumbered to correspond to XynB full length sequence (Uniprot ID P77853). Thus residue 1 in the structure was renumbered to be residue 29. This was done using the domain insertion module of Rosetta++ v2.3 (1). The intein Tth homology model was generated using SwissModel Three dimensional models of the Tth DnaE-1 intein (Tth) inserted into the S158 and T134 sites of the enzyme XynB from endo-1,4-beta xylanase from α atoms) were refined using the high-resolution refine module. The lowest energy model for each the T134 and S158 insertion site was chosen as the best approximate model.With the structure of XynB and Tth, structural models of the XynB-T134 and -S158 Tth intein modified enzymes were generated. To generate these models we aligned the two domains such that the intein insertion site of the extein was close to and facing the N- and C- termini of the intein. This was done by first identifying the Cα atoms of the +1 position (extC) and the −1 position extN and the N-terminal (intN) and the C-terminal intein position (intC). The intein and the extein were aligned to minimize the extC-intC and the extN-intN distances as well as maximize (closest to 180°) the angle formed by the center of mass of the intein, the midpoint (MP) of the positions extC, intC, extN and intN and the center of mass of the extein. The intein was then moved away along the axis formed by the center of mass of the intein and MP in 0.5 Å increments until no backbone atom clashes occurred between the intein and the extein. For each insertion site, domain insertion were performed using the program and protocol as described in Berrondo et al. Figure S1ROC curve of the prediction of Tth intein insertion sites in XynB. The true-positive rate and the false-positive rate was calculated for the 4 features used to predict the Tth intein insertion into 20 different sites of XynB: (blue) distance to the active site or dimer interface, (green) SVM score, (red) distance to SS/Loop junction and (cyan) sequence conservation. These rates were determined over a range of cutoff values which were: for the distance to the active site or dimer interface site the cutoff value was varied from 0 to 30 Å, for the SVM scoring the cutoff was varied from −10 to 10, for the distance to the SS/Loop junction the cutoff was varied from 0 to 5 amino acids and for the conservation rank the cutoff was varied from 0 to 1. The points marked with circles are the maximum enrichment points of the true positive rate versus the false positive rate for the native intein insertion sites. The black dashed line indicates a random prediction.(TIF)Click here for additional data file.Table S1List of inteins, exteins, insertion sites and insertion site cassettes. All sequence numbering is based upon the full length precursor protein.(DOC)Click here for additional data file.


S-nitrosylation substrates, JNK1 and IKKβ. Inhibition of JNK1 by NO reduces Bcl-2 phosphorylation and increases the Bcl-2–Beclin 1 interaction, thereby disrupting hVps34/Beclin 1 complex formation. Additionally, NO inhibits IKKβ and reduces AMPK phosphorylation, leading to mTORC1 activation via TSC2. Overexpression of nNOS, iNOS, or eNOS impairs autophagosome formation primarily via the JNK1–Bcl-2 pathway. Conversely, NOS inhibition enhances the clearance of autophagic substrates and reduces neurodegeneration in models of Huntington's disease. Our data suggest that nitrosative stress-mediated protein aggregation in neurodegenerative diseases may be, in part, due to autophagy inhibition.Autophagy, a major degradation process for long-lived and aggregate-prone proteins, affects various human processes, such as development, immunity, cancer, and neurodegeneration. Several autophagy regulators have been identified in recent years. Here we show that nitric oxide (NO), a potent cellular messenger, inhibits autophagosome synthesis via a number of mechanisms. NO impairs autophagy by inhibiting the activity of  ► NO inhibits autophagy by independently inhibiting JNK1 and IKKβ ► NO inhibits autophagic flux via mTOR and mTOR-independent routes ► NOS overexpression impairs autophagosome synthesis via JNK1–Bcl-2 pathway ► NOS inhibition induces autophagy and protects against neurodegeneration ATG (AuTophaGy related) genes, many of which have mammalian orthologs. The only known mammalian protein that specifically associates with autophagosome membranes throughout their lifespan is microtubule-associated protein 1 (MAP1) light chain 3 (LC3), the mammalian ortholog of yeast Atg8. LC3 normally exists in the cytosol as LC3-I. Upon autophagy induction, LC3-I conjugates with phosphatidylethanolamine to form the autophagosome-associated LC3-II. LC3-II levels (relative to actin/tubulin loading controls) correlate with autophagosome numbers (Macroautophagy (henceforth referred to as “autophagy”) is an intracellular bulk degradation process involved in the clearance of long-lived proteins and organelles, which affects various physiological and pathological processes, including development, immunity, longevity, cancer, and neurodegenerative diseases . Autopha numbers .The classical pathway regulating mammalian autophagy involves the serine/threonine kinase, mammalian target of rapamycin (mTOR) . This prStarvation also regulates autophagy by activating c-Jun N-terminal kinase 1 (JNK1), which in turn phosphorylates Bcl-2 at multiple sites . Bcl-2 normally inhibits autophagy by interacting with the autophagy protein Beclin 1, whereas Bcl-2 phosphorylation inhibits this interaction to stimulate autophagy . Beclin Autophagy can also be regulated independently of mTOR . Recent S-nitrosylation, which is the redox-based modification of Cys thiol side chains, is a common mechanism of NO-mediated signal transduction  also regulate autophagy under amino acid and serum starvation conditions, where superoxide has been suggested to be the major ROS species involved in ROS-mediated autophagy . Howeversduction . Proteinneration . An exceneration .S-nitrosylates JNK1 and IKKβ, and our data suggest that these events affect autophagy, as NO decreases JNK1 activity and Bcl-2 phosphorylation and activates mTORC1 in an IKKβ- and TSC2-dependent manner. Overexpression of NOS isoforms also impairs autophagic flux. Conversely, inhibition of NO synthesis induces autophagy and protects against neurodegeneration in models of Huntington's disease (HD). Our data delineate the role of NO in regulating autophagy, which may have underlying implications in its myriad of cellular functions.Here we show that NO inhibits autophagic flux in mammalian cells. NO 1   A, indicaWe next analyzed autophagic flux in a stable HeLa cell line expressing mRFP-GFP-LC3 reporter, in which autophagosomes have both mRFP and GFP signals, whereas the autolysosomes emit only mRFP signal because of quenching of the GFP in the acidic lysosomal environment . NO dono+/+Atg5 (autophagy-competent) MEFs, but not in Atg5−/− (autophagy-deficient) MEFs, which is consistent with an effect on autophagy inhibition , a well-established autophagy substrate, where the proportion of cells with aggregates formed by this protein correlates linearly with expression levels . NO donohibition B. Furthehibition G and 1H.hibition , these d1–treated HeLa cells, suggesting that NO-mediated autophagy inhibition was independent of the cGMP pathway , which, in turn, activates cGMP-dependent protein kinase G (PKG) . To test pathway F.S-nitrosylation at C116, leading to reduced JNK1 phosphorylation   , we founA Bcl-2) C and S2DBeclin 1 regulates the initiation of autophagosome formation as a part of the hVps34/PI3K complex . ConsistWT Bcl-2) or AAA mutant Bcl-2 (AAA Bcl-2)  . Interesl-2 MEFs A. NO donl-2 MEFs B–3D.jnk1 or jnk2 genes , at T389, and that of the ribosomal S6 protein (S6), a substrate of S6K, at S235/236 . NO donol-2 MEFs H. An eleLa cells B and S3CThe regulation of mTORC1 activity by amino acids has been correlated with the localization of mTORC1 on late endosomes/lysosomes via the Rag GTPases . Althoug+/+Tsc2 and Tsc2−/− MEFs , consisting of a heterodimer of TSC1 and TSC2, is an upstream negative regulator of mTORC1. TSC1/2 is the GTPase-activating protein for the Ras-family GTP-binding protein Rheb, which directly binds and activates mTORC1 . Loss of−/− MEFs . We foun/−, MEFs A, suggesS-nitrosylates and inhibits IKKβ. However, the active IKK complex normally regulates autophagy by enhancing AMPK phosphorylation (possibly indirectly), which, in turn, inhibits mTOR via TSC1/2  by the JNK1–Bcl-2 and mTORC1 pathways , inducible NOS (iNOS or NOS2), and endothelial NOS (eNOS or NOS3). Although nNOS and eNOS are Ca induced .1, compared to the control cells (ωN-nitro-L-arginine methyl ester hydrochloride (L-NAME)  F–S5J. Fu(L-NAME) G and 5H.We next tested the NO signaling pathways regulating autophagy in the HEK293 NOS stable cells under basal (FM) and starvation (HBSS) conditions. We found that the phosphorylation of JNK1 and Bcl-2 were reduced in all the NOS-expressing cells compared to the control cells cultured in FM or HBSS A–6F, com1-treated mouse primary cortical neurons and HeLa cells under basal conditions  in bafilomycin Anditions A. It alsnditions A and S7Bnditions B and 7C.nditions C–S7E.In contrast to the autophagy signaling pathways perturbed by NO donors, induction of autophagy by L-NAME was independent of mTORC1 activity or Bcl-2 phosphorylation, because HeLa cells treated with L-NAME exhibiting elevated LC3-II levels had no change in the phosphorylation of S6K, S6, or Bcl-2 F. Moreov+/+Atg5 MEFs, but not in autophagy-deficient Atg5−/− MEFs  in their photoreceptors exhibit retinal degeneration, a phenomenon that is not observed in flies expressing the wild-type protein with 23 glutamines (Q23)  . The phoes (Q23) E. Moreovgulation . The progulation . Using tgulation F.cu5] by assessing mutant huntingtin aggregates in the retina where the transgene is driven by the rhodopsin promoter (4Cl) increased aggregate count promoter . We founte count J. The abependent I.S-nitrosylates and inactivates JNK1, thereby reducing Bcl-2 phosphorylation, which increases Bcl-2–Beclin 1 interaction and disrupts Beclin 1–hVps34 association. Second, NO activates mTORC1 in a manner dependent on TSC2 and IKKβ and is associated with decreased AMPK phosphorylation by IKK, consistent with the previously described inhibitory effects of NO on IKKβ activity  in the presence of bafilomycin Aescribed .Autolysosomes were quantified with an automated Cellomics microscope, as previously described .The percentage of EGFP- or mCherry-positive cells with >5 EGFP-LC3 or mCherry-LC3 vesicles was assessed with a fluorescence microscope, as previously described .∗∗∗, p < 0.001; ∗∗, p < 0.01; ∗, p < 0.05; NS, nonsignificant).Statistical analyses were performed using STATVIEW v4.53 and SPSS 9, as previously described . Statist


In cardiac muscle, the sarcolemmal sodium/potassium ATPase is the principal quantitative means of active transport at the myocyte cell surface, and its activity is essential for maintaining the trans-sarcolemmal sodium gradient that drives ion exchange and transport processes that are critical for cardiac function. The 72-residue phosphoprotein phospholemman regulates the sodium pump in the heart: unphosphorylated phospholemman inhibits the pump, and phospholemman phosphorylation increases pump activity. Phospholemman is subject to a remarkable plethora of post-translational modifications for such a small protein: the combination of three phosphorylation sites, two palmitoylation sites, and one glutathionylation site means that phospholemman integrates multiple signaling events to control the cardiac sodium pump. Since misregulation of cytosolic sodium contributes to contractile and metabolic dysfunction during cardiac failure, a complete understanding of the mechanisms that control the cardiac sodium pump is vital. This review explores our current understanding of these mechanisms.The online version of this article (doi:10.1007/s00018-012-1134-y) contains supplementary material, which is available to authorized users. In 1997, a share of the Nobel Prize in Chemistry was awarded to Jens Christian Skou for his 1957 discovery of the Na, K ATPase [While the use of Na pump-inhibiting cardiotonic steroids for the management of heart failure is now restricted to only a small subset of patients , our undThe Na pump is a multi-subunit enzyme with a minimum requirement for an α and β subunit to form a functional pump. The ~100-kDa α subunit is the catalytic core of the enzyme, containing the binding sites for sodium, potassium, and ATP as well as cardiotonic steroids such as ouabain. It requires an obligatory association with a β subunit to traffic through the secretory pathway to the plasma membrane , 9. The Although four isoforms of the α subunit have been identified, only α1 and α2 are reportedly expressed at significant levels in cardiac myocytes , 14. ThaIn the mouse, approximately 70 % of functional α2 and 40 % of α1 subunits are t-tubular, even though the t-tubule membranes represent only 30 % of total surface area . The conThe principle β subunit found in cardiac muscle is β1 , and thIn excitable tissues, the activity of the plasmalemmal Na pump is vital for the maintenance of normal electrical activity and ion gradients. In cardiac muscle, the transarcolemmal sodium gradient established by Na pump activity is essential not only for generating the rapid upstroke of the action potential but also for driving a number of ion exchange and transport processes critical for normal cellular function, ion homeostasis, and the control of cell volume. These sodium-dependent membrane transporters include those responsible for the regulation of other ions , as welKm of the cardiac Na pump for intracellular ATP has variously been estimated to range from 0.46 mM (embryonic chick hearts [The k hearts ) to 0.21k hearts ) to 94 μk hearts ). In ratk hearts . Interesk hearts , much ask hearts , althougKm for sodium has variously been estimated as: 9 mM , 14 mM vesicles ), 11 mM vesicles ), 19 mM vesicles ), 19 mM vesicles ). Inter-vesicles , but whaThe idea that sub-sarcolemmal sodium gradients not only exist but may dynamically change to influence E–C coupling was first proposed by Lederer et al.  and LeblWhile the pump may functionally interact with NCX, the situation may be more complex. As discussed above “”, α1 andIK1). However, during the plateau of the action potential, the input impedance of the membrane is significantly raised (due to the inward rectification of IK1) and hence the pump current, which is itself increased by depolarization, may have a more significant impact on membrane potential with pump stimulation shortening, and inhibition prolonging, the duration of the action potential [The activity of the Na pump is strongly influenced by membrane voltage , 38, 54.otential .The influence of voltage on pump current differs between the two main cardiac isoforms , 56. WhiThe functional link between the adrenergic system and the cardiac pump is well established, but remarkably, after many decades of research, there is still disagreement about the functional consequences of adrenoceptor activation on pump activity. In general activation of PKA (via β1-adrenoceptors linked to adenylate cyclase) and PKC (via α1 adrenoceptors linked to phospholipase C) increases stroke volume in ventricular muscle via phosphorylation of L-type calcium channels, the ryanodine receptor and phospholamban . Pump inMost laboratories report activation of the pump by PKA agonists in the heart , 59–68, Vmax, presumably through an effect on these PKCs [In some tissues there is convincing evidence that PKC phosphorylation of the pump α subunit is a signal for pump internalization and degradation (for example –78), butese PKCs .There is near-universal agreement that nitric oxide (NO) stimulates the cardiac Na pump –89, althAs a major target for hormone-stimulated phosphorylation in the heart, the physiological function of phospholemman is likely to be an important one [Phospholemman (PLM) was first identified as an abundant phosphoprotein in the cardiac sarcolemma in 1985 , and it tant one : it was tant one  of whichtant one , 100–102PLM FXYD1) is unique in the FXYD family in possessing phosphorylation sites in its carboxyl terminus that are conserved across all vertebrate sequences cloned to date , to phyUnphosphorylated PLM inhibits the cardiac Na pump. The exact nature of the inhibitory effect has been reported to be via a reduction in sodium affinity with no alteration in maximum transport rate  and Cam kinase (at T17), with essentially identical effects on SERCA activity of phosphorylation at either site . The anaOne complication in the investigations into the relationship between PLM and the Na pump is the relatively high basal phosphorylation of PLM in ventricular muscle. In rat ventricular myocytes, ~30 % of PLM is phosphorylated at S68 and ~50 % is phosphorylated at S63  . This baThe relative contributions of basally phosphorylated and unphosphorylated PLM to pump regulation in wild-type myocytes was addressed by Pavlovic et al. . A peptiVmax [Vmax), and activating PKC after PKA causes further phosphorylation of S68 and increases pump Vmax [may reflect a pump isoform-specific effect of PLM phosphorylation on the pump, in the mouse, almost 90 % of pump is α1-containing [Vmax observed when PKC is activated in the mouse ventricular myocytes [Na pump activation by PKC in the heart also requires PLM . In mousVmax , with noVmax , and an Vmax . The effmp Vmax . Taken tntaining . Hence, ntaining , 113, itmyocytes . Xenopus oocyte expression system, PKA phosphorylation of PLM at S68 increases the sodium affinity of both α1 and α2-containing pumps, whereas PKC phosphorylation of PLM increases the turnover rate only of α2 containing pump (to a level above the activity of pump expressed without PLM) [Vmax induced by PKC phosphorylation is abolished by mutation of PLM S68 [That PKA and PKC are functionally linked to different isoforms of the catalytic subunit of the pump in cardiac muscle was first proposed even before the role of PLM in regulating the pump had been discovered . In the out PLM) , closelyout PLM) . The funout PLM) . Importaout PLM) . Insteadout PLM) , the sit PLM S68 , it is l PLM S68 . That saVmax must reflect an increase in the rate of this partial reaction. Doubtless the mechanism underlying this will become clearer when more structural information is available.It is perhaps significant that there are notable sequence differences between α1 and α2 subunits in a region of the N domain that may be capable of interacting with PLM, which might explain the isoform-specific effects of PLM phosphorylation. Figure Vmax of α2-containing pumps [The elegant transgenic model in which the ouabain affinities of Na pump α1 and α2 subunits are swapped (SWAP mice ) has allng pumps . For nowng pumps  that couRelatively little research has been conducted to date on the pathways leading to PLM dephosphorylation. Shortly after the cloning of PLM, adenosine receptor agonists were reported to attenuate adrenoceptor agonist-induced phosphorylation of PLM independent of cellular cAMP . SubsequIn the context of adrenoceptor activation increasing myocardial contractility, it is pertinent to ask why hearts need PLM. On the face of it, enhanced Na pump activity, by increasing the driving force for calcium efflux through NCX, will tend to oppose the positive inotropy achieved through activation of L-type calcium channels, SERCA, and the ryanodine receptor by PKA. Genetic deletion of PLM slightly reduces cardiac contractility in vivo, although this is partly offset by a (possibly adaptive) reduction in pump subunit expression . It turnS-palmitoylation is the reversible covalent post-translational attachment of the fatty acid palmitic acid to the thiol group of cysteine, via an acyl-thioester linkage [S-palmitoylation has emerged as an important and common post-translational modification in a variety of tissues [S-palmitoylation is catalyzed by palmitoyl acyltransferases, reversed by protein thioesterases, and occurs dynamically and reversibly throughout the secretory pathway in a manner analogous to protein phosphorylation [ linkage . In rece tissues . Proteinrylation . Many dirylation , 129, iorylation , transporylation , receptorylation , and prorylation , 134. Ofrylation , 135: itPLM is palmitoylated at two intracellular cysteines, C40 and C42, just beyond the transmembrane domain . NotablyAlthough the palmitoyl acyltransferase that palmitoylates PLM is yet to be identified, one important regulator of PLM palmitoylation is its phosphorylation status. Paradoxically, phosphorylation of PLM at S68 by PKA increases PLM palmitoylation . Hence, Although beyond the scope of this review, it is important to note that several other functional roles are ascribed to PLM in the heart. Phosphorylation of PLM at S68 is associated with inhibition of NCX , 140. ItAlthough the original proposal  that PLMIn contrast to the apparent contradictions in the literature regarding the effect of PKA and PKC agonists on the cardiac pump, the exquisite sensitivity of Na pump activity in the heart to oxidant stress is widely accepted. Shattock and Matsuura  investiggenerally non-enzymatic, and occurs by direct reaction between oxidized/modified protein sulfhydryls and cellular glutathione, direct reaction between protein sulfhydryls and modified  glutathione, disulfide exchange between protein sulfhydryls and glutathionylated proteins, or direct reaction between protein sulfhydryls and glutathione in the presence of oxidants [Oxidant modification and regulation of the pump has also been the subject of more recent investigation. Glutathionylation is the reversible conjugation of the tripeptide glutathione to protein cysteines in a mixed disulfide. Many classes of protein have been found to be regulated by glutathionylation, including metabolic enzymes such as glyceraldehyde-3-phosphate dehydrogenase , kinasesoxidants . The lacoxidants .The α subunit of the cardiac Na pump has recently been reported to be glutathionylated to regulate its activity . ExposurVmax of the pump through a reduction of the rate constant for the E2 to E1 transition in the reaction cycle [The Na pump in the heart is also reported to be regulated by glutathionylation of its β rather than α subunit . The caron cycle . The cyson cycle . Recent on cycle . Ouabainon cycle .inhibition of the cardiac pump by PKA [A similar signaling mechanism has been proposed to mediate p by PKA . In rabbp by PKA , 125, bup by PKA , 84, 161p by PKA  . Nor can redox and phosphoregulation by PLM be easily separated since it is now well established that oxidizing species activate PKA and PKG in ventricular muscle –167. IndTo date, remarkably few studies have investigated the effects of endogenous agonists of adrenoceptors on Na pump activity in ventricular muscle. Although a considerable amount of evidence points to the involvement of pathways linked to β1, β3 and α1 adrenoceptors, the additional activation of β2 adrenoceptors by adrenalin/noradrenalin must also be considered, as this generates a cAMP signal localized to the sarcolemma by caveolae  , 169. ThWhile perhaps not the subject of recent research, it is noteworthy that the cardiac Na pump is regulated by lipid species. Palmitoyl carnitine and lysophosphatidylcholine are potent pump inhibitors , 173, whWhile the stimulatory effect of long-chain acyl CoA on the pump is thought to be due to specific binding of the acyl-CoA to an intracellular domain of the pump, rather than non-specific effects on the bilayer , the mecAlthough the cardiotonic effects of Na pump inhibitors such as digoxin and ouabain have been established for centuries, consensus on the finer molecular details of this phenomenon has been hard to reach. That the cardiotonic steroid binding site in the pump α subunit is physiologically important is demonstrated by its conservation across all vertebrates , althougPositive inotropy following pump inhibition by cardiotonic steroids is as a result of reduced NCX activity increasing the calcium content of the SR. The pump isoform responsible for this phenomenon in the heart has been the subject of some debate. In rat ventricular myocytes, selective inhibition of α2-containing pumps  increases contractility without a global increase in intracellular sodium through the sodium-loading of a sub-sarcolemmal compartment functionally linked to NCX . ThroughPrecise control of the subcellular location of the Na pump is essential for co-ordinated control of its activity. In cardiac muscle essentially all active pump is found in caveolae . CaveolaApart from the Na pump, a considerable number of cardiac ion transporters are resident in cardiac caveolae: voltage-gated sodium channels , L-type XXXXφXXφ at the N terminus, or φXφXXXXφ at the C terminus, where φ represents an aromatic amino acid [The presence of the cardiac pump in caveolae is achieved through the presence of a caveolin binding motif : either ino acid . These mino acid , essentiino acid . Given tino acid .The relative functional concentration of pump isoforms in cardiac t-tubules , 24 mustIn addition to the well-characterized interaction between the cardiac Na pump and NCX1 , 190, thmay reflect an increase in sodium influx [A reduction of the transarcolemmal Na gradient in cardiac myocytes has been implicated in a variety of pathologies including ischemia/reperfusion , 224, hym influx , there im influx –227 and m influx .In hypertrophy and HF many aspects of E–C coupling are clearly altered, however, the elevation in intracellular sodium may contribute to (1) the negative force-frequency relationship, (2) slowed relaxation (3) arrhythmias, and (4) impaired mitochondrial energetics , 230. Inincrease myocyte sodium efflux, and therefore restore the sodium gradient, may be an effective means to restore mitochondrial ‘supply–demand’ matching in hypertrophy and failure. The positive effect on the Na pump of agents used in the treatment of cardiac hypertrophy and failure has been noted by others: ACE inhibitors, angiotensin receptor blockers, aldosterone antagonists, NO-donors and insulin are all known to stimulate the Na pump and, in clinical trials, these agents have all been shown to be protective [and α2-pump inhibition to calcium-load the SR via NCX without imposing a significant sodium load [This raises the interesting paradigm that strategies that otective . Clearlyotective . Whetherium load , 190. Tiwhy is it essential to fine-tune intracellular sodium so precisely?’ and ‘which regulatory pathways are the most important?’. It is undoubtedly not necessary to fine-tune intracellular sodium to the nearest micromolar, but it is perhaps not precision but accuracy that is important for the cardiac Na pump. That is, the set-point for intracellular sodium and Na pump activity may not need to be precisely clamped, but rather need to vary depending on prevailing physiological conditions . As for which are the most important regulatory pathways, the simplest answer may be that it depends on the state of the heart when it receives a particular signal whether that signal leads to Na pump activation or inhibition. Hence one can conceive of adrenoceptor activation leading to kinase-induced pump activation via PLM in the absence of redox stress, and oxidant-induced pump inhibition in a heart with reduced anti-oxidant reserve. That said, the most compelling information is perhaps that PLM knockout myocytes are more prone to calcium-overload induced arrhythmias when treated with a β adrenoceptor agonist [In conclusion, all manner of regulatory pathways converge on the cardiac Na pump, and it sits exquisitely poised to respond to the changing demands of the cardiovascular system. To some extent a number of these regulatory pathways seem to cancel each other out, while others complement each other, and this raises questions such as ‘ agonist . This unAnother crucial factor in understanding the control of the cardiac pump is to appreciate the importance of local signaling networks and local ion gradients. This is not simply distinguishing t-tubular from surface sarcolemmal pumps, or α1 from α2 pumps, but drilling down into identifying subpopulations of pumps, the ion transporters with which they co-localize, the regulatory mechanisms that control them, their physiological significance, and measuring the sodium concentration ‘seen’ by each subpopulation. In other words our existing experimental tools must be improved so that we do not simply rely on whole cell pump currents and fluorescence measurements as indices of activity and substrate availability. Cardiac myocytes are complicated beasts: we are already accumulating evidence for local ion gradients and functional coupling of some but not all pumps to other sarcolemmal ion transporters. To continue to investigate global rather than local pump control is to ignore the reductionist approach that has served so well in the first 50 years of investigating this remarkable, ubiquitous enzyme complex.Supplementary material 1 (DOC 213 kb)Below is the link to the electronic supplementary material.


Receptor phosphorylation is thought to be tightly regulated because phosphorylated receptors initiate signaling cascades leading to cellular activation. The T cell antigen receptor (TCR) on the surface of T cells is phosphorylated by the kinase Lck and dephosphorylated by the phosphatase CD45 on multiple immunoreceptor tyrosine-based activation motifs (ITAMs). Intriguingly, Lck sequentially phosphorylates ITAMs and ZAP-70, a cytosolic kinase, binds to phosphorylated ITAMs with differential affinities. The purpose of multiple ITAMs, their sequential phosphorylation, and the differential ZAP-70 affinities are unknown. Here, we use a systems model to show that this signaling architecture produces emergent ultrasensitivity resulting in switch-like responses at the scale of individual TCRs. Importantly, this switch-like response is an emergent property, so that removal of multiple ITAMs, sequential phosphorylation, or differential affinities abolishes the switch. We propose that highly regulated TCR phosphorylation is achieved by an emergent switch-like response and use the systems model to design novel chimeric antigen receptors for therapy. Recognition of antigen by the T cell antigen receptor (TCR) is a central event in the initiation of adaptive immune responses and for this reason the TCR has been extensively studied. Multiple studies performed over the past 20 years have revealed intriguing findings that include the observation that the TCR has multiple phosphorylation sites that are sequentially phosphorylated by the kinase Lck and that ZAP-70, a cytosolic kinase, binds to these sites with different affinities. The purpose of multiple sites, their sequential phosphorylation by Lck, and the differential binding affinities of ZAP-70 are unknown. Using a novel mechanistic model that incorporates a high level of molecular detail, we find, unexpectedly, that all factors are critical for producing ultrasensitivity (switch-like response) and therefore this signaling architecture exhibits systems-level emergent ultrasensitivity. We use the model to study existing therapeutic chimeric antigen receptors and in the design of novel ones. The work also has direct implications to the study of many other immune receptors. T cells patrol the body in search of infection and cancer derived antigens The importance of the TCR in initiating and regulating adaptive immune responses has meant that TCR proximal proteins have been extensively studied. The TCR itself is a multi-subunit receptor that contains subunits for ligand binding  . As expeMs (XX3) , reflectMs (XX3)  and sensMs (XX3) . These eWe next investigated the contribution of ZAP-70 binding to potency and sensitivity. To do this, we calculated the total concentration of phosphorylated f ZAP-70 .The increase in potency  not the more commonly performed assay that involves titrating the number of target cells (with a fixed antigen concentration). Optimal CARs should exhibit low potency ,The profiles of ITAM-bound ZAP-70 and total TCR Figure S1Alignment of the TCR (EPS)Click here for additional data file.Figure S2Ultrasensitivity in the absence of ZAP-70 can be mediated by differential affinities of Lck, CD45, or both. A) The total phosphorylation of TCR (EPS)Click here for additional data file.Figure S3An alternate measure of sensitivity for the heat map in (EPS)Click here for additional data file.Text S1BioNetGen source code used to generate the wild-type systems model of T cell receptor proximal signaling.(PDF)Click here for additional data file.