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Biophysics
A Chimeric Egfr Protein Reporter Mouse Reveals Egfr Localization and Trafficking In Vivo
May 12, 2017   Cell Reports
Yang YP, Ma H, Starchenko A, Huh WJ, Li W, Hickman FE, Zhang Q, Franklin JL, Mortlock DP, Fuhrmann S, Carter BD, Ihrie RA, Coffey RJ
A Chimeric Egfr Protein Reporter Mouse Reveals Egfr Localization and Trafficking In Vivo
May 12, 2017
Cell Reports
EGF receptor (EGFR) is a critical signaling node throughout life. However, it has not been possible to directly visualize endogenous Egfr in mice. Using CRISPR/Cas9 genome editing, we appended a fluorescent reporter to the C terminus of the Egfr. Homozygous reporter mice appear normal and EGFR signaling is intact in vitro and in vivo. We detect distinct patterns of Egfr expression in progenitor and differentiated compartments in embryonic and adult mice. Systemic delivery of EGF or amphiregulin results in markedly different patterns of Egfr internalization and trafficking in hepatocytes. In the normal intestine, Egfr localizes to the crypt rather than villus compartment, expression is higher in adjacent epithelium than in intestinal tumors, and following colonic injury expression appears in distinct cell populations in the stroma. This reporter, under control of its endogenous regulatory elements, enables in vivo monitoring of the dynamics of Egfr localization and trafficking in normal and disease states. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.
Protein Truncating Variants at the Cholesteryl Ester Transfer Protein Gene and Risk for Coronary Heart Disease
May 16, 2017   Circulation Research
Nomura A, Won HH, Khera AV, Takeuchi F, Ito K,   . . . . . .   , Kubo M, Kato N, Chen YI, Dewey F, Kathiresan S
Protein Truncating Variants at the Cholesteryl Ester Transfer Protein Gene and Risk for Coronary Heart Disease
May 16, 2017
Circulation Research
Rationale: Therapies which inhibit cholesteryl ester transfer protein (CETP) have failed to demonstrate a reduction in risk for coronary heart disease (CHD). Human deoxyribonucleic acid sequence variants that truncate the CETP gene may provide insight into the efficacy of CETP inhibition. Objective: To test whether protein truncating variants (PTVs) at the CETP gene were associated with plasma lipid levels and CHD. Methods and Results: We sequenced the exons of the CETP gene in 58,469 participants from 12 case-control studies (18,817 CHD cases, 39,652 CHD-free controls). We defined PTV as those that lead to a premature stop, disrupt canonical splice-sites, or lead to insertions/deletions that shift frame. We also genotyped one Japanese-specific PTV in 27,561 participants from three case-control studies (14,286 CHD cases, 13,275 CHD-free controls). We tested association of CETP PTV carrier status with both plasma lipids and CHD. Among 58,469 participants with CETP gene sequencing data available, average age was 51.5 years and 43% were female; 1 in 975 participants carried a PTV at the CETP gene. Compared to non-carriers, carriers of PTV at CETP had higher high-density lipoprotein cholesterol (HDL-C; effect size, 22.6 mg/dL; 95% confidence interval [CI], 18 to 27; P < 1.0x10-4), lower low-density lipoprotein cholesterol (LDL-C; -12.2 mg/dL; 95% CI, -23 to -0.98; P = 0.033), and lower triglycerides (-6.3%; 95% CI, -12 to -0.22, P = 0.043). CETP PTV carrier status was associated with reduced risk for CHD (summary odds ratio, 0.70; 95% CI, 0.54 to 0.90; P = 5.1x10-3). Conclusions: Compared with non-carriers, carriers of PTV at CETP displayed higher HDL-C, lower LDL-C, lower triglycerides, and lower risk for CHD.
Store-operated Ca2+ entry supports contractile function in hearts of hibernators
May 22, 2017   PloS One
Nakipova OV, Averin AS, Evdokimovskii EV, Pimenov OY, Kosarski L, Ignat'ev D, Anufriev A, Kokoz YM, Reyes S, Terzic A, Alekseev AE
Store-operated Ca2+ entry supports contractile function in hearts of hibernators
May 22, 2017
PloS One
Hibernators have a distinctive ability to adapt to seasonal changes of body temperature in a range between 37°C and near freezing, exhibiting, among other features, a unique reversibility of cardiac contractility. The adaptation of myocardial contractility in hibernation state relies on alterations of excitation contraction coupling, which becomes less-dependent from extracellular Ca2+ entry and is predominantly controlled by Ca2+ release from sarcoplasmic reticulum, replenished by the Ca2+-ATPase (SERCA). We found that the specific SERCA inhibitor cyclopiazonic acid (CPA), in contrast to its effect in papillary muscles (PM) from rat hearts, did not reduce but rather potentiated contractility of PM from hibernating ground squirrels (GS). In GS ventricles we identified drastically elevated, compared to rats, expression of Orai1, Stim1 and Trpc1/3/4/5/6/7 mRNAs, putative components of store operated Ca2+ channels (SOC). Trpc3 protein levels were found increased in winter compared to summer GS, yet levels of Trpc5, Trpc6 or Trpc7 remained unchanged. Under suppressed voltage-dependent K+, Na+ and Ca2+ currents, the SOC inhibitor 2-aminoethyl diphenylborinate (2-APB) diminished whole-cell membrane currents in isolated cardiomyocytes from hibernating GS, but not from rats. During cooling-reheating cycles (30°C-7°C-30°C) of ground squirrel PM, 2-APB did not affect typical CPA-sensitive elevation of contractile force at low temperatures, but precluded the contractility at 30°C before and after the cooling. Wash-out of 2-APB reversed PM contractility to control values. Thus, we suggest that SOC play a pivotal role in governing the ability of hibernator hearts to maintain their function during the transition in and out of hibernating states.
Thermal stability and kinetic constants for 129 variants of a family 1 glycoside hydrolase reveal that enzyme activity and stability can be separately designed
May 22, 2017   PloS One
Carlin DA, Hapig-Ward S, Chan BW, Damrau N, Riley M, Caster RW, Bethards B, Siegel JB
Thermal stability and kinetic constants for 129 variants of a family 1 glycoside hydrolase reveal that enzyme activity and stability can be separately designed
May 22, 2017
PloS One
Accurate modeling of enzyme activity and stability is an important goal of the protein engineering community. However, studies seeking to evaluate current progress are limited by small data sets of quantitative kinetic constants and thermal stability measurements. Here, we report quantitative measurements of soluble protein expression in E. coli, thermal stability, and Michaelis-Menten constants (kcat, KM, and kcat/KM) for 129 designed mutants of a glycoside hydrolase. Statistical analyses reveal that functional Tm is independent of kcat, KM, and kcat/KM in this system, illustrating that an individual mutation can modulate these functional parameters independently. In addition, this data set is used to evaluate computational predictions of protein stability using the established Rosetta and FoldX algorithms. Predictions for both are found to correlate only weakly with experimental measurements, suggesting improvements are needed in the underlying algorithms.
A Schizophrenia-Related Deletion Leads to KCNQ2-Dependent Abnormal Dopaminergic Modulation of Prefrontal Cortical Interneuron Activity
May 19, 2017   Cerebral Cortex (New York, N.Y. : 1991)
Choi SJ, Mukai J, Kvajo M, Xu B, Diamantopoulou A, Pitychoutis PM, Gou B, Gogos JA, Zhang H
A Schizophrenia-Related Deletion Leads to KCNQ2-Dependent Abnormal Dopaminergic Modulation of Prefrontal Cortical Interneuron Activity
May 19, 2017
Cerebral Cortex (New York, N.Y. : 1991)
Altered prefrontal cortex function is implicated in schizophrenia (SCZ) pathophysiology and could arise from imbalance between excitation and inhibition (E/I) in local circuits. It remains unclear whether and how such imbalances relate to genetic etiologies. We used a mouse model of the SCZ-predisposing 22q11.2 deletion (Df(16)A+/- mice) to evaluate how this genetic lesion affects the excitability of layer V prefrontal pyramidal neurons and its modulation by dopamine (DA). Df(16)A+/- mice have normal balance between E/I at baseline but are unable to maintain it upon dopaminergic challenge. Specifically, in wild-type mice, D1 receptor (D1R) activation enhances excitability of layer V prefrontal pyramidal neurons and D2 receptor (D2R) activation reduces it. Whereas the excitatory effect upon D1R activation is enhanced in Df(16)A+/- mice, the inhibitory effect upon D2R activation is reduced. The latter is partly due to the inability of mutant mice to activate GABAergic parvalbumin (PV)+ interneurons through D2Rs. We further demonstrate that reduced KCNQ2 channel function in PV+ interneurons in Df(16)A+/- mice renders them less capable of inhibiting pyramidal neurons upon D2 modulation. Thus, DA modulation of PV+ interneurons and control of E/I are altered in Df(16)A+/- mice with a higher excitation and lower inhibition during dopaminergic modulation. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.
Evolutionary enhancement of Zika virus infectivity in Aedes aegypti mosquitoes
May 17, 2017   Nature Add nature.com free-link Cancel
Liu Y, Liu J, Du S, Shan C, Nie K, Zhang R, Li XF, Zhang R, Wang T, Qin CF, Wang P, Shi PY, Cheng G
Evolutionary enhancement of Zika virus infectivity in Aedes aegypti mosquitoes
May 17, 2017
Nature
Zika virus (ZIKV) remained obscure until the recent explosive outbreaks in French Polynesia (2013-2014) and South America (2015-2016). Phylogenetic studies have shown that ZIKV has evolved into African and Asian lineages. The Asian lineage of ZIKV was responsible for the recent epidemics in the Americas. However, the underlying mechanisms through which ZIKV rapidly and explosively spread from Asia to the Americas are unclear. Non-structural protein 1 (NS1) facilitates flavivirus acquisition by mosquitoes from an infected mammalian host and subsequently enhances viral prevalence in mosquitoes. Here we show that NS1 antigenaemia determines ZIKV infectivity in its mosquito vector Aedes aegypti, which acquires ZIKV via a blood meal. Clinical isolates from the most recent outbreak in the Americas were much more infectious in mosquitoes than the FSS13025 strain, which was isolated in Cambodia in 2010. Further analyses showed that these epidemic strains have higher NS1 antigenaemia than the FSS13025 strain because of an alanine-to-valine amino acid substitution at residue 188 in NS1. ZIKV infectivity was enhanced by this amino acid substitution in the ZIKV FSS13025 strain in mosquitoes that acquired ZIKV from a viraemic C57BL/6 mouse deficient in type I and II interferon (IFN) receptors (AG6 mouse). Our results reveal that ZIKV evolved to acquire a spontaneous mutation in its NS1 protein, resulting in increased NS1 antigenaemia. Enhancement of NS1 antigenaemia in infected hosts promotes ZIKV infectivity and prevalence in mosquitoes, which could have facilitated transmission during recent ZIKV epidemics.
DNA recognition by an RNA-guided bacterial Argonaute
May 18, 2017   PloS One
Doxzen KW, Doudna JA
DNA recognition by an RNA-guided bacterial Argonaute
May 18, 2017
PloS One
Argonaute (Ago) proteins are widespread in prokaryotes and eukaryotes and share a four-domain architecture capable of RNA- or DNA-guided nucleic acid recognition. Previous studies identified a prokaryotic Argonaute protein from the eubacterium Marinitoga piezophila (MpAgo), which binds preferentially to 5'-hydroxylated guide RNAs and cleaves single-stranded RNA (ssRNA) and DNA (ssDNA) targets. Here we present a 3.2 Å resolution crystal structure of MpAgo bound to a 21-nucleotide RNA guide and a complementary 21-nucleotide ssDNA substrate. Comparison of this ternary complex to other target-bound Argonaute structures reveals a unique orientation of the N-terminal domain, resulting in a straight helical axis of the entire RNA-DNA heteroduplex through the central cleft of the protein. Additionally, mismatches introduced into the heteroduplex reduce MpAgo cleavage efficiency with a symmetric profile centered around the middle of the helix. This pattern differs from the canonical mismatch tolerance of other Argonautes, which display decreased cleavage efficiency for substrates bearing sequence mismatches to the 5' region of the guide strand. This structural analysis of MpAgo bound to a hybrid helix advances our understanding of the diversity of target recognition mechanisms by Argonaute proteins.
Chemicogenetic Restoration of the Prefrontal Cortex to Amygdala Pathway Ameliorates Stress-Induced Deficits
May 12, 2017   Cerebral Cortex (New York, N.Y. : 1991)
Wei J, Zhong P, Qin L, Tan T, Yan Z
Chemicogenetic Restoration of the Prefrontal Cortex to Amygdala Pathway Ameliorates Stress-Induced Deficits
May 12, 2017
Cerebral Cortex (New York, N.Y. : 1991)
Corticosteroid stress hormones exert a profound impact on cognitive and emotional processes. Understanding the neuronal circuits that are altered by chronic stress is important for counteracting the detrimental effects of stress in a brain region- and cell type-specific manner. Using the chemogenetic tool, Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), which enables the remote, noninvasive and long-lasting modulation of cellular activity and signal transduction in discrete neuronal populations in vivo, we sought to identify the specific pathways that play an essential role in stress responses. We found that prolonged severe stress induced the diminished glutamatergic projection from pyramidal neurons in prefrontal cortex (PFC) to GABAergic interneurons in basolateral amygdala (BLA), leading to the loss of feedforward inhibition and ensuing hyperexcitability of BLA principal neurons, which caused a variety of behavioral abnormalities. Activating PFC pyramidal neurons with hM3D(Gq) DREADD restored the functional connection between PFC and BLA in stressed animals, resulting in the rescue of recognition memory, normalization of locomotor activity and reduction of aggressive behaviors. Inhibiting BLA principal neurons directly with hM4D(Gi) DREADD also blocked BLA hyperactivity and aggressive behaviors in stressed animals. These results have offered an effective avenue to counteract the stress-induced disruption of circuitry homeostasis. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.
Surround Integration Organizes a Spatial Map during Active Sensation
May 15, 2017   Neuron
Pluta SR, Lyall EH, Telian GI, Ryapolova-Webb E, Adesnik H
Surround Integration Organizes a Spatial Map during Active Sensation
May 15, 2017
Neuron
During active sensation, sensors scan space in order to generate a representation of the outside world. However, since spatial coding in sensory systems is typically addressed by measuring receptive fields in a fixed, sensor-based coordinate frame, the cortical representation of scanned space is poorly understood. To address this question, we probed spatial coding in the rodent whisker system using a combination of two-photon imaging and electrophysiology during active touch. We found that surround whiskers powerfully transform the cortical representation of scanned space. On the single-neuron level, surround input profoundly alters response amplitude and modulates spatial preference in the cortex. On the population level, surround input organizes the spatial preference of neurons into a continuous map of the space swept out by the whiskers. These data demonstrate how spatial summation over a moving sensor array is critical to generating population codes of sensory space. Copyright © 2017 Elsevier Inc. All rights reserved.
Spatial Asymmetry and Short-Term Suppression Underlie Direction Selectivity of Synaptic Excitation in the Mouse Visual Cortex
May 12, 2017   Cerebral Cortex (New York, N.Y. : 1991)
Li YT, Fang Q, Zhang LI, Tao HW
Spatial Asymmetry and Short-Term Suppression Underlie Direction Selectivity of Synaptic Excitation in the Mouse Visual Cortex
May 12, 2017
Cerebral Cortex (New York, N.Y. : 1991)
Direction selectivity (DS) of neuronal responses is fundamental for motion detection. With in vivo whole-cell voltage-clamp recordings from layer (L)4 neurons in the mouse visual cortex, we observed a strong correlation between DS and spatial asymmetry in the distribution of excitatory input strengths. This raises an interesting possibility that the latter may contribute to DS. The preferred direction of excitatory input was found from the stronger to weaker side of its spatial receptive field. A simple linear summation of asymmetrically distributed excitatory responses to stationary flash stimuli however failed to predict the correct directionality: it at best resulted in weak DS with preferred direction opposite to what was observed experimentally. Further studies with sequential 2 flash-bar stimulation revealed a short-term suppression of excitatory input evoked by the late bar. More importantly, the level of the suppression positively correlated with the relative amplitude of the early-bar response. Implementing this amplitude-dependent suppressive interaction can successfully predict DS of excitatory input. Our results suggest that via nonlinear temporal interactions, the spatial asymmetry can be transformed into differential temporal integration of inputs under opposite directional movements. This mechanism may contribute to the DS of excitatory inputs to L4 neurons. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.
Predicting accurate contacts in thousands of Pfam domain families using PconsC3
May 23, 2017   Bioinformatics (Oxford, England)
Michel M, Skwark MJ, Menéndez Hurtado D, Ekeberg M, Elofsson A
Predicting accurate contacts in thousands of Pfam domain families using PconsC3
May 23, 2017
Bioinformatics (Oxford, England)
A fewyears ago it was shown that by using amaximumentropy approach to describe couplings between columns in a multiple sequence alignment it is possible to significantly increase the accuracy of residue contact predictions. For very large protein families with more than 1000 effective sequences the accuracy is sufficient to produce accurate models of proteins as well as complexes. Today, for about half of all Pfam domain families no structure is known, but unfortunately most of these families have at most a few hundred members, i.e. are too small for such contact prediction methods. To extend accurate contact predictions to the thousands of smaller protein families we present PconsC3, a fast and improved method for protein contact predictions that can be used for families with even 100 effective sequence members. PconsC3 outperforms direct coupling analysis (DCA) methods significantly independent on family size, secondary structure content, contact range, or the number of selected contacts. PconsC3 is available as a web server and downloadable version at http://c3.pcons.net . The downloadable version is free for all to use and licensed under the GNU General Public License, version 2. At this site contact predictions for most Pfam families are also available. We do estimate that more than 4000 contact maps for Pfam families of unknown structure have more than 50% of the top-ranked contacts predicted correctly. arne@bioinfo.se. Supplementary data are available at Bioinformatics online.
Rules of engagement between αvβ6 integrin and foot-and-mouth disease virus
May 23, 2017   Nature Communications
Kotecha A, Wang Q, Dong X, Ilca SL, Ondiviela M, Zihe R, Seago J, Charleston B, Fry EE, Abrescia NGA, Springer TA, Huiskonen JT, Stuart DI
Rules of engagement between αvβ6 integrin and foot-and-mouth disease virus
May 23, 2017
Nature Communications
Foot-and-mouth disease virus (FMDV) mediates cell entry by attachment to an integrin receptor, generally αvβ6, via a conserved arginine-glycine-aspartic acid (RGD) motif in the exposed, antigenic, GH loop of capsid protein VP1. Infection can also occur in tissue culture adapted virus in the absence of integrin via acquired basic mutations interacting with heparin sulphate (HS); this virus is attenuated in natural infections. HS interaction has been visualized at a conserved site in two serotypes suggesting a propensity for sulfated-sugar binding. Here we determined the interaction between αvβ6 and two tissue culture adapted FMDV strains by cryo-electron microscopy. In the preferred mode of engagement, the fully open form of the integrin, hitherto unseen at high resolution, attaches to an extended GH loop via interactions with the RGD motif plus downstream hydrophobic residues. In addition, an N-linked sugar of the integrin attaches to the previously identified HS binding site, suggesting a functional role.
Efficient protein production inspired by how spiders make silk
May 23, 2017   Nature Communications
Kronqvist N, Sarr M, Lindqvist A, Nordling K, Otikovs M,   . . . . . .   , Hebert H, Jaudzems K, Curstedt T, Rising A, Johansson J
Efficient protein production inspired by how spiders make silk
May 23, 2017
Nature Communications
Membrane proteins are targets of most available pharmaceuticals, but they are difficult to produce recombinantly, like many other aggregation-prone proteins. Spiders can produce silk proteins at huge concentrations by sequestering their aggregation-prone regions in micellar structures, where the very soluble N-terminal domain (NT) forms the shell. We hypothesize that fusion to NT could similarly solubilize non-spidroin proteins, and design a charge-reversed mutant (NT*) that is pH insensitive, stabilized and hypersoluble compared to wild-type NT. NT*-transmembrane protein fusions yield up to eight times more of soluble protein in Escherichia coli than fusions with several conventional tags. NT* enables transmembrane peptide purification to homogeneity without chromatography and manufacture of low-cost synthetic lung surfactant that works in an animal model of respiratory disease. NT* also allows efficient expression and purification of non-transmembrane proteins, which are otherwise refractory to recombinant production, and offers a new tool for reluctant proteins in general.
BLISS is a versatile and quantitative method for genome-wide profiling of DNA double-strand breaks
May 12, 2017   Nature Communications
Yan WX, Mirzazadeh R, Garnerone S, Scott D, Schneider MW,   . . . . . .   , Federova Y, Zetsche B, Zhang F, Bienko M, Crosetto N
BLISS is a versatile and quantitative method for genome-wide profiling of DNA double-strand breaks
May 12, 2017
Nature Communications
Precisely measuring the location and frequency of DNA double-strand breaks (DSBs) along the genome is instrumental to understanding genomic fragility, but current methods are limited in versatility, sensitivity or practicality. Here we present Breaks Labeling In Situ and Sequencing (BLISS), featuring the following: (1) direct labelling of DSBs in fixed cells or tissue sections on a solid surface; (2) low-input requirement by linear amplification of tagged DSBs by in vitro transcription; (3) quantification of DSBs through unique molecular identifiers; and (4) easy scalability and multiplexing. We apply BLISS to profile endogenous and exogenous DSBs in low-input samples of cancer cells, embryonic stem cells and liver tissue. We demonstrate the sensitivity of BLISS by assessing the genome-wide off-target activity of two CRISPR-associated RNA-guided endonucleases, Cas9 and Cpf1, observing that Cpf1 has higher specificity than Cas9. Our results establish BLISS as a versatile, sensitive and efficient method for genome-wide DSB mapping in many applications.
Sirt1 carboxyl-domain is an ATP-repressible domain that is transferrable to other proteins
May 15, 2017   Nature Communications
Kang H, Oka S, Lee DY, Park J, Aponte AM,   . . . . . .   , Tjandra N, Lee SB, Kim MK, Sadoshima J, Chung JH
Sirt1 carboxyl-domain is an ATP-repressible domain that is transferrable to other proteins
May 15, 2017
Nature Communications
Sirt1 is an NAD+-dependent protein deacetylase that regulates many physiological functions, including stress resistance, adipogenesis, cell senescence and energy production. Sirt1 can be activated by energy deprivation, but the mechanism is poorly understood. Here, we report that Sirt1 is negatively regulated by ATP, which binds to the C-terminal domain (CTD) of Sirt1. ATP suppresses Sirt1 activity by impairing the CTD's ability to bind to the deacetylase domain as well as its ability to function as the substrate recruitment site. ATP, but not NAD+, causes a conformational shift to a less compact structure. Mutations that prevent ATP binding increase Sirt1's ability to promote stress resistance and inhibit adipogenesis under high-ATP conditions. Interestingly, the CTD can be attached to other proteins, thereby converting them into energy-regulated proteins. These discoveries provide insight into how extreme energy deprivation can impact Sirt1 activity and underscore the complex nature of Sirt1 structure and regulation.
Structural basis for conductance through TRIC cation channels
May 19, 2017   Nature Communications
Su M, Gao F, Yuan Q, Mao Y, Li DL,   . . . . . .   , Zeng Y, Zhang FB, Marks AR, Hendrickson WA, Chen YH
Structural basis for conductance through TRIC cation channels
May 19, 2017
Nature Communications
Mammalian TRICs function as K+-permeable cation channels that provide counter ions for Ca2+ handling in intracellular stores. Here we describe the structures of two prokaryotic homologues, archaeal SaTRIC and bacterial CpTRIC, showing that TRIC channels are symmetrical trimers with transmembrane pores through each protomer. Each pore holds a string of water molecules centred at kinked helices in two inverted-repeat triple-helix bundles (THBs). The pores are locked in a closed state by a hydrogen bond network at the C terminus of the THBs, which is lost when the pores assume an open conformation. The transition between the open and close states seems to be mediated by cation binding to conserved residues along the three-fold axis. Electrophysiology and mutagenesis studies show that prokaryotic TRICs have similar functional properties to those of mammalian TRICs and implicate the three-fold axis in the allosteric regulation of the channel.
Concerted regulation of retinal pigment epithelium basement membrane and barrier function by angiocrine factors
May 19, 2017   Nature Communications
Benedicto I, Lehmann GL, Ginsberg M, Nolan DJ, Bareja R,   . . . . . .   , Rabbany SY, Maminishkis A, Miller SS, Rafii S, Rodriguez-Boulan E
Concerted regulation of retinal pigment epithelium basement membrane and barrier function by angiocrine factors
May 19, 2017
Nature Communications
The outer blood-retina barrier is established through the coordinated terminal maturation of the retinal pigment epithelium (RPE), fenestrated choroid endothelial cells (ECs) and Bruch's membrane, a highly organized basement membrane that lies between both cell types. Here we study the contribution of choroid ECs to this process by comparing their gene expression profile before (P5) and after (P30) the critical postnatal period when mice acquire mature visual function. Transcriptome analyses show that expression of extracellular matrix-related genes changes dramatically over this period. Co-culture experiments support the existence of a novel regulatory pathway: ECs secrete factors that remodel RPE basement membrane, and integrin receptors sense these changes triggering Rho GTPase signals that modulate RPE tight junctions and enhance RPE barrier function. We anticipate our results will spawn a search for additional roles of choroid ECs in RPE physiology and disease.
Temporal dynamics of gene expression and histone marks at the Arabidopsis shoot meristem during flowering
May 17, 2017   Nature Communications
You Y, Sawikowska A, Neumann M, Posé D, Capovilla G, Langenecker T, Neher RA, Krajewski P, Schmid M
Temporal dynamics of gene expression and histone marks at the Arabidopsis shoot meristem during flowering
May 17, 2017
Nature Communications
Plants can produce organs throughout their entire life from pluripotent stem cells located at their growing tip, the shoot apical meristem (SAM). At the time of flowering, the SAM of Arabidopsis thaliana switches fate and starts producing flowers instead of leaves. Correct timing of flowering in part determines reproductive success, and is therefore under environmental and endogenous control. How epigenetic regulation contributes to the floral transition has eluded analysis so far, mostly because of the poor accessibility of the SAM. Here we report the temporal dynamics of the chromatin modifications H3K4me3 and H3K27me3 and their correlation with transcriptional changes at the SAM in response to photoperiod-induced flowering. Emphasizing the importance of tissue-specific epigenomic analyses we detect enrichments of chromatin states in the SAM that were not apparent in whole seedlings. Furthermore, our results suggest that regulation of translation might be involved in adjusting meristem function during the induction of flowering.
An allosteric site in the T-cell receptor Cβ domain plays a critical signalling role
May 16, 2017   Nature Communications
Natarajan K, McShan AC, Jiang J, Kumirov VK, Wang R, Zhao H, Schuck P, Tilahun ME, Boyd LF, Ying J, Bax A, Margulies DH, Sgourakis NG
An allosteric site in the T-cell receptor Cβ domain plays a critical signalling role
May 16, 2017
Nature Communications
The molecular mechanism through which the interaction of a clonotypic αβ T-cell receptor (TCR) with a peptide-loaded major histocompatibility complex (p/MHC) leads to T-cell activation is not yet fully understood. Here we exploit a high-affinity TCR (B4.2.3) to examine the structural changes that accompany binding to its p/MHC ligand (P18-I10/H2-Dd). In addition to conformational changes in complementarity-determining regions (CDRs) of the TCR seen in comparison of unliganded and bound X-ray structures, NMR characterization of the TCR β-chain dynamics reveals significant chemical shift effects in sites removed from the MHC-binding site. Remodelling of electrostatic interactions near the Cβ H3 helix at the membrane-proximal face of the TCR, a region implicated in interactions with the CD3 co-receptor, suggests a possible role for an allosteric mechanism in TCR signalling. The contribution of these TCR residues to signal transduction is supported by mutagenesis and T-cell functional assays.
Structure of the quaternary complex between SRP, SR, and translocon bound to the translating ribosome
May 19, 2017   Nature Communications
Jomaa A, Fu YH, Boehringer D, Leibundgut M, Shan SO, Ban N
Structure of the quaternary complex between SRP, SR, and translocon bound to the translating ribosome
May 19, 2017
Nature Communications
During co-translational protein targeting, the signal recognition particle (SRP) binds to the translating ribosome displaying the signal sequence to deliver it to the SRP receptor (SR) on the membrane, where the signal peptide is transferred to the translocon. Using electron cryo-microscopy, we have determined the structure of a quaternary complex of the translating Escherichia coli ribosome, the SRP-SR in the 'activated' state and the translocon. Our structure, supported by biochemical experiments, reveals that the SRP RNA adopts a kinked and untwisted conformation to allow repositioning of the 'activated' SRP-SR complex on the ribosome. In addition, we observe the translocon positioned through interactions with the SR in the vicinity of the ribosome exit tunnel where the signal sequence is extending beyond its hydrophobic binding groove of the SRP M domain towards the translocon. Our study provides new insights into the mechanism of signal sequence transfer from the SRP to the translocon.
ATP controls the crowd
May 19, 2017   Science (New York, N.Y.)
Rice AM, Rosen MK
ATP controls the crowd
May 19, 2017
Science (New York, N.Y.)
Macrophage function in tissue repair and remodeling requires IL-4 or IL-13 with apoptotic cells
May 12, 2017   Science (New York, N.Y.)
Bosurgi L, Cao YG, Cabeza-Cabrerizo M, Tucci A, Hughes LD,   . . . . . .   , Gagliani N, Craft JE, Flavell RA, Ghosh S, Rothlin CV
Macrophage function in tissue repair and remodeling requires IL-4 or IL-13 with apoptotic cells
May 12, 2017
Science (New York, N.Y.)
Tissue repair is a subset of a broad repertoire of IL-4/IL-13-dependent host responses during helminth infection. Here, we show that IL-4/IL-13 alone were not sufficient, but IL-4/IL-13 together with apoptotic cells induced the tissue repair program in macrophages. Genetic ablation of sensors of apoptotic cells impaired the proliferation of tissue-resident macrophages and the induction of anti-inflammatory/tissue repair genes in the lung following helminth infection or in the gut following induction of colitis. In contrast, the recognition of apoptotic cells was dispensable for cytokine-dependent induction of pattern recognition receptor, cell adhesion or chemotaxis genes in macrophages. Detection of apoptotic cells can therefore spatially compartmentalize or prevent premature or ectopic activity of pleiotropic, soluble cytokines, such as IL-4/IL-13. Copyright © 2017, American Association for the Advancement of Science.
Markovian Analysis of the Sequential Behavior of the Spontaneous Spinal Cord Dorsum Potentials Induced by Acute Nociceptive Stimulation in the Anesthetized Cat
May 16, 2017   Frontiers In Computational Neuroscience
Martin M, Béjar J, Esposito G, Chávez D, Contreras-Hernández E, Glusman S, Cortés U, Rudomín P
Markovian Analysis of the Sequential Behavior of the Spontaneous Spinal Cord Dorsum Potentials Induced by Acute Nociceptive Stimulation in the Anesthetized Cat
May 16, 2017
Frontiers In Computational Neuroscience
In a previous study we developed a Machine Learning procedure for the automatic identification and classification of spontaneous cord dorsum potentials (CDPs). This study further supported the proposal that in the anesthetized cat, the spontaneous CDPs recorded from different lumbar spinal segments are generated by a distributed network of dorsal horn neurons with structured (non-random) patterns of functional connectivity and that these configurations can be changed to other non-random and stable configurations after the noceptive stimulation produced by the intradermic injection of capsaicin in the anesthetized cat. Here we present a study showing that the sequence of identified forms of the spontaneous CDPs follows a Markov chain of at least order one. That is, the system has memory in the sense that the spontaneous activation of dorsal horn neuronal ensembles producing the CDPs is not independent of the most recent activity. We used this markovian property to build a procedure to identify portions of signals as belonging to a specific functional state of connectivity among the neuronal networks involved in the generation of the CDPs. We have tested this procedure during acute nociceptive stimulation produced by the intradermic injection of capsaicin in intact as well as spinalized preparations. Altogether, our results indicate that CDP sequences cannot be generated by a renewal stochastic process. Moreover, it is possible to describe some functional features of activity in the cord dorsum by modeling the CDP sequences as generated by a Markov order one stochastic process. Finally, these Markov models make possible to determine the functional state which produced a CDP sequence. The proposed identification procedures appear to be useful for the analysis of the sequential behavior of the ongoing CDPs recorded from different spinal segments in response to a variety of experimental procedures including the changes produced by acute nociceptive stimulation. They are envisaged as a useful tool to examine alterations of the patterns of functional connectivity between dorsal horn neurons under normal and different pathological conditions, an issue of potential clinical concern.
Angular measurements of the dynein ring reveal a stepping mechanism dependent on a flexible stalk
May 23, 2017   Proceedings Of The National Academy Of Sciences Of The United States Of America
Lippert LG, Dadosh T, Hadden JA, Karnawat V, Diroll BT, Murray CB, Holzbaur ELF, Schulten K, Reck-Peterson SL, Goldman YE
Angular measurements of the dynein ring reveal a stepping mechanism dependent on a flexible stalk
May 23, 2017
Proceedings Of The National Academy Of Sciences Of The United States Of America
The force-generating mechanism of dynein differs from the force-generating mechanisms of other cytoskeletal motors. To examine the structural dynamics of dynein's stepping mechanism in real time, we used polarized total internal reflection fluorescence microscopy with nanometer accuracy localization to track the orientation and position of single motors. By measuring the polarized emission of individual quantum nanorods coupled to the dynein ring, we determined the angular position of the ring and found that it rotates relative to the microtubule (MT) while walking. Surprisingly, the observed rotations were small, averaging only 8.3°, and were only weakly correlated with steps. Measurements at two independent labeling positions on opposite sides of the ring showed similar small rotations. Our results are inconsistent with a classic power-stroke mechanism, and instead support a flexible stalk model in which interhead strain rotates the rings through bending and hinging of the stalk. Mechanical compliances of the stalk and hinge determined based on a 3.3-μs molecular dynamics simulation account for the degree of ring rotation observed experimentally. Together, these observations demonstrate that the stepping mechanism of dynein is fundamentally different from the stepping mechanisms of other well-studied MT motors, because it is characterized by constant small-scale fluctuations of a large but flexible structure fully consistent with the variable stepping pattern observed as dynein moves along the MT.
Evolutionary steps involving counterion displacement in a tunicate opsin
May 23, 2017   Proceedings Of The National Academy Of Sciences Of The United States Of America
Kojima K, Yamashita T, Imamoto Y, Kusakabe TG, Tsuda M, Shichida Y
Evolutionary steps involving counterion displacement in a tunicate opsin
May 23, 2017
Proceedings Of The National Academy Of Sciences Of The United States Of America
Ci-opsin1 is a visible light-sensitive opsin present in the larval ocellus of an ascidian, Ciona intestinalis This invertebrate opsin belongs to the vertebrate visual and nonvisual opsin groups in the opsin phylogenetic tree. Ci-opsin1 contains candidate counterions (glutamic acid residues) at positions 113 and 181; the former is a newly acquired position in the vertebrate visual opsin lineage, whereas the latter is an ancestral position widely conserved among invertebrate opsins. Here, we show that Glu113 and Glu181 in Ci-opsin1 act synergistically as counterions, which imparts molecular properties to Ci-opsin1 intermediate between those of vertebrate- and invertebrate-type opsins. Synergy between the counterions in Ci-opsin1 was demonstrated by E113Q and E181Q mutants that exhibit a pH-dependent spectral shift, whereas only the E113Q mutation in vertebrate rhodopsin yields this spectral shift. On absorbing light, Ci-opsin1 forms an equilibrium between two intermediates with protonated and deprotonated Schiff bases, namely the MI-like and MII-like intermediates, respectively. Adding G protein caused the equilibrium to shift toward the MI-like intermediate, indicating that Ci-opsin1 has a protonated Schiff base in its active state, like invertebrate-type opsins. Ci-opsin1's G protein activation efficiency is between the efficiencies of vertebrate- and invertebrate-type opsins. Interestingly, the E113Y and E181S mutations change the molecular properties of Ci-opsin1 into those resembling invertebrate-type or bistable opsins and vertebrate ancient/vertebrate ancient-long or monostable opsins, respectively. These results strongly suggest that acquisition of counterion Glu113 changed the molecular properties of visual opsin in a vertebrate/tunicate common ancestor as a crucial step in the evolution of vertebrate visual opsins.

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