Expression of transmembrane 4 superfamily (TM4SF) proteins and their role in hepatic stellate cell motility and wound healing migration.

PubMed

Mazzocca, Antonio; Carloni, Vinicio; Sciammetta, Silvia; Cordella, Claudia; Pantaleo, Pietro; Caldini, Anna; Gentilini, Paolo; Pinzani, Massimo

2002-09-01

Migration of activated hepatic stellate cells (HSC) is a key event in the progression of liver fibrosis. Little is known about transmembrane proteins involved in HSC motility. Tetraspanins (TM4SF) have been implicated in cell development, differentiation, motility and tumor cell invasion. We evaluated the expression and function of four TM4SF, namely CD9, CD81, CD63 and CD151, and their involvement in HSC migration, adhesion, and proliferation. All TM4SF investigated were highly expressed at the human HSC surface with different patterns of intracellular distribution. Monoclonal antibodies directed against the four TM4SF inhibited HSC migration induced by extracellular matrix proteins in both wound healing and haptotaxis assays. This inhibition was independent of the ECM substrates employed (collagen type I or IV, laminin), and was comparable to that obtained by incubating the cells with an anti-beta1 blocking mAb. Importantly, cell adhesion was unaffected by the incubation with the same antibodies. Co-immunoprecipitation studies revealed different patterns of association between the four TM4SF studied and beta1 integrin. Finally, anti-TM4SF antibodies did not affect HSC growth. These findings provide the first characterization of tetraspanins expression and of their role in HSC migration, a key event in liver tissue wound healing and fibrogenesis.

N-TERMINALLY ELONGATED SpliInx2 AND SpliInx3 REDUCE BACULOVIRUS-TRIGGERED APOPTOSIS VIA HEMICHANNEL CLOSURE.

PubMed

Chen, Ya-Bin; Xiao, Wei; Li, Ming; Zhang, Yan; Yang, Yang; Hu, Jian-Sheng; Luo, Kai-Jun

2016-05-01

The hemichannel and gap junction channel are major portals for the release of factors responsible for the effects of apoptotic cells on the spread of apoptosis to neighboring cells and apoptotic corpse clearance, typically by phagocytes. The N-terminal cytoplasmic domain in the connexins, gap junction proteins in vertebrate, has been implicated in regulating channel closure. However, little is known about how the hemichannel close responds to apoptotic signaling transduction leading to the reduction of neighboring cellular apoptosis in an invertebrate. An insect Bac-to-Bac expression system, pFastBac(TM) HT A, allows us to construct an N-terminally elongated SpliInx2 (Nte-Inx2) and SpliInx3 (Nte-Inx3). Here, we demonstrated that recombinant baculovirus Bac-Nte-Inx2 (reBac-Net-Inx2) and Bac-Nte-Inx3 (reBac-Nte-Inx3) closed the endogenous hemichannel on the Sf9 cell surface. Importantly, primary baculovirus infections significantly caused early apoptosis, and this apoptosis was reduced by hemichannel-closed Sf9 cells at 24-h post-infection (PI). Although N-terminal-elongated residue led to the increase in the phosphorylated sites in both Nte-Inx2 and Nte-Inx3 and an additional transmembrane domain in Nte-Inx3, both the proteins localized on the cell surface, suggesting Nte-Inxs proteins could mediate hemichannel closure. Further supporting evidence showed that hemichannel closure was dependent on N-Inxs expressed by baculovirus polyhedrin promoter, which began to express at 18-24 h PI. These results identify an unconventional function of N-terminal-elongated innexins that could act as a plug to manipulate hemichannel closure and provide a mechanism connecting the effect of hemichannel closure directly to apoptotic signaling transduction from intracellular to extracellular compartment. © 2016 Wiley Periodicals, Inc.

Measurement of the 169Tm (n ,3 n ) 167Tm cross section and the associated branching ratios in the decay of 167Tm

NASA Astrophysics Data System (ADS)

Champine, B.; Gooden, M. E.; Krishichayan, Norman, E. B.; Scielzo, N. D.; Stoyer, M. A.; Thomas, K. J.; Tonchev, A. P.; Tornow, W.; Wang, B. S.

2016-01-01

The cross section for the 169Tm(n ,3 n ) 167Tm reaction was measured from 17 to 22 MeV using quasimonoenergetic neutrons produced by the 2H(d ,n ) 3He reaction. This energy range was studied to resolve the discrepancy between previous (n ,3 n ) cross-section measurements. In addition, the absolute γ -ray branching ratios following the electron-capture decay of 167Tm were measured. These results provide more reliable nuclear data for an important diagnostic that is used at the National Ignition Facility to estimate the yield of reaction-in-flight neutrons produced via the inertial-confinement-fusion plasma in deuterium-tritium capsules.

Measurements of the 169Tm(n ,2 n )168Tm cross section from threshold to 15 MeV

NASA Astrophysics Data System (ADS)

Soter, J.; Bhike, M.; Finch, S. W.; Krishichayan, Tornow, W.

2017-12-01

Measurements of the 169Tm(n ,2 n )168Tm cross section have been performed via the activation technique at 13 energies between 8.5 and 15.0 MeV. The purpose of this comprehensive data set is to provide an alternative diagnostic tool for obtaining subtle information on the neutron energy distribution produced in inertial confinement deuterium-tritium fusion experiments at the National Ignition Facility (NIF) at Lawrence Livermore National Laboratory. The 169Tm(n ,2 n )168Tm reaction not only provides the primary 14-MeV neutron fluence, but also the important down-scattered neutron fluence, the latter providing information on the density achieved in the deuterium-tritium plasma during a laser shot.

Measurements of the Tm 169 ( n , 2 n ) Tm 168 cross section from threshold to 15 MeV

DOE PAGES

Soter, J.; Bhike, M.; Finch, S. W.; ...

2017-12-27

Measurements of the 169Tm(n,2n) 168Tm cross section have been performed via the activation technique at 13 energies between 8.5 and 15.0 MeV. The purpose of this comprehensive data set is to provide an alternative diagnostic tool for obtaining subtle information on the neutron energy distribution produced in inertial confinement deuterium-tritium fusion experiments at the National Ignition Facility (NIF) at Lawrence Livermore National Laboratory. In conclusion, the 169Tm(n,2n) 168Tm reaction not only provides the primary 14-MeV neutron fluence, but also the important down-scattered neutron fluence, the latter providing information on the density achieved in the deuterium-tritium plasma during a laser shot.

Effect of the Southeast Asian Ovalocytosis Deletion on the Conformational Dynamics of Signal-Anchor Transmembrane Segment 1 of Red Cell Anion Exchanger 1 (AE1, Band 3, or SLC4A1)

PubMed Central

2017-01-01

The first transmembrane (TM1) helix in the red cell anion exchanger (AE1, Band 3, or SLC4A1) acts as an internal signal anchor that binds the signal recognition particle and directs the nascent polypeptide chain to the endoplasmic reticulum (ER) membrane where it moves from the translocon laterally into the lipid bilayer. The sequence N-terminal to TM1 forms an amphipathic helix that lies at the membrane interface and is connected to TM1 by a bend at Pro403. Southeast Asian ovalocytosis (SAO) is a red cell abnormality caused by a nine-amino acid deletion (Ala400–Ala408) at the N-terminus of TM1. Here we demonstrate, by extensive (∼4.5 μs) molecular dynamics simulations of TM1 in a model 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membrane, that the isolated TM1 peptide is highly dynamic and samples the structure of TM1 seen in the crystal structure of the membrane domain of AE1. The SAO deletion not only removes the proline-induced bend but also causes a “pulling in” of the part of the amphipathic helix into the hydrophobic phase of the bilayer, as well as the C-terminal of the peptide. The dynamics of the SAO peptide very infrequently resembles the structure of TM1 in AE1, demonstrating the disruptive effect the SAO deletion has on AE1 folding. These results provide a precise molecular view of the disposition and dynamics of wild-type and SAO TM1 in a lipid bilayer, an important early biosynthetic intermediate in the insertion of AE1 into the ER membrane, and extend earlier results of cell-free translation experiments. PMID:28068080

Effect of the Southeast Asian Ovalocytosis Deletion on the Conformational Dynamics of Signal-Anchor Transmembrane Segment 1 of Red Cell Anion Exchanger 1 (AE1, Band 3, or SLC4A1).

PubMed

Fowler, Philip W; Sansom, Mark S P; Reithmeier, Reinhart A F

2017-02-07

The first transmembrane (TM1) helix in the red cell anion exchanger (AE1, Band 3, or SLC4A1) acts as an internal signal anchor that binds the signal recognition particle and directs the nascent polypeptide chain to the endoplasmic reticulum (ER) membrane where it moves from the translocon laterally into the lipid bilayer. The sequence N-terminal to TM1 forms an amphipathic helix that lies at the membrane interface and is connected to TM1 by a bend at Pro403. Southeast Asian ovalocytosis (SAO) is a red cell abnormality caused by a nine-amino acid deletion (Ala400-Ala408) at the N-terminus of TM1. Here we demonstrate, by extensive (∼4.5 μs) molecular dynamics simulations of TM1 in a model 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membrane, that the isolated TM1 peptide is highly dynamic and samples the structure of TM1 seen in the crystal structure of the membrane domain of AE1. The SAO deletion not only removes the proline-induced bend but also causes a "pulling in" of the part of the amphipathic helix into the hydrophobic phase of the bilayer, as well as the C-terminal of the peptide. The dynamics of the SAO peptide very infrequently resembles the structure of TM1 in AE1, demonstrating the disruptive effect the SAO deletion has on AE1 folding. These results provide a precise molecular view of the disposition and dynamics of wild-type and SAO TM1 in a lipid bilayer, an important early biosynthetic intermediate in the insertion of AE1 into the ER membrane, and extend earlier results of cell-free translation experiments.

NMR resolved multiple anesthetic binding sites in the TM domains of the α4β2 nAChR

PubMed Central

Bondarenko, Vasyl; Mowrey, David; Liu, Lu Tian; Xu, Yan; Tang, Pei

2012-01-01

The α4β2 nicotinic acetylcholine receptor (nAChR) has significant roles in nervous system function and disease. It is also a molecular target of general anesthetics. Anesthetics inhibit the α4β2 nAChR at clinically relevant concentrations, but their binding sites in α4β2 remain unclear. The recently determined NMR structures of the α4β2 nAChR transmembrane (TM) domains provide valuable frameworks for identifying the binding sites. In this study, we performed solution NMR experiments on the α4β2 TM domains in the absence and presence of halothane and ketamine. Both anesthetics were found in an intra-subunit cavity near the extracellular end of the 2 transmembrane helices, homologous to a common anesthetic binding site observed in X-ray structures of anesthetic-bound GLIC (Nury, et. al. 2011). Halothane, but not ketamine, was also found in cavities adjacent to the common anesthetic site at the interface of α4 and β2. In addition, both anesthetics bound to cavities near the ion selectivity filter at the intracellular end of the TM domains. Anesthetic binding induced profound changes in protein conformational exchanges. A number of residues, close to or remote from the binding sites, showed resonance signal splitting from single to double peaks, signifying that anesthetics decreased conformation exchange rates. It was also evident that anesthetics shifted population of two conformations. Altogether, the study comprehensively resolved anesthetic binding sites in the α4β2 nAChR. Furthermore, the study provided compelling experimental evidence of anesthetic-induced changes in protein dynamics, especially near regions of the hydrophobic gate and ion selectivity filter that directly regulate channel functions. PMID:23000369

NMR resolved multiple anesthetic binding sites in the TM domains of the α4β2 nAChR.

PubMed

Bondarenko, Vasyl; Mowrey, David; Liu, Lu Tian; Xu, Yan; Tang, Pei

2013-02-01

The α4β2 nicotinic acetylcholine receptor (nAChR) has significant roles in nervous system function and disease. It is also a molecular target of general anesthetics. Anesthetics inhibit the α4β2 nAChR at clinically relevant concentrations, but their binding sites in α4β2 remain unclear. The recently determined NMR structures of the α4β2 nAChR transmembrane (TM) domains provide valuable frameworks for identifying the binding sites. In this study, we performed solution NMR experiments on the α4β2 TM domains in the absence and presence of halothane and ketamine. Both anesthetics were found in an intra-subunit cavity near the extracellular end of the β2 transmembrane helices, homologous to a common anesthetic binding site observed in X-ray structures of anesthetic-bound GLIC (Nury et al., [32]). Halothane, but not ketamine, was also found in cavities adjacent to the common anesthetic site at the interface of α4 and β2. In addition, both anesthetics bound to cavities near the ion selectivity filter at the intracellular end of the TM domains. Anesthetic binding induced profound changes in protein conformational exchanges. A number of residues, close to or remote from the binding sites, showed resonance signal splitting from single to double peaks, signifying that anesthetics decreased conformation exchange rates. It was also evident that anesthetics shifted population of two conformations. Altogether, the study comprehensively resolved anesthetic binding sites in the α4β2 nAChR. Furthermore, the study provided compelling experimental evidence of anesthetic-induced changes in protein dynamics, especially near regions of the hydrophobic gate and ion selectivity filter that directly regulate channel functions. Copyright © 2012 Elsevier B.V. All rights reserved.

Crystal structure of the complete integrin αVβ3 ectodomain plus an α/β transmembrane fragment

PubMed Central

Xiong, Jian-Ping; Mahalingham, Bhuvaneshwari; Alonso, Jose Luis; Borrelli, Laura Ann; Rui, Xianliang; Anand, Saurabh; Hyman, Bradley T.; Rysiok, Thomas; Müller-Pompalla, Dirk; Goodman, Simon L.

2009-01-01

We determined the crystal structure of 1TM-αVβ3, which represents the complete unconstrained ectodomain plus short C-terminal transmembrane stretches of the αV and β3 subunits. 1TM-αVβ3 is more compact and less active in solution when compared with ΔTM-αVβ3, which lacks the short C-terminal stretches. The structure reveals a bent conformation and defines the α–β interface between IE2 (EGF-like 2) and the thigh domains. Modifying this interface by site-directed mutagenesis leads to robust integrin activation. Fluorescent lifetime imaging microscopy of inactive full-length αVβ3 on live cells yields a donor–membrane acceptor distance, which is consistent with the bent conformation and does not change in the activated integrin. These data are the first direct demonstration of conformational coupling of the integrin leg and head domains, identify the IE2–thigh interface as a critical steric barrier in integrin activation, and suggest that inside-out activation in intact cells may involve conformational changes other than the postulated switch to a genu-linear state. PMID:19704023

Conformational Changes in the Epidermal Growth Factor Receptor: Role of the Transmembrane Domain Investigated by Coarse-Grained MetaDynamics Free Energy Calculations

PubMed Central

2016-01-01

The epidermal growth factor receptor (EGFR) is a dimeric membrane protein that regulates key aspects of cellular function. Activation of the EGFR is linked to changes in the conformation of the transmembrane (TM) domain, brought about by changes in interactions of the TM helices of the membrane lipid bilayer. Using an advanced computational approach that combines Coarse-Grained molecular dynamics and well-tempered MetaDynamics (CG-MetaD), we characterize the large-scale motions of the TM helices, simulating multiple association and dissociation events between the helices in membrane, thus leading to a free energy landscape of the dimerization process. The lowest energy state of the TM domain is a right-handed dimer structure in which the TM helices interact through the N-terminal small-X3-small sequence motif. In addition to this state, which is thought to correspond to the active form of the receptor, we have identified further low-energy states that allow us to integrate with a high level of detail a range of previous experimental observations. These conformations may lead to the active state via two possible activation pathways, which involve pivoting and rotational motions of the helices, respectively. Molecular dynamics also reveals correlation between the conformational changes of the TM domains and of the intracellular juxtamembrane domains, paving the way for a comprehensive understanding of EGFR signaling at the cell membrane. PMID:27459426

Conformational Changes in the Epidermal Growth Factor Receptor: Role of the Transmembrane Domain Investigated by Coarse-Grained MetaDynamics Free Energy Calculations.

PubMed

Lelimousin, Mickaël; Limongelli, Vittorio; Sansom, Mark S P

2016-08-24

The epidermal growth factor receptor (EGFR) is a dimeric membrane protein that regulates key aspects of cellular function. Activation of the EGFR is linked to changes in the conformation of the transmembrane (TM) domain, brought about by changes in interactions of the TM helices of the membrane lipid bilayer. Using an advanced computational approach that combines Coarse-Grained molecular dynamics and well-tempered MetaDynamics (CG-MetaD), we characterize the large-scale motions of the TM helices, simulating multiple association and dissociation events between the helices in membrane, thus leading to a free energy landscape of the dimerization process. The lowest energy state of the TM domain is a right-handed dimer structure in which the TM helices interact through the N-terminal small-X3-small sequence motif. In addition to this state, which is thought to correspond to the active form of the receptor, we have identified further low-energy states that allow us to integrate with a high level of detail a range of previous experimental observations. These conformations may lead to the active state via two possible activation pathways, which involve pivoting and rotational motions of the helices, respectively. Molecular dynamics also reveals correlation between the conformational changes of the TM domains and of the intracellular juxtamembrane domains, paving the way for a comprehensive understanding of EGFR signaling at the cell membrane.

Transmembrane Domains of Attraction on the TSH Receptor

PubMed Central

Ali, M. Rejwan; Mezei, Mihaly; Davies, Terry F.

2015-01-01

The TSH receptor (TSHR) has the propensity to form dimers and oligomers. Our data using ectodomain-truncated TSHRs indicated that the predominant interfaces for oligomerization reside in the transmembrane (TM) domain. To map the potentially interacting residues, we first performed in silico studies of the TSHR transmembrane domain using a homology model and using Brownian dynamics (BD). The cluster of dimer conformations obtained from BD analysis indicated that TM1 made contact with TM4 and two residues in TM2 made contact with TM5. To confirm the proximity of these contact residues, we then generated cysteine mutants at all six contact residues predicted by the BD analysis and performed cysteine cross-linking studies. These results showed that the predicted helices in the protomer were indeed involved in proximity interactions. Furthermore, an alternative experimental approach, receptor truncation experiments and LH receptor sequence substitution experiments, identified TM1 harboring a major region involved in TSHR oligomerization, in agreement with the conclusion from the cross-linking studies. Point mutations of the predicted interacting residues did not yield a substantial decrease in oligomerization, unlike the truncation of the TM1, so we concluded that constitutive oligomerization must involve interfaces forming domains of attraction in a cooperative manner that is not dominated by interactions between specific residues. PMID:25406938

Influence of the C-terminus of the glycophorin A transmembrane fragment on the dimerization process.

PubMed Central

Orzáez, M.; Pérez-Payá, E.; Mingarro, I.

2000-01-01

The monomer-dimer equilibrium of the glycophorin A (GpA) transmembrane (TM) fragment has been used as a model system to investigate the amino acid sequence requirements that permit an appropriate helix-helix packing in a membrane-mimetic environment. In particular, we have focused on a region of the helix where no crucial residues for packing have been yet reported. Various deletion and replacement mutants in the C-terminal region of the TM fragment showed that the distance between the dimerization motif and the flanking charged residues from the cytoplasmic side of the protein is important for helix packing. Furthermore, selected GpA mutants have been used to illustrate the rearrangement of TM fragments that takes place when leucine repeats are introduced in such protein segments. We also show that secondary structure of GpA derivatives was independent from dimerization, in agreement with the two-stage model for membrane protein folding and oligomerization. PMID:10892817

Conductance and amantadine binding of a pore formed by a lysine-flanked transmembrane domain of SARS coronavirus envelope protein

PubMed Central

Torres, Jaume; Maheswari, Uma; Parthasarathy, Krupakar; Ng, Lifang; Liu, Ding Xiang; Gong, Xiandi

2007-01-01

The coronavirus responsible for the severe acute respiratory syndrome (SARS-CoV) contains a small envelope protein, E, with putative involvement in host cell apoptosis and virus morphogenesis. It has been suggested that E protein can form a membrane destabilizing transmembrane (TM) hairpin, or homooligomerize to form a regular TM α-helical bundle. We have shown previously that the topology of the α-helical putative TM domain of E protein (ETM), flanked by two lysine residues at C and N termini to improve solubility, is consistent with a regular TM α-helix, with orientational parameters in lipid bilayers that are consistent with a homopentameric model. Herein, we show that this peptide, reconstituted in lipid bilayers, shows sodium conductance. Channel activity is inhibited by the anti-influenza drug amantadine, which was found to bind our preparation with moderate affinity. Results obtained from single or double mutants indicate that the organization of the transmembrane pore is consistent with our previously reported pentameric α-helical bundle model. PMID:17766393

New Insights into Molecular Organization of Human Neuraminidase-1: Transmembrane Topology and Dimerization Ability

NASA Astrophysics Data System (ADS)

Maurice, Pascal; Baud, Stéphanie; Bocharova, Olga V.; Bocharov, Eduard V.; Kuznetsov, Andrey S.; Kawecki, Charlotte; Bocquet, Olivier; Romier, Beatrice; Gorisse, Laetitia; Ghirardi, Maxime; Duca, Laurent; Blaise, Sébastien; Martiny, Laurent; Dauchez, Manuel; Efremov, Roman G.; Debelle, Laurent

2016-12-01

Neuraminidase 1 (NEU1) is a lysosomal sialidase catalyzing the removal of terminal sialic acids from sialyloconjugates. A plasma membrane-bound NEU1 modulating a plethora of receptors by desialylation, has been consistently documented from the last ten years. Despite a growing interest of the scientific community to NEU1, its membrane organization is not understood and current structural and biochemical data cannot account for such membrane localization. By combining molecular biology and biochemical analyses with structural biophysics and computational approaches, we identified here two regions in human NEU1 - segments 139-159 (TM1) and 316-333 (TM2) - as potential transmembrane (TM) domains. In membrane mimicking environments, the corresponding peptides form stable α-helices and TM2 is suited for self-association. This was confirmed with full-size NEU1 by co-immunoprecipitations from membrane preparations and split-ubiquitin yeast two hybrids. The TM2 region was shown to be critical for dimerization since introduction of point mutations within TM2 leads to disruption of NEU1 dimerization and decrease of sialidase activity in membrane. In conclusion, these results bring new insights in the molecular organization of membrane-bound NEU1 and demonstrate, for the first time, the presence of two potential TM domains that may anchor NEU1 in the membrane, control its dimerization and sialidase activity.

Structural Basis for Toughness and Flexibility in the C-terminal Passenger Domain of an Acinetobacter Trimeric Autotransporter Adhesin*

PubMed Central

Koiwai, Kotaro; Hartmann, Marcus D.; Linke, Dirk; Lupas, Andrei N.; Hori, Katsutoshi

2016-01-01

Trimeric autotransporter adhesins (TAAs) on the cell surface of Gram-negative pathogens mediate bacterial adhesion to host cells and extracellular matrix proteins. However, AtaA, a TAA in the nonpathogenic Acinetobacter sp. strain Tol 5, shows nonspecific high adhesiveness to abiotic material surfaces as well as to biotic surfaces. It consists of a passenger domain secreted by the C-terminal transmembrane anchor domain (TM), and the passenger domain contains an N-terminal head, N-terminal stalk, C-terminal head (Chead), and C-terminal stalk (Cstalk). The Chead-Cstalk-TM fragment, which is conserved in many Acinetobacter TAAs, has by itself the head-stalk-anchor architecture of a complete TAA. Here, we show the crystal structure of the Chead-Cstalk fragment, AtaA_C-terminal passenger domain (CPSD), providing the first view of several conserved TAA domains. The YadA-like head (Ylhead) of the fragment is capped by a unique structure (headCap), composed of three β-hairpins and a connector motif; it also contains a head insert motif (HIM1) before its last inner β-strand. The headCap, Ylhead, and HIM1 integrally form a stable Chead structure. Some of the major domains of the CPSD fragment are inherently flexible and provide bending sites for the fiber between segments whose toughness is ensured by topological chain exchange and hydrophobic core formation inside the trimer. Thus, although adherence assays using in-frame deletion mutants revealed that the characteristic adhesive sites of AtaA reside in its N-terminal part, the flexibility and toughness of the CPSD part provide the resilience that enables the adhesive properties of the full-length fiber across a wide range of conditions. PMID:26698633

Structural and Functional Importance of Transmembrane Domain 3 (TM3) in the Aspartate:Alanine Antiporter AspT: Topology and Function of the Residues of TM3 and Oligomerization of AspT▿

PubMed Central

Nanatani, Kei; Maloney, Peter C.; Abe, Keietsu

2009-01-01

AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, a membrane protein of 543 amino acids with 10 putative transmembrane (TM) helices, is the prototype of the aspartate:alanine exchanger (AAE) family of transporters. Because TM3 (isoleucine 64 to methionine 85) has many amino acid residues that are conserved among members of the AAE family and because TM3 contains two charged residues and four polar residues, it is thought to be located near (or to form part of) the substrate translocation pathway that includes the binding site for the substrates. To elucidate the role of TM3 in the transport process, we carried out cysteine-scanning mutagenesis. The substitutions of tyrosine 75 and serine 84 had the strongest inhibitory effects on transport (initial rates of l-aspartate transport were below 15% of the rate for cysteine-less AspT). Considerable but less-marked effects were observed upon the replacement of methionine 70, phenylalanine 71, glycine 74, arginine 76, serine 83, and methionine 85 (initial rates between 15% and 30% of the rate for cysteine-less AspT). Introduced cysteine residues at the cytoplasmic half of TM3 could be labeled with Oregon green maleimide (OGM), whereas cysteines close to the periplasmic half (residues 64 to 75) were not labeled. These results suggest that TM3 has a hydrophobic core on the periplasmic half and that hydrophilic residues on the cytoplasmic half of TM3 participate in the formation of an aqueous cavity in membranes. Furthermore, the presence of l-aspartate protected the cysteine introduced at glycine 62 against a reaction with OGM. In contrast, l-aspartate stimulated the reactivity of the cysteine introduced at proline 79 with OGM. These results demonstrate that TM3 undergoes l-aspartate-induced conformational alterations. In addition, nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses and a glutaraldehyde cross-linking assay suggest that functional AspT forms homo-oligomers as a

Structural and functional importance of transmembrane domain 3 (TM3) in the aspartate:alanine antiporter AspT: topology and function of the residues of TM3 and oligomerization of AspT.

PubMed

Nanatani, Kei; Maloney, Peter C; Abe, Keietsu

2009-04-01

AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, a membrane protein of 543 amino acids with 10 putative transmembrane (TM) helices, is the prototype of the aspartate:alanine exchanger (AAE) family of transporters. Because TM3 (isoleucine 64 to methionine 85) has many amino acid residues that are conserved among members of the AAE family and because TM3 contains two charged residues and four polar residues, it is thought to be located near (or to form part of) the substrate translocation pathway that includes the binding site for the substrates. To elucidate the role of TM3 in the transport process, we carried out cysteine-scanning mutagenesis. The substitutions of tyrosine 75 and serine 84 had the strongest inhibitory effects on transport (initial rates of l-aspartate transport were below 15% of the rate for cysteine-less AspT). Considerable but less-marked effects were observed upon the replacement of methionine 70, phenylalanine 71, glycine 74, arginine 76, serine 83, and methionine 85 (initial rates between 15% and 30% of the rate for cysteine-less AspT). Introduced cysteine residues at the cytoplasmic half of TM3 could be labeled with Oregon green maleimide (OGM), whereas cysteines close to the periplasmic half (residues 64 to 75) were not labeled. These results suggest that TM3 has a hydrophobic core on the periplasmic half and that hydrophilic residues on the cytoplasmic half of TM3 participate in the formation of an aqueous cavity in membranes. Furthermore, the presence of l-aspartate protected the cysteine introduced at glycine 62 against a reaction with OGM. In contrast, l-aspartate stimulated the reactivity of the cysteine introduced at proline 79 with OGM. These results demonstrate that TM3 undergoes l-aspartate-induced conformational alterations. In addition, nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses and a glutaraldehyde cross-linking assay suggest that functional AspT forms homo-oligomers as a

AR-C155858 is a potent inhibitor of monocarboxylate transporters MCT1 and MCT2 that binds to an intracellular site involving transmembrane helices 7-10.

PubMed

Ovens, Matthew J; Davies, Andrew J; Wilson, Marieangela C; Murray, Clare M; Halestrap, Andrew P

2010-01-15

In the present study we characterize the properties of the potent MCT1 (monocarboxylate transporter 1) inhibitor AR-C155858. Inhibitor titrations of L-lactate transport by MCT1 in rat erythrocytes were used to determine the Ki value and number of AR-C155858-binding sites (Et) on MCT1 and the turnover number of the transporter (kcat). Derived values were 2.3+/-1.4 nM, 1.29+/-0.09 nmol per ml of packed cells and 12.2+/-1.1 s-1 respectively. When expressed in Xenopus laevis oocytes, MCT1 and MCT2 were potently inhibited by AR-C155858, whereas MCT4 was not. Inhibition of MCT1 was shown to be time-dependent, and the compound was also active when microinjected, suggesting that AR-C155858 probably enters the cell before binding to an intracellular site on MCT1. Measurement of the inhibitor sensitivity of several chimaeric transporters combining different domains of MCT1 and MCT4 revealed that the binding site for AR-C155858 is contained within the C-terminal half of MCT1, and involves TM (transmembrane) domains 7-10. This is consistent with previous data identifying Phe360 (in TM10) and Asp302 plus Arg306 (TM8) as key residues in substrate binding and translocation by MCT1. Measurement of the Km values of the chimaeras for L-lactate and pyruvate demonstrate that both the C- and N-terminal halves of the molecule influence transport kinetics consistent with our proposed molecular model of MCT1 and its translocation mechanism that requires Lys38 in TM1 in addition to Asp302 and Arg306 in TM8 [Wilson, Meredith, Bunnun, Sessions and Halestrap (2009) J. Biol. Chem. 284, 20011-20021].

Insights into the transmembrane helix associations of kit ligand by molecular dynamics simulation and TOXCAT.

PubMed

Chai, Mengya; Liu, Bo; Sun, Fude; Wei, Peng; Chen, Peng; Xu, Lida; Luo, Shi-Zhong

2017-07-01

Kit ligand (KITL) plays important roles in cell proliferation, differentiation, and survival via interaction with its receptor Kit. The previous studies demonstrated that KITL formed a noncovalent homodimer through transmembrane (TM) domain; however, the undergoing mechanism of transmembrane association that determines KITL TM dimerization is still not clear. Herein, molecular dynamics (MD) simulation strategy and TOXCAT assay were combined to characterize the dimerization interface and structure of KITL TM in details. KITL TM formed a more energetically favorable noncovalent dimer through a conserved SxxxGxxxG motif in the MD simulation. Furthermore, the TOXCAT results demonstrated that KITL TM self-associated strongly in the bilayer membrane environment. Mutating any one of the small residues Ser11, Gly15 or Gly19 to Ile disrupted KITL TM dimerization dramatically, which further validated our MD simulation results. In addition, our results showed that Tyr22 could help to stabilize the TM interactions via interacting with the phosphoric group in the bilayer membrane. Pro7 did not induce helix kinks or swivel angles in KITL TM, but it was related with the pitch of the turn around this residue so as to affect the dimer formation. Combining the results of computer modeling and experimental mutagenesis studies on the KITL TM provide new insights for the transmembrane helix association of KITL dimerization. Proteins 2017; 85:1362-1370. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Measurements of the 169Tm(n,2n)168Tm cross section between 9.0 and 17.5 MeV

NASA Astrophysics Data System (ADS)

Soter, J.; Bhike, Megha; Krishichayan, Fnu; Finch, S. W.; Tornow, W.

2016-09-01

Measurements of the 169Tm(n,2n)168Tm cross section have been performed in 0.5 MeV intervals for neutron energies ranging from 9.0 MeV to 17.5 MeV in order to resolve discrepancies in the current literature data. The neutron activation technique was used with 90Zr and 197Au as monitor foils. After irradiation, de-excitation gamma rays were recorded off-line with High-Purity Germanium (HPGE) detectors in TUNL's Low-Background Counting Facility. In addition, data for the 169Tm(n,3n)167Tm reaction have also been obtained from 15.5 MeV to 17.5 MeV. The results of these measurements provide the basis for investigating properties of the interial confinement fusion plasma in deuterium-tritium (DT) capsules at the National Ignition Facility located at Lawrence Livermore National Laboratory.

Contribution of Kunitz protease inhibitor and transmembrane domains to amyloid precursor protein homodimerization.

PubMed

Ben Khalifa, N; Tyteca, D; Courtoy, P J; Renauld, J C; Constantinescu, S N; Octave, J N; Kienlen-Campard, P

2012-01-01

The two major isoforms of the human amyloid precursor protein (APP) are APP695 and APP751. They differ by the insertion of a Kunitz-type protease inhibitor (KPI) sequence in the extracellular domain of APP751. APP-KPI isoforms are increased in Alzheimer's disease brains, and they could be associated with disease progression. Recent studies have shown that APP processing to Aβ is regulated by homodimerization, which involves both extracellular and juxtamembrane/transmembrane (JM/TM) regions. Our aim is to understand the mechanisms controlling APP dimerization and the contribution of the ectodomain and JM/TM regions to this process. We used bimolecular fluorescence complementation approaches coupled to fluorescence-activated cell sorting analysis to measure the dimerization level of different APP isoforms and APP C-terminal fragments (C99) mutated in their JM/TM region. APP751 was found to form significantly more homodimers than APP695. Mutation of dimerization motifs in the TM domain of APP or C99 did not significantly affect fluorescence complementation. These findings indicate that the KPI domain plays a major role in APP dimerization. They set the basis for further investigation of the relation between dimerization, metabolism and function of APP. Copyright © 2012 S. Karger AG, Basel.

A Trigger Residue for Transmembrane Signaling in the Escherichia coli Serine Chemoreceptor

PubMed Central

Kitanovic, Smiljka; Ames, Peter

2015-01-01

ABSTRACT The transmembrane Tsr protein of Escherichia coli mediates chemotactic responses to environmental serine gradients. Serine binds to the periplasmic domain of the homodimeric Tsr molecule, promoting a small inward displacement of one transmembrane helix (TM2). TM2 piston displacements, in turn, modulate the structural stability of the Tsr-HAMP domain on the cytoplasmic side of the membrane to control the autophosphorylation activity of the signaling CheA kinase bound to the membrane-distal cytoplasmic tip of Tsr. A five-residue control cable segment connects TM2 to the AS1 helix of HAMP and transmits stimulus and sensory adaptation signals between them. To explore the possible role of control cable helicity in transmembrane signaling by Tsr, we characterized the signaling properties of mutant receptors with various control cable alterations. An all-alanine control cable shifted Tsr output toward the kinase-on state, whereas an all-glycine control cable prevented Tsr from reaching either a fully on or fully off output state. Restoration of the native isoleucine (I214) in these synthetic control cables largely alleviated their signaling defects. Single amino acid replacements at Tsr-I214 shifted output toward the kinase-off (L, N, H, and R) or kinase-on (A and G) states, whereas other control cable residues tolerated most amino acid replacements with little change in signaling behavior. These findings indicate that changes in control cable helicity might mediate transitions between the kinase-on and kinase-off states during transmembrane signaling by chemoreceptors. Moreover, the Tsr-I214 side chain plays a key role, possibly through interaction with the membrane interfacial environment, in triggering signaling changes in response to TM2 piston displacements. IMPORTANCE The Tsr protein of E. coli mediates chemotactic responses to environmental serine gradients. Stimulus signals from the Tsr periplasmic sensing domain reach its cytoplasmic kinase control

Characterization, cell-surface expression and ligand-binding properties of different truncated N-terminal extracellular domains of the ionotropic glutamate receptor subunit GluR1.

PubMed

McIlhinney, R A; Molnár, E

1996-04-01

To identify the location of the first transmembrane segment of the GluR1 glutamate receptor subunit artificial stop codons have been introduced into the N-terminal domain at amino acid positions 442, 510, and 563, namely just before and spanning the proposed first two transmembrane regions. The resultant truncated N-terminal fragments of GluR1, termed NT1, NT2, and NT3 respectively were expressed in Cos-7 cells and their cellular distribution and cell-surface expression analysed using an N-terminal antibody to GluR1. All of the fragments were fully glycosylated and were found to be associated with cell membranes but none was secreted. Differential extraction of the cell membranes indicated that both NT1 and NT2 behave as peripheral membrane proteins. In contrast NT3, like the full subunit, has integral membrane protein properties. Furthermore only NT3 is expressed at the cell surface as determined by immunofluorescence and cell-surface biotinylation. Protease protection assays indicated that only NT3 had a cytoplasmic tail. Binding studies using the selective ligand [(3)H]alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate ([(3)H]AMPA) demonstrated that NT3 does not bind ligand. Together these results indicate that the first transmembrane domain of the GluR1 subunit lies between residues 509 and 562, that the N-terminal domain alone cannot form a functional ligand-binding site and that this domain can be targeted to the cell surface provided that it has a transmembrane-spanning region.

Differential Effects of TM4 Tryptophan Mutations on Inhibition of N-Methyl-D-Aspartate Receptors by Ethanol and Toluene

PubMed Central

Smothers, C. Thetford; Woodward, John J.

2017-01-01

The voluntary use and abuse of alcohol and inhalants is a recognized health problem throughout the world. Previous studies have shown that these agents affect brain function in a variety of ways including direct inhibition of key ion channels that regulate neuronal excitability. Among these, the N-methyl-D-aspartate (NMDA) receptor is particularly important given its key role in glutamatergic synaptic transmission, neuronal plasticity and learning and memory. Previous studies from this laboratory and others have identified key residues within transmembrane (TM) domains of the NMDA receptor that appear to regulate its sensitivity to alcohol and anesthetics. In this study, we extend these findings and examine the role of a TM4 residue in modulating sensitivity of recombinant NMDA receptors to ethanol and toluene. HEK293 cells were transfected with GluN1-1a and either wild-type or tryptophan-substituted GluN2(A–D) subunits and whole-cell currents were recorded using patch-clamp electrophysiology in the absence or presence of ethanol or toluene. Both ethanol (100 mM) and toluene (1 or 3 mM) reversibly inhibited glutamate-activated currents from wild-type NMDARs with GluN2B containing receptors showing heightened sensitivity to either agent. Substitution of tryptophan (W) at positions 825, 826, 823 or 850 in the TM4 domain of GluN2A, GluN2B, GluN2C or GluN2D subunits; respectively, significantly reduced the degree of inhibition by ethanol. In contrast, toluene inhibition of glutamate-activated currents in cells expressing the TM4-W mutants was not different from that of the wild-type controls. These data suggest that despite similarities in their action on NMDARs, ethanol and toluene may act at different sites to reduce ion flux through NMDA receptors. PMID:27814790

Differential effects of TM4 tryptophan mutations on inhibition of N-methyl-d-aspartate receptors by ethanol and toluene.

PubMed

Smothers, C Thetford; Woodward, John J

2016-11-01

The voluntary use and abuse of alcohol and inhalants is a recognized health problem throughout the world. Previous studies have shown that these agents affect brain function in a variety of ways including direct inhibition of key ion channels that regulate neuronal excitability. Among these, the N-methyl-d-aspartate (NMDA) receptor is particularly important given its key role in glutamatergic synaptic transmission, neuronal plasticity and learning and memory. Previous studies from this laboratory and others have identified key residues within transmembrane (TM) domains of the NMDA receptor that appear to regulate its sensitivity to alcohol and anesthetics. In this study, we extend these findings and examine the role of a TM4 residue in modulating sensitivity of recombinant NMDA receptors to ethanol and toluene. HEK293 cells were transfected with GluN1-1a and either wild-type or tryptophan-substituted GluN2(A-D) subunits and whole-cell currents were recorded using patch-clamp electrophysiology in the absence or presence of ethanol or toluene. Both ethanol (100 mM) and toluene (1 or 3 mM) reversibly inhibited glutamate-activated currents from wild-type NMDARs with GluN2B containing receptors showing heightened sensitivity to either agent. Substitution of tryptophan (W) at positions 825, 826, 823 or 850 in the TM4 domain of GluN2A, GluN2B, GluN2C or GluN2D subunits; respectively, significantly reduced the degree of inhibition by ethanol. In contrast, toluene inhibition of glutamate-activated currents in cells expressing the TM4-W mutants was not different from that of the wild-type controls. These data suggest that despite similarities in their action on NMDARs, ethanol and toluene may act at different sites to reduce ion flux through NMDA receptors. Copyright © 2016 Elsevier Inc. All rights reserved.

Influence of proline upon the folding and geometry of the WALP19 transmembrane peptide.

PubMed

Thomas, Rachel; Vostrikov, Vitaly V; Greathouse, Denise V; Koeppe, Roger E

2009-12-22

The orientations, geometries, and lipid interactions of designed transmembrane (TM) peptides have attracted significant experimental and theoretical interest. Because the amino acid proline will introduce a known discontinuity into an alpha helix, we have sought to measure the extent of helix kinking caused by a single proline within the isolated TM helical domain of WALP19. For this purpose, we synthesized acetyl-GWWLALALAP(10)ALALALWWA-ethanolamide and included pairs of deuterated alanines by using 60-100% Fmoc-l-Ala-d(4) at selected sequence positions. Solid-state deuterium ((2)H) magnetic resonance spectra from oriented, hydrated samples (1/40, peptide/lipid; using several lipids) reveal signals from many of the alanine backbone C(alpha) deuterons as well as the alanine side-chain C(beta) methyl groups, whereas signals from C(alpha) deuterons generally have not been observed for similar peptides without proline. It is conceivable that altered peptide dynamics may be responsible for the apparent "unmasking" of the backbone resonances in the presence of the proline. Data analysis using the geometric analysis of labeled alanines (GALA) method reveals that the peptide helix is distorted due to the presence of the proline. To provide additional data points for evaluating the segmental tilt angles of the two halves of the peptide, we substituted selected leucines with l-Ala-d(4). Using this approach, we were able to deduce that the apparent average tilt of the C-terminal increases from approximately 4 degrees to approximately 12 degrees when Pro(10) is introduced. The segment N-terminal to proline is more complex and possibly is more dynamically flexible; Leu to Ala mutations within the N-terminal segment alter the average orientations of alanines in both segments. Nevertheless, in DOPC, we could estimate an apparent kink angle of approximately 19 degrees . Together, the results suggest that the central proline influences not only the geometry but also the dynamics

TM4SF5-mediated protein-protein networks and tumorigenic roles

PubMed Central

Lee, Jung Weon

2014-01-01

Transmembrane 4 L six family member 5 (TM4SF5), as a membrane glycoprotein with 4 transmembrane domains, is similar to the tetraspanins in terms of membrane topology and plays important roles in tumorigenesis and tumor metastasis. Especially, TM4SF5 appears to form a massive protein-protein complex consisting of diverse membrane proteins and/or receptors in addition to cytosolic signaling molecules to regulate their signaling activities during the pathological processes. TM4SF5 is shown to interact with integrins α2, α5, and β1, EGFR, IL6R, CD151, focal adhesion kinase (FAK), and c-Src. This review focuses on the significance of the interactions with regards to TM4SF5-positive tumorigenesis and metastasis. [BMB Reports 2014; 47(9): 483-487] PMID:25027595

Transmembrane protein topology prediction using support vector machines.

PubMed

Nugent, Timothy; Jones, David T

2009-05-26

Alpha-helical transmembrane (TM) proteins are involved in a wide range of important biological processes such as cell signaling, transport of membrane-impermeable molecules, cell-cell communication, cell recognition and cell adhesion. Many are also prime drug targets, and it has been estimated that more than half of all drugs currently on the market target membrane proteins. However, due to the experimental difficulties involved in obtaining high quality crystals, this class of protein is severely under-represented in structural databases. In the absence of structural data, sequence-based prediction methods allow TM protein topology to be investigated. We present a support vector machine-based (SVM) TM protein topology predictor that integrates both signal peptide and re-entrant helix prediction, benchmarked with full cross-validation on a novel data set of 131 sequences with known crystal structures. The method achieves topology prediction accuracy of 89%, while signal peptides and re-entrant helices are predicted with 93% and 44% accuracy respectively. An additional SVM trained to discriminate between globular and TM proteins detected zero false positives, with a low false negative rate of 0.4%. We present the results of applying these tools to a number of complete genomes. Source code, data sets and a web server are freely available from http://bioinf.cs.ucl.ac.uk/psipred/. The high accuracy of TM topology prediction which includes detection of both signal peptides and re-entrant helices, combined with the ability to effectively discriminate between TM and globular proteins, make this method ideally suited to whole genome annotation of alpha-helical transmembrane proteins.

Conserved Allosteric Hot Spots in the Transmembrane Domains of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channels and Multidrug Resistance Protein (MRP) Pumps*

PubMed Central

Wei, Shipeng; Roessler, Bryan C.; Chauvet, Sylvain; Guo, Jingyu; Hartman, John L.; Kirk, Kevin L.

2014-01-01

ATP-binding cassette (ABC) transporters are an ancient family of transmembrane proteins that utilize ATPase activity to move substrates across cell membranes. The ABCC subfamily of the ABC transporters includes active drug exporters (the multidrug resistance proteins (MRPs)) and a unique ATP-gated ion channel (cystic fibrosis transmembrane conductance regulator (CFTR)). The CFTR channel shares gating principles with conventional ligand-gated ion channels, but the allosteric network that couples ATP binding at its nucleotide binding domains (NBDs) with conformational changes in its transmembrane helices (TMs) is poorly defined. It is also unclear whether the mechanisms that govern CFTR gating are conserved with the thermodynamically distinct MRPs. Here we report a new class of gain of function (GOF) mutation of a conserved proline at the base of the pore-lining TM6. Multiple substitutions of this proline promoted ATP-free CFTR activity and activation by the weak agonist, 5′-adenylyl-β,γ-imidodiphosphate (AMP-PNP). TM6 proline mutations exhibited additive GOF effects when combined with a previously reported GOF mutation located in an outer collar of TMs that surrounds the pore-lining TMs. Each TM substitution allosterically rescued the ATP sensitivity of CFTR gating when introduced into an NBD mutant with defective ATP binding. Both classes of GOF mutations also rescued defective drug export by a yeast MRP (Yor1p) with ATP binding defects in its NBDs. We conclude that the conserved TM6 proline helps set the energy barrier to both CFTR channel opening and MRP-mediated drug efflux and that CFTR channels and MRP pumps utilize similar allosteric mechanisms for coupling conformational changes in their translocation pathways to ATP binding at their NBDs. PMID:24876383

A Trigger Residue for Transmembrane Signaling in the Escherichia coli Serine Chemoreceptor.

PubMed

Kitanovic, Smiljka; Ames, Peter; Parkinson, John S

2015-08-01

The transmembrane Tsr protein of Escherichia coli mediates chemotactic responses to environmental serine gradients. Serine binds to the periplasmic domain of the homodimeric Tsr molecule, promoting a small inward displacement of one transmembrane helix (TM2). TM2 piston displacements, in turn, modulate the structural stability of the Tsr-HAMP domain on the cytoplasmic side of the membrane to control the autophosphorylation activity of the signaling CheA kinase bound to the membrane-distal cytoplasmic tip of Tsr. A five-residue control cable segment connects TM2 to the AS1 helix of HAMP and transmits stimulus and sensory adaptation signals between them. To explore the possible role of control cable helicity in transmembrane signaling by Tsr, we characterized the signaling properties of mutant receptors with various control cable alterations. An all-alanine control cable shifted Tsr output toward the kinase-on state, whereas an all-glycine control cable prevented Tsr from reaching either a fully on or fully off output state. Restoration of the native isoleucine (I214) in these synthetic control cables largely alleviated their signaling defects. Single amino acid replacements at Tsr-I214 shifted output toward the kinase-off (L, N, H, and R) or kinase-on (A and G) states, whereas other control cable residues tolerated most amino acid replacements with little change in signaling behavior. These findings indicate that changes in control cable helicity might mediate transitions between the kinase-on and kinase-off states during transmembrane signaling by chemoreceptors. Moreover, the Tsr-I214 side chain plays a key role, possibly through interaction with the membrane interfacial environment, in triggering signaling changes in response to TM2 piston displacements. The Tsr protein of E. coli mediates chemotactic responses to environmental serine gradients. Stimulus signals from the Tsr periplasmic sensing domain reach its cytoplasmic kinase control domain through piston

Conserved allosteric hot spots in the transmembrane domains of cystic fibrosis transmembrane conductance regulator (CFTR) channels and multidrug resistance protein (MRP) pumps.

PubMed

Wei, Shipeng; Roessler, Bryan C; Chauvet, Sylvain; Guo, Jingyu; Hartman, John L; Kirk, Kevin L

2014-07-18

ATP-binding cassette (ABC) transporters are an ancient family of transmembrane proteins that utilize ATPase activity to move substrates across cell membranes. The ABCC subfamily of the ABC transporters includes active drug exporters (the multidrug resistance proteins (MRPs)) and a unique ATP-gated ion channel (cystic fibrosis transmembrane conductance regulator (CFTR)). The CFTR channel shares gating principles with conventional ligand-gated ion channels, but the allosteric network that couples ATP binding at its nucleotide binding domains (NBDs) with conformational changes in its transmembrane helices (TMs) is poorly defined. It is also unclear whether the mechanisms that govern CFTR gating are conserved with the thermodynamically distinct MRPs. Here we report a new class of gain of function (GOF) mutation of a conserved proline at the base of the pore-lining TM6. Multiple substitutions of this proline promoted ATP-free CFTR activity and activation by the weak agonist, 5'-adenylyl-β,γ-imidodiphosphate (AMP-PNP). TM6 proline mutations exhibited additive GOF effects when combined with a previously reported GOF mutation located in an outer collar of TMs that surrounds the pore-lining TMs. Each TM substitution allosterically rescued the ATP sensitivity of CFTR gating when introduced into an NBD mutant with defective ATP binding. Both classes of GOF mutations also rescued defective drug export by a yeast MRP (Yor1p) with ATP binding defects in its NBDs. We conclude that the conserved TM6 proline helps set the energy barrier to both CFTR channel opening and MRP-mediated drug efflux and that CFTR channels and MRP pumps utilize similar allosteric mechanisms for coupling conformational changes in their translocation pathways to ATP binding at their NBDs. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Peptidoglycan-associated outer membrane protein Mep45 of rumen anaerobe Selenomonas ruminantium forms a non-specific diffusion pore via its C-terminal transmembrane domain.

PubMed

Kojima, Seiji; Hayashi, Kanako; Tochigi, Saeko; Kusano, Tomonobu; Kaneko, Jun; Kamio, Yoshiyuki

2016-10-01

The major outer membrane protein Mep45 of Selenomonas ruminantium, an anaerobic Gram-negative bacterium, comprises two distinct domains: the N-terminal S-layer homologous (SLH) domain that protrudes into the periplasm and binds to peptidoglycan, and the remaining C-terminal transmembrane domain, whose function has been unknown. Here, we solubilized and purified Mep45 and characterized its function using proteoliposomes reconstituted with Mep45. We found that Mep45 forms a nonspecific diffusion channel via its C-terminal region. The channel was permeable to solutes smaller than a molecular weight of roughly 600, and the estimated pore radius was 0.58 nm. Truncation of the SLH domain did not affect the channel property. On the basis of the fact that Mep45 is the most abundant outer membrane protein in S. ruminantium, we conclude that Mep45 serves as a main pathway through which small solutes diffuse across the outer membrane of this bacterium.

Anti-cancer Activity of Novel TM4SF5-Targeting Antibodies through TM4SF5 Neutralization and Immune Cell-Mediated Cytotoxicity

PubMed Central

Ahn, Hye-Mi; Ryu, Jihye; Song, Jin Myeong; Lee, Yunhee; Kim, Hye-Jin; Ko, Dongjoon; Choi, Inpyo; Kim, Sang Jick; Lee, Jung Weon; Kim, Semi

2017-01-01

The transmembrane four L6 family member 5 (TM4SF5) protein is a novel molecular target for the prevention and treatment of hepatocellular carcinoma. TM4SF5 is highly expressed in liver, colon, esophageal, and pancreatic cancers and is implicated in tumor progression. Here, we screened monoclonal antibodies that specifically bound to the extracellular loop 2 (EC2) of TM4SF5 from a phage-displayed murine antibody (single-chain variable fragment; scFv) library. We constructed and characterized chimeric antibodies, Ab27 and Ab79, of scFv fused with Fc domain of human IgG1. The affinity (KD) of Ab27 and Ab79 for soluble EC2 was approximately 9.2 nM and 16.9 nM, respectively, as determined by surface plasmon resonance analysis. Ab27 and Ab79 efficiently bound to native TM4SF5 on the cell surface were internalized into the cancer cells, leading to a decrease in cell surface TM4SF5. Ab27 and Ab79 inhibited the proliferation and invasion of TM4SF5-positive liver and colon cancer cells and reduced FAK and c-Src phosphorylation. Ab27 and Ab79 also enhanced anoikis sensitivity and reduced survivin. Ab27 mediated antibody-dependent cell-mediated cytotoxicity in vitro. Ab27 and Ab79 efficiently inhibited tumor growth in a liver cancer xenograft model. These results strongly support the further development of Ab27 as a novel anti-cancer agent in the clinic. PMID:28255353

Trans-membrane transport of n-octadecane by Pseudomonas sp. DG17.

PubMed

Hua, Fei; Wang, Hong Qi; Li, Yi; Zhao, Yi Cun

2013-12-01

The trans-membrane transport of hydrocarbons is an important and complex aspect of the process of biodegradation of hydrocarbons by microorganisms. The mechanism of transport of (14)C n-octadecane by Pseudomonas sp. DG17, an alkane-degrading bacterium, was studied by the addition of ATP inhibitors and different substrate concentrations. When the concentration of n-octadecane was higher than 4.54 μmol/L, the transport of (14)C n-octadecane was driven by a facilitated passive mechanism following the intra/extra substrate concentration gradient. However, when the cells were grown with a low concentration of the substrate, the cellular accumulation of n-octadecane, an energy-dependent process, was dramatically decreased by the presence of ATP inhibitors, and n-octadecane accumulation continually increased against its concentration gradient. Furthermore, the presence of non-labeled alkanes blocked (14)C n-octadecane transport only in the induced cells, and the trans-membrane transport of n-octadecane was specific with an apparent dissociation constant K t of 11.27 μmol/L and V max of 0.96 μmol/min/mg protein. The results indicated that the trans-membrane transport of n-octadecane by Pseudomonas sp. DG17 was related to the substrate concentration and ATP.

Multiscale simulations on conformational dynamics and membrane interactions of the non-structural 2 (NS2) transmembrane domain.

PubMed

Hung, Huynh Minh; Hang, Tran Dieu; Nguyen, Minh Tho

2016-09-09

Hepatitis C virus (HCV) is one of the most crucial global health issues, in which the HCV non-structural protein 2 (NS2), particularly its three transmembrane segments, plays a crucial role in HCV assembly. In this context, multiscale MD simulations have been applied to investigate the preferred orientation of transmembrane domain of NS2 protein (TNS2) in a POPC bilayer, structural stability and characteristic of intramembrane protein-lipid and protein-protein interaction. Our study indicates that NS2 protein adopts three trans-membrane segments with highly stable α-helix structure in a POPC bilayer and a short helical luminal segment. While the first and second TM segment involved in continuous helical domain, the third TM segment is however cleaved into two sub-segments with different tilt angles via a kink at L87G88. Salt bridges K81-E45, R32-PO4 and R43-PO4 are determined as the key factor to stabilize the structure of TM2 and TM3 which consist of charged residues located in the hydrophobic region of the membrane. Copyright © 2016 Elsevier Inc. All rights reserved.

The Intracytoplasmic Domain of the Env Transmembrane Protein Is a Locus for Attenuation of Simian Immunodeficiency Virus SIVmac in Rhesus Macaques

PubMed Central

Shacklett, Barbara L.; Weber, Claudia Jo; Shaw, Karen E. S.; Keddie, Elise M.; Gardner, Murray B.; Sonigo, Pierre; Luciw, Paul A.

2000-01-01

The human and simian immunodeficiency virus (HIV-1 and SIVmac) transmembrane proteins contain unusually long intracytoplasmic domains (ICD-TM). These domains are suggested to play a role in envelope fusogenicity, interaction with the viral matrix protein during assembly, viral infectivity, binding of intracellular calmodulin, disruption of membranes, and induction of apoptosis. Here we describe a novel mutant virus, SIVmac-M4, containing multiple mutations in the coding region for the ICD-TM of pathogenic molecular clone SIVmac239. Parental SIVmac239-Nef+ produces high-level persistent viremia and simian AIDS in both juvenile and newborn rhesus macaques. The ICD-TM region of SIVmac-M4 contains three stop codons, a +1 frameshift, and mutation of three highly conserved, charged residues in the conserved C-terminal alpha-helix referred to as lentivirus lytic peptide 1 (LLP-1). Overlapping reading frames for tat, rev, and nef are not affected by these changes. In this study, four juvenile macaques received SIVmac-M4 by intravenous injection. Plasma viremia, as measured by branched-DNA (bDNA) assay, reached a peak at 2 weeks postinoculation but dropped to below detectable levels by 12 weeks. At over 1.5 years postinoculation, all four juvenile macaques remain healthy and asymptomatic. In a subsequent experiment, four neonatal rhesus macaques were given SIVmac-M4 intravenously. These animals exhibited high levels of viremia in the acute phase (2 weeks postinoculation) but are showing a relatively low viral load in the chronic phase of infection, with no clinical signs of disease for 1 year. These findings demonstrated that the intracytoplasmic domain of the transmembrane Env (Env-TM) is a locus for attenuation in rhesus macaques. PMID:10846063

Recombinant influenza H7 hemagglutinin containing CFLLC minidomain in the transmembrane domain showed enhanced cross-protection in mice.

PubMed

Wang, Yang; Zhang, Yun; Wu, Jialing; Lin, Ying; Wu, Zhihui; Wei, Ying; Wei, Xiaona; Qin, Jianru; Xue, Chunyi; Liu, George Dacai; Cao, Yongchang

2017-10-15

Since February 2013, H7N9 influenza virus, causing human infections with high mortality in China, has been a potential pandemic threat. The H7N9 viruses are found to diverge into distinct genotypes as other influenza viruses; thus a vaccine that can provide sufficient cross-protection against different genotypes of H7N9 viruses is urgently needed. Our previous studies demonstrated that the HA-based structural design approach by introducing a CFLLC minidomain into transmembrane domain (TM) of H1, H5 or H9 hemagglutinin (HA) proteins by replacing with H3 subtype HA TM could enhance their cross-protection. In this study, we used Sf9 insect cell expression system to express recombinant H7 HA proteins H7-53WT, in which HA gene was derived from H7N9-53 strain, and H7-53TM containing CFLLC minidomian by replacing its TM domain with H3 HA TM. We investigated whether introduction of CFLLC minidomain into H7 HA (H7-53TM) could increase its cross-reactivity and cross-protection against different genotypes of H7N9 viruses. The results showed that the H7-53TM either with or without squalene adjuvant induced increased HI antibodies, serum IgG antibodies, and IFN-γ production to a panel of 7 H7N9 viruses in mice. Vaccinated animals with H7-53TM alone showed complete protection against challenge with heterologous H7N9-MCX strain, while H7-53WT alone showed incomplete protection (80%). Furthermore, mice vaccinated with H7-53TM HA showed less body weight loss and less pulmonary lesions and inflammation after challenge with homologous or heterologous H7N9 viruses, comparing to H7-53WT. In summary, this study presents a better subunit vaccine candidate (H7-53TM) against potential H7N9 pandemic. Copyright © 2017 Elsevier B.V. All rights reserved.

Synaptobrevin Transmembrane Domain Dimerization Studied by Multiscale Molecular Dynamics Simulations

PubMed Central

Han, Jing; Pluhackova, Kristyna; Wassenaar, Tsjerk A.; Böckmann, Rainer A.

2015-01-01

Synaptic vesicle fusion requires assembly of the SNARE complex composed of SNAP-25, syntaxin-1, and synaptobrevin-2 (sybII) proteins. The SNARE proteins found in vesicle membranes have previously been shown to dimerize via transmembrane (TM) domain interactions. While syntaxin homodimerization is supposed to promote the transition from hemifusion to complete fusion, the role of synaptobrevin’s TM domain association in the fusion process remains poorly understood. Here, we combined coarse-grained and atomistic simulations to model the homodimerization of the sybII transmembrane domain and of selected TM mutants. The wild-type helix is shown to form a stable, right-handed dimer with the most populated helix-helix interface, including key residues predicted in a previous mutagenesis study. In addition, two alternative binding interfaces were discovered, which are essential to explain the experimentally observed higher-order oligomerization of sybII. In contrast, only one dimerization interface was found for a fusion-inactive poly-Leu mutant. Moreover, the association kinetics found for this mutant is lower as compared to the wild-type. These differences in dimerization between the wild-type and the poly-Leu mutant are suggested to be responsible for the reported differences in fusogenic activity between these peptides. This study provides molecular insight into the role of TM sequence specificity for peptide aggregation in membranes. PMID:26287628

Decreasing transmembrane segment length greatly decreases perfringolysin O pore size

DOE PAGES

Lin, Qingqing; Li, Huilin; Wang, Tong; ...

2015-04-08

Perfringolysin O (PFO) is a transmembrane (TM) β-barrel protein that inserts into mammalian cell membranes. Once inserted into membranes, PFO assembles into pore-forming oligomers containing 30–50 PFO monomers. These form a pore of up to 300 Å, far exceeding the size of most other proteinaceous pores. In this study, we found that altering PFO TM segment length can alter the size of PFO pores. A PFO mutant with lengthened TM segments oligomerized to a similar extent as wild-type PFO, and exhibited pore-forming activity and a pore size very similar to wild-type PFO as measured by electron microscopy and a leakagemore » assay. In contrast, PFO with shortened TM segments exhibited a large reduction in pore-forming activity and pore size. This suggests that the interaction between TM segments can greatly affect the size of pores formed by TM β-barrel proteins. PFO may be a promising candidate for engineering pore size for various applications.« less

Trimeric Transmembrane Domain Interactions in Paramyxovirus Fusion Proteins

PubMed Central

Smith, Everett Clinton; Smith, Stacy E.; Carter, James R.; Webb, Stacy R.; Gibson, Kathleen M.; Hellman, Lance M.; Fried, Michael G.; Dutch, Rebecca Ellis

2013-01-01

Paramyxovirus fusion (F) proteins promote membrane fusion between the viral envelope and host cell membranes, a critical early step in viral infection. Although mutational analyses have indicated that transmembrane (TM) domain residues can affect folding or function of viral fusion proteins, direct analysis of TM-TM interactions has proved challenging. To directly assess TM interactions, the oligomeric state of purified chimeric proteins containing the Staphylococcal nuclease (SN) protein linked to the TM segments from three paramyxovirus F proteins was analyzed by sedimentation equilibrium analysis in detergent and buffer conditions that allowed density matching. A monomer-trimer equilibrium best fit was found for all three SN-TM constructs tested, and similar fits were obtained with peptides corresponding to just the TM region of two different paramyxovirus F proteins. These findings demonstrate for the first time that class I viral fusion protein TM domains can self-associate as trimeric complexes in the absence of the rest of the protein. Glycine residues have been implicated in TM helix interactions, so the effect of mutations at Hendra F Gly-508 was assessed in the context of the whole F protein. Mutations G508I or G508L resulted in decreased cell surface expression of the fusogenic form, consistent with decreased stability of the prefusion form of the protein. Sedimentation equilibrium analysis of TM domains containing these mutations gave higher relative association constants, suggesting altered TM-TM interactions. Overall, these results suggest that trimeric TM interactions are important driving forces for protein folding, stability and membrane fusion promotion. PMID:24178297

Structure of a Double Transmembrane Fragment of a G-Protein-Coupled Receptor in Micelles

PubMed Central

Neumoin, Alexey; Cohen, Leah S.; Arshava, Boris; Tantry, Subramanyam; Becker, Jeffrey M.; Zerbe, Oliver; Naider, Fred

2009-01-01

Abstract The structure and dynamic properties of an 80-residue fragment of Ste2p, the G-protein-coupled receptor for α-factor of Saccharomyces cerevisiae, was studied in LPPG micelles with the use of solution NMR spectroscopy. The fragment Ste2p(G31-T110) (TM1-TM2) consisted of 19 residues from the N-terminal domain, the first TM helix (TM1), the first cytoplasmic loop, the second TM helix (TM2), and seven residues from the first extracellular loop. Multidimensional NMR experiments on [15N], [15N, 13C], [15N, 13C, 2H]-labeled TM1-TM2 and on protein fragments selectively labeled at specific amino acid residues or protonated at selected methyl groups resulted in >95% assignment of backbone and side-chain nuclei. The NMR investigation revealed the secondary structure of specific residues of TM1-TM2. TALOS constraints and NOE connectivities were used to calculate a structure for TM1-TM2 that was highlighted by the presence of three α-helices encompassing residues 39–47, 49–72, and 80–103, with higher flexibility around the internal Arg58 site of TM1. RMSD values of individually superimposed helical segments 39–47, 49–72, and 80–103 were 0.25 ± 0.10 Å, 0.40 ± 0.13 Å, and 0.57 ± 0.19 Å, respectively. Several long-range interhelical connectivities supported the folding of TM1-TM2 into a tertiary structure typified by a crossed helix that splays apart toward the extracellular regions and contains considerable flexibility in the G56VRSG60 region. 15N-relaxation and hydrogen-deuterium exchange data support a stable fold for the TM parts of TM1-TM2, whereas the solvent-exposed segments are more flexible. The NMR structure is consistent with the results of biochemical experiments that identified the ligand-binding site within this region of the receptor. PMID:19383463

Growth and optical characteristics of Tm-doped AlGaN layer grown by organometallic vapor phase epitaxy

NASA Astrophysics Data System (ADS)

Takatsu, J.; Fuji, R.; Tatebayashi, J.; Timmerman, D.; Lesage, A.; Gregorkiewicz, T.; Fujiwara, Y.

2018-04-01

We report on the growth and optical properties of Tm-doped AlGaN layers by organometallic vapor phase epitaxy (OMVPE). The morphological and optical properties of Tm-doped GaN (GaN:Tm) and Tm-doped AlGaN (AlGaN:Tm) were investigated by Nomarski differential interference contrast microscopy and photoluminescence (PL) characterization. Nomarski images reveal an increase of surface roughness upon doping Tm into both GaN and AlGaN layers. The PL characterization of GaN:Tm shows emission in the near-infrared range originating from intra-4f shell transitions of Tm3+ ions. In contrast, AlGaN:Tm also exhibits blue light emission from Tm3+ ions. In that case, the wider band gap of the AlGaN host allows energy transfer to higher states of the Tm3+ ions. With time-resolved PL measurements, we could distinguish three types of luminescent sites of Tm3+ in the AlGaN:Tm layer, having different decay times. Our results confirm that Tm ions can be doped into GaN and AlGaN by OMVPE, and show potential for the fabrication of novel high-color-purity blue light emitting diodes.

A novel transmembrane Ser/Thr kinase complexes with protein phosphatase-1 and inhibitor-2.

PubMed

Wang, Hong; Brautigan, David L

2002-12-20

Protein kinases and protein phosphatases exert coordinated control over many essential cellular processes. Here, we describe the cloning and characterization of a novel human transmembrane protein KPI-2 (Kinase/Phosphatase/Inhibitor-2) that was identified by yeast two-hybrid using protein phosphatase inhibitor-2 (Inh2) as bait. KPI-2 mRNA was predominantly expressed in skeletal muscle. KPI-2 is a 1503-residue protein with two predicted transmembrane helices at the N terminus, a kinase domain, followed by a C-terminal domain. The transmembrane helices were sufficient for targeting proteins to the membrane. KPI-2 kinase domain has about 60% identity with its closest relative, a tyrosine kinase. However, it only exhibited serine/threonine kinase activity in autophosphorylation reactions or with added substrates. KPI-2 kinase domain phosphorylated protein phosphatase-1 (PP1C) at Thr(320), which attenuated PP1C activity. KPI-2 C-terminal domain directly associated with PP1C, and this required a VTF motif. Inh2 associated with KPI-2 C-terminal domain with and without PP1C. Thus, KPI-2 is a kinase with sites to associate with PP1C and Inh2 to form a regulatory complex that is localized to membranes.

Receptor-mediated protein kinase activation and the mechanism of transmembrane signaling in bacterial chemotaxis.

PubMed Central

Liu, Y; Levit, M; Lurz, R; Surette, M G; Stock, J B

1997-01-01

Chemotaxis responses of Escherichia coli and Salmonella are mediated by type I membrane receptors with N-terminal extracytoplasmic sensing domains connected by transmembrane helices to C-terminal signaling domains in the cytoplasm. Receptor signaling involves regulation of an associated protein kinase, CheA. Here we show that kinase activation by a soluble signaling domain construct involves the formation of a large complex, with approximately 14 receptor signaling domains per CheA dimer. Electron microscopic examination of these active complexes indicates a well defined bundle composed of numerous receptor filaments. Our findings suggest a mechanism for transmembrane signaling whereby stimulus-induced changes in lateral packing interactions within an array of receptor-sensing domains at the cell surface perturb an equilibrium between active and inactive receptor-kinase complexes within the cytoplasm. PMID:9405352

Transmembrane helix prediction: a comparative evaluation and analysis.

PubMed

Cuthbertson, Jonathan M; Doyle, Declan A; Sansom, Mark S P

2005-06-01

The prediction of transmembrane (TM) helices plays an important role in the study of membrane proteins, given the relatively small number (approximately 0.5% of the PDB) of high-resolution structures for such proteins. We used two datasets (one redundant and one non-redundant) of high-resolution structures of membrane proteins to evaluate and analyse TM helix prediction. The redundant (non-redundant) dataset contains structure of 434 (268) TM helices, from 112 (73) polypeptide chains. Of the 434 helices in the dataset, 20 may be classified as 'half-TM' as they are too short to span a lipid bilayer. We compared 13 TM helix prediction methods, evaluating each method using per segment, per residue and termini scores. Four methods consistently performed well: SPLIT4, TMHMM2, HMMTOP2 and TMAP. However, even the best methods were in error by, on average, about two turns of helix at the TM helix termini. The best and worst case predictions for individual proteins were analysed. In particular, the performance of the various methods and of a consensus prediction method, were compared for a number of proteins (e.g. SecY, ClC, KvAP) containing half-TM helices. The difficulties of predicting half-TM helices suggests that current prediction methods successfully embody the two-state model of membrane protein folding, but do not accommodate a third stage in which, e.g., short helices and re-entrant loops fold within a bundle of stable TM helices.

Activation gating kinetics of GIRK channels are mediated by cytoplasmic residues adjacent to transmembrane domains.

PubMed

Sadja, Rona; Reuveny, Eitan

2009-01-01

G-protein-coupled inwardly rectifying potassium channels (GIRK/Kir3.x) are involved in neurotransmission-mediated reduction of excitability. The gating mechanism following G protein activation of these channels likely proceeds from movement of inner transmembrane helices to allow K(+) ions movement through the pore of the channel. There is limited understanding of how the binding of G-protein betagamma subunits to cytoplasmic regions of the channel transduces the signal to the transmembrane regions. In this study, we examined the molecular basis that governs the activation kinetics of these channels, using a chimeric approach. We identified two regions as being important in determining the kinetics of activation. One region is the bottom of the outer transmembrane helix (TM1) and the cytoplasmic domain immediately adjacent (the slide helix); and the second region is the bottom of the inner transmembrane helix (TM2) and the cytoplasmic domain immediately adjacent. Interestingly, both of these regions are sufficient in mediating the kinetics of fast activation gating. This result suggests that there is a cooperative movement of either one of these domains to allow fast and efficient activation gating of GIRK channels.

Large Multiple Transmembrane Domain Fragments of a G Protein-Coupled Receptor: Biosynthesis, Purification, and Biophysical Studies

PubMed Central

Potetinova, Zhanna; Tantry, Subramanyam; Cohen, Leah S.; Caroccia, Katrina E.; Arshava, Boris; Becker, Jeffrey M.; Naider, Fred

2013-01-01

To conduct biophyiscal analyses on large domains of GPCRs, multi-milligram quantities of highly homogeneous proteins are necessary. This communication discusses the biosynthesis of 4 transmembrane and 5 transmembrane-containing fragments of Ste2p, a GPCR recognizing the Saccharomyces cerevisiae tridecapeptide pheromone α-factor. The target fragments contained the predicted four N-terminal Ste2p[G31-A198] (4TMN), four C-terminal Ste2p[T155-L340] (4TMC) or five C-terminal Ste2p[I120-L340] (5TMC) transmembrane segments of Ste2p. 4TMN was expressed as a fusion protein using a modified pMMHa vector in L-arabinose-induced Escherichia coli BL21-AI, and cleaved with cyanogen bromide. 4TMC and 5TMC were obtained by direct expression using a pET21a vector in IPTG-induced Escherichia coli BL21(DE3) cells. 4TMC and 5TMC were biosynthesized on a preparative scale, isolated in multi-milligram amounts, characterized by MS and investigated by biophysical methods. CD spectroscopy indicated the expected highly α-helical content for 4TMC and 5TMC in membrane mimetic environments. Tryptophan fluorescence showed that 5TMC integrated into the nonpolar region of 1-stearoyl-2-hydroxy-sn-glycero-3-phospho-(1′-rac-glycerol) micelles. HSQC-TROSY investigations revealed that [15N]-labeled 5TMC in 50% trifluoroethanol-d2/H2O/0.05% trifluoroacetic acid was stable enough to conduct long multidimensional NMR measurements. The entire Ste2p GPCR was not readily reconstituted from the first two and last five or first three and last four transmembrane domains. PMID:23203693

N-terminal dual lipidation-coupled molecular targeting into the primary cilium.

PubMed

Kumeta, Masahiro; Panina, Yulia; Yamazaki, Hiroya; Takeyasu, Kunio; Yoshimura, Shige H

2018-06-13

The primary cilium functions as an "antenna" for cell signaling, studded with characteristic transmembrane receptors and soluble protein factors, raised above the cell surface. In contrast to the transmembrane proteins, targeting mechanisms of nontransmembrane ciliary proteins are poorly understood. We focused on a pathogenic mutation that abolishes ciliary localization of retinitis pigmentosa 2 protein and revealed a dual acylation-dependent ciliary targeting pathway. Short N-terminal sequences which contain myristoylation and palmitoylation sites are sufficient to target a marker protein into the cilium in a palmitoylation-dependent manner. A Golgi-localized palmitoyltransferase DHHC-21 was identified as the key enzyme controlling this targeting pathway. Rapid turnover of the targeted protein was ensured by cholesterol-dependent membrane fluidity, which balances highly and less-mobile populations of the molecules within the cilium. This targeting signal was found in a set of signal transduction molecules, suggesting a general role of this pathway in proper ciliary organization, and dysfunction in ciliary disorders. © 2018 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Neutron Scattering Cross Section Measurements for 169Tm via the (n,n') Technique

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alimeti, Afrim; Kegel, Gunter H.R.; Egan, James J.

2005-05-24

The neutron physics group at the University of Massachusetts Lowell (UML) has been involved in a program of scattering cross-section measurements for highly deformed nuclei such as 159Tb, 169Tm, 232Th, 235U, 238U, and 239Pu. Ko et al. have reported neutron inelastic scattering data from 169Tm for states above 100 keV via the (n,n'{gamma}) reaction at incident energies in the 0.2 MeV to 1.0 MeV range. In the present research, in which the time-of-flight method was employed, direct (n,n') measurements of neutrons scattered from 169Tm in the 0.2 to 1.0 MeV range were taken. It requires that our 5.5-MeV Van demore » Graaff accelerator be operated in the pulsed and bunched beam mode producing subnanosecond pulses at a 5-MHz repetition frequency. Neutrons are produced by the 7Li(p,n)7Be reaction using a thin metallic elemental lithium target.« less

GPR30: a seven-transmembrane-spanning estrogen receptor that triggers EGF release.

PubMed

Filardo, Edward J; Thomas, Peter

2005-10-01

Heterotrimeric G proteins and seven-transmembrane-spanning (7TM) receptors are implicated in rapid estrogen signaling. The orphan 7TM receptor GPR30 is linked to estrogen-mediated activation of adenylyl cyclase, release of epidermal growth factor (EGF)-related ligands, and specific estrogen binding. GPR30 acts independently of estrogen receptors, ERalpha and ERbeta, and probably functions as a heptahelical ER. 7TM receptors elicit signals that stimulate second messengers, and convey intracellular signals via EGF receptors. Identification of GPR30 as a Gs-coupled 7TM receptor that triggers release of heparin-binding EGF establishes its role in cell signaling cascades initiated by estrogens, and explains their capacity to activate second messengers and promote EGF-like effects. Thus, estrogen can signal by the same mechanism as various other hormones, through a specific 7TM receptor.

Synthesis, structure and reactivity of [Tm(Bu(t))]ZnH, a monomeric terminal zinc hydride compound in a sulfur-rich coordination environment: access to a heterobimetallic compound.

PubMed

Kreider-Mueller, Ava; Quinlivan, Patrick J; Rauch, Michael; Owen, Jonathan S; Parkin, Gerard

2016-02-07

The first terminal zinc hydride complex that features a sulfur-rich coordination environment, namely the tris(2-mercapto-1-tert-butylimidazolyl)hydroborato compound, [Tm(Bu(t))]ZnH, has been synthesized via the reaction of [Tm(Bu(t))]ZnOPh with PhSiH3. The Zn-H bond of [Tm(Bu(t))]ZnH is subject to insertion of CO2 and facile protolytic cleavage, of which the latter provides access to heterobimetallic [Tm(Bu(t))]ZnMo(CO)3Cp.

Structure and ion channel activity of the human respiratory syncytial virus (hRSV) small hydrophobic protein transmembrane domain

PubMed Central

Gan, Siok Wan; Ng, Lifang; Lin, Xin; Gong, Xiandi; Torres, Jaume

2008-01-01

The small hydrophobic (SH) protein from the human respiratory syncytial virus (hRSV) is a glycoprotein of ∼64 amino acids with one putative α-helical transmembrane domain. Although SH protein is important for viral infectivity, its exact role during viral infection is not clear. Herein, we have studied the secondary structure, orientation, and oligomerization of the transmembrane domain of SH (SH-TM) in the presence of lipid bilayers. Only one oligomer, a pentamer, was observed in PFO-PAGE. Using polarized attenuated total reflection-Fourier transform infrared (PATR-FTIR) spectroscopy, we show that the SH-TM is α-helical. The rotational orientation of SH-TM was determined by site-specific infrared dichroism (SSID) at two consecutive isotopically labeled residues. This orientation is consistent with that of an evolutionary conserved pentameric model obtained from a global search protocol using 13 homologous sequences of RSV. Conductance studies of SH-TM indicate ion channel activity, which is cation selective, and inactive below the predicted pKa of histidine. Thus, our results provide experimental evidence that the transmembrane domain of SH protein forms pentameric α-helical bundles that form cation-selective ion channels in planar lipid bilayers. We provide a model for this pore, which should be useful in mutagenesis studies to elucidate its role during the virus cycle. PMID:18369195

Uncoupling GP1 and GP2 Expression in the Lassa Virus Glycoprotein Complex: Implications for GPI Ectodomain Shedding

DTIC Science & Technology

2008-12-23

glycoprotein precursor (GPC) signal peptide (SP) or human IgG signal sequences (s.s.). GP2 was secreted from cells only when (1) the transmembrane (TM) domain...consistent with viral TM fusion proteins [9,10]. GPC con- tains a 58 residue hydrophobic N-terminal signal peptide (SP), which directs the precursor to the...including GPC, GP1, and GP2. Various signal peptides , purification tags, and modifications to internal domains were employed for the generation and

The positive inside rule is stronger when followed by a transmembrane helix.

PubMed

Virkki, Minttu T; Peters, Christoph; Nilsson, Daniel; Sörensen, Therese; Cristobal, Susana; Wallner, Björn; Elofsson, Arne

2014-08-12

The translocon recognizes transmembrane helices with sufficient level of hydrophobicity and inserts them into the membrane. However, sometimes less hydrophobic helices are also recognized. Positive inside rule, orientational preferences of and specific interactions with neighboring helices have been shown to aid in the recognition of these helices, at least in artificial systems. To better understand how the translocon inserts marginally hydrophobic helices, we studied three naturally occurring marginally hydrophobic helices, which were previously shown to require the subsequent helix for efficient translocon recognition. We find no evidence for specific interactions when we scan all residues in the subsequent helices. Instead, we identify arginines located at the N-terminal part of the subsequent helices that are crucial for the recognition of the marginally hydrophobic transmembrane helices, indicating that the positive inside rule is important. However, in two of the constructs, these arginines do not aid in the recognition without the rest of the subsequent helix; that is, the positive inside rule alone is not sufficient. Instead, the improved recognition of marginally hydrophobic helices can here be explained as follows: the positive inside rule provides an orientational preference of the subsequent helix, which in turn allows the marginally hydrophobic helix to be inserted; that is, the effect of the positive inside rule is stronger if positively charged residues are followed by a transmembrane helix. Such a mechanism obviously cannot aid C-terminal helices, and consequently, we find that the terminal helices in multi-spanning membrane proteins are more hydrophobic than internal helices. Copyright © 2014 Elsevier Ltd. All rights reserved.

[RDBH-method and big DyeTM terminator technology in accurate diagnosis of β-thalassemia and the allelic polymorphism of β-globin cluster].

PubMed

Akperova, G A

2014-11-01

IThe purpose of this study was to evaluate of the efficiency of RDBH-method and Big DyeTM Terminator technology in an accurate diagnosis of β-thalassemia and the allelic polymorphism of β-globin cluster. It was done a complete hematology analysis (HB, MCH, MCV, MCHC, RBC, Hct, HbA2, HbF, Serum iron, Serum ferritin at four children (males, 6-10 years old) and their parents. Molecular analysis included Reverse Dot-Blot Hybridization StripAssay (RDBH) and DNA sequencing on ABI PRISM Big DyeTM Terminator. Hematologic and molecular parameters were contradictory. The homozygosity for β0-thalassemia (β0IVS2.1[G>A] and β0codon 8[-AA]) at three boys with the mild clinical manifestation and heterozygosity of their parents for mutations, and the absence of β-globin mutations at parents and a boy who holds monthly transfusion was established by RDBH-analysis. DNA sequencing by technology Big DyeTM Terminator showed polymorphism at positions -551 and -521 of Cap5'-region (-650-250) - (AT)7(T)7 and (AT)8(T)5. Application of the integrated clinical-molecular approach is an ideal method for an accurate diagnosis, identification of asymptomatic carriers and a reduce of the risk of complications from β-thalassemia, moreover screening of γG-gene and the level of fetal hemoglobin in early childhood will help manage of β-thalassemia clinic and prevent heavy consequences of the disease.

PSOFuzzySVM-TMH: identification of transmembrane helix segments using ensemble feature space by incorporated fuzzy support vector machine.

PubMed

Hayat, Maqsood; Tahir, Muhammad

2015-08-01

Membrane protein is a central component of the cell that manages intra and extracellular processes. Membrane proteins execute a diversity of functions that are vital for the survival of organisms. The topology of transmembrane proteins describes the number of transmembrane (TM) helix segments and its orientation. However, owing to the lack of its recognized structures, the identification of TM helix and its topology through experimental methods is laborious with low throughput. In order to identify TM helix segments reliably, accurately, and effectively from topogenic sequences, we propose the PSOFuzzySVM-TMH model. In this model, evolutionary based information position specific scoring matrix and discrete based information 6-letter exchange group are used to formulate transmembrane protein sequences. The noisy and extraneous attributes are eradicated using an optimization selection technique, particle swarm optimization, from both feature spaces. Finally, the selected feature spaces are combined in order to form ensemble feature space. Fuzzy-support vector Machine is utilized as a classification algorithm. Two benchmark datasets, including low and high resolution datasets, are used. At various levels, the performance of the PSOFuzzySVM-TMH model is assessed through 10-fold cross validation test. The empirical results reveal that the proposed framework PSOFuzzySVM-TMH outperforms in terms of classification performance in the examined datasets. It is ascertained that the proposed model might be a useful and high throughput tool for academia and research community for further structure and functional studies on transmembrane proteins.

Substrate dependence of TM-polarized light emission characteristics of BAlGaN/AlN quantum wells

NASA Astrophysics Data System (ADS)

Park, Seoung-Hwan; Ahn, Doyeol

2018-06-01

To study the substrate dependence of light emission characteristics of transverse-magnetic (TM)-polarized light emitted from BAlGaN/AlN quantum wells (QWs) grown on GaN and AlN substrates were investigated theoretically. It is found that the topmost valence subband for QW structures grown on AlN substrate, is heavy hole state (HH1) while that for QW structures grown on GaN substrate is crystal-field split off light hole state (CL1), irrespective of the boron content. Since TM-polarized light emission is associated with the light hole state, the TM-polarized emission peak of BAlGaN/AlN QW structures grown on GaN substrate is expected to be much larger than that of the QW structure grown on AlN substrate. Also, both QW structures show that the spontaneous emission peak of BAlGaN/AlN QW structures would be improved with the inclusion of the boron. However, it rapidly begins to decrease when the boron content exceeds a critical value.

TM9/Phg1 and SadA proteins control surface expression and stability of SibA adhesion molecules in Dictyostelium.

PubMed

Froquet, Romain; le Coadic, Marion; Perrin, Jackie; Cherix, Nathalie; Cornillon, Sophie; Cosson, Pierre

2012-02-01

TM9 proteins form a family of conserved proteins with nine transmembrane domains essential for cellular adhesion in many biological systems, but their exact role in this process remains unknown. In this study, we found that genetic inactivation of the TM9 protein Phg1A dramatically decreases the surface levels of the SibA adhesion molecule in Dictyostelium amoebae. This is due to a decrease in sibA mRNA levels, in SibA protein stability, and in SibA targeting to the cell surface. A similar phenotype was observed in cells devoid of SadA, a protein that does not belong to the TM9 family but also exhibits nine transmembrane domains and is essential for cellular adhesion. A contact site A (csA)-SibA chimeric protein comprising only the transmembrane and cytosolic domains of SibA and the extracellular domain of the Dictyostelium surface protein csA also showed reduced stability and relocalization to endocytic compartments in phg1A knockout cells. These results indicate that TM9 proteins participate in cell adhesion by controlling the levels of adhesion proteins present at the cell surface.

Structural Model of the Bilitranslocase Transmembrane Domain Supported by NMR and FRET Data.

PubMed

Roy Choudhury, Amrita; Sikorska, Emilia; van den Boom, Johannes; Bayer, Peter; Popenda, Łukasz; Szutkowski, Kosma; Jurga, Stefan; Bonomi, Massimiliano; Sali, Andrej; Zhukov, Igor; Passamonti, Sabina; Novič, Marjana

2015-01-01

We present a 3D model of the four transmembrane (TM) helical regions of bilitranslocase (BTL), a structurally uncharacterized protein that transports organic anions across the cell membrane. The model was computed by considering helix-helix interactions as primary constraints, using Monte Carlo simulations. The interactions between the TM2 and TM3 segments have been confirmed by Förster resonance energy transfer (FRET) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy, increasing our confidence in the model. Several insights into the BTL transport mechanism were obtained by analyzing the model. For example, the observed cis-trans Leu-Pro peptide bond isomerization in the TM3 fragment may indicate a key conformational change during anion transport by BTL. Our structural model of BTL may facilitate further studies, including drug discovery.

Overexpression, crystallization and preliminary X-ray crystallographic analysis of β-N-acetylglucosaminidase from Thermotoga maritima encoded by the Tm0809 gene.

PubMed

Lee, Hyung Ho; Jung, Sang Taek

2013-02-01

β-N-acetylglucosaminidase (NagA) protein hs a chitin-degrading activity and chitin is one of the most abundant polymers in nature. NagA contains a family 3 glycoside (GH3)-type N-terminal domain and a unique C-terminal domain. The structurally uncharacterized C-terminal domain of NagA may be involved in substrate specificity. To provide a structural basis for the substrate specificity of NagA, structural analysis of NagA from Thermotoga maritima encoded by the Tm0809 gene was initiated. NagA from T. maritima has been overexpressed in Escherichia coli and crystallized at 296 K using ammonium sulfate as a precipitant. Crystals of T. maritima NagA diffracted to 3.80 Å resolution and belonged to the monoclinic space group C2, with unit-cell parameters a = 231.15, b = 133.62, c = 140.88 Å, β = 89.97°. The crystallization of selenomethionyl-substituted protein is in progress to solve the crystal structure of T. maritima NagA.

Structure and function of transmembrane segment XII in osmosensor and osmoprotectant transporter ProP of Escherichia coli.

PubMed

Liu, Feng; Culham, Doreen E; Vernikovska, Yaroslava I; Keates, Robert A B; Boggs, Joan M; Wood, Janet M

2007-05-15

Escherichia coli transporter ProP acts as both an osmosensor and an osmoregulator. As medium osmolality rises, ProP is activated and mediates H+-coupled uptake of osmolytes like proline. A homology model of ProP with 12-transmembrane (TM) helices and cytoplasmic termini was created, and the protein's topology was substantiated experimentally. Residues 468-497, at the end of the C-terminal domain and linked to TM XII, form an intermolecular, homodimeric alpha-helical coiled-coil that tunes the transporter's response to osmolality. We aim to further define the structure and function of ProP residues Q415-E440, predicted to include TM XII. Each residue was replaced with cysteine (Cys) in a histidine-tagged, Cys-less ProP variant (ProP*). Cys at positions 415-418 and 438-440 were most reactive with Oregon Green Maleimide (OGM), suggesting that residues 419 through 437 are in the membrane. Except for V429-I433, reactivity of those Cys varied with helical periodicity. Cys predicted to face the interior of ProP were more reactive than Cys predicted to face the lipid. The former may be exposed to hydrated polar residues in the protein interior, particularly on the periplasmic side. Intermolecular cross-links formed when ProP* variants with Cys at positions 419, 420, 422, and 439 were treated with DTME. Thus TM XII can participate, along its entire length, in the dimer interface of ProP. Cys substitution E440C rendered ProP* inactive. All other variants retained more than 30% of the proline uptake activity of ProP* at high osmolality. Most variants with Cys substitutions in the periplasmic half of TM XII activated at lower osmolalities than ProP*. Variants with Cys substitutions on one face of the cytoplasmic half of TM XII required a higher osmolality to activate. They included elements of a GXXXG motif that are predicted to form the interface of TM XII with TM VII. These studies define the position of ProP TM XII within the membrane, further support the predicted

Mutational Analysis of the Control Cable That Mediates Transmembrane Signaling in the Escherichia coli Serine Chemoreceptor▿

PubMed Central

Kitanovic, Smiljka; Ames, Peter; Parkinson, John S.

2011-01-01

During transmembrane signaling by Escherichia coli Tsr, changes in ligand occupancy in the periplasmic serine-binding domain promote asymmetric motions in a four-helix transmembrane bundle. Piston displacements of the signaling TM2 helix in turn modulate the HAMP bundle on the cytoplasmic side of the membrane to control receptor output signals to the flagellar motors. A five-residue control cable joins TM2 to the HAMP AS1 helix and mediates conformational interactions between them. To explore control cable structural features important for signal transmission, we constructed and characterized all possible single amino acid replacements at the Tsr control cable residues. Only a few lesions abolished Tsr function, indicating that the chemical nature and size of the control cable side chains are not individually critical for signal control. Charged replacements at I214 mimicked the signaling consequences of attractant or repellent stimuli, most likely through aberrant structural interactions of the mutant side chains with the membrane interfacial environment. Prolines at residues 214 to 217 also caused signaling defects, suggesting that the control cable has helical character. However, proline did not disrupt function at G213, the first control cable residue, which might serve as a structural transition between the TM2 and AS1 helix registers. Hydrophobic amino acids at S217, the last control cable residue, produced attractant-mimic effects, most likely by contributing to packing interactions within the HAMP bundle. These results suggest a helix extension mechanism of Tsr transmembrane signaling in which TM2 piston motions influence HAMP stability by modulating the helicity of the control cable segment. PMID:21803986

Functional reconstitution of the human serotonin receptor 5-HT(6) using synthetic transmembrane peptides.

PubMed

Lee, Won-Kyu; Han, Jason J; Jin, Bong-Suk; Boo, Doo Wan; Yu, Yeon Gyu

2009-12-18

Seven transmembrane (7TM) synthetic peptides mimicking the alpha-helical TM domains of the human serotonin receptor subtype-6 (5-HT(6)) were autonomously reconstituted in detergent micelle and liposome environments. The degree of assembly of the 7TM peptides was characterized by monitoring the fluorescence resonance energy transfer (FRET) between donor and acceptor probes labeled at the amino termini of the second and fourth TM-peptides, respectively. The FRET efficiency of these peptides significantly increased when the 7TM peptides were reconstituted in liposome compare to detergent micelles. Furthermore, the 7TM peptides reconstituted in liposomes selectively bound to free serotonin and serotonin-conjugated magnetic beads, yielding a dissociation constant of 0.84 microM. These results show that the seven individual TM domains of 5-HT(6) can spontaneously assemble into liposomes in a conformation that mimics a native structure, and further demonstrate that specific interactions between TM helices play a critical role in the folding and stabilizing of GPCRs. The autonomous assembly of 7TM-peptides can be applied to the screening of agonists for GPCRs that are difficult to manipulate.

Molecular mechanisms for generating transmembrane proton gradients

PubMed Central

Gunner, M.R.; Amin, Muhamed; Zhu, Xuyu; Lu, Jianxun

2013-01-01

Membrane proteins use the energy of light or high energy substrates to build a transmembrane proton gradient through a series of reactions leading to proton release into the lower pH compartment (P-side) and proton uptake from the higher pH compartment (N-side). This review considers how the proton affinity of the substrates, cofactors and amino acids are modified in four proteins to drive proton transfers. Bacterial reaction centers (RCs) and photosystem II (PSII) carry out redox chemistry with the species to be oxidized on the P-side while reduction occurs on the N-side of the membrane. Terminal redox cofactors are used which have pKas that are strongly dependent on their redox state, so that protons are lost on oxidation and gained on reduction. Bacteriorhodopsin is a true proton pump. Light activation triggers trans to cis isomerization of a bound retinal. Strong electrostatic interactions within clusters of amino acids are modified by the conformational changes initiated by retinal motion leading to changes in proton affinity, driving transmembrane proton transfer. Cytochrome c oxidase (CcO) catalyzes the reduction of O2 to water. The protons needed for chemistry are bound from the N-side. The reduction chemistry also drives proton pumping from N- to P-side. Overall, in CcO the uptake of 4 electrons to reduce O2 transports 8 charges across the membrane, with each reduction fully coupled to removal of two protons from the N-side, the delivery of one for chemistry and transport of the other to the P-side. PMID:23507617

Arrestin-related proteins mediate pH signaling in fungi.

PubMed

Herranz, Silvia; Rodríguez, José M; Bussink, Henk-Jan; Sánchez-Ferrero, Juan C; Arst, Herbert N; Peñalva, Miguel A; Vincent, Olivier

2005-08-23

Metazoan arrestins bind to seven-transmembrane (7TM) receptors to regulate function. Aspergillus nidulans PalF, a protein involved in the fungal ambient pH signaling pathway, contains arrestin N-terminal and C-terminal domains and binds strongly to two different regions within the C-terminal cytoplasmic tail of the 7TM, putative pH sensor PalH. Upon exposure to alkaline ambient pH, PalF is phosphorylated and, like mammalian beta-arrestins, ubiquitinated in a signal-dependent and 7TM protein-dependent manner. Substitution in PalF of a highly conserved arrestin N-terminal domain Ser residue prevents PalF-PalH interaction and pH signaling in vivo. Thus, PalF is the first experimentally documented fungal arrestin-related protein, dispelling the notion that arrestins are restricted to animal proteomes. Epistasis analyses demonstrate that PalF posttranslational modification is partially dependent on the 4TM protein PalI but independent of the remaining pH signal transduction pathway proteins PalA, PalB, and PalC, yielding experimental evidence bearing on the order of participation of the six components of the pH signal transduction pathway. Our data strongly implicate PalH as an ambient pH sensor, possibly with the cooperation of PalI.

Importance of the short cytoplasmic domain of the feline immunodeficiency virus transmembrane glycoprotein for fusion activity and envelope glycoprotein incorporation into virions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Celma, Cristina C.P.; Paladino, Monica G.; Gonzalez, Silvia A.

2007-09-30

The mature form of the envelope (Env) glycoprotein of lentiviruses is a heterodimer composed of the surface (SU) and transmembrane (TM) subunits. Feline immunodeficiency virus (FIV) possesses a TM glycoprotein with a cytoplasmic tail of approximately 53 amino acids which is unusually short compared with that of the other lentiviral glycoproteins (more than 100 residues). To investigate the relevance of the FIV TM cytoplasmic domain to Env-mediated viral functions, we characterized the biological properties of a series of Env glycoproteins progressively shortened from the carboxyl terminus. All the mutant Env proteins were efficiently expressed in feline cells and processed intomore » the SU and TM subunits. Deletion of 5 or 11 amino acids from the TM C-terminus did not significantly affect Env surface expression, fusogenic activity or Env incorporation into virions, whereas removal of 17 or 23 residues impaired Env-mediated cell-to-cell fusion. Further truncation of the FIV TM by 29 residues resulted in an Env glycoprotein that was poorly expressed at the cell surface, exhibited only 20% of the wild-type Env fusogenic capacity and was inefficiently incorporated into virions. Remarkably, deletion of the TM C-terminal 35 or 41 amino acids restored or even enhanced Env biological functions. Indeed, these mutant Env glycoproteins bearing cytoplasmic domains of 18 or 12 amino acids were found to be significantly more fusogenic than the wild-type Env and were efficiently incorporated into virions. Interestingly, truncation of the TM cytoplasmic domain to only 6 amino acids did not affect Env incorporation into virions but abrogated Env fusogenicity. Finally, removal of the entire TM cytoplasmic tail or deletion of as many as 6 amino acids into the membrane-spanning domain led to a complete loss of Env functions. Our results demonstrate that despite its relatively short length, the FIV TM cytoplasmic domain plays an important role in modulating Env-mediated viral

Poxvirus-encoded TNF decoy receptors inhibit the biological activity of transmembrane TNF.

PubMed

Pontejo, Sergio M; Alejo, Ali; Alcami, Antonio

2015-10-01

Poxviruses encode up to four different soluble TNF receptors, named cytokine response modifier B (CrmB), CrmC, CrmD and CrmE. These proteins mimic the extracellular domain of the cellular TNF receptors to bind and inhibit the activity of TNF and, in some cases, other TNF superfamily ligands. Most of these ligands are released after the enzymic cleavage of a membrane precursor. However, transmembrane TNF (tmTNF) is not only a precursor of soluble TNF but also exerts specific pro-inflammatory and immunological activities. Here, we report that viral TNF receptors bound and inhibited tmTNF and describe some interesting differences in their activity against the soluble cytokine. Thus, CrmE, which does not inhibit mouse soluble TNF, could block murine tmTNF-induced cytotoxicity. We propose that this anti-tmTNF effect should be taken into consideration when assessing the role of viral TNF decoy receptors in the pathogenesis of poxvirus.

Impact of charged amino acid substitution in the transmembrane domain of L-alanine exporter, AlaE, of Escherichia coli on the L-alanine export.

PubMed

Kim, Seryoung; Ihara, Kohei; Katsube, Satoshi; Ando, Tasuke; Isogai, Emiko; Yoneyama, Hiroshi

2017-01-01

The Escherichia coli alaE gene encodes the L-alanine exporter, AlaE, that catalyzes active export of L-alanine using proton electrochemical potential. The transporter comprises only 149 amino acid residues and four predicted transmembrane domains (TMs), which contain three charged amino acid residues. The AlaE-deficient L-alanine non-metabolizing cells (ΔalaE cells) appeared hypersusceptible to L-alanyl-L-alanine showing a minimum inhibitory concentration (MIC) of 2.5 µg/ml for the dipeptide due to a toxic accumulation of L-alanine. To elucidate the mechanism by which AlaE exports L-alanine, we replaced charged amino acid residues in the TMs, glutamic acid-30 (TM-I), arginine-45 (TM-II), and aspartic acid-84 (TM-III) with their respective charge-conserved amino acid or a net neutral cysteine. The ΔalaE cells producing R45K or R45C appeared hypersusceptible to the dipeptide, indicating that arginine-45 is essential for AlaE activity. MIC of the dipeptide in the ΔalaE cells expressing E30D and E30C was 156 µg/ml and >10,000 µg/ml, respectively, thereby suggesting that a negative charge at this position is not essential. The ΔalaE cells expressing D84E or D84C showed an MIC >10,000 and 78 µg/ml, respectively, implying that a negative charge is required at this position. These results were generally consistent with that of the L-alanine accumulation experiments in intact cells. We therefore concluded that charged amino acid residues (R45 and D84) in the AlaE transmembrane domain play a pivotal role in L-alanine export. Replacement of three cysteine residues at C22, C28 (both in TM-I), and C135 (C-terminal region) with alanine showed only a marginal effect on L-alanine export.

The Cucumber leaf spot virus p25 auxiliary replicase protein binds and modifies the endoplasmic reticulum via N-terminal transmembrane domains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ghoshal, Kankana; Theilmann, Jane; Reade, Ron

2014-11-15

Cucumber leaf spot virus (CLSV) is a member of the Aureusvirus genus, family Tombusviridae. The auxiliary replicase of Tombusvirids has been found to localize to endoplasmic reticulum (ER), peroxisomes or mitochondria; however, localization of the auxiliary replicase of aureusviruses has not been determined. We have found that the auxiliary replicase of CLSV (p25) fused to GFP colocalizes with ER and that three predicted transmembrane domains (TMDs) at the N-terminus of p25 are sufficient for targeting, although the second and third TMDs play the most prominent roles. Confocal analysis of CLSV infected 16C plants shows that the ER becomes modified includingmore » the formation of punctae at connections between ER tubules and in association with the nucleus. Ultrastructural analysis shows that the cytoplasm contains numerous vesicles which are also found between the perinuclear ER and nuclear membrane. It is proposed that these vesicles correspond to modified ER used as sites for CLSV replication. - Highlights: • The CLSV p25 auxiliary replicase targets the endoplasmic reticulum (ER). • Targeting of CLSV p25 is associated with ER restructuring. • Restructuring of the ER occurs during CLSV infection. • CLSV p25 contains 3 predicted transmembrane domains 2 of which are required for ER targeting. • Vesicles derived from the ER may be sites of CLSV replication.« less

Processing, fusogenicity, virion incorporation and CXCR4-binding activity of a feline immunodeficiency virus envelope glycoprotein lacking the two conserved N-glycosylation sites at the C-terminus of the V3 domain.

PubMed

González, Silvia A; Affranchino, José L

2016-07-01

The process of feline immunodeficiency virus (FIV) entry into its target cells is initiated by the association of the surface (SU) subunit of the viral envelope glycoprotein (Env) with the cellular receptors CD134 and CXCR4. This event is followed by the fusion of the viral and cellular membranes, which is mediated by the transmembrane (TM) subunit of Env. We and others have previously demonstrated that the V3 domain of the SU subunit of Env is essential for CXCR4 binding. Of note, there are two contiguous and highly conserved potential N-glycosylation sites ((418)NST(420) and (422)NLT(424)) located at the C-terminal side of the V3 domain. We therefore decided to study the relevance for Env functions of these N-glycosylation motifs and found that disruption of both of them by introducing the N418Q/N422Q double amino acid substitution drastically impairs Env processing into the SU and TM subunits. Moreover, the simultaneous mutation of these N-glycosylation sites prevents Env incorporation into virions and Env-mediated cell-to-cell fusion. Notably, a recombinant soluble version of the SU glycoprotein carrying the double amino acid replacement N418Q/N422Q at the V3 C-terminal side binds to CXCR4 with an efficiency similar to that of wild-type SU.

An L319F mutation in transmembrane region 3 (TM3) selectively reduces sensitivity to okaramine B of the Bombyx mori l-glutamate-gated chloride channel.

PubMed

Furutani, Shogo; Okuhara, Daiki; Hashimoto, Anju; Ihara, Makoto; Kai, Kenji; Hayashi, Hideo; Sattelle, David B; Matsuda, Kazuhiko

2017-10-01

Okaramines produced by Penicillium simplicissimum AK-40 activate l-glutamate-gated chloride channels (GluCls) and thus paralyze insects. However, the okaramine binding site on insect GluCls is poorly understood. Sequence alignment shows that the equivalent of residue Leucine319 of the okaramine B sensitive Bombyx mori (B. mori) GluCl is a phenylalanine in the okaramine B insensitive B. mori γ-aminobutyric acid-gated chloride channel of the same species. This residue is located in the third transmembrane (TM3) region, a location which in a nematode GluCl is close to the ivermectin binding site. The B. mori GluCl containing the L319F mutation retained its sensitivity to l-glutamate, but responses to ivermectin were reduced and those to okaramine B were completely blocked.

Important Role for the Transmembrane Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Protein during Entry

PubMed Central

Broer, Rene; Boson, Bertrand; Spaan, Willy; Cosset, François-Loïc; Corver, Jeroen

2006-01-01

The spike protein (S) of severe acute respiratory syndrome coronavirus (SARS-CoV) is responsible for receptor binding and membrane fusion. It contains a highly conserved transmembrane domain that consists of three parts: an N-terminal tryptophan-rich domain, a central domain, and a cysteine-rich C-terminal domain. The cytoplasmic tail of S has previously been shown to be required for assembly. Here, the roles of the transmembrane and cytoplasmic domains of S in the infectivity and membrane fusion activity of SARS-CoV have been studied. SARS-CoV S-pseudotyped retrovirus (SARSpp) was used to measure S-mediated infectivity. In addition, the cell-cell fusion activity of S was monitored by a Renilla luciferase-based cell-cell fusion assay. Svsv-cyt, an S chimera with a cytoplasmic tail derived from vesicular stomatitis virus G protein (VSV-G), and Smhv-tmdcyt, an S chimera with the cytoplasmic and transmembrane domains of mouse hepatitis virus, displayed wild-type-like activity in both assays. Svsv-tmdcyt, a chimera with the cytoplasmic and transmembrane domains of VSV-G, was impaired in the SARSpp and cell-cell fusion assays, showing 3 to 25% activity compared to the wild type, depending on the assay and the cells used. Examination of the oligomeric state of the chimeric S proteins in SARSpp revealed that Svsv-tmdcyt trimers were less stable than wild-type S trimers, possibly explaining the lowered fusogenicity and infectivity. PMID:16415007

Transmembrane proteins in the Protein Data Bank: identification and classification.

PubMed

Tusnády, Gábor E; Dosztányi, Zsuzsanna; Simon, István

2004-11-22

Integral membrane proteins play important roles in living cells. Although these proteins are estimated to constitute 25% of proteins at a genomic scale, the Protein Data Bank (PDB) contains only a few hundred membrane proteins due to the difficulties with experimental techniques. The presence of transmembrane proteins in the structure data bank, however, is quite invisible, as the annotation of these entries is rather poor. Even if a protein is identified as a transmembrane one, the possible location of the lipid bilayer is not indicated in the PDB because these proteins are crystallized without their natural lipid bilayer, and currently no method is publicly available to detect the possible membrane plane using the atomic coordinates of membrane proteins. Here, we present a new geometrical approach to distinguish between transmembrane and globular proteins using structural information only and to locate the most likely position of the lipid bilayer. An automated algorithm (TMDET) is given to determine the membrane planes relative to the position of atomic coordinates, together with a discrimination function which is able to separate transmembrane and globular proteins even in cases of low resolution or incomplete structures such as fragments or parts of large multi chain complexes. This method can be used for the proper annotation of protein structures containing transmembrane segments and paves the way to an up-to-date database containing the structure of all known transmembrane proteins and fragments (PDB_TM) which can be automatically updated. The algorithm is equally important for the purpose of constructing databases purely of globular proteins.

Transmembrane domain dependent inhibitory function of FcγRIIB.

PubMed

Wang, Junyi; Li, Zongyu; Xu, Liling; Yang, Hengwen; Liu, Wanli

2018-03-01

FcγRIIB, the only inhibitory IgG Fc receptor, functions to suppress the hyper-activation of immune cells. Numerous studies have illustrated its inhibitory function through the ITIM motif in the cytoplasmic tail of FcγRIIB. However, later studies revealed that in addition to the ITIM, the transmembrane (TM) domain of FcγRIIB is also indispensable for its inhibitory function. Indeed, recent epidemiological studies revealed that a non-synonymous single nucleotide polymorphism (rs1050501) within the TM domain of FcγRIIB, responsible for the I232T substitution, is associated with the susceptibility to systemic lupus erythematosus (SLE). In this review, we will summarize these epidemiological and functional studies of FcγRIIB-I232T in the past few years, and will further discuss the mechanisms accounting for the functional loss of FcγRIIB-I232T. Our review will help the reader gain a deeper understanding of the importance of the TM domain in mediating the inhibitory function of FcγRIIB and may provide insights to a new therapeutic target for the associated diseases.

Uncoupling GP1 and GP2 Expression in the Lassa Virus Glycoprotein Complex: Implications for GP1 Ectodomain Shedding

DTIC Science & Technology

2008-12-23

glycoprotein precursor (GPC) signal peptide (SP) or human IgG signal sequences (s.s.). GP2 was secreted from cells only when (1) the transmembrane (TM) domain... peptide (SP) or human IgG signal sequences (s.s.). GP2 was secreted from cells only when (1) the transmembrane (TM) domain was deleted, the...terminal signal peptide (SP), which directs the precursor to the endoplasmic retic- ulum (ER) for further processing [11]. The SP, which has been

Type II integral membrane protein, TM of J paramyxovirus promotes cell-to-cell fusion.

PubMed

Li, Zhuo; Hung, Cher; Paterson, Reay G; Michel, Frank; Fuentes, Sandra; Place, Ryan; Lin, Yuan; Hogan, Robert J; Lamb, Robert A; He, Biao

2015-10-06

Paramyxoviruses include many important animal and human pathogens. Most paramyxoviruses have two integral membrane proteins: fusion protein (F) and attachment proteins hemagglutinin, hemagglutinin-neuraminidase, or glycoprotein (G), which are critical for viral entry into cells. J paramyxovirus (JPV) encodes four integral membrane proteins: F, G, SH, and transmembrane (TM). The function of TM is not known. In this work, we have generated a viable JPV lacking TM (JPV∆TM). JPV∆TM formed opaque plaques compared with JPV. Quantitative syncytia assays showed that JPV∆TM was defective in promoting cell-to-cell fusion (i.e., syncytia formation) compared with JPV. Furthermore, cells separately expressing F, G, TM, or F plus G did not form syncytia whereas cells expressing F plus TM formed some syncytia. However, syncytia formation was much greater with coexpression of F, G, and TM. Biochemical analysis indicates that F, G, and TM interact with each other. A small hydrophobic region in the TM ectodomain from amino acid residues 118 to 132, the hydrophobic loop (HL), was important for syncytial promotion, suggesting that the TM HL region plays a critical role in cell-to-cell fusion.

Studies on the structures of the Tm, Sj, M1, Can, Sext and Hu blood group antigens.

PubMed

Dahr, W; Knuppertz, G; Beyreuther, K; Moulds, J J; Moulds, M; Wilkinson, S; Capon, C; Fournet, B; Issitt, P D

1991-08-01

The Glycophorins (GPs = sialoglycoproteins) in erythrocyte membranes from various Black individuals, some of which exhibit the M1, Can, Sj, Tm, Sext and/or Hu antigens, and several Caucasian donors, including pooled fetal red cells, were studied. Using agglutination inhibition assays with GP fractions, GP fragments and chemically modified GPs as well as trypsin treatment of intact red cells, the antigens defined by anti-M1, anti-M+M1, anti-Can and anti-Tm sera were found to be located on the N-terminal tryptic peptide (T2, residues 1-31) of the major GP (GP A = MN sialoglycoprotein). Evidence was obtained that the N-terminal amino-acid residue, NeuNAc and/or (a) different sugar residue(s) are involved in the antigens. Amino-acid sequence and composition analyses excluded an amino-acid exchange within the N-terminal region (residues 1-31) of GP A. Carbohydrate analyses revealed the attachment of GlcNAc residues (up to about five, dependent on the strength of the above-mentioned antigens) to O-glycosidically linked oligosaccharides within the N-terminal portion (residues 1-31) of GP A. As judged from the carbohydrate compositions of peptides, the alteration of the O-glycosidic oligosaccharides is associated with a slight increase of the Gal and Fuc contents and a slight decrease of the NeuNAc level. Analyses of small, secondary cyanogen bromide and V8 proteinase peptides from the N-terminal region of GP A from Blacks, Caucasians and Caucasian fetal cells suggest that the variable attachment of small quantities of GlcNAc (about 0.03 to about 0.2 residues per peptide molecule) accounts, at least in part, for the polymorphisms detected by anti-Can and the original anti-Tm (serum Sheerin). Remarkably, the GlcNAc-containing O-glycosidic oligosaccharides occur only in small quantities, or not all at, within the positions 32-61 of GP A and the glycosylated domains of GP B and GP C.(ABSTRACT TRUNCATED AT 400 WORDS)

Intracellular distribution of TM4SF1 and internalization of TM4SF1-antibody complex in vascular endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sciuto, Tracey E.; Merley, Anne; Lin, Chi-Iou

2015-09-25

Transmembrane-4 L-six family member-1 (TM4SF1) is a small plasma membrane-associated glycoprotein that is highly and selectively expressed on the plasma membranes of tumor cells, cultured endothelial cells, and, in vivo, on tumor-associated endothelium. Immunofluorescence microscopy also demonstrated TM4SF1 in cytoplasm and, tentatively, within nuclei. With monoclonal antibody 8G4, and the finer resolution afforded by immuno-nanogold transmission electron microscopy, we now demonstrate TM4SF1 in uncoated cytoplasmic vesicles, nuclear pores and nucleoplasm. Because of its prominent surface location on tumor cells and tumor-associated endothelium, TM4SF1 has potential as a dual therapeutic target using an antibody drug conjugate (ADC) approach. For ADC to bemore » successful, antibodies reacting with cell surface antigens must be internalized for delivery of associated toxins to intracellular targets. We now report that 8G4 is efficiently taken up into cultured endothelial cells by uncoated vesicles in a dynamin-dependent, clathrin-independent manner. It is then transported along microtubules through the cytoplasm and passes through nuclear pores into the nucleus. These findings validate TM4SF1 as an attractive candidate for cancer therapy with antibody-bound toxins that have the capacity to react with either cytoplasmic or nuclear targets in tumor cells or tumor-associated vascular endothelium. - Highlights: • Anti-TM4SF1 antibody 8G4 was efficiently taken up by cultured endothelial cells. • TM4SF1–8G4 internalization is dynamin-dependent but clathrin-independent. • TM4SF1–8G4 complexes internalize along microtubules to reach the perinuclear region. • Internalized TM4SF1–8G4 complexes pass through nuclear pores into the nucleus. • TM4SF1 is an attractive candidate for ADC cancer therapy.« less

PSI/TM-Coffee: a web server for fast and accurate multiple sequence alignments of regular and transmembrane proteins using homology extension on reduced databases.

PubMed

Floden, Evan W; Tommaso, Paolo D; Chatzou, Maria; Magis, Cedrik; Notredame, Cedric; Chang, Jia-Ming

2016-07-08

The PSI/TM-Coffee web server performs multiple sequence alignment (MSA) of proteins by combining homology extension with a consistency based alignment approach. Homology extension is performed with Position Specific Iterative (PSI) BLAST searches against a choice of redundant and non-redundant databases. The main novelty of this server is to allow databases of reduced complexity to rapidly perform homology extension. This server also gives the possibility to use transmembrane proteins (TMPs) reference databases to allow even faster homology extension on this important category of proteins. Aside from an MSA, the server also outputs topological prediction of TMPs using the HMMTOP algorithm. Previous benchmarking of the method has shown this approach outperforms the most accurate alignment methods such as MSAProbs, Kalign, PROMALS, MAFFT, ProbCons and PRALINE™. The web server is available at http://tcoffee.crg.cat/tmcoffee. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Location of the β4 transmembrane helices in the BK potassium channel

PubMed Central

Wu, Roland S.; Chudasama, Neelesh; Zakharov, Sergey I.; Doshi, Darshan; Motoike, Howard; Liu, Guoxia; Yao, Yongneng; Niu, Xiaowei; Deng, Shi-Xian; Landry, Donald W.; Karlin, Arthur; Marx, Steven O.

2009-01-01

Large-conductance, voltage- and Ca2+-gated potassium (BK) channels control excitability in a number of cell types. BK channels are composed of α subunits, which contain the voltage-sensor domains and the Ca2+- sensor domains, and form the pore, and often one of four types of β subunits, which modulate the channel in a cell-specific manner. β4 is expressed in neurons throughout the brain. Deletion of β4 in mice causes temporal lobe epilepsy. Compared to channels composed of α alone, channels composed of α and β4 activate and deactivate more slowly. We inferred the locations of the two β4 transmembrane (TM) helices, TM1 and TM2, relative to the seven αTM helices, S0-S6, from the extent of disulfide bond formation between cysteines substituted in the extracellular flanks of these TM helices. We found that β4 TM2 is close to α S0 and that β4 TM1 is close to both α S1 and S2. At least at their extracellular ends, TM1 and TM2 are not close to S3 through S6. In six of eight of the most highly crosslinked cysteine pairs, four crosslinks from TM2 to S0 and one each from TM1 to S1 and S2 had small effects on the V50 and on the rates of activation and deactivation. That disulfide crosslinking caused only small functional perturbations is consistent with the proximity of the extracellular ends of TM2 to S0 and of TM1 to S1 and to S2, in both the open and closed states. PMID:19571123

All-Atom Structural Models of the Transmembrane Domains of Insulin and Type 1 Insulin-Like Growth Factor Receptors

PubMed Central

Mohammadiarani, Hossein; Vashisth, Harish

2016-01-01

The receptor tyrosine kinase superfamily comprises many cell-surface receptors including the insulin receptor (IR) and type 1 insulin-like growth factor receptor (IGF1R) that are constitutively homodimeric transmembrane glycoproteins. Therefore, these receptors require ligand-triggered domain rearrangements rather than receptor dimerization for activation. Specifically, binding of peptide ligands to receptor ectodomains transduces signals across the transmembrane domains for trans-autophosphorylation in cytoplasmic kinase domains. The molecular details of these processes are poorly understood in part due to the absence of structures of full-length receptors. Using MD simulations and enhanced conformational sampling algorithms, we present all-atom structural models of peptides containing 51 residues from the transmembrane and juxtamembrane regions of IR and IGF1R. In our models, the transmembrane regions of both receptors adopt helical conformations with kinks at Pro961 (IR) and Pro941 (IGF1R), but the C-terminal residues corresponding to the juxtamembrane region of each receptor adopt unfolded and flexible conformations in IR as opposed to a helix in IGF1R. We also observe that the N-terminal residues in IR form a kinked-helix sitting at the membrane–solvent interface, while homologous residues in IGF1R are unfolded and flexible. These conformational differences result in a larger tilt-angle of the membrane-embedded helix in IGF1R in comparison to IR to compensate for interactions with water molecules at the membrane–solvent interfaces. Our metastable/stable states for the transmembrane domain of IR, observed in a lipid bilayer, are consistent with a known NMR structure of this domain determined in detergent micelles, and similar states in IGF1R are consistent with a previously reported model of the dimerized transmembrane domains of IGF1R. Our all-atom structural models suggest potentially unique structural organization of kinase domains in each receptor. PMID

The transmembrane domain of the p75 neurotrophin receptor stimulates phosphorylation of the TrkB tyrosine kinase receptor.

PubMed

Saadipour, Khalil; MacLean, Michael; Pirkle, Sean; Ali, Solav; Lopez-Redondo, Maria-Luisa; Stokes, David L; Chao, Moses V

2017-10-06

The function of protein products generated from intramembraneous cleavage by the γ-secretase complex is not well defined. The γ-secretase complex is responsible for the cleavage of several transmembrane proteins, most notably the amyloid precursor protein that results in Aβ, a transmembrane (TM) peptide. Another protein that undergoes very similar γ-secretase cleavage is the p75 neurotrophin receptor. However, the fate of the cleaved p75 TM domain is unknown. p75 neurotrophin receptor is highly expressed during early neuronal development and regulates survival and process formation of neurons. Here, we report that the p75 TM can stimulate the phosphorylation of TrkB (tyrosine kinase receptor B). In vitro phosphorylation experiments indicated that a peptide representing p75 TM increases TrkB phosphorylation in a dose- and time-dependent manner. Moreover, mutagenesis analyses revealed that a valine residue at position 264 in the rat p75 neurotrophin receptor is necessary for the ability of p75 TM to induce TrkB phosphorylation. Because this residue is just before the γ-secretase cleavage site, we then investigated whether the p75(αγ) peptide, which is a product of both α- and γ-cleavage events, could also induce TrkB phosphorylation. Experiments using TM domains from other receptors, EGFR and FGFR1, failed to stimulate TrkB phosphorylation. Co-immunoprecipitation and biochemical fractionation data suggested that p75 TM stimulates TrkB phosphorylation at the cell membrane. Altogether, our results suggest that TrkB activation by p75(αγ) peptide may be enhanced in situations where the levels of the p75 receptor are increased, such as during brain injury, Alzheimer's disease, and epilepsy. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Structure of the human smoothened receptor 7TM bound to an antitumor agent

PubMed Central

Wang, Chong; Wu, Huixian; Katritch, Vsevolod; Han, Gye Won; Huang, Xi-Ping; Liu, Wei; Siu, Fai Yiu; Roth, Bryan L.; Cherezov, Vadim; Stevens, Raymond C.

2013-01-01

The smoothened (SMO) receptor, a key signal transducer in the Hedgehog (Hh) signaling pathway is both responsible for the maintenance of normal embryonic development and implicated in carcinogenesis. The SMO receptor is classified as a class Frizzled (class F) G protein-coupled receptor (GPCR), although the canonical Hh signaling pathway involves the transcription factor Gli and the sequence similarity with class A GPCRs is less than 10%. Here we report the crystal structure at 2.5 Å resolution of the transmembrane domain of the human SMO receptor bound to the small molecule antagonist LY2940680. Although the SMO receptor shares the seven transmembrane helical (7TM) fold, most conserved motifs for class A GPCRs are absent, and the structure reveals an unusually complex arrangement of long extracellular loops stabilized by four disulfide bonds. The ligand binds at the extracellular end of the 7TM bundle and forms extensive contacts with the loops. PMID:23636324

'2TM proteins': an antigenically diverse superfamily with variable functions and export pathways.

PubMed

Kaur, Jasweer; Hora, Rachna

2018-01-01

Malaria is a disease that affects millions of people annually. An intracellular habitat and lack of protein synthesizing machinery in erythrocytes pose numerous difficulties for survival of the human pathogen Plasmodium falciparum . The parasite refurbishes the infected red blood cell (iRBC) by synthesis and export of several proteins in an attempt to suffice its metabolic needs and evade the host immune response. Immune evasion is largely mediated by surface display of highly polymorphic protein families known as variable surface antigens. These include the two trans-membrane (2TM) superfamily constituted by multicopy repetitive interspersed family (RIFINs), subtelomeric variable open reading frame (STEVORs) and Plasmodium falciparum Maurer's cleft two trans-membrane proteins present only in P. falciparum and some simian infecting Plasmodium species. Their hypervariable region flanked by 2TM domains exposed on the iRBC surface is believed to generate antigenic diversity. Though historically named "2TM superfamily," several A-type RIFINs and some STEVORs assume one trans-membrane topology. RIFINs and STEVORs share varied functions in different parasite life cycle stages like rosetting, alteration of iRBC rigidity and immune evasion. Additionally, a member of the STEVOR family has been implicated in merozoite invasion. Differential expression of these families in laboratory strains and clinical isolates propose them to be important for host cell survival and defense. The role of RIFINs in modulation of host immune response and presence of protective antibodies against these surface exposed molecules in patient sera highlights them as attractive targets of antimalarial therapies and vaccines. 2TM proteins are Plasmodium export elements positive, and several of these are exported to the infected erythrocyte surface after exiting through the classical secretory pathway within parasites. Cleaved and modified proteins are trafficked after packaging in vesicles to reach

Effects of Hydrophilic Residues and Hydrophobic Length on Flip-Flop Promotion by Transmembrane Peptides.

PubMed

Nakao, Hiroyuki; Hayashi, Chihiro; Ikeda, Keisuke; Saito, Hiroaki; Nagao, Hidemi; Nakano, Minoru

2018-04-19

Peptide-induced phospholipid flip-flop (scrambling) was evaluated using transmembrane model peptides in which the central residue was substituted with various amino acid residues (sequence: Ac-GKK(LA) n XW(LA) n LKKA-CONH 2 ). Peptides with a strongly hydrophilic residue (X = Q, N, or H) had higher scramblase activity than that of other peptides, and the activity was also dependent on the length of the peptides. Peptides with a hydrophobic stretch of 17 residues showed high flip-promotion propensity, whereas those of 21 and 25 residues did not, suggesting that membrane thinning under negative mismatch conditions promotes the flipping. Interestingly, a hydrophobic stretch of 19 residues intensively promoted phospholipid scrambling and membrane leakage. The distinctive characteristics of the peptide were ascribed by long-term molecular dynamics simulation to the arrangement of central glutamine and terminal four lysine residues on the same side of the helix. The combination of simulated and experimental data enables understanding of the mechanisms by which transmembrane helices, and ultimately unidentified scramblases in biomembranes, cause lipid scrambling.

A Sidekick for Membrane Simulations: Automated Ensemble Molecular Dynamics Simulations of Transmembrane Helices

PubMed Central

Hall, Benjamin A; Halim, Khairul Abd; Buyan, Amanda; Emmanouil, Beatrice; Sansom, Mark S P

2016-01-01

The interactions of transmembrane (TM) α-helices with the phospholipid membrane and with one another are central to understanding the structure and stability of integral membrane proteins. These interactions may be analysed via coarse-grained molecular dynamics (CGMD) simulations. To obtain statistically meaningful analysis of TM helix interactions, large (N ca. 100) ensembles of CGMD simulations are needed. To facilitate the running and analysis of such ensembles of simulations we have developed Sidekick, an automated pipeline software for performing high throughput CGMD simulations of α-helical peptides in lipid bilayer membranes. Through an end-to-end approach, which takes as input a helix sequence and outputs analytical metrics derived from CGMD simulations, we are able to predict the orientation and likelihood of insertion into a lipid bilayer of a given helix of family of helix sequences. We illustrate this software via analysis of insertion into a membrane of short hydrophobic TM helices containing a single cationic arginine residue positioned at different positions along the length of the helix. From analysis of these ensembles of simulations we estimate apparent energy barriers to insertion which are comparable to experimentally determined values. In a second application we use CGMD simulations to examine self-assembly of dimers of TM helices from the ErbB1 receptor tyrosine kinase, and analyse the numbers of simulation repeats necessary to obtain convergence of simple descriptors of the mode of packing of the two helices within a dimer. Our approach offers proof-of-principle platform for the further employment of automation in large ensemble CGMD simulations of membrane proteins. PMID:26580541

Sidekick for Membrane Simulations: Automated Ensemble Molecular Dynamics Simulations of Transmembrane Helices.

PubMed

Hall, Benjamin A; Halim, Khairul Bariyyah Abd; Buyan, Amanda; Emmanouil, Beatrice; Sansom, Mark S P

2014-05-13

The interactions of transmembrane (TM) α-helices with the phospholipid membrane and with one another are central to understanding the structure and stability of integral membrane proteins. These interactions may be analyzed via coarse grained molecular dynamics (CGMD) simulations. To obtain statistically meaningful analysis of TM helix interactions, large (N ca. 100) ensembles of CGMD simulations are needed. To facilitate the running and analysis of such ensembles of simulations, we have developed Sidekick, an automated pipeline software for performing high throughput CGMD simulations of α-helical peptides in lipid bilayer membranes. Through an end-to-end approach, which takes as input a helix sequence and outputs analytical metrics derived from CGMD simulations, we are able to predict the orientation and likelihood of insertion into a lipid bilayer of a given helix of a family of helix sequences. We illustrate this software via analyses of insertion into a membrane of short hydrophobic TM helices containing a single cationic arginine residue positioned at different positions along the length of the helix. From analyses of these ensembles of simulations, we estimate apparent energy barriers to insertion which are comparable to experimentally determined values. In a second application, we use CGMD simulations to examine the self-assembly of dimers of TM helices from the ErbB1 receptor tyrosine kinase and analyze the numbers of simulation repeats necessary to obtain convergence of simple descriptors of the mode of packing of the two helices within a dimer. Our approach offers a proof-of-principle platform for the further employment of automation in large ensemble CGMD simulations of membrane proteins.

Evolution of the α-Subunit of Na/K-ATPase from Paramecium to Homo sapiens: Invariance of Transmembrane Helix Topology.

PubMed

Morrill, Gene A; Kostellow, Adele B; Liu, Lijun; Gupta, Raj K; Askari, Amir

2016-05-01

Na/K-ATPase is a key plasma membrane enzyme involved in cell signaling, volume regulation, and maintenance of electrochemical gradients. The α-subunit, central to these functions, belongs to a large family of P-type ATPases. Differences in transmembrane (TM) helix topology, sequence homology, helix-helix contacts, cell signaling, and protein domains of Na/K-ATPase α-subunit were compared in fungi (Beauveria), unicellular organisms (Paramecia), primitive multicellular organisms (Hydra), and vertebrates (Xenopus, Homo sapiens), and correlated with evolution of physiological functions in the α-subunit. All α-subunits are of similar length, with groupings of four and six helices in the N- and C-terminal regions, respectively. Minimal homology was seen for protein domain patterns in Paramecium and Hydra, with high correlation between Hydra and vertebrates. Paramecium α-subunits display extensive disorder, with minimal helix contacts. Increases in helix contacts in Hydra approached vertebrates. Protein motifs known to be associated with membrane lipid rafts and cell signaling reveal significant positional shifts between Paramecium and Hydra vulgaris, indicating that regional membrane fluidity changes occur during evolution. Putative steroid binding sites overlapping TM-3 occurred in all species. Sites associated with G-protein-receptor stimulation occur both in vertebrates and amphibia but not in Hydra or Paramecia. The C-terminus moiety "KETYY," necessary for the Na(+) activation of pump phosphorylation, is not present in unicellular species indicating the absence of classical Na(+)/K(+)-pumps. The basic protein topology evolved earliest, followed by increases in protein domains and ordered helical arrays, correlated with appearance of α-subunit regions known to involve cell signaling, membrane recycling, and ion channel formation.

The EspF N-Terminal of Enterohemorrhagic Escherichia coli O157:H7 EDL933w Imparts Stronger Toxicity Effects on HT-29 Cells than the C-Terminal.

PubMed

Wang, Xiangyu; Du, Yanli; Hua, Ying; Fu, Muqing; Niu, Cong; Zhang, Bao; Zhao, Wei; Zhang, Qiwei; Wan, Chengsong

2017-01-01

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 EspF is an important multifunctional protein that destroys the tight junctions of intestinal epithelial cells and promotes host cell apoptosis. However, its molecular mechanism remains elusive. We knocked out the espF sequence (747 bp, Δ espF ), N-terminal sequence (219 bp, Δ espF N ), and C-terminal sequence (528 bp, Δ espF C ) separately using the pKD46-mediated λ Red homologous recombination system. Then, we built the corresponding complementation strains, namely, Δ espF/pespF , Δ espF N /pespF N , and Δ espF C /pespF C by overlap PCR, which were used in infecting HT-29 cells and BALB/C mice. The level of reactive oxygen species, cell apoptosis, mitochondrial trans-membrane potential, inflammatory factors, transepithelial electrical resistance (TER), and animal mortality were evaluated by DCFH-DA, double staining of Annexin V-FITC/PI, JC-1 staining, ELISA kit, and a mouse assay. The wild-type (WT), Δ espF , Δ espF/pespF , Δ espF C , Δ espF C /pespF C , Δ espF N , and Δ espF N /pespF N groups exhibited apoptotic rates of 68.3, 27.9, 64.9, 65.7, 73.4, 41.3, and 35.3% respectively, and mean TNF-α expression levels of 428 pg/mL, 342, 466, 446, 381, 383, and 374 pg/mL, respectively. In addition, the apoptotic rates and TNF-α levels of the WT, Δ espF/pespF , and Δ espF C were significantly higher than that of Δ espF , Δ espF N , Δ espF C /pespF C , and Δ espF N /pespF N group ( p < 0.05). The N-terminal of EspF resulted in an increase in the number of apoptotic cells, TNF-α secretion, ROS generation, mitochondria apoptosis, and pathogenicity in BalB/c mice. In conclusion, the N-terminal domain of the Enterohemorrhagic E. coli O157:H7 EspF more strongly promotes apoptosis and inflammation than the C-terminal domain.

Trimeric transmembrane domain interactions in paramyxovirus fusion proteins: roles in protein folding, stability, and function.

PubMed

Smith, Everett Clinton; Smith, Stacy E; Carter, James R; Webb, Stacy R; Gibson, Kathleen M; Hellman, Lance M; Fried, Michael G; Dutch, Rebecca Ellis

2013-12-13

Paramyxovirus fusion (F) proteins promote membrane fusion between the viral envelope and host cell membranes, a critical early step in viral infection. Although mutational analyses have indicated that transmembrane (TM) domain residues can affect folding or function of viral fusion proteins, direct analysis of TM-TM interactions has proved challenging. To directly assess TM interactions, the oligomeric state of purified chimeric proteins containing the Staphylococcal nuclease (SN) protein linked to the TM segments from three paramyxovirus F proteins was analyzed by sedimentation equilibrium analysis in detergent and buffer conditions that allowed density matching. A monomer-trimer equilibrium best fit was found for all three SN-TM constructs tested, and similar fits were obtained with peptides corresponding to just the TM region of two different paramyxovirus F proteins. These findings demonstrate for the first time that class I viral fusion protein TM domains can self-associate as trimeric complexes in the absence of the rest of the protein. Glycine residues have been implicated in TM helix interactions, so the effect of mutations at Hendra F Gly-508 was assessed in the context of the whole F protein. Mutations G508I or G508L resulted in decreased cell surface expression of the fusogenic form, consistent with decreased stability of the prefusion form of the protein. Sedimentation equilibrium analysis of TM domains containing these mutations gave higher relative association constants, suggesting altered TM-TM interactions. Overall, these results suggest that trimeric TM interactions are important driving forces for protein folding, stability and membrane fusion promotion.

A Novel Topology of Proline-rich Transmembrane Protein 2 (PRRT2)

PubMed Central

Rossi, Pia; Sterlini, Bruno; Castroflorio, Enrico; Marte, Antonella; Onofri, Franco; Valtorta, Flavia; Maragliano, Luca; Corradi, Anna; Benfenati, Fabio

2016-01-01

Proline-rich transmembrane protein 2 (PRRT2) has been identified as the single causative gene for a group of paroxysmal syndromes of infancy, including epilepsy, paroxysmal movement disorders, and migraine. On the basis of topology predictions, PRRT2 has been assigned to the recently characterized family of Dispanins, whose members share the two-transmembrane domain topology with a large N terminus and short C terminus oriented toward the outside of the cell. Because PRRT2 plays a role at the synapse, it is important to confirm the exact orientation of its N and C termini with respect to the plasma membrane to get clues regarding its possible function. Using a combination of different experimental approaches, including live immunolabeling, immunogold electron microscopy, surface biotinylation and computational modeling, we demonstrate a novel topology for this protein. PRRT2 is a type II transmembrane protein in which only the second hydrophobic segment spans the plasma membrane, whereas the first one is associated with the internal surface of the membrane and forms a helix-loop-helix structure without crossing it. Most importantly, the large proline-rich N-terminal domain is not exposed to the extracellular space but is localized intracellularly, and only the short C terminus is extracellular (Ncyt/Cexo topology). Accordingly, we show that PRRT2 interacts with the Src homology 3 domain-bearing protein Intersectin 1, an intracellular protein involved in synaptic vesicle cycling. These findings will contribute to the clarification of the role of PRRT2 at the synapse and the understanding of pathogenic mechanisms on the basis of PRRT2-related neurological disorders. PMID:26797119

Hendra virus fusion protein transmembrane domain contributes to pre-fusion protein stability

PubMed Central

Webb, Stacy; Nagy, Tamas; Moseley, Hunter; Fried, Michael; Dutch, Rebecca

2017-01-01

Enveloped viruses utilize fusion (F) proteins studding the surface of the virus to facilitate membrane fusion with a target cell membrane. Fusion of the viral envelope with a cellular membrane is required for release of viral genomic material, so the virus can ultimately reproduce and spread. To drive fusion, the F protein undergoes an irreversible conformational change, transitioning from a metastable pre-fusion conformation to a more thermodynamically stable post-fusion structure. Understanding the elements that control stability of the pre-fusion state and triggering to the post-fusion conformation is important for understanding F protein function. Mutations in F protein transmembrane (TM) domains implicated the TM domain in the fusion process, but the structural and molecular details in fusion remain unclear. Previously, analytical ultracentrifugation was utilized to demonstrate that isolated TM domains of Hendra virus F protein associate in a monomer-trimer equilibrium (Smith, E. C., Smith, S. E., Carter, J. R., Webb, S. R., Gibson, K. M., Hellman, L. M., Fried, M. G., and Dutch, R. E. (2013) J. Biol. Chem. 288, 35726–35735). To determine factors driving this association, 140 paramyxovirus F protein TM domain sequences were analyzed. A heptad repeat of β-branched residues was found, and analysis of the Hendra virus F TM domain revealed a heptad repeat leucine-isoleucine zipper motif (LIZ). Replacement of the LIZ with alanine resulted in dramatically reduced TM-TM association. Mutation of the LIZ in the whole protein resulted in decreased protein stability, including pre-fusion conformation stability. Together, our data suggest that the heptad repeat LIZ contributed to TM-TM association and is important for F protein function and pre-fusion stability. PMID:28213515

Cooperative folding of a polytopic α-helical membrane protein involves a compact N-terminal nucleus and nonnative loops

PubMed Central

Paslawski, Wojciech; Lillelund, Ove K.; Kristensen, Julie Veje; Schafer, Nicholas P.; Baker, Rosanna P.; Urban, Sinisa; Otzen, Daniel E.

2015-01-01

Despite the ubiquity of helical membrane proteins in nature and their pharmacological importance, the mechanisms guiding their folding remain unclear. We performed kinetic folding and unfolding experiments on 69 mutants (engineered every 2–3 residues throughout the 178-residue transmembrane domain) of GlpG, a membrane-embedded rhomboid protease from Escherichia coli. The only clustering of significantly positive ϕ-values occurs at the cytosolic termini of transmembrane helices 1 and 2, which we identify as a compact nucleus. The three loops flanking these helices show a preponderance of negative ϕ-values, which are sometimes taken to be indicative of nonnative interactions in the transition state. Mutations in transmembrane helices 3–6 yielded predominantly ϕ-values near zero, indicating that this part of the protein has denatured-state–level structure in the transition state. We propose that loops 1–3 undergo conformational rearrangements to position the folding nucleus correctly, which then drives folding of the rest of the domain. A compact N-terminal nucleus is consistent with the vectorial nature of cotranslational membrane insertion found in vivo. The origin of the interactions in the transition state that lead to a large number of negative ϕ-values remains to be elucidated. PMID:26056273

The mitochondrial intermembrane loop region of rat carnitine palmitoyltransferase 1A is a major determinant of its malonyl-CoA sensitivity.

PubMed

Borthwick, Karen; Jackson, Vicky N; Price, Nigel T; Zammit, Victor A

2006-11-03

Carnitine palmitoyltransferase (CPT) 1A adopts a polytopic conformation within the mitochondrial outer membrane, having both the N- and C-terminal segments on the cytosolic aspect of the membrane and a loop region connecting the two transmembrane (TM) segments protruding into the inter membrane space. In this study we demonstrate that the loop exerts major effects on the sensitivity of the enzyme to its inhibitor, malonyl-CoA. Insertion of a 16-residue spacer between the C-terminal part of the loop sequence (i.e. between residues 100 and 101) and TM2 (which is predicted to start at residue 102) increased the sensitivity to malonyl-CoA inhibition of the resultant mutant protein by more than 10-fold. By contrast, the same insertion made between TM1 and the loop had no effects on the kinetic properties of the enzyme, indicating that effects on the catalytic C-terminal segment were specifically induced by loop-TM2 interactions. Enhanced sensitivity was also observed in all mutants in which the native TM2-loop pairing was disrupted either by making chimeras in which the loops and TM2 segments of CPT 1A and CPT 1B were exchanged or by deleting successive 9-residue segments from the loop sequence. The data suggest that the sequence spanning the loop-TM2 boundary determines the disposition of this TM in the membrane so as to alter the conformation of the C-terminal segment and thus affect its interaction with malonyl-CoA.

[Research advances in CKLF-like MARVEL transmembrane domain containing member 5].

PubMed

Yuan, Ye-qing; Xiao, Yun-bei; Liu, Zhen-hua; Zhang, Xiao-wei; Xu, Tao; Wang, Xiao-feng

2012-12-01

CKLF-like MARVEL transmembrane domain containing member(CMTM)is a novel generic family firstly reported by Peking University Center for Human Disease Genomics. CMTM5 belongs to this family and has exhibited tumor-inhibiting activities. It can encode proteins approaching to the transmembrane 4 superfamily(TM4SF). CMTM5 is broadly expressed in normal adult and fetal human tissues, but is undetectable or down-regulated in most carcinoma cell lines and tissues. Restoration of CMTM5 may inhibit the proliferation, migration, and invasion of carcinoma cells. Although the exact mechanism of its anti-tumor activity remains unclear, CMTM5 may be involved in various signaling pathways governing the occurrence and development of tumors. CMTM5 may be a new target in the gene therapies for tumors, while further studies on CMTM5 and its anti-tumor mechanisms are warranted.

Human lysozyme possesses novel antimicrobial peptides within its N-terminal domain that target bacterial respiration.

PubMed

Ibrahim, Hisham R; Imazato, Kenta; Ono, Hajime

2011-09-28

Human milk lysozyme is thought to be a key defense factor in protecting the gastrointestinal tract of newborns against bacterial infection. Recently, evidence was found that pepsin, under conditions relevant to the newborn stomach, cleaves chicken lysozyme (cLZ) at specific loops to generate five antimicrobial peptide motifs. This study explores the antimicrobial role of the corresponding peptides of human lysozyme (hLZ), the actual protein in breast milk. Five peptide motifs of hLZ, one helix-loop-helix (HLH), its two helices (H1 and H2), and two helix-sheet motifs, H2-β-strands 1-2 (H2-S12) or H2-β-strands 1-3 (H2-S13), were synthesized and examined for antimicrobial action. The five peptides of hLZ exhibit microbicidal activity to various degrees against several bacterial strains. The HLH peptide and its N-terminal helix (H1) were significantly the most potent bactericidal to Gram-positive and Gram-negative bacteria and the fungus Candida albicans . Outer and inner membrane permeabilization studies, as well as measurements of transmembrane electrochemical potentials, provided evidence that HLH peptide and its N-terminal helix (H1) kill bacteria by crossing the outer membrane of Gram-negative bacteria via self-promoted uptake and are able to dissipate the membrane potential-dependent respiration of Gram-positive bacteria. This finding is the first to describe that hLZ possesses multiple antimicrobial peptide motifs within its N-terminal domain, providing insight into new classes of antibiotic peptides with potential use in the treatment of infectious diseases.

Glycines from the APP GXXXG/GXXXA Transmembrane Motifs Promote Formation of Pathogenic Aβ Oligomers in Cells

PubMed Central

Decock, Marie; Stanga, Serena; Octave, Jean-Noël; Dewachter, Ilse; Smith, Steven O.; Constantinescu, Stefan N.; Kienlen-Campard, Pascal

2016-01-01

Alzheimer’s disease (AD) is the most common neurodegenerative disorder characterized by progressive cognitive decline leading to dementia. The amyloid precursor protein (APP) is a ubiquitous type I transmembrane (TM) protein sequentially processed to generate the β-amyloid peptide (Aβ), the major constituent of senile plaques that are typical AD lesions. There is a growing body of evidence that soluble Aβ oligomers correlate with clinical symptoms associated with the disease. The Aβ sequence begins in the extracellular juxtamembrane region of APP and includes roughly half of the TM domain. This region contains GXXXG and GXXXA motifs, which are critical for both TM protein interactions and fibrillogenic properties of peptides derived from TM α-helices. Glycine-to-leucine mutations of these motifs were previously shown to affect APP processing and Aβ production in cells. However, the detailed contribution of these motifs to APP dimerization, their relation to processing, and the conformational changes they can induce within Aβ species remains undefined. Here, we describe highly resistant Aβ42 oligomers that are produced in cellular membrane compartments. They are formed in cells by processing of the APP amyloidogenic C-terminal fragment (C99), or by direct expression of a peptide corresponding to Aβ42, but not to Aβ40. By a point-mutation approach, we demonstrate that glycine-to-leucine mutations in the G29XXXG33 and G38XXXA42 motifs dramatically affect the Aβ oligomerization process. G33 and G38 in these motifs are specifically involved in Aβ oligomerization; the G33L mutation strongly promotes oligomerization, while G38L blocks it with a dominant effect on G33 residue modification. Finally, we report that the secreted Aβ42 oligomers display pathological properties consistent with their suggested role in AD, but do not induce toxicity in survival assays with neuronal cells. Exposure of neurons to these Aβ42 oligomers dramatically affects neuronal

Hendra virus fusion protein transmembrane domain contributes to pre-fusion protein stability.

PubMed

Webb, Stacy; Nagy, Tamas; Moseley, Hunter; Fried, Michael; Dutch, Rebecca

2017-04-07

Enveloped viruses utilize fusion (F) proteins studding the surface of the virus to facilitate membrane fusion with a target cell membrane. Fusion of the viral envelope with a cellular membrane is required for release of viral genomic material, so the virus can ultimately reproduce and spread. To drive fusion, the F protein undergoes an irreversible conformational change, transitioning from a metastable pre-fusion conformation to a more thermodynamically stable post-fusion structure. Understanding the elements that control stability of the pre-fusion state and triggering to the post-fusion conformation is important for understanding F protein function. Mutations in F protein transmembrane (TM) domains implicated the TM domain in the fusion process, but the structural and molecular details in fusion remain unclear. Previously, analytical ultracentrifugation was utilized to demonstrate that isolated TM domains of Hendra virus F protein associate in a monomer-trimer equilibrium (Smith, E. C., Smith, S. E., Carter, J. R., Webb, S. R., Gibson, K. M., Hellman, L. M., Fried, M. G., and Dutch, R. E. (2013) J. Biol. Chem. 288, 35726-35735). To determine factors driving this association, 140 paramyxovirus F protein TM domain sequences were analyzed. A heptad repeat of β-branched residues was found, and analysis of the Hendra virus F TM domain revealed a heptad repeat leucine-isoleucine zipper motif (LIZ). Replacement of the LIZ with alanine resulted in dramatically reduced TM-TM association. Mutation of the LIZ in the whole protein resulted in decreased protein stability, including pre-fusion conformation stability. Together, our data suggest that the heptad repeat LIZ contributed to TM-TM association and is important for F protein function and pre-fusion stability. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The N- and C-terminal carbohydrate recognition domains of Haemonchus contortus galectin bind to distinct receptors of goat PBMC and contribute differently to its immunomodulatory functions in host-parasite interactions.

PubMed

Lu, MingMin; Tian, XiaoWei; Yang, XinChao; Yuan, Cheng; Ehsan, Muhammad; Liu, XinChao; Yan, RuoFeng; Xu, LiXin; Song, XiaoKai; Li, XiangRui

2017-09-05

Hco-gal-m is a tandem-repeat galectin isolated from the adult worm of Haemonchus contortus. A growing body of studies have demonstrated that Hco-gal-m could exert its immunomodulatory effects on host peripheral blood mononuclear cells (PBMC) to facilitate the immune evasion. Our previous work revealed that C-terminal and N-terminal carbohydrate recognition domains (CRD) of Hco-gal-m had different sugar binding abilities. However, whether different domains of Hco-gal-m account differently for its multiple immunomodulatory functions in the host-parasite interaction remains to be elucidated. We found that the N-terminal CRD of Hco-gal-m (MNh) and the C-terminal CRD (MCh) could bind to goat peripheral blood mononuclear cells by distinct receptors: transmembrane protein 63A (TMEM63A) was a binding receptor of MNh, while transmembrane protein 147 (TMEM147) was a binding receptor of MCh. In addition, MCh was much more potent than MNh in inhibiting cell proliferation and inducing apoptosis, while MNh was much more effective in inhibiting NO production. Moreover, MNh could suppress the transcription of interferon-γ (IFN-γ), but MCh not. Our data suggested that these two CRDs of Hco-gal-m bind to distinct receptors and contributed differently to its ability to downregulate host immune response. These results will improve our understanding of galectins from parasitic nematodes contributing to the mechanism of parasitic immune evasion and continue to illustrate the diverse range of biological activities attributable to the galectin family.

Transmembrane helices containing a charged arginine are thermodynamically stable.

PubMed

Ulmschneider, Martin B; Ulmschneider, Jakob P; Freites, J Alfredo; von Heijne, Gunnar; Tobias, Douglas J; White, Stephen H

2017-10-01

Hydrophobic amino acids are abundant in transmembrane (TM) helices of membrane proteins. Charged residues are sparse, apparently due to the unfavorable energetic cost of partitioning charges into nonpolar phases. Nevertheless, conserved arginine residues within TM helices regulate vital functions, such as ion channel voltage gating and integrin receptor inactivation. The energetic cost of arginine in various positions along hydrophobic helices has been controversial. Potential of mean force (PMF) calculations from atomistic molecular dynamics simulations predict very large energetic penalties, while in vitro experiments with Sec61 translocons indicate much smaller penalties, even for arginine in the center of hydrophobic TM helices. Resolution of this conflict has proved difficult, because the in vitro assay utilizes the complex Sec61 translocon, while the PMF calculations rely on the choice of simulation system and reaction coordinate. Here we present the results of computational and experimental studies that permit direct comparison with the Sec61 translocon results. We find that the Sec61 translocon mediates less efficient membrane insertion of Arg-containing TM helices compared with our computational and experimental bilayer-insertion results. In the simulations, a combination of arginine snorkeling, bilayer deformation, and peptide tilting is sufficient to lower the penalty of Arg insertion to an extent such that a hydrophobic TM helix with a central Arg residue readily inserts into a model membrane. Less favorable insertion by the translocon may be due to the decreased fluidity of the endoplasmic reticulum (ER) membrane compared with pure palmitoyloleoyl-phosphocholine (POPC). Nevertheless, our results provide an explanation for the differences between PMF- and experiment-based penalties for Arg burial.

TM4SF5-Mediated Roles in the Development of Fibrotic Phenotypes

PubMed Central

Ryu, Jihye

2017-01-01

Transmembrane 4 L six family member 5 (TM4SF5) can form tetraspanin-enriched microdomains (TERMs) on the cell's surface. TERMs contain protein-protein complexes comprised of tetraspanins, growth factor receptors, and integrins. These complexes regulate communication between extracellular and intracellular spaces to control diverse cellular functions. TM4SF5 influences the epithelial-mesenchymal transition (EMT), aberrant multilayer cellular growth, drug resistance, enhanced migration and invasion, circulation through the bloodstream, tumor-initiation property, metastasis, and muscle development in zebrafish. Here, current data on TM4SF5's roles in the development of fibrotic phenotypes are reviewed. TM4SF5 is induced by transforming growth factor β1 (TGFβ1) signaling via a collaboration with epidermal growth factor receptor (EGFR) activation. TM4SF5, by itself or in concert with other receptors, transduces signals intracellularly. In hepatocytes, TM4SF5 expression regulates cell cycle progression, migration, and expression of extracellular matrix components. In CCl4-treated mice, TM4SF5, α-smooth muscle actin (α-SMA), and collagen I expression are observed together along the fibrotic septa regions of the liver. These fibrotic phenotypes are diminished by anti-TM4SF5 reagents, such as a specific small compound [TSAHC, 4′-(p-toluenesulfonylamido)-4-hydroxychalcone] or a chimeric antibody. This review discusses the antifibrotic strategies that target TM4SF5 and its associated protein networks that regulate the intracellular signaling necessary for fibrotic functions of hepatocytes. PMID:28458469

Amino acid screening based on structural modeling identifies critical residues for the function, ion selectivity and structure of Arabidopsis MTP1.

PubMed

Kawachi, Miki; Kobae, Yoshihiro; Kogawa, Sayaka; Mimura, Tetsuro; Krämer, Ute; Maeshima, Masayoshi

2012-07-01

Arabidopsis thaliana MTP1 is a vacuolar membrane Zn(2+)/H(+) antiporter of the cation diffusion facilitator family. Here we present a structure-function analysis of AtMTP1-mediated transport and its remarkable Zn(2+) selectivity by functional complementation tests of more than 50 mutant variants in metal-sensitive yeast strains. This was combined with homology modeling of AtMTP1 based on the crystal structure of the Escherichia coli broad-specificity divalent cation transporter YiiP. The Zn(2+)-binding sites of EcYiiP in the cytoplasmic C-terminus, and the pore formed by transmembrane helices TM2 and TM5, are conserved in AtMTP1. Although absent in EcYiiP, Cys31 and Cys36 in the extended N-terminal cytosolic domain of AtMTP1 are necessary for complementation of a Zn-sensitive yeast strain. On the cytosolic side of the active Zn(2+)-binding site inside the transmembrane pore, Ala substitution of either Asn258 in TM5 or Ser101 in TM2 non-selectively enhanced the metal tolerance conferred by AtMTP1. Modeling predicts that these residues obstruct the movement of cytosolic Zn(2+) into the intra-membrane Zn(2+)-binding site of AtMTP1. A conformational change in the immediately preceding His-rich cytosolic loop may displace Asn258 and permit Zn(2+) entry into the pore. This would allow dynamic coupling of Zn(2+) transport to the His-rich loop, thus acting as selectivity filter or sensor of cytoplasmic Zn(2+) levels. Individual mutations at diverse sites within AtMTP1 conferred Co and Cd tolerance in yeast, and included deletions in N-terminal and His-rich intra-molecular cytosolic domains, and mutations of single residues flanking the transmembrane pore or participating in intra- or inter-molecular domain interactions, all of which are not conserved in the non-selective EcYiiP. © 2012 The Authors Journal compilation © 2012 FEBS.

Lipids and topological rules of membrane protein assembly: balance between long and short range lipid-protein interactions.

PubMed

Vitrac, Heidi; Bogdanov, Mikhail; Heacock, Phil; Dowhan, William

2011-04-29

The N-terminal six-transmembrane domain (TM) bundle of lactose permease of Escherichia coli is uniformly inverted when assembled in membranes lacking phosphatidylethanolamine (PE). Inversion is dependent on the net charge of cytoplasmically exposed protein domains containing positive and negative residues, net charge of the membrane surface, and low hydrophobicity of TM VII acting as a molecular hinge between the two halves of lactose permease (Bogdanov, M., Xie, J., Heacock, P., and Dowhan, W. (2008) J. Cell Biol. 182, 925-935). Net neutral lipids suppress the membrane translocation potential of negatively charged amino acids, thus increasing the cytoplasmic retention potential of positively charged amino acids. Herein, TM organization of sucrose permease (CscB) and phenylalanine permease (PheP) as a function of membrane lipid composition was investigated to extend these principles to other proteins. For CscB, topological dependence on PE only becomes evident after a significant increase in the net negative charge of the cytoplasmic surface of the N-terminal TM bundle. High negative charge is required to overcome the thermodynamic block to inversion due to the high hydrophobicity of TM VII. Increasing the positive charge of the cytoplasmic surface of the N-terminal TM hairpin of PheP, which is misoriented in PE-lacking cells, favors native orientation in the absence of PE. PheP and CscB also display co-existing dual topologies dependent on changes in the charge balance between protein domains and the membrane lipids. Therefore, the topology of both permeases is dependent on PE. However, CscB topology is governed by thermodynamic balance between opposing lipid-dependent electrostatic and hydrophobic interactions.

Transmembrane Domains of Highly Pathogenic Viral Fusion Proteins Exhibit Trimeric Association In Vitro

PubMed Central

Webb, Stacy R.; Smith, Stacy E.; Fried, Michael G.

2018-01-01

ABSTRACT Enveloped viruses require viral fusion proteins to promote fusion of the viral envelope with a target cell membrane. To drive fusion, these proteins undergo large conformational changes that must occur at the right place and at the right time. Understanding the elements which control the stability of the prefusion state and the initiation of conformational changes is key to understanding the function of these important proteins. The construction of mutations in the fusion protein transmembrane domains (TMDs) or the replacement of these domains with lipid anchors has implicated the TMD in the fusion process. However, the structural and molecular details of the role of the TMD in these fusion events remain unclear. Previously, we demonstrated that isolated paramyxovirus fusion protein TMDs associate in a monomer-trimer equilibrium, using sedimentation equilibrium analytical ultracentrifugation. Using a similar approach, the work presented here indicates that trimeric interactions also occur between the fusion protein TMDs of Ebola virus, influenza virus, severe acute respiratory syndrome coronavirus (SARS CoV), and rabies virus. Our results suggest that TM-TM interactions are important in the fusion protein function of diverse viral families. IMPORTANCE Many important human pathogens are enveloped viruses that utilize membrane-bound glycoproteins to mediate viral entry. Factors that contribute to the stability of these glycoproteins have been identified in the ectodomain of several viral fusion proteins, including residues within the soluble ectodomain. Although it is often thought to simply act as an anchor, the transmembrane domain of viral fusion proteins has been implicated in protein stability and function as well. Here, using a biophysical approach, we demonstrated that the fusion protein transmembrane domains of several deadly pathogens—Ebola virus, influenza virus, SARS CoV, and rabies virus—self-associate. This observation across various viral

TM6SF2 is a regulator of liver fat metabolism influencing triglyceride secretion and hepatic lipid droplet content

PubMed Central

Mahdessian, Hovsep; Taxiarchis, Apostolos; Popov, Sergej; Silveira, Angela; Franco-Cereceda, Anders; Hamsten, Anders; Eriksson, Per; van't Hooft, Ferdinand

2014-01-01

Genome-wide association studies have identified a locus on chromosome 19 associated with plasma triglyceride (TG) concentration and nonalcoholic fatty liver disease. However, the identity and functional role of the gene(s) responsible for these associations remain unknown. Of 19 expressed genes contained in this locus, none has previously been implicated in lipid metabolism. We performed gene expression studies and expression quantitative trait locus analysis in 206 human liver samples to identify the putative causal gene. Transmembrane 6 superfamily member 2 (TM6SF2), a gene with hitherto unknown function, expressed predominantly in liver and intestine, was identified as the putative causal gene. TM6SF2 encodes a protein of 351 amino acids with 7–10 predicted transmembrane domains. Otherwise, no other protein features were identified which could help to elucidate the function of TM6SF2. Protein subcellular localization studies with confocal microscopy demonstrated that TM6SF2 is localized in the endoplasmic reticulum and the ER-Golgi intermediate compartment of human liver cells. Functional studies for secretion of TG-rich lipoproteins (TRLs) and lipid droplet content were performed in human hepatoma Huh7 and HepG2 cells using confocal microscopy and siRNA inhibition and overexpression techniques. In agreement with the genome-wide association data, it was found that TM6SF2 siRNA inhibition was associated with reduced secretion of TRLs and increased cellular TG concentration and lipid droplet content, whereas TM6SF2 overexpression reduced liver cell steatosis. We conclude that TM6SF2 is a regulator of liver fat metabolism with opposing effects on the secretion of TRLs and hepatic lipid droplet content. PMID:24927523

Mouse Hepatitis Virus Strain A59 and Blocking Antireceptor Monoclonal Antibody Bind to the N-Terminal Domain of Cellular Receptor

NASA Astrophysics Data System (ADS)

Dveksler, Gabriela S.; Pensiero, Michael N.; Dieffenbach, Carl W.; Cardellichio, Christine B.; Basile, Alexis A.; Elia, Patrick E.; Holmes, Kathryn V.

1993-03-01

Mouse hepatitis virus (MHV) strain A59 uses as cellular receptors members of the carcinoembryonic antigen family in the immunoglobulin superfamily. Recombinant receptor proteins with deletions of whole or partial immunoglobulin domains were used to identify the regions of receptor glycoprotein recognized by virus and by antireceptor monoclonal antibody CC1, which blocks infection of murine cells. Monoclonal antibody CC1 and MHV-A59 virions bound only to recombinant proteins containing the entire first domain of MHV receptor. To determine which of the proteins could serve as functional virus receptors, receptor-negative hamster cells were transfected with recombinant deletion clones and then challenged with MHV-A59 virions. Receptor activity required the entire N-terminal domain with either the second or the fourth domain and the transmembrane and cytoplasmic domains. Recombinant proteins lacking the first domain or its C-terminal portion did not serve as viral receptors. Thus, like other virus receptors in the immunoglobulin superfamily, including CD4, poliovirus receptor, and intercellular adhesion molecule 1, the N-terminal domain of MHV receptor is recognized by the virus and the blocking monoclonal antibody.

Structure, Dynamics, and Allosteric Potential of Ionotropic Glutamate Receptor N-Terminal Domains

PubMed Central

Krieger, James; Bahar, Ivet; Greger, Ingo H.

2015-01-01

Ionotropic glutamate receptors (iGluRs) are tetrameric cation channels that mediate synaptic transmission and plasticity. They have a unique modular architecture with four domains: the intracellular C-terminal domain (CTD) that is involved in synaptic targeting, the transmembrane domain (TMD) that forms the ion channel, the membrane-proximal ligand-binding domain (LBD) that binds agonists such as L-glutamate, and the distal N-terminal domain (NTD), whose function is the least clear. The extracellular portion, comprised of the LBD and NTD, is loosely arranged, mediating complex allosteric regulation and providing a rich target for drug development. Here, we briefly review recent work on iGluR NTD structure and dynamics, and further explore the allosteric potential for the NTD in AMPA-type iGluRs using coarse-grained simulations. We also investigate mechanisms underlying the established NTD allostery in NMDA-type iGluRs, as well as the fold-related metabotropic glutamate and GABAB receptors. We show that the clamshell motions intrinsically favored by the NTD bilobate fold are coupled to dimeric and higher-order rearrangements that impact the iGluR LBD and ultimately the TMD. Finally, we explore the dynamics of intact iGluRs and describe how it might affect receptor operation in a synaptic environment. PMID:26255587

Dynamic and coordinated single-molecular interactions at TM4SF5-enriched microdomains guide invasive behaviors in 2- and 3-dimensional environments.

PubMed

Kim, Hye-Jin; Kwon, Sojung; Nam, Seo Hee; Jung, Jae Woo; Kang, Minkyung; Ryu, Jihye; Kim, Ji Eon; Cheong, Jin-Gyu; Cho, Chang Yun; Kim, Somi; Song, Dae-Geun; Kim, Yong-Nyun; Kim, Tai Young; Jung, Min-Kyo; Lee, Kyung-Min; Pack, Chan-Gi; Lee, Jung Weon

2017-04-01

Membrane proteins sense extracellular cues and transduce intracellular signaling to coordinate directionality and speed during cellular migration. They are often localized to specific regions, as with lipid rafts or tetraspanin-enriched microdomains; however, the dynamic interactions of tetraspanins with diverse receptors within tetraspanin-enriched microdomains on cellular surfaces remain largely unexplored. Here, we investigated effects of tetraspan(in) TM4SF5 (transmembrane 4 L6 family member 5)-enriched microdomains (T 5 ERMs) on the directionality of cell migration. Physical association of TM4SF5 with epidermal growth factor receptor (EGFR) and integrin α5 was visualized by live fluorescence cross-correlation spectroscopy and higher-resolution microscopy at the leading edge of migratory cells, presumably forming TM4SF5-enriched microdomains. Whereas TM4SF5 and EGFR colocalized at the migrating leading region more than at the rear, TM4SF5 and integrin α5 colocalized evenly throughout cells. Cholesterol depletion and disruption in TM4SF5 post-translational modifications, including N -glycosylation and palmitoylation, altered TM4SF5 interactions and cellular localization, which led to less cellular migration speed and directionality in 2- or 3-dimensional conditions. TM4SF5 controlled directional cell migration and invasion, and importantly, these TM4SF5 functions were dependent on cholesterol, TM4SF5 post-translational modifications, and EGFR and integrin α5 activity. Altogether, we showed that TM4SF5 dynamically interacted with EGFR and integrin α5 in migratory cells to control directionality and invasion.-Kim, H.-J., Kwon, S., Nam, S. H., Jung, J. W., Kang, M., Ryu, J., Kim, J. E., Cheong, J.-G., Cho, C. Y., Kim, S., Song, D.-G., Kim, Y.-N., Kim, T. Y., Jung, M.-K., Lee, K.-M., Pack, C.-G., Lee, J. W. Dynamic and coordinated single-molecular interactions at TM4SF5-enriched microdomains guide invasive behaviors in 2- and 3-dimensional environments. © FASEB.

A three amino acid deletion in the transmembrane domain of the nicotinic acetylcholine receptor α6 subunit confers high-level resistance to spinosad in Plutella xylostella

PubMed Central

Wang, Jing; Wang, Xingliang; Lansdell, Stuart J.; Zhang, Jianheng; Millar, Neil S.; Wu, Yidong

2016-01-01

Spinosad is a macrocyclic lactone insecticide that acts primarily at the nicotinic acetylcholine receptors (nAChRs) of target insects. Here we describe evidence that high levels of resistance to spinosad in the diamondback moth (Plutella xylostella) are associated with a three amino acid (3-aa) deletion in the fourth transmembrane domain (TM4) of the nAChR α6 subunit (Pxα6). Following laboratory selection with spinosad, the SZ-SpinR strain of P. xylostella exhibited 940-fold resistance to spinosad. In addition, the selected insect population had 1060-fold cross-resistance to spinetoram but, in contrast, no cross-resistance to abamectin was observed. Genetic analysis indicates that spinosad resistance in SZ-SpinR is inherited as a recessive and autosomal trait, and that the 3-aa deletion (IIA) in TM4 of Pxα6 is tightly linked to spinosad resistance. Because of well-established difficulties in functional expression of cloned insect nAChRs, the analogous resistance-associated deletion mutation was introduced into a prototype nAChR (the cloned human α7 subunit). Two-electrode voltage-clamp recording with wild-type and mutated nAChRs expressed in Xenopus laevis oocytes indicated that the mutation causes a complete loss of agonist activation. In addition, radioligand binding studies indicated that the 3-aa deletion resulted in significantly lower-affinity binding of the extracellular neurotransmitter-binding site. These findings are consistent with the 3-amino acid (IIA) deletion within the transmembrane domain of Pxα6 being responsible for target-site resistance to spinosad in the SZ-SpinR strain of P. xylostella. PMID:26855198

Magnetic properties of TM3[Cr(CN)6]2.n H2O

NASA Astrophysics Data System (ADS)

Zentková, M.; Mihalik, M.; Ková, J.; Zentko, A.; Mitróová, Z.; Lukáová, M.; Kaveanský, V.; Kiss, L. F.

2006-01-01

Magnetization measurements performed on Prussian blue analogs TM2+3[CrIII(CN)6]2.n H2O (TM = Cr, Mn, Fe, Co, Ni, Cu) confirmed the dual character of the exchange interaction (antiferromagnetic AFM and ferromagnetic FM) in this system. AFM interaction dominates for the Cr2+ sample and with rising atomic number Z the FM interaction becomes more important reaching pure FM character for the Cu2+ sample.

Functional role of the extracellular N-terminal domain of neuropeptide Y subfamily receptors in membrane integration and agonist-stimulated internalization.

PubMed

Lindner, Diana; Walther, Cornelia; Tennemann, Anja; Beck-Sickinger, Annette G

2009-01-01

The N terminus is the most variable element in G protein-coupled receptors (GPCRs), ranging from seven residues up to approximately 5900 residues. For family B and C GPCRs it is described that at least part of the ligand binding site is located within the N terminus. Here we investigated the role of the N terminus in the neuropeptide Y receptor family, which belongs to the class A of GPCRs. We cloned differentially truncated Y receptor mutants, in which the N terminus was partially or completely deleted. We found, that eight amino acids are sufficient for full ligand binding and signal transduction activity. Interestingly, we could show that no specific amino acids but rather the extension of the first transmembrane helix by any residues is sufficient for receptor activity but also for membrane integration in case of the hY(1) and the hY(4) receptors. In contrast, the complete deletion of the N terminus in the hY(2) receptors resulted in a mutant that is fully integrated in the membrane but does not bind the ligand very well and internalizes much slower compared to the wild type receptor. Interestingly, also these effects could be reverted by any N-terminal extension. Accordingly, the most important function of the N termini seems to be the stabilization of the first transmembrane helix to ensure the correct receptor structure, which obviously is essential for ligand binding, integration into the cell membrane and receptor internalization.

Stabilization of the μ-opioid receptor by truncated single transmembrane splice variants through a chaperone-like action.

PubMed

Xu, Jin; Xu, Ming; Brown, Taylor; Rossi, Grace C; Hurd, Yasmin L; Inturrisi, Charles E; Pasternak, Gavril W; Pan, Ying-Xian

2013-07-19

The μ-opioid receptor gene, OPRM1, undergoes extensive alternative pre-mRNA splicing, as illustrated by the identification of an array of splice variants generated by both 5' and 3' alternative splicing. The current study reports the identification of another set of splice variants conserved across species that are generated through exon skipping or insertion that encodes proteins containing only a single transmembrane (TM) domain. Using a Tet-Off system, we demonstrated that the truncated single TM variants can dimerize with the full-length 7-TM μ-opioid receptor (MOR-1) in the endoplasmic reticulum, leading to increased expression of MOR-1 at the protein level by a chaperone-like function that minimizes endoplasmic reticulum-associated degradation. In vivo antisense studies suggested that the single TM variants play an important role in morphine analgesia, presumably through modulation of receptor expression levels. Our studies suggest the functional roles of truncated receptors in other G protein-coupled receptor families.

Molecular modeling of biomembranes and their complexes with protein transmembrane α-helices

NASA Astrophysics Data System (ADS)

Kuznetsov, Andrey S.; Smirnov, Kirill V.; Antonov, Mikhail Yu.; Nikolaev, Ivan N.; Efremov, Roman G.

2017-11-01

Helical segments are common structural elements of membrane proteins. Dimerization and oligomerization of transmembrane (TM) α-helices provides the framework for spatial structure formation and protein-protein interactions. The membrane itself also takes part in the protein functioning. There are some examples of the mutual influence of the lipid bilayer properties and embedded membrane proteins. This work aims at the detail investigation of protein-lipid interactions using model systems: TM peptides corresponding to native protein segments. Three peptides were considered corresponding to TM domains of human glycophorin A (GpA), epidermal growth factor receptor (EGFR) and proposed TM-segment of human neuraminidase-1 (Neu1). A computational analysis of structural and dynamical properties was performed using molecular dynamics method. Monomers of peptides were considered incorporated into hydrated lipid bilayers. It was confirmed, that all these TM peptides have stable helical conformation in lipid environment, and the mutual adaptation of peptides and membrane was observed. It was shown that incorporation of the peptide into membrane results in the modulation of local and mean structural properties of the bilayer. Each peptide interacts with lipid acyl chains having special binding sites on the surface of central part of α-helix that exist for at least 200 ns. However, lipid acyl chains substitute each other faster occupying the same site. The formation of a special pattern of protein-lipid interactions may modulate the association of TM domains of membrane proteins, so membrane environment should be considered when proposing new substances targeting cell receptors.

Tryptophan Scanning Reveals Dense Packing of Connexin Transmembrane Domains in Gap Junction Channels Composed of Connexin32.

PubMed

Brennan, Matthew J; Karcz, Jennifer; Vaughn, Nicholas R; Woolwine-Cunningham, Yvonne; DePriest, Adam D; Escalona, Yerko; Perez-Acle, Tomas; Skerrett, I Martha

2015-07-10

Tryptophan was substituted for residues in all four transmembrane domains of connexin32. Function was assayed using dual cell two-electrode voltage clamp after expression in Xenopus oocytes. Tryptophan substitution was poorly tolerated in all domains, with the greatest impact in TM1 and TM4. For instance, in TM1, 15 substitutions were made, six abolished coupling and five others significantly reduced function. Only TM2 and TM3 included a distinct helical face that lacked sensitivity to tryptophan substitution. Results were visualized on a comparative model of Cx32 hemichannel. In this model, a region midway through the membrane appears highly sensitive to tryptophan substitution and includes residues Arg-32, Ile-33, Met-34, and Val-35. In the modeled channel, pore-facing regions of TM1 and TM2 were highly sensitive to tryptophan substitution, whereas the lipid-facing regions of TM3 and TM4 were variably tolerant. Residues facing a putative intracellular water pocket (the IC pocket) were also highly sensitive to tryptophan substitution. Although future studies will be required to separate trafficking-defective mutants from those that alter channel function, a subset of interactions important for voltage gating was identified. Interactions important for voltage gating occurred mainly in the mid-region of the channel and focused on TM1. To determine whether results could be extrapolated to other connexins, TM1 of Cx43 was scanned revealing similar but not identical sensitivity to TM1 of Cx32. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Cell Activation Mediated by Glycosylphosphatidylinositol-Anchored or Transmembrane Forms of CD14†

PubMed Central

Pugin, J.; Kravchenko, V. V.; Lee, J.-D.; Kline, L.; Ulevitch, R. J.; Tobias, P. S.

1998-01-01

CD14 is a glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein which functions as a receptor on myeloid cells for ligands derived from microbial pathogens such as lipopolysaccharide (LPS). We have studied the importance of the GPI tail of CD14 in signalling with the promonocytic cell line THP-1 expressing recombinant CD14 in a GPI-anchored form (THP1-wtCD14 cells) or in a transmembrane form (THP1-tmCD14). We found that, like other GPI-anchored molecules, GPI-anchored CD14 was recovered mainly from a Triton X-100-insoluble fraction, whereas transmembrane CD14 was fully soluble in Triton X-100. LPS induced cell activation of THP1-wtCD14 and of THP1-tmCD14 (protein tyrosine kinase phosphorylation, NF-κB activation, and cytokine production) in a very similar manner. However, anti-CD14 antibody-induced cross-linking caused a rapid calcium mobilization signal only in GPI-anchored CD14 cells. Studies with pharmacologic inhibitors of intracellular signalling events implicate phospholipase C and protein tyrosine kinases in the genesis of this antibody-induced calcium signal. Our results suggest that GPI anchoring and CD14 targeting to glycolipid-rich membrane microdomains are not required for LPS-mediated myeloid cell activation. GPI anchoring may however be important for other signalling functions, such as those events reflected by antibody cross-linking. PMID:9488411

On the self-association potential of transmembrane tight junction proteins.

PubMed

Blasig, I E; Winkler, L; Lassowski, B; Mueller, S L; Zuleger, N; Krause, E; Krause, G; Gast, K; Kolbe, M; Piontek, J

2006-02-01

Tight junctions seal intercellular clefts via membrane-related strands, hence, maintaining important organ functions. We investigated the self-association of strand-forming transmembrane tight junction proteins. The regulatory tight junction protein occludin was differently tagged and cotransfected in eucaryotic cells. These occludins colocalized within the plasma membrane of the same cell, coprecipitated and exhibited fluorescence resonance energy transfer. Differently tagged strand-forming claudin-5 also colocalized in the plasma membrane of the same cell and showed fluorescence resonance energy transfer. This demonstrates self-association in intact cells both of occludin and claudin-5 in one plasma membrane. In search of dimerizing regions of occludin, dimerization of its cytosolic C-terminal coiledcoil domain was identified. In claudin-5, the second extracellular loop was detected as a dimer. Since the transmembrane junctional adhesion molecule also is known to dimerize, the assumption that homodimerization of transmembrane tight junction proteins may serve as a common structural feature in tight junction assembly is supported.

Concurrent Validity of LibQUAL+[TM] Scores: What Do LibQUAL+[TM] Scores Measure?

ERIC Educational Resources Information Center

Thompson, Bruce; Cook, Colleen; Kyrillidou, Martha

2005-01-01

The present study investigated the validity of LibQUAL+[TM] scores, and specifically how total and subscale LibQUAL+[TM] scores are associated with self-reported, library-related satisfaction and outcomes scores. Participants included 88,664 students and faculty who completed the American English (n[AE] = 69,494) or the British English (n[BE] =…

Molecular contacts in the transmembrane c-subunit oligomer of F-ATPases identified by tryptophan substitution mutagenesis.

PubMed

Schnick, C; Forrest, L R; Sansom, M S; Groth, G

2000-07-20

When isolated in its monomeric form, subunit c of the proton transporting ATP synthase of Escherichia coli was shown to fold in a hairpin-like structure consisting of two hydrophobic membrane spanning helices and a short connecting hydrophilic loop. In the plasma membrane of Escherichia coli, however, about 9-12 c-subunit monomers form an oligomeric complex that functions in transmembrane proton conduction and in energy transduction to the catalytic F1 domain. The arrangement of the monomers and the molecular architecture of the complex were studied by tryptophan scanning mutagenesis and restrained MD simulations. Residues 12-24 of the N-terminal transmembrane segment of subunit c were individually substituted by the large and moderately hydrophobic tryptophan side chain. Effects on the activity of the mutant proteins were studied in selective growth experiments and various ATP synthase specific activity assays. The results identify potential intersubunit contacts and structurally non-distorted, accessible residues in the c-oligomer and add constraints to the arrangement of monomers in the oligomeric complex. Results from our mutagenesis experiments were interpreted in structural models of the c-oligomer that have been obtained by restrained MD simulations. Different stoichiometries and monomer orientations were applied in these calculations. A cylindrical complex consisting of 10 monomers that are arranged in two concentric rings with the N-terminal helices of the monomers located at the periphery shows the best match with the experimental data.

A three amino acid deletion in the transmembrane domain of the nicotinic acetylcholine receptor α6 subunit confers high-level resistance to spinosad in Plutella xylostella.

PubMed

Wang, Jing; Wang, Xingliang; Lansdell, Stuart J; Zhang, Jianheng; Millar, Neil S; Wu, Yidong

2016-04-01

Spinosad is a macrocyclic lactone insecticide that acts primarily at the nicotinic acetylcholine receptors (nAChRs) of target insects. Here we describe evidence that high levels of resistance to spinosad in the diamondback moth (Plutella xylostella) are associated with a three amino acid (3-aa) deletion in the fourth transmembrane domain (TM4) of the nAChR α6 subunit (Pxα6). Following laboratory selection with spinosad, the SZ-SpinR strain of P. xylostella exhibited 940-fold resistance to spinosad. In addition, the selected insect population had 1060-fold cross-resistance to spinetoram but, in contrast, no cross-resistance to abamectin was observed. Genetic analysis indicates that spinosad resistance in SZ-SpinR is inherited as a recessive and autosomal trait, and that the 3-aa deletion (IIA) in TM4 of Pxα6 is tightly linked to spinosad resistance. Because of well-established difficulties in functional expression of cloned insect nAChRs, the analogous resistance-associated deletion mutation was introduced into a prototype nAChR (the cloned human α7 subunit). Two-electrode voltage-clamp recording with wild-type and mutated nAChRs expressed in Xenopus laevis oocytes indicated that the mutation causes a complete loss of agonist activation. In addition, radioligand binding studies indicated that the 3-aa deletion resulted in significantly lower-affinity binding of the extracellular neurotransmitter-binding site. These findings are consistent with the 3-amino acid (IIA) deletion within the transmembrane domain of Pxα6 being responsible for target-site resistance to spinosad in the SZ-SpinR strain of P. xylostella. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

The EspF N-Terminal of Enterohemorrhagic Escherichia coli O157:H7 EDL933w Imparts Stronger Toxicity Effects on HT-29 Cells than the C-Terminal

PubMed Central

Wang, Xiangyu; Du, Yanli; Hua, Ying; Fu, Muqing; Niu, Cong; Zhang, Bao; Zhao, Wei; Zhang, Qiwei; Wan, Chengsong

2017-01-01

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 EspF is an important multifunctional protein that destroys the tight junctions of intestinal epithelial cells and promotes host cell apoptosis. However, its molecular mechanism remains elusive. We knocked out the espF sequence (747 bp, ΔespF), N-terminal sequence (219 bp, ΔespFN), and C-terminal sequence (528 bp, ΔespFC) separately using the pKD46-mediated λ Red homologous recombination system. Then, we built the corresponding complementation strains, namely, ΔespF/pespF, ΔespFN/pespFN, and ΔespFC/pespFC by overlap PCR, which were used in infecting HT-29 cells and BALB/C mice. The level of reactive oxygen species, cell apoptosis, mitochondrial trans-membrane potential, inflammatory factors, transepithelial electrical resistance (TER), and animal mortality were evaluated by DCFH-DA, double staining of Annexin V-FITC/PI, JC-1 staining, ELISA kit, and a mouse assay. The wild-type (WT), ΔespF, ΔespF/pespF, ΔespFC, ΔespFC/pespFC, ΔespFN, and ΔespFN/pespFN groups exhibited apoptotic rates of 68.3, 27.9, 64.9, 65.7, 73.4, 41.3, and 35.3% respectively, and mean TNF-α expression levels of 428 pg/mL, 342, 466, 446, 381, 383, and 374 pg/mL, respectively. In addition, the apoptotic rates and TNF-α levels of the WT, ΔespF/pespF, and ΔespFC were significantly higher than that of ΔespF, ΔespFN, ΔespFC/pespFC, and ΔespFN/pespFN group (p < 0.05). The N-terminal of EspF resulted in an increase in the number of apoptotic cells, TNF-α secretion, ROS generation, mitochondria apoptosis, and pathogenicity in BalB/c mice. In conclusion, the N-terminal domain of the Enterohemorrhagic E. coli O157:H7 EspF more strongly promotes apoptosis and inflammation than the C-terminal domain. PMID:28983470

Structure and genetic variability of envelope glycoproteins of two antigenic variants of caprine arthritis-encephalitis lentivirus.

PubMed

Knowles, D P; Cheevers, W P; McGuire, T C; Brassfield, A L; Harwood, W G; Stem, T A

1991-11-01

To define the structure of the caprine arthritis-encephalitis virus (CAEV) env gene and characterize genetic changes which occur during antigenic variation, we sequenced the env genes of CAEV-63 and CAEV-Co, two antigenic variants of CAEV defined by serum neutralization. The deduced primary translation product of the CAEV env gene consists of a 60- to 80-amino-acid signal peptide followed by an amino-terminal surface protein (SU) and a carboxy-terminal transmembrane protein (TM) separated by an Arg-Lys-Lys-Arg cleavage site. The signal peptide cleavage site was verified by amino-terminal amino acid sequencing of native CAEV-63 SU. In addition, immunoprecipitation of [35S]methionine-labeled CAEV-63 proteins by sera from goats immunized with recombinant vaccinia virus expressing the CAEV-63 env gene confirmed that antibodies induced by env-encoded recombinant proteins react specifically with native virion SU and TM. The env genes of CAEV-63 and CAEV-Co encode 28 conserved cysteines and 25 conserved potential N-linked glycosylation sites. Nucleotide sequence variability results in 62 amino acid changes and one deletion within the SU and 34 amino acid changes within the TM.

Structure and genetic variability of envelope glycoproteins of two antigenic variants of caprine arthritis-encephalitis lentivirus.

PubMed Central

Knowles, D P; Cheevers, W P; McGuire, T C; Brassfield, A L; Harwood, W G; Stem, T A

1991-01-01

To define the structure of the caprine arthritis-encephalitis virus (CAEV) env gene and characterize genetic changes which occur during antigenic variation, we sequenced the env genes of CAEV-63 and CAEV-Co, two antigenic variants of CAEV defined by serum neutralization. The deduced primary translation product of the CAEV env gene consists of a 60- to 80-amino-acid signal peptide followed by an amino-terminal surface protein (SU) and a carboxy-terminal transmembrane protein (TM) separated by an Arg-Lys-Lys-Arg cleavage site. The signal peptide cleavage site was verified by amino-terminal amino acid sequencing of native CAEV-63 SU. In addition, immunoprecipitation of [35S]methionine-labeled CAEV-63 proteins by sera from goats immunized with recombinant vaccinia virus expressing the CAEV-63 env gene confirmed that antibodies induced by env-encoded recombinant proteins react specifically with native virion SU and TM. The env genes of CAEV-63 and CAEV-Co encode 28 conserved cysteines and 25 conserved potential N-linked glycosylation sites. Nucleotide sequence variability results in 62 amino acid changes and one deletion within the SU and 34 amino acid changes within the TM. Images PMID:1656067

Unprecedented multiplicity of Ig transmembrane and secretory mRNA forms in the cartilaginous fish.

PubMed

Rumfelt, Lynn L; Diaz, Marilyn; Lohr, Rebecca L; Mochon, Evonne; Flajnik, Martin F

2004-07-15

In most jawed vertebrates including cartilaginous fish, membrane-bound IgM is expressed as a five Ig superfamily (Igsf)-domain H chain attached to a transmembrane (Tm) region. Heretofore, bony fish IgM was the one exception with IgM mRNA spliced to produce a four-domain Tm H chain. We now demonstrate that the Tm and secretory (Sec) mRNAs of the novel cartilaginous fish Ig isotypes, IgW and IgNAR, are present in multiple forms, most likely generated by alternative splicing. In the nurse shark, Ginglymostoma cirratum, and horn shark, Heterodontus francisci, alternative splicing of Tm exons to the second or the fourth constant (C(H)) exons produces two distinct IgW Tm cDNAs. Although the seven-domain IgW Sec cDNA form contains a canonical secretory tail shared with IgM, IgNAR, and IgA, we report a three-domain cDNA form of shark IgW (IgW(short)) having an unusual Sec tail, which is orthologous to skate IgX(short) cDNA. The IgW and IgW(short) Sec transcripts are restricted in their tissue distribution and expression levels vary among individual sharks, with all forms expressed early in ontogeny. IgNAR mRNA is alternatively spliced to produce a truncated four-domain Tm cDNA and a second Tm cDNA is expressed identical in Igsf domains as the Sec form. PBL is enriched in the Tm cDNA of these Igs. These molecular data suggest that cartilaginous fish have augmented their humoral immune repertoire by diversifying the sizes of their Ig isotypes. Furthermore, these Tm cDNAs are prototypical and the truncated variants may translate as more stable protein at the cell surface.

Structure, Dynamics, and Allosteric Potential of Ionotropic Glutamate Receptor N-Terminal Domains.

PubMed

Krieger, James; Bahar, Ivet; Greger, Ingo H

2015-09-15

Ionotropic glutamate receptors (iGluRs) are tetrameric cation channels that mediate synaptic transmission and plasticity. They have a unique modular architecture with four domains: the intracellular C-terminal domain (CTD) that is involved in synaptic targeting, the transmembrane domain (TMD) that forms the ion channel, the membrane-proximal ligand-binding domain (LBD) that binds agonists such as L-glutamate, and the distal N-terminal domain (NTD), whose function is the least clear. The extracellular portion, comprised of the LBD and NTD, is loosely arranged, mediating complex allosteric regulation and providing a rich target for drug development. Here, we briefly review recent work on iGluR NTD structure and dynamics, and further explore the allosteric potential for the NTD in AMPA-type iGluRs using coarse-grained simulations. We also investigate mechanisms underlying the established NTD allostery in NMDA-type iGluRs, as well as the fold-related metabotropic glutamate and GABAB receptors. We show that the clamshell motions intrinsically favored by the NTD bilobate fold are coupled to dimeric and higher-order rearrangements that impact the iGluR LBD and ultimately the TMD. Finally, we explore the dynamics of intact iGluRs and describe how it might affect receptor operation in a synaptic environment. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Immunising with the transmembrane envelope proteins of different retroviruses including HIV-1

PubMed Central

Denner, Joachim

2013-01-01

The induction of neutralizing antibodies is a promising way to prevent retrovirus infections. Neutralizing antibodies are mainly directed against the envelope proteins, which consist of two molecules, the surface envelope (SU) protein and the transmembrane envelope (TM) protein. Antibodies broadly neutralizing the human immunodeficiencvy virus-1 (HIV-1) and binding to the TM protein gp41 of the virus have been isolated from infected individuals. Their epitopes are located in the membrane proximal external region (MPER). Since there are difficulties to induce such neutralizing antibodies as basis for an effective AIDS vaccine, we performed a comparative analysis immunising with the TM proteins of different viruses from the family Retroviridae. Both subfamilies, the Orthoretrovirinae and the Spumaretrovirinae were included. In this study, the TM proteins of three gammaretroviruses including (1) the porcine endogenous retrovirus (PERV), (2) the Koala retrovirus (KoRV), (3) the feline leukemia virus (FeLV), of two lentiviruses, HIV-1, HIV-2, and of two spumaviruses, the feline foamy virus (FFV) and the primate foamy virus (PFV) were used for immunisation. Whereas in all immunisation studies binding antibodies were induced, neutralizing antibodies were only found in the case of the gammaretroviruses. The induced antibodies were directed against the MPER and the fusion peptide proximal region (FPPR) of their TM proteins; however only the antibodies against the MPER were neutralizing. Most importantly, the epitopes in the MPER were localized in the same position as the epitopes of the antibodies broadly neutralizing HIV-1 in the TM protein gp41 of HIV-1, indicating that the MPER is an effective target for the neutralization of retroviruses. PMID:23249763

Functions of transmembrane domain 3 of human melanocortin-4 receptor.

PubMed

Mo, Xiu-Lei; Yang, Rui; Tao, Ya-Xiong

2012-12-01

The melanocortin-4 receptor (MC4R) is a G protein-coupled receptor critical for maintaining energy homeostasis. Transmembrane domain 3 (TM3) of MC4R contains residues that were suggested to be essential in ligand binding and signaling. Several MC4R mutations in TM3 are associated with human obesity. To gain a better understanding of the functions of TM3, we analyzed the functions of 26 residues in TM3 using alanine-scanning mutagenesis. We showed that all mutants had normal cell-surface expression. Four mutants were defective in ligand binding and signaling and six mutants had normal ligand binding but impaired cAMP production. L140A had increased basal cAMP level. To further characterize the function of L140, we generated 17 additional L140 mutants. Fifteen L140 mutants had significantly decreased cell-surface expression, with L140R and L140V expressed normally. Ten L140 mutants had increased basal cAMP activities. Four L140 mutants were defective in ligand-stimulated cAMP generation. Interestingly, with the ERK1/2 pathway, we showed that nine constitutively active mutants had similar levels of basal pERK1/2 as that of WT, and two signaling defective mutants had similar levels of pERK1/2 as that of WT upon agonist stimulation, different from their cAMP signaling properties, suggesting biased signaling in these mutant receptors. In summary, we identified 13 residues in TM3 that were essential for ligand binding and/or signaling. Moreover, L140 was critical for locking MC4R in inactive conformation and several mutants showed biased signaling in cAMP and ERK1/2 signaling pathways.

N-terminal nesprin-2 variants regulate β-catenin signalling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Qiuping; Minaisah, Rose-Marie; Ferraro, Elisa

2016-07-15

The spatial compartmentalisation of biochemical signalling pathways is essential for cell function. Nesprins are a multi-isomeric family of proteins that have emerged as signalling scaffolds, herein, we investigate the localisation and function of novel nesprin-2 N-terminal variants. We show that these nesprin-2 variants display cell specific distribution and reside in both the cytoplasm and nucleus. Immunofluorescence microscopy revealed that nesprin-2 N-terminal variants colocalised with β-catenin at cell-cell junctions in U2OS cells. Calcium switch assays demonstrated that nesprin-2 and β-catenin are lost from cell-cell junctions in low calcium conditions whereas emerin localisation at the NE remained unaltered, furthermore, an N-terminal fragmentmore » of nesprin-2 was sufficient for cell-cell junction localisation and interacted with β-catenin. Disruption of these N-terminal nesprin-2 variants, using siRNA depletion resulted in loss of β-catenin from cell-cell junctions, nuclear accumulation of active β-catenin and augmented β-catenin transcriptional activity. Importantly, we show that U2OS cells lack nesprin-2 giant, suggesting that the N-terminal nesprin-2 variants regulate β-catenin signalling independently of the NE. Together, these data identify N-terminal nesprin-2 variants as novel regulators of β-catenin signalling that tether β-catenin to cell-cell contacts to inhibit β-catenin transcriptional activity. - Highlights: • N-terminal nesprin-2 variants display cell specific expression patterns. • N-terminal spectrin repeats of nesprin-2 interact with β-catenin. • N-terminal nesprin-2 variants scaffold β-catenin at cell-cell junctions.. • Nesprin-2 variants play multiple roles in β-catenin signalling.« less

Targeting of GLUT1-GLUT5 chimeric proteins in the polarized cell line Caco-2.

PubMed

Inukai, K; Takata, K; Asano, T; Katagiri, H; Ishihara, H; Nakazaki, M; Fukushima, Y; Yazaki, Y; Kikuchi, M; Oka, Y

1997-04-01

Caco-2, a human differentiated intestinal epithelial cell line, is a promising model for investigating the mechanism of polarized targeting of apical and basolateral membrane proteins. We stably transfected rat GLUT5 cDNA and rabbit GLUT1 cDNA into Caco-2 cells with an expression vector. Immunohistochemical study revealed that the GLUT5 protein expressed was localized at apical membranes and that the GLUT1 expressed was present primarily in the basolateral membranes of cells grown on permeable support. Next, to investigate the domain responsible for determining apical vs. basolateral sorting in glucose transporters, we prepared several GLUT1-GLUT5 chimeric cDNAs and transfected them into Caco-2 cells. A GLUT1 [N terminus approximately sixth transmembrane domain (TM6)]-GLUT5 [intracellular loop (IL) approximately C terminus] chimera was observed exclusively at the apical membrane, while GLUT1 (N terminus approximately IL)-GLUT5 (TM7 approximately C terminus) and GLUT1 (N terminus approximately TM12)-GLUT5 (C-terminal domain) chimeras were observed mainly at the basolateral membrane, a localization similar to that of GLUT1. Moreover, using a recombinant adenovirus expression system, we expressed a GLUT5 (N terminus approximately TM6)-GLUT1(IL)-GLUT5(TM7 approximately C-terminus) chimera, which was observed at the basolateral membrane. Based on these results, the C-terminal domain does not determine isoform-specific targeting of GLUT1 and GLUT5. Rather, it is the intracellular loop in glucose transporters that appears to play a pivotal role in apical-basolateral sorting signals in Caco-2 cells.

Tetraspan TM4SF5-dependent direct activation of FAK and metastatic potential of hepatocarcinoma cells

PubMed Central

Jung, Oisun; Choi, Suyong; Jang, Sun-Bok; Lee, Sin-Ae; Lim, Ssang-Taek; Choi, Yoon-Ju; Kim, Hye-Jin; Kim, Do-Hee; Kwak, Tae Kyoung; Kim, Hyeonjung; Kang, Minkyung; Lee, Mi-Sook; Park, Sook Young; Ryu, Jihye; Jeong, Doyoung; Cheong, Hae-Kap; Kim, Hyun Jeong; Park, Ki Hun; Lee, Bong-Jin; Schlaepfer, David D.; Lee, Jung Weon

2012-01-01

Summary Transmembrane 4 L six family member 5 (TM4SF5) plays an important role in cell migration, and focal adhesion kinase (FAK) activity is essential for homeostatic and pathological migration of adherent cells. However, it is unclear how TM4SF5 signaling mediates the activation of cellular migration machinery, and how FAK is activated during cell adhesion. Here, we showed that direct and adhesion-dependent binding of TM4SF5 to FAK causes a structural alteration that may release the inhibitory intramolecular interaction in FAK. In turn, this may activate FAK at the cell's leading edge, to promote migration/invasion and in vivo metastasis. TM4SF5-mediated FAK activation occurred during integrin-mediated cell adhesion. TM4SF5 was localized at the leading edge of the cells, together with FAK and actin-organizing molecules, indicating a signaling link between TM4SF5/FAK and actin reorganization machinery. Impaired interactions between TM4SF5 and FAK resulted in an attenuated FAK phosphorylation (the signaling link to actin organization machinery) and the metastatic potential. Our findings demonstrate that TM4SF5 directly binds to and activates FAK in an adhesion-dependent manner, to regulate cell migration and invasion, suggesting that TM4SF5 is a promising target in the treatment of metastatic cancer. PMID:23077174

Bimolecular fluorescence complementation: lighting up seven transmembrane domain receptor signalling networks

PubMed Central

Rose, Rachel H; Briddon, Stephen J; Holliday, Nicholas D

2010-01-01

There is increasing complexity in the organization of seven transmembrane domain (7TM) receptor signalling pathways, and in the ability of their ligands to modulate and direct this signalling. Underlying these events is a network of protein interactions between the 7TM receptors themselves and associated effectors, such as G proteins and β-arrestins. Bimolecular fluorescence complementation, or BiFC, is a technique capable of detecting these protein–protein events essential for 7TM receptor function. Fluorescent proteins, such as those from Aequorea victoria, are split into two non-fluorescent halves, which then tag the proteins under study. On association, these fragments refold and regenerate a mature fluorescent protein, producing a BiFC signal indicative of complex formation. Here, we review the experimental criteria for successful application of BiFC, considered in the context of 7TM receptor signalling events such as receptor dimerization, G protein and β-arrestin signalling. The advantages and limitations of BiFC imaging are compared with alternative resonance energy transfer techniques. We show that the essential simplicity of the fluorescent BiFC measurement allows high-content and advanced imaging applications, and that it can probe more complex multi-protein interactions alone or in combination with resonance energy transfer. These capabilities suggest that BiFC techniques will become ever more useful in the analysis of ligand and 7TM receptor pharmacology at the molecular level of protein–protein interactions. This article is part of a themed section on Imaging in Pharmacology. To view the editorial for this themed section visit http://dx.doi.org/10.1111/j.1476-5381.2010.00685.x PMID:20015298

Improved growth response of antibody/receptor chimera attained by the engineering of transmembrane domain.

PubMed

Kawahara, Masahiro; Ogo, Yuko; Ueda, Hiroshi; Nagamune, Teruyuki

2004-10-01

Structure-based design of antibody/cytokine receptor chimeras has permitted a growth signal transduction in response to non-natural ligands such as fluorescein-conjugated BSA as mimicry of cytokine-cytokine receptor systems. However, while tight on/off regulation is observed in the natural cytokine receptor systems, many chimeras constructed to date showed residual growth-promoting activity in the absence of ligands. Here we tried to reduce the basal growth signal intensity from a chimera by engineering the transmembrane domain (TM) that is thought to be involved in the interchain interaction of natural cytokine receptors. When the retroviral vectors encoding the chimeras with either the wild-type erythropoietin receptor (EpoR) TM or the one bearing two mutations in the leucine zipper motif were transduced to non-strictly interleukin-6-dependent 7TD1 cells, a tight antigen-dependent on/off regulation was attained, also demonstrating the first antigen-mediated genetically modified cell amplification of non-strictly factor-dependent cells. The results clearly indicate that the TM mutation is an effective means to improve the growth response of the antibody/receptor chimera.

The cytoplasmic end of transmembrane domain 3 regulates the activity of the Saccharomyces cerevisiae G-protein-coupled alpha-factor receptor.

PubMed Central

Parrish, William; Eilers, Markus; Ying, Weiwen; Konopka, James B

2002-01-01

The binding of alpha-factor to its receptor (Ste2p) activates a G-protein-signaling pathway leading to conjugation of MATa cells of the budding yeast S. cerevisiae. We conducted a genetic screen to identify constitutively activating mutations in the N-terminal region of the alpha-factor receptor that includes transmembrane domains 1-5. This approach identified 12 unique constitutively activating mutations, the strongest of which affected polar residues at the cytoplasmic ends of transmembrane domains 2 and 3 (Asn84 and Gln149, respectively) that are conserved in the alpha-factor receptors of divergent yeast species. Targeted mutagenesis, in combination with molecular modeling studies, suggested that Gln149 is oriented toward the core of the transmembrane helix bundle where it may be involved in mediating an interaction with Asn84. These residues appear to play specific roles in maintaining the inactive conformation of the protein since a variety of mutations at either position cause constitutive receptor signaling. Interestingly, the activity of many mammalian G-protein-coupled receptors is also regulated by conserved polar residues (the E/DRY motif) at the cytoplasmic end of transmembrane domain 3. Altogether, the results of this study suggest a conserved role for the cytoplasmic end of transmembrane domain 3 in regulating the activity of divergent G-protein-coupled receptors. PMID:11861550

Systemic GLIPR1-ΔTM protein as a novel therapeutic approach for prostate cancer.

PubMed

Karantanos, Theodoros; Tanimoto, Ryuta; Edamura, Kohei; Hirayama, Takahiro; Yang, Guang; Golstov, Alexei A; Wang, Jianxiang; Kurosaka, Shinji; Park, Sanghee; Thompson, Timothy C

2014-04-15

GLIPR1 is a p53 target gene known to be downregulated in prostate cancer, and increased endogenous GLIPR1 expression has been associated with increased production of reactive oxygen species, increased apoptosis, decreased c-Myc protein levels and increased cell cycle arrest. Recently, we found that upregulation of GLIPR1 in prostate cancer cells increases mitotic catastrophe through interaction with heat shock cognate protein 70 (Hsc70) and downregulation of Aurora kinase A and TPX2. In this study, we evaluated the mechanisms of recombinant GLIPR1 protein (glioma pathogenesis-related protein 1-transmembrane domain deleted [GLIPR1-ΔTM]) uptake by prostate cancer cells and the efficacy of systemic GLIPR1-ΔTM administration in a prostate cancer xenograft mouse model. GLIPR1-ΔTM was selectively internalized by prostate cancer cells, leading to increased apoptosis through reactive oxygen species production and to decreased c-Myc protein levels. Interestingly, GLIPR1-ΔTM was internalized through clathrin-mediated endocytosis in association with Hsc70. Systemic administration of GLIPR1-ΔTM significantly inhibited VCaP xenograft growth. GLIPR1-ΔTM showed no evidence of toxicity following elimination from mouse models 8 hr after injection. Our results demonstrate that GLIPR1-ΔTM is selectively endocytosed by prostate cancer cells, leading to increased reactive oxygen species production and apoptosis, and that systemic GLIPR1-ΔTM significantly inhibits growth of VCaP xenografts without substantial toxicity. © 2013 UICC.

Role of the Lipid Environment in the Dimerization of Transmembrane Domains of Glycophorin A

PubMed Central

Kuznetsov, A. S.; Volynsky, P. E.; Efremov, R. G.

2015-01-01

An efficient computational approach is developed to quantify the free energy of a spontaneous association of the α-helices of proteins in the membrane environment. The approach is based on the numerical decomposition of the free energy profiles of the transmembrane (TM) helices into components corresponding to protein-protein, protein-lipid, and protein-water interactions. The method was tested for the TM segments of human glycophorin A (GpA) and two mutant forms, Gly83Ala and Thr87Val. It was shown that lipids make a significant negative contribution to the free energy of dimerization, while amino acid residues forming the interface of the helix-helix contact may be unfavorable in terms of free energy. The detailed balance between different energy contributions is highly dependent on the amino acid sequence of the TM protein segment. The results show the dominant role of the environment in the interaction of membrane proteins that is changing our notion of the driving force behind the spontaneous association of TM α-helices. Adequate estimation of the contribution of the water-lipid environment thus becomes an extremely urgent task for a rational design of new molecules targeting bitopic membrane proteins, including receptor tyrosine kinases. PMID:26798499

Probing structure, thermochemistry, electron affinity, and magnetic moment of thulium-doped silicon clusters TmSi n (n = 3-10) and their anions with density functional theory.

PubMed

Huang, Xintao; Yang, Jucai

2017-12-26

The most stable structures and electronic properties of TmSi n (n = 3-10) clusters and their anions have been probed by using the ABCluster global search technique combined with the PBE, TPSSh, and B3LYP density functional methods. The results revealed that the most stable structures of neutral TmSi n and their anions can be regarded as substituting a Si atom of the ground state structure of Si n + 1 with a Tm atom. The reliable AEAs, VDEs and simulated PES of TmSi n (n = 3-10) are presented. Calculations of HOMO-LUMO gap revealed that introducing Tm atom to Si cluster can improve photochemical reactivity of the cluster. The NPA analyses indicated that the 4f electron of Tm atom in TmSi n (n = 3-10) and their anions do not participate in bonding. The total magnetic moments of TmSi n are mainly provided by the 4f electrons of Tm atom. The dissociation energy of Tm atom from the most stable structure of TmSi n and their anions has been calculated to examine relative stability.

Computational Identification and Comparative Analysis of Secreted and Transmembrane Proteins in Six Burkholderia Species.

PubMed

Nguyen, Thao Thi; Lee, Hyun-Hee; Park, Jungwook; Park, Inmyoung; Seo, Young-Su

2017-04-01

As a step towards discovering novel pathogenesis-related proteins, we performed a genome scale computational identification and characterization of secreted and transmembrane (TM) proteins, which are mainly responsible for bacteria-host interactions and interactions with other bacteria, in the genomes of six representative Burkholderia species. The species comprised plant pathogens ( B. glumae BGR1, B. gladioli BSR3), human pathogens ( B. pseudomallei K96243, B. cepacia LO6), and plant-growth promoting endophytes ( Burkholderia sp. KJ006, B. phytofirmans PsJN). The proportions of putative classically secreted proteins (CSPs) and TM proteins among the species were relatively high, up to approximately 20%. Lower proportions of putative type 3 non-classically secreted proteins (T3NCSPs) (~10%) and unclassified non-classically secreted proteins (NCSPs) (~5%) were observed. The numbers of TM proteins among the three clusters (plant pathogens, human pathogens, and endophytes) were different, while the distribution of these proteins according to the number of TM domains was conserved in which TM proteins possessing 1, 2, 4, or 12 TM domains were the dominant groups in all species. In addition, we observed conservation in the protein size distribution of the secreted protein groups among the species. There were species-specific differences in the functional characteristics of these proteins in the various groups of CSPs, T3NCSPs, and unclassified NCSPs. Furthermore, we assigned the complete sets of the conserved and unique NCSP candidates of the collected Burkholderia species using sequence similarity searching. This study could provide new insights into the relationship among plant-pathogenic, human-pathogenic, and endophytic bacteria.

Distinct Neurobehavioural Effects of Cannabidiol in Transmembrane Domain Neuregulin 1 Mutant Mice

PubMed Central

Long, Leonora E.; Chesworth, Rose; Huang, Xu-Feng; Wong, Alexander; Spiro, Adena; McGregor, Iain S.; Arnold, Jonathon C.; Karl, Tim

2012-01-01

The cannabis constituent cannabidiol (CBD) possesses anxiolytic and antipsychotic properties. We have previously shown that transmembrane domain neuregulin 1 mutant (Nrg1 TM HET) mice display altered neurobehavioural responses to the main psychoactive constituent of cannabis, Δ9-tetrahydrocannabinol. Here we investigated whether Nrg1 TM HET mice respond differently to CBD and whether CBD reverses schizophrenia-related phenotypes expressed by these mice. Adult male Nrg1 TM HET and wild type-like littermates (WT) received vehicle or CBD (1, 50 or 100 mg/kg i.p.) for 21 days. During treatment and 48 h after withdrawal we measured behaviour, whole blood CBD concentrations and autoradiographic receptor binding. Nrg1 HET mice displayed locomotor hyperactivity, PPI deficits and reduced 5-HT2A receptor binding density in the substantia nigra, but these phenotypes were not reversed by CBD. However, long-term CBD (50 and 100 mg/kg) selectively enhanced social interaction in Nrg1 TM HET mice. Furthermore, acute CBD (100 mg/kg) selectively increased PPI in Nrg1 TM HET mice, although tolerance to this effect was manifest upon repeated CBD administration. Long-term CBD (50 mg/kg) also selectively increased GABAA receptor binding in the granular retrosplenial cortex in Nrg1 TM HET mice and reduced 5-HT2A binding in the substantia nigra in WT mice. Nrg1 appears necessary for CBD-induced anxiolysis since only WT mice developed decreased anxiety-related behaviour with repeated CBD treatment. Altered pharmacokinetics in mutant mice could not explain our findings since no genotype differences existed in CBD blood concentrations. Here we demonstrate that Nrg1 modulates acute and long-term neurobehavioural effects of CBD, which does not reverse the schizophrenia-relevant phenotypes. PMID:22509273

Distinct neurobehavioural effects of cannabidiol in transmembrane domain neuregulin 1 mutant mice.

PubMed

Long, Leonora E; Chesworth, Rose; Huang, Xu-Feng; Wong, Alexander; Spiro, Adena; McGregor, Iain S; Arnold, Jonathon C; Karl, Tim

2012-01-01

The cannabis constituent cannabidiol (CBD) possesses anxiolytic and antipsychotic properties. We have previously shown that transmembrane domain neuregulin 1 mutant (Nrg1 TM HET) mice display altered neurobehavioural responses to the main psychoactive constituent of cannabis, Δ(9)-tetrahydrocannabinol. Here we investigated whether Nrg1 TM HET mice respond differently to CBD and whether CBD reverses schizophrenia-related phenotypes expressed by these mice. Adult male Nrg1 TM HET and wild type-like littermates (WT) received vehicle or CBD (1, 50 or 100 mg/kg i.p.) for 21 days. During treatment and 48 h after withdrawal we measured behaviour, whole blood CBD concentrations and autoradiographic receptor binding. Nrg1 HET mice displayed locomotor hyperactivity, PPI deficits and reduced 5-HT(2A) receptor binding density in the substantia nigra, but these phenotypes were not reversed by CBD. However, long-term CBD (50 and 100 mg/kg) selectively enhanced social interaction in Nrg1 TM HET mice. Furthermore, acute CBD (100 mg/kg) selectively increased PPI in Nrg1 TM HET mice, although tolerance to this effect was manifest upon repeated CBD administration. Long-term CBD (50 mg/kg) also selectively increased GABA(A) receptor binding in the granular retrosplenial cortex in Nrg1 TM HET mice and reduced 5-HT(2A) binding in the substantia nigra in WT mice. Nrg1 appears necessary for CBD-induced anxiolysis since only WT mice developed decreased anxiety-related behaviour with repeated CBD treatment. Altered pharmacokinetics in mutant mice could not explain our findings since no genotype differences existed in CBD blood concentrations. Here we demonstrate that Nrg1 modulates acute and long-term neurobehavioural effects of CBD, which does not reverse the schizophrenia-relevant phenotypes.

Conformational states of mutant M13 coat proteins are regulated by transmembrane residues.

PubMed

Li, Z; Glibowicka, M; Joensson, C; Deber, C M

1993-03-05

Mutational and structural analysis of the 28 viable bacteriophage M13 mutants obtained by randomized mutagenesis of the effective transmembrane (TM) segment of the 50-residue major coat (gene VIII) protein (residues 21-39) demonstrated that M13 coat protein functionality, as reflected by phage viability, is incompatible with an increase in Gly + beta-branched residue content in its TM core. SDS-polyacrylamide gel electrophoresis and circular dichroism spectroscopy performed in membrane environments on purified mutant coat proteins revealed that these proteins exist in a range of state(s), identified as helical monomers and dimers and polymeric (alpha-helical and/or beta-sheet) species, of which relative populations, and thermally induced conformational transitions, were dependent uniquely upon mutation type and locus. Mutations to relatively polar residues (e.g. G23D, Y24D, Y24H, A27E, I32T, and T36S) stabilized principally monomeric species, while mutants with decreased beta-branched content in the protein TM hydrophobic core (e.g. V29A, V30A, V31A, V31L, and V33A) displayed mainly dimeric species. Mutation of Ile37-->Thr within a "Sternberg-Gullick" consensus sequence of the coat protein TM segment led to a highly oligomerized/polymerized protein. The overall results suggest that TM residues in M13 coat protein are not universal components of a hydrophobic anchor segment per se, but are further selected (i) to impart conformational flexibility to the TM segment through helix destabilization and (ii) to retain the capacity to regulate protein-protein association and packing motifs within membranes.

A trans-membrane segment inside the ribosome exit tunnel triggers RAMP4 recruitment to the Sec61p translocase

PubMed Central

2009-01-01

Membrane protein integration occurs predominantly at the endoplasmic reticulum and is mediated by the translocon, which is formed by the Sec61p complex. The translocon binds to the ribosome at the polypeptide exit site such that integration occurs in a cotranslational manner. Ribosomal protein Rpl17 is positioned such that it contacts both the ribosome exit tunnel and the surface of the ribosome near the exit site, where it is intimately associated with the translocon. The presence of a trans-membrane (TM) segment inside the ribosomal exit tunnel leads to the recruitment of RAMP4 to the translocon at a site adjacent to Rpl17. This suggests a signaling function for Rpl17 such that it can recognize a TM segment inside the ribosome and triggers rearrangements of the translocon, priming it for subsequent TM segment integration. PMID:19468070

Recombinant Expression Screening of P. aeruginosa Bacterial Inner Membrane Proteins

PubMed Central

2010-01-01

Background Transmembrane proteins (TM proteins) make up 25% of all proteins and play key roles in many diseases and normal physiological processes. However, much less is known about their structures and molecular mechanisms than for soluble proteins. Problems in expression, solubilization, purification, and crystallization cause bottlenecks in the characterization of TM proteins. This project addressed the need for improved methods for obtaining sufficient amounts of TM proteins for determining their structures and molecular mechanisms. Results Plasmid clones were obtained that encode eighty-seven transmembrane proteins with varying physical characteristics, for example, the number of predicted transmembrane helices, molecular weight, and grand average hydrophobicity (GRAVY). All the target proteins were from P. aeruginosa, a gram negative bacterial opportunistic pathogen that causes serious lung infections in people with cystic fibrosis. The relative expression levels of the transmembrane proteins were measured under several culture growth conditions. The use of E. coli strains, a T7 promoter, and a 6-histidine C-terminal affinity tag resulted in the expression of 61 out of 87 test proteins (70%). In this study, proteins with a higher grand average hydrophobicity and more transmembrane helices were expressed less well than less hydrophobic proteins with fewer transmembrane helices. Conclusions In this study, factors related to overall hydrophobicity and the number of predicted transmembrane helices correlated with the relative expression levels of the target proteins. Identifying physical characteristics that correlate with protein expression might aid in selecting the "low hanging fruit", or proteins that can be expressed to sufficient levels using an E. coli expression system. The use of other expression strategies or host species might be needed for sufficient levels of expression of transmembrane proteins with other physical characteristics. Surveys like this

Non-equivalent role of TM2 gating hinges in heteromeric Kir4.1/Kir5.1 potassium channels.

PubMed

Shang, Lijun; Tucker, Stephen J

2008-02-01

Comparison of the crystal structures of the KcsA and MthK potassium channels suggests that the process of opening a K(+) channel involves pivoted bending of the inner pore-lining helices at a highly conserved glycine residue. This bending motion is proposed to splay the transmembrane domains outwards to widen the gate at the "helix-bundle crossing". However, in the inwardly rectifying (Kir) potassium channel family, the role of this "hinge" residue in the second transmembrane domain (TM2) and that of another putative glycine gating hinge at the base of TM2 remain controversial. We investigated the role of these two positions in heteromeric Kir4.1/Kir5.1 channels, which are unique amongst Kir channels in that both subunits lack a conserved glycine at the upper hinge position. Contrary to the effect seen in other channels, increasing the potential flexibility of TM2 by glycine substitutions at the upper hinge position decreases channel opening. Furthermore, the contribution of the Kir4.1 subunit to this process is dominant compared to Kir5.1, demonstrating a non-equivalent contribution of these two subunits to the gating process. A homology model of heteromeric Kir4.1/Kir5.1 shows that these upper "hinge" residues are in close contact with the base of the pore alpha-helix that supports the selectivity filter. Our results also indicate that the highly conserved glycine at the "lower" gating hinge position is required for tight packing of the TM2 helices at the helix-bundle crossing, rather than acting as a hinge residue.

Proline residues in transmembrane segment IV are critical for activity, expression and targeting of the Na+/H+ exchanger isoform 1.

PubMed Central

Slepkov, Emily R; Chow, Signy; Lemieux, M Joanne; Fliegel, Larry

2004-01-01

NHE1 (Na+/H+ exchanger isoform 1) is a ubiquitously expressed integral membrane protein that regulates intracellular pH in mammalian cells. Proline residues within transmembrane segments have unusual properties, acting as helix breakers and increasing flexibility of membrane segments, since they lack an amide hydrogen. We examined the importance of three conserved proline residues in TM IV (transmembrane segment IV) of NHE1. Pro167 and Pro168 were mutated to Gly, Ala or Cys, and Pro178 was mutated to Ala. Pro168 and Pro178 mutant proteins were expressed at levels similar to wild-type NHE1 and were targeted to the plasma membrane. However, the mutants P167G (Pro167-->Gly), P167A and P167C were expressed at lower levels compared with wild-type NHE1, and a significant portion of P167G and P167C were retained intracellularly, possibly indicating induced changes in the structure of TM IV. P167G, P167C, P168A and P168C mutations abolished NHE activity, and P167A and P168G mutations caused markedly decreased activity. In contrast, the activity of the P178A mutant was not significantly different from that of wild-type NHE1. The results indicate that both Pro167 and Pro168 in TM IV of NHE1 are required for normal NHE activity. In addition, mutation of Pro167 affects the expression and membrane targeting of the exchanger. Thus both Pro167 and Pro168 are strictly required for NHE function and may play critical roles in the structure of TM IV of the NHE. PMID:14680478

Tetraspanin TM4SF5 mediates loss of contact inhibition through epithelial-mesenchymal transition in human hepatocarcinoma

PubMed Central

Lee, Sin-Ae; Lee, Sung-Yul; Cho, Ik-Hyun; Oh, Min-A; Kang, Eun-Sil; Kim, Yong-Bae; Seo, Woo Duck; Choi, Suyong; Nam, Ju-Ock; Tamamori-Adachi, Mimi; Kitajima, Shigetaka; Ye, Sang-Kyu; Kim, Semi; Hwang, Yoon-Jin; Kim, In-San; Park, Ki Hun; Lee, Jung Weon

2008-01-01

The growth of normal cells is arrested when they come in contact with each other, a process known as contact inhibition. Contact inhibition is lost during tumorigenesis, resulting in uncontrolled cell growth. Here, we investigated the role of the tetraspanin transmembrane 4 superfamily member 5 (TM4SF5) in contact inhibition and tumorigenesis. We found that TM4SF5 was overexpressed in human hepatocarcinoma tissue. TM4SF5 expression in clinical samples and in human hepatocellular carcinoma cell lines correlated with enhanced p27Kip1 expression and cytosolic stabilization as well as morphological elongation mediated by RhoA inactivation. These TM4SF5-mediated effects resulted in epithelial-mesenchymal transition (EMT) via loss of E-cadherin expression. The consequence of this was aberrant cell growth, as assessed by S-phase transition in confluent conditions, anchorage-independent growth, and tumor formation in nude mice. The TM4SF5-mediated effects were abolished by suppressing the expression of either TM4SF5 or cytosolic p27Kip1, as well as by reconstituting the expression of E-cadherin. Our observations have revealed a role for TM4SF5 in causing uncontrolled growth of human hepatocarcinoma cells through EMT. PMID:18357344

Synthesis and assembly of Hepatitis B virus envelope protein-derived particles in Escherichia coli.

PubMed

Li, Hao; Onbe, Keisuke; Liu, Qiushi; Iijima, Masumi; Tatematsu, Kenji; Seno, Masaharu; Tada, Hiroko; Kuroda, Shun' Ichi

2017-08-19

Hepatitis B virus (HBV) envelope particles have been synthesized in eukaryotic cells (e.g., mammalian cells, insect cells, and yeast cells) as an HB vaccine immunogen and drug delivery system (DDS) nanocarrier. Many researchers had made attempts to synthesize the particles in Escherichia coli for minimize the cost and time for producing HBV envelope particles, but the protein was too deleterious to be synthesized in E. coli. In this study, we generated deletion mutants of HBV envelope L protein (389 amino acid residues (aa)) containing three transmembrane domains (TM1, TM2, TM3). The ΔNC mutant spanning from TM2 to N-terminal half of TM3 (from 237 aa to 335 aa) was found as a shortest form showing spontaneous particle formation. After the N-terminal end of ΔNC mutant was optimized by the N-end rule for E. coli expression, the modified ΔNC mutant (mΔNC) was efficiently expressed as particles in E. coli. The molecular mass of mΔNC particle was approx. 670 kDa, and the diameter was 28.5 ± 6.2 nm (mean ± SD, N = 61). The particle could react with anti-HBV envelope S protein antibody, indicating the particles exhibited S antigenic domain outside as well as HBV envelope particles. Taken together, the E. coli-derived mΔNC particles could be used as a substitute of eukaryotic cell-derived HBV envelope particles for versatile applications. Copyright © 2017 Elsevier Inc. All rights reserved.

Generation of proton-motive force by an archaeal terminal quinol oxidase from Sulfolobus acidocaldarius.

PubMed

Gleissner, M; Elferink, M G; Driessen, A J; Konings, W N; Anemüller, S; Schäfer, G

1994-09-15

The terminal quinol oxidase of the cytochrome aa3 type was isolated from the extreme thermoacidophilic archaeon Sulfolobus acidocaldarius. In micellar solution, the enzyme oxidized various quinols and exerted the highest activity with the physiological substrate caldariella quinol. The enzyme was functionally reconstituted into monolayer liposomes composed of archaeal tetraether lipids also derived from S. acidocaldarius. With the electron donor system ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine, the reconstituted enzyme was more active in the archaeal lipids as compared to lipids derived from Escherichia coli at temperatures above 50 degrees C. Due to the low proton permeability of the tetraether lipids, it was possible to generate a steady-state transmembrane electrical potential (delta psi, interior negative), and transmembrane pH gradient (delta pH, interior alkaline) at temperatures up to 70 degrees C. The successful functional reconstitution of the cytochrome aa3-type quinol oxidase from Sulfolobus identifies it as the key energy converter in the respiratory system of this hyperthermophilic archaeon.

Engineering a thermostable fungal GH10 xylanase, importance of N-terminal amino acids.

PubMed

Song, Letian; Tsang, Adrian; Sylvestre, Michel

2015-06-01

Xylanases are used in many industrial processes including pulp bleaching, baking, detergent, and the hydrolysis of plant cell wall in biofuels production. In this work we have evolved a single domain GH10 xylanase, Xyn10A_ASPNG, from Aspergillus niger to improve its thermostability. We introduced a rational approach involving as the first step a computational analysis to guide the design of a mutagenesis library in targeted regions which identified thermal important residues that were subsequently randomly mutagenized through rounds of iterative saturation mutagenesis (ISM). Focusing on five residues, four rounds of ISM had generated a quintuple mutant 4S1 (R25W/V29A/I31L/L43F/T58I) which exhibited thermal inactivation half-life (t1/2 ) at 60°C that was prolonged by 30 folds in comparison with wild-type enzyme. Whereas the wild-type enzyme retained 0.2% of its initial activity after a heat treatment of 10 min at 60°C and was completely inactivated after 2 min at 65°C, 4S1 mutant retained 30% of its initial activity after 15 min heating at 65°C. Furthermore, the mutant melting temperature (Tm ) increased by 17.4°C compared to the wild type. Each of the five mutations in 4S1 was found to contribute to thermoresistance, but the dramatic improvement of enzyme thermoresistance of 4S1 was attributed to the synergistic effects of the five mutations. Comparison of biochemical data and model structure between 4S1 and the wild-type enzyme suggested that the N-terminal coil of the enzyme is important in stabilizing GH10 xylanase structure. Based on model structure analyses, we propose that enforced hydrophobic interactions within N-terminal elements and between N- and C-terminal ends are responsible for the improved thermostability of Xyn10A_ASPNG. © 2015 Wiley Periodicals, Inc.

Global functions of extracellular, transmembrane and cytoplasmic domains of organic solute transporter β-subunit.

PubMed

Christian, Whitney V; Hinkle, Patricia M

2017-05-25

Transport of bile acids across the basolateral membrane of the intestinal enterocyte is carried out by the organic solute transporter (Ost) composed of a seven-transmembrane domain (TMD) subunit (Ostα) and an ancillary single TMD subunit (Ostβ). Although previous investigations have demonstrated the importance of the TMD of Ostβ for its activity, further studies were conducted to assess the contributions of other regions of the Ostβ subunit. Transport activity was retained when Ostβ was truncated to contain only the TMD with 15 additional residues on each side and co-expressed with Ostα, whereas shorter fragments were inactive. To probe the broader functions of Ostβ segments, chimeric proteins were constructed in which N-terminal, TMD or C-terminal regions of Ostβ were fused to corresponding regions of receptor activity-modifying protein (RAMP1), a single TMD protein required by several seven-TMD G-protein-coupled receptors including the calcitonin receptor-like receptor (CLR). Ostβ/RAMP1 chimeras were expressed with Ostα and CLR. As expected, replacing the Ostβ TMD abolished transport activity; however, replacing either the entire N-terminal or entire C-terminal domain of Ostβ with RAMP1 sequences did not prevent plasma membrane localization or the ability to support [ 3 H]taurocholate uptake. Co-immunoprecipitation experiments revealed that the C-terminus of Ostβ is a previously unrecognized site of interaction with Ostα. All chimeras containing N-terminal RAMP1 segments allowed co-expressed CLR to respond to agonists with strong increases in cyclic AMP. These results provide new insights into the structure and function of the heteromeric Ost transporter complex. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

Crystal Structure of Oligomeric β1-Adrenergic G Protein- Coupled Receptors in Ligand-Free Basal State

PubMed Central

Huang, Jianyun; Chen, Shuai; Zhang, J. Jillian; Huang, Xin-Yun

2013-01-01

G protein-coupled receptors (GPCRs) mediate transmembrane signaling. Before ligand binding, GPCRs exist in a basal state. Crystal structures of several GPCRs bound with antagonists or agonists have been solved. However, the crystal structure of the ligand-free basal state of a GPCR, the starting point of GPCR activation and function, has not been determined. Here we report the X-ray crystal structure of the first ligand-free basal state of a GPCR in a lipid membrane-like environment. Oligomeric turkey β1-adrenergic receptors display two alternating dimer interfaces. One interface involves the transmembrane domain (TM) 1, TM2, the C-terminal H8, and the extracellular loop 1. The other interface engages residues from TM4, TM5, the intracellular loop 2 and the extracellular loop 2. Structural comparisons show that this ligand-free state is in an inactive conformation. This provides the structural information regarding GPCR dimerization and oligomerization. PMID:23435379

The stability of the three transmembrane and the four transmembrane human vitamin K epoxide reductase models

NASA Astrophysics Data System (ADS)

Wu, Sangwook

2016-04-01

The three transmembrane and the four transmembrane helix models are suggested for human vitamin K epoxide reductase (VKOR). In this study, we investigate the stability of the human three transmembrane/four transmembrane VKOR models by employing a coarse-grained normal mode analysis and molecular dynamics simulation. Based on the analysis of the mobility of each transmembrane domain, we suggest that the three transmembrane human VKOR model is more stable than the four transmembrane human VKOR model.

N-terminal RASSF family

PubMed Central

Underhill-Day, Nicholas; Hill, Victoria

2011-01-01

Epigenetic inactivation of tumor suppressor genes is a hallmark of cancer development. RASSF1A (Ras Association Domain Family 1 isoform A) tumor suppressor gene is one of the most frequently epigenetically inactivated genes in a wide range of adult and children's cancers and could be a useful molecular marker for cancer diagnosis and prognosis. RASSF1A has been shown to play a role in several biological pathways, including cell cycle control, apoptosis and microtubule dynamics. RASSF2, RASSF4, RASSF5 and RASSF6 are also epigenetically inactivated in cancer but have not been analyzed in as wide a range of malignancies as RASSF1A. Recently four new members of the RASSF family were identified these are termed N-Terminal RASSF genes (RASSF7–RASSF10). Molecular and biological analysis of these newer members has just begun. This review highlights what we currently know in respects to structural, functional and molecular properties of the N-Terminal RASSFs. PMID:21116130

Spectroscopic analysis of the NIR emission in Tm implanted Al{sub x}Ga{sub 1-x}N layers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rodrigues, J., E-mail: joana.catarina@ua.pt; Esteves, T. C.; Santos, N. F.

2016-08-28

Al{sub x}Ga{sub 1-x}N samples, with different AlN molar fractions, x = 0, 0.15, 0.77, and 1, grown by halide vapor phase epitaxy were implanted with Tm ions. Photoluminescence (PL) measurements revealed that after thermal annealing all the samples exhibit intraionic Tm{sup 3+} luminescence. In samples with x > 0, the low temperature emission is dominated by the lines that appear in the near infrared (NIR) spectral region, corresponding to the overlapped {sup 1}G{sub 4} → {sup 3}H{sub 5} and {sup 3}H{sub 4} → {sup 3}H{sub 6} multiplet transitions. A detailed spectroscopic analysis of NIR emission of the thulium implanted and annealed Al{sub x}Ga{sub 1-x}Nmore » layers is presented by using temperature dependent steady-state PL, room temperature PL excitation, and time resolved PL. The results indicate that the excitonic features sensitive to the alloy disorder are involved in the excitation population processes of the Tm{sup 3+} luminescence and the highest thermal stability for the NIR emission occurs for the AlN:Tm sample.« less

All-atom 3D structure prediction of transmembrane β-barrel proteins from sequences.

PubMed

Hayat, Sikander; Sander, Chris; Marks, Debora S; Elofsson, Arne

2015-04-28

Transmembrane β-barrels (TMBs) carry out major functions in substrate transport and protein biogenesis but experimental determination of their 3D structure is challenging. Encouraged by successful de novo 3D structure prediction of globular and α-helical membrane proteins from sequence alignments alone, we developed an approach to predict the 3D structure of TMBs. The approach combines the maximum-entropy evolutionary coupling method for predicting residue contacts (EVfold) with a machine-learning approach (boctopus2) for predicting β-strands in the barrel. In a blinded test for 19 TMB proteins of known structure that have a sufficient number of diverse homologous sequences available, this combined method (EVfold_bb) predicts hydrogen-bonded residue pairs between adjacent β-strands at an accuracy of ∼70%. This accuracy is sufficient for the generation of all-atom 3D models. In the transmembrane barrel region, the average 3D structure accuracy [template-modeling (TM) score] of top-ranked models is 0.54 (ranging from 0.36 to 0.85), with a higher (44%) number of residue pairs in correct strand-strand registration than in earlier methods (18%). Although the nonbarrel regions are predicted less accurately overall, the evolutionary couplings identify some highly constrained loop residues and, for FecA protein, the barrel including the structure of a plug domain can be accurately modeled (TM score = 0.68). Lower prediction accuracy tends to be associated with insufficient sequence information and we therefore expect increasing numbers of β-barrel families to become accessible to accurate 3D structure prediction as the number of available sequences increases.

Measurement of the 209Bi(n ,4 n )206Bi and 169Tm(n ,3 n )167Tm cross sections between 23.5 and 30.5 MeV relevant to reaction-in-flight neutron studies at the National Ignition Facility

NASA Astrophysics Data System (ADS)

Gooden, M. E.; Bredeweg, T. A.; Champine, B.; Combs, D. C.; Finch, S.; Hayes-Sterbenz, A.; Henry, E.; Krishichayan, Rundberg, R.; Tornow, W.; Wilhelmy, J.; Yeamans, C.

2017-08-01

At the National Ignition Facility, experiments are being performed to measure charged-particle stopping powers in the previously unexplored warm dense plasma regime. These measurements are done using reaction-in-flight (RIF) neutrons from an inertial confinement fusion system. RIF neutrons are produced with a continuum of energies up to 30 MeV. By making activation measurements utilizing threshold reactions for neutrons in the energy range of 15 Tm(n ,3 n )167Tm reaction has been used. However, in an effort to provide a secondary complimentary measurement, efforts are underway to make use of the 209Bi(n ,4 n )206Bi reaction, with a threshold of 22.5 MeV. The cross sections were measured at the 10 MV tandem Van De Graaff accelerator at the Triangle Universities Nuclear Laboratory with quasimonoenergetic neutrons between 23.5 and 30.5 MeV, where few previous measurements have been made. Cross-section data are compared to calculations and other available measurements.

Molecular insights into the m-AAA protease-mediated dislocation of transmembrane helices in the mitochondrial inner membrane.

PubMed

Lee, Seoeun; Lee, Hunsang; Yoo, Suji; Kim, Hyun

2017-12-08

Protein complexes involved in respiration, ATP synthesis, and protein import reside in the mitochondrial inner membrane; thus, proper regulation of these proteins is essential for cell viability. The m -AAA protease, a conserved hetero-hexameric AAA (ATPase associated with diverse cellular activities) protease, composed of the Yta10 and Yta12 proteins, regulates mitochondrial proteostasis by mediating protein maturation and degradation. It also recognizes and mediates the dislocation of membrane-embedded substrates, including foreign transmembrane (TM) segments, but the molecular mechanism involved in these processes remains elusive. This study investigated the role of the TM domains in the m -AAA protease by systematic replacement of one TM domain at a time in yeast. Our data indicated that replacement of the Yta10 TM2 domain abolishes membrane dislocation for only a subset of substrates, whereas replacement of the Yta12 TM2 domain impairs membrane dislocation for all tested substrates, suggesting different roles of the TM domains in each m -AAA protease subunit. Furthermore, m -AAA protease-mediated membrane dislocation was impaired in the presence of a large downstream hydrophilic moiety in a membrane substrate. This finding suggested that the m -AAA protease cannot dislocate large hydrophilic domains across the membrane, indicating that the membrane dislocation probably occurs in a lipid environment. In summary, this study highlights previously underappreciated biological roles of TM domains of the m -AAA proteases in mediating the recognition and dislocation of membrane-embedded substrates. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

TMDIM: an improved algorithm for the structure prediction of transmembrane domains of bitopic dimers

NASA Astrophysics Data System (ADS)

Cao, Han; Ng, Marcus C. K.; Jusoh, Siti Azma; Tai, Hio Kuan; Siu, Shirley W. I.

2017-09-01

α-Helical transmembrane proteins are the most important drug targets in rational drug development. However, solving the experimental structures of these proteins remains difficult, therefore computational methods to accurately and efficiently predict the structures are in great demand. We present an improved structure prediction method TMDIM based on Park et al. (Proteins 57:577-585, 2004) for predicting bitopic transmembrane protein dimers. Three major algorithmic improvements are introduction of the packing type classification, the multiple-condition decoy filtering, and the cluster-based candidate selection. In a test of predicting nine known bitopic dimers, approximately 78% of our predictions achieved a successful fit (RMSD <2.0 Å) and 78% of the cases are better predicted than the two other methods compared. Our method provides an alternative for modeling TM bitopic dimers of unknown structures for further computational studies. TMDIM is freely available on the web at https://cbbio.cis.umac.mo/TMDIM. Website is implemented in PHP, MySQL and Apache, with all major browsers supported.

The conserved glycine residues in the transmembrane domain of the Semliki Forest virus fusion protein are not required for assembly and fusion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liao Maofu; Kielian, Margaret

2005-02-05

The alphavirus Semliki Forest virus (SFV) infects cells via a low pH-triggered fusion reaction mediated by the viral E1 protein. Both the E1 fusion peptide and transmembrane (TM) domain are essential for membrane fusion, but the functional requirements for the TM domain are poorly understood. Here we explored the role of the five TM domain glycine residues, including the highly conserved glycine pair at E1 residues 415/416. SFV mutants with alanine substitutions for individual or all five glycine residues (5G/A) showed growth kinetics and fusion pH dependence similar to those of wild-type SFV. Mutants with increasing substitution of glycine residuesmore » showed an increasingly more stringent requirement for cholesterol during fusion. The 5G/A mutant showed decreased fusion kinetics and extent in fluorescent lipid mixing assays. TM domain glycine residues thus are not required for efficient SFV fusion or assembly but can cause subtle effects on the properties of membrane fusion.« less

Survival of Human Multiple Myeloma Cells Is Dependent on MUC1 C-Terminal Transmembrane Subunit Oncoprotein Function

PubMed Central

Yin, Li; Ahmad, Rehan; Kosugi, Michio; Kufe, Turner; Vasir, Baldev; Avigan, David; Kharbanda, Surender

2010-01-01

The MUC1 C-terminal transmembrane subunit (MUC1-C) oncoprotein is a direct activator of the canonical nuclear factor-κB (NF-κB) RelA/p65 pathway and is aberrantly expressed in human multiple myeloma cells. However, it is not known whether multiple myeloma cells are sensitive to the disruption of MUC1-C function for survival. The present studies demonstrate that peptide inhibitors of MUC1-C oligomerization block growth of human multiple myeloma cells in vitro. Inhibition of MUC1-C function also blocked the interaction between MUC1-C and NF-κB p65 and activation of the NF-κB pathway. In addition, inhibition of MUC1-C in multiple myeloma cells was associated with activation of the intrinsic apoptotic pathway and induction of late apoptosis/necrosis. Primary multiple myeloma cells, but not normal B-cells, were also sensitive to MUC1-C inhibition. Significantly, treatment of established U266 multiple myeloma xenografts growing in nude mice with a lead candidate MUC1-C inhibitor resulted in complete tumor regression and lack of recurrence. These findings indicate that multiple myeloma cells are dependent on intact MUC1-C function for constitutive activation of the canonical NF-κB pathway and for their growth and survival. PMID:20444960

Measurement of the Bi 209 ( n , 4 n ) Bi 206 and Tm 169 ( n , 3 n ) Tm 167 cross sections between 23.5 and 30.5 MeV relevant to reaction-in-flight neutron studies at the National Ignition Facility

DOE PAGES

Gooden, M. E.; Bredeweg, T. A.; Champine, B.; ...

2017-08-01

At the National Ignition Facility, experiments are being performed to measure charged-particle stopping powers in the previously unexplored warm dense plasma regime. These measurements are done using reaction-in-flight (RIF) neutrons from an inertial confinement fusion system. RIF neutrons are produced with a continuum of energies up to 30 MeV. By making activation measurements utilizing threshold reactions for neutrons in the energy range of 15 < E n < 30 MeV , the number of RIF neutrons can be determined and from this the stopping power of the deuterium and tritium ions that produced the RIF neutrons can be inferred. Currently,more » the 169 Tm ( n , 3 n ) 167 Tm reaction has been used. However, in an effort to provide a secondary complimentary measurement, efforts are underway to make use of the 209 Bi ( n , 4 n ) 206 Bi reaction, with a threshold of 22.5 MeV. The cross sections were measured at the 10 MV tandem Van De Graaff accelerator at the Triangle Universities Nuclear Laboratory with quasimonoenergetic neutrons between 23.5 and 30.5 MeV, where few previous measurements have been made. Cross-section data are compared to calculations and other available measurements.« less

Measurement of the Bi 209 ( n , 4 n ) Bi 206 and Tm 169 ( n , 3 n ) Tm 167 cross sections between 23.5 and 30.5 MeV relevant to reaction-in-flight neutron studies at the National Ignition Facility

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gooden, M. E.; Bredeweg, T. A.; Champine, B.

At the National Ignition Facility, experiments are being performed to measure charged-particle stopping powers in the previously unexplored warm dense plasma regime. These measurements are done using reaction-in-flight (RIF) neutrons from an inertial confinement fusion system. RIF neutrons are produced with a continuum of energies up to 30 MeV. By making activation measurements utilizing threshold reactions for neutrons in the energy range of 15 < E n < 30 MeV , the number of RIF neutrons can be determined and from this the stopping power of the deuterium and tritium ions that produced the RIF neutrons can be inferred. Currently,more » the 169 Tm ( n , 3 n ) 167 Tm reaction has been used. However, in an effort to provide a secondary complimentary measurement, efforts are underway to make use of the 209 Bi ( n , 4 n ) 206 Bi reaction, with a threshold of 22.5 MeV. The cross sections were measured at the 10 MV tandem Van De Graaff accelerator at the Triangle Universities Nuclear Laboratory with quasimonoenergetic neutrons between 23.5 and 30.5 MeV, where few previous measurements have been made. Cross-section data are compared to calculations and other available measurements.« less

Ser/Thr Motifs in Transmembrane Proteins: Conservation Patterns and Effects on Local Protein Structure and Dynamics

PubMed Central

del Val, Coral; White, Stephen H.

2014-01-01

We combined systematic bioinformatics analyses and molecular dynamics simulations to assess the conservation patterns of Ser and Thr motifs in membrane proteins, and the effect of such motifs on the structure and dynamics of α-helical transmembrane (TM) segments. We find that Ser/Thr motifs are often present in β-barrel TM proteins. At least one Ser/Thr motif is present in almost half of the sequences of α-helical proteins analyzed here. The extensive bioinformatics analyses and inspection of protein structures led to the identification of molecular transporters with noticeable numbers of Ser/Thr motifs within the TM region. Given the energetic penalty for burying multiple Ser/Thr groups in the membrane hydrophobic core, the observation of transporters with multiple membrane-embedded Ser/Thr is intriguing and raises the question of how the presence of multiple Ser/Thr affects protein local structure and dynamics. Molecular dynamics simulations of four different Ser-containing model TM peptides indicate that backbone hydrogen bonding of membrane-buried Ser/Thr hydroxyl groups can significantly change the local structure and dynamics of the helix. Ser groups located close to the membrane interface can hydrogen bond to solvent water instead of protein backbone, leading to an enhanced local solvation of the peptide. PMID:22836667

Non-equivalent role of TM2 gating hinges in heteromeric Kir4.1/Kir5.1 potassium channels

PubMed Central

Shang, Lijun

2007-01-01

Comparison of the crystal structures of the KcsA and MthK potassium channels suggests that the process of opening a K+ channel involves pivoted bending of the inner pore-lining helices at a highly conserved glycine residue. This bending motion is proposed to splay the transmembrane domains outwards to widen the gate at the “helix-bundle crossing”. However, in the inwardly rectifying (Kir) potassium channel family, the role of this “hinge” residue in the second transmembrane domain (TM2) and that of another putative glycine gating hinge at the base of TM2 remain controversial. We investigated the role of these two positions in heteromeric Kir4.1/Kir5.1 channels, which are unique amongst Kir channels in that both subunits lack a conserved glycine at the upper hinge position. Contrary to the effect seen in other channels, increasing the potential flexibility of TM2 by glycine substitutions at the upper hinge position decreases channel opening. Furthermore, the contribution of the Kir4.1 subunit to this process is dominant compared to Kir5.1, demonstrating a non-equivalent contribution of these two subunits to the gating process. A homology model of heteromeric Kir4.1/Kir5.1 shows that these upper “hinge” residues are in close contact with the base of the pore α-helix that supports the selectivity filter. Our results also indicate that the highly conserved glycine at the “lower” gating hinge position is required for tight packing of the TM2 helices at the helix-bundle crossing, rather than acting as a hinge residue. PMID:17657484

Corrector VX-809 stabilizes the first transmembrane domain of CFTR.

PubMed

Loo, Tip W; Bartlett, M Claire; Clarke, David M

2013-09-01

Processing mutations that inhibit folding and trafficking of CFTR are the main cause of cystic fibrosis (CF). A potential CF therapy would be to repair CFTR processing mutants. It has been demonstrated that processing mutants of P-glycoprotein (P-gp), CFTR's sister protein, can be efficiently repaired by a drug-rescue mechanism. Many arginine suppressors that mimic drug-rescue have been identified in the P-gp transmembrane (TM) domains (TMDs) that rescue by forming hydrogen bonds with residues in adjacent helices to promote packing of the TM segments. To test if CFTR mutants could be repaired by a drug-rescue mechanism, we used truncation mutants to test if corrector VX-809 interacted with the TMDs. VX-809 was selected for study because it is specific for CFTR, it is the most effective corrector identified to date, but it has limited clinical benefit. Identification of the VX-809 target domain will help to develop correctors with improved clinical benefits. It was found that VX-809 rescued truncation mutants lacking the NBD2 and R domains. When the remaining domains (TMD1, NBD1, TMD2) were expressed as separate polypeptides, VX-809 only increased the stability of TMD1. We then performed arginine mutagenesis on TM6 in TMD1. Although the results showed that TM6 had distinct lipid and aqueous faces, CFTR was different from P-gp as no arginine promoted maturation of CFTR processing mutants. The results suggest that TMD1 contains a VX-809 binding site, but its mechanism differed from P-gp drug-rescue. We also report that V510D acts as a universal suppressor to rescue CFTR processing mutants. Copyright © 2013 Elsevier Inc. All rights reserved.

TM grating coupler on low-loss LPCVD based Si3N4 waveguide platform

NASA Astrophysics Data System (ADS)

Dabos, G.; Manolis, A.; Giesecke, A. L.; Porschatis, C.; Chmielak, B.; Wahlbrink, T.; Pleros, N.; Tsiokos, D.

2017-12-01

We demonstrate, for the first time to our knowledge, a fully etched TM grating coupler for low-loss Low-Pressure-Chemical-Vapor-Deposition (LPCVD) based silicon nitride platform with a coupling loss of 6.5 dB at 1541 nm and a 1 dB bandwidth of 55 nm, addressing applications where TM polarization is a pre-requisite. The proposed GC and the 360 nm × 800 nm strip based Si3N4 waveguides have been fabricated by optical projection lithography using an i-line stepper tool enabling low-cost and mass manufacturing of photonic-integrated-circuits.

Effect of Phosphorylation on Interactions between Transmembrane Domains of SERCA and Phospholamban.

PubMed

Martin, Peter D; James, Zachary M; Thomas, David D

2018-06-05

We have used site-directed spin labeling and electron paramagnetic resonance (EPR) to map interactions between the transmembrane (TM) domains of the sarcoplasmic reticulum Ca 2+ -ATPase (SERCA) and phospholamban (PLB) as affected by PLB phosphorylation. In the cardiac sarcoplasmic reticulum, PLB binding to SERCA results in Ca-dependent enzyme inhibition, which is reversed by PLB phosphorylation at Ser16. Previous spectroscopic studies on SERCA-PLB have largely focused on the cytoplasmic domain of PLB, showing that phosphorylation induces a structural shift in this domain relative to SERCA. However, SERCA inhibition is due entirely to TM domain interactions. Therefore, we focus here on PLB's TM domain, attaching Cys-reactive spin labels at five different positions. In each case, continuous-wave EPR indicated moderate spin-label mobility, with the addition of SERCA revealing two populations, one indistinguishable from PLB alone and another with more restricted rotational mobility, presumably due to SERCA-binding. Phosphorylation had no effect on the rotational mobility of either component but significantly decreased the mole fraction of the restricted component. Solvent-accessibility experiments using power-saturation EPR and saturation-recovery EPR confirmed that these two spectral components were SERCA-bound and unbound PLB and showed that phosphorylation increased the overall lipid accessibility of the TM domain by increasing the fraction of unbound PLB. However-based on these results-at physiological levels of SERCA and PLB, most SERCA would have bound PLB even after phosphorylation. Additionally, no structural shift in the TM domain of SERCA-bound PLB was detected, as there were no significant changes in membrane insertion depth or its accessibility. Therefore, we conclude that under physiological conditions, the phosphorylation of PLB induces little or no change in the interaction of the TM domain with SERCA, so relief of inhibition is predominantly due to the

A Novel Topology of Proline-rich Transmembrane Protein 2 (PRRT2): HINTS FOR AN INTRACELLULAR FUNCTION AT THE SYNAPSE.

PubMed

Rossi, Pia; Sterlini, Bruno; Castroflorio, Enrico; Marte, Antonella; Onofri, Franco; Valtorta, Flavia; Maragliano, Luca; Corradi, Anna; Benfenati, Fabio

2016-03-18

Proline-rich transmembrane protein 2 (PRRT2) has been identified as the single causative gene for a group of paroxysmal syndromes of infancy, including epilepsy, paroxysmal movement disorders, and migraine. On the basis of topology predictions, PRRT2 has been assigned to the recently characterized family of Dispanins, whose members share the two-transmembrane domain topology with a large N terminus and short C terminus oriented toward the outside of the cell. Because PRRT2 plays a role at the synapse, it is important to confirm the exact orientation of its N and C termini with respect to the plasma membrane to get clues regarding its possible function. Using a combination of different experimental approaches, including live immunolabeling, immunogold electron microscopy, surface biotinylation and computational modeling, we demonstrate a novel topology for this protein. PRRT2 is a type II transmembrane protein in which only the second hydrophobic segment spans the plasma membrane, whereas the first one is associated with the internal surface of the membrane and forms a helix-loop-helix structure without crossing it. Most importantly, the large proline-rich N-terminal domain is not exposed to the extracellular space but is localized intracellularly, and only the short C terminus is extracellular (N cyt/C exo topology). Accordingly, we show that PRRT2 interacts with the Src homology 3 domain-bearing protein Intersectin 1, an intracellular protein involved in synaptic vesicle cycling. These findings will contribute to the clarification of the role of PRRT2 at the synapse and the understanding of pathogenic mechanisms on the basis of PRRT2-related neurological disorders. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Response of GWALP Transmembrane Peptides to Changes in the Tryptophan Anchor Positions†

PubMed Central

Vostrikov, Vitaly V.; Koeppe, Roger E.

2011-01-01

Trp side chains. A Trp side chain near the C-terminus adopts a different orientation and undergoes somewhat faster dynamics than a corresponding Trp side chain located an equivalent distance from the N-terminus. In contrast, as the inter-Trp distance changes, the variations among the average orientations of the Trp indole rings at either terminus are systematic yet fairly small. We conclude that subtle adjustments to the peptide tilt, and to the N- and C-terminal Trp side-chain torsion angles, permit the GWx,yALP23 peptides to maintain preferred transmembrane orientations while adapting to lipid bilayers of differing hydrophobic thickness. PMID:21800919

Fluorophores, environments, and quantification techniques in the analysis of transmembrane helix interaction using FRET.

PubMed

Khadria, Ambalika S; Senes, Alessandro

2015-07-01

Förster resonance energy transfer (FRET) has been widely used as a spectroscopic tool in vitro to study the interactions between transmembrane (TM) helices in detergent and lipid environments. This technique has been instrumental to many studies that have greatly contributed to quantitative understanding of the physical principles that govern helix-helix interactions in the membrane. These studies have also improved our understanding of the biological role of oligomerization in membrane proteins. In this review, we focus on the combinations of fluorophores used, the membrane mimetic environments, and measurement techniques that have been applied to study model systems as well as biological oligomeric complexes in vitro. We highlight the different formalisms used to calculate FRET efficiency and the challenges associated with accurate quantification. The goal is to provide the reader with a comparative summary of the relevant literature for planning and designing FRET experiments aimed at measuring TM helix-helix associations. © 2015 Wiley Periodicals, Inc.

Interacting helical surfaces of the transmembrane segments of subunits a and c' of the yeast V-ATPase defined by disulfide-mediated cross-linking.

PubMed

Kawasaki-Nishi, Shoko; Nishi, Tsuyoshi; Forgac, Michael

2003-10-24

Proton translocation by the vacuolar (H+)-ATPase (or V-ATPase) has been shown by mutagenesis to be dependent upon charged residues present within transmembrane segments of subunit a as well as the three proteolipid subunits (c, c', and c"). Interaction between R735 in TM7 of subunit a and the glutamic acid residue in the middle of TM4 of subunits c and c' or TM2 of subunit c" has been proposed to be essential for proton release to the luminal compartment. In order to determine whether the helical face of TM7 of subunit a containing R735 is capable of interacting with the helical face of TM4 of subunit c' containing the essential glutamic acid residue (Glu-145), cysteine-mediated cross-linking between these subunits in yeast has been performed. Cys-less forms of subunits a and c' as well as forms containing unique cysteine residues were constructed, introduced together into a strain disrupted in both endogenous subunits, and tested for growth at neutral pH, for assembly competence and for cross-linking in the presence of cupric-phenanthroline by SDS-PAGE and Western blot analysis. Four different cysteine mutants of subunit a were each tested pairwise with ten different unique cysteine mutants of subunit c'. Strong cross-linking was observed for the pairs aS728C/c'I142C, aA731C/c'E145C, aA738C/c'F143C, aA738C/c'L147C, and aL739C/c'L147C. Partial cross-linking was observed for an additional 13 of 40 pairs analyzed. When arrayed on a helical wheel diagram, the results suggest that the helical face of TM7 of subunit a containing Arg-735 interacts with the helical face of TM4 of subunit c' centered on Val-146 and bounded by Glu-145 and Leu-147. The results are consistent with a possible rotational flexibility of one or both of these transmembrane segments as well as some flexibility of movement perpendicular to the membrane.

Ends of the line for tmRNA-SmpB

DOE PAGES

Hudson, Corey M.; Lau, Britney Y.; Williams, Kelly P.

2014-08-13

Genes for the RNA tmRNA and protein SmpB, partners in the trans-translation process that rescues stalled ribosomes, have previously been found in all bacteria and some organelles. We validate recent identification of tmRNA homologs in oomycete mitochondria by finding partner genes from oomycete nuclei that target SmpB to the mitochondrion. Exhaustive search now identifies a small number of complete, often highly derived, bacterial genomes that appear to lack a functional copy of one or the other partner gene (but not both). Three groups with reduced genomes have lost the central loop of SmpB, which is thought to improve alanylation andmore » EF-Tu activation: Carsonella, Hodgkinia and the hemplasmas (hemotropic Mycoplasma). Carsonella has also lost the SmpB C-terminal tail, thought to stimulate the decoding center of the ribosome. Carsonella moreover exhibits gene overlap such that tmRNA maturation should produce a non-stop smpB mRNA, and one isolate exhibits complete degradation of the tmRNA gene yet its smpB shows no evidence for relaxed selective constraint. After loss of the SmpB central loop in the hemoplasmas, a subclade apparently lost tmRNA. At least some of the tmRNA/SmpB-deficient strains appear to further lack the ArfA and ArfB backup systems for ribosome rescue. The most frequent neighbors of smpB are the tmRNA gene, a ratA/rnfH unit, and the gene for RNaseR, a known physical and functional partner of tmRNA-SmpB. The tmRNA Website has moved and been updated, adding an SmpB sequence database (http://bioinformatics.sandia.gov/tmrna).« less

Diverse Reactivity of ECp* (E = Al, Ga) toward Low-Coordinate Transition Metal Amides [TM(N(SiMe3)2)2] (TM = Fe, Co, Zn): Insertion, Cp* Transfer, and Orthometalation.

PubMed

Weßing, Jana; Göbel, Christoph; Weber, Birgit; Gemel, Christian; Fischer, Roland A

2017-03-20

The reactivity of the carbenoid group 13 metal ligands ECp* (E = Al, Ga) toward low valent transition metal complexes [TM(btsa) 2 ] (TM = Fe, Co, Zn; btsa = bis(trimethylsilyl)amide) was investigated, revealing entirely different reaction patterns for E = Al and Ga. Treatment of [Co(btsa) 2 ] with AlCp* yields [Cp*Co(μ-H)(Al(κ 2 -(CH 2 SiMe 2 )NSiMe 3 )(btsa))] (1) featuring an unusual heterometallic bicyclic structure that results from the insertion of AlCp* into the TM-N bond with concomitant ligand rearrangement including C-H activation at one amide ligand. For [Fe(btsa) 2 ], complete ligand exchange gives FeCp* 2 , irrespective of the employed stoichiometric ratio of the reactants. In contrast, treatment of [TM(btsa) 2 ] (TM = Fe, Co) with GaCp* forms the 1:1 and 1:2 adducts [(GaCp*)Co(btsa) 2 ] (2) and [(GaCp*) 2 Fe(btsa) 2 ] (3), respectively. The tendency of AlCp* to undergo Cp* transfer to the TM center appears to be dependent on the nature of the TM center: For [Zn(btsa) 2 ], no Cp* transfer is observed on reaction with AlCp*; instead, the insertion product [Zn(Al(η 2 -Cp*)(btsa)) 2 ] (4) is formed. In the reaction of [Co(btsa) 2 ] with the trivalent [Cp*AlH 2 ], transfer of the amide ligands without further ligand rearrangement is observed, leading to [Co(μ-H) 4 (Al(η 2 -Cp*)(btsa)) 2 ] (5).

Magnetism, structures and stabilities of cluster assembled TM@Si nanotubes (TM = Cr, Mn and Fe): a density functional study.

PubMed

Dhaka, Kapil; Bandyopadhyay, Debashis

2016-08-02

The present study reports transition metal (TM = Cr, Mn and Fe) doped silicon nanotubes with tunable band structures and magnetic properties by careful selection of cluster assemblies as building blocks using the first-principles density functional theory. We found that the transition metal doping and in addition, the hydrogen termination process can stabilize the pure silicon nanoclusters or cluster assemblies and then it could be extended as magnetic nanotubes with finite magnetic moments. Study of the band structures and density of states (DOS) of different empty and TM doped nanotubes (Type 1 to Type 4) show that these nanotubes are useful as metals, semiconductors, semi-metals and half-metals. These designer magnetic materials could be useful in spintronics and magnetic devices of nanoscale order.

Functional characterization of transmembrane adenylyl cyclases from the honeybee brain.

PubMed

Balfanz, Sabine; Ehling, Petra; Wachten, Sebastian; Jordan, Nadine; Erber, Joachim; Mujagic, Samir; Baumann, Arnd

2012-06-01

The second messenger cAMP has a pivotal role in animals' physiology and behavior. Intracellular concentrations of cAMP are balanced by cAMP-synthesizing adenylyl cyclases (ACs) and cAMP-cleaving phosphodiesterases. Knowledge about ACs in the honeybee (Apis mellifera) is rather limited and only an ortholog of the vertebrate AC3 isoform has been functionally characterized, so far. Employing bioinformatics and functional expression we characterized two additional honeybee genes encoding membrane-bound (tm)ACs. The proteins were designated AmAC2t and AmAC8. Unlike the common structure of tmACs, AmAC2t lacks the first transmembrane domain. Despite this unusual topography, AmAC2t-activity could be stimulated by norepinephrine and NKH477 with EC(50s) of 0.07 μM and 3 μM. Both ligands stimulated AmAC8 with EC(50s) of 0.24 μM and 3.1 μM. In brain cryosections, intensive staining of mushroom bodies was observed with specific antibodies against AmAC8, an expression pattern highly reminiscent of the Drosophila rutabaga AC. In a current release of the honeybee genome database we identified three additional tmAC- and one soluble AC-encoding gene. These results suggest that (1) the AC-gene family in honeybees is comparably large as in other species, and (2) based on the restricted expression of AmAC8 in mushroom bodies, this enzyme might serve important functions in honeybee behavior. Copyright © 2012 Elsevier Ltd. All rights reserved.

Persistence of evolutionary memory: primordial six-transmembrane helical domain mu opiate receptors selectively linked to endogenous morphine signaling.

PubMed

Kream, Richard M; Sheehan, Melinda; Cadet, Patrick; Mantione, Kirk J; Zhu, Wei; Casares, Federico; Stefano, George B

2007-12-01

Biochemical, molecular and pharmacological evidence for two unique six-transmembrane helical (TMH) domain opiate receptors expressed from the micro opioid receptor (MOR) gene have been shown. Designated micro3 and micro4 receptors, both protein species are Class A rhodopsin-like members of the superfamily of G-protein coupled receptors but are selectively tailored to mediate the cellular regulatory effects of endogenous morphine and related morphinan alkaloids via stimulation of nitric oxide (NO) production and release. Both micro3 and micro4 receptors lack an amino acid sequence of approximately 90 amino acids that constitute the extracellular N-terminal and TMH1 domains and part of the first intracellular loop of the micro1 receptor, but retain the empirically defined ligand binding pocket distributed across conserved TMH2, TMH3, and TMH7 domains of the micro1 sequence. Additionally, the receptor proteins are terminated by unique intracellular C-terminal amino acid sequences that serve as putative coupling or docking domains required for constitutive NO synthase activation. Because the recognition profile of micro3 and micro4 receptors is restricted to rigid benzylisoquinoline alkaloids typified by morphine and its extended family of chemical congeners, it is hypothesized that conformational stabilization provided by interaction of extended extracellular N-terminal protein domains and the extracellular loops is required for binding of endogenous opioid peptides as well as synthetic flexible opiate alkaloids.

Topology of transmembrane channel-like gene 1 protein.

PubMed

Labay, Valentina; Weichert, Rachel M; Makishima, Tomoko; Griffith, Andrew J

2010-10-05

Mutations of transmembrane channel-like gene 1 (TMC1) cause hearing loss in humans and mice. TMC1 is the founding member of a family of genes encoding proteins of unknown function that are predicted to contain multiple transmembrane domains. The goal of our study was to define the topology of mouse TMC1 expressed heterologously in tissue culture cells. TMC1 was retained in the endoplasmic reticulum (ER) membrane of five tissue culture cell lines that we tested. We used anti-TMC1 and anti-HA antibodies to probe the topologic orientation of three native epitopes and seven HA epitope tags along full-length TMC1 after selective or complete permeabilization of transfected cells with digitonin or Triton X-100, respectively. TMC1 was present within the ER as an integral membrane protein containing six transmembrane domains and cytosolic N- and C-termini. There is a large cytoplasmic loop, between the fourth and fifth transmembrane domains, with two highly conserved hydrophobic regions that might associate with or penetrate, but do not span, the plasma membrane. Our study is the first to demonstrate that TMC1 is a transmembrane protein. The topologic organization revealed by this study shares some features with that of the shaker-TRP superfamily of ion channels.

An ankyrin-like protein with transmembrane domains is specifically lost after oncogenic transformation of human fibroblasts.

PubMed

Jaquemar, D; Schenker, T; Trueb, B

1999-03-12

We have identified a novel transformation-sensitive mRNA, which is present in cultured fibroblasts but is lacking in SV40 transformed cells as well as in many mesenchymal tumor cell lines. The corresponding gene is located on human chromosome 8 in band 8q13. The open reading frame of the mRNA encodes a protein of 1119 amino acids forming two distinct domains. The N-terminal domain consists of 18 repeats that are related to the cytoskeletal protein ankyrin. The C-terminal domain contains six putative transmembrane segments that resemble many ion channels. This overall structure is reminiscent of TRP-like proteins that function as store-operated calcium channels. The novel protein with an Mr of 130 kDa is expressed at a very low level in human fibroblasts and at a moderate level in liposarcoma cells. Overexpression in eukaryotic cells appears to interfere with normal growth, suggesting that it might play a direct or indirect role in signal transduction and growth control.

Modulating Transmembrane α-Helix Interactions through pH-Sensitive Boundary Residues.

PubMed

Ng, Derek P; Deber, Charles M

2016-08-09

Changes in pH can alter the structure and activity of proteins and may be used by the cell to control molecular function. This coupling can also be used in non-native applications through the design of pH-sensitive biomolecules. For example, the pH (low) insertion peptide (pHLIP) can spontaneously insert into a lipid bilayer when the pH decreases. We have previously shown that the α-helicity and helix-helix interactions of the TM2 α-helix of the proteolipid protein (PLP) are sensitive to the local hydrophobicity at its C-terminus. Given that there is an ionizable residue (Glu-88) at the C-terminus of this transmembrane (TM) segment, we hypothesized that changing the ionization state of this residue through pH may alter the local hydrophobicity of the peptide enough to affect both its secondary structure and helix-helix interactions. To examine this phenomenon, we synthesized peptide analogues of the PLP TM2 α-helix (wild-type sequence (66)AFQYVIYGTASFFFLYGALLLAEGF(90)). Using circular dichroism and Förster resonance energy transfer in the membrane-mimetic detergent sodium dodecyl sulfate, we found that a decrease in pH increases both peptide α-helicity and the extent of self-association. This pH-dependent effect is due specifically to the presence of Glu-88 at the C-terminus. Additional experiments in which Phe-90 was mutated to residues of varying hydrophobicities indicated that the strength of this effect is dependent on the local hydrophobicity near Glu-88. Our results have implications for the design of TM peptide switches and improve our understanding of how membrane protein structure and activity can be regulated through local molecular environmental changes.

Cytoskeletal tropomyosin Tm5NM1 is required for normal excitation-contraction coupling in skeletal muscle.

PubMed

Vlahovich, Nicole; Kee, Anthony J; Van der Poel, Chris; Kettle, Emma; Hernandez-Deviez, Delia; Lucas, Christine; Lynch, Gordon S; Parton, Robert G; Gunning, Peter W; Hardeman, Edna C

2009-01-01

The functional diversity of the actin microfilaments relies in part on the actin binding protein tropomyosin (Tm). The muscle-specific Tms regulate actin-myosin interactions and hence contraction. However, there is less known about the roles of the numerous cytoskeletal isoforms. We have shown previously that a cytoskeletal Tm, Tm5NM1, defines a Z-line adjacent cytoskeleton in skeletal muscle. Recently, we identified a second cytoskeletal Tm in this region, Tm4. Here we show that Tm4 and Tm5NM1 define separate actin filaments; the former associated with the terminal sarcoplasmic reticulum (SR) and other tubulovesicular structures. In skeletal muscles of Tm5NM1 knockout (KO) mice, Tm4 localization was unchanged, demonstrating the specificity of the membrane association. Tm5NM1 KO muscles exhibit potentiation of T-system depolarization and decreased force rundown with repeated T-tubule depolarizations consistent with altered T-tubule function. These results indicate that a Tm5NM1-defined actin cytoskeleton is required for the normal excitation-contraction coupling in skeletal muscle.

Cytoskeletal Tropomyosin Tm5NM1 Is Required for Normal Excitation–Contraction Coupling in Skeletal Muscle

PubMed Central

Vlahovich, Nicole; Kee, Anthony J.; Van der Poel, Chris; Kettle, Emma; Hernandez-Deviez, Delia; Lucas, Christine; Lynch, Gordon S.; Parton, Robert G.; Gunning, Peter W.

2009-01-01

The functional diversity of the actin microfilaments relies in part on the actin binding protein tropomyosin (Tm). The muscle-specific Tms regulate actin-myosin interactions and hence contraction. However, there is less known about the roles of the numerous cytoskeletal isoforms. We have shown previously that a cytoskeletal Tm, Tm5NM1, defines a Z-line adjacent cytoskeleton in skeletal muscle. Recently, we identified a second cytoskeletal Tm in this region, Tm4. Here we show that Tm4 and Tm5NM1 define separate actin filaments; the former associated with the terminal sarcoplasmic reticulum (SR) and other tubulovesicular structures. In skeletal muscles of Tm5NM1 knockout (KO) mice, Tm4 localization was unchanged, demonstrating the specificity of the membrane association. Tm5NM1 KO muscles exhibit potentiation of T-system depolarization and decreased force rundown with repeated T-tubule depolarizations consistent with altered T-tubule function. These results indicate that a Tm5NM1-defined actin cytoskeleton is required for the normal excitation–contraction coupling in skeletal muscle. PMID:19005216

The influenza virus neuraminidase protein transmembrane and head domains have coevolved.

PubMed

da Silva, Diogo V; Nordholm, Johan; Dou, Dan; Wang, Hao; Rossman, Jeremy S; Daniels, Robert

2015-01-15

Transmembrane domains (TMDs) from single-spanning membrane proteins are commonly viewed as membrane anchors for functional domains. Influenza virus neuraminidase (NA) exemplifies this concept, as it retains enzymatic function upon proteolytic release from the membrane. However, the subtype 1 NA TMDs have become increasingly more polar in human strains since 1918, which suggests that selection pressure exists on this domain. Here, we investigated the N1 TMD-head domain relationship by exchanging a prototypical "old" TMD (1933) with a "recent" (2009), more polar TMD and an engineered hydrophobic TMD. Each exchange altered the TMD association, decreased the NA folding efficiency, and significantly reduced viral budding and replication at 37°C compared to at 33°C, at which NA folds more efficiently. Passaging the chimera viruses at 37°C restored the NA folding efficiency, viral budding, and infectivity by selecting for NA TMD mutations that correspond with their polar or hydrophobic assembly properties. These results demonstrate that single-spanning membrane protein TMDs can influence distal domain folding, as well as membrane-related processes, and suggest the NA TMD in H1N1 viruses has become more polar to maintain compatibility with the evolving enzymatic head domain. The neuraminidase (NA) protein from influenza A viruses (IAVs) functions to promote viral release and is one of the major surface antigens. The receptor-destroying activity in NA resides in the distal head domain that is linked to the viral membrane by an N-terminal hydrophobic transmembrane domain (TMD). Over the last century, the subtype 1 NA TMDs (N1) in human H1N1 viruses have become increasingly more polar, and the head domains have changed to alter their antigenicity. Here, we provide the first evidence that an "old" N1 head domain from 1933 is incompatible with a "recent" (2009), more polar N1 TMD sequence and that, during viral replication, the head domain drives the selection of TMD

The influence of allosteric modulators and transmembrane mutations on desensitisation and activation of α7 nicotinic acetylcholine receptors

PubMed Central

Chatzidaki, Anna; D'Oyley, Jarryl M.; Gill-Thind, JasKiran K.; Sheppard, Tom D.; Millar, Neil S.

2015-01-01

Acetylcholine activates nicotinic acetylcholine receptors (nAChRs) by binding at an extracellular orthosteric site. Previous studies have described several positive allosteric modulators (PAMs) that are selective for homomeric α7 nAChRs. These include type I PAMs, which exert little or no effect on the rate of receptor desensitisation, and type II PAMs, which cause a dramatic loss of agonist-induced desensitisation. Here we report evidence that transmembrane mutations in α7 nAChRs have diverse effects on receptor activation and desensitisation by allosteric ligands. It has been reported previously that the L247T mutation, located toward the middle of the second transmembrane domain (at the 9′ position), confers reduced levels of desensitisation. In contrast, the M260L mutation, located higher up in the TM2 domain (at the 22′ position), does not show any difference in desensitisation compared to wild-type receptors. We have found that in receptors containing the L247T mutation, both type I PAMs and type II PAMs are converted into non-desensitising agonists. In contrast, in receptors containing the M260L mutation, this effect is seen only with type II PAMs. These findings, indicating that the M260L mutation has a selective effect on type II PAMs, have been confirmed both with previously described PAMs and also with a series of novel α7-selective PAMs. The novel PAMs examined in this study have close chemical similarity but diverse pharmacological properties. For example, they include compounds displaying effects on receptor desensitisation that are typical of classical type I and type II PAMs but, in addition, they include compounds with intermediate properties. PMID:25998276

[Bacterial synthesis, purification, and solubilization of transmembrane segments of ErbB family members].

PubMed

Goncharuk, M V; Shul'ga, A A; Ermoliuk, Ia S; Tkach, E N; Goncharuk, S A; Pustovalova, Iu E; Mineev, K S; Bocharov, É V; Maslennikov, I V; Arsen'ev, A S; Kirpichnikov, M P

2011-01-01

A family of epidermal growth factor receptors, ErbB, represents an important class of receptor tyrosine kinases, playing a leading role in cellular growth, development and differentiation. Transmembrane domains of these receptors transduce biochemical signals across plasma membrane via lateral homo- and heterodimerization. Relatively small size of complexes of ErbB transmembrane domains with detergents or lipids allows one to study their detailed spatial structure using three-dimensional heteronuclear high-resolution NMR spectroscopy. Here, we describe the effective expression system and purification procedure for preparative-scale production of transmembrane peptides from four representatives of ErbB family, ErbB1, ErbB2, ErbB3, ErbB4, for structural studies. The recombinant peptides were produced in Escherichia coli BL21(DE3)pLysS as C-terminal extensions of thioredoxin A. The fusion protein cleavage was accomplished with the light subunit of human enterokinase. Several (10-30) milligrams of purified isotope-labeled transmembrane peptides were isolated with the use of a simple and convenient procedure, which consists of consecutive steps of immobilized metal affinity chromatography and cation-exchange chromatography. The purified peptides were reconstituted in lipid/detergent environment (micelles or bicelles) and characterized using dynamic light scattering, CD and NMR spectroscopy. The data obtained indicate that the purified ErbB transmembrane peptides are suitable for structural and dynamic studies of their homo- and heterodimer complexes using high resolution NMR spectroscopy.

Structural basis for substrate recognition by the human N-terminal methyltransferase 1

DOE PAGES

Dong, Cheng; Mao, Yunfei; Tempel, Wolfram; ...

2015-11-05

α-N-terminal methylation represents a highly conserved and prevalent post-translational modification, yet its biological function has remained largely speculative. The recent discovery of α-N-terminal methyltransferase 1 (NTMT1) and its physiological substrates propels the elucidation of a general role of α-N-terminal methylation in mediating DNA-binding ability of the modified proteins. The phenotypes, observed from both NTMT1 knockdown in breast cancer cell lines and knockout mouse models, suggest the potential involvement of α-N-terminal methylation in DNA damage response and cancer development. In this study, we report the first crystal structures of human NTMT1 in complex with cofactor S-adenosyl-L-homocysteine (SAH) and six substrate peptides,more » respectively, and reveal that NTMT1 contains two characteristic structural elements (a β hairpin and an N-terminal extension) that contribute to its substrate specificity. Our complex structures, coupled with mutagenesis, binding, and enzymatic studies, also present the key elements involved in locking the consensus substrate motif XPK (X indicates any residue type other than D/E) into the catalytic pocket for α-N-terminal methylation and explain why NTMT1 prefers an XPK sequence motif. We propose a catalytic mechanism for α-N-terminal methylation. Overall, this study gives us the first glimpse of the molecular mechanism of α-N-terminal methylation and potentially contributes to the advent of therapeutic agents for human diseases associated with deregulated α-N-terminal methylation.« less

Neutralizing Epitopes in the Membrane-Proximal External Region of HIV-1 gp41 Are Influenced by the Transmembrane Domain and the Plasma Membrane

PubMed Central

Montero, Marinieve; Klaric, Kristina-Ana; Donald, Jason E.; Lepik, Christa; Wu, Sampson; Tsai, Sue; Julien, Jean-Philippe; Hessell, Ann J.; Wang, Shixia; Lu, Shan; Burton, Dennis R.; Pai, Emil F.; DeGrado, William F.

2012-01-01

Failure to elicit broadly neutralizing (bNt) antibodies (Abs) against the membrane-proximal external region of HIV-1 gp41 (MPER) reflects the difficulty of mimicking its neutralization-competent structure (NCS). Here, we analyzed MPER antigenicity in the context of the plasma membrane and identified a role for the gp41 transmembrane domain (TM) in exposing the epitopes of three bNt monoclonal Abs (MAbs) (2F5, 4E10, and Z13e1). We transiently expressed DNA constructs encoding gp41 ectodomain fragments fused to either the TM of the platelet-derived growth factor receptor (PDGFR) or the gp41 TM and cytoplasmic tail domain (CT). Constructs encoding the MPER tethered to the gp41 TM followed by a 27-residue CT fragment (MPER-TM1) produced optimal MAb binding. Critical binding residues for the three Nt MAbs were identified using a panel of 24 MPER-TM1 mutants bearing single amino acid substitutions in the MPER; many were previously shown to affect MAb-mediated viral neutralization. Moreover, non-Nt mutants of MAbs 2F5 and 4E10 exhibited a reduction in binding to MPER-TM1 and yet maintained binding to synthetic MPER peptides, indicating that MPER-TM1 better approximates the MPER NCS than peptides. Replacement of the gp41 TM and CT of MPER-TM1 with the PDGFR TM reduced binding by MAb 4E10, but not 2F5, indicating that the gp41 TM plays a pivotal role in orienting the 4E10 epitope, and more globally, in affecting MPER exposure. PMID:22238313

Oxidative Folding and N-terminal Cyclization of Onconase+

PubMed Central

Welker, Ervin; Hathaway, Laura; Xu, Guoqiang; Narayan, Mahesh; Pradeep, Lovy; Shin, Hang-Cheol; Scheraga, Harold A.

2008-01-01

Cyclization of the N-terminal glutamine residue to pyroglutamic acid in onconase, an anti-cancer chemotherapeutic agent, increases the activity and stability of the protein. Here, we examine the correlated effects of the folding/unfolding process and the formation of this N-terminal pyroglutamic acid. The results in this study indicate that cyclization of the N-terminal glutamine has no significant effect on the rate of either reductive unfolding or oxidative folding of the protein. Both the cyclized and uncyclized proteins seem to follow the same oxidative folding pathways; however, cyclization altered the relative flux of the protein in these two pathways by increasing the rate of formation of a kinetically trapped intermediate. Glutaminyl cyclase (QC) catalyzed the cyclization of the unfolded, reduced protein, but had no effect on the disulfide-intact, uncyclized, folded protein. The structured intermediates of uncyclized onconase were also resistant to QC-catalysis, consistent with their having a native-like fold. These observations suggest that, in vivo, cyclization takes place during the initial stages of oxidative folding, specifically, before the formation of structured intermediates. The competition between oxidative folding and QC-mediated cyclization suggests that QC-catalyzed cyclization of the N-terminal glutamine in onconase occurs in the endoplasmic reticulum, probably co-translationally. PMID:17439243

ε-Poly-l-Lysine Peptide Chain Length Regulated by the Linkers Connecting the Transmembrane Domains of ε-Poly-l-Lysine Synthetase

PubMed Central

Kito, Naoko; Kita, Akihiro; Imokawa, Yuuki; Yamanaka, Kazuya; Maruyama, Chitose; Katano, Hajime

2014-01-01

ε-Poly-l-lysine (ε-PL), consisting of 25 to 35 l-lysine residues with linkages between the α-carboxyl groups and ε-amino groups, is produced by Streptomyces albulus NBRC14147. ε-PL synthetase (Pls) is a membrane protein with six transmembrane domains (TM1 to TM6) as well as both an adenylation domain and a thiolation domain, characteristic of the nonribosomal peptide synthetases. Pls directly generates ε-PL chain length diversity (25- to 35-mer), but the processes that control the chain length of ε-PL during the polymerization reaction are still not fully understood. Here, we report on the identification of Pls amino acid residues involved in the regulation of the ε-PL chain length. From approximately 12,000 variants generated by random mutagenesis, we found 8 Pls variants that produced shorter chains of ε-PL. These variants have one or more mutations in two linker regions connecting the TM1 and TM2 domains and the TM3 and TM4 domains. In the Pls catalytic mechanism, the growing chain of ε-PL is not tethered to the enzyme, implying that the enzyme must hold the growing chain until the polymerization reaction is complete. Our findings reveal that the linker regions are important contributors to grasp the growing chain of ε-PL. PMID:24907331

ε-Poly-L-lysine peptide chain length regulated by the linkers connecting the transmembrane domains of ε-Poly-L-lysine synthetase.

PubMed

Hamano, Yoshimitsu; Kito, Naoko; Kita, Akihiro; Imokawa, Yuuki; Yamanaka, Kazuya; Maruyama, Chitose; Katano, Hajime

2014-08-01

ε-Poly-l-lysine (ε-PL), consisting of 25 to 35 l-lysine residues with linkages between the α-carboxyl groups and ε-amino groups, is produced by Streptomyces albulus NBRC14147. ε-PL synthetase (Pls) is a membrane protein with six transmembrane domains (TM1 to TM6) as well as both an adenylation domain and a thiolation domain, characteristic of the nonribosomal peptide synthetases. Pls directly generates ε-PL chain length diversity (25- to 35-mer), but the processes that control the chain length of ε-PL during the polymerization reaction are still not fully understood. Here, we report on the identification of Pls amino acid residues involved in the regulation of the ε-PL chain length. From approximately 12,000 variants generated by random mutagenesis, we found 8 Pls variants that produced shorter chains of ε-PL. These variants have one or more mutations in two linker regions connecting the TM1 and TM2 domains and the TM3 and TM4 domains. In the Pls catalytic mechanism, the growing chain of ε-PL is not tethered to the enzyme, implying that the enzyme must hold the growing chain until the polymerization reaction is complete. Our findings reveal that the linker regions are important contributors to grasp the growing chain of ε-PL. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

A Membrane-Bound NAC Transcription Factor, ANAC017, Mediates Mitochondrial Retrograde Signaling in Arabidopsis[W][OPEN

PubMed Central

Ng, Sophia; Ivanova, Aneta; Duncan, Owen; Law, Simon R.; Van Aken, Olivier; De Clercq, Inge; Wang, Yan; Carrie, Chris; Xu, Lin; Kmiec, Beata; Walker, Hayden; Van Breusegem, Frank; Whelan, James; Giraud, Estelle

2013-01-01

Plants require daily coordinated regulation of energy metabolism for optimal growth and survival and therefore need to integrate cellular responses with both mitochondrial and plastid retrograde signaling. Using a forward genetic screen to characterize regulators of alternative oxidase1a (rao) mutants, we identified RAO2/Arabidopsis NAC domain-containing protein17 (ANAC017) as a direct positive regulator of AOX1a. RAO2/ANAC017 is targeted to connections and junctions in the endoplasmic reticulum (ER) and F-actin via a C-terminal transmembrane (TM) domain. A consensus rhomboid protease cleavage site is present in ANAC017 just prior to the predicted TM domain. Furthermore, addition of the rhomboid protease inhibitor N-p-Tosyl-l-Phe chloromethyl abolishes the induction of AOX1a upon antimycin A treatment. Simultaneous fluorescent tagging of ANAC017 with N-terminal red fluorescent protein (RFP) and C-terminal green fluorescent protein (GFP) revealed that the N-terminal RFP domain migrated into the nucleus, while the C-terminal GFP tag remained in the ER. Genome-wide analysis of the transcriptional network regulated by RAO2/ANAC017 under stress treatment revealed that RAO2/ANAC017 function was necessary for >85% of the changes observed as a primary response to cytosolic hydrogen peroxide (H2O2), but only ∼33% of transcriptional changes observed in response to antimycin A treatment. Plants with mutated rao2/anac017 were more stress sensitive, whereas a gain-of-function mutation resulted in plants that had lower cellular levels of H2O2 under untreated conditions. PMID:24045017

TM4SF5 promotes metastatic behavior of cells in 3D extracellular matrix gels by reducing dependency on environmental cues

PubMed Central

Nam, Seo Hee; Cheong, Jin-Gyu; Jeong, Doyoung; Lee, Seo-Jin; Pan, Cheol-Ho; Jung, Jae Woo; Kim, Hye-Jin; Ryu, Jihye; Kim, Ji Eon; Kim, Somi; Cho, Chang Yun; Kang, Min-Kyung; Lee, Kyung-Min; Lee, Jung Weon

2017-01-01

Transmembrane 4 L six family member 5 (TM4SF5) is highly expressed in hepatocellular carcinoma tissues and enhances migration in two-dimensional environments. Here, we investigated how TM4SF5 is involved in diverse pro-metastatic phenotypes in in vivo-like three-dimensional (3D) extracellular matrix gels. TM4SF5-positive cells aggressively formed invasive foci in 3D Matrigel, depending on TM4SF5-mediated signaling activity, cytoskeletal organization, and matrix metallopeptidase (MMP) 2-mediated extracellular remodeling, whereas TM4SF5-null cells did not. The TM4SF5-null cells did, however, form invasive foci in 3D Matrigel following inhibition of Rho-associated protein kinase or addition of collagen I, suggesting that collagen I compensated for TM4SF5 expression. Similarly, TM4SF5-positive cells expressing vascular endothelial-cadherin formed network-like vasculogenic mimicry in 3D Matrigel and collagen I mixture gels, whereas TM4SF5-negative cells in the mixture gels displayed the network structures only upon further treatment with epidermal growth factor. The foci formation also required MMP2-mediated remodeling of the extracellular matrix. Co-cultures exhibited TM4SF5-positive or cancer-associated fibroblasts at the outward edges of TM4SF5-null cell clusters. Compared with TM4SF5-null cells, TM4SF5-positive cells in 3D collagen gels showed a more invasive outgrowth with dramatic invadopodia. These observations suggest that TM4SF5 plays roles in the promotion of diverse metastatic properties with fewer environmental requirements than TM4SF5-negative cells. PMID:29137358

TMDIM: an improved algorithm for the structure prediction of transmembrane domains of bitopic dimers.

PubMed

Cao, Han; Ng, Marcus C K; Jusoh, Siti Azma; Tai, Hio Kuan; Siu, Shirley W I

2017-09-01

[Formula: see text]-Helical transmembrane proteins are the most important drug targets in rational drug development. However, solving the experimental structures of these proteins remains difficult, therefore computational methods to accurately and efficiently predict the structures are in great demand. We present an improved structure prediction method TMDIM based on Park et al. (Proteins 57:577-585, 2004) for predicting bitopic transmembrane protein dimers. Three major algorithmic improvements are introduction of the packing type classification, the multiple-condition decoy filtering, and the cluster-based candidate selection. In a test of predicting nine known bitopic dimers, approximately 78% of our predictions achieved a successful fit (RMSD <2.0 Å) and 78% of the cases are better predicted than the two other methods compared. Our method provides an alternative for modeling TM bitopic dimers of unknown structures for further computational studies. TMDIM is freely available on the web at https://cbbio.cis.umac.mo/TMDIM . Website is implemented in PHP, MySQL and Apache, with all major browsers supported.

Core Transmembrane Domain 6 Plays a Pivotal Role in the Transport Cycle of the Sodium/Proline Symporter PutP*

PubMed Central

Bracher, Susanne; Schmidt, Claudia C.; Dittmer, Sophie I.; Jung, Heinrich

2016-01-01

Crystal structures of transporters with a LeuT-type structural fold assign core transmembrane domain 6 (TM6′) a central role in substrate binding and translocation. Here, the function of TM6′ in the sodium/proline symporter PutP, a member of the solute/sodium symporter family, was investigated. A complete scan of TM6′ identified eight amino acids as particularly important for PutP function. Of these residues, Tyr-248, His-253, and Arg-257 impact sodium binding, whereas Arg-257 and Ala-260 may participate in interactions leading to closure of the inner gate. Furthermore, the previous suggestion of an involvement of Trp-244, Tyr-248, and Pro-252 in proline binding is further supported. In addition, substitution of Gly-245, Gly-247, and Gly-250 affects the amount of PutP in the membrane. A Cys accessibility analysis suggests an involvement of the inner half of TM6′ in the formation of a hydrophilic pathway that is open to the inside in the absence of ligands and closed in the presence of sodium and proline. In conclusion, the results demonstrate that TM6′ plays a central role in substrate binding and release on the inner side of the membrane also in PutP and extend the knowledge on functionally relevant amino acids in transporters with a LeuT-type structural fold. PMID:27793991

Obif, a Transmembrane Protein, Is Required for Bone Mineralization and Spermatogenesis in Mice

PubMed Central

Mizuhashi, Koji; Chaya, Taro; Kanamoto, Takashi; Omori, Yoshihiro; Furukawa, Takahisa

2015-01-01

Background Various kinds of transmembrane and secreted proteins play pivotal roles in development through cell-cell communication. We previously reported that Obif (Osteoblast induction factor, Tmem119), encoding a single transmembrane protein, is expressed in differentiating osteoblasts, and that Obif−/− mice exhibit significantly reduced bone volume in the femur. In the current study, we characterized the Obif protein and further investigated the biological phenotypes of a variety of tissues in Obif−/− mice. Results First, we found that O-glycosylation of the Obif protein occurs at serine residue 36 in the Obif extracellular domain. Next, we observed that Obif−/− mice exhibit bone dysplasia in association with significantly increased osteoid volume per osteoid surface (OV/OS) and osteoid maturation time (Omt), and significantly decreased mineral apposition rate (MAR) and bone formation rate per bone surface (BFR/BS). In addition, we observed that Obif−/− mice show a significant decrease in testis weight as well as in sperm number. By histological analysis, we found that Obif is expressed in spermatocytes and spermatids in the developing testis and that spermatogenesis is halted at the round spermatid stage in the Obif−/− testis that lacks sperm. However, the number of litters fathered by male mice was slightly reduced in Obif−/− mice compared with wild-type mice, although this was not statistically significant. Conclusions Our results, taken together with previous observations, indicate that Obif is a type Ia transmembrane protein whose N-terminal region is O-glycosylated. In addition, we found that Obif is required for normal bone mineralization and late testicular differentiation in vivo. These findings suggest that Obif plays essential roles in the development of multiple tissues. PMID:26207632

Obif, a Transmembrane Protein, Is Required for Bone Mineralization and Spermatogenesis in Mice.

PubMed

Mizuhashi, Koji; Chaya, Taro; Kanamoto, Takashi; Omori, Yoshihiro; Furukawa, Takahisa

2015-01-01

Various kinds of transmembrane and secreted proteins play pivotal roles in development through cell-cell communication. We previously reported that Obif (Osteoblast induction factor, Tmem119), encoding a single transmembrane protein, is expressed in differentiating osteoblasts, and that Obif-/- mice exhibit significantly reduced bone volume in the femur. In the current study, we characterized the Obif protein and further investigated the biological phenotypes of a variety of tissues in Obif-/- mice. First, we found that O-glycosylation of the Obif protein occurs at serine residue 36 in the Obif extracellular domain. Next, we observed that Obif-/- mice exhibit bone dysplasia in association with significantly increased osteoid volume per osteoid surface (OV/OS) and osteoid maturation time (Omt), and significantly decreased mineral apposition rate (MAR) and bone formation rate per bone surface (BFR/BS). In addition, we observed that Obif-/- mice show a significant decrease in testis weight as well as in sperm number. By histological analysis, we found that Obif is expressed in spermatocytes and spermatids in the developing testis and that spermatogenesis is halted at the round spermatid stage in the Obif-/- testis that lacks sperm. However, the number of litters fathered by male mice was slightly reduced in Obif-/- mice compared with wild-type mice, although this was not statistically significant. Our results, taken together with previous observations, indicate that Obif is a type Ia transmembrane protein whose N-terminal region is O-glycosylated. In addition, we found that Obif is required for normal bone mineralization and late testicular differentiation in vivo. These findings suggest that Obif plays essential roles in the development of multiple tissues.

Transmembrane Tumor Necrosis Factor Controls Myeloid-Derived Suppressor Cell Activity via TNF Receptor 2 and Protects from Excessive Inflammation during BCG-Induced Pleurisy

PubMed Central

Chavez-Galan, Leslie; Vesin, Dominique; Uysal, Husnu; Blaser, Guillaume; Benkhoucha, Mahdia; Ryffel, Bernhard; Quesniaux, Valérie F. J.; Garcia, Irene

2017-01-01

Pleural tuberculosis (TB) is a form of extra-pulmonary TB observed in patients infected with Mycobacterium tuberculosis. Accumulation of myeloid-derived suppressor cells (MDSC) has been observed in animal models of TB and in human patients but their role remains to be fully elucidated. In this study, we analyzed the role of transmembrane TNF (tmTNF) in the accumulation and function of MDSC in the pleural cavity during an acute mycobacterial infection. Mycobacterium bovis BCG-induced pleurisy was resolved in mice expressing tmTNF, but lethal in the absence of tumor necrosis factor. Pleural infection induced MDSC accumulation in the pleural cavity and functional MDSC required tmTNF to suppress T cells as did pleural wild-type MDSC. Interaction of MDSC expressing tmTNF with CD4 T cells bearing TNF receptor 2 (TNFR2), but not TNFR1, was required for MDSC suppressive activity on CD4 T cells. Expression of tmTNF attenuated Th1 cell-mediated inflammatory responses generated by the acute pleural mycobacterial infection in association with effective MDSC expressing tmTNF and interacting with CD4 T cells expressing TNFR2. In conclusion, this study provides new insights into the crucial role played by the tmTNF/TNFR2 pathway in MDSC suppressive activity required during acute pleural infection to attenuate excessive inflammation generated by the infection. PMID:28890718

The N-terminal strand modulates immunoglobulin light chain fibrillogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pozo-Yauner, Luis del, E-mail: ldelpozo@inmegen.gob.mx; Wall, Jonathan S.; González Andrade, Martín

2014-01-10

Highlights: •We evaluated the impact of mutations in the N-terminal strand of 6aJL2 protein. •Mutations destabilized the protein in a position-dependent manner. •Destabilizing mutations accelerated the fibrillogenesis by shortening the lag time. •The effect on the kinetic of fibril elongation by seeding was of different nature. •The N-terminal strand is buried in the fibrillar state of 6aJL2 protein. -- Abstract: It has been suggested that the N-terminal strand of the light chain variable domain (V{sub L}) protects the molecule from aggregation by hindering spurious intermolecular contacts. We evaluated the impact of mutations in the N-terminal strand on the thermodynamic stabilitymore » and kinetic of fibrillogenesis of the V{sub L} protein 6aJL2. Mutations in this strand destabilized the protein in a position-dependent manner, accelerating the fibrillogenesis by shortening the lag time; an effect that correlated with the extent of destabilization. In contrast, the effect on the kinetics of fibril elongation, as assessed in seeding experiments was of different nature, as it was not directly dependant on the degree of destabilization. This finding suggests different factors drive the nucleation-dependent and elongation phases of light chain fibrillogenesis. Finally, taking advantage of the dependence of the Trp fluorescence upon environment, four single Trp substitutions were made in the N-terminal strand, and changes in solvent exposure during aggregation were evaluated by acrylamide-quenching. The results suggest that the N-terminal strand is buried in the fibrillar state of 6aJL2 protein. This finding suggest a possible explanation for the modulating effect exerted by the mutations in this strand on the aggregation behavior of 6aJL2 protein.« less

WRF-TMH: predicting transmembrane helix by fusing composition index and physicochemical properties of amino acids.

PubMed

Hayat, Maqsood; Khan, Asifullah

2013-05-01

Membrane protein is the prime constituent of a cell, which performs a role of mediator between intra and extracellular processes. The prediction of transmembrane (TM) helix and its topology provides essential information regarding the function and structure of membrane proteins. However, prediction of TM helix and its topology is a challenging issue in bioinformatics and computational biology due to experimental complexities and lack of its established structures. Therefore, the location and orientation of TM helix segments are predicted from topogenic sequences. In this regard, we propose WRF-TMH model for effectively predicting TM helix segments. In this model, information is extracted from membrane protein sequences using compositional index and physicochemical properties. The redundant and irrelevant features are eliminated through singular value decomposition. The selected features provided by these feature extraction strategies are then fused to develop a hybrid model. Weighted random forest is adopted as a classification approach. We have used two benchmark datasets including low and high-resolution datasets. tenfold cross validation is employed to assess the performance of WRF-TMH model at different levels including per protein, per segment, and per residue. The success rates of WRF-TMH model are quite promising and are the best reported so far on the same datasets. It is observed that WRF-TMH model might play a substantial role, and will provide essential information for further structural and functional studies on membrane proteins. The accompanied web predictor is accessible at http://111.68.99.218/WRF-TMH/ .

The Transmembrane Region of Guard Cell SLAC1 Channels Perceives CO2 Signals via an ABA-Independent Pathway in Arabidopsis

PubMed Central

Yamamoto, Yoshiko; Negi, Juntaro; Isogai, Yasuhiro; Schroeder, Julian I.; Iba, Koh

2016-01-01

The guard cell S-type anion channel, SLOW ANION CHANNEL1 (SLAC1), a key component in the control of stomatal movements, is activated in response to CO2 and abscisic acid (ABA). Several amino acids existing in the N-terminal region of SLAC1 are involved in regulating its activity via phosphorylation in the ABA response. However, little is known about sites involved in CO2 signal perception. To dissect sites that are necessary for the stomatal CO2 response, we performed slac1 complementation experiments using transgenic plants expressing truncated SLAC1 proteins. Measurements of gas exchange and stomatal apertures in the truncated transgenic lines in response to CO2 and ABA revealed that sites involved in the stomatal CO2 response exist in the transmembrane region and do not require the SLAC1 N and C termini. CO2 and ABA regulation of S-type anion channel activity in guard cells of the transgenic lines confirmed these results. In vivo site-directed mutagenesis experiments targeted to amino acids within the transmembrane region of SLAC1 raise the possibility that two tyrosine residues exposed on the membrane are involved in the stomatal CO2 response. PMID:26764376

COMSAT: Residue contact prediction of transmembrane proteins based on support vector machines and mixed integer linear programming.

PubMed

Zhang, Huiling; Huang, Qingsheng; Bei, Zhendong; Wei, Yanjie; Floudas, Christodoulos A

2016-03-01

In this article, we present COMSAT, a hybrid framework for residue contact prediction of transmembrane (TM) proteins, integrating a support vector machine (SVM) method and a mixed integer linear programming (MILP) method. COMSAT consists of two modules: COMSAT_SVM which is trained mainly on position-specific scoring matrix features, and COMSAT_MILP which is an ab initio method based on optimization models. Contacts predicted by the SVM model are ranked by SVM confidence scores, and a threshold is trained to improve the reliability of the predicted contacts. For TM proteins with no contacts above the threshold, COMSAT_MILP is used. The proposed hybrid contact prediction scheme was tested on two independent TM protein sets based on the contact definition of 14 Å between Cα-Cα atoms. First, using a rigorous leave-one-protein-out cross validation on the training set of 90 TM proteins, an accuracy of 66.8%, a coverage of 12.3%, a specificity of 99.3% and a Matthews' correlation coefficient (MCC) of 0.184 were obtained for residue pairs that are at least six amino acids apart. Second, when tested on a test set of 87 TM proteins, the proposed method showed a prediction accuracy of 64.5%, a coverage of 5.3%, a specificity of 99.4% and a MCC of 0.106. COMSAT shows satisfactory results when compared with 12 other state-of-the-art predictors, and is more robust in terms of prediction accuracy as the length and complexity of TM protein increase. COMSAT is freely accessible at http://hpcc.siat.ac.cn/COMSAT/. © 2016 Wiley Periodicals, Inc.

Crystallization and preliminary X-ray diffraction analysis of P30, the transmembrane domain of pertactin, an autotransporter from Bordetella pertussis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, Yanshi; Black, Isobel; Roszak, Aleksander W.

2007-07-01

P30, the transmembrane C-terminal domain of pertactin from B. pertussis has been crystallized after refolding in vitro. Preliminary X-ray crystallographic data are reported. P30, the 32 kDa transmembrane C-terminal domain of pertactin from Bordetella pertussis, is supposed to form a β-barrel inserted into the outer membrane for the translocation of the passenger domain. P30 was cloned and expressed in inclusion bodies in Escherichia coli. After refolding and purification, the protein was crystallized using the sitting-drop vapour-diffusion method at 292 K. The crystals diffract to a resolution limit of 3.5 Å using synchrotron radiation and belong to the hexagonal space groupmore » P6{sub 1}22, with unit-cell parameters a = b = 123.27, c = 134.43 Å.« less

Identification and Functional Characterization of N-Terminally Acetylated Proteins in Drosophila melanogaster

PubMed Central

Gerrits, Bertran; Roschitzki, Bernd; Mohanty, Sonali; Niederer, Eva M.; Laczko, Endre; Timmerman, Evy; Lange, Vinzenz; Hafen, Ernst; Aebersold, Ruedi; Vandekerckhove, Joël; Basler, Konrad; Ahrens, Christian H.; Gevaert, Kris; Brunner, Erich

2009-01-01

Protein modifications play a major role for most biological processes in living organisms. Amino-terminal acetylation of proteins is a common modification found throughout the tree of life: the N-terminus of a nascent polypeptide chain becomes co-translationally acetylated, often after the removal of the initiating methionine residue. While the enzymes and protein complexes involved in these processes have been extensively studied, only little is known about the biological function of such N-terminal modification events. To identify common principles of N-terminal acetylation, we analyzed the amino-terminal peptides from proteins extracted from Drosophila Kc167 cells. We detected more than 1,200 mature protein N-termini and could show that N-terminal acetylation occurs in insects with a similar frequency as in humans. As the sole true determinant for N-terminal acetylation we could extract the (X)PX rule that indicates the prevention of acetylation under all circumstances. We could show that this rule can be used to genetically engineer a protein to study the biological relevance of the presence or absence of an acetyl group, thereby generating a generic assay to probe the functional importance of N-terminal acetylation. We applied the assay by expressing mutated proteins as transgenes in cell lines and in flies. Here, we present a straightforward strategy to systematically study the functional relevance of N-terminal acetylations in cells and whole organisms. Since the (X)PX rule seems to be of general validity in lower as well as higher eukaryotes, we propose that it can be used to study the function of N-terminal acetylation in all species. PMID:19885390

A conserved residue, PomB-F22, in the transmembrane segment of the flagellar stator complex, has a critical role in conducting ions and generating torque.

PubMed

Terauchi, Takashi; Terashima, Hiroyuki; Kojima, Seiji; Homma, Michio

2011-08-01

Bacterial flagellar motors exploit the electrochemical potential gradient of a coupling ion (H(+) or Na(+)) as their energy source, and are composed of stator and rotor proteins. Sodium-driven and proton-driven motors have the stator proteins PomA and PomB or MotA and MotB, respectively, which interact with each other in their transmembrane (TM) regions to form an ion channel. The single TM region of PomB or MotB, which forms the ion-conduction pathway together with TM3 and TM4 of PomA or MotA, respectively, has a highly conserved aspartate residue that is the ion binding site and is essential for rotation. To investigate the ion conductivity and selectivity of the Na(+)-driven PomA/PomB stator complex, we replaced conserved residues predicted to be near the conserved aspartate with H(+)-type residues, PomA-N194Y, PomB-F22Y and/or PomB-S27T. Motility analysis revealed that the ion specificity was not changed by either of the PomB mutations. PomB-F22Y required a higher concentration of Na(+) to exhibit swimming, but this effect was suppressed by additional mutations, PomA-N194Y or PomB-S27T. Moreover, the motility of the PomB-F22Y mutant was resistant to phenamil, a specific inhibitor for the Na(+) channel. When PomB-F22 was changed to other amino acids and the effects on swimming ability were investigated, replacement with a hydrophilic residue decreased the maximum swimming speed and conferred strong resistance to phenamil. From these results, we speculate that the Na(+) flux is reduced by the PomB-F22Y mutation, and that PomB-F22 is important for the effective release of Na(+) from PomB-D24.

N-Terminal Acetylation Inhibits Protein Targeting to the Endoplasmic Reticulum

PubMed Central

Forte, Gabriella M. A.; Pool, Martin R.; Stirling, Colin J.

2011-01-01

Amino-terminal acetylation is probably the most common protein modification in eukaryotes with as many as 50%–80% of proteins reportedly altered in this way. Here we report a systematic analysis of the predicted N-terminal processing of cytosolic proteins versus those destined to be sorted to the secretory pathway. While cytosolic proteins were profoundly biased in favour of processing, we found an equal and opposite bias against such modification for secretory proteins. Mutations in secretory signal sequences that led to their acetylation resulted in mis-sorting to the cytosol in a manner that was dependent upon the N-terminal processing machinery. Hence N-terminal acetylation represents an early determining step in the cellular sorting of nascent polypeptides that appears to be conserved across a wide range of species. PMID:21655302

Factors influencing the temporal growth rate of the high order TM{sub 0n} modes in the Ka-band overmoded Cherenkov oscillator

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Dapeng, E-mail: vipbenjamin@163.com; Shu, Ting; Ju, Jinchuan

2015-06-15

When the wavelength of overmoded Cherenkov oscillator goes into Ka-band, power handling capacity becomes an essential issue. Using the TM{sub 02} mode or higher order TM{sub 0n} modes as the operating mode is a potential solution. This paper is aimed to find some proper parameters to make the temporal growth rate of the TM{sub 02} mode higher in our previously studied Gigawatt (GW)-class Ka band oscillator. An accurate and fast calculation method of the “hot” dispersion equation is derived for rectangular corrugated SWSs, which are widely used in the high frequency Cherenkov devices. Then, factors that affect the temporal growthmore » rate of the high order TM{sub 0n} modes are analyzed, including the depth of corrugation, the radius of drift tube, and the diode voltage. Results show that, when parameters are chosen properly, the temporal growth rate of the TM{sub 02} mode can be as high as 0.3 ns{sup −1}.« less

Blue and white light emission in Tm3+ and Tm3+/Dy3+ doped zinc phosphate glasses upon UV light excitation

NASA Astrophysics Data System (ADS)

Meza-Rocha, A. N.; Speghini, A.; Lozada-Morales, R.; Caldiño, U.

2016-08-01

A spectroscopic study based on photoluminescence spectra and decay time profiles in Tm3+ and Tm3+/Dy3+ doped Zn(PO3)2 glasses is reported. The Tm3+ doped Zn(PO3)2 glass, upon 357 nm excitation, exhibits blue emission with CIE1931 chromaticity coordinates, x = 0.157 and y = 0.030, and color purity of about 96%. Under excitations at 348, 352 and 363 nm, which match with the emissions of AlGaN and GaN based LEDs, the Tm3+/Dy3+ co-doped Zn(PO3)2 glass displays natural white, bluish white and cool white overall emissions, with correlated color temperature values of 4523, 10700 and 7788 K, respectively, depending strongly on the excitation wavelength. The shortening of the Dy3+ emission decay time in presence of Tm3+ suggests that Dy3+→Tm3+ non-radiative energy transfer occurs. By using the Inokuti-Hirayama model, it is inferred that an electric quadrupole-quadrupole interaction might be the dominant mechanism involved in the energy transfer. The efficiency and probability of this energy transfer are 0.12 and 126.70 s-1, respectively.

Synthesis and Characterization of Modified Phenylethynyl Terminated Polyimides

NASA Technical Reports Server (NTRS)

Chang, Alice C.

1998-01-01

As an ongoing effort to develop structural adhesives for high performance aerospace applications, recent work has focused on phenylethynyl terminated imide (PETI) oligomers. The work reported herein involves the synthesis and characterization of a series of phenylethynyl containing oligomers designated LARC (TM) MPEI (Modified Phenylethynyl Terminated Polyimide). These oligomers contain mixtures of linear, branched and star-shaped molecules. The fully imidized polymers exhibited minimum melt viscosity as low as 600 poise at 335 C. Ti/Ti lap shear specimens processed at 288 C under 15 psi showed tensile shear strength of approx. 6000 psi and 5200 psi at ambient and 350 F temperatures, respectively. The chemistry and properties of these new MPEIs are presented and compared to an optimized linear PETI, LaRC(Tm) -PETI-5.

Thermal lensing and microchip laser performance of N g-cut Tm3+:KY(WO4)2 crystal

NASA Astrophysics Data System (ADS)

Gaponenko, M. S.; Loiko, P. A.; Gusakova, N. V.; Yumashev, K. V.; Kuleshov, N. V.; Pavlyuk, A. A.

2012-09-01

The thermal lensing effect was characterized in the diode-pumped monoclinic N g-cut Tm:KYW crystal under laser operation conditions at the wavelength of 1.94 μm. The thermal lens was found to be slightly astigmatic; its optical power D being positive for rays lying in all meridional planes. Thermal lens sensitivity factors M= dD/ dP abs equal 11.8 m-1/W and 8.8 m-1/W (with respect to the absorbed pump power P abs) for principal meridional planes containing N p and N m axes. Nearly athermal behavior of N g-cut crystal is associated with the mutual compensation of different impacts to the thermal lens optical power that arise from temperature dependence of the refractive index dn/ dT and anisotropic thermal expansion. It was utilized to produce passively cooled diode-pumped 0.65 W cw Tm:KYW microchip laser with slope efficiency of 44 % and low thermo-optic aberrations.

The influence of allosteric modulators and transmembrane mutations on desensitisation and activation of α7 nicotinic acetylcholine receptors.

PubMed

Chatzidaki, Anna; D'Oyley, Jarryl M; Gill-Thind, JasKiran K; Sheppard, Tom D; Millar, Neil S

2015-10-01

Acetylcholine activates nicotinic acetylcholine receptors (nAChRs) by binding at an extracellular orthosteric site. Previous studies have described several positive allosteric modulators (PAMs) that are selective for homomeric α7 nAChRs. These include type I PAMs, which exert little or no effect on the rate of receptor desensitisation, and type II PAMs, which cause a dramatic loss of agonist-induced desensitisation. Here we report evidence that transmembrane mutations in α7 nAChRs have diverse effects on receptor activation and desensitisation by allosteric ligands. It has been reported previously that the L247T mutation, located toward the middle of the second transmembrane domain (at the 9' position), confers reduced levels of desensitisation. In contrast, the M260L mutation, located higher up in the TM2 domain (at the 22' position), does not show any difference in desensitisation compared to wild-type receptors. We have found that in receptors containing the L247T mutation, both type I PAMs and type II PAMs are converted into non-desensitising agonists. In contrast, in receptors containing the M260L mutation, this effect is seen only with type II PAMs. These findings, indicating that the M260L mutation has a selective effect on type II PAMs, have been confirmed both with previously described PAMs and also with a series of novel α7-selective PAMs. The novel PAMs examined in this study have close chemical similarity but diverse pharmacological properties. For example, they include compounds displaying effects on receptor desensitisation that are typical of classical type I and type II PAMs but, in addition, they include compounds with intermediate properties. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Lack of P4H-TM in mice results in age-related retinal and renal alterations.

PubMed

Leinonen, Henri; Rossi, Maarit; Salo, Antti M; Tiainen, Päivi; Hyvärinen, Jaana; Pitkänen, Marja; Sormunen, Raija; Miinalainen, Ilkka; Zhang, Chi; Soininen, Raija; Kivirikko, Kari I; Koskelainen, Ari; Tanila, Heikki; Myllyharju, Johanna; Koivunen, Peppi

2016-09-01

Age-related macular degeneration (AMD), affecting the retinal pigment epithelium (RPE), is the leading cause of blindness in middle-aged and older people in developed countries. Genetic and environmental risk factors have been identified, but no effective cure exists. Using a mouse model we show that a transmembrane prolyl 4-hydroxylase (P4H-TM), which participates in the oxygen-dependent regulation of the hypoxia-inducible factor (HIF), is a potential novel candidate gene for AMD. We show that P4h-tm had its highest expression levels in the mouse RPE and brain, heart, lung, skeletal muscle and kidney. P4h-tm -/- mice were fertile and had a normal life span. Lack of P4h-tm stabilized HIF-1α in cortical neurons under normoxia, while in hypoxia it increased the expression of certain HIF target genes in tissues with high endogenous P4h-tm expression levels more than in wild-type mice. Renal erythropoietin levels increased in P4h-tm -/- mice with aging, but the resulting ∼2-fold increase in erythropoietin serum levels did not lead to erythrocytosis. Instead, accumulation of lipid-containing lamellar bodies in renal tubuli was detected in P4h-tm -/- mice with aging, resulting in inflammation and fibrosis, and later glomerular sclerosis and albuminuria. Lack of P4h-tm was associated with retinal thinning, rosette-like infoldings and drusen-like structure accumulation in RPE with aging, as is characteristic of AMD. Photoreceptor recycling was compromised, and electroretinograms revealed functional impairment of the cone pathway in adult P4h-tm -/- mice and cone and rod deficiency in middle-aged mice. P4H-TM is therefore imperative for normal vision, and potentially a novel candidate for age-induced diseases, such as AMD. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

SH3-like motif-containing C-terminal domain of staphylococcal teichoic acid transporter suggests possible function.

PubMed

Ko, Tzu-Ping; Tseng, Shih-Ting; Lai, Shu-Jung; Chen, Sheng-Chia; Guan, Hong-Hsiang; Shin Yang, Chia; Jung Chen, Chun; Chen, Yeh

2016-09-01

The negatively charged bacterial polysaccharides-wall teichoic acids (WTAs) are synthesized intracellularly and exported by a two-component transporter, TagGH, comprising a transmembrane subunit TagG and an ATPase subunit TagH. We determined the crystal structure of the C-terminal domain of TagH (TagH-C) to investigate its function. The structure shows an N-terminal SH3-like subdomain wrapped by a C-terminal subdomain with an anti-parallel β-sheet and an outer shell of α-helices. A stretch of positively charged surface across the subdomain interface is flanked by two negatively charged regions, suggesting a potential binding site for negatively charged polymers, such as WTAs or acidic peptide chains. Proteins 2016; 84:1328-1332. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Structure and Regulatory Interactions of the Cytoplasmic Terminal Domains of Serotonin Transporter

PubMed Central

2014-01-01

Uptake of neurotransmitters by sodium-coupled monoamine transporters of the NSS family is required for termination of synaptic transmission. Transport is tightly regulated by protein–protein interactions involving the small cytoplasmic segments at the amino- and carboxy-terminal ends of the transporter. Although structures of homologues provide information about the transmembrane regions of these transporters, the structural arrangement of the terminal domains remains largely unknown. Here, we combined molecular modeling, biochemical, and biophysical approaches in an iterative manner to investigate the structure of the 82-residue N-terminal and 30-residue C-terminal domains of human serotonin transporter (SERT). Several secondary structures were predicted in these domains, and structural models were built using the Rosetta fragment-based methodology. One-dimensional 1H nuclear magnetic resonance and circular dichroism spectroscopy supported the presence of helical elements in the isolated SERT N-terminal domain. Moreover, introducing helix-breaking residues within those elements altered the fluorescence resonance energy transfer signal between terminal cyan fluorescent protein and yellow fluorescent protein tags attached to full-length SERT, consistent with the notion that the fold of the terminal domains is relatively well-defined. Full-length models of SERT that are consistent with these and published experimental data were generated. The resultant models predict confined loci for the terminal domains and predict that they move apart during the transport-related conformational cycle, as predicted by structures of homologues and by the “rocking bundle” hypothesis, which is consistent with spectroscopic measurements. The models also suggest the nature of binding to regulatory interaction partners. This study provides a structural context for functional and regulatory mechanisms involving SERT terminal domains. PMID:25093911

The role of receptor topology in the vitamin D3 uptake and Ca{sup 2+} response systems

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morrill, Gene A., E-mail: gene.morrill@einstein.yu.edu; Kostellow, Adele B.; Gupta, Raj K.

The steroid hormone, vitamin D{sub 3}, regulates gene transcription via at least two receptors and initiates putative rapid response systems at the plasma membrane. The vitamin D receptor (VDR) binds vitamin D{sub 3} and a second receptor, importin-4, imports the VDR-vitamin D{sub 3} complex into the nucleus via nuclear pores. Here we present evidence that the Homo sapiens VDR homodimer contains two transmembrane (TM) helices ({sup 327}E – D{sup 342}), two TM “half-helix” ({sup 264}K − N{sup 276}), one or more large channels, and 16 cholesterol binding (CRAC/CARC) domains. The importin-4 monomer exhibits 3 pore-lining regions ({sup 226}E – L{supmore » 251}; {sup 768}V – G{sup 783}; {sup 876}S – A{sup 891}) and 16 CRAC/CARC domains. The MEMSAT algorithm indicates that VDR and importin-4 may not be restricted to cytoplasm and nucleus. VDR homodimer TM helix-topology predicts insertion into the plasma membrane, with two 84 residue C-terminal regions being extracellular. Similarly, MEMSAT predicts importin-4 insertion into the plasma membrane with 226 residue extracellular N-terminal regions and 96 residue C-terminal extracellular loops; with the pore-lining regions contributing gated Ca{sup 2+} channels. The PoreWalker algorithm indicates that, of the 427 residues in each VDR monomer, 91 line the largest channel, including two vitamin D{sub 3} binding sites and residues from both the TM helix and “half-helix”. Cholesterol-binding domains also extend into the channel within the ligand binding region. Programmed changes in bound cholesterol may regulate both membrane Ca{sup 2+} response systems and vitamin D{sub 3} uptake as well as receptor internalization by the endomembrane system culminating in uptake of the vitamin D{sub 3}-VDR-importin-4 complex into the nucleus.« less

N-terminal acetylation modulates Bax targeting to mitochondria.

PubMed

Alves, Sara; Neiri, Leire; Chaves, Susana Rodrigues; Vieira, Selma; Trindade, Dário; Manon, Stephen; Dominguez, Veronica; Pintado, Belen; Jonckheere, Veronique; Van Damme, Petra; Silva, Rui Duarte; Aldabe, Rafael; Côrte-Real, Manuela

2018-02-01

The pro-apoptotic Bax protein is the main effector of mitochondrial permeabilization during apoptosis. Bax is controlled at several levels, including post-translational modifications such as phosphorylation and S-palmitoylation. However, little is known about the contribution of other protein modifications to Bax activity. Here, we used heterologous expression of human Bax in yeast to study the involvement of N-terminal acetylation by yNaa20p (yNatB) on Bax function. We found that human Bax is N-terminal (Nt-)acetylated by yNaa20p and that Nt-acetylation of Bax is essential to maintain Bax in an inactive conformation in the cytosol of yeast and Mouse Embryonic Fibroblast (MEF) cells. Bax accumulates in the mitochondria of yeast naa20Δ and Naa25 -/- MEF cells, but does not promote cytochrome c release, suggesting that an additional step is required for full activation of Bax. Altogether, our results show that Bax N-terminal acetylation by NatB is involved in its mitochondrial targeting. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Transmembrane Region of Guard Cell SLAC1 Channels Perceives CO2 Signals via an ABA-Independent Pathway in Arabidopsis.

PubMed

Yamamoto, Yoshiko; Negi, Juntaro; Wang, Cun; Isogai, Yasuhiro; Schroeder, Julian I; Iba, Koh

2016-02-01

The guard cell S-type anion channel, SLOW ANION CHANNEL1 (SLAC1), a key component in the control of stomatal movements, is activated in response to CO2 and abscisic acid (ABA). Several amino acids existing in the N-terminal region of SLAC1 are involved in regulating its activity via phosphorylation in the ABA response. However, little is known about sites involved in CO2 signal perception. To dissect sites that are necessary for the stomatal CO2 response, we performed slac1 complementation experiments using transgenic plants expressing truncated SLAC1 proteins. Measurements of gas exchange and stomatal apertures in the truncated transgenic lines in response to CO2 and ABA revealed that sites involved in the stomatal CO2 response exist in the transmembrane region and do not require the SLAC1 N and C termini. CO2 and ABA regulation of S-type anion channel activity in guard cells of the transgenic lines confirmed these results. In vivo site-directed mutagenesis experiments targeted to amino acids within the transmembrane region of SLAC1 raise the possibility that two tyrosine residues exposed on the membrane are involved in the stomatal CO2 response. © 2016 American Society of Plant Biologists. All rights reserved.

Autoinhibition of a calmodulin-dependent calcium pump involves a structure in the stalk that connects the transmembrane domain to the ATPase catalytic domain

NASA Technical Reports Server (NTRS)

Curran, A. C.; Hwang, I.; Corbin, J.; Martinez, S.; Rayle, D.; Sze, H.; Harper, J. F.; Evans, M. L. (Principal Investigator)

2000-01-01

The regulation of Ca(2+)-pumps is important for controlling [Ca(2+)] in the cytosol and organelles of all eukaryotes. Here, we report a genetic strategy to identify residues that function in autoinhibition of a novel calmodulin-activated Ca(2+)-pump with an N-terminal regulatory domain (isoform ACA2 from Arabidopsis). Mutant pumps with constitutive activity were identified by complementation of a yeast (K616) deficient in two Ca(2+)-pumps. Fifteen mutations were found that disrupted a segment of the N-terminal autoinhibitor located between Lys(23) and Arg(54). Three mutations (E167K, D219N, and E341K) were found associated with the stalk that connects the ATPase catalytic domain (head) and with the transmembrane domain. Enzyme assays indicated that the stalk mutations resulted in calmodulin-independent activity, with V(max), K(mATP), and K(mCa(2+)) similar to that of a pump in which the N-terminal autoinhibitor had been deleted. A highly conservative substitution at Asp(219) (D219E) still produced a deregulated pump, indicating that the autoinhibitory structure in the stalk is highly sensitive to perturbation. In plasma membrane H(+)-ATPases from yeast and plants, similarly positioned mutations resulted in hyperactive pumps. Together, these results suggest that a structural feature of the stalk is of general importance in regulating diverse P-type ATPases.

Identification of novel key amino acids at the interface of the transmembrane domains of human BST-2 and HIV-1 Vpu.

PubMed

Pang, Xiaojing; Hu, Siqi; Li, Jian; Xu, Fengwen; Mei, Shan; Zhou, Jinming; Cen, Shan; Jin, Qi; Guo, Fei

2013-08-06

BST-2 (bone marrow stromal cell antigen 2) is an interferon-inducible protein that inhibits virus release by tethering viral particles to the cell surface. This antiviral activity of BST-2 is antagonized by HIV-1 accessory protein Vpu. Vpu physically interacts with BST-2 through their mutual transmembrane (TM) domains. In this study, we utilized the BRET assay and molecular dynamics (MD) simulation method to further characterize the interaction of BST-2 and Vpu. Amino acids I34, L37, P40 and L41 in the TM domain of BST-2, and L11, A18 and W22 in the TM domain of Vpu were identified to be critical for the interaction between BST-2 and Vpu. The residues P40 in the TM domain of BST-2 and L11 in the TM domain of Vpu were shown, for the first time, to be important for their interaction. Furthermore, triple-amino-acid substitutions, 14-16 (AII to VAA) and 26-28 (IIE to AAA) in Vpu TM, not the single-residue mutation, profoundly disrupted BST-2/Vpu interaction. The results of MD simulation revealed significant conformational changes of the BST-2/Vpu complex as a result of mutating P40 of BST-2 and L11, 14-16 (AII to VAA) and 26-28 (IIE to AAA) of Vpu. In addition, disrupting the interaction between BST-2 and Vpu rendered BST-2 resistant to Vpu antagonization. Through use of the BRET assay, we identified novel key residues P40 in the TM domain of BST-2 and L11 in the TM domain of Vpu that are important for their interaction. These results add new insights into the molecular mechanism behind BST-2 antagonization by HIV-1 Vpu.

Identification of novel key amino acids at the interface of the transmembrane domains of human BST-2 and HIV-1 Vpu

PubMed Central

2013-01-01

Background BST-2 (bone marrow stromal cell antigen 2) is an interferon-inducible protein that inhibits virus release by tethering viral particles to the cell surface. This antiviral activity of BST-2 is antagonized by HIV-1 accessory protein Vpu. Vpu physically interacts with BST-2 through their mutual transmembrane (TM) domains. In this study, we utilized the BRET assay and molecular dynamics (MD) simulation method to further characterize the interaction of BST-2 and Vpu. Results Amino acids I34, L37, P40 and L41 in the TM domain of BST-2, and L11, A18 and W22 in the TM domain of Vpu were identified to be critical for the interaction between BST-2 and Vpu. The residues P40 in the TM domain of BST-2 and L11 in the TM domain of Vpu were shown, for the first time, to be important for their interaction. Furthermore, triple-amino-acid substitutions, 14–16 (AII to VAA) and 26–28 (IIE to AAA) in Vpu TM, not the single-residue mutation, profoundly disrupted BST-2/Vpu interaction. The results of MD simulation revealed significant conformational changes of the BST-2/Vpu complex as a result of mutating P40 of BST-2 and L11, 14–16 (AII to VAA) and 26–28 (IIE to AAA) of Vpu. In addition, disrupting the interaction between BST-2 and Vpu rendered BST-2 resistant to Vpu antagonization. Conclusions Through use of the BRET assay, we identified novel key residues P40 in the TM domain of BST-2 and L11 in the TM domain of Vpu that are important for their interaction. These results add new insights into the molecular mechanism behind BST-2 antagonization by HIV-1 Vpu. PMID:23919512

Performance and Applications of the first HVE 5MV Tandetron{sup TM} at the University of Madrid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mous, D.J.W.; Gottdang, A.; Haitsma, R.G.

2003-08-26

The first HVE Tandetron{sup TM} with a nominal terminal voltage of 5 MV has been put into operation at the Universidad Autonoma de Madrid (Spain) as part of their new IBA facility. The accelerator features a coaxial structure in which the all-solid-state power supply is constructed around the high-energy acceleration tube, thereby avoiding the T-shaped tank that has characterized the HVE Tandetrons{sup TM} so far. The new IBA facility covers a number of different ion beam analysis techniques including ERD using heavy-element time-of-flight, RBS, as well as an external micro-beam for PIXE. During installation, tests have shown a stable terminalmore » voltage of 5.5 MV. The terminal voltage ripple was deduced to be below 6 x 10-6 (RMS) for terminal voltages above 800 kV. Terminal voltage undershoot was measured to be 1.4 x 10-3 for a {approx}1 kW beam at 3 MV and recovered to 1 x 10-4 within 800 ms. IBA experiments that require low energy hydrogen beams are supported by a stable terminal voltage down to 100 kV.« less

The N-terminal strand modulates immunoglobulin light chain fibrillogenesis.

PubMed

del Pozo-Yauner, Luis; Wall, Jonathan S; González Andrade, Martín; Sánchez-López, Rosana; Rodríguez-Ambriz, Sandra L; Pérez Carreón, Julio I; Ochoa-Leyva, Adrián; Fernández-Velasco, D Alejandro

2014-01-10

It has been suggested that the N-terminal strand of the light chain variable domain (V(L)) protects the molecule from aggregation by hindering spurious intermolecular contacts. We evaluated the impact of mutations in the N-terminal strand on the thermodynamic stability and kinetic of fibrillogenesis of the V(L) protein 6aJL2. Mutations in this strand destabilized the protein in a position-dependent manner, accelerating the fibrillogenesis by shortening the lag time; an effect that correlated with the extent of destabilization. In contrast, the effect on the kinetics of fibril elongation, as assessed in seeding experiments was of different nature, as it was not directly dependant on the degree of destabilization. This finding suggests different factors drive the nucleation-dependent and elongation phases of light chain fibrillogenesis. Finally, taking advantage of the dependence of the Trp fluorescence upon environment, four single Trp substitutions were made in the N-terminal strand, and changes in solvent exposure during aggregation were evaluated by acrylamide-quenching. The results suggest that the N-terminal strand is buried in the fibrillar state of 6aJL2 protein. This finding suggest a possible explanation for the modulating effect exerted by the mutations in this strand on the aggregation behavior of 6aJL2 protein. Copyright © 2013 Elsevier Inc. All rights reserved.

NMR assignments of the N-terminal domain of Nephila clavipes spidroin 1

PubMed Central

Parnham, Stuart; Gaines, William A.; Duggan, Brendan M.; Marcotte, William R.

2011-01-01

The building blocks of spider dragline silk are two fibrous proteins secreted from the major ampullate gland named spidroins 1 and 2 (MaSp1, MaSp2). These proteins consist of a large central domain composed of approximately 100 tandem copies of a 35–40 amino acid repeat sequence. Non-repetitive N and C-terminal domains, of which the C-terminal domain has been implicated to transition from soluble and insoluble states during spinning, flank the repetitive core. The N-terminal domain until recently has been largely unknown due to difficulties in cloning and expression. Here, we report nearly complete assignment for all 1H, 13C, and 15N resonances in the 14 kDa N-terminal domain of major ampullate spidroin 1 (MaSp1-N) of the golden orb-web spider Nephila clavipes. PMID:21152998

Specific Binding of Adamantane Drugs and Direction of their Polar Amines in the Pore of the Influenza M2 Transmembrane Domain in Lipid Bilayers and Dodecylphosphocholine Micelles Determined by NMR Spectroscopy

PubMed Central

Cady, Sarah D.; Wang, Jun; Wu, Yibing; DeGrado, William F.; Hong, Mei

2011-01-01

The transmembrane domain of the influenza M2 protein (M2TM) forms a tetrameric proton channel important for the virus lifecycle. The proton-channel activity is inhibited by amine-containing adamantyl drugs amantadine and rimantadine, which have been shown to bind specifically to the pore of M2TM near Ser31. However, whether the polar amine points to the N- or C-terminus of the channel has not yet been determined. Elucidating the polar group direction will shed light on the mechanism by which drug binding inhibits this proton channel and will facilitate rational design of new inhibitors. In this study, we determine the polar amine direction using M2TM reconstituted in lipid bilayers as well as DPC micelles. 13C-2H rotational-echo double-resonance NMR experiments of 13C-labeled M2TM and methyl-deuterated rimantadine in lipid bilayers showed that the polar amine pointed to the C-terminus of the channel, with the methyl group close to Gly34. Solution NMR experiments of M2TM in dodecylphosphocholine (DPC) micelles indicate that drug binding causes significant chemical shift perturbations of the protein that are very similar to those seen for M2TM and M2(18–60) bound to lipid bilayers. Specific 2H-labeling of the drugs permitted the assignment of drug-protein cross peaks, which indicate that amantadine and rimantadine bind to the pore in the same fashion as for bilayer-bound M2TM. These results strongly suggest that adamantyl inhibition of M2TM is achieved not only by direct physical occlusion of the pore but also by perturbing the equilibrium constant of the proton-sensing residue His37. The reproduction of the pharmacologically relevant specific pore-binding site in DPC micelles, which was not observed with a different detergent, DHPC, underscores the significant influence of the detergent environment on the functional structure of membrane proteins. PMID:21381693

Structure and function of the C-terminal domain of MrpA in the Bacillus subtilis Mrp-antiporter complex--the evolutionary progenitor of the long horizontal helix in complex I.

PubMed

Virzintiene, Egle; Moparthi, Vamsi K; Al-Eryani, Yusra; Shumbe, Leonard; Górecki, Kamil; Hägerhäll, Cecilia

2013-10-11

MrpA and MrpD are homologous to NuoL, NuoM and NuoN in complex I over the first 14 transmembrane helices. In this work, the C-terminal domain of MrpA, outside this conserved area, was investigated. The transmembrane orientation was found to correspond to that of NuoJ in complex I. We have previously demonstrated that the subunit NuoK is homologous to MrpC. The function of the MrpA C-terminus was tested by expression in a previously used Bacillus subtilis model system. At neutral pH, the truncated MrpA still worked, but at pH 8.4, where Mrp-complex formation is needed for function, the C-terminal domain of MrpA was absolutely required. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

First-principles calculations of electronic, magnetic and optical properties of HoN doped with TM (Ti, V, Cr, Mn, Co and Ni)

NASA Astrophysics Data System (ADS)

Rouchdi, M.; Salmani, E.; Dehmani, M.; Ez-Zahraouy, H.; Hassanain, N.; Benyoussef, A.; Mzerd, A.

2018-02-01

Using the first-principles calculations within the Korringa-Kohn-Rostoker (KKR) method combined with the coherent potential approximation (CPA), the structural, optical and magnetic properties of rare-earth nitride Ho0.95TM0.05N doped with transition metal (TM) atoms (Ti, V, Cr, Mn, Co and Ni) are investigated as a function the generalized gradient approximation and self-interaction correction (GGA-SIC) approximation. The optical properties are studied in detail by using ab-initio calculations. Using GGA-SIC we have showed that the bandgap value is in good agreement with the experimental value. Using GGA-SIC approximation for HoN, we have obtained a bandgap of 0.9 eV. Some of the dilute magnetic semiconductors (DMS) like Ho0.95TM0.05N under study exhibit a half-metallic behavior, which makes them suitable for spintronic applications. Moreover, the optical absorption spectra confirm the ferromagnetic stability based on the charge state of magnetic impurities.

Transmembrane Polyproline Helix.

PubMed

Kubyshkin, Vladimir; Grage, Stephan L; Bürck, Jochen; Ulrich, Anne S; Budisa, Nediljko

2018-05-03

The third most abundant polypeptide conformation in nature, the polyproline-II helix, is a polar, extended secondary structure with a local organization stabilized by intercarbonyl interactions within the peptide chain. Here we design a hydrophobic polyproline-II helical peptide based on an oligomeric octahydroindole-2-carboxylic acid scaffold and demonstrate its transmembrane alignment in model lipid bilayers by means of solid-state 19 F NMR. As result, we provide a first example of a purely artificial transmembrane peptide with a structural organization that is not based on hydrogen-bonding.

Bidirectional signaling between TM4SF5 and IGF1R promotes resistance to EGFR kinase inhibitors.

PubMed

Choi, Jungeun; Kang, Minkyung; Nam, Seo Hee; Lee, Gyu-Ho; Kim, Hye-Jin; Ryu, Jihye; Cheong, Jin Gyu; Jung, Jae Woo; Kim, Tai Young; Lee, Ho-Young; Lee, Jung Weon

2015-10-01

The membrane glycoprotein TM4SF5 (transmembrane 4 L6 family member 5), which is similar to the tetraspanins, is highly expressed in different cancers and causes epithelial-mesenchymal transition (EMT). TM4SF5 interacts with other membrane proteins during its pro-tumorigenic roles, presumably at tetraspanin-enriched microdomains (TEMs/TERMs). Here, we explored TM4SF5-mediated resistance against the clinically important EGFR kinase inhibitors, with regards to cooperation with other membrane proteins, particularly the insulin-like growth factor 1 receptor (IGF1R). Using cancer cells including NSCLC with TM4SF5 overexpression or IGF1R suppression in either normal 2 dimensional (2D), 3D aqueous spheroids, or 3D collagen I gels systems, the sensitivity to tyrosine kinase inhibitors (TKIs) were evaluated. We found that TM4SF5 and IGF1R transcriptionally modulated one another, with each protein promoting the expressions of the other. Expression of TM4SF5 in gefitinib-sensitive HCC827 cells caused resistance to erlotinib and gefitinib, but not to sorafenib [a platelet derived growth factor receptor (PDGFR) inhibitor]; whereas suppression of IGF1R from gefitinib-resistant NCI-H1299 cells caused enhanced sensitization to the inhibitors. Expression of TM4SF5 and IGF1R in the drug-sensitive cells promoted signaling activities of extracellular signal-regulated kinases (ERKs), protein kinase B (Akt), and S6 kinase (S6K), and resulted in a higher residual EGFR activity, even after EGFR kinase inhibitor treatment. Complex formation between TM4SF5 and IGF1R was observed, and also included EGFR, dependent on TM4SF5 expression. The TM4SF5-mediated drug resistance was further confirmed in an aqueous 3D spheroid system or upon being embedded in 3D extracellular matrix (ECM)-surrounded gel systems. Collectively, these data suggest that anti-TM4SF5 reagents may be combined with the EGFR kinase inhibitors to enhance the efficacy of chemotherapies against NSCLC. Copyright © 2015 Elsevier

N-terminal Proteomics Assisted Profiling of the Unexplored Translation Initiation Landscape in Arabidopsis thaliana *

PubMed Central

Ndah, Elvis; Jonckheere, Veronique

2017-01-01

Proteogenomics is an emerging research field yet lacking a uniform method of analysis. Proteogenomic studies in which N-terminal proteomics and ribosome profiling are combined, suggest that a high number of protein start sites are currently missing in genome annotations. We constructed a proteogenomic pipeline specific for the analysis of N-terminal proteomics data, with the aim of discovering novel translational start sites outside annotated protein coding regions. In summary, unidentified MS/MS spectra were matched to a specific N-terminal peptide library encompassing protein N termini encoded in the Arabidopsis thaliana genome. After a stringent false discovery rate filtering, 117 protein N termini compliant with N-terminal methionine excision specificity and indicative of translation initiation were found. These include N-terminal protein extensions and translation from transposable elements and pseudogenes. Gene prediction provided supporting protein-coding models for approximately half of the protein N termini. Besides the prediction of functional domains (partially) contained within the newly predicted ORFs, further supporting evidence of translation was found in the recently released Araport11 genome re-annotation of Arabidopsis and computational translations of sequences stored in public repositories. Most interestingly, complementary evidence by ribosome profiling was found for 23 protein N termini. Finally, by analyzing protein N-terminal peptides, an in silico analysis demonstrates the applicability of our N-terminal proteogenomics strategy in revealing protein-coding potential in species with well- and poorly-annotated genomes. PMID:28432195

N-terminal Proteomics Assisted Profiling of the Unexplored Translation Initiation Landscape in Arabidopsis thaliana.

PubMed

Willems, Patrick; Ndah, Elvis; Jonckheere, Veronique; Stael, Simon; Sticker, Adriaan; Martens, Lennart; Van Breusegem, Frank; Gevaert, Kris; Van Damme, Petra

2017-06-01

Proteogenomics is an emerging research field yet lacking a uniform method of analysis. Proteogenomic studies in which N-terminal proteomics and ribosome profiling are combined, suggest that a high number of protein start sites are currently missing in genome annotations. We constructed a proteogenomic pipeline specific for the analysis of N-terminal proteomics data, with the aim of discovering novel translational start sites outside annotated protein coding regions. In summary, unidentified MS/MS spectra were matched to a specific N-terminal peptide library encompassing protein N termini encoded in the Arabidopsis thaliana genome. After a stringent false discovery rate filtering, 117 protein N termini compliant with N-terminal methionine excision specificity and indicative of translation initiation were found. These include N-terminal protein extensions and translation from transposable elements and pseudogenes. Gene prediction provided supporting protein-coding models for approximately half of the protein N termini. Besides the prediction of functional domains (partially) contained within the newly predicted ORFs, further supporting evidence of translation was found in the recently released Araport11 genome re-annotation of Arabidopsis and computational translations of sequences stored in public repositories. Most interestingly, complementary evidence by ribosome profiling was found for 23 protein N termini. Finally, by analyzing protein N-terminal peptides, an in silico analysis demonstrates the applicability of our N-terminal proteogenomics strategy in revealing protein-coding potential in species with well- and poorly-annotated genomes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Two Different Conformations in Hepatitis C Virus p7 Protein Account for Proton Transport and Dye Release

PubMed Central

Vararattanavech, Ardcharaporn; Torres, Jaume

2014-01-01

The p7 protein from the hepatitis C virus (HCV) is a 63 amino acid long polypeptide that is essential for replication, and is involved in protein trafficking and proton transport. Therefore, p7 is a possible target for antivirals. The consensus model for the channel formed by p7 protein is a hexameric or heptameric oligomer of α-helical hairpin monomers, each having two transmembrane domains, TM1 and TM2, where the N-terminal TM1 would face the lumen of this channel. A reported high-throughput functional assay to search for p7 channel inhibitors is based on carboxyfluorescein (CF) release from liposomes after p7 addition. However, the rationale for the dual ability of p7 to serve as an ion or proton channel in the infected cell, and to permeabilize membranes to large molecules like CF is not clear. We have recreated both activities in vitro, examining the conformation present in these assays using infrared spectroscopy. Our results indicate that an α-helical form of p7, which can transport protons, is not able to elicit CF release. In contrast, membrane permeabilization to CF is observed when p7 contains a high percentage of β-structure, or when using a C-terminal fragment of p7, encompassing TM2. We propose that the reported inhibitory effect of some small compounds, e.g., rimantadine, on both CF release and proton transport can be explained via binding to the membrane-inserted C-terminal half of p7, increasing its rigidity, in a similar way to the influenza A M2-rimantadine interaction. PMID:24409277

Agonist activation of α7 nicotinic acetylcholine receptors via an allosteric transmembrane site

PubMed Central

Gill, JasKiran K.; Savolainen, Mari; Young, Gareth T.; Zwart, Ruud; Sher, Emanuele; Millar, Neil S.

2011-01-01

Conventional nicotinic acetylcholine receptor (nAChR) agonists, such as acetylcholine, act at an extracellular “orthosteric” binding site located at the interface between two adjacent subunits. Here, we present evidence of potent activation of α7 nAChRs via an allosteric transmembrane site. Previous studies have identified a series of nAChR-positive allosteric modulators (PAMs) that lack agonist activity but are able to potentiate responses to orthosteric agonists, such as acetylcholine. It has been shown, for example, that TQS acts as a conventional α7 nAChR PAM. In contrast, we have found that a compound with close chemical similarity to TQS (4BP-TQS) is a potent allosteric agonist of α7 nAChRs. Whereas the α7 nAChR antagonist metyllycaconitine acts competitively with conventional nicotinic agonists, metyllycaconitine is a noncompetitive antagonist of 4BP-TQS. Mutation of an amino acid (M253L), located in a transmembrane cavity that has been proposed as being the binding site for PAMs, completely blocks agonist activation by 4BP-TQS. In contrast, this mutation had no significant effect on agonist activation by acetylcholine. Conversely, mutation of an amino acid located within the known orthosteric binding site (W148F) has a profound effect on agonist potency of acetylcholine (resulting in a shift of ∼200-fold in the acetylcholine dose-response curve), but had little effect on the agonist dose-response curve for 4BP-TQS. Computer docking studies with an α7 homology model provides evidence that both TQS and 4BP-TQS bind within an intrasubunit transmembrane cavity. Taken together, these findings provide evidence that agonist activation of nAChRs can occur via an allosteric transmembrane site. PMID:21436053

Structure of Thermotoga maritima TM0439: implications for the mechanism of bacterial GntR transcription regulators with Zn{sup 2+}-binding FCD domains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, Meiying; Cooper, David R.; Grossoehme, Nickolas E.

2009-04-01

Here, the crystal structure of TM0439, a GntR regulator with an FCD domain found in the Thermotoga maritima genome, is described. The GntR superfamily of dimeric transcription factors, with more than 6200 members encoded in bacterial genomes, are characterized by N-terminal winged-helix DNA-binding domains and diverse C-terminal regulatory domains which provide a basis for the classification of the constituent families. The largest of these families, FadR, contains nearly 3000 proteins with all-α-helical regulatory domains classified into two related Pfam families: FadR-C and FCD. Only two crystal structures of FadR-family members, those of Escherichia coli FadR protein and LldR from Corynebacteriummore » glutamicum, have been described to date in the literature. Here, the crystal structure of TM0439, a GntR regulator with an FCD domain found in the Thermotoga maritima genome, is described. The FCD domain is similar to that of the LldR regulator and contains a buried metal-binding site. Using atomic absorption spectroscopy and Trp fluorescence, it is shown that the recombinant protein contains bound Ni{sup 2+} ions but that it is able to bind Zn{sup 2+} with K{sub d} < 70 nM. It is concluded that Zn{sup 2+} is the likely physiological metal and that it may perform either structural or regulatory roles or both. Finally, the TM0439 structure is compared with two other FadR-family structures recently deposited by structural genomics consortia. The results call for a revision in the classification of the FadR family of transcription factors.« less

Conformational changes induced by a single amino acid substitution in the trans-membrane domain of Vpu: implications for HIV-1 susceptibility to channel blocking drugs.

PubMed

Park, Sang Ho; Opella, Stanley J

2007-10-01

The channel-forming trans-membrane domain of Vpu (Vpu TM) from HIV-1 is known to enhance virion release from the infected cells and is a potential target for ion-channel blockers. The substitution of alanine at position 18 by a histidine (A18H) has been shown to render HIV-1 infections susceptible to rimantadine, a channel blocker of M2 protein from the influenza virus. In order to describe the influence of the mutation on the structure and rimantadine susceptibility of Vpu, we determined the structure of A18H Vpu TM, and compared it to those of wild-type Vpu TM and M2 TM. Both isotropic and orientationally dependent NMR frequencies of the backbone amide resonance of His18 were perturbed by rimantadine, and those of Ile15 and Trp22 were also affected, suggesting that His18 is the key residue for rimantadine binding and that residues located on the same face of the TM helix are also involved. A18H Vpu TM has an ideal, straight alpha-helix spanning residues 6-27 with an average tilt angle of 41 degrees in C14 phospholipid bicelles, indicating that the tilt angle is increased by 11 degrees compared to that of wild-type Vpu TM. The longer helix formed by the A18H mutation has a larger tilt angle to compensate for the hydrophobic mismatch with the length of the phospholipids in the bilayer. These results demonstrate that the local change of the primary structure plays an important role in secondary and tertiary structures of Vpu TM in lipid bilayers and affects its ability to interact with channel blockers.

Expression of TM4SF10, a Claudin/EMP/PMP22 family cell junction protein, during mouse kidney development and podocyte differentiation.

PubMed

Bruggeman, Leslie A; Martinka, Scott; Simske, Jeffrey S

2007-02-01

Cell junctions in the nephron are highly specialized to perform specific and distinct filtration and reabsorption functions. The mature kidney forms complex cell junctions including slit diaphragms that prevent the passage of serum proteins into the filtrate, and tubule cell junctions that regulate specific paracellular ion reuptake. We have investigated the expression of TM4SF10 (Trans-Membrane tetra(4)-Span Family 10) in mouse kidneys. TM4SF10 is the vertebrate orthologue of Caenorhabditis elegans VAB-9, a tetraspan adherens junction protein in the PMP22/EMP/Claudin family of proteins. We found that TM4SF10 localizes at the basal-most region of podocyte precursors before the capillary loop stage, at some tubule precursors, and at the ureteric bud junction with S-shaped bodies. Overall expression of TM4SF10 peaked at postnatal day 4 and was virtually absent in adult kidneys. The very limited expression of TM4SF10 protein that persisted into adulthood was restricted to a few tubule segments but remained localized to the basal region of lateral membranes. In undifferentiated cultured podocytes, TM4SF10 localized to the perinuclear region and translocated to the cell membrane after Cadherin appearance at cell-cell contacts. TM4SF10 colocalized with ZO1 and p120ctn in undifferentiated confluent podocytes and also colocalized with the tips of actin filaments at cell contacts. Upon differentiation of cultured podocytes, TM4SF10 protein disappeared from cell contacts and expression ceased. These results suggest that TM4SF10 functions during differentiation of podocytes and may participate in the maturation of cell junctions from simple adherens junctions to elaborate slit diaphragms. TM4SF10 may define a new class of Claudin-like proteins that function during junctional development.

Conformational Plasticity of the Influenza A M2 Transmembrane Helix in Lipid Bilayers Under Varying pH, Drug Binding and Membrane Thickness

PubMed Central

Hu, Fanghao; Luo, Wenbin; Cady, Sarah D.; Hong, Mei

2010-01-01

Membrane proteins change their conformations to respond to environmental cues, thus conformational plasticity is important for function. The influenza A M2 protein forms an acid-activated proton channel important for the virus lifecycle. Here we have used solid-state NMR spectroscopy to examine the conformational plasticity of membrane-bound transmembrane domain of M2 (M2TM). 13C and 15N chemical shifts indicate coupled conformational changes of several pore-facing residues due to changes in bilayer thickness, drug binding and pH. The structural changes are attributed to the formation of a well-defined helical kink at G34 in the drug-bound state and in thick lipid bilayers, non-ideal backbone conformation of the secondary-gate residue V27 in the presence of drug, and non-ideal conformation of the proton-sensing residue H37 at high pH. The chemical shifts constrained the (ϕ, ψ) torsion angles for three basis states, the equilibrium among which explains the multiple resonances per site in the NMR spectra under different combinations of bilayer thickness, drug binding and pH conditions. Thus, conformational plasticity is important for the proton conduction and inhibition of M2TM. The study illustrates the utility of NMR chemical shifts for probing the structural plasticity and folding of membrane proteins. PMID:20883664

Blocking an N-terminal acetylation–dependent protein interaction inhibits an E3 ligase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Scott, Daniel C.; Hammill, Jared T.; Min, Jaeki

N-terminal acetylation is an abundant modification influencing protein functions. Because ~80% of mammalian cytosolic proteins are N-terminally acetylated, this modification is potentially an untapped target for chemical control of their functions. Structural studies have revealed that, like lysine acetylation, N-terminal acetylation converts a positively charged amine into a hydrophobic handle that mediates protein interactions; hence, this modification may be a druggable target. We report the development of chemical probes targeting the N-terminal acetylation–dependent interaction between an E2 conjugating enzyme (UBE2M or UBC12) and DCN1 (DCUN1D1), a subunit of a multiprotein E3 ligase for the ubiquitin-like protein NEDD8. The inhibitors aremore » highly selective with respect to other protein acetyl-amide–binding sites, inhibit NEDD8 ligation in vitro and in cells, and suppress anchorage-independent growth of a cell line with DCN1 amplification. Overall, our data demonstrate that N-terminal acetyl-dependent protein interactions are druggable targets and provide insights into targeting multiprotein E2–E3 ligases.« less

Duck Interferon-Inducible Transmembrane Protein 3 Mediates Restriction of Influenza Viruses.

PubMed

Blyth, Graham A D; Chan, Wing Fuk; Webster, Robert G; Magor, Katharine E

2016-01-01

Interferon-inducible transmembrane proteins (IFITMs) can restrict the entry of a wide range of viruses. IFITM3 localizes to endosomes and can potently restrict the replication of influenza A viruses (IAV) and several other viruses that also enter host cells through the endocytic pathway. Here, we investigate whether IFITMs are involved in protection in ducks, the natural host of influenza virus. We identify and sequence duck IFITM1, IFITM2, IFITM3, and IFITM5. Using quantitative PCR (qPCR), we demonstrate the upregulation of these genes in lung tissue in response to highly pathogenic IAV infection by 400-fold, 30-fold, 30-fold, and 5-fold, respectively. We express each IFITM in chicken DF-1 cells and show duck IFITM1 localizes to the cell surface, while IFITM3 localizes to LAMP1-containing compartments. DF-1 cells stably expressing duck IFITM3 (but not IFITM1 or IFITM2) show increased restriction of replication of H1N1, H6N2, and H11N9 IAV strains but not vesicular stomatitis virus. Although duck and human IFITM3 share only 38% identity, critical residues for viral restriction are conserved. We generate chimeric and mutant IFITM3 proteins and show duck IFITM3 does not require its N-terminal domain for endosomal localization or antiviral function; however, this N-terminal end confers endosomal localization and antiviral function on IFITM1. In contrast to mammalian IFITM3, the conserved YXXθ endocytosis signal sequence in the N-terminal domain of duck IFITM3 is not essential for correct endosomal localization. Despite significant structural and amino acid divergence, presumably due to host-virus coevolution, duck IFITM3 is functional against IAV. Immune IFITM genes are poorly conserved across species, suggesting that selective pressure from host-specific viruses has driven this divergence. We wondered whether coevolution between viruses and their natural host would result in the evasion of IFITM restriction. Ducks are the natural host of avian influenza A viruses

N-terminal Domains Elicit Formation of Functional Pmel17 Amyloid Fibrils*

PubMed Central

Watt, Brenda; van Niel, Guillaume; Fowler, Douglas M.; Hurbain, Ilse; Luk, Kelvin C.; Stayrook, Steven E.; Lemmon, Mark A.; Raposo, Graça; Shorter, James; Kelly, Jeffery W.; Marks, Michael S.

2009-01-01

Pmel17 is a transmembrane protein that mediates the early steps in the formation of melanosomes, the subcellular organelles of melanocytes in which melanin pigments are synthesized and stored. In melanosome precursor organelles, proteolytic fragments of Pmel17 form insoluble, amyloid-like fibrils upon which melanins are deposited during melanosome maturation. The mechanism(s) by which Pmel17 becomes competent to form amyloid are not fully understood. To better understand how amyloid formation is regulated, we have defined the domains within Pmel17 that promote fibril formation in vitro. Using purified recombinant fragments of Pmel17, we show that two regions, an N-terminal domain of unknown structure and a downstream domain with homology to a polycystic kidney disease-1 repeat, efficiently form amyloid in vitro. Analyses of fibrils formed in melanocytes confirm that the polycystic kidney disease-1 domain forms at least part of the physiological amyloid core. Interestingly, this same domain is also required for the intracellular trafficking of Pmel17 to multivesicular compartments within which fibrils begin to form. Although a domain of imperfect repeats (RPT) is required for fibril formation in vivo and is a component of fibrils in melanosomes, RPT is not necessary for fibril formation in vitro and in isolation is unable to adopt an amyloid fold in a physiologically relevant time frame. These data define the structural core of Pmel17 amyloid, imply that the RPT domain plays a regulatory role in timing amyloid conversion, and suggest that fibril formation might be physically linked with multivesicular body sorting. PMID:19840945

Effect of Environment on Fatigue Behavior of a Nicalon(TM)/Si-N-C Ceramic Matrix Composite

NASA Technical Reports Server (NTRS)

Kalluri, Sreeramesh; Ojard, Greg C.; Verrilli, Michael J.; Kiraly, Louis J. (Technical Monitor)

2002-01-01

The effect of environmental exposure on the fatigue life of Nicalon(TM) /Si-N-C composite was investigated in this study. Test specimens with arrays of 1.8 mm diameter holes and two different open areas, 25 and 35%, were machined. Three environmental conditions were studied: 1) continuous fatigue cycling in air, 2) fatigue cycling in air alternating with humidity exposure, and 3) fatigue cycling in air alternating with exposure to a salt-fog environment. All fatigue testing on specimens with holes was performed with a load ratio, R = 0.05, and at a temperature of 910 C. In general, fatigue lives were shortest for specimens subjected to salt-fog exposure and longest for specimens subjected to continuous fatigue cycling in air. The fatigue data generated on the specimens with holes were compared with fatigue data generated in air on specimens with no holes. Fatigue strength reduction factors for different environmental conditions and open areas investigated in the study were calculated for the Nicalon(TM) /Si-N-C composite.

Role of protein kinase D in Golgi exit and lysosomal targeting of the transmembrane protein, Mcoln1

PubMed Central

Marks, David L.; Holicky, Eileen L.; Wheatley, Christine L.; Frumkin, Ayala; Bach, Gideon; Pagano, Richard E.

2012-01-01

The targeting of lysosomal transmembrane proteins from the Golgi apparatus to lysosomes is a complex process that is only beginning to be understood. Here, the lysosomal targeting of Mcoln1, the transmembrane protein defective in the autosomal recessive disease, Mucolipidosis, type IV, was studied by over-expressing full length and truncated forms of the protein in human cells, followed by detection using immunofluorescence and immunoblotting. We demonstrated that a 53 amino acid C-terminal region of Mcoln1 is required for efficient exit from the Golgi. Truncations lacking this region exhibited reduced delivery to lysosomes and decreased proteolytic cleavage of Mcoln1 into characteristic ~35 kDa fragments, suggesting that this cleavage occurs in lysosomes. In addition, we found that co-expression of full length Mcoln1 with kinase-inactive protein kinase D (PKD) 1 or 2 inhibited Mcoln1 Golgi exit and transport to lysosomes and decreased Mcoln1 cleavage. These studies suggest that PKDs play a role in the delivery of some lysosomal resident transmembrane proteins from the Golgi to the lysosomes. PMID:22268962

Insights into seven and single transmembrane-spanning domain receptors and their signaling pathways in human natural killer cells.

PubMed

Maghazachi, Azzam A

2005-09-01

Human natural killer (NK) cells are important cells of the innate immune system. These cells perform two prominent functions: the first is recognizing and destroying virally infected cells and transformed cells; the second is secreting various cytokines that shape up the innate and adaptive immune re-sponses. For these cells to perform these activities, they express different sets of receptors. The receptors used by NK cells to extravasate into sites of injury belong to the seven transmembrane (7TM) family of receptors, which characteristically bind heterotrimeric G proteins. These receptors allow NK cells to sense the chemotactic gradients and activate second messengers, which aid NK cells in polarizing and migrating toward the sites of injured tissues. In addition, these receptors determine how and why human resting NK cells are mainly found in the bloodstream, whereas activated NK cells extravasate into inflammatory sites. Receptors for chemokines and lysophospholipids belong to the 7TM family. On the other hand, NK cells recognize invading or transformed cells through another set of receptors that belong to the single transmembrane-spanning domain family. These receptors are either inhibitory or activating. Inhibitory receptors contain the immune receptor tyrosine-based inhibitory motif, and activating receptors belong to either those that associate with adaptor molecules containing the immune receptor tyrosine-based activating motif (ITAM) or those that associate with adaptor molecules containing motifs other than ITAM. This article will describe the nature of these receptors and examine the intracellular signaling pathways induced in NK cells after ligating both types of receptors. These pathways are crucial for NK cell biology, development, and functions.

Quantitative x-ray diffraction analysis of bimodal damage distributions in Tm implanted Al0.15Ga0.85N

NASA Astrophysics Data System (ADS)

Magalhães, S.; Fialho, M.; Peres, M.; Lorenz, K.; Alves, E.

2016-04-01

In this work radial symmetric x-ray diffraction scans of Al0.15Ga0.85N thin films implanted with Tm ions were measured to determine the lattice deformation and crystal quality as functions of depth. The alloys were implanted with 300 keV Tm with 10° off-set to the sample normal to avoid channelling, with fluences varying between 1013 Tm cm-2 and 5  ×  1015 Tm cm-2. Simulations of the radial 2θ-ω scans were performed under the frame of the dynamical theory of x-ray diffraction assuming Gaussian distributions of the lattice strain induced by implantation defects. The structure factor of the individual layers is multiplied by a static Debye-Waller factor in order to take into account the effect of lattice disorder due to implantation. For higher fluences two asymmetric Gaussians are required to describe well the experimental diffractograms, although a single asymmetric Gaussian profile for the deformation is found in the sample implanted with 1013 Tm cm-2. After thermal treatment at 1200 °C, the crystal quality partially recovers as seen in a reduction of the amplitude of the deformation maximum as well as the total thickness of the deformed layer. Furthermore, no evidence of changes with respect to the virgin crystal mosaicity is found after implantation and annealing.

Effects of clinically relevant MPL mutations in the transmembrane domain revealed at the atomic level through computational modeling.

PubMed

Lee, Tai-Sung; Kantarjian, Hagop; Ma, Wanlong; Yeh, Chen-Hsiung; Giles, Francis; Albitar, Maher

2011-01-01

Mutations in the thrombopoietin receptor (MPL) may activate relevant pathways and lead to chronic myeloproliferative neoplasms (MPNs). The mechanisms of MPL activation remain elusive because of a lack of experimental structures. Modern computational biology techniques were utilized to explore the mechanisms of MPL protein activation due to various mutations. Transmembrane (TM) domain predictions, homology modeling, ab initio protein structure prediction, and molecular dynamics (MD) simulations were used to build structural dynamic models of wild-type and four clinically observed mutants of MPL. The simulation results suggest that S505 and W515 are important in keeping the TM domain in its correct position within the membrane. Mutations at either of these two positions cause movement of the TM domain, altering the conformation of the nearby intracellular domain in unexpected ways, and may cause the unwanted constitutive activation of MPL's kinase partner, JAK2. Our findings represent the first full-scale molecular dynamics simulations of the wild-type and clinically observed mutants of the MPL protein, a critical element of the MPL-JAK2-STAT signaling pathway. In contrast to usual explanations for the activation mechanism that are based on the relative translational movement between rigid domains of MPL, our results suggest that mutations within the TM region could result in conformational changes including tilt and rotation (azimuthal) angles along the membrane axis. Such changes may significantly alter the conformation of the adjacent and intrinsically flexible intracellular domain. Hence, caution should be exercised when interpreting experimental evidence based on rigid models of cytokine receptors or similar systems.

A Conserved Asparagine Residue in Transmembrane Segment 1 (TM1) of Serotonin Transporter Dictates Chloride-coupled Neurotransmitter Transport*

PubMed Central

Henry, L. Keith; Iwamoto, Hideki; Field, Julie R.; Kaufmann, Kristian; Dawson, Eric S.; Jacobs, Miriam T.; Adams, Chelsea; Felts, Bruce; Zdravkovic, Igor; Armstrong, Vanessa; Combs, Steven; Solis, Ernesto; Rudnick, Gary; Noskov, Sergei Y.; DeFelice, Louis J.; Meiler, Jens; Blakely, Randy D.

2011-01-01

Na+- and Cl−-dependent uptake of neurotransmitters via transporters of the SLC6 family, including the human serotonin transporter (SLC6A4), is critical for efficient synaptic transmission. Although residues in the human serotonin transporter involved in direct Cl− coordination of human serotonin transport have been identified, the role of Cl− in the transport mechanism remains unclear. Through a combination of mutagenesis, chemical modification, substrate and charge flux measurements, and molecular modeling studies, we reveal an unexpected role for the highly conserved transmembrane segment 1 residue Asn-101 in coupling Cl− binding to concentrative neurotransmitter uptake. PMID:21730057

Targeted mass spectrometric analysis of N-terminally truncated isoforms generated via alternative translation initiation.

PubMed

Kobayashi, Ryuji; Patenia, Rebecca; Ashizawa, Satoshi; Vykoukal, Jody

2009-07-21

Alternative translation initiation is a mechanism whereby functionally altered proteins are produced from a single mRNA. Internal initiation of translation generates N-terminally truncated protein isoforms, but such isoforms observed in immunoblot analysis are often overlooked or dismissed as degradation products. We identified an N-terminally truncated isoform of human Dok-1 with N-terminal acetylation as seen in the wild-type. This Dok-1 isoform exhibited distinct perinuclear localization whereas the wild-type protein was distributed throughout the cytoplasm. Targeted analysis of blocked N-terminal peptides provides rapid identification of protein isoforms and could be widely applied for the general evaluation of perplexing immunoblot bands.

Predicting the transmembrane secondary structure of ligand-gated ion channels.

PubMed

Bertaccini, E; Trudell, J R

2002-06-01

Recent mutational analyses of ligand-gated ion channels (LGICs) have demonstrated a plausible site of anesthetic action within their transmembrane domains. Although there is a consensus that the transmembrane domain is formed from four membrane-spanning segments, the secondary structure of these segments is not known. We utilized 10 state-of-the-art bioinformatics techniques to predict the transmembrane topology of the tetrameric regions within six members of the LGIC family that are relevant to anesthetic action. They are the human forms of the GABA alpha 1 receptor, the glycine alpha 1 receptor, the 5HT3 serotonin receptor, the nicotinic AChR alpha 4 and alpha 7 receptors and the Torpedo nAChR alpha 1 receptor. The algorithms utilized were HMMTOP, TMHMM, TMPred, PHDhtm, DAS, TMFinder, SOSUI, TMAP, MEMSAT and TOPPred2. The resulting predictions were superimposed on to a multiple sequence alignment of the six amino acid sequences created using the CLUSTAL W algorithm. There was a clear statistical consensus for the presence of four alpha helices in those regions experimentally thought to span the membrane. The consensus of 10 topology prediction techniques supports the hypothesis that the transmembrane subunits of the LGICs are tetrameric bundles of alpha helices.

Cystic fibrosis transmembrane conductance regulator: temperature-dependent cysteine reactivity suggests different stable conformers of the conduction pathway.

PubMed

Liu, Xuehong; Dawson, David C

2011-11-29

Cysteine scanning has been widely used to identify pore-lining residues in mammalian ion channels, including the cystic fibrosis transmembrane conductance regulator (CFTR). These studies, however, have been typically conducted at room temperature rather than human body temperature. Reports of substantial effects of temperature on gating and anion conduction in CFTR channels as well as an unexpected pattern of cysteine reactivity in the sixth transmembrane segment (TM6) prompted us to investigate the effect of temperature on the reactivity of cysteines engineered into TM6 of CFTR. We compared reaction rates at temperatures ranging from 22 to 37 °C for cysteines placed on either side of an apparent size-selective accessibility barrier previously defined by comparing reactivity toward channel-permeant and channel-impermeant, thiol-directed reagents. The results indicate that the reactivity of cysteines at three positions extracellular to the position of the accessibility barrier, 334, 336, and 337, is highly temperature-dependent. At 37 °C, cysteines at these positions were highly reactive toward MTSES(-), whereas at 22 °C, the reaction rates were 2-6-fold slower to undetectable. An activation energy of 157 kJ/mol for the reaction at position 337 is consistent with the hypothesis that, at physiological temperature, the extracellular portion of the CFTR pore can adopt conformations that differ significantly from those that can be accessed at room temperature. However, the position of the accessibility barrier defined empirically by applying channel-permeant and channel-impermeant reagents to the extracellular aspect of the pore is not altered. The results illuminate previous scanning results and indicate that the assay temperature is a critical variable in studies designed to use chemical modification to test structural models for the CFTR anion conduction pathway.

OncoLogicTM

EPA Science Inventory

OncoLogic^TM - A Computer System to Evaluate the Carcinogenic Potential of Chemicals
OncoLogic^TM is a software program that evaluates the likelihood that a chemical may cause cancer. OncoLogic^TM has been peer reviewed and is being rele...

Warfarin resistance associated with genetic polymorphism of VKORC1: linking clinical response to molecular mechanism using computational modeling.

PubMed

Lewis, Benjamin C; Nair, Pramod C; Heran, Subash S; Somogyi, Andrew A; Bowden, Jeffrey J; Doogue, Matthew P; Miners, John O

2016-01-01

The variable response to warfarin treatment often has a genetic basis. A protein homology model of human vitamin K epoxide reductase, subunit 1 (VKORC1), was generated to elucidate the mechanism of warfarin resistance observed in a patient with the Val66Met mutation. The VKORC1 homology model comprises four transmembrane (TM) helical domains and a half helical lid domain. Cys132 and Cys135, located in the N-terminal end of TM-4, are linked through a disulfide bond. Two distinct binding sites for warfarin were identified. Site-1, which binds vitamin K epoxide (KO) in a catalytically favorable orientation, shows higher affinity for S-warfarin compared with R-warfarin. Site-2, positioned in the domain occupied by the hydrophobic tail of KO, binds both warfarin enantiomers with similar affinity. Displacement of Arg37 occurs in the Val66Met mutant, blocking access of warfarin (but not KO) to Site-1, consistent with clinical observation of warfarin resistance.

Diode-pumped SrMoO4:Tm3+ crystal lasing near 1500 nm

NASA Astrophysics Data System (ADS)

Doroshenko, M. E.; Sulc, J.; Jelinkova, H.; Nemec, M.; Ivleva, L. I.; Dunaeva, E. E.

2018-04-01

Diode-pumped lasing of Tm3+ ions in SrMoO4 crystal at wavelength near 1500 nm was obtained for the first time to our best knowledge. Two laser lines with orthogonal polarizations were observed at 1452 and 1492 nm. The laser pulse was self-terminated about 500 µs after the pump start.

Positions of the cytoplasmic end of BK α S0 helix relative to S1–S6 and of β1 TM1 and TM2 relative to S0–S6

PubMed Central

Liu, Guoxia; Zakharov, Sergey I.; Yao, Yongneng

2015-01-01

The large-conductance, voltage- and Ca2+-gated K+ (BK) channel consists of four α subunits, which form a voltage- and Ca2+-gated channel, and up to four modulatory β subunits. The β1 subunit is expressed in smooth muscle, where it slows BK channel kinetics and shifts the conductance–voltage (G-V) curve to the left at [Ca2+] > 2 µM. In addition to the six transmembrane (TM) helices, S1–S6, conserved in all voltage-dependent K+ channels, BK α has a unique seventh TM helix, S0, which may contribute to the unusual rightward shift in the G-V curve of BK α in the absence of β1 and to a leftward shift in its presence. Such a role is supported by the close proximity of S0 to S3 and S4 in the voltage-sensing domain. Furthermore, on the extracellular side of the membrane, one of the two TM helices of β1, TM2, is adjacent to S0. We have now analyzed induced disulfide bond formation between substituted Cys residues on the cytoplasmic side of the membrane. There, in contrast, S0 is closest to the S2–S3 loop, from which position it is displaced on the addition of β1. The cytoplasmic ends of β1 TM1 and TM2 are adjacent and are located between the S2–S3 loop of one α subunit and S1 of a neighboring α subunit and are not adjacent to S0; i.e., S0 and TM2 have different trajectories through the membrane. In the absence of β1, 70% of disulfide bonding of W43C (S0) and L175C (S2–S3) has no effect on V50 for activation, implying that the cytoplasmic end of S0 and the S2–S3 loop move in concert, if at all, during activation. Otherwise, linking them together in one state would obstruct the transition to the other state, which would certainly change V50. PMID:25667410

Understanding the effects on constitutive activation and drug binding of a D130N mutation in the β2 adrenergic receptor via molecular dynamics simulation.

PubMed

Zhu, Yanyan; Yuan, Yuan; Xiao, Xiuchan; Zhang, Liyun; Guo, Yanzhi; Pu, Xuemei

2014-11-01

G-protein-coupled receptors (GPCRs) are currently one of the largest families of drug targets. The constitutive activation induced by mutation of key GPCR residues is associated closely with various diseases. However, the structural basis underlying such activation and its role in drug binding has remained unclear. Herein, we used all-atom molecular dynamics simulations and free energy calculations to study the effects of a D130N mutation on the structure of β2 adrenergic receptor (β2AR) and its binding of the agonist salbutamol. The results indicate that the mutation caused significant changes in some key helices. In particular, the mutation leads to the departure of transmembrane 3 (TM3) from transmembrane 6 (TM6) and marked changes in the NPxxY region as well as the complete disruption of a key ionic lock, all of which contribute to the observed constitutive activation. In addition, the D130N mutation weakens some important H-bonds, leading to structural changes in these regions. Binding free energy calculations indicate that van der Waals and electrostatic interactions are the main driving forces in binding salbutamol; however, binding strength in the mutant β2AR is significantly enhanced mainly through modifying electrostatic interactions. Further analysis revealed that the increase in binding energy upon mutation stems mainly from the H-bonds formed between the hydroxyl group of salbutamol and the serine residues of TM5. This observation suggests that modifications of the H-bond groups of this drug could significantly influence drug efficacy in the treatment of diseases associated with this mutation.

A single amino acid substitution within the transmembrane domain of the human immunodeficiency virus type 1 Vpu protein renders simian-human immunodeficiency virus (SHIV{sub KU-1bMC33}) susceptible to rimantadine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hout, David R.; Gomez, Lisa M.; Pacyniak, Erik

2006-05-10

Previous studies from our laboratory have shown that the transmembrane domain (TM) of the Vpu protein of human immunodeficiency virus type 1 (HIV-1) contributes to the pathogenesis of SHIV{sub KU-1bMC33} in macaques and that the TM domain of Vpu could be replaced with the M2 protein viroporin from influenza A virus. Recently, we showed that the replacement of the TM domain of Vpu with that of the M2 protein of influenza A virus resulted in a virus (SHIV{sub M2}) that was sensitive to rimantadine [Hout, D.R., Gomez, M.L., Pacyniak, E., Gomez, L.M., Inbody, S.H., Mulcahy, E.R., Culley, N., Pinson, D.M.,more » Powers, M.F., Wong, S.W., Stephens, E.B., 2006. Substitution of the transmembrane domain of Vpu in simian human immunodeficiency virus (SHIV{sub KU-1bMC33}) with that of M2 of influenza A results in a virus that is sensitive to inhibitors of the M2 ion channel and is pathogenic for pig-tailed macaques. Virology 344, 541-558]. Based on previous studies of the M2 protein which have shown that the His-X-X-X-Trp motif within the M2 is essential to the function of the M2 proton channel, we have constructed a novel SHIV in which the alanine at position 19 of the TM domain was replaced with a histidine residue resulting in the motif His-Ile-Leu-Val-Trp. The SHIV{sub VpuA19H} replicated with similar kinetics as the parental SHIV{sub KU-1bMC33} and pulse-chase analysis revealed that the processing of viral proteins was similar to SHIV{sub KU-1bMC33}. This SHIV{sub VpuA19H} virus was found to be more sensitive to the M2 ion channel blocker rimantadine than SHIV{sub M2}. Electron microscopic examination of SHIV{sub VpuA19H}-infected cells treated with rimantadine revealed an accumulation of viral particles at the cell surface and within intracellular vesicles, which was similar to that previously observed to SHIV{sub M2}-infected cells treated with rimantadine. These data indicate that the Vpu protein of HIV-1 can be converted into a rimantadine-sensitive ion channel

Structure of Thermotoga maritima TM0439: implications for the mechanism of bacterial GntR transcription regulators with Zn2+-binding FCD domains

PubMed Central

Zheng, Meiying; Cooper, David R.; Grossoehme, Nickolas E.; Yu, Minmin; Hung, Li-Wei; Cieslik, Marcin; Derewenda, Urszula; Lesley, Scott A.; Wilson, Ian A.; Giedroc, David P.; Derewenda, Zygmunt S.

2009-01-01

The GntR superfamily of dimeric transcription factors, with more than 6200 members encoded in bacterial genomes, are characterized by N-terminal winged-helix DNA-binding domains and diverse C-terminal regulatory domains which provide a basis for the classification of the constituent families. The largest of these families, FadR, contains nearly 3000 proteins with all-α-helical regulatory domains classified into two related Pfam families: FadR_C and FCD. Only two crystal structures of FadR-family members, those of Escherichia coli FadR protein and LldR from Corynebacterium glutamicum, have been described to date in the literature. Here, the crystal structure of TM0439, a GntR regulator with an FCD domain found in the Thermotoga maritima genome, is described. The FCD domain is similar to that of the LldR regulator and contains a buried metal-binding site. Using atomic absorption spectroscopy and Trp fluorescence, it is shown that the recombinant protein contains bound Ni2+ ions but that it is able to bind Zn2+ with K d < 70 nM. It is concluded that Zn2+ is the likely physiological metal and that it may perform either structural or regulatory roles or both. Finally, the TM0439 structure is compared with two other FadR-family structures recently deposited by structural genomics consortia. The results call for a revision in the classification of the FadR family of transcription factors. PMID:19307717

Semithiobambus[6]uril is a transmembrane anion transporter.

PubMed

Lang, Chao; Mohite, Amar; Deng, Xiaoli; Yang, Feihu; Dong, Zeyuan; Xu, Jiayun; Liu, Junqiu; Keinan, Ehud; Reany, Ofer

2017-07-04

Semithiobambus[6]uril is shown to be an efficient transmembrane anion transporter. Although all bambusuril analogs (having either O, S or N atoms in their portals) are excellent anion binders, only the sulfur analog is also an effective anion transporter capable of polarizing lipid membranes through selective anion uniport. This notable divergence reflects significant differences in the lipophilic character of the bambusuril analogs.

A Convenient Approach to Synthesizing Peptide C-Terminal N-Alkyl Amides

PubMed Central

Fang, Wei-Jie; Yakovleva, Tatyana; Aldrich, Jane V.

2014-01-01

Peptide C-terminal N-alkyl amides have gained more attention over the past decade due to their biological properties, including improved pharmacokinetic and pharmacodynamic profiles. However, the synthesis of this type of peptide on solid phase by current available methods can be challenging. Here we report a convenient method to synthesize peptide C-terminal N-alkyl amides using the well-known Fukuyama N-alkylation reaction on a standard resin commonly used for the synthesis of peptide C-terminal primary amides, the PAL-PEG-PS (Peptide Amide Linker-polyethylene glycol-polystyrene) resin. The alkylation and oNBS deprotection were conducted under basic conditions and were therefore compatible with this acid labile resin. The alkylation reaction was very efficient on this resin with a number of different alkyl iodides or bromides, and the synthesis of model enkephalin N-alkyl amide analogs using this method gave consistently high yields and purities, demonstrating the applicability of this methodology. The synthesis of N-alkyl amides was more difficult on a Rink amide resin, especially the coupling of the first amino acid to the N-alkyl amine, resulting in lower yields for loading the first amino acid onto the resin. This method can be widely applied in the synthesis of peptide N-alkyl amides. PMID:22252422

The C- and N-Terminal Residues of Synthetic Heptapeptide Ion Channels Influence Transport Efficacy Through Phospholipid Bilayers

PubMed Central

Djedovič, Natasha; Ferdani, Riccardo; Harder, Egan; Pajewska, Jolanta; Pajewski, Robert; Weber, Michelle E.; Schlesinger, Paul H.; Gokel, George W.

2008-01-01

The synthetic peptide, R2N-COCH2OCH2CO-Gly-Gly-Gly-Pro-Gly-Gly-Gly-OR’, was shown to be selective for Cl- over K+ when R is n-octadecyl and R’ is benzyl. Nineteen heptapeptides have now been prepared in which the N-terminal and C-terminal residues have been varied. All of the N-terminal residues are dialkyl but the C-terminal chains are esters, 2° amides, or 3° amides. The compounds having varied N-terminal anchors and C-terminal benzyl groups are as follows: 1, R = n-propyl; 2, R = n-hexyl; 3, R = n-octyl; 4, R = n-decyl; 5, R = n-dodecyl; 6, R = n-tetradecyl; 7, R = n-hexadecyl; 8, R = n-octadecyl. Compounds 9-19 have R = n-octadecyl and C-terminal residues as follows: 9, OR’ = OCH2CH3; 10, OR’ = OCH(CH3)2; 11, OR’ = O(CH2)6CH3; 12, OR’ = OCH2-c-C6H11; 13, OR’ = O(CH2)9CH3; 14, OR’ = O (CH2)17CH3; 15, NR’2 = N[(CH2)6CH3]2; 16, NHR’ = NH(CH2)9CH3; 17, NR’2 = N[(CH2)9CH3]2; 18, NHR’ = NH(CH2)17CH3; 19, NR’2 = N[(CH2)17CH3]2. The highest anion transport activities were observed as follows. For the benzyl esters whose N-terminal residues were varied, i.e. 1-8, compound 3 was most active. For the C18 anchored esters 10-14, n-heptyl ester 11 was most active. For the C18 anchored, C-terminal amides 15-19, di-n-decylamide 17 was most active. It was concluded that both the C- and N-terminal anchors were important for channel function in the bilayer but that activity was lost unless only one of the two anchoring groups was dominant. PMID:19633728

Grafting PNIPAAm from β-barrel shaped transmembrane nanopores.

PubMed

Charan, Himanshu; Kinzel, Julia; Glebe, Ulrich; Anand, Deepak; Garakani, Tayebeh Mirzaei; Zhu, Leilei; Bocola, Marco; Schwaneberg, Ulrich; Böker, Alexander

2016-11-01

The research on protein-polymer conjugates by grafting from the surface of proteins has gained significant interest in the last decade. While there are many studies with globular proteins, membrane proteins have remained untouched to the best of our knowledge. In this study, we established the conjugate formation with a class of transmembrane proteins and grow polymer chains from the ferric hydroxamate uptake protein component A (FhuA; a β-barrel transmembrane protein of Escherichia coli). As the lysine residues of naturally occurring FhuA are distributed over the whole protein, FhuA was reengineered to have up to 11 lysines, distributed symmetrically in a rim on the membrane exposed side (outside) of the protein channel and exclusively above the hydrophobic region. Reengineering of FhuA ensures a polymer growth only on the outside of the β-barrel and prevents blockage of the channel as a result of the polymerization. A water-soluble initiator for controlled radical polymerization (CRP) was consecutively linked to the lysine residues of FhuA and N-isopropylacrylamide (NIPAAm) polymerized under copper-mediated CRP conditions. The conjugate formation was analyzed by using MALDI-ToF mass spectrometry, SDS-PAGE, circular dichroism spectroscopy, analytical ultracentrifugation, dynamic light scattering, transmission electron microscopy and size exclusion chromatography. Such conjugates combine the specific functions of the transmembrane proteins, like maintaining membrane potential gradients or translocation of substrates with the unique properties of synthetic polymers such as temperature and pH stimuli handles. FhuA-PNIPAAm conjugates will serve as functional nanosized building blocks for applications in targeted drug delivery, self-assembly systems, functional membranes and transmembrane protein gated nanoreactors. Copyright © 2016 Elsevier Ltd. All rights reserved.

Construction of pTM series plasmids for gene expression in Brucella species.

PubMed

Tian, Mingxing; Qu, Jing; Bao, Yanqing; Gao, Jianpeng; Liu, Jiameng; Wang, Shaohui; Sun, Yingjie; Ding, Chan; Yu, Shengqing

2016-04-01

Brucellosis, the most common widespread zoonotic disease, is caused by Brucella spp., which are facultative, intracellular, Gram-negative bacteria. With the development of molecular biology techniques, more and more virulence-associated factors have been identified in Brucella spp. A suitable plasmid system is an important tool to study virulence genes in Brucella. In this study, we constructed three constitutive replication plasmids (pTM1-Cm, pTM2-Amp, and pTM3-Km) using the replication origin (rep) region derived from the pBBR1-MCS vector. Also, a DNA fragment containing multiple cloning sites (MCSs) and a terminator sequence derived from the pCold vector were produced for complementation of the deleted genes. Besides pGH-6×His, a plasmid containing the groE promoter of Brucella spp. was constructed to express exogenous proteins in Brucella with high efficiency. Furthermore, we constructed the inducible expression plasmid pZT-6×His, containing the tetracycline-inducible promoter pzt1, which can induce expression by the addition of tetracycline in the Brucella culture medium. The constructed pTM series plasmids will play an important role in the functional investigation of Brucella spp. Copyright © 2016 Elsevier B.V. All rights reserved.

Involvement of the N-terminal region in alpha-crystallin-lens membrane recognition

NASA Technical Reports Server (NTRS)

Ifeanyi, F.; Takemoto, L.; Spooner, B. S. (Principal Investigator)

1991-01-01

Previous studies have demonstrated that alpha-crystallin binds specifically, in a saturable manner, to lens membrane. To determine the region of the alpha-crystallin molecule that might be involved in this binding, native alpha-crystallin from the bovine lens has been treated by limited digestion with trypsin, to produce alpha-A molecules with an intact C-terminal region, and a nicked N-terminal region. Compared to intact alpha-crystallin, trypsin-treated alpha-crystallin binds less avidly to lens membrane, suggesting that the N-terminal region of the alpha-A molecule may play a key role in the recognition between lens membrane and crystallin.

In-Operando Spatial Imaging of Edge Termination Electric Fields in GaN Vertical p-n Junction Diodes

DOE PAGES

Leonard, Francois; Dickerson, J. R.; King, M. P.; ...

2016-05-03

Control of electric fields with edge terminations is critical to maximize the performance of high-power electronic devices. We proposed a variety of edge termination designs which makes the optimization of such designs challenging due to many parameters that impact their effectiveness. And while modeling has recently allowed new insight into the detailed workings of edge terminations, the experimental verification of the design effectiveness is usually done through indirect means, such as the impact on breakdown voltages. In this letter, we use scanning photocurrent microscopy to spatially map the electric fields in vertical GaN p-n junction diodes in operando. We alsomore » reveal the complex behavior of seemingly simple edge termination designs, and show how the device breakdown voltage correlates with the electric field behavior. Modeling suggests that an incomplete compensation of the p-type layer in the edge termination creates a bilayer structure that leads to these effects, with variations that significantly impact the breakdown voltage.« less

Releasing N-glycan from peptide N-terminus by N-terminal succinylation assisted enzymatic deglycosylation.

PubMed

Weng, Yejing; Sui, Zhigang; Jiang, Hao; Shan, Yichu; Chen, Lingfan; Zhang, Shen; Zhang, Lihua; Zhang, Yukui

2015-04-22

Due to the important roles of N-glycoproteins in various biological processes, the global N-glycoproteome analysis has been paid much attention. However, by current strategies for N-glycoproteome profiling, peptides with glycosylated Asn at N-terminus (PGANs), generated by protease digestion, could hardly be identified, due to the poor deglycosylation capacity by enzymes. However, theoretically, PGANs occupy 10% of N-glycopeptides in the typical tryptic digests. Therefore, in this study, we developed a novel strategy to identify PGANs by releasing N-glycans through the N-terminal site-selective succinylation assisted enzymatic deglycosylation. The obtained PGANs information is beneficial to not only achieve the deep coverage analysis of glycoproteomes, but also discover the new biological functions of such modification.

Charged Residues at the First Transmembrane Region Contribute to the Voltage Dependence of the Slow Gate of Connexins*

PubMed Central

Pinto, Bernardo I.; García, Isaac E.; Pupo, Amaury; Retamal, Mauricio A.; Martínez, Agustín D.; Latorre, Ramón; González, Carlos

2016-01-01

Connexins (Cxs) are a family of membrane-spanning proteins that form gap junction channels and hemichannels. Connexin-based channels exhibit two distinct voltage-dependent gating mechanisms termed slow and fast gating. Residues located at the C terminus of the first transmembrane segment (TM-1) are important structural components of the slow gate. Here, we determined the role of the charged residues at the end of TM-1 in voltage sensing in Cx26, Cx46, and Cx50. Conductance/voltage curves obtained from tail currents together with kinetics analysis reveal that the fast and slow gates of Cx26 involves the movement of two and four charges across the electric field, respectively. Primary sequence alignment of different Cxs shows the presence of well conserved glutamate residues in the C terminus of TM-1; only Cx26 contains a lysine in that position (lysine 41). Neutralization of lysine 41 in Cx26 increases the voltage dependence of the slow gate. Swapping of lysine 41 with glutamate 42 maintains the voltage dependence. In Cx46, neutralization of negative charges or addition of a positive charge in the Cx26 equivalent region reduced the slow gate voltage dependence. In Cx50, the addition of a glutamate in the same region decreased the voltage dependence, and the neutralization of a negative charge increased it. These results indicate that the charges at the end of TM-1 are part of the slow gate voltage sensor in Cxs. The fact that Cx42, which has no charge in this region, still presents voltage-dependent slow gating suggests that charges still unidentified also contribute to the slow gate voltage sensitivity. PMID:27143357

Expression cloning and chromosomal mapping of the leukocyte activation antigen CD97, a new seven-span transmembrane molecule of the secretin receptor superfamily with an unusual extracellular domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hamann, J.; Hamann, D.; Lier, R.A.W.

1995-08-15

CD97 is a monomeric glycoprotein of 75 to 85 kDa that is induced rapidly on the surface of most leukocytes upon activation. We herein report the isolation of a cDNA encoding human CD97 by expression cloning in COS cells. The 3-kb cDNA clone encodes a mature polypeptide chain of 722 amino acids with a predicted molecular mass of 79 kDa. Within the C-terminal part of the protein, a region with seven hydrophobic segments was identified, suggesting that CD97 is a seven-span transmembrane molecule. Sequence comparison indicates that CD97 is the first leukocyte Ag in a recently described superfamily that includesmore » the receptors for secretin, calcitonin, and other mammalian and insect peptide hormones. Different from these receptors, CD97 has an extended extracellular region of 433 amino acids that possesses three N-terminal epidermal growth factor-like domains, two of them with a calcium-binding site, and single Arg-Gly-Asp (RGD) motif. The existence of structural elements characteristic for extracellular matrix proteins in a seven-span transmembrane molecule makes CD97 a receptor potentially involved in both adhesion and signaling processes early after leukocyte activation. The gene encoding CD97 is localized on chromosome 19 (19p13.12-13.2).« less

Structure-Based Sequence Alignment of the Transmembrane Domains of All Human GPCRs: Phylogenetic, Structural and Functional Implications

PubMed Central

Cvicek, Vaclav; Goddard, William A.; Abrol, Ravinder

2016-01-01

The understanding of G-protein coupled receptors (GPCRs) is undergoing a revolution due to increased information about their signaling and the experimental determination of structures for more than 25 receptors. The availability of at least one receptor structure for each of the GPCR classes, well separated in sequence space, enables an integrated superfamily-wide analysis to identify signatures involving the role of conserved residues, conserved contacts, and downstream signaling in the context of receptor structures. In this study, we align the transmembrane (TM) domains of all experimental GPCR structures to maximize the conserved inter-helical contacts. The resulting superfamily-wide GpcR Sequence-Structure (GRoSS) alignment of the TM domains for all human GPCR sequences is sufficient to generate a phylogenetic tree that correctly distinguishes all different GPCR classes, suggesting that the class-level differences in the GPCR superfamily are encoded at least partly in the TM domains. The inter-helical contacts conserved across all GPCR classes describe the evolutionarily conserved GPCR structural fold. The corresponding structural alignment of the inactive and active conformations, available for a few GPCRs, identifies activation hot-spot residues in the TM domains that get rewired upon activation. Many GPCR mutations, known to alter receptor signaling and cause disease, are located at these conserved contact and activation hot-spot residue positions. The GRoSS alignment places the chemosensory receptor subfamilies for bitter taste (TAS2R) and pheromones (Vomeronasal, VN1R) in the rhodopsin family, known to contain the chemosensory olfactory receptor subfamily. The GRoSS alignment also enables the quantification of the structural variability in the TM regions of experimental structures, useful for homology modeling and structure prediction of receptors. Furthermore, this alignment identifies structurally and functionally important residues in all human GPCRs

The human dopamine transporter forms a tetramer in the plasma membrane: cross-linking of a cysteine in the fourth transmembrane segment is sensitive to cocaine analogs.

PubMed

Hastrup, Hanne; Sen, Namita; Javitch, Jonathan A

2003-11-14

Using cysteine cross-linking, we demonstrated previously that the dopamine transporter (DAT) is at least a homodimer, with the extracellular end of transmembrane segment (TM) 6 at a symmetrical dimer interface. We have now explored the possibility that DAT exists as a higher order oligomer in the plasma membrane. Cysteine cross-linking of wild type DAT resulted in bands on SDS-PAGE consistent with dimer, trimer, and tetramer, suggesting that DAT forms a tetramer in the plasma membrane. A cysteine-depleted DAT (CD-DAT) into which only Cys243 or Cys306 was reintroduced was cross-linked to dimer, suggesting that these endogenous cysteines in TM4 and TM6, respectively, were cross-linked at a symmetrical dimer interface. Reintroduction of both Cys243 and Cys306 into CD-DAT led to a pattern of cross-linking indistinguishable from that of wild type, with dimer, trimer, and tetramer bands. This indicated that the TM4 interface and the TM6 interface are distinct and further suggested that DAT may exist in the plasma membrane as a dimer of dimers, with two symmetrical homodimer interfaces. The cocaine analog MFZ 2-12 and other DAT inhibitors, including benztropine and mazindol, protected Cys243 against cross-linking. In contrast, two substrates of DAT, dopamine and tyramine, did not significantly impact cross-linking. We propose that the impairment of cross-linking produced by the inhibitors results from a conformational change at the TM4 interface, further demonstrating that these compounds are not neutral blockers but by themselves have effects on the structure of the transporter.

R76 in transmembrane domain 3 of the aspartate:alanine transporter AspT is involved in substrate transport.

PubMed

Suzuki, Satomi; Nanatani, Kei; Abe, Keietsu

2016-01-01

The L-aspartate:L-alanine antiporter of Tetragenococcus halophilus (AspT) possesses an arginine residue (R76) within the GxxxG motif in the central part of transmembrane domain 3 (TM3)-a residue that has been estimated to transport function. In this study, we carried out amino acid substitutions of R76 and used proteoliposome reconstitution for analyzing the transport function of each substitution. Both l-aspartate and l-alanine transport assays showed that R76K has higher activity than the AspT-WT (R76), whereas R76D and R76E have lower activity than the AspT-WT. These results suggest that R76 is involved in AspT substrate transport.

Transmembrane adenylyl cyclase regulates amphibian sperm motility through Protein Kinase A activation

PubMed Central

O’Brien, Emma D.; Krapf, Darío; Cabada, Marcelo O.; Visconti, Pablo E.; Arranz, Silvia E.

2014-01-01

Sperm motility is essential for achieving fertilization. In animals with external fertilization as amphibians, spermatozoa are stored in a quiescent state in the testis. Spermiation to hypotonic fertilization media triggers activation of sperm motility. Bufo arenarum sperm are immotile in artificial seminal plasma (ASP) but acquire in situ flagellar beating upon dilution. In addition to the effect of low osmolarity on sperm motility activation, we report that diffusible factors of the egg jelly coat (EW) regulate motility patterns, switching from in situ to progressive movement. The signal transduction pathway involved in amphibian sperm motility activation is mostly unknown. In the present study, we show a correlation between motility activation triggered by low osmotic pressure and activation of protein kinase A (PKA). Moreover, this is the first study to present strong evidences that point toward a role of a transmembrane adenyl-cyclase (tmAC) in the regulation of amphibian sperm motility through PKA activation. PMID:21126515

Differential structural properties of GLP-1 and exendin-4 determine their relative affinity for the GLP-1 receptor N-terminal extracellular domain.

PubMed

Runge, Steffen; Schimmer, Susann; Oschmann, Jan; Schiødt, Christine Bruun; Knudsen, Sanne Möller; Jeppesen, Claus Bekker; Madsen, Kjeld; Lau, Jesper; Thøgersen, Henning; Rudolph, Rainer

2007-05-15

Glucagon-like peptide-1 (GLP-1) and exendin-4 (Ex4) are homologous peptides with established potential for treatment of type 2 diabetes. They bind and activate the pancreatic GLP-1 receptor (GLP-1R) with similar affinity and potency and thereby promote insulin secretion in a glucose-dependent manner. GLP-1R belongs to family B of the seven transmembrane G-protein coupled receptors. The N-terminal extracellular domain (nGLP-1R) is a ligand binding domain with differential affinity for Ex4 and GLP-1: low affinity for GLP-1 and high affinity for exendin-4. The superior affinity of nGLP-1R for Ex4 was previously explained by an additional interaction between nGLP-1R and the C-terminal Trp-cage of Ex4. In this study we have combined biophysical and pharmacological approaches thus relating structural properties of the ligands in solution to their relative binding affinity for nGLP-1R. We used both a tracer competition assay and ligand-induced thermal stabilization of nGLP-1R to measure the relative affinity of full length, truncated, and chimeric ligands for soluble refolded nGLP-1R. The ligands in solution and the conformational consequences of ligand binding to nGLP-1R were characterized by circular dichroism and fluorescence spectroscopy. We found a correlation between the helical content of the free ligands and their relative binding affinity for nGLP-1R, supporting the hypothesis that the ligands are helical at least in the segment that binds to nGLP-1R. The Trp-cage of Ex4 was not necessary to maintain a superior helicity of Ex4 compared to GLP-1. The results suggest that the differential affinity of nGLP-1R is explained almost entirely by divergent residues in the central part of the ligands: Leu10-Gly30 of Ex4 and Val16-Arg36 of GLP-1. In view of our results it appears that the Trp-cage plays only a minor role for the interaction between Ex4 and nGLP-1R and for the differential affinity of nGLP-1R for GLP-1 and Ex4.

N-terminal pro-brain natriuretic peptide in acute Kawasaki disease correlates with coronary artery involvement.

PubMed

Adjagba, Philippe M; Desjardins, Laurent; Fournier, Anne; Spigelblatt, Linda; Montigny, Martine; Dahdah, Nagib

2015-10-01

We have lately documented the importance of N-terminal pro-brain natriuretic peptide in aiding the diagnosis of Kawasaki disease. We sought to investigate the potential value of N-terminal pro-brain natriuretic peptide pertaining to the prediction of coronary artery dilatation (Z-score>2.5) and/or of resistance to intravenous immunoglobulin therapy. We hypothesised that increased serum N-terminal pro-brain natriuretic peptide level correlates with increased coronary artery dilatation and/or resistance to intravenous immunoglobulin. We carried out a prospective study involving newly diagnosed patients treated with 2 g/kg intravenous immunoglobulin within 5-10 days of onset of fever. Echocardiography was performed in all patients at onset, then weekly for 3 weeks, then at month 2, and month 3. Coronary arteries were measured at each visit, and coronary artery Z-score was calculated. All the patients had N-terminal pro-brain natriuretic peptide serum level measured at onset, and the Z-score calculated. There were 109 patients enrolled at 6.58±2.82 days of fever, age 3.79±2.92 years. High N-terminal pro-brain natriuretic peptide level was associated with coronary artery dilatation at onset in 22.2 versus 5.6% for normal N-terminal pro-brain natriuretic peptide levels (odds ratio 4.8 [95% confidence interval 1.05-22.4]; p=0.031). This was predictive of cumulative coronary artery dilatation for the first 3 months (p=0.04-0.02), but not during convalescence at 2-3 months (odds ratio 1.28 [95% confidence interval 0.23-7.3]; p=non-significant). Elevated N-terminal pro-brain natriuretic peptide levels did not predict intravenous immunoglobulin resistance, 15.3 versus 13.5% (p=1). Elevated N-terminal pro-brain natriuretic peptide level correlates with acute coronary artery dilatation in treated Kawasaki disease, but not with intravenous immunoglobulin resistance.

Evidence for an unusual transmembrane configuration of AGG3, a class C Gγ subunit of Arabidopsis

DOE PAGES

Wolfenstetter, Susanne; Chakravorty, David; Kula, Ryan; ...

2014-12-22

Heterotrimeric G proteins are crucial for the perception of external signals and subsequent signal transduction in animal and plant cells. In both model systems, the complex is comprised of one Gα, one Gβ and one Gγ subunit. However, in addition to the canonical Gγ subunits (Class A), plants also possess two unusual, plant-specific classes of Gγ subunits (Classes B and C) not yet found in animals. These include Gγ subunits lacking the C-terminal CaaX motif (Class B) which is important for membrane anchoring of the protein, and thus give rise to a flexible subpopulation of Gβ/γ heterodimers that is notmore » necessarily restricted to the plasma membrane. Even more interesting, plants also contain Class C Gγ subunits which are twice the size of canonical Gγs, with a predicted transmembrane domain, and a large cysteine-rich, extracellular C-terminus. However, neither the presence of the transmembrane domain nor the membrane topology has been unequivocally demonstrated. Finally, we provide compelling evidence that AGG3, a Class C Ggamma subunit of Arabidopsis, contains a functional transmembrane domain, which is sufficient but not essential for plasma membrane localization, and that the cysteine-rich C-terminus is extracellular.« less

c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Sclerosis

DTIC Science & Technology

2014-10-01

AWARD NUMBER: W81XWH-12-1-0431 TITLE: “c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Sclerosis ” PRINCIPAL INVESTIGATOR...TITLE AND SUBTITLE “c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Scelerosis” 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH

The tmRNA website

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hudson, Corey M.; Williams, Kelly P.

We report that the transfer-messenger RNA (tmRNA) and its partner protein SmpB act together in resolving problems arising when translating bacterial ribosomes reach the end of mRNA with no stop codon. Their genes have been found in nearly all bacterial genomes and in some organelles. The tmRNA Website serves tmRNA sequences, alignments and feature annotations, and has recently moved to http: //bioinformatics.sandia.gov/tmrna/. New features include software used to find the sequences, an update raising the number of unique tmRNA sequences from 492 to 1716, and a database of SmpB sequences which are served along with the tmRNA sequence from themore » same organism.« less

The tmRNA website

DOE PAGES

Hudson, Corey M.; Williams, Kelly P.

2014-11-05

We report that the transfer-messenger RNA (tmRNA) and its partner protein SmpB act together in resolving problems arising when translating bacterial ribosomes reach the end of mRNA with no stop codon. Their genes have been found in nearly all bacterial genomes and in some organelles. The tmRNA Website serves tmRNA sequences, alignments and feature annotations, and has recently moved to http: //bioinformatics.sandia.gov/tmrna/. New features include software used to find the sequences, an update raising the number of unique tmRNA sequences from 492 to 1716, and a database of SmpB sequences which are served along with the tmRNA sequence from themore » same organism.« less

COPT6 Is a Plasma Membrane Transporter That Functions in Copper Homeostasis in Arabidopsis and Is a Novel Target of SQUAMOSA Promoter-binding Protein-like 7*

PubMed Central

Jung, Ha-il; Gayomba, Sheena R.; Rutzke, Michael A.; Craft, Eric; Kochian, Leon V.; Vatamaniuk, Olena K.

2012-01-01

Among the mechanisms controlling copper homeostasis in plants is the regulation of its uptake and tissue partitioning. Here we characterized a newly identified member of the conserved CTR/COPT family of copper transporters in Arabidopsis thaliana, COPT6. We showed that COPT6 resides at the plasma membrane and mediates copper accumulation when expressed in the Saccharomyces cerevisiae copper uptake mutant. Although the primary sequence of COPT6 contains the family conserved domains, including methionine-rich motifs in the extracellular N-terminal domain and a second transmembrane helix (TM2), it is different from the founding family member, S. cerevisiae Ctr1p. This conclusion was based on the finding that although the positionally conserved Met106 residue in the TM2 of COPT6 is functionally essential, the conserved Met27 in the N-terminal domain is not. Structure-function studies revealed that the N-terminal domain is dispensable for COPT6 function in copper-replete conditions but is important under copper-limiting conditions. In addition, COPT6 interacts with itself and with its homolog, COPT1, unlike Ctr1p, which interacts only with itself. Analyses of the expression pattern showed that although COPT6 is expressed in different cell types of different plant organs, the bulk of its expression is located in the vasculature. We also show that COPT6 expression is regulated by copper availability that, in part, is controlled by a master regulator of copper homeostasis, SPL7. Finally, studies using the A. thaliana copt6-1 mutant and plants overexpressing COPT6 revealed its essential role during copper limitation and excess. PMID:22865877

Hsp90 N- and C-terminal double inhibition synergistically suppresses Bcr-Abl-positive human leukemia cells

PubMed Central

Chen, Xianling; Chen, Xiaole; Li, Ding; Fan, Yingjuan; Xu, Jianhua; Chen, Yuanzhong; Wu, Lixian

2017-01-01

Heat shock protein 90 (Hsp90) contains amino (N)–terminal domain, carboxyl(C)-terminal domain, and middle domains, which activate Hsp90 chaperone function cooperatively in tumor cells. One terminal occupancy might influence another terminal binding with inhibitor. The Bcr-Abl kinase is one of the Hsp90 clients implicated in the pathogenesis of chronic myeloid leukemia (CML). Present studies demonstrate that double inhibition of the N- and C-terminal termini can disrupt Hsp90 chaperone function synergistically, but not antagonistically, in Bcr-Abl-positive human leukemia cells. Furthermore, both the N-terminal inhibitor 17-AAG and the C-terminal inhibitor cisplatin (CP) have the capacity to suppress progenitor cells; however, only CP is able to inhibit leukemia stem cells (LSCs) significantly, which implies that the combinational treatment is able to suppress human leukemia in different mature states. PMID:28036294

Identification of amino acids in the human tetherin transmembrane domain responsible for HIV-1 Vpu interaction and susceptibility.

PubMed

Kobayashi, Tomoko; Ode, Hirotaka; Yoshida, Takeshi; Sato, Kei; Gee, Peter; Yamamoto, Seiji P; Ebina, Hirotaka; Strebel, Klaus; Sato, Hironori; Koyanagi, Yoshio

2011-01-01

Tetherin, also known as BST-2/CD317/HM1.24, is an antiviral cellular protein that inhibits the release of HIV-1 particles from infected cells. HIV-1 viral protein U (Vpu) is a specific antagonist of human tetherin that might contribute to the high virulence of HIV-1. In this study, we show that three amino acid residues (I34, L37, and L41) in the transmembrane (TM) domain of human tetherin are critical for the interaction with Vpu by using a live cell-based assay. We also found that the conservation of an additional amino acid at position 45 and two residues downstream of position 22, which are absent from monkey tetherins, are required for the antagonism by Vpu. Moreover, computer-assisted structural modeling and mutagenesis studies suggest that an alignment of these four amino acid residues (I34, L37, L41, and T45) on the same helical face in the TM domain is crucial for the Vpu-mediated antagonism of human tetherin. These results contribute to the molecular understanding of human tetherin-specific antagonism by HIV-1 Vpu.

The diagnostic value of plasma N-terminal connective tissue growth factor levels in children with heart failure.

PubMed

Li, Gang; Song, Xueqing; Xia, Jiyi; Li, Jing; Jia, Peng; Chen, Pengyuan; Zhao, Jian; Liu, Bin

2017-01-01

The aim of this study was to assess the diagnostic value of plasma N-terminal connective tissue growth factor in children with heart failure. Methods and results Plasma N-terminal connective tissue growth factor was determined in 61 children, including 41 children with heart failure, 20 children without heart failure, and 30 healthy volunteers. The correlations between plasma N-terminal connective tissue growth factor levels and clinical parameters were investigated. Moreover, the diagnostic value of N-terminal connective tissue growth factor levels was evaluated. Compared with healthy volunteers and children without heart failure, plasma N-terminal connective tissue growth factor levels were significantly elevated in those with heart failure (p0.05), but it obviously improved the ability of diagnosing heart failure in children, as demonstrated by the integrated discrimination improvement (6.2%, p=0.013) and net re-classification improvement (13.2%, p=0.017) indices. Plasma N-terminal connective tissue growth factor is a promising diagnostic biomarker for heart failure in children.

Structural Insights into Triglyceride Storage Mediated by Fat Storage-Inducing Transmembrane (FIT) Protein 2

PubMed Central

Gross, David A.; Snapp, Erik L.; Silver, David L.

2010-01-01

Fat storage-Inducing Transmembrane proteins 1 & 2 (FIT1/FITM1 and FIT2/FITM2) belong to a unique family of evolutionarily conserved proteins localized to the endoplasmic reticulum that are involved in triglyceride lipid droplet formation. FIT proteins have been shown to mediate the partitioning of cellular triglyceride into lipid droplets, but not triglyceride biosynthesis. FIT proteins do not share primary sequence homology with known proteins and no structural information is available to inform on the mechanism by which FIT proteins function. Here, we present the experimentally-solved topological models for FIT1 and FIT2 using N-glycosylation site mapping and indirect immunofluorescence techniques. These methods indicate that both proteins have six-transmembrane-domains with both N- and C-termini localized to the cytosol. Utilizing this model for structure-function analysis, we identified and characterized a gain-of-function mutant of FIT2 (FLL(157-9)AAA) in transmembrane domain 4 that markedly augmented the total number and mean size of lipid droplets. Using limited-trypsin proteolysis we determined that the FLL(157-9)AAA mutant has enhanced trypsin cleavage at K86 relative to wild-type FIT2, indicating a conformational change. Taken together, these studies indicate that FIT2 is a 6 transmembrane domain-containing protein whose conformation likely regulates its activity in mediating lipid droplet formation. PMID:20520733

LANDSAT D local user terminal study

NASA Technical Reports Server (NTRS)

Alexander, L.; Louie, M.; Spencer, R.; Stow, W. K.

1976-01-01

The effect of the changes incorporated in the LANDSAT D system on the ability of a local user terminal to receive, record and process data in real time was studied. Alternate solutions to the problems raised by these changes were evaluated. A loading analysis was performed in order to determine the quantities of data that a local user terminal (LUT) would be interested in receiving and processing. The number of bits in an MSS and a TM scene were calculated along with the number of scenes per day that an LUT might require for processing. These then combined to a total number of processed bits/day for an LUT as a function of sensor and coverage circle radius.

Transmembrane Segment Five Serines of the D4 Dopamine Receptor Uniquely Influence the Interactions of Dopamine, Norepinephrine, and Ro10-4548

PubMed Central

Cummings, David F.; Ericksen, Spencer S.; Goetz, Angela

2010-01-01

Conserved serines of transmembrane segment (TM) five (TM5) are critical for the interactions of endogenous catecholamines with α1- and α2-adrenergic, β2-adrenergic, and D1, D2, and D3 dopamine receptors. The unique high-affinity interaction of the D4 dopamine receptor subtype with both norepinephrine and dopamine, and the fact that TM5 serine interactions have never been studied for this receptor subtype, led us to investigate the interactions of ligands with D4 receptor TM5 serines. Serine-to-alanine mutations at positions 5.42 and 5.46 drastically decreased affinities of dopamine and norepinephrine for the D4 receptor. The D4-S5.43A receptor mutant had substantially reduced affinity for norepinephrine, but a modest loss of affinity for dopamine. In functional assays of cAMP accumulation, norephinephrine was unable to activate any of the mutant receptors, even though the agonist quinpirole displayed wild-type functional properties for all of them. Dopamine was unable to activate the S5.46A mutant and had reduced potency for the S5.43A mutant and reduced potency and efficacy for the S5.42A mutant. In contrast, Ro10-4548 [RAC-2′-2-hydroxy-3-4-(4-hydroxy-2-methoxyphenyl)-1-piperazinyl-propoxy-acetanilide], a catechol-like antagonist of the wild-type receptor unexpectedly functions as an agonist of the S5.43A mutant. Other noncatechol ligands had similar properties for mutant and wild-type receptors. This is the first example of a dopamine receptor point mutation selectively changing the receptor's interaction with a specific antagonist to that of an agonist, and together with other data, provides evidence, supported by molecular modeling, that catecholamine-type agonism is induced by different ligand-specific configurations of intermolecular H-bonds with the TM5 conserved serines. PMID:20215412

MarvelD3 regulates the c-Jun N-terminal kinase pathway during eye development in Xenopus

PubMed Central

Vacca, Barbara; Sanchez-Heras, Elena; Steed, Emily; Balda, Maria S.; Ohnuma, Shin-Ichi; Sasai, Noriaki; Mayor, Roberto

2016-01-01

ABSTRACT Ocular morphogenesis requires several signalling pathways controlling the expression of transcription factors and cell-cycle regulators. However, despite a well-known mechanism, the dialogue between those signals and factors remains to be unveiled. Here, we identify a requirement for MarvelD3, a tight junction transmembrane protein, in eye morphogenesis in Xenopus. MarvelD3 depletion led to an abnormally pigmented eye or even an eye-less phenotype, which was rescued by ectopic MarvelD3 expression. Altering MarvelD3 expression led to deregulated expression of cell-cycle regulators and transcription factors required for eye development. The eye phenotype was rescued by increased c-Jun terminal Kinase activation. Thus, MarvelD3 links tight junctions and modulation of the JNK pathway to eye morphogenesis. PMID:27870636

Altering the N-terminal arms of the polymerase manager protein UmuD modulates protein interactions.

PubMed

Murison, David A; Ollivierre, Jaylene N; Huang, Qiuying; Budil, David E; Beuning, Penny J

2017-01-01

Escherichia coli cells that are exposed to DNA damaging agents invoke the SOS response that involves expression of the umuD gene products, along with more than 50 other genes. Full-length UmuD is expressed as a 139-amino-acid protein, which eventually cleaves its N-terminal 24 amino acids to form UmuD'. The N-terminal arms of UmuD are dynamic and contain recognition sites for multiple partner proteins. Cleavage of UmuD to UmuD' dramatically affects the function of the protein and activates UmuC for translesion synthesis (TLS) by forming DNA Polymerase V. To probe the roles of the N-terminal arms in the cellular functions of the umuD gene products, we constructed additional N-terminal truncated versions of UmuD: UmuD 8 (UmuD Δ1-7) and UmuD 18 (UmuD Δ1-17). We found that the loss of just the N-terminal seven (7) amino acids of UmuD results in changes in conformation of the N-terminal arms, as determined by electron paramagnetic resonance spectroscopy with site-directed spin labeling. UmuD 8 is cleaved as efficiently as full-length UmuD in vitro and in vivo, but expression of a plasmid-borne non-cleavable variant of UmuD 8 causes hypersensitivity to UV irradiation, which we determined is the result of a copy-number effect. UmuD 18 does not cleave to form UmuD', but confers resistance to UV radiation. Moreover, removal of the N-terminal seven residues of UmuD maintained its interactions with the alpha polymerase subunit of DNA polymerase III as well as its ability to disrupt interactions between alpha and the beta processivity clamp, whereas deletion of the N-terminal 17 residues resulted in decreases in binding to alpha and in the ability to disrupt the alpha-beta interaction. We find that UmuD 8 mimics full-length UmuD in many respects, whereas UmuD 18 lacks a number of functions characteristic of UmuD.

Charged Residues at the First Transmembrane Region Contribute to the Voltage Dependence of the Slow Gate of Connexins.

PubMed

Pinto, Bernardo I; García, Isaac E; Pupo, Amaury; Retamal, Mauricio A; Martínez, Agustín D; Latorre, Ramón; González, Carlos

2016-07-22

Connexins (Cxs) are a family of membrane-spanning proteins that form gap junction channels and hemichannels. Connexin-based channels exhibit two distinct voltage-dependent gating mechanisms termed slow and fast gating. Residues located at the C terminus of the first transmembrane segment (TM-1) are important structural components of the slow gate. Here, we determined the role of the charged residues at the end of TM-1 in voltage sensing in Cx26, Cx46, and Cx50. Conductance/voltage curves obtained from tail currents together with kinetics analysis reveal that the fast and slow gates of Cx26 involves the movement of two and four charges across the electric field, respectively. Primary sequence alignment of different Cxs shows the presence of well conserved glutamate residues in the C terminus of TM-1; only Cx26 contains a lysine in that position (lysine 41). Neutralization of lysine 41 in Cx26 increases the voltage dependence of the slow gate. Swapping of lysine 41 with glutamate 42 maintains the voltage dependence. In Cx46, neutralization of negative charges or addition of a positive charge in the Cx26 equivalent region reduced the slow gate voltage dependence. In Cx50, the addition of a glutamate in the same region decreased the voltage dependence, and the neutralization of a negative charge increased it. These results indicate that the charges at the end of TM-1 are part of the slow gate voltage sensor in Cxs. The fact that Cx42, which has no charge in this region, still presents voltage-dependent slow gating suggests that charges still unidentified also contribute to the slow gate voltage sensitivity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

FoldGPCR: structure prediction protocol for the transmembrane domain of G protein-coupled receptors from class A.

PubMed

Michino, Mayako; Chen, Jianhan; Stevens, Raymond C; Brooks, Charles L

2010-08-01

Building reliable structural models of G protein-coupled receptors (GPCRs) is a difficult task because of the paucity of suitable templates, low sequence identity, and the wide variety of ligand specificities within the superfamily. Template-based modeling is known to be the most successful method for protein structure prediction. However, refinement of homology models within 1-3 A C alpha RMSD of the native structure remains a major challenge. Here, we address this problem by developing a novel protocol (foldGPCR) for modeling the transmembrane (TM) region of GPCRs in complex with a ligand, aimed to accurately model the structural divergence between the template and target in the TM helices. The protocol is based on predicted conserved inter-residue contacts between the template and target, and exploits an all-atom implicit membrane force field. The placement of the ligand in the binding pocket is guided by biochemical data. The foldGPCR protocol is implemented by a stepwise hierarchical approach, in which the TM helical bundle and the ligand are assembled by simulated annealing trials in the first step, and the receptor-ligand complex is refined with replica exchange sampling in the second step. The protocol is applied to model the human beta(2)-adrenergic receptor (beta(2)AR) bound to carazolol, using contacts derived from the template structure of bovine rhodopsin. Comparison with the X-ray crystal structure of the beta(2)AR shows that our protocol is particularly successful in accurately capturing helix backbone irregularities and helix-helix packing interactions that distinguish rhodopsin from beta(2)AR. (c) 2010 Wiley-Liss, Inc.

β-Subunit of the Ostα-Ostβ Organic Solute Transporter Is Required Not Only for Heterodimerization and Trafficking but Also for Function*

PubMed Central

Christian, Whitney V.; Li, Na; Hinkle, Patricia M.; Ballatori, Nazzareno

2012-01-01

The organic solute transporter, Ost/Slc51, is composed of two distinct proteins that must heterodimerize to generate transport activity, but the role of the individual subunits in mediating transport activity is unknown. The present study identified regions in Ostβ required for heterodimerization with Ostα, trafficking of the Ostα-Ostβ complex to the plasma membrane, and bile acid transport activity in HEK293 cells. Bimolecular fluorescence complementation analysis revealed that a 25-amino acid peptide containing the Ostβ transmembrane (TM) domain heterodimerized with Ostα, although the resulting complex failed to reach the plasma membrane and generate cellular [3H]taurocholate transport activity. Deletion of the single TM domain of Ostβ abolished interaction with Ostα, demonstrating that the TM segment is necessary and sufficient for formation of a heteromeric complex with Ostα. Mutation of the highly conserved tryptophan-asparagine sequence within the TM domain of Ostβ to alanines did not prevent cell surface trafficking, but abolished transport activity. Removal of the N-terminal 27 amino acids of Ostβ resulted in a transporter complex that reached the plasma membrane and exhibited transport activity at 30 °C. Complete deletion of the C terminus of Ostβ abolished [3H]taurocholate transport activity, but reinsertion of two native arginines immediately C-terminal to the TM domain rescued this defect. These positively charged residues establish the correct Nexo/Ccyt topology of the peptide, in accordance with the positive inside rule. Together, the results demonstrate that Ostβ is required for both proper trafficking of Ostα and formation of the functional transport unit, and identify specific residues of Ostβ critical for these processes. PMID:22535958

Regulation of presynaptic Ca2+, synaptic plasticity and contextual fear conditioning by a N-terminal β-amyloid fragment.

PubMed

Lawrence, James L M; Tong, Mei; Alfulaij, Naghum; Sherrin, Tessi; Contarino, Mark; White, Michael M; Bellinger, Frederick P; Todorovic, Cedomir; Nichols, Robert A

2014-10-22

Soluble β-amyloid has been shown to regulate presynaptic Ca(2+) and synaptic plasticity. In particular, picomolar β-amyloid was found to have an agonist-like action on presynaptic nicotinic receptors and to augment long-term potentiation (LTP) in a manner dependent upon nicotinic receptors. Here, we report that a functional N-terminal domain exists within β-amyloid for its agonist-like activity. This sequence corresponds to a N-terminal fragment generated by the combined action of α- and β-secretases, and resident carboxypeptidase. The N-terminal β-amyloid fragment is present in the brains and CSF of healthy adults as well as in Alzheimer's patients. Unlike full-length β-amyloid, the N-terminal β-amyloid fragment is monomeric and nontoxic. In Ca(2+) imaging studies using a model reconstituted rodent neuroblastoma cell line and isolated mouse nerve terminals, the N-terminal β-amyloid fragment proved to be highly potent and more effective than full-length β-amyloid in its agonist-like action on nicotinic receptors. In addition, the N-terminal β-amyloid fragment augmented theta burst-induced post-tetanic potentiation and LTP in mouse hippocampal slices. The N-terminal fragment also rescued LTP inhibited by elevated levels of full-length β-amyloid. Contextual fear conditioning was also strongly augmented following bilateral injection of N-terminal β-amyloid fragment into the dorsal hippocampi of intact mice. The fragment-induced augmentation of fear conditioning was attenuated by coadministration of nicotinic antagonist. The activity of the N-terminal β-amyloid fragment appears to reside largely in a sequence surrounding a putative metal binding site, YEVHHQ. These findings suggest that the N-terminal β-amyloid fragment may serve as a potent and effective endogenous neuromodulator. Copyright © 2014 the authors 0270-6474/14/3414210-09$15.00/0.

Aberrant termination of reproduction-related TMEM30C transcripts in the hominoids.

PubMed

Osada, Naoki; Hashimoto, Katsuyuki; Hirai, Momoki; Kusuda, Jun

2007-05-01

Finding genetic novelties that may contribute to human-specific physiology and diseases is a key issue of current biomedical studies. TMEM30C is a gene containing two transmembrane (TM) domains and homologous to the yeast CDC50 family, which is related to polarized cell division. It is conserved among mammals along with two other paralogs, TMEM30A and TMEM30B. We found that TMEM30C is expressed specifically in the testis of mammals, in contrast to the relatively wide expression distributions of the other paralogs. While macaques expressed two alternative splicing isoforms which include one or two TM domains, humans and chimpanzees predominantly expressed truncated transcripts because of the mutations in the splicing and/or poly(A) signal sites. The major transcript in humans harbored non-stop ORF (open reading frame) while the chimpanzee counterpart encoded a protein with one TM domain. The difference was due to the 1-bp indel upstream of the poly(A) signal site. In addition, both the hominoids expressed minor transcripts encoding short proteins with one TM domain. Phylogenetic analysis has showed the acceleration of amino acid substitution after the human and chimpanzee divergence, which may have been caused by a recent relaxation in functional constraints or positive selection on TMEM30C. Elucidating the precise reproductive function of TMEM30C in mammals will be important to the foundation of divergence in higher primates at a molecular level.

A conserved αβ transmembrane interface forms the core of a compact T-cell receptor–CD3 structure within the membrane

PubMed Central

Krshnan, Logesvaran; Park, Soohyung; Im, Wonpil; Call, Melissa J.; Call, Matthew E.

2016-01-01

The T-cell antigen receptor (TCR) is an assembly of eight type I single-pass membrane proteins that occupies a central position in adaptive immunity. Many TCR-triggering models invoke an alteration in receptor complex structure as the initiating event, but both the precise subunit organization and the pathway by which ligand-induced alterations are transferred to the cytoplasmic signaling domains are unknown. Here, we show that the receptor complex transmembrane (TM) domains form an intimately associated eight-helix bundle organized by a specific interhelical TCR TM interface. The salient features of this core structure are absolutely conserved between αβ and γδ TCR sequences and throughout vertebrate evolution, and mutations at key interface residues caused defects in the formation of stable TCRαβ:CD3δε:CD3γε:ζζ complexes. These findings demonstrate that the eight TCR–CD3 subunits form a compact and precisely organized structure within the membrane and provide a structural basis for further investigation of conformationally regulated models of transbilayer TCR signaling. PMID:27791034

A conserved αβ transmembrane interface forms the core of a compact T-cell receptor-CD3 structure within the membrane.

PubMed

Krshnan, Logesvaran; Park, Soohyung; Im, Wonpil; Call, Melissa J; Call, Matthew E

2016-10-25

The T-cell antigen receptor (TCR) is an assembly of eight type I single-pass membrane proteins that occupies a central position in adaptive immunity. Many TCR-triggering models invoke an alteration in receptor complex structure as the initiating event, but both the precise subunit organization and the pathway by which ligand-induced alterations are transferred to the cytoplasmic signaling domains are unknown. Here, we show that the receptor complex transmembrane (TM) domains form an intimately associated eight-helix bundle organized by a specific interhelical TCR TM interface. The salient features of this core structure are absolutely conserved between αβ and γδ TCR sequences and throughout vertebrate evolution, and mutations at key interface residues caused defects in the formation of stable TCRαβ:CD3δε:CD3γε:ζζ complexes. These findings demonstrate that the eight TCR-CD3 subunits form a compact and precisely organized structure within the membrane and provide a structural basis for further investigation of conformationally regulated models of transbilayer TCR signaling.

Modeling Cr-to-Tm and Cr-to-Tm-to-Ho energy transfer in YAG crystals

NASA Technical Reports Server (NTRS)

Swetits, John J.

1991-01-01

A systematic analysis of energy transfer processes in crystals of YAG doped with varying concentrations of Cr and Tm is described. Both spectral measurements and measurements of the temporal response to pulsed excitation are used to give independent determinations of the microscopic interaction parameter for Cr to Tm transfer. The different factors in influencing the temperature dependence of the Cr to Tm transfer are discussed. The dependence of the Tm cross-relaxation rate on Tm concentration is determined.

Molecular cloning and characterization of autophagy-related gene TmATG8 in Listeria-invaded hemocytes of Tenebrio molitor.

PubMed

Tindwa, Hamisi; Jo, Yong Hun; Patnaik, Bharat Bhusan; Lee, Yong Seok; Kang, Sang Sun; Han, Yeon Soo

2015-07-01

Macroautophagy (hereinafter called autophagy) is a highly regulated process used by eukaryotic cells to digest portions of the cytoplasm that remodels and recycles nutrients and disposes of unwanted cytoplasmic constituents. Currently 36 autophagy-related genes (ATG) and their homologs have been characterized in yeast and higher eukaryotes, including insects. In the present study, we identified and functionally characterized the immune function of an ATG8 homolog in a coleopteran insect, Tenebrio molitor (TmATG8). The cDNA of TmATG8 comprises of an ORF of 363 bp that encodes a protein of 120 amino acid residues. TmATG8 transcripts are detected in all the developmental stages analyzed. TmAtg8 protein contains a highly conserved C-terminal glycine residue (Gly116) and shows high amino acid sequence identity (98%) to its Tribolium castaneum homolog, TcAtg8. Loss of function of TmATG8 by RNAi led to a significant increase in the mortality rates of T. molitor larvae against Listeria monocytogenes. Unlike dsEGFP-treated control larvae, TmATG8-silenced larvae failed to turn-on autophagy in hemocytes after injection with L. monocytogenes. These data suggest that TmATG8 play a role in mediating autophagy-based clearance of Listeria in T. molitor. Copyright © 2015 Elsevier Ltd. All rights reserved.

Aspergillus fumigatus generates an enhanced Th2-biased immune response in mice with defective cystic fibrosis transmembrane conductance regulator.

PubMed

Allard, Jenna B; Poynter, Matthew E; Marr, Kieren A; Cohn, Lauren; Rincon, Mercedes; Whittaker, Laurie A

2006-10-15

Cystic fibrosis (CF) lung disease is characterized by persistent airway inflammation and airway infection that ultimately leads to respiratory failure. Aspergillus sp. are present in the airways of 20-40% of CF patients and are of unclear clinical significance. In this study, we demonstrate that CF transmembrane conductance regulator (CFTR)-deficient (CFTR knockout, Cftr(tm1Unc-)TgN(fatty acid-binding protein)CFTR) and mutant (DeltaF508) mice develop profound lung inflammation in response to Aspergillus fumigatus hyphal Ag exposure. CFTR-deficient mice also develop an enhanced Th2 inflammatory response to A. fumigatus, characterized by elevated IL-4 in the lung and IgE and IgG1 in serum. In contrast, CFTR deficiency does not promote a Th1 immune response. Furthermore, we demonstrate that CD4+ T cells from naive CFTR-deficient mice produce higher levels of IL-4 in response to TCR ligation than wild-type CD4+ T cells. The Th2 bias of CD4+ T cells in the absence of functional CFTR correlates with elevated nuclear levels of NFAT. Thus, CFTR is important to maintain the Th1/Th2 balance in CD4+ T cells.

Structural determinants of Actinomyces sortase SrtC2 required for membrane localization and assembly of type 2 fimbriae for interbacterial coaggregation and oral biofilm formation.

PubMed

Wu, Chenggang; Mishra, Arunima; Reardon, Melissa E; Huang, I-Hsiu; Counts, Sarah C; Das, Asis; Ton-That, Hung

2012-05-01

As a pioneer colonizer of the oral cavity, Actinomyces oris expresses proteinaceous pili (also called fimbriae) to mediate the following two key events in biofilm formation: adherence to saliva deposits on enamel and interbacterial associations. Assembly of type 2 fimbriae that directly facilitate coaggregation with oral streptococci and Actinomyces biofilm development requires the class C sortase SrtC2. Although the general sortase-associated mechanisms have been elucidated, several structural attributes unique to the class C sortases require functional investigation. Mutational studies reported here suggest that the N-terminal transmembrane (TM) region of SrtC2, predicted to contain a signal peptide sequence, is cleaved off the mature protein and that this processing is critical for the proper integration of the enzyme at the cytoplasmic membrane, which is mediated by the extended hydrophobic C terminus containing a TM domain and a cytoplasmic tail. Deletion of this putative TM or the entire cytoplasmic domain abolished the enzyme localization and functionality. Alanine substitution of the conserved catalytic Cys-His dyad abrogated the SrtC2 enzymatic activity. In contrast, mutations designed to alter a "lid" domain that covers the catalytic pocket of a class C sortase showed no effect on enzyme activity. Finally, each of the deleterious mutations that affected SrtC2 activity or membrane localization also eliminated Actinomyces species biofilm development and bacterial coaggregation with streptococci. We conclude that the N terminus of SrtC2, which contains the signal sequence, is required for proper protein translocation and maturation, while the extended C-terminal hydrophobic region serves as a stable membrane anchor for proper enzyme functionality.

Structural Determinants of Actinomyces sortase SrtC2 Required for Membrane Localization and Assembly of Type 2 Fimbriae for Interbacterial Coaggregation and Oral Biofilm Formation

PubMed Central

Wu, Chenggang; Mishra, Arunima; Reardon, Melissa E.; Huang, I-Hsiu; Counts, Sarah C.; Das, Asis

2012-01-01

As a pioneer colonizer of the oral cavity, Actinomyces oris expresses proteinaceous pili (also called fimbriae) to mediate the following two key events in biofilm formation: adherence to saliva deposits on enamel and interbacterial associations. Assembly of type 2 fimbriae that directly facilitate coaggregation with oral streptococci and Actinomyces biofilm development requires the class C sortase SrtC2. Although the general sortase-associated mechanisms have been elucidated, several structural attributes unique to the class C sortases require functional investigation. Mutational studies reported here suggest that the N-terminal transmembrane (TM) region of SrtC2, predicted to contain a signal peptide sequence, is cleaved off the mature protein and that this processing is critical for the proper integration of the enzyme at the cytoplasmic membrane, which is mediated by the extended hydrophobic C terminus containing a TM domain and a cytoplasmic tail. Deletion of this putative TM or the entire cytoplasmic domain abolished the enzyme localization and functionality. Alanine substitution of the conserved catalytic Cys-His dyad abrogated the SrtC2 enzymatic activity. In contrast, mutations designed to alter a “lid” domain that covers the catalytic pocket of a class C sortase showed no effect on enzyme activity. Finally, each of the deleterious mutations that affected SrtC2 activity or membrane localization also eliminated Actinomyces species biofilm development and bacterial coaggregation with streptococci. We conclude that the N terminus of SrtC2, which contains the signal sequence, is required for proper protein translocation and maturation, while the extended C-terminal hydrophobic region serves as a stable membrane anchor for proper enzyme functionality. PMID:22447896

Membrane interaction of the N-terminal domain of chemokine receptor CXCR1.

PubMed

Haldar, Sourav; Raghuraman, H; Namani, Trishool; Rajarathnam, Krishna; Chattopadhyay, Amitabha

2010-06-01

The N-terminal domain of chemokine receptors constitutes one of the two critical ligand binding sites, and plays important roles by mediating binding affinity, receptor selectivity, and regulating function. In this work, we monitored the organization and dynamics of a 34-mer peptide of the CXC chemokine receptor 1 (CXCR1) N-terminal domain and its interaction with membranes by utilizing a combination of fluorescence-based approaches and surface pressure measurements. Our results show that the CXCR1 N-domain 34-mer peptide binds vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and upon binding, the tryptophan residues of the peptide experience motional restriction and exhibit red edge excitation shift (REES) of 19nm. These results are further supported by increase in fluorescence anisotropy and mean fluorescence lifetime upon membrane binding. These results constitute one of the first reports demonstrating membrane interaction of the N-terminal domain of CXCR1 and gain relevance in the context of the emerging role of cellular membranes in chemokine signaling.

Comparative study of high power Tm:YLF and Tm:LLF slab lasers in continuous wave regime.

PubMed

Berrou, Antoine; Collett, Oliver J P; Morris, Daniel; Esser, M J Daniel

2018-04-16

We report on Tm:YLF and Tm:LLF slab lasers (1.5 x 11 x 20 mm 3 ) end pumped from one end with a high-brightness 792 nm laser diode stack. These two lasers are compared under identical pump conditions in continuous-wave regime. A stronger negative thermal lens in Tm:LLF than in Tm:YLF is highlighted, making it more difficult to operate the Tm:LLF laser under stable lasing conditions. In a configuration where the high reflectivity cavity mirror has a radius of curvature of r = 150 mm, the Tm:YLF (Tm:LLF) laser produces a maximum output power of 150 W (143 W) for 428 W of incident pump power (respectively). For a second cavity configuration where the high reflectivity cavity mirror has a radius of curvature of r = 500 mm, the Tm:YLF laser produces a maximum output power of 164 W for 412 W of incident pump power and a 57% slope efficiency with respect to the absorbed pump power. The emitted wavelength of these two lasers are measured as a function of the output coupler reflectivity and it shows that Tm:LLF laser emits at a longer wavelength than Tm:YLF.

Effect and mode of action of the Texel muscling QTL (TM-QTL) on carcass traits in purebred Texel lambs.

PubMed

Macfarlane, J M; Lambe, N R; Matika, O; Johnson, P L; Wolf, B T; Haresign, W; Bishop, S C; Bünger, L

2014-07-01

TM-QTL is a quantitative trait locus (QTL) on ovine chromosome 18 (OAR18) known to affect loin muscling in Texel sheep. Previous work suggested that its mode of inheritance is consistent with paternal polar overdominance, but this has yet to be formally demonstrated. This study used purebred Texel sheep segregating for TM-QTL to confirm its presence in the chromosomal region in which it was first reported and to determine its pattern of inheritance. To do so, this study used the first available data from a Texel flock, which included homozygote TM-QTL carriers (TM/TM; n=34) in addition to homozygote non-carriers (+/+; n=40 and, heterozygote TM-QTL-carriers inheriting TM-QTL from their sire (TM/+; n=53) or their dam (+/TM; n=17). Phenotypes included a wide range of loin muscling, carcass composition and tissue distribution traits. The presence of a QTL affecting ultrasound muscle depth on OAR18 was confirmed with a paternal QTL effect ranging from +0.54 to +2.82 mm UMD (s.e. 0.37 to 0.57 mm) across the sires segregating for TM-QTL. Loin muscle width, depth and area, loin muscle volume and dissected M. longissimus lumborum weight were significantly greater for TM/+ than +/+ lambs (+2.9% to +7.9%; P<0.05). There was significant evidence that the effect of TM-QTL on the various loin muscling traits measured was paternally polar overdominant (P<0.05). In contrast, there was an additive effect of TM-QTL on both live weight at 20 weeks and carcass weight; TM/TM animals were significantly (P<0.05) heavier than +/+ (+11.1% and +7.3%, respectively) and +/TM animals (+11.9% and +11.7%, respectively), with TM/+ intermediate. Weights of the leg, saddle and shoulder region (corrected for carcass weight) were similar in the genotypic groups. There was a tendency for lambs inheriting TM-QTL from their sire to be less fat with slightly more muscle than non-carriers. For example, carcass muscle weight measured by live animal CT-scanning was 2.8% higher in TM/TM than +/+ lambs (P<0

Design, Synthesis, and Evaluation of N- and C-Terminal Protein Bioconjugates as G Protein-Coupled Receptor Agonists.

PubMed

Healey, Robert D; Wojciechowski, Jonathan P; Monserrat-Martinez, Ana; Tan, Susan L; Marquis, Christopher P; Sierecki, Emma; Gambin, Yann; Finch, Angela M; Thordarson, Pall

2018-02-21

A G protein-coupled receptor (GPCR) agonist protein, thaumatin, was site-specifically conjugated at the N- or C-terminus with a fluorophore for visualization of GPCR:agonist interactions. The N-terminus was specifically conjugated using a synthetic 2-pyridinecarboxyaldehyde reagent. The interaction profiles observed for N- and C-terminal conjugates were varied; N-terminal conjugates interacted very weakly with the GPCR of interest, whereas C-terminal conjugates bound to the receptor. These chemical biology tools allow interactions of therapeutic proteins:GPCR to be monitored and visualized. The methodology used for site-specific bioconjugation represents an advance in application of 2-pyridinecarboxyaldehydes for N-terminal specific bioconjugations.

Identification of Amino Acids in the Human Tetherin Transmembrane Domain Responsible for HIV-1 Vpu Interaction and Susceptibility▿ †

PubMed Central

Kobayashi, Tomoko; Ode, Hirotaka; Yoshida, Takeshi; Sato, Kei; Gee, Peter; Yamamoto, Seiji P.; Ebina, Hirotaka; Strebel, Klaus; Sato, Hironori; Koyanagi, Yoshio

2011-01-01

Tetherin, also known as BST-2/CD317/HM1.24, is an antiviral cellular protein that inhibits the release of HIV-1 particles from infected cells. HIV-1 viral protein U (Vpu) is a specific antagonist of human tetherin that might contribute to the high virulence of HIV-1. In this study, we show that three amino acid residues (I34, L37, and L41) in the transmembrane (TM) domain of human tetherin are critical for the interaction with Vpu by using a live cell-based assay. We also found that the conservation of an additional amino acid at position 45 and two residues downstream of position 22, which are absent from monkey tetherins, are required for the antagonism by Vpu. Moreover, computer-assisted structural modeling and mutagenesis studies suggest that an alignment of these four amino acid residues (I34, L37, L41, and T45) on the same helical face in the TM domain is crucial for the Vpu-mediated antagonism of human tetherin. These results contribute to the molecular understanding of human tetherin-specific antagonism by HIV-1 Vpu. PMID:21068238

Apoptosis-like cell death in Leishmania donovani treated with KalsomeTM10, a new liposomal amphotericin B

PubMed Central

Shadab, Md.; Jha, Baijayanti; Asad, Mohammad; Deepthi, Makaraju; Kamran, Mohd.; Ali, Nahid

2017-01-01

Objective The present study aimed to elucidate the cell death mechanism in Leishmania donovani upon treatment with KalsomeTM10, a new liposomal amphotericin B. Methodology/Principal findings We studied morphological alterations in promastigotes through phase contrast and scanning electron microscopy. Phosphatidylserine (PS) exposure, loss of mitochondrial membrane potential and disruption of mitochondrial integrity was determined by flow cytometry using annexinV-FITC, JC-1 and mitotraker, respectively. For analysing oxidative stress, generation of H2O2 (bioluminescence kit) and mitochondrial superoxide O2− (mitosox) were measured. DNA fragmentation was evaluated using terminal deoxyribonucleotidyl transferase mediated dUTP nick-end labelling (TUNEL) and DNA laddering assay. We found that KalsomeTM10 is more effective then Ambisome against the promastigote as well as intracellular amastigote forms. The mechanistic study showed that KalsomeTM10 induced several morphological alterations in promastigotes typical of apoptosis. KalsomeTM10 treatment showed a dose- and time-dependent exposure of PS in promastigotes. Further, study on mitochondrial pathway revealed loss of mitochondrial membrane potential as well as disruption in mitochondrial integrity with depletion of intracellular pool of ATP. KalsomeTM10 treated promastigotes showed increased ROS production, diminished GSH levels and increased caspase-like activity. DNA fragmentation and cell cycle arrest was observed in KalsomeTM10 treated promastigotes. Apoptotic DNA fragmentation was also observed in KalsomeTM10 treated intracellular amastigotes. KalsomeTM10 induced generation of ROS and nitric oxide leads to the killing of the intracellular parasites. Moreover, endocytosis is indispensable for KalsomeTM10 mediated anti-leishmanial effect in host macrophage. Conclusions KalsomeTM10 induces apoptotic-like cell death in L. donovani parasites to exhibit its anti-leishmanial function. PMID:28170432

The Structure of Barmah Forest Virus as Revealed by Cryo-Electron Microscopy at a 6-Angstrom Resolution Has Detailed Transmembrane Protein Architecture and Interactions ▿ †

PubMed Central

Kostyuchenko, Victor A.; Jakana, Joanita; Liu, Xiangan; Haddow, Andrew D.; Aung, Myint; Weaver, Scott C.; Chiu, Wah; Lok, Shee-Mei

2011-01-01

Barmah Forest virus (BFV) is a mosquito-borne alphavirus that infects humans. A 6-Å-resolution cryo-electron microscopy three-dimensional structure of BFV exhibits a typical alphavirus organization, with RNA-containing nucleocapsid surrounded by a bilipid membrane anchored with the surface proteins E1 and E2. The map allows details of the transmembrane regions of E1 and E2 to be seen. The C-terminal end of the E2 transmembrane helix binds to the capsid protein. Following the E2 transmembrane helix, a short α-helical endodomain lies on the inner surface of the lipid envelope. The E2 endodomain interacts with E1 transmembrane helix from a neighboring E1-E2 trimeric spike, thereby acting as a spacer and a linker between spikes. In agreement with previous mutagenesis studies, the endodomain plays an important role in recruiting other E1-E2 spikes to the budding site during virus assembly. The E2 endodomain may thus serve as a target for antiviral drug design. PMID:21752915

15N and 31P solid-state NMR study of transmembrane domain alignment of M2 protein of influenza A virus in hydrated cylindrical lipid bilayers confined to anodic aluminum oxide nanopores.

PubMed

Chekmenev, Eduard Y; Hu, Jun; Gor'kov, Peter L; Brey, William W; Cross, Timothy A; Ruuge, Andres; Smirnov, Alex I

2005-04-01

This communication reports the first example of a high resolution solid-state 15N 2D PISEMA NMR spectrum of a transmembrane peptide aligned using hydrated cylindrical lipid bilayers formed inside nanoporous anodic aluminum oxide (AAO) substrates. The transmembrane domain SSDPLVVA(A-15N)SIIGILHLILWILDRL of M2 protein from influenza A virus was reconstituted in hydrated 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine bilayers that were macroscopically aligned by a conventional micro slide glass support or by the AAO nanoporous substrate. 15N and 31P NMR spectra demonstrate that both the phospholipids and the protein transmembrane domain are uniformly aligned in the nanopores. Importantly, nanoporous AAO substrates may offer several advantages for membrane protein alignment in solid-state NMR studies compared to conventional methods. Specifically, higher thermal conductivity of aluminum oxide is expected to suppress thermal gradients associated with inhomogeneous radio frequency heating. Another important advantage of the nanoporous AAO substrate is its excellent accessibility to the bilayer surface for exposure to solute molecules. Such high accessibility achieved through the substrate nanochannel network could facilitate a wide range of structure-function studies of membrane proteins by solid-state NMR.

Deletion of the N-terminal Domain (NTD) Alters the Ethanol Inhibition of NMDA Receptors in a Subunit-Dependent Manner

PubMed Central

Smothers, C. Thetford; Jin, Chun; Woodward, John J.

2013-01-01

Background Ethanol inhibition of NMDA receptors is poorly understood due in part to the organizational complexity of the receptor that provides ample locations for sites of action. Among these the N-terminal domain of NMDA receptor subunits contains binding sites for a variety of modulatory agents including zinc, protons and GluN2B selective antagonists such as ifenprodil or Ro-25–6981. Ethanol inhibition of neuronal NMDA receptors expressed in some brain areas has been reported to be occluded by the presence of ifenprodil or similar compounds suggesting that the N-terminal domain may be important in regulating the ethanol sensitivity of NMDA receptors. Methods Wild-type GluN1 and GluN2 subunits and those in which the coding sequence for the N-terminal domain was deleted were expressed in HEK293 cells. Whole-cell voltage-clamp recording was used to assess ethanol inhibition of wild-type and mutant receptors lacking the N-terminal domain. Results As compared to wild-type GluN1/GluN2A receptors, ethanol inhibition was slightly greater in cells expressing GluN2A subunits lacking the N-terminal domain. In contrast, GluN2B N-terminal deletion mutants showed normal ethanol inhibition while those lacking the N-terminal domain in both GluN1 and GluN2B subunits had decreased ethanol inhibition as compared to wild-type receptors. N-terminal domain lacking GluN2B receptors were insensitive to ifenprodil but retained normal sensitivity to ethanol. Conclusions These findings indicate that the N-terminal domain modestly influences the ethanol sensitivity of NMDA receptors in a subunit-dependent manner. They also show that ifenprodil’s actions on GluN2B containing receptors can be dissociated from those of ethanol. These results suggest that while the N-terminal domain is not a primary site of action for ethanol on NMDA receptors, it likely affects sensitivity via actions on intrinsic channel properties. PMID:23905549

Distinctive functions of Syk N-terminal and C-terminal SH2 domains in the signaling cascade elicited by oxidative stress in B cells.

PubMed

Ding, J; Takano, T; Hermann, P; Gao, S; Han, W; Noda, C; Yanagi, S; Yamamura, H

2000-05-01

Syk plays a crucial role in the transduction of oxidative stress signaling. In this paper, we investigated the roles of Src homology 2 (SH2) domains of Syk in oxidative stress signaling, using Syk-negative DT40 cells expressing the N- or C-terminal SH2 domain mutant [mSH2(N) or mSH2(C)] of Syk. Tyrosine phosphorylation of Syk in cells expressing mSH2(N) Syk after H(2)O(2) treatment was higher than that in cells expressing wild-type Syk or mSH2(C) Syk. The tyrosine phosphorylation of wild-type Syk and mSH2(C) Syk, but not that of mSH2(N), was sensitive to PP2, a specific inhibitor of Src-family protein-tyrosine kinase. In oxidative stress, the C-terminal SH2 domain of Syk was demonstrated to be required for induction of tyrosine phosphorylation of cellular proteins, phospholipase C (PLC)-gamma2 phosphorylation, inositol 1,4, 5-triphosphate (IP(3)) generation, Ca(2)(+) release from intracellular stores, and c-Jun N-terminal kinase activation. In contrast, in mSH2(N) Syk-expressing cells, tyrosine phosphorylation of intracellular proteins including PLC-gamma2 was markedly induced in oxidative stress. The enhanced phosphorylation of mSH2(N) Syk and PLC-gamma2, however, did not link to Ca(2)(+) mobilization from intracellular pools and IP(3) generation. Thus, the N- and C-terminal SH2 domains of Syk possess distinctive functions in oxidative stress signaling.

Oxidation of the N-terminal methionine of lens alpha-A crystallin

NASA Technical Reports Server (NTRS)

Takemoto, L.; Horwitz, J.; Emmons, T.; Spooner, B. S. (Principal Investigator)

1992-01-01

Antiserum against the N-terminal peptide of bovine alpha-A crystallin has been used to monitor purification of two different seropositive peptides (i.e. T1a and T1b) from a tryptic digest of bovine lens proteins. Both these peptides have similar amino acid compositions, but peptide T1b has a molecular weight 16 atomic mass units larger than T1a, suggesting posttranslational modification. Analysis of ionization fragments of the T1b peptide by mass spectrometry demonstrates that this difference in molecular weight is due to the in vivo oxidation of the N-terminal met residue of the alpha-A crystallin molecule.

Identifying and quantifying proteolytic events and the natural N terminome by terminal amine isotopic labeling of substrates.

PubMed

Kleifeld, Oded; Doucet, Alain; Prudova, Anna; auf dem Keller, Ulrich; Gioia, Magda; Kizhakkedathu, Jayachandran N; Overall, Christopher M

2011-09-22

Analysis of the sequence and nature of protein N termini has many applications. Defining the termini of proteins for proteome annotation in the Human Proteome Project is of increasing importance. Terminomics analysis of protease cleavage sites in degradomics for substrate discovery is a key new application. Here we describe the step-by-step procedures for performing terminal amine isotopic labeling of substrates (TAILS), a 2- to 3-d (depending on method of labeling) high-throughput method to identify and distinguish protease-generated neo-N termini from mature protein N termini with all natural modifications with high confidence. TAILS uses negative selection to enrich for all N-terminal peptides and uses primary amine labeling-based quantification as the discriminating factor. Labeling is versatile and suited to many applications, including biochemical and cell culture analyses in vitro; in vivo analyses using tissue samples from animal and human sources can also be readily performed. At the protein level, N-terminal and lysine amines are blocked by dimethylation (formaldehyde/sodium cyanoborohydride) and isotopically labeled by incorporating heavy and light dimethylation reagents or stable isotope labeling with amino acids in cell culture labels. Alternatively, easy multiplex sample analysis can be achieved using amine blocking and labeling with isobaric tags for relative and absolute quantification, also known as iTRAQ. After tryptic digestion, N-terminal peptide separation is achieved using a high-molecular-weight dendritic polyglycerol aldehyde polymer that binds internal tryptic and C-terminal peptides that now have N-terminal alpha amines. The unbound naturally blocked (acetylation, cyclization, methylation and so on) or labeled mature N-terminal and neo-N-terminal peptides are recovered by ultrafiltration and analyzed by tandem mass spectrometry (MS/MS). Hierarchical substrate winnowing discriminates substrates from the background proteolysis products and

Resin-assisted Enrichment of N-terminal Peptides for Characterizing Proteolytic Processing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Jong Seo; Dai, Ziyu; Aryal, Uma K.

2013-06-17

Proteolytic processing is a ubiquitous, irreversible posttranslational modification that plays an important role in cellular regulation in all living organisms. Herein we report a resin-assisted positive selection method for specifically enriching protein N-terminal peptides to facilitate the characterization of proteolytic processing events by liquid chromatography-tandem mass spectrometry. In this approach, proteins are initially reduced and alkylated and their lysine residues are converted to homoarginines. Then, protein N-termini are selectively converted to reactive thiol groups. We demonstrate that these sequential reactions were achieved with nearly quantitative efficiencies. Thiol-containing N-terminal peptides are then captured (>98% efficiency) by a thiol-affinity resin, a significantmore » improvement over the traditional avidin/biotin enrichment. Application to cell lysates of Aspergillus niger, a filamentous fungus of interest for biomass degradation, enabled the identification of 1672 unique protein N-termini and proteolytic cleavage sites from 690 unique proteins.« less

Magnetic ordering in TmGa.

PubMed

Cadogan, J M; Stewart, G A; Muñoz Pérez, S; Cobas, R; Hansen, B R; Avdeev, M; Hutchison, W D

2014-03-19

We have determined the magnetic structure of the intermetallic compound TmGa by high-resolution neutron powder diffraction and (169)Tm Mössbauer spectroscopy. This compound crystallizes in the orthorhombic (Cmcm) CrB-type structure and its magnetic structure is characterized by magnetic order of the Tm sublattice along the a-axis. The initial magnetic ordering occurs at 15(1) K and yields an incommensurate antiferromagnetic structure described by the propagation vector k1 = [0 0.275(2) 0]. At 12 K the dominant ferromagnetic ordering of the Tm sublattice along the a-axis develops in what appears to be a first-order transition. At 3 K the magnetic structure of TmGa is predominantly ferromagnetic but a weakened incommensurate component remains. The ferromagnetic Tm moment reaches 6.7(2) μB at 3 K and the amplitude of the remaining incommensurate component is 2.7(4) μB. The (169)Tm hyperfine magnetic field at 5 K is 631(1) T.

Plastic Muscles TM as lightweight, low voltage actuators and sensors

NASA Astrophysics Data System (ADS)

Bennett, Matthew; Leo, Donald; Duncan, Andrew

2008-03-01

Using proprietary technology, Discover Technologies has developed ionomeric polymer transducers that are capable of long-term operation in air. These "Plastic Muscle TM" transducers are useful as soft distributed actuators and sensors and have a wide range of applications in the aerospace, robotics, automotive, electronics, and biomedical industries. Discover Technologies is developing novel fabrication methods that allow the Plastic Muscles TM to be manufactured on a commercial scale. The Plastic Muscle TM transducers are capable of generating more than 0.5% bending strain at a peak strain rate of over 0.1 %/s with a 3 V input. Because the Plastic Muscles TM use an ionic liquid as a replacement solvent for water, they are able to operate in air for long periods of time. Also, the Plastic Muscles TM do not exhibit the characteristic "back relaxation" phenomenon that is common in water-swollen devices. The elastic modulus of the Plastic Muscle TM transducers is estimated to be 200 MPa and the maximum generated stress is estimated to be 1 MPa. Based on these values, the maximum blocked force at the tip of a 6 mm wide, 35 mm long actuator is estimated to be 19 mN. Modeling of the step response with an exponential series reveals nonlinearity in the transducers' behavior.

Molecular properties of the N-terminal extension of the fission yeast kinesin-5, Cut7.

PubMed

Edamatsu, M

2016-02-11

Kinesin-5 plays an essential role in spindle formation and function, and serves as a potential target for anti-cancer drugs. The aim of this study was to elucidate the molecular properties of the N-terminal extension of the Schizosaccharomyces pombe kinesin-5, Cut7. This extension is rich in charged amino acids and predicted to be intrinsically disordered. In S. pombe cells, a Cut7 construct lacking half the N-terminal extension failed to localize along the spindle microtubules and formed a monopolar spindle. However, a construct lacking the entire N-terminal extension exhibited normal localization and formed a typical bipolar spindle. In addition, in vitro analyses revealed that the truncated Cut7 constructs demonstrated similar motile velocities and directionalities as the wild-type motor protein, but the microtubule landing rates were significantly reduced. These findings suggest that the N-terminal extension is not required for normal Cut7 intracellular localization or function, but alters the microtubule-binding properties of this protein in vitro.

Imaging the Impact of Proton Irradiation on Edge Terminations in Vertical GaN pin Diodes

DOE PAGES

Collins, Kimberlee C.; King, Michael P.; Dickerson, Jeramy R.; ...

2017-05-29

Devices based on GaN have shown great promise for high power electronics, including their potential use as radiation tolerant components. An important step to realizing high power diodes is the design and implementation of an edge termination to mitigate field crowding, which can lead to premature breakdown. However, little is known about the effects of radiation on edge termination functionality. We experimentally examine the effects of proton irradiation on multiple field ring edge terminations in high power vertical GaN pin diodes using in operando imaging with electron beam induced current (EBIC). We find that exposure to proton irradiation influences fieldmore » spreading in the edge termination as well as carrier transport near the anode. By using depth-dependent EBIC measurements of hole diffusion length in homoepitaxial n-GaN we demonstrate that the carrier transport effect is due to a reduction in hole diffusion length following proton irradiation.« less

Imaging the Impact of Proton Irradiation on Edge Terminations in Vertical GaN pin Diodes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Collins, Kimberlee C.; King, Michael P.; Dickerson, Jeramy R.

Devices based on GaN have shown great promise for high power electronics, including their potential use as radiation tolerant components. An important step to realizing high power diodes is the design and implementation of an edge termination to mitigate field crowding, which can lead to premature breakdown. However, little is known about the effects of radiation on edge termination functionality. We experimentally examine the effects of proton irradiation on multiple field ring edge terminations in high power vertical GaN pin diodes using in operando imaging with electron beam induced current (EBIC). We find that exposure to proton irradiation influences fieldmore » spreading in the edge termination as well as carrier transport near the anode. By using depth-dependent EBIC measurements of hole diffusion length in homoepitaxial n-GaN we demonstrate that the carrier transport effect is due to a reduction in hole diffusion length following proton irradiation.« less

Seroepidemiology of TmPV1 infection in captive and wild Florida manatees (Trichechus manatus latirostris)

USGS Publications Warehouse

Dona, Maria Gabriella; Rehtanz, Manuela; Adimey, Nicole M.; Bossart, Gregory D.; Jenson, Alfred B.; Bonde, Robert K.; Ghim, Shin-je

2011-01-01

In 1997, cutaneous papillomatosis caused by Florida manatee (Trichechus manatus latirostris [Tm]) papillomavirus 1 (TmPV1) was detected in seven captive manatees at the Homosassa Springs Wildlife State Park, Florida, USA, and, subsequently, in two wild manatees from the adjacent Homosassa River. Since then, papillomatosis has been reported in captive manatees housed in other locations, but not in wild animals. To determine TmPV1 antibody prevalence in captive and wild manatees sampled at various locations throughout Florida coastal regions, virus-like particles, composed of the L1 capsid protein of TmPV1, were generated with a baculovirus expression system and used to measure anti-TmPV1 antibodies in an enzyme-linked immunosorbent assay. Serologic analysis of 156 manatees revealed a TmPV1 antibody prevalence of 26.3%, with no significant difference between captive (n=39) and wild (n=117) manatees (28.2% and 25.6%, respectively). No antibody-positive wild animal showed PV-induced cutaneous lesions, whereas papillomatosis was observed in 72.7% of antibody-positive captive manatees. Our data indicate that Florida manatees living in the wild are naturally infected by TmPV1 but rarely show TmPV1-induced papillomatosis. Hence, it appears that the wild population would not be harmed in a case of contact with captive animals without visible lesions and productive infections, which could be thus released into the wild.

Seroepidemiology of TmPV1 infection in captive and wild Florida manatees (Trichechus manatus latirostris).

PubMed

Donà, Maria Gabriella; Rehtanz, Manuela; Adimey, Nicole M; Bossart, Gregory D; Jenson, Alfred B; Bonde, Robert K; Ghim, Shin-je

2011-07-01

In 1997, cutaneous papillomatosis caused by Florida manatee (Trichechus manatus latirostris [Tm]) papillomavirus 1 (TmPV1) was detected in seven captive manatees at the Homosassa Springs Wildlife State Park, Florida, USA, and, subsequently, in two wild manatees from the adjacent Homosassa River. Since then, papillomatosis has been reported in captive manatees housed in other locations, but not in wild animals. To determine TmPV1 antibody prevalence in captive and wild manatees sampled at various locations throughout Florida coastal regions, virus-like particles, composed of the L1 capsid protein of TmPV1, were generated with a baculovirus expression system and used to measure anti-TmPV1 antibodies in an enzyme-linked immunosorbent assay. Serologic analysis of 156 manatees revealed a TmPV1 antibody prevalence of 26.3%, with no significant difference between captive (n=39) and wild (n=117) manatees (28.2% and 25.6%, respectively). No antibody-positive wild animal showed PV-induced cutaneous lesions, whereas papillomatosis was observed in 72.7% of antibody-positive captive manatees. Our data indicate that Florida manatees living in the wild are naturally infected by TmPV1 but rarely show TmPV1-induced papillomatosis. Hence, it appears that the wild population would not be harmed in a case of contact with captive animals without visible lesions and productive infections, which could be thus released into the wild.

Prototype foamy virus envelope glycoprotein leader peptide processing is mediated by a furin-like cellular protease, but cleavage is not essential for viral infectivity.

PubMed

Duda, Anja; Stange, Annett; Lüftenegger, Daniel; Stanke, Nicole; Westphal, Dana; Pietschmann, Thomas; Eastman, Scott W; Linial, Maxine L; Rethwilm, Axel; Lindemann, Dirk

2004-12-01

Analogous to cellular glycoproteins, viral envelope proteins contain N-terminal signal sequences responsible for targeting them to the secretory pathway. The prototype foamy virus (PFV) envelope (Env) shows a highly unusual biosynthesis. Its precursor protein has a type III membrane topology with both the N and C terminus located in the cytoplasm. Coexpression of FV glycoprotein and interaction of its leader peptide (LP) with the viral capsid is essential for viral particle budding and egress. Processing of PFV Env into the particle-associated LP, surface (SU), and transmembrane (TM) subunits occur posttranslationally during transport to the cell surface by yet-unidentified cellular proteases. Here we provide strong evidence that furin itself or a furin-like protease and not the signal peptidase complex is responsible for both processing events. N-terminal protein sequencing of the SU and TM subunits of purified PFV Env-immunoglobulin G immunoadhesin identified furin consensus sequences upstream of both cleavage sites. Mutagenesis analysis of two overlapping furin consensus sequences at the PFV LP/SU cleavage site in the wild-type protein confirmed the sequencing data and demonstrated utilization of only the first site. Fully processed SU was almost completely absent in viral particles of mutants having conserved arginine residues replaced by alanines in the first furin consensus sequence, but normal processing was observed upon mutation of the second motif. Although these mutants displayed a significant loss in infectivity as a result of reduced particle release, no correlation to processing inhibition was observed, since another mutant having normal LP/SU processing had a similar defect.

The α-Secretase-derived N-terminal Product of Cellular Prion, N1, Displays Neuroprotective Function in Vitro and in Vivo*

PubMed Central

Guillot-Sestier, Marie-Victoire; Sunyach, Claire; Druon, Charlotte; Scarzello, Sabine; Checler, Frédéric

2009-01-01

Cellular prion protein (PrPc) undergoes a disintegrin-mediated physiological cleavage, generating a soluble amino-terminal fragment (N1), the function of which remained unknown. Recombinant N1 inhibits staurosporine-induced caspase-3 activation by modulating p53 transcription and activity, whereas the PrPc-derived pathological fragment (N2) remains biologically inert. Furthermore, N1 protects retinal ganglion cells from hypoxia-induced apoptosis, reduces the number of terminal deoxynucleotidyltransferase-mediated biotinylated UTP nick end labeling-positive and p53-immunoreactive neurons in a pressure-induced ischemia model of the rat retina and triggers a partial recovery of b-waves but not a-waves of rat electroretinograms. Our work is the first demonstration that the α-secretase-derived PrPc fragment N1, but not N2, displays in vivo and in vitro neuroprotective function by modulating p53 pathway. It further demonstrates that distinct N-terminal cleavage products of PrPc harbor different biological activities underlying the various phenotypes linking PrPc to cell survival. PMID:19850936

Hidden markov model for the prediction of transmembrane proteins using MATLAB.

PubMed

Chaturvedi, Navaneet; Shanker, Sudhanshu; Singh, Vinay Kumar; Sinha, Dhiraj; Pandey, Paras Nath

2011-01-01

Since membranous proteins play a key role in drug targeting therefore transmembrane proteins prediction is active and challenging area of biological sciences. Location based prediction of transmembrane proteins are significant for functional annotation of protein sequences. Hidden markov model based method was widely applied for transmembrane topology prediction. Here we have presented a revised and a better understanding model than an existing one for transmembrane protein prediction. Scripting on MATLAB was built and compiled for parameter estimation of model and applied this model on amino acid sequence to know the transmembrane and its adjacent locations. Estimated model of transmembrane topology was based on TMHMM model architecture. Only 7 super states are defined in the given dataset, which were converted to 96 states on the basis of their length in sequence. Accuracy of the prediction of model was observed about 74 %, is a good enough in the area of transmembrane topology prediction. Therefore we have concluded the hidden markov model plays crucial role in transmembrane helices prediction on MATLAB platform and it could also be useful for drug discovery strategy. The database is available for free at bioinfonavneet@gmail.comvinaysingh@bhu.ac.in.

Acetylation within the N- and C-Terminal Domains of Src Regulates Distinct Roles of STAT3-Mediated Tumorigenesis.

PubMed

Huang, Chao; Zhang, Zhe; Chen, Lihan; Lee, Hank W; Ayrapetov, Marina K; Zhao, Ting C; Hao, Yimei; Gao, Jinsong; Yang, Chunzhang; Mehta, Gautam U; Zhuang, Zhengping; Zhang, Xiaoren; Hu, Guohong; Chin, Y Eugene

2018-06-01

Posttranslational modifications of mammalian c-Src N-terminal and C-terminal domains regulate distinct functions. Myristoylation of G 2 controls its cell membrane association and phosphorylation of Y419/Y527 controls its activation or inactivation, respectively. We provide evidence that Src-cell membrane association-dissociation and catalytic activation-inactivation are both regulated by acetylation. In EGF-treated cells, CREB binding protein (CBP) acetylates an N-terminal lysine cluster (K5, K7, and K9) of c-Src to promote dissociation from the cell membrane. CBP also acetylates the C-terminal K401, K423, and K427 of c-Src to activate intrinsic kinase activity for STAT3 recruitment and activation. N-terminal domain phosphorylation (Y14, Y45, and Y68) of STAT3 by c-Src activates transcriptionally active dimers of STAT3. Moreover, acetyl-Src translocates into nuclei, where it forms the Src-STAT3 enhanceosome for gene regulation and cancer cell proliferation. Thus, c-Src acetylation in the N-terminal and C-terminal domains play distinct roles in Src activity and regulation. Significance: CBP-mediated acetylation of lysine clusters in both the N-terminal and C-terminal regions of c-Src provides additional levels of control over STAT3 transcriptional activity. Cancer Res; 78(11); 2825-38. ©2018 AACR . ©2018 American Association for Cancer Research.

Structure of the Tropomyosin Overlap Complex from Chicken Smooth Muscle: Insight into the Diversity of N-Terminal Recognition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Frye, Jeremiah; Klenchin, Vadim A.; Rayment, Ivan

Tropomyosin is a stereotypical {alpha}-helical coiled coil that polymerizes to form a filamentous macromolecular assembly that lies on the surface of F-actin. The interaction between the C-terminal and N-terminal segments on adjacent molecules is known as the overlap region. We report here two X-ray structures of the chicken smooth muscle tropomyosin overlap complex. A novel approach was used to stabilize the C-terminal and N-terminal fragments. Globular domains from both the human DNA ligase binding protein XRCC4 and bacteriophage {phi}29 scaffolding protein Gp7 were fused to 37 and 28 C-terminal amino acid residues of tropomyosin, respectively, whereas the 29 N-terminal aminomore » acids of tropomyosin were fused to the C-terminal helix bundle of microtubule binding protein EB1. The structures of both the XRCC4 and Gp7 fusion proteins complexed with the N-terminal EB1 fusion contain a very similar helix bundle in the overlap region that encompasses {approx}15 residues. The C-terminal coiled coil opens to allow formation of the helix bundle, which is stabilized by hydrophobic interactions. These structures are similar to that observed in the NMR structure of the rat skeletal overlap complex [Greenfield, N. J., et al. (2006) J. Mol. Biol. 364, 80-96]. The interactions between the N- and C-terminal coiled coils of smooth muscle tropomyosin show significant curvature, which differs somewhat between the two structures and implies flexibility in the overlap complex, at least in solution. This is likely an important attribute that allows tropomyosin to assemble around the actin filaments. These structures provide a molecular explanation for the role of N-acetylation in the assembly of native tropomyosin.« less

N-terminal domain of the dual-targeted pea glutathione reductase signal peptide controls organellar targeting efficiency.

PubMed

Rudhe, Charlotta; Clifton, Rachel; Whelan, James; Glaser, Elzbieta

2002-12-06

Import of nuclear-encoded proteins into mitochondria and chloroplasts is generally organelle specific and its specificity depends on the N-terminal signal peptide. Yet, a group of proteins known as dual-targeted proteins have a targeting peptide capable of leading the mature protein to both organelles. We have investigated the domain structure of the dual-targeted pea glutathione reductase (GR) signal peptide by using N-terminal truncations. A mutant of the GR precursor (pGR) starting with the second methionine residue of the targeting peptide, pGRdelta2-4, directed import into both organelles, negating the possibility that dual import was controlled by the nature of the N terminus. The deletion of the 30 N-terminal residues (pGRdelta2-30) inhibited import efficiency into chloroplasts substantially and almost completely into mitochondria, whereas the removal of only 16 N-terminal amino acid residues (pGRdelta2-16) resulted in the strongly stimulated mitochondrial import without significantly affecting chloroplast import. Furthermore, N-terminal truncations of the signal peptide (pGRdelta2-16 and pGRdelta2-30) greatly stimulated the mitochondrial processing activity measured with the isolated processing peptidase. These results suggest a domain structure for the dual-targeting peptide of pGR and the existence of domains controlling organellar import efficiency therein.

Proteolytic interconversion and N-terminal sequences of the Citrobacter diversus major beta-lactamases.

PubMed Central

Franceschini, N; Amicosante, G; Perilli, M; Maccarrone, M; Oratore, A; van Beeumen, J; Frère, J M

1991-01-01

The N-terminal sequences of the two major beta-lactamases produced by Citrobacter diversus differed only by the absence of the first residue in form II and the loss of five amino acid residues at the C-terminal end. Limited proteolysis of the homogeneous form I protein yielded a variety of enzymatically active products. In the major product obtained after the action of papain, the first three N-terminal residues of form I had been cleaved, whereas at the C-terminal end the treated enzyme lacked five residues. However, this cannot explain the different behaviours of form I, form II and papain digestion product upon chromatofocusing. Form I, which was sequenced up to position 56, exhibited a very high degree of similarity with a Klebsiella oxytoca beta-lactamase. The determined sequence, which contained the active serine residue, demonstrated that the chromosome-encoded beta-lactamase of Citrobacter diversus belong to class A. Images Fig. 2. PMID:2039443

Miro's N-Terminal GTPase Domain Is Required for Transport of Mitochondria into Axons and Dendrites

PubMed Central

Babic, Milos; Russo, Gary J.; Wellington, Andrea J.; Sangston, Ryan M.; Gonzalez, Migdalia

2015-01-01

Mitochondria are dynamically transported in and out of neuronal processes to maintain neuronal excitability and synaptic function. In higher eukaryotes, the mitochondrial GTPase Miro binds Milton/TRAK adaptor proteins linking microtubule motors to mitochondria. Here we show that Drosophila Miro (dMiro), which has previously been shown to be required for kinesin-driven axonal transport, is also critically required for the dynein-driven distribution of mitochondria into dendrites. In addition, we used the loss-of-function mutations dMiroT25N and dMiroT460N to determine the significance of dMiro's N-terminal and C-terminal GTPase domains, respectively. Expression of dMiroT25N in the absence of endogenous dMiro caused premature lethality and arrested development at a pupal stage. dMiroT25N accumulated mitochondria in the soma of larval motor and sensory neurons, and prevented their kinesin-dependent and dynein-dependent distribution into axons and dendrites, respectively. dMiroT25N mutant mitochondria also were severely fragmented and exhibited reduced kinesin and dynein motility in axons. In contrast, dMiroT460N did not impair viability, mitochondrial size, or the distribution of mitochondria. However, dMiroT460N reduced dynein motility during retrograde mitochondrial transport in axons. Finally, we show that substitutions analogous to the constitutively active Ras-G12V mutation in dMiro's N-terminal and C-terminal GTPase domains cause neomorphic phenotypic effects that are likely unrelated to the normal function of each GTPase domain. Overall, our analysis indicates that dMiro's N-terminal GTPase domain is critically required for viability, mitochondrial size, and the distribution of mitochondria out of the neuronal soma regardless of the employed motor, likely by promoting the transition from a stationary to a motile state. PMID:25855186

The lysosomal potassium channel TMEM175 adopts a novel tetrameric architecture.

PubMed

Lee, Changkeun; Guo, Jiangtao; Zeng, Weizhong; Kim, Sunghoon; She, Ji; Cang, Chunlei; Ren, Dejian; Jiang, Youxing

2017-07-27

TMEM175 is a lysosomal K + channel that is important for maintaining the membrane potential and pH stability in lysosomes. It contains two homologous copies of a six-transmembrane-helix (6-TM) domain, which has no sequence homology to the canonical tetrameric K + channels and lacks the TVGYG selectivity filter motif found in these channels. The prokaryotic TMEM175 channel, which is present in a subset of bacteria and archaea, contains only a single 6-TM domain and functions as a tetramer. Here, we present the crystal structure of a prokaryotic TMEM175 channel from Chamaesiphon minutus, CmTMEM175, the architecture of which represents a completely different fold from that of canonical K + channels. All six transmembrane helices of CmTMEM175 are tightly packed within each subunit without undergoing domain swapping. The highly conserved TM1 helix acts as the pore-lining inner helix, creating an hourglass-shaped ion permeation pathway in the channel tetramer. Three layers of hydrophobic residues on the carboxy-terminal half of the TM1 helices form a bottleneck along the ion conduction pathway and serve as the selectivity filter of the channel. Mutagenesis analysis suggests that the first layer of the highly conserved isoleucine residues in the filter is primarily responsible for channel selectivity. Thus, the structure of CmTMEM175 represents a novel architecture of a tetrameric cation channel whose ion selectivity mechanism appears to be distinct from that of the classical K + channel family.

An automated system for the analysis of G protein-coupled receptor transmembrane binding pockets: alignment, receptor-based pharmacophores, and their application.

PubMed

Kratochwil, Nicole A; Malherbe, Pari; Lindemann, Lothar; Ebeling, Martin; Hoener, Marius C; Mühlemann, Andreas; Porter, Richard H P; Stahl, Martin; Gerber, Paul R

2005-01-01

G protein-coupled receptors (GPCRs) share a common architecture consisting of seven transmembrane (TM) domains. Various lines of evidence suggest that this fold provides a generic binding pocket within the TM region for hosting agonists, antagonists, and allosteric modulators. Here, a comprehensive and automated method allowing fast analysis and comparison of these putative binding pockets across the entire GPCR family is presented. The method relies on a robust alignment algorithm based on conservation indices, focusing on pharmacophore-like relationships between amino acids. Analysis of conservation patterns across the GPCR family and alignment to the rhodopsin X-ray structure allows the extraction of the amino acids lining the TM binding pocket in a so-called ligand binding pocket vector (LPV). In a second step, LPVs are translated to simple 3D receptor pharmacophore models, where each amino acid is represented by a single spherical pharmacophore feature and all atomic detail is omitted. Applications of the method include the assessment of selectivity issues, support of mutagenesis studies, and the derivation of rules for focused screening to identify chemical starting points in early drug discovery projects. Because of the coarseness of this 3D receptor pharmacophore model, however, meaningful scoring and ranking procedures of large sets of molecules are not justified. The LPV analysis of the trace amine-associated receptor family and its experimental validation is discussed as an example. The value of the 3D receptor model is demonstrated for a class C GPCR family, the metabotropic glutamate receptors.

c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Sclerosis

DTIC Science & Technology

2015-03-01

1 AWARD NUMBER: W81XWH-12-1-0431 TITLE: “c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Sclerosis ” PRINCIPAL...TITLE AND SUBTITLE “c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Scelerosis” 5a. CONTRACT NUMBER 5b. GRANT NUMBER... Lateral   Sclerosis ”   Final  Report:  Project  Period  Sept  2012-­‐Dec  2014     Personnel  List:     Feng,  Yangbo

The Topology of the l-Arginine Exporter ArgO Conforms to an Nin-Cout Configuration in Escherichia coli: Requirement for the Cytoplasmic N-Terminal Domain, Functional Helical Interactions, and an Aspartate Pair for ArgO Function.

PubMed

Pathania, Amit; Gupta, Arvind Kumar; Dubey, Swati; Gopal, Balasubramanian; Sardesai, Abhijit A

2016-12-01

ArgO and LysE are members of the LysE family of exporter proteins and ordinarily mediate the export of l-arginine (Arg) in Escherichia coli and l-lysine (Lys) and Arg in Corynebacterium glutamicum, respectively. Under certain conditions, ArgO also mediates Lys export. To delineate the arrangement of ArgO in the cytoplasmic membrane of E. coli, we have employed a combination of cysteine accessibility in situ, alkaline phosphatase fusion reporters, and protein modeling to arrive at a topological model of ArgO. Our studies indicate that ArgO assumes an N in -C out configuration, potentially forming a five-transmembrane helix bundle flanked by a cytoplasmic N-terminal domain (NTD) comprising roughly its first 38 to 43 amino acyl residues and a short periplasmic C-terminal region (CTR). Mutagenesis studies indicate that the CTR, but not the NTD, is dispensable for ArgO function in vivo and that a pair of conserved aspartate residues, located near the opposing edges of the cytoplasmic membrane, may play a pivotal role in facilitating transmembrane Arg flux. Additional studies on amino acid substitutions that impair ArgO function in vivo and their derivatives bearing compensatory amino acid alterations indicate a role for intramolecular interactions in the Arg export mechanism, and some interactions are corroborated by normal-mode analyses. Lastly, our studies suggest that ArgO may exist as a monomer in vivo, thus highlighting the requirement for intramolecular interactions in ArgO, as opposed to interactions across multiple ArgO monomers, in the formation of an Arg-translocating conduit. The orthologous proteins LysE of C. glutamicum and ArgO of E. coli function as exporters of the basic amino acids l-arginine and l-lysine and the basic amino acid l-arginine, respectively, and LysE can functionally substitute for ArgO when expressed in E. coli Notwithstanding this functional equivalence, studies reported here show that ArgO possesses a membrane topology that is distinct

Coding variants in PNPLA3 and TM6SF2 are risk factors for hepatic steatosis and elevated serum alanine aminotransferases caused by a glucagon receptor antagonist

PubMed Central

Guzman, Cristina B.; Duvvuru, Suman; Akkari, Anthony; Bhatnagar, Pallav; Battioui, Chakib; Foster, Wendra; Zhang, Xiaotian Michelle; Shankar, Sudha S.; Deeg, Mark A.; Hardy, Thomas A.; Kazda, Christof M.

2018-01-01

LY2409021 is a glucagon receptor antagonist that was associated with hepatic steatosis and elevated aminotransferases in phase 2 diabetes studies. We investigated the relationship between selected genetic variants and hepatic steatosis and elevated alanine aminotransferases (ALTs) associated with LY2409021. Patients participated in a 6‐week placebo‐controlled trial (I1R‐MC‐GLDI [GLDI], n = 246) and a 52‐week placebo‐ and active comparator‐controlled trial (I1R‐MC‐GLDJ [GLDJ], n = 158). GLDJ had endpoints at 6 months, including measures of hepatic fat fraction (HFF) by magnetic resonance imaging. The five genes tested were patatin‐like phospholipase domain containing 3 (PNPLA3) (rs738409 and rs738491), transmembrane 6 superfamily member 2 (TM6SF2) (rs58542926), peroxisome proliferative activated receptor gamma coactivator 1 alpha (PPARGC1A) (rs4361373, rs3774921, rs2970849), adenylate cyclase 3 (ADCY3) (rs713586), and insulin‐like growth factor 1 (IGF‐1) (rs1520220). In GLDI, PNPLA3 I148M (P = 0.001) and TM6SF2 E167K (P = 0.001) were significantly associated with an increase in ALT at 6 weeks for LY2409021 but not for placebo. In GLDJ, PNPLA3 I148M showed the same effect (P = 0.007) on ALT at 6 months but the placebo or sitagliptin did not. In GLDJ, both PNPLA3 and TM6SF2 risk‐allele carriers showed increases in HFF that were numerically greater but not statistically significant. The carriers of PNPLA3 and/or TM6SF2 risk alleles showed significantly increased ALT (GLDI, +13.28 U/L in carriers versus +4.84 U/L in noncarriers, P = 4 × 10–5; GLDJ, +14.6 U/L in carriers versus +1.7 in noncarriers, P = 0.0018) and HFF (GLDJ, +5.35% in carriers versus 2.38% in noncarriers, P = 0.048). Elevation of transaminase and HFF were also noted in the noncarriers but at a significantly lower degree. Conclusion: The carriers of PNPLA3 and/or TM6SF2 variant alleles are at risk for hepatic steatosis and elevated ALT levels caused by LY2409021, a glucagon

Coding variants in PNPLA3 and TM6SF2 are risk factors for hepatic steatosis and elevated serum alanine aminotransferases caused by a glucagon receptor antagonist.

PubMed

Guzman, Cristina B; Duvvuru, Suman; Akkari, Anthony; Bhatnagar, Pallav; Battioui, Chakib; Foster, Wendra; Zhang, Xiaotian Michelle; Shankar, Sudha S; Deeg, Mark A; Chalasani, Naga; Hardy, Thomas A; Kazda, Christof M; Pillai, Sreekumar G

2018-05-01

LY2409021 is a glucagon receptor antagonist that was associated with hepatic steatosis and elevated aminotransferases in phase 2 diabetes studies. We investigated the relationship between selected genetic variants and hepatic steatosis and elevated alanine aminotransferases (ALTs) associated with LY2409021. Patients participated in a 6-week placebo-controlled trial (I1R-MC-GLDI [GLDI], n = 246) and a 52-week placebo- and active comparator-controlled trial (I1R-MC-GLDJ [GLDJ], n = 158). GLDJ had endpoints at 6 months, including measures of hepatic fat fraction (HFF) by magnetic resonance imaging. The five genes tested were patatin-like phospholipase domain containing 3 ( PNPLA3 ) (rs738409 and rs738491), transmembrane 6 superfamily member 2 ( TM6SF2 ) (rs58542926), peroxisome proliferative activated receptor gamma coactivator 1 alpha ( PPARGC1A ) (rs4361373, rs3774921, rs2970849), adenylate cyclase 3 ( ADCY3 ) ( rs713586), and insulin-like growth factor 1 ( IGF-1 ) (rs1520220). In GLDI, PNPLA3 I148M ( P = 0.001) and TM6SF2 E167K ( P = 0.001) were significantly associated with an increase in ALT at 6 weeks for LY2409021 but not for placebo. In GLDJ, PNPLA3 I148M showed the same effect ( P = 0.007) on ALT at 6 months but the placebo or sitagliptin did not. In GLDJ, both PNPLA3 and TM6SF2 risk-allele carriers showed increases in HFF that were numerically greater but not statistically significant. The carriers of PNPLA3 and/or TM6SF2 risk alleles showed significantly increased ALT (GLDI, +13.28 U/L in carriers versus +4.84 U/L in noncarriers, P = 4 × 10 -5 ; GLDJ, +14.6 U/L in carriers versus +1.7 in noncarriers, P = 0.0018) and HFF (GLDJ, +5.35% in carriers versus 2.38% in noncarriers, P = 0.048). Elevation of transaminase and HFF were also noted in the noncarriers but at a significantly lower degree. Conclusion: The carriers of PNPLA3 and/or TM6SF2 variant alleles are at risk for hepatic steatosis and elevated ALT levels caused by LY2409021, a glucagon receptor

Comparative study of optical and scintillation properties of Tm3+:YAG, and Tm3+:LuAG single crystals

NASA Astrophysics Data System (ADS)

Fujimoto, Yutaka; Sugiyama, Makoto; Yanagida, Takayuki; Wakahara, Shingo; Suzuki, Shotaro; Kurosawa, Shunsuke; Chani, Valery; Yoshikawa, Akira

2013-09-01

The optical and scintillation properties of Tm3+-doped yttrium aluminum garnet Y3Al5O12 (YAG) and Tm3+-doped lutetium aluminum garnet Lu3Al5O12 (LuAG) are compared. The Tm3+-doped single crystals were grown by the micro-pulling down (μ-PD) technique. Both crystals demonstrated some emission peaks originated from 4f-4f forbidden transition of Tm3+ under 241Am alpha-ray excitation. The scintillation decay time of Tm3+-doped YAG was similar to that of LuAG. When irradiated by the gamma-rays from a 137Cs source, the relative scintillation light yields of Tm:YAG was 90% greater than that of Tm:LuAG.

Ligand field splittings in core level transitions for transition metal (TM) oxides: Tanabe-Sugano diagrams and (TM) dangling bonds in vacated O-atom defects

NASA Astrophysics Data System (ADS)

Lucovsky, Gerry; Wu, Kun; Pappas, Brian; Whitten, Jerry

2013-04-01

Defect states in the forbidden band-gap below the conduction band edge are active as electron traps in nano-grain high-) transition metal (TM) oxides with thickness >0.3 nm, e.g., ZrO2 and HfO2. These oxides have received considerable attention as gate-dielectrics in complementary metal oxide semiconductor (CMOS) devices, and more recently are emerging as candidates for charge storage and memory devices. To provide a theoretical basis for device functionality, ab-initio many-electron theory is combined with X-ray absorption spectroscopy (XAS) to study O K edge and TM core level transitions. These studies identify ligand field splittings (ΔLF) for defect state features,. When compared with those obtained from O-atom and TM-atom core spectroscopic transitions, this provides direct information about defect state sun-nm bonding arrangements. comparisons are made for (i) elemental TiO2 and Ti2O3 with different formal ionic charges, Ti4+ and Ti3+ and for (ii) Magneli Phase alloys, TinO2n-1, n is an integer 9>=n>3, and (TiO2)x(HfO2)1-x alloys. The alloys display multi-valent behavior from (i) different ionic-charge states, (ii} local bond-strain, and (iii) metallic hopping transport. The intrinsic bonding defects in TM oxides are identified as pairs of singly occupied dangling bonds. For 6-fold coordinated Ti-oxides defect excited states in 2nd derivative O K pre-edge spectra are essentially the same as single Ti-atom d2 transitions in Tanabe-Sugano (T-S) diagrams. O-vacated site defects in 8-fold coordinated ZrO2 and HfO2 are described by d8 T-S diagrams. T-S defect state ordering and splittings are functions of the coordination and symmetry of vacated site bordering TM atoms. ΔLF values from the analysis of T-S diagrams indicate medium range order (MRO) extending to 3rd and 4th nearest-neighbor (NN) TM-atoms. Values are different for 6-fold Ti, and 8-fold ZrO2 and HfO2, and scale inversely with differences in respective formal ionic radii. O-vacated site bonding

Dual Role of Jun N-Terminal Kinase Activity in Bone Morphogenetic Protein-Mediated Drosophila Ventral Head Development.

PubMed

Park, Sung Yeon; Stultz, Brian G; Hursh, Deborah A

2015-12-01

The Drosophila bone morphogenetic protein encoded by decapentaplegic (dpp) controls ventral head morphogenesis by expression in the head primordia, eye-antennal imaginal discs. These are epithelial sacs made of two layers: columnar disc proper cells and squamous cells of the peripodial epithelium. dpp expression related to head formation occurs in the peripodial epithelium; cis-regulatory mutations disrupting this expression display defects in sensory vibrissae, rostral membrane, gena, and maxillary palps. Here we document that disruption of this dpp expression causes apoptosis in peripodial cells and underlying disc proper cells. We further show that peripodial Dpp acts directly on the disc proper, indicating that Dpp must cross the disc lumen to act. We demonstrate that palp defects are mechanistically separable from the other mutant phenotypes; both are affected by the c-Jun N-terminal kinase pathway but in opposite ways. Slight reduction of both Jun N-terminal kinase and Dpp activity in peripodial cells causes stronger vibrissae, rostral membrane, and gena defects than Dpp alone; additionally, strong reduction of Jun N-terminal kinase activity alone causes identical defects. A more severe reduction of dpp results in similar vibrissae, rostral membrane, and gena defects, but also causes mutant maxillary palps. This latter defect is correlated with increased peripodial Jun N-terminal kinase activity and can be caused solely by ectopic activation of Jun N-terminal kinase. We conclude that formation of sensory vibrissae, rostral membrane, and gena tissue in head morphogenesis requires the action of Jun N-terminal kinase in peripodial cells, while excessive Jun N-terminal kinase signaling in these same cells inhibits the formation of maxillary palps. Copyright © 2015 by the Genetics Society of America.

Ethanol Inhibition of Constitutively Open N-Methyl-d-Aspartate Receptors

PubMed Central

Xu, Minfu; Smothers, C. Thetford; Trudell, James

2012-01-01

N-Methyl-d-aspartate (NMDA) receptors gate a slow and calcium-rich component of the postsynaptic glutamate response. Like all ionotropic glutamate receptors, NMDA subunits contain a highly conserved motif (SYTANLAAF) in the transmembrane (TM) 3 domain that is critically involved in channel gating. Mutation of an alanine in this domain (A7; underlined above) results in constitutively open receptors that show reduced sensitivity to several allosteric modulators. In this study, we examined the effects of ethanol, a substance that inhibits NMDA currents via an unknown mechanism, on tonically active NMDA receptors expressed in human embryonic kidney 293 cells. Ethanol (100 mM) inhibited currents from GluN1(A7R)/GluN2A and GluN1(A7R)/GluN2B receptors by approximately 50%, whereas those from GluN1/GluN2B(A7R) receptors were reduced by less than 10%. In cysteine-substituted GluN1 and GluN2 A7 mutants, estimated ethanol IC50 values for agonist-gated currents were 101, 117, 103, and 69 mM for GluN1(A7C)/GluN2A, GluN1(A7C)/GluN2B, GluN1/GluN2A(A7C), and GluN1/GluN2B(A7C) receptors, respectively. After exposure to the thiol-modifying reagent 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET), A7C mutants showed robust agonist-independent currents and reduced sensitivity to ethanol (IC50 values of 371, 256, 715, and 958 mM, respectively, as above). In contrast, cysteine modification of the ligand-binding domain resulted in constitutively open receptors that showed robust ethanol inhibition. Ethanol inhibition of MTSET-treated GluN1(A7C) receptors was further reduced by TM3/TM4 mutations previously shown to reduce ethanol sensitivity of agonist-gated receptors. Overall, these results show that ethanol affects NMDA receptor function at a site distal from agonist binding and appears to exert greater effects via perturbation of GluN2 subunits. PMID:22005043

An improved stable isotope N-terminal labeling approach with light/heavy TMPP to automate proteogenomics data validation: dN-TOP.

PubMed

Bertaccini, Diego; Vaca, Sebastian; Carapito, Christine; Arsène-Ploetze, Florence; Van Dorsselaer, Alain; Schaeffer-Reiss, Christine

2013-06-07

In silico gene prediction has proven to be prone to errors, especially regarding precise localization of start codons that spread in subsequent biological studies. Therefore, the high throughput characterization of protein N-termini is becoming an emerging challenge in the proteomics and especially proteogenomics fields. The trimethoxyphenyl phosphonium (TMPP) labeling approach (N-TOP) is an efficient N-terminomic approach that allows the characterization of both N-terminal and internal peptides in a single experiment. Due to its permanent positive charge, TMPP labeling strongly affects MS/MS fragmentation resulting in unadapted scoring of TMPP-derivatized peptide spectra by classical search engines. This behavior has led to difficulties in validating TMPP-derivatized peptide identifications with usual score filtering and thus to low/underestimated numbers of identified N-termini. We present herein a new strategy (dN-TOP) that overwhelmed the previous limitation allowing a confident and automated N-terminal peptide validation thanks to a combined labeling with light and heavy TMPP reagents. We show how this double labeling allows increasing the number of validated N-terminal peptides. This strategy represents a considerable improvement to the well-established N-TOP method with an enhanced and accelerated data processing making it now fully compatible with high-throughput proteogenomics studies.

Ligand binding to the human MT2 melatonin receptor: The role of residues in transmembrane domains 3, 6, and 7

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mazna, Petr; Berka, Karel; Jelinkova, Irena

To better understand the mechanism of interactions between G-protein-coupled melatonin receptors and their ligands, our previously reported homology model of human MT2 receptor with docked 2-iodomelatonin was further refined and used to select residues within TM3, TM6, and TM7 potentially important for receptor-ligand interactions. Selected residues were mutated and radioligand-binding assay was used to test the binding affinities of hMT2 receptors transiently expressed in HEK293 cells. Our data demonstrate that residues N268 and A275 in TM6 as well as residues V291 and L295 in TM7 are essential for 2-iodomelatonin binding to the hMT2 receptor, while TM3 residues M120, G121, V124,more » and I125 may participate in binding of other receptor agonists and/or antagonists. Presented data also hint at possible specific interaction between the side-chain of Y188 in second extracellular loop and N-acetyl group of 2-iodomelatonin.« less

A systematic search method for the identification of tightly packed transmembrane parallel alpha-helices.

PubMed

Akula, Nagaraju; Pattabiraman, Nagarajan

2005-06-01

Membrane proteins play a major role in number of biological processes such as signaling pathways. The determination of the three-dimensional structure of these proteins is increasingly important for our understanding of their structure-function relationships. Due to the difficulty in isolating membrane proteins for X-ray diffraction studies, computational techniques are being developed to generate the 3D structures of TM domains. Here, we present a systematic search method for the identification of energetically favorable and tightly packed transmembrane parallel alpha-helices. The first step in our systematic search method is the generation of 3D models for pairs of parallel helix bundles with all possible orientations followed by an energy-based filter to eliminate structures with severe non-bonded contacts. Then, a RMS-based filter was used to cluster these structures into families. Furthermore, these dimers were energy minimized using molecular mechanics force field. Finally, we identified the tightly packed parallel alpha-helices by using an interface surface area. To validate our search method, we compared our predicted GlycophorinA dimer structures with the reported NMR structures. With our search method, we are able to reproduce NMR structures of GPA with 0.9A RMSD. In addition, by considering the reported mutational data on GxxxG motif interactions, twenty percent of our predicted dimers are within in the 2.0A RMSD. The dimers obtained from our method were used to generate parallel trimeric and tetramer TM structures of GPA and found that the structure of GPA might exist only in a dimer form as reported earlier.

Characterization of a novel human sperm-associated antigen 9 (SPAG9) having structural homology with c-Jun N-terminal kinase-interacting protein

PubMed Central

Jagadish, Nirmala; Rana, Ritu; Selvi, Ramasamy; Mishra, Deepshikha; Garg, Manoj; Yadav, Shikha; Herr, John C.; Okumura, Katsuzumi; Hasegawa, Akiko; Koyama, Koji; Suri, Anil

2005-01-01

We report a novel SPAG9 (sperm-associated antigen 9) protein having structural homology with JNK (c-Jun N-terminal kinase)-interacting protein 3. SPAG9, a single copy gene mapped to the human chromosome 17q21.33 syntenic with location of mouse chromosome 11, was earlier shown to be expressed exclusively in testis [Shankar, Mohapatra and Suri (1998) Biochem. Biophys. Res. Commun. 243, 561–565]. The SPAG9 amino acid sequence analysis revealed identity with the JNK-binding domain and predicted coiled-coil, leucine zipper and transmembrane domains. The secondary structure analysis predicted an α-helical structure for SPAG9 that was confirmed by CD spectra. Microsequencing of higher-order aggregates of recombinant SPAG9 by tandem MS confirmed the amino acid sequence and mono atomic mass of 83.9 kDa. Transient expression of SPAG9 and its deletion mutants revealed that both leucine zipper with extended coiled-coil domains and transmembrane domain of SPAG9 were essential for dimerization and proper localization. Studies of MAPK (mitogenactivated protein kinase) interactions demonstrated that SPAG9 interacted with higher binding affinity to JNK3 and JNK2 compared with JNK1. No interaction was observed with p38α or extracellular-signal-regulated kinase pathways. Polyclonal antibodies raised against recombinant SPAG9 recognized native protein in human sperm extracts and localized specifically on the acrosomal compartment of intact human spermatozoa. Acrosome-reacted spermatozoa demonstrated SPAG9 immunofluorescence, indicating its retention on the equatorial segment after the acrosome reaction. Further, anti-SPAG9 antibodies inhibited the binding of human spermatozoa to intact human oocytes as well as to matched hemizona. This is the first report of sperm-associated JNK-binding protein that may have a role in spermatozoa–egg interaction. PMID:15693750

Characterization of a novel human sperm-associated antigen 9 (SPAG9) having structural homology with c-Jun N-terminal kinase-interacting protein.

PubMed

Jagadish, Nirmala; Rana, Ritu; Selvi, Ramasamy; Mishra, Deepshikha; Garg, Manoj; Yadav, Shikha; Herr, John C; Okumura, Katsuzumi; Hasegawa, Akiko; Koyama, Koji; Suri, Anil

2005-07-01

We report a novel SPAG9 (sperm-associated antigen 9) protein having structural homology with JNK (c-Jun N-terminal kinase)-interacting protein 3. SPAG9, a single copy gene mapped to the human chromosome 17q21.33 syntenic with location of mouse chromosome 11, was earlier shown to be expressed exclusively in testis [Shankar, Mohapatra and Suri (1998) Biochem. Biophys. Res. Commun. 243, 561-565]. The SPAG9 amino acid sequence analysis revealed identity with the JNK-binding domain and predicted coiled-coil, leucine zipper and transmembrane domains. The secondary structure analysis predicted an alpha-helical structure for SPAG9 that was confirmed by CD spectra. Microsequencing of higher-order aggregates of recombinant SPAG9 by tandem MS confirmed the amino acid sequence and mono atomic mass of 83.9 kDa. Transient expression of SPAG9 and its deletion mutants revealed that both leucine zipper with extended coiled-coil domains and transmembrane domain of SPAG9 were essential for dimerization and proper localization. Studies of MAPK (mitogenactivated protein kinase) interactions demonstrated that SPAG9 interacted with higher binding affinity to JNK3 and JNK2 compared with JNK1. No interaction was observed with p38alpha or extracellular-signal-regulated kinase pathways. Polyclonal antibodies raised against recombinant SPAG9 recognized native protein in human sperm extracts and localized specifically on the acrosomal compartment of intact human spermatozoa. Acrosome-reacted spermatozoa demonstrated SPAG9 immunofluorescence, indicating its retention on the equatorial segment after the acrosome reaction. Further, anti-SPAG9 antibodies inhibited the binding of human spermatozoa to intact human oocytes as well as to matched hemizona. This is the first report of sperm-associated JNK-binding protein that may have a role in spermatozoa-egg interaction.

Near-IR Resonance Raman Spectroscopy of Archaerhodopsin 3: Effects of Transmembrane Potential

PubMed Central

Saint Clair, Erica C.; Ogren, John I.; Mamaev, Sergey; Russano, Daniel; Kralj, Joel M.; Rothschild, Kenneth J.

2013-01-01

Archaerhodopsin 3 (AR3) is a light driven proton pump from Halorubrum sodomense that has been used as a genetically targetable neuronal silencer and an effective fluorescent sensor of transmembrane potential. Unlike the more extensively studied bacteriorhodopsin (BR) from Halobacterium salinarum, AR3 readily incorporates into the plasma membrane of both E. coli and mammalian cells. Here, we used near-IR resonance Raman confocal microscopy to study the effects of pH and membrane potential on the AR3 retinal chromophore structure. Measurements were performed both on AR3 reconstituted into E. coli polar lipids and in vivo in E. coli expressing AR3 in the absence and presence of a negative transmembrane potential. The retinal chromophore structure of AR3 is in an all-trans configuration almost identical to BR over the entire pH range from 3–11. Small changes are detected in the retinal ethylenic stretching frequency and Schiff Base (SB) hydrogen bonding strength relative to BR which may be related to a different water structure near the SB. In the case of the AR3 mutant D95N, at neutral pH an all-trans retinal O-like species (Oall-trans) is found. At higher pH a second 13-cis retinal N-like species (N13-cis) is detected which is attributed to a slowly decaying intermediate in the red-light photocycle of D95N. However, the amount of N13-cis detected is less in E. coli cells but is restored upon addition of carbonyl cyanide m-chlorophenyl hydrazone (CCCP) or sonication, both of which dissipate the normal negative membrane potential. We postulate that these changes are due to the effect of membrane potential on the N13-cis to M13-cis levels accumulated in the D95N red-light photocycle and on a molecular level by the effects of the electric field on the protonation/deprotonation of the cytoplasmic accessible SB. This mechanism also provides a possible explanation for the observed fluorescence dependence of AR3 and other microbial rhodopsins on transmembrane potential

Role of Transmembrane Domain 8 in Substrate Selectivity and Translocation of SteT, a Member of the l-Amino Acid Transporter (LAT) Family*

PubMed Central

Bartoccioni, Paola; del Rio, César; Ratera, Merce; Kowalczyk, Lukasz; Baldwin, Jocelyn M.; Zorzano, Antonio; Quick, Matthias; Baldwin, Stephen A.; Vázquez-Ibar, José Luis; Palacín, Manuel

2010-01-01

System l-amino acid transporters (LAT) belong to the amino acid, polyamine, and organic cation superfamily of transporters and include the light subunits of heteromeric amino acid transporters and prokaryotic homologues. Cysteine reactivity of SteT (serine/threonine antiporter) has been used here to study the substrate-binding site of LAT transporters. Residue Cys-291, in transmembrane domain 8 (TM8), is inactivated by thiol reagents in a substrate protectable manner. Surprisingly, DTT activated the transporter by reducing residue Cys-291. Cysteine-scanning mutagenesis of TM8 showed DTT activation in the single-cysteine mutants S287C, G294C, and S298C, lining the same α-helical face. S-Thiolation in Escherichia coli cells resulted in complete inactivation of the single-cysteine mutant G294C. l-Serine blocked DTT activation with an EC50 similar to the apparent KM of this mutant. Thus, S-thiolation abolished substrate translocation but not substrate binding. Mutation of Lys-295, to Cys (K295C) broadened the profile of inhibitors and the spectrum of substrates with the exception of imino acids. A structural model of SteT based on the structural homologue AdiC (arginine/agmatine antiporter) positions residues Cys-291 and Lys-295 in the putative substrate binding pocket. All this suggests that Lys-295 is a main determinant in the recognition of the side chain of SteT substrates. In contrast, Gly-294 is not facing the surface, suggesting conformational changes involving TM8 during the transport cycle. Our results suggest that TM8 sculpts the substrate-binding site and undergoes conformational changes during the transport cycle of SteT. PMID:20610400

Site-directed fluorescence labeling reveals a revised N-terminal membrane topology and functional periplasmic residues in the Escherichia coli cell division protein FtsK.

PubMed

Berezuk, Alison M; Goodyear, Mara; Khursigara, Cezar M

2014-08-22

In Escherichia coli, FtsK is a large integral membrane protein that coordinates chromosome segregation and cell division. The N-terminal domain of FtsK (FtsKN) is essential for division, and the C terminus (FtsKC) is a well characterized DNA translocase. Although the function of FtsKN is unknown, it is suggested that FtsK acts as a checkpoint to ensure DNA is properly segregated before septation. This may occur through modulation of protein interactions between FtsKN and other division proteins in both the periplasm and cytoplasm; thus, a clear understanding of how FtsKN is positioned in the membrane is required to characterize these interactions. The membrane topology of FtsKN was initially determined using site-directed reporter fusions; however, questions regarding this topology persist. Here, we report a revised membrane topology generated by site-directed fluorescence labeling. The revised topology confirms the presence of four transmembrane segments and reveals a newly identified periplasmic loop between the third and fourth transmembrane domains. Within this loop, four residues were identified that, when mutated, resulted in the appearance of cellular voids. High resolution transmission electron microscopy of these voids showed asymmetric division of the cytoplasm in the absence of outer membrane invagination or visible cell wall ingrowth. This uncoupling reveals a novel role for FtsK in linking cell envelope septation events and yields further evidence for FtsK as a critical checkpoint of cell division. The revised topology of FtsKN also provides an important platform for future studies on essential interactions required for this process. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Genetic variants in PNPLA3 and TM6SF2 predispose to the development of hepatocellular carcinoma in individuals with alcohol-related cirrhosis.

PubMed

Stickel, Felix; Buch, Stephan; Nischalke, Hans Dieter; Weiss, Karl Heinz; Gotthardt, Daniel; Fischer, Janett; Rosendahl, Jonas; Deltenre, Pierre; Marot, Astrid; Elamly, Mona; Casper, Markus; Lammert, Frank; McQuillin, Andrew; Zopf, Steffen; Spengler, Ulrich; Marhenke, Silke; Kirstein, Martha M; Vogel, Arndt; Eyer, Florian; von Felden, Johann; Wege, Henning; Buch, Thorsten; Schafmayer, Clemens; Braun, Felix; Berg, Thomas; Morgan, Marsha Y; Hampe, Jochen

2018-03-13

Variants in patatin-like phospholipase domain-containing 3 (PNPLA3; rs738409), transmembrane 6 superfamily member 2 (TM6SF2; rs58542926), and membrane bound O-acyltransferase domain containing 7 (MBOAT7; rs641738) are risk factors for the development of alcohol-related cirrhosis. Within this population, PNPLA3 rs738409 is also an established risk factor for the development of hepatocellular carcinoma (HCC). The aim of this study was to explore possible risk associations of TM6SF2 rs58542926 and MBOAT7 rs641738 with HCC. Risk variants in PNPLA3, TM6SF2, and MBOAT7 were genotyped in 751 cases with alcohol-related cirrhosis and HCC and in 1165 controls with alcohol-related cirrhosis without HCC. Association with the risk of developing HCC was analyzed using multivariate logistic regression. The development of HCC was independently associated with PNPLA3 rs738409 (OR adjusted 1.84 [95% CI 1.55-2.18], p = 1.85 × 10 -12 ) and TM6SF2 rs58542926 (OR adjusted 1.66 [1.30-2.13], p = 5.13 × 10 -05 ), using an additive model, and controlling the sex, age, body mass index, and type 2 diabetes mellitus; the risk associated with carriage of MBOAT7 rs641738 (OR adjusted 1.04 [0.88-1.24], p = 0.61) was not significant. The population-attributable fractions were 43.5% for PNPLA3 rs738409, 11.5% for TM6SF2 rs58542926, and 49.9% for the carriage of both the variants combined. Carriage of TM6SF2 rs58542926 is an additional risk factor for the development of HCC in people with alcohol-related cirrhosis. Carriage of both PNPLA3 rs738409 and TM6SF2 rs58542926 accounts for half of the attributable risk for HCC in this population. Genotyping will allow for more precise HCC risk-stratification of patients with alcohol-related cirrhosis, and genotype-guided screening algorithms would optimize patient care.

Engineering of a Novel Saccharomyces cerevisiae Wine Strain with a Respiratory Phenotype at High External Glucose Concentrations

PubMed Central

Henricsson, C.; de Jesus Ferreira, M. C.; Hedfalk, K.; Elbing, K.; Larsson, C.; Bill, R. M.; Norbeck, J.; Hohmann, S.; Gustafsson, L.

2005-01-01

The recently described respiratory strain Saccharomyces cerevisiae KOY.TM6*P is, to our knowledge, the only reported strain of S. cerevisiae which completely redirects the flux of glucose from ethanol fermentation to respiration, even at high external glucose concentrations (27). In the KOY.TM6*P strain, portions of the genes encoding the predominant hexose transporter proteins, Hxt1 and Hxt7, were fused within the regions encoding transmembrane (TM) domain 6. The resulting chimeric gene, TM6*, encoded a chimera composed of the amino-terminal half of Hxt1 and the carboxy-terminal half of Hxt7. It was subsequently integrated into the genome of an hxt null strain. In this study, we have demonstrated the transferability of this respiratory phenotype to the V5 hxt1-7Δ strain, a derivative of a strain used in enology. We also show by using this mutant that it is not necessary to transform a complete hxt null strain with the TM6* construct to obtain a non-ethanol-producing phenotype. The resulting V5.TM6*P strain, obtained by transformation of the V5 hxt1-7Δ strain with the TM6* chimeric gene, produced only minor amounts of ethanol when cultured on external glucose concentrations as high as 5%. Despite the fact that glucose flux was reduced to 30% in the V5.TM6*P strain compared with that of its parental strain, the V5.TM6*P strain produced biomass at a specific rate as high as 85% that of the V5 wild-type strain. Even more relevant for the potential use of such a strain for the production of heterologous proteins and also of low-alcohol beverages is the observation that the biomass yield increased 50% with the mutant compared to its parental strain. PMID:16204537

Engineering of a novel Saccharomyces cerevisiae wine strain with a respiratory phenotype at high external glucose concentrations.

PubMed

Henricsson, C; de Jesus Ferreira, M C; Hedfalk, K; Elbing, K; Larsson, C; Bill, R M; Norbeck, J; Hohmann, S; Gustafsson, L

2005-10-01

The recently described respiratory strain Saccharomyces cerevisiae KOY.TM6*P is, to our knowledge, the only reported strain of S. cerevisiae which completely redirects the flux of glucose from ethanol fermentation to respiration, even at high external glucose concentrations (27). In the KOY.TM6*P strain, portions of the genes encoding the predominant hexose transporter proteins, Hxt1 and Hxt7, were fused within the regions encoding transmembrane (TM) domain 6. The resulting chimeric gene, TM6*, encoded a chimera composed of the amino-terminal half of Hxt1 and the carboxy-terminal half of Hxt7. It was subsequently integrated into the genome of an hxt null strain. In this study, we have demonstrated the transferability of this respiratory phenotype to the V5 hxt1-7Delta strain, a derivative of a strain used in enology. We also show by using this mutant that it is not necessary to transform a complete hxt null strain with the TM6* construct to obtain a non-ethanol-producing phenotype. The resulting V5.TM6*P strain, obtained by transformation of the V5 hxt1-7Delta strain with the TM6* chimeric gene, produced only minor amounts of ethanol when cultured on external glucose concentrations as high as 5%. Despite the fact that glucose flux was reduced to 30% in the V5.TM6*P strain compared with that of its parental strain, the V5.TM6*P strain produced biomass at a specific rate as high as 85% that of the V5 wild-type strain. Even more relevant for the potential use of such a strain for the production of heterologous proteins and also of low-alcohol beverages is the observation that the biomass yield increased 50% with the mutant compared to its parental strain.

Molecular determinants and thermodynamics of the amyloid precursor protein transmembrane domain implicated in Alzheimer's disease

PubMed Central

Wang, Hao; Barreyro, Laura; Provasi, Davide; Djemil, Imane; Torres-Arancivia, Celia; Filizola, Marta; Ubarretxena-Belandia, Iban

2011-01-01

The deposition of toxic amyloid-β peptide (Aβ) aggregates in the brain is a hallmark of Alzheimer's disease. The intramembrane proteolysis by γ-secretase of the amyloid precursor protein carboxy-terminal fragment (APP-βCTF) constitutes the final step in the production of Aβs. Mounting evidence suggests that APP-βCTF is a transmembrane domain (TMD) dimer, and that dimerization might modulate the production of Aβ species that are prone to aggregation, and therefore most toxic. We combined experimental and computational approaches to study the molecular determinants and thermodynamics of APP-βCTF dimerization, and produced a unifying structural model that reconciles much of the published data. Using a cell assay, which exploits a dimerization-dependent activator of transcription, we identified specific dimerization-disrupting mutations located mostly at the N-terminus of the TMD of APP-βCTF. The ability of selected mutants to disrupt the dimerization of full length APP-βCTF was confirmed by fluorescence resonance energy transfer experiments. Free-energy estimates of wild-type (WT) and mutants of the TMD of APP-βCTF derived from enhanced molecular dynamics simulations showed that the dimeric state is comprised of different arrangements, in which either 709GXXXA713 or 700GXXXG704GXXXG708 interaction motifs can engage in symmetric or asymmetric associations. Mutations along the TMD of APP-βCTF were found to modulate the relative free energy of the dimeric configurations, and to differently affect the distribution of interfaces within the dimeric state. This observation might have important biological implications, since dimers with a different arrangement of the transmembrane helices are likely to be recognized differently by γ-secretase and lead to a variation of Aβ levels. PMID:21440556

NMR detection of pH-dependent histidine-water proton exchange reveals the conduction mechanism of a transmembrane proton channel.

PubMed

Hu, Fanghao; Schmidt-Rohr, Klaus; Hong, Mei

2012-02-29

The acid-activated proton channel formed by the influenza M2 protein is important for the life cycle of the virus. A single histidine, His37, in the M2 transmembrane domain (M2TM) is responsible for pH activation and proton selectivity of the channel. Recent studies suggested three models for how His37 mediates proton transport: a shuttle mechanism involving His37 protonation and deprotonation, a H-bonded imidazole-imidazolium dimer model, and a transporter model involving large protein conformational changes in synchrony with proton conduction. Using magic-angle-spinning (MAS) solid-state NMR spectroscopy, we examined the proton exchange and backbone conformational dynamics of M2TM in a virus-envelope-mimetic membrane. At physiological temperature and pH, (15)N NMR spectra show fast exchange of the imidazole (15)N between protonated and unprotonated states. To quantify the proton exchange rates, we measured the (15)N T(2) relaxation times and simulated them for chemical-shift exchange and fluctuating N-H dipolar fields under (1)H decoupling and MAS. The exchange rate is 4.5 × 10(5) s(-1) for Nδ1 and 1.0 × 10(5) s(-1) for Nε2, which are approximately synchronized with the recently reported imidazole reorientation. Binding of the antiviral drug amantadine suppressed both proton exchange and ring motion, thus interfering with the proton transfer mechanism. By measuring the relative concentrations of neutral and cationic His as a function of pH, we determined the four pK(a) values of the His37 tetrad in the viral membrane. Fitting the proton current curve using the charge-state populations from these pK(a)'s, we obtained the relative conductance of the five charge states, which showed that the +3 channel has the highest time-averaged unitary conductance. At physiologically relevant pH, 2D correlation spectra indicated that the neutral and cationic histidines do not have close contacts, ruling out the H-bonded dimer model. Moreover, a narrowly distributed nonideal

The TDP-43 N-terminal domain structure at high resolution.

PubMed

Mompeán, Miguel; Romano, Valentina; Pantoja-Uceda, David; Stuani, Cristiana; Baralle, Francisco E; Buratti, Emanuele; Laurents, Douglas V

2016-04-01

Transactive response DNA-binding protein 43 kDa (TDP-43) is an RNA transporting and processing protein whose aberrant aggregates are implicated in neurodegenerative diseases. The C-terminal domain of this protein plays a key role in mediating this process. However, the N-terminal domain (residues 1-77) is needed to effectively recruit TDP-43 monomers into this aggregate. In the present study, we report, for the first time, the essentially complete (1) H, (15) N and (13) C NMR assignments and the structure of the N-terminal domain determined on the basis of 26 hydrogen-bond, 60 torsion angle and 1058 unambiguous NOE structural restraints. The structure consists of an α-helix and six β-strands. Two β-strands form a β-hairpin not seen in the ubiquitin fold. All Pro residues are in the trans conformer and the two Cys are reduced and distantly separated on the surface of the protein. The domain has a well defined hydrophobic core composed of F35, Y43, W68, Y73 and 17 aliphatic side chains. The fold is topologically similar to the reported structure of axin 1. The protein is stable and no denatured species are observed at pH 4 and 25 °C. At 4 kcal·mol(-1) , the conformational stability of the domain, as measured by hydrogen/deuterium exchange, is comparable to ubiquitin (6 kcal·mol(-1) ). The β-strands, α-helix, and three of four turns are generally rigid, although the loop formed by residues 47-53 is mobile, as determined by model-free analysis of the (15) N{(1) H}NOE, as well as the translational and transversal relaxation rates. Structural data have been deposited in the Protein Data Bank under accession code: 2n4p. The NMR assignments have been deposited in the BMRB database under access code: 25675. © 2016 Federation of European Biochemical Societies.

Glutamic Acid as a Precursor to N-Terminal Pyroglutamic Acid in Mouse Plasmacytoma Protein

PubMed Central

Twardzik, Daniel R.; Peterkofsky, Alan

1972-01-01

Cell suspensions derived from a mouse plasmacytoma (RPC-20) that secretes an immunoglobulin light chain containing N-terminal pyroglutamic acid can synthesize protein in vitro. Chromatographic examination of an enzymatic digest of protein labeled with glutamic acid shows only labeled glutamic acid and pyroglutamic acid; hydrolysis of protein from cells labeled with glutamine, however, yields substantial amounts of glutamic acid in addition to glutamine and pyroglutamic acid. The absence of glutamine synthetase and presence of glutaminase in plasmacytoma homogenates is consistent with these findings. These data indicate that N-terminal pyroglutamic acid can be derived from glutamic acid without prior conversion of glutamic acid to glutamine. Since free or bound forms of glutamine cyclize nonezymatically to pyroglutamate with ease, while glutamic acid does not, the data suggest that N-terminal pyroglutamic acid formation from glutamic acid is enzymatic rather than spontaneous. Images PMID:4400295

157 nm Photodissociation of Dipeptide Ions Containing N-Terminal Arginine

NASA Astrophysics Data System (ADS)

Webber, Nathaniel; He, Yi; Reilly, James P.

2014-02-01

Twenty singly-charged dipeptide ions with N-terminal arginine were photodissociated using 157 nm light in both a linear ion-trap mass spectrometer and a MALDI-TOF-TOF mass spectrometer. Analogous to previous work on dipeptides containing C-terminal arginine, this set of samples enabled insights into the photofragmentation propensities associated with individual residues. In addition to familiar products such as a-, d-, and immonium ions, m2 and m2+13 ions were also observed. Certain side chains tended to cleave between their β and γ carbons without necessarily forming d- or w-type ions, and a few other ions were produced by the high-energy fragmentation of multiple bonds.

PRINT: A Protein Bioconjugation Method with Exquisite N-terminal Specificity

PubMed Central

Sur, Surojit; Qiao, Yuan; Fries, Anja; O’Meally, Robert N.; Cole, Robert N.; Kinzler, Kenneth W.; Vogelstein, Bert; Zhou, Shibin

2015-01-01

Chemical conjugation is commonly used to enhance the pharmacokinetics, biodistribution, and potency of protein therapeutics, but often leads to non-specific modification or loss of bioactivity. Here, we present a simple, versatile and widely applicable method that allows exquisite N-terminal specific modification of proteins. Combining reversible side-chain blocking and protease mediated cleavage of a commonly used HIS tag appended to a protein, we generate with high yield and purity exquisitely site specific and selective bio-conjugates of TNF-α by using amine reactive NHS ester chemistry. We confirm the N terminal selectivity and specificity using mass spectral analyses and show near complete retention of the biological activity of our model protein both in vitro and in vivo murine models. We believe that this methodology would be applicable to a variety of potentially therapeutic proteins and the specificity afforded by this technique would allow for rapid generation of novel biologics. PMID:26678960

Absolute calibration accuracy of L4 TM and L5 TM sensor image pairs

USGS Publications Warehouse

Chander, G.; Micijevic, E.

2006-01-01

The Landsat suite of satellites has collected the longest continuous archive of multispectral data of any land-observing space program. From the Landsat program's inception in 1972 to the present, the Earth science user community has benefited from a historical record of remotely sensed data. However, little attention has been paid to ensuring that the data are calibrated and comparable from mission to mission, Launched in 1982 and 1984 respectively, the Landsat 4 (L4) and Landsat 5 (L5) Thematic Mappers (TM) are the backbone of an extensive archive of moderate resolution Earth imagery. To evaluate the "current" absolute accuracy of these two sensors, image pairs from the L5 TM and L4 TM sensors were compared. The approach involves comparing image statistics derived from large common areas observed eight days apart by the two sensors. The average percent differences in reflectance estimates obtained from the L4 TM agree with those from the L5 TM to within 15 percent. Additional work to characterize the absolute differences between the two sensors over the entire mission is in progress.

A Novel N14Y Mutation in Connexin26 in Keratitis-Ichthyosis-Deafness Syndrome

PubMed Central

Arita, Ken; Akiyama, Masashi; Aizawa, Tomoyasu; Umetsu, Yoshitaka; Segawa, Ikuo; Goto, Maki; Sawamura, Daisuke; Demura, Makoto; Kawano, Keiichi; Shimizu, Hiroshi

2006-01-01

Connexins (Cxs) are transmembranous proteins that connect adjacent cells via channels known as gap junctions. The N-terminal 21 amino acids of Cx26 are located at the cytoplasmic side of the channel pore and are thought to be essential for the regulation of channel selectivity. We have found a novel mutation, N14Y, in the N-terminal domain of Cx26 in a case of keratitis-ichthyosis-deafness syndrome. Reduced gap junctional intercellular communication was observed in the patient’s keratinocytes by the dye transfer assay using scrape-loading methods. The effect of this mutation on molecular structure was investigated using synthetic N-terminal peptides from both wild-type and mutated Cx26. Two-dimensional 1H nuclear magnetic resonance and circular dichroism measurements demonstrated that the secondary structures of these two model peptides are similar to each other. However, several novel nuclear Overhauser effect signals appeared in the N14Y mutant, and the secondary structure of the mutant peptide was more susceptible to induction of 2,2,2-trifluoroethanol than wild type. Thus, it is likely that the N14Y mutation induces a change in local structural flexibility of the N-terminal domain, which is important for exerting the activity of the channel function, resulting in impaired gap junctional intercellular communication. PMID:16877344

Normally-off AlGaN/GaN-based MOS-HEMT with self-terminating TMAH wet recess etching

NASA Astrophysics Data System (ADS)

Son, Dong-Hyeok; Jo, Young-Woo; Won, Chul-Ho; Lee, Jun-Hyeok; Seo, Jae Hwa; Lee, Sang-Heung; Lim, Jong-Won; Kim, Ji Heon; Kang, In Man; Cristoloveanu, Sorin; Lee, Jung-Hee

2018-03-01

Normally-off AlGaN/GaN-based MOS-HEMT has been fabricated by utilizing damage-free self-terminating tetramethyl ammonium hydroxide (TMAH) recess etching. The device exhibited a threshold voltage of +2.0 V with good uniformity, extremely small hysteresis of ∼20 mV, and maximum drain current of 210 mA/mm. The device also exhibited excellent off-state performances, such as breakdown voltage of ∼800 V with off-state leakage current as low as ∼10-12 A and high on/off current ratio (Ion/Ioff) of 1010. These excellent device performances are believed to be due to the high quality recessed surface, provided by the simple self-terminating TMAH etching.

Chimeric rabies glycoprotein with a transmembrane domain and cytoplasmic tail from Newcastle disease virus fusion protein incorporates into the Newcastle disease virion at reduced levels.

PubMed

Yu, Gui Mei; Zu, Shu Long; Zhou, Wei Wei; Wang, Xi Jun; Shuai, Lei; Wang, Xue Lian; Ge, Jin Ying; Bu, Zhi Gao

2017-08-31

Rabies remains an important worldwide health problem. Newcastle disease virus (NDV) was developed as a vaccine vector in animals by using a reverse genetics approach. Previously, our group generated a recombinant NDV (LaSota strain) expressing the complete rabies virus G protein (RVG), named rL-RVG. In this study, we constructed the variant rL-RVGTM, which expresses a chimeric rabies virus G protein (RVGTM) containing the ectodomain of RVG and the transmembrane domain (TM) and a cytoplasmic tail (CT) from the NDV fusion glycoprotein to study the function of RVG's TM and CT. The RVGTM did not detectably incorporate into NDV virions, though it was abundantly expressed at the surface of infected BHK-21 cells. Both rL-RVG and rL-RVGTM induced similar levels of NDV virus-neutralizing antibody (VNA) after initial and secondary vaccination in mice, whereas rabies VNA induction by rL-RVGTM was markedly lower than that induced by rL-RVG. Though rL-RVG could spread from cell to cell like that in rabies virus, rL-RVGTM lost this ability and spread in a manner similar to the parental NDV. Our data suggest that the TM and CT of RVG are essential for its incorporation into NDV virions and for spreading of the recombinant virus from the initially infected cells to surrounding cells.

The role of proline residues in the dynamics of transmembrane helices: the case of bacteriorhodopsin.

PubMed

Perálvarez-Marín, Alex; Bourdelande, José-Luis; Querol, Enric; Padrós, Esteve

2006-01-01

Proline residues in transmembrane helices have been found to have important roles in the functioning of membrane proteins. Moreover, Pro residues occur with high frequency in transmembrane alpha-helices, as compared to alpha-helices for soluble proteins. Here, we report several properties of the bacteriorhodopsin mutants P50A (helix B), P91A (helix C) and P186A (helix F). Compared to wild type, strongly perturbed behaviour has been found for these mutants. In the resting state, increased hydroxylamine accessibility and altered Asp-85 pKa and light-dark adaptation were observed. On light activation, hydroxylamine accessibility was increased and proton transport activity, M formation kinetics and FTIR difference spectra of M and N intermediates showed clear distortions. On the basis of these alterations and the near identity of the crystalline structures of mutants with that of wild type, we conclude that the transmembrane proline residues of bacteriorhodopsin fulfil a dynamic role in both the resting and the light-activated states. Our results are consistent with the notion that mutation of Pro to Ala allows the helix to increase its flexibility towards the direction originally hindered by the steric clash between the ring Cgamma and the carbonyl O of the i-4 residue, at the same time decreasing the mobility towards the opposite direction. Due to their properties, transmembrane Pro residues may serve as transmission elements of conformational changes during the transport process. We propose that these concepts can be extended to other transmembrane proteins.

Reliability and Validity of the Zephyr[TM] BioHarness[TM] to Measure Respiratory Responses to Exercise

ERIC Educational Resources Information Center

Hailstone, Jono; Kilding, Andrew E.

2011-01-01

The Zephyr[TM] BioHarness[TM] (Zephyr Technology, Auckland, New Zealand) is a wireless physiological monitoring system that has the ability to measure respiratory rate unobtrusively. However, the ability of the BioHarness[TM] to accurately and reproducibly determine respiratory rate across a range of intensities is currently unknown. The aim of…

Orientations and Proximities of the Extracellular Ends of Transmembrane Helices S0 and S4 in Open and Closed BK Potassium Channels

PubMed Central

Wu, Roland S.; Chudasama, Neelesh; Zakharov, Sergey I.; Karlin, Arthur; Marx, Steven O.

2013-01-01

The large-conductance potassium channel (BK) α subunit contains a transmembrane (TM) helix S0 preceding the canonical TM helices S1 through S6. S0 lies between S4 and the TM2 helix of the regulatory β1 subunit. Pairs of Cys were substituted in the first helical turns in the membrane of BK α S0 and S4 and in β1 TM2. One such pair, W22C in S0 and W203C in S4, was 95% crosslinked endogenously. Under voltage-clamp conditions in outside-out patches, this crosslink was reduced by DTT and reoxidized by a membrane-impermeant bis-quaternary ammonium derivative of diamide. The rate constants for this reoxidation were not significantly different in the open and closed states of the channel. Thus, these two residues are approximately equally close in the two states. In addition, 90% crosslinking of a second pair, R20C in S0 and W203C in S4, had no effect on the V50 for opening. Taken together, these findings indicate that separation between residues at the extracellular ends of S0 and S4 is not required for voltage-sensor activation. On the contrary, even though W22C and W203C were equally likely to form a disulfide in the activated and deactivated states, relative immobilization by crosslinking of these two residues favored the activated state. Furthermore, the efficiency of recrosslinking of W22C and W203C on the cell surface was greater in the presence of the β1 subunit than in its absence, consistent with β1 acting through S0 to stabilize its immobilization relative to α S4. PMID:23472181

Contributions of the N- and C-terminal helical segments to the lipid-free structure and lipid interaction of apolipoprotein A-I.

PubMed

Tanaka, Masafumi; Dhanasekaran, Padmaja; Nguyen, David; Ohta, Shinya; Lund-Katz, Sissel; Phillips, Michael C; Saito, Hiroyuki

2006-08-29

The tertiary structure of lipid-free apolipoprotein (apo) A-I in the monomeric state comprises two domains: a N-terminal alpha-helix bundle and a less organized C-terminal domain. This study examined how the N- and C-terminal segments of apoA-I (residues 1-43 and 223-243), which contain the most hydrophobic regions in the molecule and are located in opposite structural domains, contribute to the lipid-free conformation and lipid interaction. Measurements of circular dichroism in conjunction with tryptophan and 8-anilino-1-naphthalenesulfonic acid fluorescence data demonstrated that single (L230P) or triple (L230P/L233P/Y236P) proline insertions into the C-terminal alpha helix disrupted the organization of the C-terminal domain without affecting the stability of the N-terminal helix bundle. In contrast, proline insertion into the N terminus (Y18P) disrupted the bundle structure in the N-terminal domain, indicating that the alpha-helical segment in this region is part of the helix bundle. Calorimetric and gel-filtration measurements showed that disruption of the C-terminal alpha helix significantly reduced the enthalpy and free energy of binding of apoA-I to lipids, whereas disruption of the N-terminal alpha helix had only a small effect on lipid binding. Significantly, the presence of the Y18P mutation offset the negative effects of disruption/removal of the C-terminal helical domain on lipid binding, suggesting that the alpha helix around Y18 concealed a potential lipid-binding region in the N-terminal domain, which was exposed by the disruption of the helix-bundle structure. When these results are taken together, they indicate that the alpha-helical segment in the N terminus of apoA-I modulates the lipid-free structure and lipid interaction in concert with the C-terminal domain.

An improved procedure, involving mass spectrometry, for N-terminal amino acid sequence determination of proteins which are N alpha-blocked.

PubMed Central

Rose, K; Kocher, H P; Blumberg, B M; Kolakofsky, D

1984-01-01

A modification to a previously described procedure [Gray & del Valle (1970) Biochemistry 9, 2134-2137; Rose, Simona & Offord (1983) Biochem. J. 215, 261-272] for mass-spectral identification of the N-terminal regions of proteins is shown to be useful in cases where the N-terminus is blocked. Three proteins were studied: vesicular-stomatitis-virus N protein, Sendai-virus NP protein, and a rabbit immunoglobulin lambda-light chain. These proteins, found to be blocked at the N-terminus with either the acetyl group or a pyroglutamic acid residue, had all failed to yield to attempted Edman degradation, in one case even after attempted enzymic removal of the pyroglutamic acid residue. The N-terminal regions of all three proteins were sequenced by using the new procedure. PMID:6421284

TM surface wave diffraction by a truncated dielectric slab recessed in a perfectly conducting surface. [considering flush mounted space shuttle antenna

NASA Technical Reports Server (NTRS)

Pathak, P. H.; Kouyoumjian, R. G.

1974-01-01

The diffraction of a TM sub o surface wave by a terminated dielectric slab which is flush mounted in a perfectly conducting surface is studied. The incident surface wave gives rise to waves reflected and diffracted by the termination; these reflected and diffracted fields may be expressed in terms of the geometrical theory of diffraction by introducing surface wave reflection and diffraction coefficients which are associated with the termination. In this investigation, the surface wave reflection and diffraction coefficients have been deduced from a formally exact solution to this canonical problem. The solution is obtained by a combination of the generalized scattering matrix technique and function theoretic methods.

Modified LaRC(TM)-IA Polyimides

NASA Technical Reports Server (NTRS)

St. Clair, Terry L.; Chang, Alice C.; Hou, Tan H.; Working, Dennis C.

1994-01-01

Modified versions of thermoplastic polyimide LaRC(TM)-IA incorporate various amounts of additional, rigid moieties into backbones of LaRC(TM)-IA molecules. Modified versions more resistant to solvents and exhibit higher glass-transition temperatures, yet retain melt-flow processability of unmodified LaRC(TM)-IA.

Phonon anomalies in intermediate valent TmXSe and TmSe1 - yTey

NASA Astrophysics Data System (ADS)

Boppart, H.; Treindl, A.; Wachter, P.

1981-03-01

In TmxSe and TmSe1-yTey the degree of valence mixing can be adjusted between nearly 3+ for Tm0.87Se and 2.55+ for TmSe0.7Te0.3. The measurement of sound velocities vL, vTl and vT2 and the evaluation of the Raman effect for various compositions permit the derivation of LA [111] phonon dispersion at critical points in the Brillouin zone. vL decreases with increasing valence mixing. Near the middle of the zone the LA branch gets a dip for intermediate valent compositions, resulting in a characteristic peak in the Ramn spectrum at about 60 cm-1. The elastic constant c12 has been found negative for Tm0.99Se, also at 4.2 K. For uniaxial pressures c12 exhibits strong nonlinearities and even changes sign with pressure in an intermediate valent composition. The optical phonon frequencies, LO (L) also soften proportional with the degree of valence mixing.

Divergent N-Terminal Sequences Target an Inducible Testis Deubiquitinating Enzyme to Distinct Subcellular Structures

PubMed Central

Lin, Haijiang; Keriel, Anne; Morales, Carlos R.; Bedard, Nathalie; Zhao, Qing; Hingamp, Pascal; Lefrançois, Stephane; Combaret, Lydie; Wing, Simon S.

2000-01-01

Ubiquitin-specific processing proteases (UBPs) presently form the largest enzyme family in the ubiquitin system, characterized by a core region containing conserved motifs surrounded by divergent sequences, most commonly at the N-terminal end. The functions of these divergent sequences remain unclear. We identified two isoforms of a novel testis-specific UBP, UBP-t1 and UBP-t2, which contain identical core regions but distinct N termini, thereby permitting dissection of the functions of these two regions. Both isoforms were germ cell specific and developmentally regulated. Immunocytochemistry revealed that UBP-t1 was induced in step 16 to 19 spermatids while UBP-t2 was expressed in step 18 to 19 spermatids. Immunoelectron microscopy showed that UBP-t1 was found in the nucleus while UBP-t2 was extranuclear and was found in residual bodies. For the first time, we show that the differential subcellular localization was due to the distinct N-terminal sequences. When transfected into COS-7 cells, the core region was expressed throughout the cell but the UBP-t1 and UBP-t2 isoforms were concentrated in the nucleus and the perinuclear region, respectively. Fusions of each N-terminal end with green fluorescent protein yielded the same subcellular localization as the native proteins, indicating that the N-terminal ends were sufficient for determining differential localization. Interestingly, UBP-t2 colocalized with anti-γ-tubulin immunoreactivity, indicating that like several other components of the ubiquitin system, a deubiquitinating enzyme is associated with the centrosome. Regulated expression and alternative N termini can confer specificity of UBP function by restricting its temporal and spatial loci of action. PMID:10938131

Actively mode-locked Tm-Ho:LiYF4 and Tm-Ho:BaY2F8 lasers

NASA Astrophysics Data System (ADS)

Gatti, D.; Galzerano, G.; Toncelli, A.; Tonelli, M.; Laporta, P.

2007-01-01

We report on the generation of mode-locking pulse trains with high average output powers from diode-pumped Tm-Ho:LiYF4 and Tm-Ho:BaY2F8 lasers emitting at around 2 μm. The highest output power of 365 mW was obtained with the Tm-Ho:YLF4 laser, whereas the shortest pulse duration of 120 ps and the widest tunability range of 59 nm was achieved with the Tm-Ho:BaY2F8 laser.

The prophage-encoded hyaluronate lyase has broad substrate specificity and is regulated by the N-terminal domain.

PubMed

Singh, Sudhir Kumar; Bharati, Akhilendra Pratap; Singh, Neha; Pandey, Praveen; Joshi, Pankaj; Singh, Kavita; Mitra, Kalyan; Gayen, Jiaur R; Sarkar, Jayanta; Akhtar, Md Sohail

2014-12-19

Streptococcus equi is the causative agent of the highly contagious disease "strangles" in equines and zoonotic meningitis in human. Spreading of infection in host tissues is thought to be facilitated by the bacterial gene encoded extracellular hyaluronate lyase (HL), which degrades hyaluronan (HA), chondroitin 6-sulfate, and dermatan sulfate of the extracellular matrix). The clinical strain S. equi 4047 however, lacks a functional extracellular HL. The prophages of S. equi and other streptococci encode intracellular HLs which are reported to partially degrade HA and do not cleave any other glycosaminoglycans. The phage HLs are thus thought to play a role limited to the penetration of streptococcal HA capsules, facilitating bacterial lysogenization and not in the bacterial pathogenesis. Here we systematically looked into the structure-function relationship of S. equi 4047 phage HL. Although HA is the preferred substrate, this HL has weak activity toward chondroitin 6-sulfate and dermatan sulfate and can completely degrade all of them. Even though the catalytic triple-stranded β-helix domain of phage HL is functionally independent, its catalytic efficiency and specificity is influenced by the N-terminal domain. The phage HL also interacts with human transmembrane glycoprotein CD44. The above results suggest that the streptococci can use phage HLs to degrade glycosaminoglycans of the extracellular matrix for spreading virulence factors and toxins while utilizing the disaccharides as a nutrient source for proliferation at the site of infection. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

The Prophage-encoded Hyaluronate Lyase Has Broad Substrate Specificity and Is Regulated by the N-terminal Domain*

PubMed Central

Singh, Sudhir Kumar; Bharati, Akhilendra Pratap; Singh, Neha; Pandey, Praveen; Joshi, Pankaj; Singh, Kavita; Mitra, Kalyan; Gayen, Jiaur R.; Sarkar, Jayanta; Akhtar, Md. Sohail

2014-01-01

Streptococcus equi is the causative agent of the highly contagious disease “strangles” in equines and zoonotic meningitis in human. Spreading of infection in host tissues is thought to be facilitated by the bacterial gene encoded extracellular hyaluronate lyase (HL), which degrades hyaluronan (HA), chondroitin 6-sulfate, and dermatan sulfate of the extracellular matrix). The clinical strain S. equi 4047 however, lacks a functional extracellular HL. The prophages of S. equi and other streptococci encode intracellular HLs which are reported to partially degrade HA and do not cleave any other glycosaminoglycans. The phage HLs are thus thought to play a role limited to the penetration of streptococcal HA capsules, facilitating bacterial lysogenization and not in the bacterial pathogenesis. Here we systematically looked into the structure-function relationship of S. equi 4047 phage HL. Although HA is the preferred substrate, this HL has weak activity toward chondroitin 6-sulfate and dermatan sulfate and can completely degrade all of them. Even though the catalytic triple-stranded β-helix domain of phage HL is functionally independent, its catalytic efficiency and specificity is influenced by the N-terminal domain. The phage HL also interacts with human transmembrane glycoprotein CD44. The above results suggest that the streptococci can use phage HLs to degrade glycosaminoglycans of the extracellular matrix for spreading virulence factors and toxins while utilizing the disaccharides as a nutrient source for proliferation at the site of infection. PMID:25378402

The N-terminal sequence of albumin Redhill, a variant of human serum albumin.

PubMed

Hutchinson, D W; Matejtschuk, P

1985-12-02

Albumin Redhill, a variant human albumin, has been isolated by fast protein liquid chromatofocusing. The N-terminal sequence of this protein corresponded to that of albumin A except that one additional arginine residue was attached to the N-terminus.

Accurate computational design of multipass transmembrane proteins.

PubMed

Lu, Peilong; Min, Duyoung; DiMaio, Frank; Wei, Kathy Y; Vahey, Michael D; Boyken, Scott E; Chen, Zibo; Fallas, Jorge A; Ueda, George; Sheffler, William; Mulligan, Vikram Khipple; Xu, Wenqing; Bowie, James U; Baker, David

2018-03-02

The computational design of transmembrane proteins with more than one membrane-spanning region remains a major challenge. We report the design of transmembrane monomers, homodimers, trimers, and tetramers with 76 to 215 residue subunits containing two to four membrane-spanning regions and up to 860 total residues that adopt the target oligomerization state in detergent solution. The designed proteins localize to the plasma membrane in bacteria and in mammalian cells, and magnetic tweezer unfolding experiments in the membrane indicate that they are very stable. Crystal structures of the designed dimer and tetramer-a rocket-shaped structure with a wide cytoplasmic base that funnels into eight transmembrane helices-are very close to the design models. Our results pave the way for the design of multispan membrane proteins with new functions. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

G protein-coupled receptor transmembrane binding pockets and their applications in GPCR research and drug discovery: a survey.

PubMed

Kratochwil, Nicole A; Gatti-McArthur, Silvia; Hoener, Marius C; Lindemann, Lothar; Christ, Andreas D; Green, Luke G; Guba, Wolfgang; Martin, Rainer E; Malherbe, Pari; Porter, Richard H P; Slack, Jay P; Winnig, Marcel; Dehmlow, Henrietta; Grether, Uwe; Hertel, Cornelia; Narquizian, Robert; Panousis, Constantinos G; Kolczewski, Sabine; Steward, Lucinda

2011-01-01

G protein-coupled receptors (GPCRs) share a common architecture consisting of seven transmembrane (TM) domains. Various lines of evidence suggest that this fold provides a generic binding pocket within the TM region for hosting agonists, antagonists, and allosteric modulators. Hence, an automated method was developed that allows a fast analysis and comparison of these generic ligand binding pockets across the entire GPCR family by providing the relevant information for all GPCRs in the same format. This methodology compiles amino acids lining the TM binding pocket including parts of the ECL2 loop in a so-called 1D ligand binding pocket vector and translates these 1D vectors in a second step into 3D receptor pharmacophore models. It aims to support various aspects of GPCR drug discovery in the pharmaceutical industry. Applications of pharmacophore similarity analysis of these 1D LPVs include definition of receptor subfamilies, prediction of species differences within subfamilies in regard to in vitro pharmacology and identification of nearest neighbors for GPCRs of interest to generate starting points for GPCR lead identification programs. These aspects of GPCR research are exemplified in the field of melanopsins, trace amine-associated receptors and somatostatin receptor subtype 5. In addition, it is demonstrated how 3D pharmacophore models of the LPVs can support the prediction of amino acids involved in ligand recognition, the understanding of mutational data in a 3D context and the elucidation of binding modes for GPCR ligands and their evaluation. Furthermore, guidance through 3D receptor pharmacophore modeling for the synthesis of subtype-specific GPCR ligands will be reported. Illustrative examples are taken from the GPCR family class C, metabotropic glutamate receptors 1 and 5 and sweet taste receptors, and from the GPCR class A, e.g. nicotinic acid and 5-hydroxytryptamine 5A receptor. © 2011 Bentham Science Publishers

Arabidopsis protein disulfide isomerase-8 is a type I endoplasmic reticulum transmembrane protein with thiol-disulfide oxidase activity.

PubMed

Yuen, Christen Y L; Shek, Roger; Kang, Byung-Ho; Matsumoto, Kristie; Cho, Eun Ju; Christopher, David A

2016-08-22

In eukaryotes, classical protein disulfide isomerases (PDIs) facilitate the oxidative folding of nascent secretory proteins in the endoplasmic reticulum by catalyzing the formation, breakage, and rearrangement of disulfide bonds. Terrestrial plants encode six structurally distinct subfamilies of PDIs. The novel PDI-B subfamily is unique to terrestrial plants, and in Arabidopsis is represented by a single member, PDI8. Unlike classical PDIs, which lack transmembrane domains (TMDs), PDI8 is unique in that it has a C-terminal TMD and a single N-terminal thioredoxin domain (instead of two). No PDI8 isoforms have been experimentally characterized to date. Here we describe the characterization of the membrane orientation, expression, sub-cellular localization, and biochemical function of this novel member of the PDI family. Histochemical staining of plants harboring a PDI8 promoter:β-glucuronidase (GUS) fusion revealed that the PDI8 promoter is highly active in young, expanding leaves, the guard cells of cotyledons, and in the vasculature of several organs, including roots, leaves, cotyledons, and flowers. Immunoelectron microscopy studies using a PDI8-specific antibody on root and shoot apical cells revealed that PDI8 localizes to the endoplasmic reticulum (ER). Transient expression of two PDI8 fusions to green fluorescent protein (spGFP-PDI8 and PDI8-GFP-KKED) in leaf mesophyll protoplasts also resulted in labeling of the ER. Protease-protection immunoblot analysis indicated that PDI8 is a type I membrane protein, with its catalytic domain facing the ER lumen. The lumenal portion of PDI8 was able to functionally complement the loss of the prokaryotic protein foldase, disulfide oxidase (DsbA), as demonstrated by the reconstitution of periplasmic alkaline phosphatase in Escherichia coli. The results indicate that PDI8 is a type I transmembrane protein with its catalytic domain facing the lumen of the ER and functions in the oxidation of cysteines to produce disulfide

Blue upconversion with excitation into Tm ions at 780 nm in Yb- and Tm-codoped fluoride crystals

NASA Astrophysics Data System (ADS)

Zhang, X. X.; Hong, P.; Bass, M.; Chai, B. H. T.

1995-04-01

Strong blue emissions have been observed in fluoride crystals, such as LiYF4, BaY2F8, and KYF4, codoped with Tm3+ and Yb3+ when excited into the Tm3+ 3F4 state at ~780 nm. Energy transfer from Tm3+ to Yb3+ ions followed by the transfer from Yb3+ to Tm3+ was demonstrated to be responsible for the upconversion process. A pumping scheme is proposed based on this upconversion mechanism for blue-laser applications using these materials.

The lysosomal potassium channel TMEM175 adopts a novel tetrameric architecture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Changkeun; Guo, Jiangtao; Zeng, Weizhong

TMEM175 is a lysosomal K+ channel that is important for maintaining the membrane potential and pH stability in lysosomes1. It contains two homologous copies of a six-transmembrane-helix (6-TM) domain, which has no sequence homology to the canonical tetrameric K+ channels and lacks the TVGYG selectivity filter motif found in these channels2, 3, 4. The prokaryotic TMEM175 channel, which is present in a subset of bacteria and archaea, contains only a single 6-TM domain and functions as a tetramer. Here, we present the crystal structure of a prokaryotic TMEM175 channel from Chamaesiphon minutus, CmTMEM175, the architecture of which represents a completelymore » different fold from that of canonical K+ channels. All six transmembrane helices of CmTMEM175 are tightly packed within each subunit without undergoing domain swapping. The highly conserved TM1 helix acts as the pore-lining inner helix, creating an hourglass-shaped ion permeation pathway in the channel tetramer. Three layers of hydrophobic residues on the carboxy-terminal half of the TM1 helices form a bottleneck along the ion conduction pathway and serve as the selectivity filter of the channel. Mutagenesis analysis suggests that the first layer of the highly conserved isoleucine residues in the filter is primarily responsible for channel selectivity. Thus, the structure of CmTMEM175 represents a novel architecture of a tetrameric cation channel whose ion selectivity mechanism appears to be distinct from that of the classical K+ channel family.« less

Emission and material gain spectra of polar compressive strained AlGaN quantum wells grown on virtual AlGaN substrates: Tuning emission wavelength and mixing TE and TM mode of light polarization

NASA Astrophysics Data System (ADS)

Gladysiewicz, Marta; Rudzinski, Mariusz; Hommel, Detlef; Kudrawiec, Robert

2018-07-01

It is shown that compressively strained polar AlxGa1‑xN/AlyGa1‑yN quantum wells (QWs) of various contents grown on virtual AlYGa1‑YN substrates (Y = 20, 40, 60, 80, and 100%) are able to cover the whole UV-A, -B, and -C spectral range but their contents and widths have to be carefully optimized if they are to be used as the active region of light emitting diodes and laser diodes. The emission wavelength from AlGaN multi QWs can be tuned by both the QW width and barrier thickness, but the range of QW width for which an efficient luminescence is expected is very small (2–4 nm) due to a very weak electron-hole overlap for wider QWs. The most effective method for wavelength tuning in this QW system is content engineering, i.e., lowering Al concentration in the QW region. The decrease of Al concentration in the QW shifts the emission peak to red, broadens this peak, weakens its intensity, and changes its polarization from transverse magnetic (TM) to TM mixed with transverse electric (TE). For laser diodes the optimal QW design is more rigorous concerning the QW width since this width should be below 3 nm. Moreover it is shown that the TE and TM mode of materials gain overlap and are strongly blueshifted in comparison to emission spectrum.

Structural Basis of Typhoid: Salmonella typhi Type IVb pilin (PilS) and Cystic Fibrosis Transmembrane Conductance Regulatory Interaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Balakrishna, A.; Saxena, A; Mok, H

2009-01-01

The type IVb pilus of the enteropathogenic bacteria Salmonella typhi is a major adhesion factor during the entry of this pathogen into gastrointestinal epithelial cells. Its target of adhesion is a stretch of 10 residues from the first extracellular domain of cystic fibrosis transmembrane conductance regulator (CFTR). The crystal structure of the N-terminal 25 amino acid deleted S. typhi native PilS protein (PilS), which makes the pilus, was determined at 1.9 A resolution by the multiwavelength anomalous dispersion method. Also, the structure of the complex of PilS and a target CFTR peptide, determined at 1.8 A, confirms that residues 113-117more » (NKEER) of CFTR are involved in binding with the pilin protein and gives us insight on the amino acids that are essential for binding. Furthermore, we have also explored the role of a conserved disulfide bridge in pilus formation. The subunit structure and assembly architecture are crucial for understanding pilus functions and designing suitable therapeutics against typhoid.« less

Structural basis of typhod: Salmonella typhi type IVb pilin (PilS) and cystic fibrosis transmembrane conductance regulator interaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Balakrishna, A.; Saxena, A; Mok, H

2009-01-01

The type IVb pilus of the enteropathogenic bacteria Salmonella typhi is a major adhesion factor during the entry of this pathogen into gastrointestinal epithelial cells. Its target of adhesion is a stretch of 10 residues from the first extracellular domain of cystic fibrosis transmembrane conductance regulator (CFTR). The crystal structure of the N-terminal 25 amino acid deleted S. typhi native PilS protein (PilS), which makes the pilus, was determined at 1.9 A resolution by the multiwavelength anomalous dispersion method. Also, the structure of the complex of PilS and a target CFTR peptide, determined at 1.8 A, confirms that residues 113-117more » (NKEER) of CFTR are involved in binding with the pilin protein and gives us insight on the amino acids that are essential for binding. Furthermore, we have also explored the role of a conserved disulfide bridge in pilus formation. The subunit structure and assembly architecture are crucial for understanding pilus functions and designing suitable therapeutics against typhoid.« less

Structural insights into the human RyR2 N-terminal region involved in cardiac arrhythmias

DOE Office of Scientific and Technical Information (OSTI.GOV)

Borko, Ľubomír; Bauerová-Hlinková, Vladena, E-mail: vladena.bauerova@savba.sk; Hostinová, Eva

2014-11-01

X-ray and solution structures of the human RyR2 N-terminal region were obtained under near-physiological conditions. The structure exhibits a unique network of interactions between its three domains, revealing an important stabilizing role of the central helix. Human ryanodine receptor 2 (hRyR2) mediates calcium release from the sarcoplasmic reticulum, enabling cardiomyocyte contraction. The N-terminal region of hRyR2 (amino acids 1–606) is the target of >30 arrhythmogenic mutations and contains a binding site for phosphoprotein phosphatase 1. Here, the solution and crystal structures determined under near-physiological conditions, as well as a homology model of the hRyR2 N-terminal region, are presented. The N-terminusmore » is held together by a unique network of interactions among its three domains, A, B and C, in which the central helix (amino acids 410–437) plays a prominent stabilizing role. Importantly, the anion-binding site reported for the mouse RyR2 N-terminal region is notably absent from the human RyR2. The structure concurs with the differential stability of arrhythmogenic mutations in the central helix (R420W, I419F and I419F/R420W) which are owing to disparities in the propensity of mutated residues to form energetically favourable or unfavourable contacts. In solution, the N-terminus adopts a globular shape with a prominent tail that is likely to involve residues 545–606, which are unresolved in the crystal structure. Docking the N-terminal domains into cryo-electron microscopy maps of the closed and open RyR1 conformations reveals C{sup α} atom movements of up to 8 Å upon channel gating, and predicts the location of the leucine–isoleucine zipper segment and the interaction site for spinophilin and phosphoprotein phosphatase 1 on the RyR surface.« less

Coarse-Grain Simulations Reveal Movement of the Synaptobrevin C-Terminus in Response to Piconewton Forces

PubMed Central

Lindau, Manfred; Hall, Benjamin A.; Chetwynd, Alan; Beckstein, Oliver; Sansom, Mark S.P.

2012-01-01

Fusion of neurosecretory vesicles with the plasma membrane is mediated by SNARE proteins, which transfer a force to the membranes. However, the mechanism by which this force transfer induces fusion pore formation is still unknown. The neuronal vesicular SNARE protein synaptobrevin 2 (syb2) is anchored in the vesicle membrane by a single C-terminal transmembrane (TM) helix. In coarse-grain molecular-dynamics simulations, self-assembly of the membrane occurred with the syb2 TM domain inserted, as expected from experimental data. The free-energy profile for the position of the syb2 membrane anchor in the membrane was determined using umbrella sampling. To predict the free-energy landscapes for a reaction pathway pulling syb2 toward the extravesicular side of the membrane, which is the direction of the force transfer from the SNARE complex, harmonic potentials were applied to the peptide in its unbiased position, pulling it toward new biased equilibrium positions. Application of piconewton forces to the extravesicular end of the TM helix in the simulation detached the synaptobrevin C-terminus from the vesicle's inner-leaflet lipid headgroups and pulled it deeper into the membrane. This C-terminal movement was facilitated and hindered by specific mutations in parallel with experimentally observed facilitation and inhibition of fusion. Direct application of such forces to the intravesicular end of the TM domain resulted in tilting motion of the TM domain through the membrane with an activation energy of ∼70 kJ/mol. The results suggest a mechanism whereby fusion pore formation is induced by movement of the charged syb2 C-terminus within the membrane in response to pulling and tilting forces generated by C-terminal zippering of the SNARE complex. PMID:23009845

Coarse-grain simulations reveal movement of the synaptobrevin C-terminus in response to piconewton forces.

PubMed

Lindau, Manfred; Hall, Benjamin A; Chetwynd, Alan; Beckstein, Oliver; Sansom, Mark S P

2012-09-05

Fusion of neurosecretory vesicles with the plasma membrane is mediated by SNARE proteins, which transfer a force to the membranes. However, the mechanism by which this force transfer induces fusion pore formation is still unknown. The neuronal vesicular SNARE protein synaptobrevin 2 (syb2) is anchored in the vesicle membrane by a single C-terminal transmembrane (TM) helix. In coarse-grain molecular-dynamics simulations, self-assembly of the membrane occurred with the syb2 TM domain inserted, as expected from experimental data. The free-energy profile for the position of the syb2 membrane anchor in the membrane was determined using umbrella sampling. To predict the free-energy landscapes for a reaction pathway pulling syb2 toward the extravesicular side of the membrane, which is the direction of the force transfer from the SNARE complex, harmonic potentials were applied to the peptide in its unbiased position, pulling it toward new biased equilibrium positions. Application of piconewton forces to the extravesicular end of the TM helix in the simulation detached the synaptobrevin C-terminus from the vesicle's inner-leaflet lipid headgroups and pulled it deeper into the membrane. This C-terminal movement was facilitated and hindered by specific mutations in parallel with experimentally observed facilitation and inhibition of fusion. Direct application of such forces to the intravesicular end of the TM domain resulted in tilting motion of the TM domain through the membrane with an activation energy of ∼70 kJ/mol. The results suggest a mechanism whereby fusion pore formation is induced by movement of the charged syb2 C-terminus within the membrane in response to pulling and tilting forces generated by C-terminal zippering of the SNARE complex. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

The properties of RE-TM magneto-optical films

NASA Astrophysics Data System (ADS)

Lee, Z. Y.; Miao, X. S.; Zhu, P.; Hu, Y. S.; Wan, D. F.; Dai, D. W.; Chen, S. B.; Lin, G. Q.

1992-09-01

In this paper, the magnetic, magneto-optical and galvonomagnetic properties, and their temperature dependence for LRE-TM SmCo, SmCoDy and HRE-TM TbFeCo magneto-optical films as high density recording media prepared by rf magnetron sputtering or evaporation are reported. By adding Dy to SmCo thin film, the SmCoDy thin film is more suitable for magneto-optical recording, its domain size being below 0.63 μm. The Kerr enhancement and corrosion protective effects of AIN and AlSiN for optimum design of the multi-layer structure of magneto-optical disk are described. The instruments of measuring the magneto-optical Kerr effect and magneto-optical recording domain characteristics of thin films are reviewed.

Evolutionary analysis of a novel zinc ribbon in the N-terminal region of threonine synthase.

PubMed

Kaur, Gurmeet; Subramanian, Srikrishna

2017-10-18

Threonine synthase (TS) catalyzes the terminal reaction in the biosynthetic pathway of threonine and requires pyridoxal phosphate as a cofactor. TSs share a common catalytic domain with other fold type II PALP dependent enzymes. TSs are broadly grouped into two classes based on their sequence, quaternary structure, and enzyme regulation. We report the presence of a novel zinc ribbon domain in the N-terminal region preceding the catalytic core in TS. The zinc ribbon domain is present in TSs belonging to both classes. Our sequence analysis reveals that archaeal TSs possess all zinc chelating residues to bind a metal ion that are lacking in the structurally characterized homologs. Phylogenetic analysis suggests that TSs with an N-terminal zinc ribbon likely represents the ancestral state of the enzyme while TSs without a zinc ribbon must have diverged later in specific lineages. The zinc ribbon and its N- and C-terminal extensions are important for enzyme stability, activity and regulation. It is likely that the zinc ribbon domain is involved in higher order oligomerization or mediating interactions with other biomolecules leading to formation of larger metabolic complexes.

Structure and Function of the Sterol Carrier Protein-2 N-Terminal Presequence†

PubMed Central

Martin, Gregory G.; Hostetler, Heather A.; McIntosh, Avery L.; Tichy, Shane E.; Williams, Brad J.; Russell, David H.; Berg, Jeremy M.; Spencer, Thomas A.; Ball, Judith; Kier, Ann B.; Schroeder, Friedhelm

2008-01-01

Although sterol carrier protein-2 (SCP-2) is encoded as a precursor protein (proSCP-2), little is known regarding the structure and function of the 20-amino acid N-terminal presequence. As shown herein, the presequence contains significant secondary structure and alters SCP-2: (i) secondary structure (CD), (ii) tertiary structure (aqueous exposure of Trp shown by UV absorbance, fluorescence, fluorescence quenching), (iii) ligand binding site [Trp response to ligands, peptide cross-linked by photoactivatable free cholesterol (FCBP)], (iv) selectivity for interaction with anionic phospholipid-rich membranes, (v) interaction with a peroxisomal import protein [FRET studies of Pex5p(C) binding], the N-terminal presequence increased SCP-2’s affinity for Pex5p(C) by 10-fold, and (vi) intracellular targeting in living and fixed cells (confocal microscopy). Nearly 5-fold more SCP-2 than proSCP-2 colocalized with plasma membrane lipid rafts/caveolae (AF488-CTB), 2.8-fold more SCP-2 than proSCP-2 colocalized with a mitochondrial marker (Mitotracker), but nearly 2-fold less SCP-2 than proSCP-2 colocalized with peroxisomes (AF488-antibody to PMP70). These data indicate the importance of the N-terminal presequence in regulating SCP-2 structure, cholesterol localization within the ligand binding site, membrane association, and, potentially, intracellular targeting. PMID:18465878

Beyond Anchoring: the Expanding Role of the Hendra Virus Fusion Protein Transmembrane Domain in Protein Folding, Stability, and Function

PubMed Central

Smith, Everett Clinton; Culler, Megan R.; Hellman, Lance M.; Fried, Michael G.; Creamer, Trevor P.

2012-01-01

While work with viral fusion proteins has demonstrated that the transmembrane domain (TMD) can affect protein folding, stability, and membrane fusion promotion, the mechanism(s) remains poorly understood. TMDs could play a role in fusion promotion through direct TMD-TMD interactions, and we have recently shown that isolated TMDs from three paramyxovirus fusion (F) proteins interact as trimers using sedimentation equilibrium (SE) analysis (E. C. Smith, et al., submitted for publication). Immediately N-terminal to the TMD is heptad repeat B (HRB), which plays critical roles in fusion. Interestingly, addition of HRB decreased the stability of the trimeric TMD-TMD interactions. This result, combined with previous findings that HRB forms a trimeric coiled coil in the prefusion form of the whole protein though HRB peptides fail to stably associate in isolation, suggests that the trimeric TMD-TMD interactions work in concert with elements in the F ectodomain head to stabilize a weak HRB interaction. Thus, changes in TMD-TMD interactions could be important in regulating F triggering and refolding. Alanine insertions between the TMD and HRB demonstrated that spacing between these two regions is important for protein stability while not affecting TMD-TMD interactions. Additional mutagenesis of the C-terminal end of the TMD suggests that β-branched residues within the TMD play a role in membrane fusion, potentially through modulation of TMD-TMD interactions. Our results support a model whereby the C-terminal end of the Hendra virus F TMD is an important regulator of TMD-TMD interactions and show that these interactions help hold HRB in place prior to the triggering of membrane fusion. PMID:22238302

The Topology of the l-Arginine Exporter ArgO Conforms to an Nin-Cout Configuration in Escherichia coli: Requirement for the Cytoplasmic N-Terminal Domain, Functional Helical Interactions, and an Aspartate Pair for ArgO Function

PubMed Central

Pathania, Amit; Gupta, Arvind Kumar; Dubey, Swati; Gopal, Balasubramanian

2016-01-01

ABSTRACT ArgO and LysE are members of the LysE family of exporter proteins and ordinarily mediate the export of l-arginine (Arg) in Escherichia coli and l-lysine (Lys) and Arg in Corynebacterium glutamicum, respectively. Under certain conditions, ArgO also mediates Lys export. To delineate the arrangement of ArgO in the cytoplasmic membrane of E. coli, we have employed a combination of cysteine accessibility in situ, alkaline phosphatase fusion reporters, and protein modeling to arrive at a topological model of ArgO. Our studies indicate that ArgO assumes an Nin-Cout configuration, potentially forming a five-transmembrane helix bundle flanked by a cytoplasmic N-terminal domain (NTD) comprising roughly its first 38 to 43 amino acyl residues and a short periplasmic C-terminal region (CTR). Mutagenesis studies indicate that the CTR, but not the NTD, is dispensable for ArgO function in vivo and that a pair of conserved aspartate residues, located near the opposing edges of the cytoplasmic membrane, may play a pivotal role in facilitating transmembrane Arg flux. Additional studies on amino acid substitutions that impair ArgO function in vivo and their derivatives bearing compensatory amino acid alterations indicate a role for intramolecular interactions in the Arg export mechanism, and some interactions are corroborated by normal-mode analyses. Lastly, our studies suggest that ArgO may exist as a monomer in vivo, thus highlighting the requirement for intramolecular interactions in ArgO, as opposed to interactions across multiple ArgO monomers, in the formation of an Arg-translocating conduit. IMPORTANCE The orthologous proteins LysE of C. glutamicum and ArgO of E. coli function as exporters of the basic amino acids l-arginine and l-lysine and the basic amino acid l-arginine, respectively, and LysE can functionally substitute for ArgO when expressed in E. coli. Notwithstanding this functional equivalence, studies reported here show that ArgO possesses a membrane topology

TM triple-mode microwave filter

NASA Astrophysics Data System (ADS)

Lai, S.-L.; Lin, W.-G.

1990-12-01

A novel realization of triple-mode six-pole microwave filters that use only TM modes is presented. The application involves TM triple degeneracies in cylindrical cavities using triple-mode elliptic function filter synthesis. Experimental results are reported.

Wnt transmembrane signaling and long-term spatial memory

PubMed Central

Tabatadze, Nino; Tomas, Caroline; McGonigal, Rhona; Lin, Brian; Schook, Andrew; Routtenberg, Aryeh

2011-01-01

Transmembrane signaling mechanisms are critical for regulating the plasticity of neuronal connections underlying the establishment of long-lasting memory (e.g., Linden and Routtenberg, 1989, Brain Res Rev. 14: 279–296; Sossin, 1996, Trends Neurosci 19: 215–218; Mayr and Montminy, 2001, Nat Rev Mol Cell Biol. 2: 599–609; Chen et al., 2011, Nature 469: 491–497). One signaling mechanism that has received surprisingly little attention in this regard is the well-known Wnt transmembrane signaling pathway even though this pathway in the adult plays a significant role, for example, in postsynaptic dendritic spine morphogenesis and presynaptic terminal neurotransmitter release (Inestrosa and Arenas, 2010, Nature Rev Neurosci 11: 77–86). The present report now provides the first evidence of Wnt signaling in spatial information storage processes. Importantly, this Wnt participation is specific and selective. Thus, spatial, but not cued, learning in a water maze selectively elevates the levels in hippocampus of Wnt 7 and Wnt 5a, but not the Wnt 3 isoform, indicating behavioral selectivity and isoform specificity. Wnt 7 elevation is subfield-specific: granule cells show an increase with no detectable change in CA3 neurons. Wnt 7 elevation is temporally specific: increased Wnt signaling is not observed during training, but is seen 7 days and, unexpectedly, 30 days later. If the Wnt elevation after learning is activity-dependent, then it may be possible to model this effect in primary hippocampal neurons in culture. Here we evaluate the consequence of potassium or glutamate depolarization on Wnt signaling. This represents, to our knowledge, the first demonstration of an activation-dependent elevation of Wnt levels. Additionally, the novel finding emerged of an increased number of Wnt-stained puncta in neuritis suggestive of trafficking from the cell body to neuronal processes, probably dendrites. It is proposed that Wnt signaling pathways, both canonical and non

Ribonucleocapsid Formation of SARS-COV Through Molecular Action of the N-Terminal Domain of N Protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saikatendu, K.S.; Joseph, J.S.; Subramanian, V.

Conserved amongst all coronaviruses are four structural proteins, the matrix (M), small envelope (E) and spike (S) that are embedded in the viral membrane and the nucleocapsid phosphoprotein (N), which exists in a ribonucleoprotein complex in their lumen. The N terminal domain of coronaviral N proteins (N-NTD) provides a scaffold for RNA binding while the C-terminal domain (N-CTD) mainly acts as oligomerization modules during assembly. The C-terminus of N protein anchors it to the viral membrane by associating with M protein. We characterized the structures of N-NTD from severe acute respiratory syndrome coronavirus (SARS-CoV) in two crystal forms, at 1.17Amore » (monoclinic) and 1.85 A (cubic) respectively, solved by molecular replacement using the homologous avian infectious bronchitis virus (IBV) structure. Flexible loops in the solution structure of SARS-CoV N-NTD are now shown to be well ordered around the beta-sheet core. The functionally important positively charged beta-hairpin protrudes out of the core and is oriented similar to that in the IBV N-NTD and is involved in crystal packing in the monoclinic form. In the cubic form, the monomers form trimeric units that stack in a helical array. Comparison of crystal packing of SARS-CoV and IBV N-NTDs suggest a common mode of RNA recognition, but probably associate differently in vivo during the formation of the ribonucleoprotein complex. Electrostatic potential distribution on the surface of homology models of related coronaviral N-NTDs hints that they employ different modes of both RNA recognition as well as oligomeric assembly, perhaps explaining why their nucleocapsids have different morphologies.« less

Protection against β-amyloid neurotoxicity by a non-toxic endogenous N-terminal β-amyloid fragment and its active hexapeptide core sequence.

PubMed

Forest, Kelly H; Alfulaij, Naghum; Arora, Komal; Taketa, Ruth; Sherrin, Tessi; Todorovic, Cedomir; Lawrence, James L M; Yoshikawa, Gene T; Ng, Ho-Leung; Hruby, Victor J; Nichols, Robert A

2018-01-01

High levels (μM) of beta amyloid (Aβ) oligomers are known to trigger neurotoxic effects, leading to synaptic impairment, behavioral deficits, and apoptotic cell death. The hydrophobic C-terminal domain of Aβ, together with sequences critical for oligomer formation, is essential for this neurotoxicity. However, Aβ at low levels (pM-nM) has been shown to function as a positive neuromodulator and this activity resides in the hydrophilic N-terminal domain of Aβ. An N-terminal Aβ fragment (1-15/16), found in cerebrospinal fluid, was also shown to be a highly active neuromodulator and to reverse Aβ-induced impairments of long-term potentiation. Here, we show the impact of this N-terminal Aβ fragment and a shorter hexapeptide core sequence in the Aβ fragment (Aβcore: 10-15) to protect or reverse Aβ-induced neuronal toxicity, fear memory deficits and apoptotic death. The neuroprotective effects of the N-terminal Aβ fragment and Aβcore on Aβ-induced changes in mitochondrial function, oxidative stress, and apoptotic neuronal death were demonstrated via mitochondrial membrane potential, live reactive oxygen species, DNA fragmentation and cell survival assays using a model neuroblastoma cell line (differentiated NG108-15) and mouse hippocampal neuron cultures. The protective action of the N-terminal Aβ fragment and Aβcore against spatial memory processing deficits in amyloid precursor protein/PSEN1 (5XFAD) mice was demonstrated in contextual fear conditioning. Stabilized derivatives of the N-terminal Aβcore were also shown to be fully protective against Aβ-triggered oxidative stress. Together, these findings indicate an endogenous neuroprotective role for the N-terminal Aβ fragment, while active stabilized N-terminal Aβcore derivatives offer the potential for therapeutic application. © 2017 International Society for Neurochemistry.

Decisions Regarding Pregnancy Termination Due to β-Thalassemia Major: a Mixed-Methods Study in Sistan and Baluchestan, Iran.

PubMed

Moudi, Zahra; Miri-Moghaddam, Ebrahim

2017-06-01

In the present study, an embedded design was applied in order to conduct a one-year cross-sectional audit of chorionic villus sampling (CVS) and foetal outcomes affected by β-thalassemia major (β-TM) in a prenatal diagnosis (PND) setting. In addition, we explored the decisions regarding pregnancy termination among women whose pregnancy (or child) was affected by β-TM. In the quantitative phase, the available data in the clients' medical records were analysed, while the qualitative phase was performed using a grounded theory method. Interviews were performed with nine pregnant women who had decided against pregnancy termination despite positive CVS results, 11 mothers who had admitted their child to the thalassemia ward for blood transfusion, and 19 mothers who had received positive CVS results and had decided against pregnancy termination in the preceding year. Over one year, 18.6 % of women decided against pregnancy termination despite positive CVS results. Two main themes related to decisions against pregnancy termination emerged from the qualitative data: 1) Cognitive factors (questioning the reliability of the tests or doubts about the accuracy of the results, understanding disease recurrence, curability, perceived severity of the disease, and lack of "real-life experiences"); and 2) Sociocultural responsiveness (family opposition, responsibility before God, and self-responsiveness). All of the mentioned factors could intensify fear of abortion in couples due to possible regret, and encourage a decision against pregnancy termination.

Temperature dependence of LRE-HRE-TM thin films

NASA Astrophysics Data System (ADS)

Li, Zuoyi; Cheng, Xiaomin; Lin, Gengqi; Li, Zhen; Huang, Zhixin; Jin, Fang; Wang, Xianran; Yang, Xiaofei

2003-04-01

Temperature dependence of the properties of RE-TM thin films is very important for MO recording. In this paper, we studied the temperature dependence of the magnetic and magneto-optical properties of the amorphous LRE-HRE-TM single layer thin films and LRE-HRE-TM/HRE-TM couple-bilayered thin films. For LRE-HRE-TM single layer thin films, the temperature dependence of the magnetization was investigated by using the mean field theory. The experimental and theoretical results matched very well. With the LRE substitution in HRE-TM thin film, the compensation temperature Tcomp decreased and the curie temperature Tc remained unchanged. Kerr rotation angle became larger and the saturation magnetization Ms at room temperature increased. For LRE-HRE-TM/HRE-TM couple-bilayered thin films, comparisons of the temperature dependences of the coercivities and Kerr rotation angles were made between isolated sublayers and couple-bilayered thin film.

Automated Tow Placed LaRC(TM)-PETI-5 Composites

NASA Technical Reports Server (NTRS)

Hou, T. H.; Belvin, H. L.; Johnston, N. J.

2001-01-01

LaRC(TM)-PETI-5 is a PhenylEthynyl-Terminated Imide resin developed at NASA Langley Research Center (LaRC) during the 1990s. It offers a combination of attractive composite and adhesive properties. IM7/LaRC(TM)-PETI-5 composites exhibit thermal and thermo-oxidative stability typical of polyimides, superior chemical resistance and processability, excellent mechanical properties, toughness and damage tolerance. It was selected for study in the High Speed Research program aimed at developing technologies for a future supersonic aircraft, the High Speed Civil Transport, with a projected life span of 60 000 h at a cruise speed up to Mach 2.4. Robust autoclave processing cycles for LaRC(TM)-PETI-5 composites have been thoroughly designed and demonstrated, which involved hand lay-up of solvent-ladened 'wet' prepregs. However, this type of processing is not only costly but also environmentally unfriendly. Volatile management and shrinkage could become serious problems in the fabrication of large complex airframe structural subcomponents. Robotic tow placement technology utilizing 'dry' material forms represents a new fabrication process which overcomes these deficiencies. This work evaluates and compares mechanical properties of composites fabricated by heated head automated tow placement (dry process) with those obtained by hand lay-up/autoclave fabrication (wet process). Thermal and rheological properties of the robotically as-placed uncured composites were measured. A post-cure cycle was designed due to the requirement of the PETI-5 resin for a 370 C/1 h hold to reach full cure, conditions which cannot be duplicated during heated head robotic placement. Mechanical properties such as 0 degree flexural strength and modulus, open hole tensile and compressive strength and moduli, reduced section compression dogbone compressive strength, and modified zippora-medium small (MZ-MS) tensile and compressive properties were obtained on the post-cured panels. These properties

Structure of the N-terminal fragment of Escherichia coli Lon protease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Mi; Basic Research Program, SAIC-Frederick, Frederick, MD 21702; Gustchina, Alla

2010-08-01

The medium-resolution structure of the N-terminal fragment of E. coli Lon protease shows that this part of the enzyme consists of two compact domains and a very long α-helix. The structure of a recombinant construct consisting of residues 1–245 of Escherichia coli Lon protease, the prototypical member of the A-type Lon family, is reported. This construct encompasses all or most of the N-terminal domain of the enzyme. The structure was solved by SeMet SAD to 2.6 Å resolution utilizing trigonal crystals that contained one molecule in the asymmetric unit. The molecule consists of two compact subdomains and a very longmore » C-terminal α-helix. The structure of the first subdomain (residues 1–117), which consists mostly of β-strands, is similar to that of the shorter fragment previously expressed and crystallized, whereas the second subdomain is almost entirely helical. The fold and spatial relationship of the two subdomains, with the exception of the C-terminal helix, closely resemble the structure of BPP1347, a 203-amino-acid protein of unknown function from Bordetella parapertussis, and more distantly several other proteins. It was not possible to refine the structure to satisfactory convergence; however, since almost all of the Se atoms could be located on the basis of their anomalous scattering the correctness of the overall structure is not in question. The structure reported here was also compared with the structures of the putative substrate-binding domains of several proteins, showing topological similarities that should help in defining the binding sites used by Lon substrates.« less

Lyme Disease and YouTube TM: A Cross-Sectional Study of Video Contents.

PubMed

Basch, Corey H; Mullican, Lindsay A; Boone, Kwanza D; Yin, Jingjing; Berdnik, Alyssa; Eremeeva, Marina E; Fung, Isaac Chun-Hai

2017-08-01

Lyme disease is the most common tick-borne disease. People seek health information on Lyme disease from YouTube TM videos. In this study, we investigated if the contents of Lyme disease-related YouTube TM videos varied by their sources. Most viewed English YouTube TM videos (n = 100) were identified and manually coded for contents and sources. Within the sample, 40 videos were consumer-generated, 31 were internet-based news, 16 were professional, and 13 were TV news. Compared with consumer-generated videos, TV news videos were more likely to mention celebrities (odds ratio [OR], 10.57; 95% confidence interval [CI], 2.13-52.58), prevention of Lyme disease through wearing protective clothing (OR, 5.63; 95% CI, 1.23-25.76), and spraying insecticides (OR, 7.71; 95% CI, 1.52-39.05). A majority of the most popular Lyme disease-related YouTube TM videos were not created by public health professionals. Responsible reporting and creative video-making facilitate Lyme disease education. Partnership with YouTube TM celebrities to co-develop educational videos may be a future direction.

Reduction of Caloric Intake Might Override the Prosteatotic Effects of the PNPLA3 p.I148M and TM6SF2 p.E167K Variants in Patients with Fatty Liver: Ultrasound-Based Prospective Study.

PubMed

Krawczyk, Marcin; Stachowska, Ewa; Milkiewicz, Piotr; Lammert, Frank; Milkiewicz, Malgorzata

2016-01-01

The adiponutrin (PNPLA3) p.I148M and transmembrane 6 superfamily member 2 (TM6SF2) p.E167K variants represent risk factors for non-alcoholic fatty liver disease (NAFLD). In this study, we assessed the effects of caloric restriction on liver phenotypes in NAFLD patients in relation to the PNPLA3 and TM6SF2 genotypes. We genotyped both variants in 143 individuals with NAFLD (55 females, age 18-74 years) and 180 controls (85 females, age 33-66 years). Liver steatosis was assessed using the ultrasound-based Hamaguchi score. A 4-month dietetic intervention, consisting of restriction of daily caloric intake without changes in physical activity, was performed. Both PNPLA3 (p = 0.002) and TM6SF2 (p = 0.041) variants were associated with NAFLD before intervention. Overall, 88 patients completed the intervention, which led to a significant decrease of steatosis, ALT activities, body mass index, hip and waist circumferences, and waist-hip ratio (all p < 0.0001). Hepatic steatosis and anthropometric traits improved significantly (p < 0.05) in carriers of either PNPLA3 or TM6SF2 risk genotype. The improvement of phenotypic traits, apart from WHR (p = 0.02), was not modified by the presence of PNPLA3 or TM6SF2 variants. The PNPLA3 and TM6SF2 polymorphisms are associated with NAFLD assessed by the Hamaguchi score. Neither PNPLA3 nor TM6SF2 risk alleles impair the response to dietetic intervention in NAFLD. © 2016 S. Karger AG, Basel.

Missense Mutations in the N-Terminal Domain of Human Phenylalanine Hydroxylase Interfere with Binding of Regulatory Phenylalanine

PubMed Central

Gjetting, Torben; Petersen, Marie; Guldberg, Per; Güttler, Flemming

2001-01-01

Hyperphenylalaninemia due to a deficiency of phenylalanine hydroxylase (PAH) is an autosomal recessive disorder caused by >400 mutations in the PAH gene. Recent work has suggested that the majority of PAH missense mutations impair enzyme activity by causing increased protein instability and aggregation. In this study, we describe an alternative mechanism by which some PAH mutations may render PAH defective. Database searches were used to identify regions in the N-terminal domain of PAH with homology to the regulatory domain of prephenate dehydratase (PDH), the rate-limiting enzyme in the bacterial phenylalanine biosynthesis pathway. Naturally occurring N-terminal PAH mutations are distributed in a nonrandom pattern and cluster within residues 46–48 (GAL) and 65–69 (IESRP), two motifs highly conserved in PDH. To examine whether N-terminal PAH mutations affect the ability of PAH to bind phenylalanine at the regulatory domain, wild-type and five mutant (G46S, A47V, T63P/H64N, I65T, and R68S) forms of the N-terminal domain (residues 2–120) of human PAH were expressed as fusion proteins in Escherichia coli. Binding studies showed that the wild-type form of this domain specifically binds phenylalanine, whereas all mutations abolished or significantly reduced this phenylalanine-binding capacity. Our data suggest that impairment of phenylalanine-mediated activation of PAH may be an important disease-causing mechanism of some N-terminal PAH mutations, which may explain some well-documented genotype-phenotype discrepancies in PAH deficiency. PMID:11326337

A novel calmodulin-regulated Ca2+-ATPase (ACA2) from Arabidopsis with an N-terminal autoinhibitory domain

NASA Technical Reports Server (NTRS)

Harper, J. F.; Hong, B.; Hwang, I.; Guo, H. Q.; Stoddard, R.; Huang, J. F.; Palmgren, M. G.; Sze, H.; Evans, M. L. (Principal Investigator)

1998-01-01

To study transporters involved in regulating intracellular Ca2+, we isolated a full-length cDNA encoding a Ca2+-ATPase from a model plant, Arabidopsis, and named it ACA2 (Arabidopsis Ca2+-ATPase, isoform 2). ACA2p is most similar to a "plasma membrane-type" Ca2+-ATPase, but is smaller (110 kDa), contains a unique N-terminal domain, and is missing a long C-terminal calmodulin-binding regulatory domain. In addition, ACA2p is localized to an endomembrane system and not the plasma membrane, as shown by aqueous-two phase fractionation of microsomal membranes. ACA2p was expressed in yeast as both a full-length protein (ACA2-1p) and an N-terminal truncation mutant (ACA2-2p; Delta residues 2-80). Only the truncation mutant restored the growth on Ca2+-depleted medium of a yeast mutant defective in both endogenous Ca2+ pumps, PMR1 and PMC1. Although basal Ca2+-ATPase activity of the full-length protein was low, it was stimulated 5-fold by calmodulin (50% activation around 30 nM). In contrast, the truncated pump was fully active and insensitive to calmodulin. A calmodulin-binding sequence was identified within the first 36 residues of the N-terminal domain, as shown by calmodulin gel overlays on fusion proteins. Thus, ACA2 encodes a novel calmodulin-regulated Ca2+-ATPase distinguished by a unique N-terminal regulatory domain and a non-plasma membrane localization.

Formation of pyroglutamic acid from N-terminal glutamic acid in immunoglobulin gamma antibodies.

PubMed

Chelius, Dirk; Jing, Kay; Lueras, Alexis; Rehder, Douglas S; Dillon, Thomas M; Vizel, Alona; Rajan, Rahul S; Li, Tiansheng; Treuheit, Michael J; Bondarenko, Pavel V

2006-04-01

The status of the N-terminus of proteins is important for amino acid sequencing by Edman degradation, protein identification by shotgun and top-down techniques, and to uncover biological functions, which may be associated with modifications. In this study, we investigated the pyroglutamic acid formation from N-terminal glutamic acid residues in recombinant monoclonal antibodies. Almost half the antibodies reported in the literature contain a glutamic acid residue at the N-terminus of the light or the heavy chain. Our reversed-phase high-performance liquid chromatography-mass spectrometry method could separate the pyroglutamic acid-containing light chains from the native light chains of reduced and alkylated recombinant monoclonal antibodies. Tryptic peptide mapping and tandem mass spectrometry of the reduced and alkylated proteins was used for the identification of the pyroglutamic acid. We identified the formation of pyroglutamic acid from N-terminal glutamic acid in the heavy chains and light chains of several antibodies, indicating that this nonenzymatic reaction does occur very commonly and can be detected after a few weeks of incubation at 37 and 45 degrees C. The rate of this reaction was measured in several aqueous buffers with different pH values, showing minimal formation of pyroglutamic acid at pH 6.2 and increased formation of pyroglutamic acid at pH 4 and pH 8. The half-life of the N-terminal glutamic acid was approximately 9 months in a pH 4.1 buffer at 45 degrees C. To our knowledge, we showed for the first time that glutamic acid residues located at the N-terminus of proteins undergo pyroglutamic acid formation in vitro.

Cryogenic-cooled Tm:SBN tunable laser

NASA Astrophysics Data System (ADS)

Švejkar, Richard; Šulc, Jan; Němec, Michal; Jelínková, Helena; Doroshenko, Maxim E.; Papashvili, Alexander G.; Batygov, Sergei H.; Osiko, Vyacheslav V.

2017-12-01

In this work the temperature dependence of spectroscopic and laser properties of new ac- tive medium Tm:SBN (Strontium-Barium Niobate, SrxBa1-xNb2O6, x = 0.61). The tested sample of Tm:SBN (2 wt. % of Tm2O3) appropriate for generation of laser radiation at 1.88 μm had plan-parallel polished faces without anti-reflection (thickness 6.65 mm). During spectroscopy and laser experiments the Tm:SBN was at- tached to temperature-controlled copper holder and was placed in a vacuum chamber. The transmission and emission spectra of Tm:SBN and the fluorescence decay time were measured depending on temperature range 80 - 350 K. The fluorescence decay time was measured to be 3.5 ms and 2.8 ms at 80 and 350 K, respectively. Longitudinal excitation of Tm:SBN was carried out by a fibre-coupled laser diode (pulse duration 10 ms, rep- etition rate 10 Hz, pump wavelength 793 nm). The laser resonator was hemispherical, 146 mm long, with flat pumping mirror (HR @1.8 - 2.1 μm) and spherical output coupler (r = 150 mm, R = 97.5 % @1.8 - 2.1 μm). The Tm:SBN laser properties were investigated at temperature range 80 - 300 K. The highest slope efficiency with respect to absorbed pumped power was 3 % at 80 K. The maximum output peak amplitude power was 0.12 W at 80 K, i.e. 3.2 times higher than it was measured at 200 K. Tunability of laser wavelength at 80 K in the range of 1827 - 1962 nm was obtained by using SiO2 birefringent filter. At 300 K, wavelength tunability reached 1859 - 1970 nm. Thus, the new Tm:SBN crystal can be an useful laser material in the region of 2 μm.

Synthesis of 3-iodoindoles by the Pd/Cu-catalyzed coupling of N,N-dialkyl-2-iodoanilines and terminal acetylenes, followed by electrophilic cyclization.

PubMed

Yue, Dawei; Yao, Tuanli; Larock, Richard C

2006-01-06

[reaction: see text] 3-Iodoindoles have been prepared in excellent yields by coupling terminal acetylenes with N,N-dialkyl-o-iodoanilines in the presence of a Pd/Cu catalyst, followed by an electrophilic cyclization of the resulting N,N-dialkyl-o-(1-alkynyl)anilines using I2 in CH2Cl2. Aryl-, vinylic-, alkyl-, and silyl-substituted terminal acetylenes undergo this process to produce excellent yields of 3-iodoindoles. The reactivity of the carbon-nitrogen bond cleavage during cyclization follows the following order: Me > n-Bu, Me > Ph, and cyclohexyl > Me. Subsequent palladium-catalyzed Sonogashira, Suzuki, and Heck reactions of the resulting 3-iodoindoles proceed smoothly in good yields.