RSM Based Optimization of Chemical and Enzymatic Transesterification of Palm Oil: Biodiesel Production and Assessment of Exhaust Emission Levels

PubMed Central

Mumtaz, Muhammad Waseem; Anwar, Farooq; Saari, Nazamid

2014-01-01

Current study presents RSM based optimized production of biodiesel from palm oil using chemical and enzymatic transesterification. The emission behavior of biodiesel and its blends, namely, POB-5, POB-20, POB-40, POB-50, POB-80, and POB-100 was examined using diesel engine (equipped with tube well). Optimized palm oil fatty acid methyl esters (POFAMEs) yields were depicted to be 47.6 ± 1.5, 92.7 ± 2.5, and 95.4 ± 2.0% for chemical transesterification catalyzed by NaOH, KOH, and NaOCH3, respectively, whereas for enzymatic transesterification reactions catalyzed by NOVOZYME-435 and A. n. lipase optimized biodiesel yields were 94.2 ± 3.1 and 62.8 ± 2.4%, respectively. Distinct decrease in particulate matter (PM) and carbon monoxide (CO) levels was experienced in exhaust emissions from engine operating on biodiesel blends POB-5, POB-20, POB-40, POB-50, POB-80, and POB-100 comparative to conventional petroleum diesel. Percentage change in CO and PM emissions for different biodiesel blends ranged from −2.1 to −68.7% and −6.2 to −58.4%, respectively, relative to conventional diesel, whereas an irregular trend was observed for NOx emissions. Only POB-5 and POB-20 showed notable reductions, whereas all other blends (POB-40 to POB-100) showed slight increase in NOx emission levels from 2.6 to 5.5% comparative to petroleum diesel. PMID:25162053

Structural and biochemical basis for ubiquitin ligase recruitment by arrestin-related domain-containing protein-3 (ARRDC3).

PubMed

Qi, Shiqian; O'Hayre, Morgan; Gutkind, J Silvio; Hurley, James H

2014-02-21

After protracted stimulation, the β2-adrenergic receptor and many other G-protein-coupled receptors are ubiquitinated and down-regulated. Arrestin-related domain-containing protein-3 (ARRDC3) has been proposed to recruit the ubiquitin ligase Nedd4 to the β2-adrenergic receptor. ARRDC3 contains two PPXY motifs that could potentially interact with any of the four WW domains of Nedd4. Here we dissect the interaction determinants. ARRDC3 PPXY-Nedd4 WW dissociation constants vary from unmeasurable to Kd = 3 μM for the third WW domain of Nedd4 binding to the first PPXY motif of ARRDC3. Structures of the uncomplexed and PPXY1-bound WW3 domain were determined at 1.1 and 1.7 Å resolution. The structures revealed conformational changes upon binding and the hydrogen bonding network in exquisite detail. Tight packing of ARRDC3 Val-352', part of a 310 helix at the C terminus of PPXY1, is important for high affinity binding to WW3. Although no single WW domain is strictly essential for the binding of Nedd4 and ARRDC3 expressed in HEK293 cells, high affinity binding of full-length ARRDC3 and Nedd4 is driven by the avid interaction of both PPXY motifs with either the WW2-WW3 or WW3-WW4 combinations, with Kd values as low as 300 nM.

Mechanism of mRNA-STAR domain interaction: Molecular dynamics simulations of Mammalian Quaking STAR protein.

PubMed

Sharma, Monika; Anirudh, C R

2017-10-03

STAR proteins are evolutionary conserved mRNA-binding proteins that post-transcriptionally regulate gene expression at all stages of RNA metabolism. These proteins possess conserved STAR domain that recognizes identical RNA regulatory elements as YUAAY. Recently reported crystal structures show that STAR domain is composed of N-terminal QUA1, K-homology domain (KH) and C-terminal QUA2, and mRNA binding is mediated by KH-QUA2 domain. Here, we present simulation studies done to investigate binding of mRNA to STAR protein, mammalian Quaking protein (QKI). We carried out conventional MD simulations of STAR domain in presence and absence of mRNA, and studied the impact of mRNA on the stability, dynamics and underlying allosteric mechanism of STAR domain. Our unbiased simulations results show that presence of mRNA stabilizes the overall STAR domain by reducing the structural deviations, correlating the 'within-domain' motions, and maintaining the native contacts information. Absence of mRNA not only influenced the essential modes of motion of STAR domain, but also affected the connectivity of networks within STAR domain. We further explored the dissociation of mRNA from STAR domain using umbrella sampling simulations, and the results suggest that mRNA binding to STAR domain occurs in multi-step: first conformational selection of mRNA backbone conformations, followed by induced fit mechanism as nucleobases interact with STAR domain.

Fourier transform coupled tryptophan scanning mutagenesis identifies a bending point on the lipid-exposed δM3 transmembrane domain of the Torpedo californica nicotinic acetylcholine receptor

PubMed Central

Caballero-Rivera, Daniel; Cruz-Nieves, Omar A; Oyola-Cintrón, Jessica; Torres-Núñez, David A; Otero-Cruz, José D

2011-01-01

The nicotinic acetylcholine receptor (nAChR) is a member of a family of ligand-gated ion channels that mediate diverse physiological functions, including fast synaptic transmission along the peripheral and central nervous systems. Several studies have made significant advances toward determining the structure and dynamics of the lipid-exposed domains of the nAChR. However, a high-resolution atomic structure of the nAChR still remains elusive. In this study, we extended the Fourier transform coupled tryptophan scanning mutagenesis (FT-TrpScanM) approach to gain insight into the secondary structure of the δM3 transmembrane domain of the Torpedo californica nAChR, to monitor conformational changes experienced by this domain during channel gating, and to identify which lipid-exposed positions are linked to the regulation of ion channel kinetics. The perturbations produced by periodic tryptophan substitutions along the δM3 transmembrane domain were characterized by two-electrode voltage clamp and 125I-labeled α-bungarotoxin binding assays. The periodicity profiles and Fourier transform spectra of this domain revealed similar helical structures for the closed- and open-channel states. However, changes in the oscillation patterns observed between positions Val-299 and Val-304 during transition between the closed- and open-channel states can be explained by the structural effects caused by the presence of a bending point introduced by a Thr-Gly motif at positions 300–301. The changes in periodicity and localization of residues between the closed-and open-channel states could indicate a structural transition between helix types in this segment of the domain. Overall, the data further demonstrate a functional link between the lipid-exposed transmembrane domain and the nAChR gating machinery. PMID:21785268

Hawking radiation from a Reisner-Nordström domain wall

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

Greenwood, Eric, E-mail: esg3@buffalo.edu

2010-01-01

We investigate the effect on the Hawking radiation given off during the time of collapse of a Reisner-Nordström domain wall. Using the functional Schrödinger formalism we are able to probe the time-dependent regime, which is out of the reach of the standard approximations like the Bogolyubov method. We calculate the occupation number of particles for a scalar field and complex scalar field. We demonstrate that the particles from the scalar field are unaffected by the charge of the Reisner-Nordström domain wall, as is expected since the scalar field doesn't carry any charge, which would couple to the charge of themore » Reisner-Nordström domain wall. Here the situation effectively reduces to the uncharged case, a spherically symmetric domain wall. To take the charge into account, we consider the complex scalar field which represents charged particles and anti-particles. Here investigate two different cases, first the non-extremal case and second the extremal case. In the non-extremal case we demonstrate that when the particle (anti-particle) carries charge opposite to that of the domain wall, the occupation number becomes suppressed during late times of the collapse. Therefore the dominate occupation number is when the particle (anti-particle) carries the same charge as the domain wall, as expected due to the Coulomb potential carried by the domain walls. In the extremal case we demonstrate that as time increases the temperature of the radiation decreases until when the domain wall reaches the horizon and the temperature then goes to zero. This is in agreement with the Hawking temperature for charged black holes.« less

Anti-staphylococcal activities of lysostaphin and LytM catalytic domain

PubMed Central

2012-01-01

Background Lysostaphin and the catalytic domain of LytM cleave pentaglycine crossbridges of Staphylococcus aureus peptidoglycan. The bacteriocin lysostaphin is secreted by Staphylococcus simulans biovar staphylolyticus and directed against the cell walls of competing S. aureus. LytM is produced by S. aureus as a latent autolysin and can be activated in vitro by the removal of an N-terminal domain and occluding region. Results We compared the efficacies of the lysostaphin and LytM catalytic domains using a newly developed model of chronic S. aureus infected eczema. Lysostaphin was effective, like in other models. In contrast, LytM was not significantly better than control. The different treatment outcomes could be correlated with in vitro properties of the proteins, including proteolytic stability, affinity to cell wall components other than peptidoglycan, and sensitivity to the ionic milieu. Conclusions Although lysostaphin and LytM cleave the same peptide bond in the peptidoglycan, the two enzymes have very different environmental requirements what is reflected in their contrasting performance in mouse eczema model. PMID:22672475

Pinning, rotation, and metastability of BiFeO 3 cycloidal domains in a magnetic field

DOE PAGES

Fishman, Randy S.

2018-01-03

Earlier models for the room-temperature multiferroic BiFeO 3 implicitly assumed that a very strong anisotropy restricts the domain wave vectors q to the threefold-symmetric axis normal to the static polarization P. However, recent measurements demonstrate that the domain wave vectors q rotate within the hexagonal plane normal to P away from the magnetic field orientation m. In this paper, we show that the previously neglected threefold anisotropy K 3 restricts the wave vectors to lie along the threefold axis in zero field. Taking m to lie along a threefold axis, the domain with q parallel to m remains metastable belowmore » B c1≈7 T. Due to the pinning of domains by nonmagnetic impurities, the wave vectors of the other two domains start to rotate away from m above 5.6 T, when the component of the torque τ=M×B along P exceeds a threshold value τ pin. Since τ=0 when m⊥q, the wave vectors of those domains never become completely perpendicular to the magnetic field. Our results explain recent measurements of the critical field as a function of field orientation, small-angle neutron scattering measurements of the wave vectors, as well as spectroscopic measurements with m along a threefold axis. Finally, the model developed in this paper also explains how the three multiferroic domains of BiFeO 3 for a fixed P can be manipulated by a magnetic field.« less

Pinning, rotation, and metastability of BiFeO 3 cycloidal domains in a magnetic field

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

Fishman, Randy S.

Earlier models for the room-temperature multiferroic BiFeO 3 implicitly assumed that a very strong anisotropy restricts the domain wave vectors q to the threefold-symmetric axis normal to the static polarization P. However, recent measurements demonstrate that the domain wave vectors q rotate within the hexagonal plane normal to P away from the magnetic field orientation m. In this paper, we show that the previously neglected threefold anisotropy K 3 restricts the wave vectors to lie along the threefold axis in zero field. Taking m to lie along a threefold axis, the domain with q parallel to m remains metastable belowmore » B c1≈7 T. Due to the pinning of domains by nonmagnetic impurities, the wave vectors of the other two domains start to rotate away from m above 5.6 T, when the component of the torque τ=M×B along P exceeds a threshold value τ pin. Since τ=0 when m⊥q, the wave vectors of those domains never become completely perpendicular to the magnetic field. Our results explain recent measurements of the critical field as a function of field orientation, small-angle neutron scattering measurements of the wave vectors, as well as spectroscopic measurements with m along a threefold axis. Finally, the model developed in this paper also explains how the three multiferroic domains of BiFeO 3 for a fixed P can be manipulated by a magnetic field.« less

M-and-C Domain Map Maker: an environment complimenting MDE with M-and-C knowledge and ensuring solution completeness

NASA Astrophysics Data System (ADS)

Patwari, Puneet; Choudhury, Subhrojyoti R.; Banerjee, Amar; Swaminathan, N.; Pandey, Shreya

2016-07-01

Model Driven Engineering (MDE) as a key driver to reduce development cost of M&C systems is beginning to find acceptance across scientific instruments such as Radio Telescopes and Nuclear Reactors. Such projects are adopting it to reduce time to integrate, test and simulate their individual controllers and increase reusability and traceability in the process. The creation and maintenance of models is still a significant challenge to realizing MDE benefits. Creating domain-specific modelling environments reduces the barriers, and we have been working along these lines, creating a domain-specific language and environment based on an M&C knowledge model. However, large projects involve several such domains, and there is still a need to interconnect the domain models, in order to ensure modelling completeness. This paper presents a knowledge-centric approach to doing that, by creating a generic system model that underlies the individual domain knowledge models. We present our vision for M&C Domain Map Maker, a set of processes and tools that enables explication of domain knowledge in terms of domain models with mutual consistency relationships to aid MDE.

Role of FAST Kinase Domains 3 (FASTKD3) in Post-transcriptional Regulation of Mitochondrial Gene Expression*

PubMed Central

Boehm, Erik; Zornoza, María; Jourdain, Alexis A.; Delmiro Magdalena, Aitor; García-Consuegra, Inés; Torres Merino, Rebeca; Orduña, Antonio; Martín, Miguel A.; Martinou, Jean-Claude; De la Fuente, Miguel A.; Simarro, María

2016-01-01

The Fas-activated serine/threonine kinase (FASTK) family of proteins has recently emerged as a central regulator of mitochondrial gene expression through the function of an unusual RNA-binding domain named RAP (for RNA-binding domain abundant in Apicomplexans), shared by all six members of the family. Here we describe the role of one of the less characterized members, FASTKD3, in mitochondrial RNA metabolism. First, we show that, in contrast to FASTK, FASTKD2, and FASTKD5, FASTKD3 does not localize in mitochondrial RNA granules, which are sites of processing and maturation of mtRNAs and ribosome biogenesis. Second, we generated FASTKD3 homozygous knock-out cell lines by homologous recombination and observed that the absence of FASTKD3 resulted in increased steady-state levels and half-lives of a subset of mature mitochondrial mRNAs: ND2, ND3, CYTB, COX2, and ATP8/6. No aberrant processing of RNA precursors was observed. Rescue experiments demonstrated that RAP domain is required for FASTKD3 function in mRNA stability. Besides, we describe that FASTKD3 is required for efficient COX1 mRNA translation without altering mRNA levels, which results in a decrease in the steady-state levels of COX1 protein. This finding is associated with reduced mitochondrial complex IV assembly and activity. Our observations suggest that the function of this family of proteins goes beyond RNA processing and ribosome assembly and includes RNA stability and translation regulation within mitochondria. PMID:27789713

Conformational changes in the expression domain of the Escherichia coli thiM riboswitch

PubMed Central

Rentmeister, Andrea; Mayer, Günter; Kuhn, Nicole; Famulok, Michael

2007-01-01

The thiM riboswitch contains an aptamer domain that adaptively binds the coenzyme thiamine pyrophosphate (TPP). The binding of TPP to the aptamer domain induces structural rearrangements that are relayed to a second domain, the so-called expression domain, thereby interfering with gene expression. The recently solved crystal structures of the aptamer domains of the thiM riboswitches in complex with TPP revealed how TPP stabilizes secondary and tertiary structures in the RNA ligand complex. To understand the global modes of reorganization between the two domains upon metabolite binding the structure of the entire riboswitch in presence and absence of TPP needs to be determined. Here we report the secondary structure of the entire thiM riboswitch from Escherichia coli in its TPP-free form and its transition into the TPP-bound variant, thereby depicting domains of the riboswitch that serve as communication links between the aptamer and the expression domain. Furthermore, structural probing provides an explanation for the lack of genetic control exerted by a riboswitch variant with mutations in the expression domain that still binds TPP. PMID:17517779

Crystallized N-terminal domain of influenza virus matrix protein M1 and method of determining and using same

NASA Technical Reports Server (NTRS)

Luo, Ming (Inventor); Sha, Bingdong (Inventor)

2000-01-01

The matrix protein, M1, of influenza virus strain A/PR/8/34 has been purified from virions and crystallized. The crystals consist of a stable fragment (18 Kd) of the M1 protein. X-ray diffraction studies indicated that the crystals have a space group of P3.sub.t 21 or P3.sub.2 21. Vm calculations showed that there are two monomers in an asymmetric unit. A crystallized N-terminal domain of M1, wherein the N-terminal domain of M1 is crystallized such that the three dimensional structure of the crystallized N-terminal domain of M1 can be determined to a resolution of about 2.1 .ANG. or better, and wherein the three dimensional structure of the uncrystallized N-terminal domain of M1 cannot be determined to a resolution of about 2.1 .ANG. or better. A method of purifying M1 and a method of crystallizing M1. A method of using the three-dimensional crystal structure of M1 to screen for antiviral, influenza virus treating or preventing compounds. A method of using the three-dimensional crystal structure of M1 to screen for improved binding to or inhibition of influenza virus M1. The use of the three-dimensional crystal structure of the M1 protein of influenza virus in the manufacture of an inhibitor of influenza virus M1. The use of the three-dimensional crystal structure of the M1 protein of influenza virus in the screening of candidates for inhibition of influenza virus M1.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of the DDX3 RNA helicase domain

PubMed Central

Rodamilans, Bernardo; Montoya, Guillermo

2007-01-01

DDX3 is a human RNA helicase that is involved in RNA processing and important human diseases. This enzyme belongs to the DEAD-box protein family, the members of which are characterized by the presence of nine conserved motifs including the Asp-Glu-Ala-Asp motif that defines the family. DDX3 has two distinct domains: an ATP-binding domain in the central region of the protein and a helicase domain in the carboxy-terminal region. The helicase domain of DDX3 was cloned and overexpressed in Escherichia coli. Crystallization experiments yielded crystals that were suitable for X-ray diffraction analysis. The final crystallization conditions were a reservoir solution consisting of 2 M ammonium sulfate, 0.1 M imidazole pH 6.4 plus 5 mM spermine tetrahydrochloride and a protein solution containing 10 mM HEPES, 500 mM ammonium sulfate pH 8.0. The crystals of the helicase domain belong to the monoclinic space group P21, with unit-cell parameters a = 43.85, b = 60.72, c = 88.39 Å, α = γ = 90, β = 101.02°, and contained three molecules per asymmetric unit. These crystals diffracted to a resolution limit of 2.2 Å using synchrotron radiation at the European Synchrotron Radiation Facility (ESRF) and the Swiss Light Source (SLS). PMID:17401195

Expression, purification, crystallization and preliminary X-ray diffraction analysis of the DDX3 RNA helicase domain.

PubMed

Rodamilans, Bernardo; Montoya, Guillermo

2007-04-01

DDX3 is a human RNA helicase that is involved in RNA processing and important human diseases. This enzyme belongs to the DEAD-box protein family, the members of which are characterized by the presence of nine conserved motifs including the Asp-Glu-Ala-Asp motif that defines the family. DDX3 has two distinct domains: an ATP-binding domain in the central region of the protein and a helicase domain in the carboxy-terminal region. The helicase domain of DDX3 was cloned and overexpressed in Escherichia coli. Crystallization experiments yielded crystals that were suitable for X-ray diffraction analysis. The final crystallization conditions were a reservoir solution consisting of 2 M ammonium sulfate, 0.1 M imidazole pH 6.4 plus 5 mM spermine tetrahydrochloride and a protein solution containing 10 mM HEPES, 500 mM ammonium sulfate pH 8.0. The crystals of the helicase domain belong to the monoclinic space group P2(1), with unit-cell parameters a = 43.85, b = 60.72, c = 88.39 A, alpha = gamma = 90, beta = 101.02 degrees , and contained three molecules per asymmetric unit. These crystals diffracted to a resolution limit of 2.2 A using synchrotron radiation at the European Synchrotron Radiation Facility (ESRF) and the Swiss Light Source (SLS).

Matrin 3 binds and stabilizes mRNA.

PubMed

Salton, Maayan; Elkon, Ran; Borodina, Tatiana; Davydov, Aleksey; Yaspo, Marie-Laure; Halperin, Eran; Shiloh, Yosef

2011-01-01

Matrin 3 (MATR3) is a highly conserved, inner nuclear matrix protein with two zinc finger domains and two RNA recognition motifs (RRM), whose function is largely unknown. Recently we found MATR3 to be phosphorylated by the protein kinase ATM, which activates the cellular response to double strand breaks in the DNA. Here, we show that MATR3 interacts in an RNA-dependent manner with several proteins with established roles in RNA processing, and maintains its interaction with RNA via its RRM2 domain. Deep sequencing of the bound RNA (RIP-seq) identified several small noncoding RNA species. Using microarray analysis to explore MATR3's role in transcription, we identified 77 transcripts whose amounts depended on the presence of MATR3. We validated this finding with nine transcripts which were also bound to the MATR3 complex. Finally, we demonstrated the importance of MATR3 for maintaining the stability of several of these mRNA species and conclude that it has a role in mRNA stabilization. The data suggest that the cellular level of MATR3, known to be highly regulated, modulates the stability of a group of gene transcripts.

Matrin 3 Binds and Stabilizes mRNA

PubMed Central

Salton, Maayan; Elkon, Ran; Borodina, Tatiana; Davydov, Aleksey; Yaspo, Marie-Laure; Halperin, Eran; Shiloh, Yosef

2011-01-01

Matrin 3 (MATR3) is a highly conserved, inner nuclear matrix protein with two zinc finger domains and two RNA recognition motifs (RRM), whose function is largely unknown. Recently we found MATR3 to be phosphorylated by the protein kinase ATM, which activates the cellular response to double strand breaks in the DNA. Here, we show that MATR3 interacts in an RNA-dependent manner with several proteins with established roles in RNA processing, and maintains its interaction with RNA via its RRM2 domain. Deep sequencing of the bound RNA (RIP-seq) identified several small noncoding RNA species. Using microarray analysis to explore MATR3′s role in transcription, we identified 77 transcripts whose amounts depended on the presence of MATR3. We validated this finding with nine transcripts which were also bound to the MATR3 complex. Finally, we demonstrated the importance of MATR3 for maintaining the stability of several of these mRNA species and conclude that it has a role in mRNA stabilization. The data suggest that the cellular level of MATR3, known to be highly regulated, modulates the stability of a group of gene transcripts. PMID:21858232

Reactor Dosimetry Applications Using RAPTOR-M3G:. a New Parallel 3-D Radiation Transport Code

NASA Astrophysics Data System (ADS)

Longoni, Gianluca; Anderson, Stanwood L.

2009-08-01

The numerical solution of the Linearized Boltzmann Equation (LBE) via the Discrete Ordinates method (SN) requires extensive computational resources for large 3-D neutron and gamma transport applications due to the concurrent discretization of the angular, spatial, and energy domains. This paper will discuss the development RAPTOR-M3G (RApid Parallel Transport Of Radiation - Multiple 3D Geometries), a new 3-D parallel radiation transport code, and its application to the calculation of ex-vessel neutron dosimetry responses in the cavity of a commercial 2-loop Pressurized Water Reactor (PWR). RAPTOR-M3G is based domain decomposition algorithms, where the spatial and angular domains are allocated and processed on multi-processor computer architectures. As compared to traditional single-processor applications, this approach reduces the computational load as well as the memory requirement per processor, yielding an efficient solution methodology for large 3-D problems. Measured neutron dosimetry responses in the reactor cavity air gap will be compared to the RAPTOR-M3G predictions. This paper is organized as follows: Section 1 discusses the RAPTOR-M3G methodology; Section 2 describes the 2-loop PWR model and the numerical results obtained. Section 3 addresses the parallel performance of the code, and Section 4 concludes this paper with final remarks and future work.

The mCpG-binding domain of human MBD3 does not bind to mCpG but interacts with NuRD/Mi2 components HDAC1 and MTA2.

PubMed

Saito, Motoki; Ishikawa, Fuyuki

2002-09-20

Although mammalian MBD3 contains the mCpG-binding domain (MBD) and is highly homologous with the authentic mCpG-binding protein MBD2, it was reported that the protein does not bind to mCpG specifically. Using recombinant human wild type and mutant MBD3 proteins, we demonstrated that atypical amino acids found in MBD3 MBD, namely, His-30 and Phe-34, are responsible for the inability of MBD3 to bind to mCpG. Interestingly, although H30K/F34Y MBD3 mutant protein binds to mCpG efficiently in vitro, it was not localized at the mCpG-rich pericentromeric regions in mouse cells. We also showed that Y34F MBD2b MBD, which possesses not the mCpG-specific DNA-binding activity but the nonspecific DNA-binding activity, was localized at the pericentromeric regions. These results suggested that the mCpG-specific DNA-binding activity is largely dispensable, and another factor(s) is required for the localization of MBD proteins in vivo. MBD3 was identified as a component of the NuRD/Mi2 complex that shows chromatin remodeling and histone deacetylase activities. We demonstrated that MBD3 MBD is necessary and sufficient for binding to HDAC1 and MTA2, two components of the NuRD/Mi2 complex. It was therefore suggested that mCpG-binding-defective MBD3 has evolutionarily conserved its MBD because of the secondary role played by the MBD in protein-protein interactions.

mTOR kinase structure, mechanism and regulation by the rapamycin-binding domain

PubMed Central

Yang, Haijuan; Rudge, Derek G.; Koos, Joseph D.; Vaidialingam, Bhamini; Yang, Hyo J.; Pavletich, Nikola P.

2015-01-01

The mammalian target of rapamycin (mTOR), a phosphoinositide 3-kinase related protein kinase, controls cell growth in response to nutrients and growth factors and is frequently deregulated in cancer. Here we report co-crystal structures of a truncated mTOR-mLST8 complex with an ATP transition state mimic and with ATP-site inhibitors. The structures reveal an intrinsically active kinase conformation, with catalytic residues and mechanism remarkably similar to canonical protein kinases. The active site is highly recessed due to the FKBP12-Rapamycin binding (FRB) domain and an inhibitory helix protruding from the catalytic cleft. mTOR activating mutations map to the structural framework that holds these elements in place, indicating the kinase is controlled by restricted access. In vitro biochemistry indicates that the FRB domain acts as a gatekeeper, with its rapamycin-binding site interacting with substrates to grant them access to the restricted active site. FKBP12-rapamycin inhibits by directly blocking substrate recruitment and by further restricting active site access. The structures also reveal active site residues and conformational changes that underlie inhibitor potency and specificity. PMID:23636326

PDZ Binding Domains, Structural Disorder and Phosphorylation: A Menage-a-trois Tailing Dcp2 mRNA Decapping Enzymes.

PubMed

Gunawardana, Dilantha

2016-01-01

Diverse cellular activities are mediated through the interaction of protein domains and their binding partners. One such protein domain widely distributed in the higher metazoan world is the PDZ domain, which facilitates abundant protein-protein interactions. The PDZ domain-PDZ binding domain interaction has been implicated in several pathologies including Alzheimer's disease, Parkinson's disease and Down syndrome. PDZ domains bind to C-terminal peptides/proteins which have either of the following combinations: S/T-X-hydrophobic-COOH for type I, hydrophobic-Xhydrophobic- COOH for type II, and D/E-X-hydrophobic-COOH for type III, although hydrophobicity in the termini form the key characteristic of the PDZ-binding domains. We identified and characterized a Dcp2 type mRNA decapping enzyme from Arabidopsis thaliana, a protein containing a putative PDZ-binding domain using mutagenesis and protein biochemistry. Now we are using bioinformatics to study the Cterminal end of mRNA decapping enzymes from complex metazoans with the aim of (1) identifying putative PDZ-binding domains (2) Correlating structural disorder with PDZ binding domains and (3) Demonstrating the presence of phosphorylation sites in C-terminal extremities of Dcp2 type mRNA decapping enzymes. It is proposed here that the trinity of PDZbinding domains, structural disorder and phosphorylation-susceptible sites are a feature of the Dcp2 family of decapping enzymes and perhaps is a wider trick in protein evolution where scaffolding/tethering is a requirement for localization and function. It is critical though laboratory-based supporting evidence is sought to back-up this bioinformatics exploration into tail regions of mRNA decapping enzymes.

Identification and specificity studies of small-molecule ligands for SH3 protein domains.

PubMed

Inglis, Steven R; Stojkoski, Cvetan; Branson, Kim M; Cawthray, Jacquie F; Fritz, Daniel; Wiadrowski, Emma; Pyke, Simon M; Booker, Grant W

2004-10-21

The Src Homology 3 (SH3) domains are small protein-protein interaction domains that bind proline-rich sequences and mediate a wide range of cell-signaling and other important biological processes. Since deregulated signaling pathways form the basis of many human diseases, the SH3 domains have been attractive targets for novel therapeutics. High-affinity ligands for SH3 domains have been designed; however, these have all been peptide-based and no examples of entirely nonpeptide SH3 ligands have previously been reported. Using the mouse Tec Kinase SH3 domain as a model system for structure-based ligand design, we have identified several simple heterocyclic compounds that selectively bind to the Tec SH3 domain. Using a combination of nuclear magnetic resonance chemical shift perturbation, structure-activity relationships, and site-directed mutagenesis, the binding of these compounds at the proline-rich peptide-binding site has been characterized. The most potent of these, 2-aminoquinoline, bound with Kd = 125 microM and was able to compete for binding with a proline-rich peptide. Synthesis of 6-substituted-2-aminoquinolines resulted in ligands with up to 6-fold improved affinity over 2-aminoquinoline and enhanced specificity for the Tec SH3 domain. Therefore, 2-aminoquinolines may potentially be useful for the development of high affinity small molecule ligands for SH3 domains.

DIMA 3.0: Domain Interaction Map.

PubMed

Luo, Qibin; Pagel, Philipp; Vilne, Baiba; Frishman, Dmitrij

2011-01-01

Domain Interaction MAp (DIMA, available at http://webclu.bio.wzw.tum.de/dima) is a database of predicted and known interactions between protein domains. It integrates 5807 structurally known interactions imported from the iPfam and 3did databases and 46,900 domain interactions predicted by four computational methods: domain phylogenetic profiling, domain pair exclusion algorithm correlated mutations and domain interaction prediction in a discriminative way. Additionally predictions are filtered to exclude those domain pairs that are reported as non-interacting by the Negatome database. The DIMA Web site allows to calculate domain interaction networks either for a domain of interest or for entire organisms, and to explore them interactively using the Flash-based Cytoscape Web software.

77 FR 54946 - Additional Designations of Individuals Pursuant to Executive Order 13581

Federal Register 2010, 2011, 2012, 2013, 2014

2012-09-06

..., Mariinskiy Posad, Chuvash Republic, Russia; DOB 8 Feb 1972; POB Rustavi, Georgia; alt. POB Kutaisi, Georgia... Emirates; DOB 3 Feb 1966; POB Raditshevo, Russia; nationality Russia (individual) [TCO]. Dated: June 6...

Conserved Domains in the Transformer Protein Act Complementary to Regulate Sex-Specific Splicing of Its Own Pre-mRNA.

PubMed

Tanaka, Arisa; Aoki, Fugaku; Suzuki, Masataka G

2018-05-26

The transformer (tra) gene, which is a female-determining master gene in the housefly Musca domestica, acts as a memory device for sex determination via its auto-regulatory function, i.e., through the contribution of the TRA protein to female-specific splicing of its own pre-mRNA. The TRA protein contains 4 small domains that are specifically conserved among TRA proteins (domains 1-4). Domain 2, also named TRA-CAM domain, is the most conserved, but its function remains unknown. To examine whether these domains are involved in the auto-regulatory function, we performed in vitro splicing assays using a tra minigene containing a partial genomic sequence of the M. domestica tra (Mdtra) gene. Co-transfection of the Mdtra minigene and an MdTRA protein expression vector into cultured insect cells strongly induced female-specific splicing of the minigene. A series of deletion mutation analyses demonstrated that these domains act complementarily to induce female-specific splicing. Domain 1 and the TRA-CAM domain were necessary for the female-specific splicing when the MdTRA protein lacked both domains 3 and 4. In this situation, mutation of the well-conserved 3 amino acids (GEG) in the TRA-CAM domain significantly reduced the female-specific splicing activity of MdTRA. GST-pull down analyses demonstrated that the MdTRA protein specifically enriched on the male-specific exonic region (exon 2b), which contains the putative TRA/TRA-2 binding sites, and that the GEG mutation disrupts this enrichment. Since the MdTRA protein interacts with its own pre-mRNA through TRA-2, our findings suggest that the conserved amino acid residues in the TRA-CAM domain may be crucial for the interaction between MdTRA and TRA-2, enhancing MdTRA recruitment on its pre-mRNA to induce female-specific splicing of tra in the housefly. © 2018 S. Karger AG, Basel.

Temperature and electric-field induced phase transitions, and full tensor properties of [011] C-poled domain-engineered tetragonal 0 .63 Pb (M g1 /3N b2 /3) -0 .37 PbTi O3 single crystals

NASA Astrophysics Data System (ADS)

Zheng, Limei; Jing, Yujia; Lu, Xiaoyan; Wang, Ruixue; Liu, Gang; Lü, Weiming; Zhang, Rui; Cao, Wenwu

2016-03-01

The phase-transition sequence of 0.67 Pb (M g1 /3N b2 /3)- 0.37 PbTi O3 (PMN-0.37PT) single crystals driven by the electric (E ) field and temperature is comprehensively studied. Based on the strain-E field loop, polarization-E field loop, and the evolution of domain configurations, the E field along the [011] C induced phase transitions have been confirmed to be as follows: tetragonal (T ) → monoclinic (MC)→ single domain orthorhombic (O ) phase. As the E field decreases, the induced O phase cannot be maintained and transformed to the MC phase, then to the coexistence state of MC and T phases. In addition, the complete sets of dielectric, piezoelectric, and elastic constants for the [011] C-poled domain-engineered PMN-0.37PT single crystal were measured at room temperature, which show high longitudinal dielectric, piezoelectric, and electromechanical properties (ɛ33T=10 661 ,d33=1052 pC /N , and k33= 0.766 ). Our results revealed that the MC phase plays an important role in the high electromechanical properties of this domain-engineered single crystal. The temperature dependence of the domain configuration revealed that the volume fraction of the MC phase decreases with temperature accompanied by the reduction of ɛ33T,d31, and k31 due to the substantially smaller intrinsic properties of the T phase.

A FYVE zinc finger domain protein specifically links mRNA transport to endosome trafficking.

PubMed

Pohlmann, Thomas; Baumann, Sebastian; Haag, Carl; Albrecht, Mario; Feldbrügge, Michael

2015-05-18

An emerging theme in cellular logistics is the close connection between mRNA and membrane trafficking. A prominent example is the microtubule-dependent transport of mRNAs and associated ribosomes on endosomes. This coordinated process is crucial for correct septin filamentation and efficient growth of polarised cells, such as fungal hyphae. Despite detailed knowledge on the key RNA-binding protein and the molecular motors involved, it is unclear how mRNAs are connected to membranes during transport. Here, we identify a novel factor containing a FYVE zinc finger domain for interaction with endosomal lipids and a new PAM2-like domain required for interaction with the MLLE domain of the key RNA-binding protein. Consistently, loss of this FYVE domain protein leads to specific defects in mRNA, ribosome, and septin transport without affecting general functions of endosomes or their movement. Hence, this is the first endosomal component specific for mRNP trafficking uncovering a new mechanism to couple mRNPs to endosomes.

Chemical Shift Assignments of the C-terminal Eps15 Homology Domain-3 EH Domain*

PubMed Central

Caplan, Steve; Sorgen, Paul L.

2013-01-01

The C-terminal Eps15 homology (EH) domain 3 (EHD3) belongs to a eukaryotic family of endocytic regulatory proteins and is involved in the recycling of various receptors from the early endosome to the endocytic recycling compartment or in retrograde transport from the endosomes to the Golgi. EH domains are highly conserved in the EHD family and function as protein-protein interaction units that bind to Asn-Pro-Phe (NPF) motif-containing proteins. The EH domain of EHD1 was the first C-terminal EH domain from the EHD family to be solved by NMR. The differences observed between this domain and proteins with N-terminal EH domains helped describe a mechanism for the differential binding of NPF-containing proteins. Here, structural studies were expanded to include the EHD3 EH domain. While the EHD1 and EHD3 EH domains are highly homologous, they have different protein partners. A comparison of these structures will help determine the selectivity in protein binding between the EHD family members and lead to a better understanding of their unique roles in endocytic regulation. PMID:23754701

Regulation of mTORC1 by PI3K signaling.

PubMed

Dibble, Christian C; Cantley, Lewis C

2015-09-01

The class I phosphoinositide 3-kinase (PI3K)-mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) signaling network directs cellular metabolism and growth. Activation of mTORC1 [composed of mTOR, regulatory-associated protein of mTOR (Raptor), mammalian lethal with SEC13 protein 8(mLST8), 40-kDa proline-rich Akt substrate (PRAS40), and DEP domain-containing mTOR-interacting protein (DEPTOR)] depends on the Ras-related GTPases (Rags) and Ras homolog enriched in brain (Rheb) GTPase and requires signals from amino acids, glucose, oxygen, energy (ATP), and growth factors (including cytokines and hormones such as insulin). Here we discuss the signal transduction mechanisms through which growth factor-responsive PI3K signaling activates mTORC1. We focus on how PI3K-dependent activation of Akt and spatial regulation of the tuberous sclerosis complex (TSC) complex (TSC complex) [composed of TSC1, TSC2, and Tre2-Bub2-Cdc16-1 domain family member 7 (TBC1D7)] switches on Rheb at the lysosome, where mTORC1 is activated. Integration of PI3K- and amino acid-dependent signals upstream of mTORC1 at the lysosome is detailed in a working model. A coherent understanding of the PI3K-mTORC1 network is imperative as its dysregulation has been implicated in diverse pathologies including cancer, diabetes, autism, and aging. Copyright © 2015 Elsevier Ltd. All rights reserved.

Structural and Biochemical Basis for Ubiquitin Ligase Recruitment by Arrestin-related Domain-containing Protein-3 (ARRDC3)*

PubMed Central

Qi, Shiqian; O'Hayre, Morgan; Gutkind, J. Silvio; Hurley, James H.

2014-01-01

After protracted stimulation, the β2-adrenergic receptor and many other G-protein-coupled receptors are ubiquitinated and down-regulated. Arrestin-related domain-containing protein-3 (ARRDC3) has been proposed to recruit the ubiquitin ligase Nedd4 to the β2-adrenergic receptor. ARRDC3 contains two PPXY motifs that could potentially interact with any of the four WW domains of Nedd4. Here we dissect the interaction determinants. ARRDC3 PPXY-Nedd4 WW dissociation constants vary from unmeasurable to Kd = 3 μm for the third WW domain of Nedd4 binding to the first PPXY motif of ARRDC3. Structures of the uncomplexed and PPXY1-bound WW3 domain were determined at 1.1 and 1.7 Å resolution. The structures revealed conformational changes upon binding and the hydrogen bonding network in exquisite detail. Tight packing of ARRDC3 Val-352′, part of a 310 helix at the C terminus of PPXY1, is important for high affinity binding to WW3. Although no single WW domain is strictly essential for the binding of Nedd4 and ARRDC3 expressed in HEK293 cells, high affinity binding of full-length ARRDC3 and Nedd4 is driven by the avid interaction of both PPXY motifs with either the WW2-WW3 or WW3-WW4 combinations, with Kd values as low as 300 nm. PMID:24379409

Monomeric CH3: A Small, Stable Antibody Domain with Therapeutic Promise | Poster

Cancer.gov

By Ashley DeVine, Staff Writer Antibody domains are emerging as promising biopharmaceuticals because of their relatively small size compared to full-sized antibodies, which are too large to effectively penetrate tumors and bind to sterically restricted therapeutic targets. In an article published in The Journal of Biological Chemistry, Tianlei Ying, Ph.D., Dimiter Dimitrov, Ph.D., and their colleagues in the Protein Interactions Group, Cancer and Inflammation Program, Center for Cancer Research, reported their design of a novel antibody domain, monomeric CH3 (mCH3).

[Effect of biopesticide Pythium oligandrum broth on gummy stem blight in cucumber seedlings, animal health and plant growth].

PubMed

Geng, Mingming; Jia, Ruilian; Sui, Zongming; Huang, Jianguo

2016-07-04

Biopesticides are safe and environment friendly. We evaluated the biocontrol effect of Pythium oligandrum broth (POB) and its toxicity to animals and plant growth. Animal, antagonist, pot, and field experiments with mice, Mycosphaerella melonis, and cucumber seedlings were carried out to study animal toxicity, control of gummy stem blight, plant growth, fruit yield and quality with POB produced from self-isolated P. oligandrum CQ2010. Mouse showed normal weight, appearances, performances and no pathogenic changes in organs and tissues with a large amount of POB supplied by lavage. The inhibition rate of POB against M. melonis was 51.95%, similar to thiophanate methy (800 times dilution) but much higher than chlorothalonil (200 times dilution). Malondialdehyde concentration was reduced whereas activities of peroxidase and superoxide dismutase were stimulated in seedling leaves irrespective of POB supplied before and after pathogenic inoculation. POB also decreased the pathogenic incidence and disease index with relative control efficacy from 54.8% to 64.1%. Thus, POB could alleviate cell membrane damage caused by pathogenic microbes, stimulate physiological reactions related to disease defense, and increase disease-resistant abilities of plants. Moreover, POB increased chlorophyll content, root activity, and uptake of nitrogen, phosphorus and potassium, resulting in growth acceleration, fruit yield increment, and quality improvement. POB is safe to animals and could control gummy stem blight of cucumber seedlings, promote plant growth, increase fruit yield, and improve the qualities.

[Anti-M3 muscarinic acetylcholine receptor antibodies and Sjögren's syndrome].

PubMed

Tsuboi, Hiroto; Iizuka, Mana; Asashima, Hiromitsu; Sumida, Takayuki

2013-01-01

Sjögren's syndrome (SS) is an autoimmune disease that affects exocrine glands including salivary and lacrimal glands. It is characterized by lymphocytic infiltration into exocrine glands, leading to dry mouth and eyes. A number of auto-antibodies are detected in patients with SS. However, no SS-specific pathologic auto-antibodies have yet been found in this condition. M3 muscarinic acetylcholine receptor (M3R) plays a crucial role in the secretion of saliva. It is reported that some patients with SS carried inhibitory auto-antibodies against M3R. To clarify the epitopes and function of anti-M3R antibodies in SS, we examined antibodies to the extracellular domains (N terminal region, the first, second, and third extracellular loop) of M3R by ELISA using synthesized peptide antigens encoding these domains in 42 SS and 42 healthy controls (HC). Titers and positivity of anti-M3R antibodies to every extracellular domain of M3R were significantly higher in SS than in HC. Our results indicated the presence of several B cell epitopes on M3R in SS. Moreover, we analyzed the functions of anti-M3R antibodies by Ca(2+)-influx assays using a human salivary gland (HSG) cell line. The functional analysis indicated that the influence of such anti-M3R antibodies on Ca(2+)-influx in HSG cells might differ based on the epitopes to which they bind. Interestingly, both IgG from anti-M3R antibodies to the second extracellular loop positive SS and anti-M3R monoclonal antibodies against the second extracellular loop of M3R, which we generated, suppressed Ca(2+)-influx in the HSG cells induced by cevimeline stimulation. These observations suggested that auto-antibodies against the second extracellular loop of M3R could be involved in salivary dysfunction in patients with SS. These results indicated the presence of several B cell epitopes on M3R in SS and the influence of anti-M3R antibodies on salivary secretion might differ based on these epitopes. Thus, anti-M3R antibodies could be not

A FYVE zinc finger domain protein specifically links mRNA transport to endosome trafficking

PubMed Central

Pohlmann, Thomas; Baumann, Sebastian; Haag, Carl; Albrecht, Mario; Feldbrügge, Michael

2015-01-01

An emerging theme in cellular logistics is the close connection between mRNA and membrane trafficking. A prominent example is the microtubule-dependent transport of mRNAs and associated ribosomes on endosomes. This coordinated process is crucial for correct septin filamentation and efficient growth of polarised cells, such as fungal hyphae. Despite detailed knowledge on the key RNA-binding protein and the molecular motors involved, it is unclear how mRNAs are connected to membranes during transport. Here, we identify a novel factor containing a FYVE zinc finger domain for interaction with endosomal lipids and a new PAM2-like domain required for interaction with the MLLE domain of the key RNA-binding protein. Consistently, loss of this FYVE domain protein leads to specific defects in mRNA, ribosome, and septin transport without affecting general functions of endosomes or their movement. Hence, this is the first endosomal component specific for mRNP trafficking uncovering a new mechanism to couple mRNPs to endosomes. DOI: http://dx.doi.org/10.7554/eLife.06041.001 PMID:25985087

The diphtheria toxin transmembrane domain as a pH sensitive membrane anchor for human interleukin-2 and murine interleukin-3.

PubMed

Liger, D; Nizard, P; Gaillard, C; vanderSpek, J C; Murphy, J R; Pitard, B; Gillet, D

1998-11-01

We have constructed two fusion proteins T-hIL-2 and T-mIL-3 in which human interleukin-2 (hIL-2) or murine interleukin-3 (mIL-3) are fused to the C-terminus of the diphtheria toxin transmembrane domain (T domain). Two additional fusion proteins, T-(Gly4-Ser)2-hIL-2 and T-(Gly4-Ser)2-mIL-3, were derived by introduction of the (Gly4-Ser)2 spacer between the T domain and cytokine components. Recognition of the hIL-2 receptor or the mIL-3 receptor by the corresponding recombinant proteins was demonstrated by their capacity to stimulate cytokine-dependent cell lines. All proteins retained the capacity of the T domain to insert into phospholipid membranes at acidic pH. Finally, anchoring of both cytokines to the membrane of lipid vesicles or living cells was assessed by specific antibody recognition. Our results show that the T domain fused to the N-terminus of a given protein can function as a pH sensitive membrane anchor for that protein.

An intermolecular binding mechanism involving multiple LysM domains mediates carbohydrate recognition by an endopeptidase

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

Wong, Jaslyn E. M. M.; Midtgaard, Søren Roi; Gysel, Kira

The crystal and solution structures of the T. thermophilus NlpC/P60 d, l-endopeptidase as well as the co-crystal structure of its N-terminal LysM domains bound to chitohexaose allow a proposal to be made regarding how the enzyme recognizes peptidoglycan. LysM domains, which are frequently present as repetitive entities in both bacterial and plant proteins, are known to interact with carbohydrates containing N-acetylglucosamine (GlcNAc) moieties, such as chitin and peptidoglycan. In bacteria, the functional significance of the involvement of multiple LysM domains in substrate binding has so far lacked support from high-resolution structures of ligand-bound complexes. Here, a structural study of themore » Thermus thermophilus NlpC/P60 endopeptidase containing two LysM domains is presented. The crystal structure and small-angle X-ray scattering solution studies of this endopeptidase revealed the presence of a homodimer. The structure of the two LysM domains co-crystallized with N-acetyl-chitohexaose revealed a new intermolecular binding mode that may explain the differential interaction between LysM domains and short or long chitin oligomers. By combining the structural information with the three-dimensional model of peptidoglycan, a model suggesting how protein dimerization enhances the recognition of peptidoglycan is proposed.« less

The crystal structure of the streptococcal collagen-like protein 2 globular domain from invasive M3-type group A Streptococcus shows significant similarity to immunomodulatory HIV protein gp41.

PubMed

Squeglia, Flavia; Bachert, Beth; De Simone, Alfonso; Lukomski, Slawomir; Berisio, Rita

2014-02-21

The arsenal of virulence factors deployed by streptococci includes streptococcal collagen-like (Scl) proteins. These proteins, which are characterized by a globular domain and a collagen-like domain, play key roles in host adhesion, host immune defense evasion, and biofilm formation. In this work, we demonstrate that the Scl2.3 protein is expressed on the surface of invasive M3-type strain MGAS315 of Streptococcus pyogenes. We report the crystal structure of Scl2.3 globular domain, the first of any Scl. This structure shows a novel fold among collagen trimerization domains of either bacterial or human origin. Despite there being low sequence identity, we observed that Scl2.3 globular domain structurally resembles the gp41 subunit of the envelope glycoprotein from human immunodeficiency virus type 1, an essential subunit for viral fusion to human T cells. We combined crystallographic data with modeling and molecular dynamics techniques to gather information on the entire lollipop-like Scl2.3 structure. Molecular dynamics data evidence a high flexibility of Scl2.3 with remarkable interdomain motions that are likely instrumental to the protein biological function in mediating adhesive or immune-modulatory functions in host-pathogen interactions. Altogether, our results provide molecular tools for the understanding of Scl-mediated streptococcal pathogenesis and important structural insights for the future design of small molecular inhibitors of streptococcal invasion.

Anomalous domain inversion in LiNbO3 single crystals investigated by scanning probe microscopy

NASA Astrophysics Data System (ADS)

Lilienblum, M.; Soergel, E.

2011-09-01

Ferroelectric domains were written in lithium niobate (LiNbO3) single crystals by applying voltage pulses to the tip of a scanning force microscope. The generated domains are subsequently imaged by piezoresponse force microscopy. As it has been previously observed not only full domains but also doughnut-shaped ones arise from tip-based domain formation. In this contribution, we present our experiments which were carried out with 10-20 μm thin LiNbO3 single crystals. We show that by choosing appropriate writing parameters, domains of predetermined shape (full or doughnut) can be reliably generated. In addition to the duration and the amplitude of the voltage pulse the moment of the retraction of the tip from the sample surface was found to be a crucial parameter for reproducible domain formation.

Orientation-resolved domain mapping in tetragonal SrTiO 3 using polarized Raman spectroscopy

DOE PAGES

Gray, Jr., Dodd J.; Merz, Tyler A.; Hikita, Yasuyuki; ...

2016-12-16

Here, we present microscopically resolved, polarized spectroscopy of Raman scattering collected from tetragonal SrTiO 3. The anisotropic response of first-order Raman peaks within a single tetragonal domain has been measured. From these data, we assign symmetries to the phonons seen in the first-order Raman spectrum which is normally complicated by uncontrolled domain structure. Using a translation stage, we map the local domain orientation of a 3–μm 3 crystal volume near the laser focus and compare it to wide-field polarized images. This technique can be performed with readily available instruments and is relevant to the study of a wide range ofmore » related materials, interfaces, and devices.« less

77 FR 40948 - Additional Designation of Persons Whose Property and Interests in Property Are Blocked Pursuant...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-07-11

..., Mombasa, Kenya; DOB 1962; alt. DOB 1967; POB Kenya; citizen Kenya (individual) [SOMALIA]. 3. GOITOM, Taeme... Jan 1969; POB Kenya; alt. POB Lamu Island, Kenya; citizen Kenya (individual) [SOMALIA]. 5. NEGASH..., Kenya; Passport A764712 (Kenya) expires 27 Mar 2013; alt. Passport B002271 (Kenya); alt. Passport...

The AvrM Effector from Flax Rust Has a Structured C-Terminal Domain and Interacts Directly with the M Resistance Protein

PubMed Central

Catanzariti, Ann-Maree; Dodds, Peter N.; Ve, Thomas; Kobe, Bostjan; Ellis, Jeffrey G.; Staskawicz, Brian J.

2011-01-01

In plant immunity, recognition of pathogen effectors by plant resistance proteins leads to the activation of plant defenses and a localized cell death response. The AvrM effector from flax rust is a small secreted protein that is recognized by the M resistance protein in flax. Here, we investigate the mechanism of M–AvrM recognition and show that these two proteins directly interact in a yeast two-hybrid assay, and that this interaction correlates with the recognition specificity observed for each of the different AvrM variants. We further characterize this interaction by demonstrating that the C-terminal domain of AvrM is required for M-dependent cell death, and show that this domain also interacts with the M protein in yeast. We investigate the role of C-terminal differences among the different AvrM proteins for their involvement in this interaction and establish that M recognition is hindered by an additional 34 amino acids present at the C terminus of several AvrM variants. Structural characterization of recombinant AvrM-A protein revealed a globular C-terminal domain that dimerizes. PMID:19958138

Characterization and receptor specific toxicity of two diphtheria toxin-related interleukin-3 fusion proteins DAB389-mIL-3 and DAB389-(Gly4Ser)2-mIL-3.

PubMed

Liger, D; vanderSpek, J C; Gaillard, C; Cansier, C; Murphy, J R; Leboulch, P; Gillet, D

1997-04-07

We have constructed two fusion proteins, DAB389-mIL-3 and DAB389-(Gly4Ser)2-mIL-3, in which the receptor-binding domain of diphtheria toxin is replaced by mouse interleukin-3 (IL-3). Cytotoxic activity of the fusion toxins was observed on three out of six cell lines assayed. This toxicity was mediated through binding to the IL-3 receptor as it was inhibited in a dose-dependent manner with murine IL-3 or anti-IL-3 neutralizing antibodies. DAB389-(Gly4Ser)2-mIL-3 was up to 5 times more toxic than DAB389-mIL-3, depending on the cell line (0.8 x 10(-10) M < IC50 < 3 x 10(-10) M). These proteins can be used for the detection of IL-3 receptors on mouse cells and should allow for the selective elimination of IL-3 receptor-positive pluripotent hematopoietic stem cells prior to bone marrow transplantation.

Drosophila Pumilio Protein Contains Multiple Autonomous Repression Domains That Regulate mRNAs Independently of Nanos and Brain Tumor

PubMed Central

Weidmann, Chase A.

2012-01-01

Drosophila melanogaster Pumilio is an RNA-binding protein that potently represses specific mRNAs. In developing embryos, Pumilio regulates a key morphogen, Hunchback, in collaboration with the cofactor Nanos. To investigate repression by Pumilio and Nanos, we created cell-based assays and found that Pumilio inhibits translation and enhances mRNA decay independent of Nanos. Nanos robustly stimulates repression through interactions with the Pumilio RNA-binding domain. We programmed Pumilio to recognize a new binding site, which garners repression of new target mRNAs. We show that cofactors Brain Tumor and eIF4E Homologous Protein are not obligatory for Pumilio and Nanos activity. The conserved RNA-binding domain of Pumilio was thought to be sufficient for its function. Instead, we demonstrate that three unique domains in the N terminus of Pumilio possess the major repressive activity and can function autonomously. The N termini of insect and vertebrate Pumilio and Fem-3 binding factors (PUFs) are related, and we show that corresponding regions of human PUM1 and PUM2 have repressive activity. Other PUF proteins lack these repression domains. Our findings suggest that PUF proteins have evolved new regulatory functions through protein sequences appended to their conserved PUF repeat RNA-binding domains. PMID:22064486

Drosophila Pumilio protein contains multiple autonomous repression domains that regulate mRNAs independently of Nanos and brain tumor.

PubMed

Weidmann, Chase A; Goldstrohm, Aaron C

2012-01-01

Drosophila melanogaster Pumilio is an RNA-binding protein that potently represses specific mRNAs. In developing embryos, Pumilio regulates a key morphogen, Hunchback, in collaboration with the cofactor Nanos. To investigate repression by Pumilio and Nanos, we created cell-based assays and found that Pumilio inhibits translation and enhances mRNA decay independent of Nanos. Nanos robustly stimulates repression through interactions with the Pumilio RNA-binding domain. We programmed Pumilio to recognize a new binding site, which garners repression of new target mRNAs. We show that cofactors Brain Tumor and eIF4E Homologous Protein are not obligatory for Pumilio and Nanos activity. The conserved RNA-binding domain of Pumilio was thought to be sufficient for its function. Instead, we demonstrate that three unique domains in the N terminus of Pumilio possess the major repressive activity and can function autonomously. The N termini of insect and vertebrate Pumilio and Fem-3 binding factors (PUFs) are related, and we show that corresponding regions of human PUM1 and PUM2 have repressive activity. Other PUF proteins lack these repression domains. Our findings suggest that PUF proteins have evolved new regulatory functions through protein sequences appended to their conserved PUF repeat RNA-binding domains.

Dual GPCR and GAG mimicry by the M3 chemokine decoy receptor

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

Alexander-Brett, Jennifer M.; Fremont, Daved H.

2008-09-23

Viruses have evolved a myriad of evasion strategies focused on undermining chemokine-mediated immune surveillance, exemplified by the mouse {gamma}-herpesvirus 68 M3 decoy receptor. Crystal structures of M3 in complex with C chemokine ligand 1/lymphotactin and CC chemokine ligand 2/monocyte chemoattractant protein 1 reveal that invariant chemokine features associated with G protein-coupled receptor binding are primarily recognized by the decoy C-terminal domain, whereas the N-terminal domain (NTD) reconfigures to engage divergent basic residue clusters on the surface of chemokines. Favorable electrostatic forces dramatically enhance the association kinetics of chemokine binding by M3, with a primary role ascribed to acidic NTD regionsmore » that effectively mimic glycosaminoglycan interactions. Thus, M3 employs two distinct mechanisms of chemical imitation to potently sequester chemokines, thereby inhibiting chemokine receptor binding events as well as the formation of chemotactic gradients necessary for directed leukocyte trafficking.« less

Topoisomerase 3β is the major topoisomerase for mRNAs and linked to neurodevelopment and mental dysfunction.

PubMed

Ahmad, Muzammil; Shen, Weiping; Li, Wen; Xue, Yutong; Zou, Sige; Xu, Dongyi; Wang, Weidong

2017-03-17

Human cells contain five topoisomerases in the nucleus and cytoplasm, but which one is the major topoisomerase for mRNAs is unclear. To date, Top3β is the only known topoisomerase that possesses RNA topoisomerase activity, binds mRNA translation machinery and interacts with an RNA-binding protein, FMRP, to promote synapse formation; and Top3β gene deletion has been linked to schizophrenia. Here, we show that Top3β is also the most abundant mRNA-binding topoisomerase in cells. Top3β, but not other topoisomerases, contains a distinctive RNA-binding domain; and deletion of this domain diminishes the amount of Top3β that associates with mRNAs, indicating that Top3β is specifically targeted to mRNAs by its RNA binding domain. Moreover, Top3β mutants lacking either its RNA-binding domain or catalytic residue fail to promote synapse formation, suggesting that Top3β requires both its mRNA-binding and catalytic activity to facilitate neurodevelopment. Notably, Top3β proteins bearing point mutations from schizophrenia and autism individuals are defective in association with FMRP; whereas one of the mutants is also deficient in binding mRNAs, catalyzing RNA topoisomerase reaction, and promoting synapse formation. Our data suggest that Top3β is the major topoisomerase for mRNAs, and requires both RNA binding and catalytic activity to promote neurodevelopment and prevent mental dysfunction. Published by Oxford University Press on behalf of Nucleic Acids Research 2016.

3D frequency-domain finite-difference modeling of acoustic wave propagation

NASA Astrophysics Data System (ADS)

Operto, S.; Virieux, J.

2006-12-01

-memory computers. The MUMPS solver is based on a multifrontal method for LU factorization. We used the METIS algorithm to perform re-ordering of the matrix coefficients before factorization. Four grid points per minimum wavelength is used for discretization. We applied our algorithm to the 3D SEG/EAGE synthetic onshore OVERTHRUST model of dimensions 20 x 20 x 4.65 km. The velocities range between 2 and 6 km/s. We performed the simulations using 192 processors with 2 Gbytes of RAM memory per processor. We performed simulations for the 5 Hz, 7 Hz and 10 Hz frequencies in some fractions of the OVERTHRUST model. The grid interval was 100 m, 75 m and 50 m respectively. The grid dimensions were 207x207x53, 275x218x71 and 409x109x102 respectively corresponding to 100, 80 and 25 percents of the model respectively. The time for factorization is 20 mn, 108 mn and 163 mn respectively. The time for resolution was 3.8, 9.3 and 10.3 s per source. The total memory used during factorization is 143, 384 and 449 Gbytes respectively. One can note the huge memory requirement for factorization and the efficiency of the direct method to compute solutions for a large number of sources. This highlights the respective drawback and merit of the frequency-domain approach with respect to the time- domain counterpart. These results show that 3D acoustic frequency-domain wave propagation modeling can be performed at low frequencies using direct solver on large clusters of Pcs. This forward modeling algorithm may be used in the future as a tool to image the first kilometers of the crust by frequency-domain full-waveform inversion. For larger problems, we will use the out-of-core memory during factorization that has been implemented by the authors of MUMPS.

The GRP1 PH domain, like the AKT1 PH domain, possesses a sentry glutamate residue essential for specific targeting to plasma membrane PI(3,4,5)P(3).

PubMed

Pilling, Carissa; Landgraf, Kyle E; Falke, Joseph J

2011-11-15

During the appearance of the signaling lipid PI(3,4,5)P(3), an important subset of pleckstrin homology (PH) domains target signaling proteins to the plasma membrane. To ensure proper pathway regulation, such PI(3,4,5)P(3)-specific PH domains must exclude the more prevalant, constitutive plasma membrane lipid PI(4,5)P(2) and bind the rare PI(3,4,5)P(3) target lipid with sufficiently high affinity. Our previous study of the E17K mutant of the protein kinase B (AKT1) PH domain, together with evidence from Carpten et al. [Carpten, J. D., et al. (2007) Nature 448, 439-444], revealed that the native AKT1 E17 residue serves as a sentry glutamate that excludes PI(4,5)P(2), thereby playing an essential role in specific PI(3,4,5)P(3) targeting [Landgraf, K. E., et al. (2008) Biochemistry 47, 12260-12269]. The sentry glutamate hypothesis proposes that an analogous sentry glutamate residue is a widespread feature of PI(3,4,5)P(3)-specific PH domains, and that charge reversal mutation at the sentry glutamate position will yield both increased PI(4,5)P(2) affinity and constitutive plasma membrane targeting. To test this hypothesis, we investigated the E345 residue, a putative sentry glutamate, of the general receptor for phosphoinositides 1 (GRP1) PH domain. The results show that incorporation of the E345K charge reversal mutation into the GRP1 PH domain enhances PI(4,5)P(2) affinity 8-fold and yields constitutive plasma membrane targeting in cells, reminiscent of the effects of the E17K mutation in the AKT1 PH domain. Hydrolysis of plasma membrane PI(4,5)P(2) releases the E345K GRP1 PH domain into the cytoplasm, and the efficiency of this release increases when Arf6 binding is disrupted. Overall, the findings provide strong support for the sentry glutamate hypothesis and suggest that the GRP1 E345K mutation will be linked to changes in cell physiology and human pathologies, as demonstrated for AKT1 E17K [Carpten, J. D., et al. (2007) Nature 448, 439-444; Lindhurst, M. J., et al

A non-chromatographic protein purification strategy using Src 3 homology domains as generalized capture domains.

PubMed

Kim, Heejae; Chen, Wilfred

2016-09-20

Protein purification using inverse phase transition of elastin-like polypeptide (ELP) domains is a useful alternative to chromatography. Genetic fusions of ELP domains to various proteins have the ability to reversibly transition between soluble monomers and micron-sized aggregates and this has been used to selectively purify many ELP fusions. Affinity domains can enhance this technology by using specific protein binding domains to enable ELP mediated affinity capture (EMAC) of proteins of interest (POI) that have been fused to corresponding affinity ligands. In this paper, we highlight the use of Src homology 3 (SH3) domains and corresponding peptide ligands in EMAC that have differential binding affinities towards SH3 for efficient capture and elution of proteins. Furthermore, differences between capture and elution of a monomeric and a multimeric protein were also studied. Copyright © 2016 Elsevier B.V. All rights reserved.

The butyrophilin 3A1 intracellular domain undergoes a conformational change involving the juxtamembrane region.

PubMed

Nguyen, Khiem; Li, Jin; Puthenveetil, Robbins; Lin, Xiaochen; Poe, Michael M; Hsiao, Chia-Hung Christine; Vinogradova, Olga; Wiemer, Andrew J

2017-11-01

Small isoprenoid diphosphates, such as ( E )-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP), are ligands of the internal domain of BTN3A1. Ligand binding in target cells promotes activation of Vγ9Vδ2 T cells. We demonstrate by small-angle X-ray scattering (SAXS) that HMBPP binding to the internal domain of BTN3A1 induces a conformational change in the position of the B30.2 domain relative to the juxtamembrane (JM) region. To better understand the molecular details of this conformational rearrangement, NMR spectroscopy was used to discover that the JM region interacts with HMBPP, specifically at the diphosphate. The spectral location of the affected amide peaks, partial NMR assignments, and JM mutants (ST 296 AA or T 304 A) investigated, confirm that the backbone amide of at least one Thr (Thr 304 ), adjacent to conserved Ser, comes close to the HMBPP diphosphate, whereas double mutation of nonconserved residues (Ser/Thr 296/297 ) may perturb the local fold. Cellular mutation of either of the identified Thr residues reduces the activation of Vγ9Vδ2 T cells by HMBPP, zoledronate, and POM 2 -C-HMBP, but not by a partial agonist BTN3 antibody. Taken together, our results show that ligand binding to BTN3A1 induces a conformational change within the intracellular domain that involves the JM region and is required for full activation.-Nguyen, K., Li, J., Puthenveetil, R., Lin, X., Poe, M. M., Hsiao, C.-H. C., Vinogradova, O., Wiemer, A. J. The butyrophilin 3A1 intracellular domain undergoes a conformational change involving the juxtamembrane region. © FASEB.

Abl N-terminal Cap stabilization of SH3 domain dynamics†

PubMed Central

Chen, Shugui; Dumitrescu, Teodora Pene; Smithgall, Thomas E.; Engen, John R.

2008-01-01

Crystal structures and other biochemical data indicate that the N-terminal cap (NCap) region of the Abelson tyrosine kinase (c-Abl) is important for maintaining the downregulated conformation of the kinase domain. The exact contributions that NCap makes in stabilizing the various intramolecular interactions within c-Abl are less clear. While the NCap appears important for locking the SH3/SH2 domains to the back of the kinase domain, there may be other more subtle elements of regulation. Hydrogen exchange (HX) and mass spectrometry (MS) were used to determine if the NCap contributes to intramolecular interactions involving the Abl SH3 domain. Under physiological conditions, the Abl SH3 domain underwent partial unfolding and its unfolding half-life was slowed during binding to the SH2-kinase linker, providing a unique assay to test NCap-induced stabilization of the SH3 domain in various constructs. The results showed that NCap stabilizes the dynamics of the SH3 domain in certain constructs but does not increase the relative affinity of the SH3 domain for the native SH2-kinase linker. The stabilization effect was absent in constructs of just NCap + SH3 but was obvious when the SH2 domain and the SH2-kinase linker were present. These results suggest that interactions between NCap and the SH3 domain can contribute to c-Abl stabilization in constructs that contain at least the SH2 domain, an effect that may partially compensate for the absence of the negative regulatory C-terminal tail found in the related Src family of kinases. PMID:18452309

Abl N-terminal cap stabilization of SH3 domain dynamics.

PubMed

Chen, Shugui; Dumitrescu, Teodora Pene; Smithgall, Thomas E; Engen, John R

2008-05-27

Crystal structures and other biochemical data indicate that the N-terminal cap (NCap) region of the Abelson tyrosine kinase (c-Abl) is important for maintaining the downregulated conformation of the kinase domain. The exact contributions that the NCap makes in stabilizing the various intramolecular interactions within c-Abl are less clear. While the NCap appears to be important for locking the SH3 and SH2 domains to the back of the kinase domain, there may be other more subtle elements of regulation. Hydrogen exchange (HX) and mass spectrometry (MS) were used to determine if the NCap contributes to intramolecular interactions involving the Abl SH3 domain. Under physiological conditions, the Abl SH3 domain underwent partial unfolding and its unfolding half-life was slowed during binding to the SH2 kinase linker, providing a unique assay for testing NCap-induced stabilization of the SH3 domain in various constructs. The results showed that the NCap stabilizes the dynamics of the SH3 domain in certain constructs but does not increase the relative affinity of the SH3 domain for the native SH2 kinase linker. The stabilization effect was absent in constructs of just the NCap and SH3 but was obvious when the SH2 domain and the SH2 kinase linker were present. These results suggest that interactions between the NCap and the SH3 domain can contribute to c-Abl stabilization in constructs that contain at least the SH2 domain, an effect that may partially compensate for the absence of the negative regulatory C-terminal tail found in the related Src family of kinases.

Dynamics of the Tec‐family tyrosine kinase SH3 domains

PubMed Central

Roberts, Justin M.; Tarafdar, Sreya; Joseph, Raji E.; Andreotti, Amy H.; Smithgall, Thomas E.; Engen, John R.

2016-01-01

Abstract The Src Homology 3 (SH3) domain is an important regulatory domain found in many signaling proteins. X‐ray crystallography and NMR structures of SH3 domains are generally conserved but other studies indicate that protein flexibility and dynamics are not. We previously reported that based on hydrogen exchange mass spectrometry (HX MS) studies, there is variable flexibility and dynamics among the SH3 domains of the Src‐family tyrosine kinases and related proteins. Here we have extended our studies to the SH3 domains of the Tec family tyrosine kinases (Itk, Btk, Tec, Txk, Bmx). The SH3 domains of members of this family augment the variety in dynamics observed in previous SH3 domains. Txk and Bmx SH3 were found to be highly dynamic in solution by HX MS and Bmx was unstructured by NMR. Itk and Btk SH3 underwent a clear EX1 cooperative unfolding event, which was localized using pepsin digestion and mass spectrometry after hydrogen exchange labeling. The unfolding was localized to peptide regions that had been previously identified in the Src‐family and related protein SH3 domains, yet the kinetics of unfolding were not. Sequence alignment does not provide an easy explanation for the observed dynamics behavior, yet the similarity of location of EX1 unfolding suggests that higher‐order structural properties may play a role. While the exact reason for such dynamics is not clear, such motions can be exploited in intra‐ and intermolecular binding assays of proteins containing the domains. PMID:26808198

From Domain Specific Languages to DEVS Components: Application to Cognitive M&S

DTIC Science & Technology

2011-04-01

AND SUBTITLE From Domain Specific Languages to DEVS Components: Application to Cognitive M&S 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ...that is devoid of any DEVS and programming language constructs (Figure 4). The key idea being domain specialists need not delve in the DEVS world to...DSL. DSLs can be created using many available tools and technologies such as: Generic Modeling Environment (GME) [23], Xtext, Ruby, Scala and many

NMR Determination of Protein Partitioning into Membrane Domains with Different Curvatures and Application to the Influenza M2 Peptide

PubMed Central

Wang, Tuo; Cady, Sarah D.; Hong, Mei

2012-01-01

The M2 protein of the influenza A virus acts both as a drug-sensitive proton channel and mediates virus budding through membrane scission. The segment responsible for causing membrane curvature is an amphipathic helix in the cytoplasmic domain of the protein. Here, we use 31P and 13C solid-state NMR to examine M2-induced membrane curvature. M2(22–46), which includes only the transmembrane (TM) helix, and M2(21–61), which contains an additional amphipathic helix, are studied. 31P chemical shift lineshapes indicate that M2(21–61) causes a high-curvature isotropic phase to both cholesterol-rich virus-mimetic membranes and 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayers, whereas M2(22–46) has minimal effect. The lamellar and isotropic domains have distinct 31P isotropic chemical shifts, indicating perturbation of the lipid headgroup conformation by the amphipathic helix. 31P- and 13C-detected 1H T2 relaxation and two-dimensional peptide-lipid correlation spectra show that M2(21–61) preferentially binds to the high-curvature domain. 31P linewidths indicate that the isotropic vesicles induced by M2(21–61) are 10–35 nm in diameter, and the virus-mimetic vesicles are smaller than the 1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles. A strong correlation is found between high membrane curvature and weak drug-binding ability of the TM helix. Thus, the M2 amphipathic helix causes membrane curvature, which in turn perturbs the TM helix conformation, abolishing drug binding. These NMR experiments are applicable to other curvature-inducing membrane proteins such as fusion proteins and antimicrobial peptides. PMID:22385849

Activation of PI3K/Akt signaling by n-terminal SH2 domain mutants of the p85α regulatory subunit of PI3K is enhanced by deletion of its c-terminal SH2 domain.

PubMed

Hofmann, Bianca T; Jücker, Manfred

2012-10-01

The phosphoinositide 3-kinase (PI3K) is frequently activated in human cancer cells due to gain of function mutations in the catalytic (p110) and the regulatory (p85) subunits. The regulatory subunit consists of an SH3 domain and two SH2 domains. An oncogenic form of p85α named p65 lacking the c-terminal SH2 domain (cSH2) has been cloned from an irradiation-induced murine thymic lymphoma and transgenic mice expressing p65 in T lymphocytes develop a lymphoproliferative disorder. We have recently detected a c-terminal truncated form of p85α named p76α in a human lymphoma cell line lacking most of the cSH2 domain due to a frame shift mutation. Here, we report that the deletion of the cSH2 domain enhances the activating effects of the n-terminal SH2 domain (nSH2) mutants K379E and R340E on the PI3K/Akt pathway and micro tumor formation in a focus assay. Further analysis revealed that this transforming effect is mediated by activation of the catalytic PI3K isoform p110α and downstream signaling through mTOR. Our data further support a mechanistic model in which mutations of the cSH2 domain of p85α can abrogate its negative regulatory function on PI3K activity via the nSH2 domain of p85α. Copyright © 2012 Elsevier Inc. All rights reserved.

An Extended Structure of the APOBEC3G Catalytic Domain Suggests a Unique Holoenzyme Model

PubMed Central

Harjes, Elena; Gross, Phillip J.; Chen, Kuan-Ming; Lu, Yongjian; Shindo, Keisuke; Nowarski, Roni; Gross, John D.; Kotler, Moshe; Harris, Reuben S.; Matsuo, Hiroshi

2009-01-01

Summary Human APOBEC3G (A3G) belongs to a family of polynucleotide cytidine deaminases. This family includes APOBEC1 and AID, which edit APOB mRNA and antibody gene DNA, respectively. A3G deaminates cytidines to uridines in single-strand DNA and inhibits the replication of HIV-1, other retroviruses and retrotransposons. Although the mechanism of A3G-catalyzed DNA deamination has been investigated genetically and biochemically, atomic details are just starting to emerge. Here, we compare the DNA cytidine deaminase activities and NMR structures of two A3G catalytic domain constructs. The longer A3G191-384 protein is considerably more active than the shorter A3G198-384 variant. The longer structure has an α1 helix (residues 201–206) that was not apparent in the shorter protein and it contributes to catalytic activity through interactions with hydrophobic core structures (β1, β3, α5 and α6). Both A3G catalytic domain solution structures have a discontinuous β2 region that is clearly different than the continuous β2 strand of another family member APOBEC2. In addition, the longer A3G191-384 structure revealed part of the N-terminal pseudo-catalytic domain including the inter-domain linker and some of the last α-helix. These structured residues (191–196) enabled a novel full-length A3G model by providing physical overlap between the N-terminal pseudo-catalytic domain and the new C-terminal catalytic domain structure. Contrary to predictions, this structurally constrained model suggested that the two domains are tethered by structured residues and that the N- and C-terminal β2 regions are too distant from one another to participate in this interaction. PMID:19389408

Calcium Domains around Single and Clustered IP3 Receptors and Their Modulation by Buffers

PubMed Central

Rüdiger, S.; Nagaiah, Ch.; Warnecke, G.; Shuai, J.W.

2010-01-01

Abstract We study Ca2+ release through single and clustered IP3 receptor channels on the ER membrane under presence of buffer proteins. Our computational scheme couples reaction-diffusion equations and a Markovian channel model and allows our investigating the effects of buffer proteins on local calcium concentrations and channel gating. We find transient and stationary elevations of calcium concentrations around active channels and show how they determine release amplitude. Transient calcium domains occur after closing of isolated channels and constitute an important part of the channel's feedback. They cause repeated openings (bursts) and mediate increased release due to Ca2+ buffering by immobile proteins. Stationary domains occur during prolonged activity of clustered channels, where the spatial proximity of IP3Rs produces a distinct [Ca2+] scale (0.5–10 μM), which is smaller than channel pore concentrations (>100 μM) but larger than transient levels. While immobile buffer affects transient levels only, mobile buffers in general reduce both transient and stationary domains, giving rise to Ca2+ evacuation and biphasic modulation of release amplitude. Our findings explain recent experiments in oocytes and provide a general framework for the understanding of calcium signals. PMID:20655827

Conformational instability of the MARK3 UBA domain compromises ubiquitin recognition and promotes interaction with the adjacent kinase domain

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

Murphy, James M.; Korzhnev, Dmitry M.; Ceccarelli, Derek F.

2012-10-23

The Par-1/MARK protein kinases play a pivotal role in establishing cellular polarity. This family of kinases contains a unique domain architecture, in which a ubiquitin-associated (UBA) domain is located C-terminal to the kinase domain. We have used a combination of x-ray crystallography and NMR dynamics experiments to understand the interaction of the human (h) MARK3 UBA domain with the adjacent kinase domain as compared with ubiquitin. The x-ray crystal structure of the linked hMARK3 kinase and UBA domains establishes that the UBA domain forms a stable intramolecular interaction with the N-terminal lobe of the kinase domain. However, solution-state NMR studiesmore » of the isolated UBA domain indicate that it is highly dynamic, undergoing conformational transitions that can be explained by a folding-unfolding equilibrium. NMR titration experiments indicated that the hMARK3 UBA domain has a detectable but extremely weak affinity for mono ubiquitin, which suggests that conformational instability of the isolated hMARK3 UBA domain attenuates binding to ubiquitin despite the presence of residues typically involved in ubiquitin recognition. Our data identify a molecular mechanism through which the hMARK3 UBA domain has evolved to bind the kinase domain, in a fashion that stabilizes an open conformation of the N- and C-terminal lobes, at the expense of its capacity to engage ubiquitin. These results may be relevant more generally to the 30% of UBA domains that lack significant ubiquitin-binding activity, and they suggest a unique mechanism by which interaction domains may evolve new binding properties.« less

Roles of the SH2 and SH3 domains in the regulation of neuronal Src kinase functions.

PubMed

Groveman, Bradley R; Xue, Sheng; Marin, Vedrana; Xu, Jindong; Ali, Mohammad K; Bienkiewicz, Ewa A; Yu, Xian-Min

2011-02-01

Previous studies demonstrated that intra-domain interactions between Src family kinases (SFKs), stabilized by binding of the phosphorylated C-terminus to the SH2 domain and/or binding of the SH2 kinase linker to the SH3 domain, lock the molecules in a closed conformation, disrupt the kinase active site, and inactivate SFKs. Here we report that the up-regulation of N-methyl-D-aspartate receptors (NMDARs) induced by expression of constitutively active neuronal Src (n-Src), in which the C-terminus tyrosine is mutated to phenylalanine (n-Src/Y535F), is significantly reduced by dysfunctions of the SH2 and/or SH3 domains of the protein. Furthermore, we found that dysfunctions of SH2 and/or SH3 domains reduce auto-phosphorylation of the kinase activation loop, depress kinase activity, and decrease NMDAR phosphorylation. The SH2 domain plays a greater regulatory role than the SH3 domain. Our data also show that n-Src binds directly to the C-terminus of the NMDAR NR2A subunit in vitro, with a K(D) of 108.2 ± 13.3 nM. This binding is not Src kinase activity-dependent, and dysfunctions of the SH2 and/or SH3 domains do not significantly affect the binding. These data indicate that the SH2 and SH3 domains may function to promote the catalytic activity of active n-Src, which is important in the regulation of NMDAR functions. © 2010 The Authors Journal compilation © 2010 FEBS.

Reconstitution of high affinity. cap alpha. /sub 2/ adrenergic agonist binding by fusion with a pertussis toxin substrate

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

Kim, M.H.; Neubig, R.R.

1986-03-05

High affinity ..cap alpha../sub 2/ adrenergic agonist binding is thought to occur via a coupling of the ..cap alpha../sub 2/ receptor with N/sub i/, the inhibitory guanyl nucleotide binding protein. Human platelet membranes pretreated at pH 11.5 exhibit a selective inactivation of agonist binding and N/sub i/. To further study the mechanism of agonist binding, alkali treated membranes (ATM) were mixed with membranes pretreated with 10 ..mu..M phenoxybenzamine to block ..cap alpha../sub 2/ receptors (POB-M). The combined membrane pellet was incubated in 50% polyethylene glycol (PEG) to promote membrane-membrane fusion and assayed for binding to the ..cap alpha../sub 2/ agonistmore » (/sup 3/H)UK 14,304 (UK) and the antagonist (/sup 3/H) yohimbine. PEG treatment resulted in a 2-4 fold enhancement of UK binding whereas yohimbine binding was unchanged. No enhancement of UK binding was observed in the absence of PEG treatment. The reconstitution was dependent on the addition of POB-M. They found that a 1:1 ratio of POB-M:ATM was optimal. Reconstituted binding was inhibited by GppNHp. Fusion of rat C6 glioma cell membranes, which do not contain ..cap alpha../sub 2/ receptors, also enhanced agonist binding to ATM. Fusion of C6 membranes from cells treated with pertussis toxin did not enhance (/sup 3/H) UK binding. These data show that a pertussis toxin sensitive membrane component, possibly N/sub i/, can reconstitute high affinity ..cap alpha../sub 2/ agonist binding.« less

Nemo-like kinase (NLK) expression in osteoblastic cells and suppression of osteoblastic differentiation

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

Nifuji, Akira, E-mail: nifuji-a@tsurumi-u.ac.jp; Department of Pharmacology, Tsurumi University School of Dental Medicine, Yokohama; Ideno, Hisashi

2010-04-15

Mitogen-activated protein kinases (MAPKs) regulate proliferation and differentiation in osteoblasts. The vertebral homologue of nemo, nemo-like kinase (NLK), is an atypical MAPK that targets several signaling components, including the T-cell factor/lymphoid enhancer factor (TCF/Lef1) transcription factor. Recent studies have shown that NLK forms a complex with the histone H3-K9 methyltransferase SETDB1 and suppresses peroxisome proliferator-activated receptor (PPAR)-gamma:: action in the mesenchymal cell line ST2. Here we investigated whether NLK regulates osteoblastic differentiation. We showed that NLK mRNA is expressed in vivo in osteoblasts at embryonic day 18.5 (E18.5) mouse calvariae. By using retrovirus vectors, we performed forced expression of NLKmore » in primary calvarial osteoblasts (pOB cells) and the mesenchymal cell line ST2. Wild-type NLK (NLK-WT) suppressed alkaline phosphatase activity and expression of bone marker genes such as alkaline phosphatase, type I procollagen, runx2, osterix, steopontin and osteocalcin in these cells. NLK-WT also decreased type I collagen protein expression in pOB and ST2 cells. Furthermore, mineralized nodule formation was reduced in pOB cells overexpressing NLK-WT. In contrast, kinase-negative form of NLK (NLK-KN) did not suppress or partially suppress ALP activity and bone marker gene expression in pOB and ST2 cells. NLK-KN did not suppress nodule formation in pOB cells. In addition to forced expression, suppression of endogenous NLK expression by siRNA increased bone marker gene expression in pOB and ST2 cells. Finally, transcriptional activity analysis of gene promoters revealed that NLK-WT suppressed Wnt1 activation of TOP flash promoter and Runx2 activation of the osteocalcin promoter. Taken together, these results suggest that NLK negatively regulates osteoblastic differentiation.« less

The Rapamycin-Binding Domain of the Protein Kinase mTOR is a Destabilizing Domain*

PubMed Central

Edwards, Sarah R.; Wandless, Thomas J.

2013-01-01

Rapamycin is an immunosuppressive drug that binds simultaneously to the 12-kDa FK506- and rapamycin-binding protein (FKBP12, or FKBP) and the FKBP-rapamycin binding domain (FRB) of the mammalian target of rapamycin (mTOR) kinase. The resulting ternary complex has been used to conditionally perturb protein function, and one such method involves perturbation of a protein of interest through its mislocalization. We synthesized two rapamycin derivatives that possess large substituents at the C16 position within the FRB-binding interface, and these derivatives were screened against a library of FRB mutants using a three-hybrid assay in Saccharomyces cerevisiae. Several FRB mutants responded to one of the rapamycin derivatives, and twenty of these mutants were further characterized in mammalian cells. The mutants most responsive to the ligand were fused to yellow fluorescent protein, and fluorescence levels in the presence and absence of the ligand were measured to determine stability of the fusion proteins. Wild-type and mutant FRB domains were expressed at low levels in the absence of the rapamycin derivative, and expression levels rose up to ten-fold upon treatment with ligand. The synthetic rapamycin derivatives were further analyzed using quantitative mass spectrometry, and one of the compounds was found to contain contaminating rapamycin. Furthermore, uncontaminated analogs retain the ability to inhibit mTOR, albeit with diminished potency relative to rapamycin. The ligand-dependent stability displayed by wildtype FRB and FRB mutants as well as the inhibitory potential and purity of the rapamycin derivatives should be considered as potentially confounding experimental variables when using these systems. PMID:17350953

Regions of very low H3K27me3 partition the Drosophila genome into topological domains

PubMed Central

Flower, Rosalyn; Choo, Siew Woh

2017-01-01

It is now well established that eukaryote genomes have a common architectural organization into topologically associated domains (TADs) and evidence is accumulating that this organization plays an important role in gene regulation. However, the mechanisms that partition the genome into TADs and the nature of domain boundaries are still poorly understood. We have investigated boundary regions in the Drosophila genome and find that they can be identified as domains of very low H3K27me3. The genome-wide H3K27me3 profile partitions into two states; very low H3K27me3 identifies Depleted (D) domains that contain housekeeping genes and their regulators such as the histone acetyltransferase-containing NSL complex, whereas domains containing moderate-to-high levels of H3K27me3 (Enriched or E domains) are associated with regulated genes, irrespective of whether they are active or inactive. The D domains correlate with the boundaries of TADs and are enriched in a subset of architectural proteins, particularly Chromator, BEAF-32, and Z4/Putzig. However, rather than being clustered at the borders of these domains, these proteins bind throughout the H3K27me3-depleted regions and are much more strongly associated with the transcription start sites of housekeeping genes than with the H3K27me3 domain boundaries. While we have not demonstrated causality, we suggest that the D domain chromatin state, characterised by very low or absent H3K27me3 and established by housekeeping gene regulators, acts to separate topological domains thereby setting up the domain architecture of the genome. PMID:28282436

Characterization of domain-peptide interaction interface: a case study on the amphiphysin-1 SH3 domain.

PubMed

Hou, Tingjun; Zhang, Wei; Case, David A; Wang, Wei

2008-02-29

Many important protein-protein interactions are mediated by peptide recognition modular domains, such as the Src homology 3 (SH3), SH2, PDZ, and WW domains. Characterizing the interaction interface of domain-peptide complexes and predicting binding specificity for modular domains are critical for deciphering protein-protein interaction networks. Here, we propose the use of an energetic decomposition analysis to characterize domain-peptide interactions and the molecular interaction energy components (MIECs), including van der Waals, electrostatic, and desolvation energy between residue pairs on the binding interface. We show a proof-of-concept study on the amphiphysin-1 SH3 domain interacting with its peptide ligands. The structures of the human amphiphysin-1 SH3 domain complexed with 884 peptides were first modeled using virtual mutagenesis and optimized by molecular mechanics (MM) minimization. Next, the MIECs between domain and peptide residues were computed using the MM/generalized Born decomposition analysis. We conducted two types of statistical analyses on the MIECs to demonstrate their usefulness for predicting binding affinities of peptides and for classifying peptides into binder and non-binder categories. First, combining partial least squares analysis and genetic algorithm, we fitted linear regression models between the MIECs and the peptide binding affinities on the training data set. These models were then used to predict binding affinities for peptides in the test data set; the predicted values have a correlation coefficient of 0.81 and an unsigned mean error of 0.39 compared with the experimentally measured ones. The partial least squares-genetic algorithm analysis on the MIECs revealed the critical interactions for the binding specificity of the amphiphysin-1 SH3 domain. Next, a support vector machine (SVM) was employed to build classification models based on the MIECs of peptides in the training set. A rigorous training-validation procedure was

A New Domain of Reactivity for High-Valent Dinuclear [M(μ-O)2 M'] Complexes in Oxidation Reactions.

PubMed

Engelmann, Xenia; Yao, Shenglai; Farquhar, Erik R; Szilvási, Tibor; Kuhlmann, Uwe; Hildebrandt, Peter; Driess, Matthias; Ray, Kallol

2017-01-02

The strikingly different reactivity of a series of homo- and heterodinuclear [(M III )(μ-O) 2 (M III )'] 2+ (M=Ni; M'=Fe, Co, Ni and M=M'=Co) complexes with β-diketiminate ligands in electrophilic and nucleophilic oxidation reactions is reported, and can be correlated to the spectroscopic features of the [(M III )(μ-O) 2 (M III )'] 2+ core. In particular, the unprecedented nucleophilic reactivity of the symmetric [Ni III (μ-O) 2 Ni III ] 2+ complex and the decay of the asymmetric [Ni III (μ-O) 2 Co III ] 2+ core through aromatic hydroxylation reactions represent a new domain for high-valent bis(μ-oxido)dimetal reactivity. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

The domain structure and distribution of Alu elements in long noncoding RNAs and mRNAs

PubMed Central

Kim, Eugene Z.; Wespiser, Adam R.; Caffrey, Daniel R.

2016-01-01

Approximately 75% of the human genome is transcribed and many of these spliced transcripts contain primate-specific Alu elements, the most abundant mobile element in the human genome. The majority of exonized Alu elements are located in long noncoding RNAs (lncRNAs) and the untranslated regions of mRNA, with some performing molecular functions. To further assess the potential for Alu elements to be repurposed as functional RNA domains, we investigated the distribution and evolution of Alu elements in spliced transcripts. Our analysis revealed that Alu elements are underrepresented in mRNAs and lncRNAs, suggesting that most exonized Alu elements arising in the population are rare or deleterious to RNA function. When mRNAs and lncRNAs retain exonized Alu elements, they have a clear preference for Alu dimers, left monomers, and right monomers. mRNAs often acquire Alu elements when their genes are duplicated within Alu-rich regions. In lncRNAs, reverse-oriented Alu elements are significantly enriched and are not restricted to the 3′ and 5′ ends. Both lncRNAs and mRNAs primarily contain the Alu J and S subfamilies that were amplified relatively early in primate evolution. Alu J subfamilies are typically overrepresented in lncRNAs, whereas the Alu S dimer is overrepresented in mRNAs. The sequences of Alu dimers tend to be constrained in both lncRNAs and mRNAs, whereas the left and right monomers are constrained within particular Alu subfamilies and classes of RNA. Collectively, these findings suggest that Alu-containing RNAs are capable of forming stable structures and that some of these Alu domains might have novel biological functions. PMID:26654912

A gain-of-function mutation in the M-domain of cardiac myosin-binding protein-C increases binding to actin.

PubMed

Bezold, Kristina L; Shaffer, Justin F; Khosa, Jaskiran K; Hoye, Elaine R; Harris, Samantha P

2013-07-26

The M-domain is the major regulatory subunit of cardiac myosin-binding protein-C (cMyBP-C) that modulates actin and myosin interactions to influence muscle contraction. However, the precise mechanism(s) and the specific residues involved in mediating the functional effects of the M-domain are not fully understood. Positively charged residues adjacent to phosphorylation sites in the M-domain are thought to be critical for effects of cMyBP-C on cross-bridge interactions by mediating electrostatic binding with myosin S2 and/or actin. However, recent structural studies revealed that highly conserved sequences downstream of the phosphorylation sites form a compact tri-helix bundle. Here we used site-directed mutagenesis to probe the functional significance of charged residues adjacent to the phosphorylation sites and conserved residues within the tri-helix bundle. Results confirm that charged residues adjacent to phosphorylation sites and residues within the tri-helix bundle are important for mediating effects of the M-domain on contraction. In addition, four missense variants within the tri-helix bundle that are associated with human hypertrophic cardiomyopathy caused either loss-of-function or gain-of-function effects on force. Importantly, the effects of the gain-of-function variant, L348P, increased the affinity of the M-domain for actin. Together, results demonstrate that functional effects of the M-domain are not due solely to interactions with charged residues near phosphorylatable serines and provide the first demonstration that the tri-helix bundle contributes to the functional effects of the M-domain, most likely by binding to actin.

Structures of Ca(V) Ca**2+/CaM-IQ Domain Complexes Reveal Binding Modes That Underlie Calcium-Dependent Inactivation And Facilitation

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

Kim, E.Y.; Rumpf, C.H.; Fujiwara, Y.

2009-05-20

Calcium influx drives two opposing voltage-activated calcium channel (Ca{sub V}) self-modulatory processes: calcium-dependent inactivation (CDI) and calcium-dependent facilitation (CDF). Specific Ca{sup 2+}/calmodulin (Ca{sup 2+}/CaM) lobes produce CDI and CDF through interactions with the Ca{sub V}{alpha}{sub 1} subunit IQ domain. Curiously, Ca{sup 2+}/CaM lobe modulation polarity appears inverted between Ca{sub V}1s and Ca{sub V}2s. Here, we present crystal structures of Ca{sub V}2.1, Ca{sub V}2.2, and Ca{sub V}2.3 Ca{sup 2+}/CaM-IQ domain complexes. All display binding orientations opposite to Ca{sub V}1.2 with a physical reversal of the CaM lobe positions relative to the IQ {alpha}-helix. Titration calorimetry reveals lobe competition for a high-affinitymore » site common to Ca{sub V}1 and Ca{sub V}2 IQ domains that is occupied by the CDI lobe in the structures. Electrophysiological experiments demonstrate that the N-terminal Ca{sub V}2 Ca{sup 2+}/C-lobe anchors affect CDF. Together, the data unveil the remarkable structural plasticity at the heart of Ca{sub V} feedback modulation and indicate that Ca{sub V}1 and Ca{sub V}2 IQ domains bear a dedicated CDF site that exchanges Ca{sup 2+}/CaM lobe occupants.« less

Inhibition of m3 muscarinic acetylcholine receptors by local anaesthetics

PubMed Central

Hollmann, Markus W; Ritter, Carsten H; Henle, Philipp; de Klaver, Manuela; Kamatchi, Ganesan L; Durieux, Marcel E

2001-01-01

Muscarinic m1 receptors are inhibited by local anaesthetics (LA) at nM concentrations. To elucidate in more detail the site(s) of LA interaction, we compared these findings with LA effects on m3 muscarinic receptors. We expressed receptors in Xenopus oocytes. Using two-electrode voltage clamp, we measured the effects of lidocaine, QX314 (permanently charged) and benzocaine (permanently uncharged) on Ca2+-activated Cl−-currents (ICl(Ca)), elicited by acetyl-β-methylcholine bromide (MCh). We also characterized the interaction of lidocaine with [3H]-quinuclydinyl benzylate ([3H]-QNB) binding to m3 receptors. Antisense-injection was used to determine the role of specific G-protein α subunits in mediating the inhibitory effects of LA. Using chimeric receptor constructs we investigated which domains of the muscarinic receptors contribute to the binding site for LA. Lidocaine inhibited m3-signalling in a concentration-dependent, reversible, non-competitive manner with an IC50 of 370 nM, approximately 21 fold higher than the IC50 (18 nM) reported for m1 receptors. Intracellular inhibition of both signalling pathways by LA was similar, and dependent on the Gq- protein α subunit. In contrast to results reported for the m1 receptor, the m3 receptor lacks the major extracellular binding site for charged LA. The N-terminus and third extracellular loop of the m1 muscarinic receptor molecule were identified as requirements to obtain extracellular inhibition by charged LA. PMID:11325812

77 FR 4400 - Additional Designations, Foreign Narcotics Kingpin Designation Act

Federal Register 2010, 2011, 2012, 2013, 2014

2012-01-27

..., DOB 04 OCT 1972; POB Guatemala City, Guatemala; nationality Guatemala; (INDIVIDUAL) [SDNTK] 2. BORRAYO...; citizen Guatemala; (INDIVIDUAL) [SDNTK] 3. FERNANDEZ CARBAJAL, Jorge Andres, DOB 26 Feb 1958; Passport..., DOB 30 Mar 1974; POB Guatemala; Passport 008818499; Nationality Guatemalan; (INDIVIDUAL) [SDNTK...

Generation and functional analysis of monoclonal antibodies against the second extracellular loop of human M3 muscarinic acetylcholine receptor.

PubMed

Tsuboi, Hiroto; Nakamura, Yumi; Iizuka, Mana; Matsuo, Naomi; Matsumoto, Isao; Sumida, Takayuki

2012-04-01

The M3 muscarinic acetylcholine receptor (M3R) plays a crucial role in the activation of salivary and lachrymal glands. The M3R contains four extracellular domains (the N-terminal, and the first, second, and third extracellular loops), and we recently detected antibodies against each of these four domains in a subgroup of patients with Sjögren's syndrome (SS). Functional analysis indicated that the influence of such anti-M3R antibodies on salivary secretion might differ based on the epitopes to which they bind. To clarify the relationship between B-cell epitopes on the M3R and its function, we generated two hybridomas producing anti-M3R monoclonal antibodies against the second extracellular loop of M3R (anti-M3R(2nd) mAbs) and analyzed their function by Ca(2+)-influx assays, using a human salivary gland (HSG) cell line. These two anti-M3R(2nd) mAbs suppressed Ca(2+)-influx in the HSG cells induced by cevimeline stimulation, suggesting that autoantibodies against the second extracellular loop of M3R could be involved in salivary dysfunction in patients with SS.

Textural domain walls in superfluid 3He-B

NASA Astrophysics Data System (ADS)

Mizushima, Takeshi

Owing to the richness of symmetry, the superfluid 3He serves as a rich repository of topological quantum phenomena. This includes the emergence of surface Majorana fermions and their quantum mass acquisition at the topological critical point. Furthermore, the marriage of the prototype topological superfluid with nanofabrication techniques brings about a rich variety of spontaneous symmetry breaking, such as the formation of the stripe order and nontrivial domain walls. In this work, we examine the possible formation of textural domain walls in the superfluid 3He-B confined to a thin slab with a sub-micron thickness. When an applied magnetic field is much higher than the dipolar field, two nearly degenerate ground states appear, which are characterized by the Ising order associated with the spontaneous breaking of a magnetic order-two symmetry, lcirc;z = + 1 and - 1 . We here discuss the structure of the textural domain wall formed by the spatial modulation of the Ising order, such as low-lying quasiparticle excitations and spontaneous spin current. We also report bosonic modes bound to the textural domain wall.

Impaired Bone Resorption by Lipopolysaccharide In Vivo in Mice Deficient in the Prostaglandin E Receptor EP4 Subtype

PubMed Central

Sakuma, Yoko; Tanaka, Kiyoshi; Suda, Michio; Komatsu, Yasato; Yasoda, Akihiro; Miura, Masako; Ozasa, Ami; Narumiya, Shuh; Sugimoto, Yukihiko; Ichikawa, Atsushi; Ushikubi, Fumitaka; Nakao, Kazuwa

2000-01-01

In a previous study we showed that the involvement of EP4 subtype of the prostaglandin E (PGE) receptor is crucial for lipopolysaccharide (LPS)-induced osteoclast formation in vitro. The present study was undertaken to test whether EP4 is actually associated with LPS-induced bone resorption in vivo. In wild-type (WT) mice, osteoclast formation in vertebrae and tibiae increased 5 days after systemic LPS injection, and urinary excretion of deoxypyridinoline, a sensitive marker for bone resorption, statistically increased 10 days after injection. In EP4 knockout (KO) mice, however, LPS injection caused no significant changes in these parameters throughout the experiment. LPS exposure for 4 h strongly induced osteoclast differentiation factor (ODF) mRNA expression in primary osteoblastic cells (POB) both from WT and EP4 KO mice, and this expression was not inhibited by indomethacin, suggesting prostaglandin (PG) independence. LPS exposure for 24 h further induced ODF expression in WT POB, but not in EP4 KO POB. Indomethacin partially inhibited ODF expression in WT POB, but not in EP4 KO POB. These data suggest that ODF is induced both PG dependently and PG independently. LPS exposure for 24 h induced slightly greater osteoclastgenesis inhibitory factor (OCIF) mRNA expression in EP4 KO than in WT POB. These findings suggest that the reduced ODF expression and apparently increased OCIF expression also are responsible for the markedly reduced LPS-induced osteoclast formation in EP4 KO mice. Our results show that the EP4 subtype of the PGE receptor is involved in LPS-induced bone resorption in vivo also. Since LPS is considered to be largely involved in bacterially induced bone loss, such as in periodontitis and osteomyelitis, our study is expected to help broaden our understanding of the pathophysiology of these conditions. PMID:11083800

Phase coexistence and domain configuration in Pb(Mg{sub 1/3}Nb{sub 2/3})O{sub 3}-0.34PbTiO{sub 3} single crystal revealed by synchrotron-based X-ray diffractive three-dimensional reciprocal space mapping and piezoresponse force microscopy

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

Wang, Ruixue; Yang, Bin, E-mail: binyang@hit.edu.cn; Sun, Enwei

The crystalline phases and domain configuration in the morphotropic phase boundary composition Pb(Mg{sub 1/3}Nb{sub 2/3})O{sub 3}-0.34PbTiO{sub 3} (PMN-0.34PT) single crystal have been investigated by synchrotron-based X-ray 3D Reciprocal Space Mapping (3D-RSM) and Piezoresponse Force Microscopy. The coexistence of tetragonal (T) and monoclinic M{sub C} phases in this PMN-0.34PT single crystal is confirmed. The affiliation of each diffraction spot in the 3D-RSM was identified with the assistance of qualitative simulation. Most importantly, the twinning structure between different domains in such a mixed phase PMN-PT crystal is firmly clarified, and the spatial distribution of different twin domains is demonstrated. In addition, themore » lattice parameters of T and M{sub C} phases in PMN-0.34PT single crystal as well as the tilting angles of crystal lattices caused by the interfacial lattice mismatch are determined.« less

Phosphatidylinositol 3,5-Bisphosphate-Rich Membrane Domains in Endosomes and Lysosomes.

PubMed

Takatori, Sho; Tatematsu, Tsuyako; Cheng, Jinglei; Matsumoto, Jun; Akano, Takuya; Fujimoto, Toyoshi

2016-02-01

Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2 ) has critical functions in endosomes and lysosomes. We developed a method to define nanoscale distribution of PtdIns(3,5)P2 using freeze-fracture electron microscopy. GST-ATG18-4×FLAG was used to label PtdIns(3,5)P2 and its binding to phosphatidylinositol 3-phosphate (PtdIns(3)P) was blocked by an excess of the p40(phox) PX domain. In yeast exposed to hyperosmotic stress, PtdIns(3,5)P2 was concentrated in intramembrane particle (IMP)-deficient domains in the vacuolar membrane, which made close contact with adjacent membranes. The IMP-deficient domain was also enriched with PtdIns(3)P, but was deficient in Vph1p, a liquid-disordered domain marker. In yeast lacking either PtdIns(3,5)P2 or its effector, Atg18p, the IMP-deficient, PtdIns(3)P-rich membranes were folded tightly to make abnormal tubular structures, thus showing where the vacuolar fragmentation process is arrested when PtdIns(3,5)P2 metabolism is defective. In HeLa cells, PtdIns(3,5)P2 was significantly enriched in the vesicular domain of RAB5- and RAB7-positive endosome/lysosomes of the tubulo-vesicular morphology. This biased distribution of PtdIns(3,5)P2 was also observed using fluorescence microscopy, which further showed enrichment of a retromer component, VPS35, in the tubular domain. This is the first report to show segregation of PtdIns(3,5)P2 -rich and -deficient domains in endosome/lysosomes, which should be important for endosome/lysosome functionality. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Disease-Homologous Mutation in the Cation Diffusion Facilitator Protein MamM Causes Single-Domain Structural Loss and Signifies Its Importance

PubMed Central

Barber-Zucker, Shiran; Uebe, René; Davidov, Geula; Navon, Yotam; Sherf, Dror; Chill, Jordan H.; Kass, Itamar; Bitton, Ronit; Schüler, Dirk; Zarivach, Raz

2016-01-01

Cation diffusion facilitators (CDF) are highly conserved, metal ion efflux transporters that maintain divalent transition metal cation homeostasis. Most CDF proteins contain two domains, the cation transporting transmembrane domain and the regulatory cytoplasmic C-terminal domain (CTD). MamM is a magnetosome-associated CDF protein essential for the biomineralization of magnetic iron-oxide particles in magnetotactic bacteria. To investigate the structure-function relationship of CDF cytoplasmic domains, we characterized a MamM M250P mutation that is synonymous with the disease-related mutation L349P of the human CDF protein ZnT-10. Our results show that the M250P exchange in MamM causes severe structural changes in its CTD resulting in abnormal reduced function. Our in vivo, in vitro and in silico studies indicate that the CTD fold is critical for CDF proteins’ proper function and support the previously suggested role of the CDF cytoplasmic domain as a CDF regulatory element. Based on our results, we also suggest a mechanism for the effects of the ZnT-10 L349P mutation in human. PMID:27550551

A novel TPR–BEN domain interaction mediates PICH–BEND3 association

PubMed Central

Pitchai, Ganesha P.; Kaulich, Manuel; Mesa, Pablo; Yao, Qi; Sarlos, Kata; Streicher, Werner W.; Nigg, Erich A.

2017-01-01

Abstract PICH is a DNA translocase required for the maintenance of chromosome stability in human cells. Recent data indicate that PICH co-operates with topoisomerase IIα to suppress pathological chromosome missegregation through promoting the resolution of ultra-fine anaphase bridges (UFBs). Here, we identify the BEN domain-containing protein 3 (BEND3) as an interaction partner of PICH in human cells in mitosis. We have purified full length PICH and BEND3 and shown that they exhibit a functional biochemical interaction in vitro. We demonstrate that the PICH–BEND3 interaction occurs via a novel interface between a TPR domain in PICH and a BEN domain in BEND3, and have determined the crystal structure of this TPR–BEN complex at 2.2 Å resolution. Based on the structure, we identified amino acids important for the TPR–BEN domain interaction, and for the functional interaction of the full-length proteins. Our data reveal a proposed new function for BEND3 in association with PICH, and the first example of a specific protein–protein interaction mediated by a BEN domain. PMID:28977671

77 FR 69707 - Additional Designations, Foreign Narcotics Kingpin Designation Act

Federal Register 2010, 2011, 2012, 2013, 2014

2012-11-20

..., Hazarjuft Area, Garmsir District, Helmand Province, Afghanistan; alt. POB Laki Village, Garmsir District, Helmand Province, Afghanistan; alt. POB Lakari Village, Garmsir District, Helmand Province, Afghanistan; alt. POB Darvishan, Garmsir District, Helmand Province, Afghanistan; alt. POB De Luy Wiyalah Village...

The C-terminal domains of NF-H and NF-M subunits maintain axonal neurofilament content by blocking turnover of the stationary neurofilament network.

PubMed

Rao, Mala V; Yuan, Aidong; Campbell, Jabbar; Kumar, Asok; Nixon, Ralph A

2012-01-01

Newly synthesized neurofilaments or protofilaments are incorporated into a highly stable stationary cytoskeleton network as they are transported along axons. Although the heavily phosphorylated carboxyl-terminal tail domains of the heavy and medium neurofilament (NF) subunits have been proposed to contribute to this process and particularly to stability of this structure, their function is still obscure. Here we show in NF-H/M tail deletion [NF-(H/M)(tailΔ)] mice that the deletion of both of these domains selectively lowers NF levels 3-6 fold along optic axons without altering either rates of subunit synthesis or the rate of slow axonal transport of NF. Pulse labeling studies carried out over 90 days revealed a significantly faster rate of disappearance of NF from the stationary NF network of optic axons in NF-(H/M)(tailΔ) mice. Faster NF disappearance was accompanied by elevated levels of NF-L proteolytic fragments in NF-(H/M)(tailΔ) axons. We conclude that NF-H and NF-M C-terminal domains do not normally regulate NF transport rates as previously proposed, but instead increase the proteolytic resistance of NF, thereby stabilizing the stationary neurofilament cytoskeleton along axons.

The C-Terminal Domains of NF-H and NF-M Subunits Maintain Axonal Neurofilament Content by Blocking Turnover of the Stationary Neurofilament Network

PubMed Central

Rao, Mala V.; Yuan, Aidong; Campbell, Jabbar; Kumar, Asok; Nixon, Ralph A.

2012-01-01

Newly synthesized neurofilaments or protofilaments are incorporated into a highly stable stationary cytoskeleton network as they are transported along axons. Although the heavily phosphorylated carboxyl-terminal tail domains of the heavy and medium neurofilament (NF) subunits have been proposed to contribute to this process and particularly to stability of this structure, their function is still obscure. Here we show in NF-H/M tail deletion [NF-(H/M)tailΔ] mice that the deletion of both of these domains selectively lowers NF levels 3–6 fold along optic axons without altering either rates of subunit synthesis or the rate of slow axonal transport of NF. Pulse labeling studies carried out over 90 days revealed a significantly faster rate of disappearance of NF from the stationary NF network of optic axons in NF-(H/M)tailΔ mice. Faster NF disappearance was accompanied by elevated levels of NF-L proteolytic fragments in NF-(H/M)tailΔ axons. We conclude that NF-H and NF-M C-terminal domains do not normally regulate NF transport rates as previously proposed, but instead increase the proteolytic resistance of NF, thereby stabilizing the stationary neurofilament cytoskeleton along axons. PMID:23028520

GRP1 PH Domain, Like AKT1 PH Domain, Possesses a Sentry Glutamate Residue Essential for Specific Targeting to Plasma Membrane PI(3,4,5)P3

PubMed Central

Pilling, Carissa; Landgraf, Kyle E.; Falke, Joseph J.

2011-01-01

During the appearance of the signaling lipid PI(3,4,5)P3, an important subset of pleckstrin homology (PH) domains target signaling proteins to the plasma membrane. To ensure proper pathway regulation, such PI(3,4,5)P3-specific PH domains must exclude the more prevalant, constitutive plasma membrane lipid PI(4,5)P2 and bind the rare PI(3,4,5)P3 target lipid with sufficiently high affinity. Our previous study of the E17K mutant of protein kinase B (AKT1) PH domain, together with evidence from Carpten et al (1), revealed that the native AKT1 E17 residue serves as a sentry glutamate that excludes PI(4,5)P2, thereby playing an essential role in specific PI(3,4,5)P3 targeting (2). The sentry glutamate hypothesis proposes that an analogous sentry glutamate residue is a widespread feature of PI(3,4,5)P3-specific PH domains, and that charge reversal mutation at the sentry glutamate position will yield both increased PI(4,5)P2 affinity and constitutive plasma membrane targeting. To test this hypothesis the present study investigates the E345 residue, a putative sentry glutamate, of General Receptor for Phosphoinositides 1 (GRP1) PH domain. The results show that incorporation of the E345K charge reversal mutation into GRP1 PH domain enhances PI(4,5)P2 affinity 8-fold and yields constitutive plasma membrane targeting in cells, reminiscent of the effects of the E17K mutation in AKT1 PH domain. Hydrolysis of plasma membrane PI(4,5)P2 releases E345K GRP1 PH domain into the cytoplasm and the efficiency of this release increases when target Arf6 binding is disrupted. Overall, the findings provide strong support for the sentry glutamate hypothesis and suggest that the GRP1 E345K mutation will be linked to changes in cell physiology and human pathologies, as demonstrated for AKT1 E17K (1, 3). Analysis of available PH domain structures suggests that a lone glutamate residue (or, in some cases an aspartate) is a common, perhaps ubiquitous, feature of PI(3,4,5)P3-specific binding

A novel TPR-BEN domain interaction mediates PICH-BEND3 association.

PubMed

Pitchai, Ganesha P; Kaulich, Manuel; Bizard, Anna H; Mesa, Pablo; Yao, Qi; Sarlos, Kata; Streicher, Werner W; Nigg, Erich A; Montoya, Guillermo; Hickson, Ian D

2017-11-02

PICH is a DNA translocase required for the maintenance of chromosome stability in human cells. Recent data indicate that PICH co-operates with topoisomerase IIα to suppress pathological chromosome missegregation through promoting the resolution of ultra-fine anaphase bridges (UFBs). Here, we identify the BEN domain-containing protein 3 (BEND3) as an interaction partner of PICH in human cells in mitosis. We have purified full length PICH and BEND3 and shown that they exhibit a functional biochemical interaction in vitro. We demonstrate that the PICH-BEND3 interaction occurs via a novel interface between a TPR domain in PICH and a BEN domain in BEND3, and have determined the crystal structure of this TPR-BEN complex at 2.2 Å resolution. Based on the structure, we identified amino acids important for the TPR-BEN domain interaction, and for the functional interaction of the full-length proteins. Our data reveal a proposed new function for BEND3 in association with PICH, and the first example of a specific protein-protein interaction mediated by a BEN domain. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Structure of the SH3 Domain of Rat Endophilin A2

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

Loll,P.; Swain, E.; Chen, Y.

2008-01-01

The crystal structure of the SH3 domain of rat endophilin A2 has been determined by the multiwavelength anomalous dispersion method and refined at a resolution of 1.70 Angstroms to R and Rfree values of 0.196 and 0.217, respectively. The structure adheres to the canonical SH3-domain fold and is highly similar to those of the corresponding domains of endophilins A1 and A3. An intermolecular packing interaction between two molecules in the lattice exploits features that are commonly observed in SH3-domain ligand recognition, including the insertion of a proline side chain into the ligand-binding groove of the protein and the recognition ofmore » a basic residue by a cluster of acidic side chains on the RT loop.« less

Deletion of uncharacterized domain from α-1,3-glucanase of Bacillus circulans KA-304 enhances heterologous enzyme production in Escherichia coli.

PubMed

Yano, Shigekazu; Suyotha, Wasana; Zanma, Sumika; Konno, Hiroyuki; Cherdvorapong, Vipavee; Wakayama, Mamoru

2018-05-08

α-1,3-Glucanase (Agl-KA) of Bacillus circulans KA-304 consists of an N-terminal discoidin domain (DS1), a carbohydrate binding module family 6 (CBM6), threonine and proline repeats (TP), a second discoidin domain (DS2), an uncharacterized conserved domain (UCD), and a C-terminal catalytic domain. Previously, we reported that DS1, CBM6, and DS2 have α-1,3-glucan-binding activity and contribute to α-1,3-glucan hydrolysis. In this study, UCD deletion mutant (AglΔUCD) was constructed, and its properties were compared with those of Agl-KA. α-1,3-Glucan hydrolyzing, α-1,3-glucan binding, and protoplast-forming activities of AglΔUCD were almost the same as those of Agl-KA. k cat /K m values of AgΔUCD and Agl-KA were 11.4 and 11.1 s -1 mg -1 mL, respectively. AglΔUCD and Agl-KA exhibited similar characteristics, such as optimal pH, pH stability, optimal temperature, and thermostability. These results suggest that UCD is not α-1,3-glucan-binding and flexible linker domain, and that deletion of UCD does not affect the affinity of N-terminal binding domains and the catalytic action of the C-terminal domain. Subsequently, heterologous UCenzyme productivity of AglΔD in Escherichia coli was compared with that of Agl-KA. The productivity of AglΔUCD was about 4-fold larger than that of Agl-KA after an 8-h induction at 30°C. In the case of induction at 20°C, the productivity of AglΔUCD was also larger than that of Agl-KA. These findings indicate that deletion of only UCD enhances the enzyme productivity in E. coli.

Single TRAM domain RNA-binding proteins in Archaea: functional insight from Ctr3 from the Antarctic methanogen Methanococcoides burtonii.

PubMed

Taha; Siddiqui, K S; Campanaro, S; Najnin, T; Deshpande, N; Williams, T J; Aldrich-Wright, J; Wilkins, M; Curmi, P M G; Cavicchioli, R

2016-09-01

TRAM domain proteins present in Archaea and Bacteria have a β-barrel shape with anti-parallel β-sheets that form a nucleic acid binding surface; a structure also present in cold shock proteins (Csps). Aside from protein structures, experimental data defining the function of TRAM domains is lacking. Here, we explore the possible functional properties of a single TRAM domain protein, Ctr3 (cold-responsive TRAM domain protein 3) from the Antarctic archaeon Methanococcoides burtonii that has increased abundance during low temperature growth. Ribonucleic acid (RNA) bound by Ctr3 in vitro was determined using RNA-seq. Ctr3-bound M. burtonii RNA with a preference for transfer (t)RNA and 5S ribosomal RNA, and a potential binding motif was identified. In tRNA, the motif represented the C loop; a region that is conserved in tRNA from all domains of life and appears to be solvent exposed, potentially providing access for Ctr3 to bind. Ctr3 and Csps are structurally similar and are both inferred to function in low temperature translation. The broad representation of single TRAM domain proteins within Archaea compared with their apparent absence in Bacteria, and scarcity of Csps in Archaea but prevalence in Bacteria, suggests they represent distinct evolutionary lineages of functionally equivalent RNA-binding proteins. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

Functional properties and structural requirements of the plasmid pMV158-encoded MobM relaxase domain.

PubMed

Fernández-López, Cris; Pluta, Radoslaw; Pérez-Luque, Rosa; Rodríguez-González, Lorena; Espinosa, Manuel; Coll, Miquel; Lorenzo-Díaz, Fabián; Boer, D Roeland

2013-07-01

A crucial element in the horizontal transfer of mobilizable and conjugative plasmids is the relaxase, a single-stranded endonuclease that nicks the origin of transfer (oriT) of the plasmid DNA. The relaxase of the pMV158 mobilizable plasmid is MobM (494 residues). In solution, MobM forms a dimer through its C-terminal domain, which is proposed to anchor the protein to the cell membrane and to participate in type 4 secretion system (T4SS) protein-protein interactions. In order to gain a deeper insight into the structural MobM requirements for efficient DNA catalysis, we studied two endonuclease domain variants that include the first 199 or 243 amino acid residues (MobMN199 and MobMN243, respectively). Our results confirmed that the two proteins behaved as monomers in solution. Interestingly, MobMN243 relaxed supercoiled DNA and cleaved single-stranded oligonucleotides harboring oriTpMV158, whereas MobMN199 was active only on supercoiled DNA. Protein stability studies using gel electrophoresis and mass spectrometry showed increased susceptibility to degradation at the domain boundary between the N- and C-terminal domains, suggesting that the domains change their relative orientation upon DNA binding. Overall, these results demonstrate that MobMN243 is capable of nicking the DNA substrate independently of its topology and that the amino acids 200 to 243 modulate substrate specificity but not the nicking activity per se. These findings suggest that these amino acids are involved in positioning the DNA for the nuclease reaction rather than in the nicking mechanism itself.

Dact1-3 mRNAs exhibit distinct expression domains during tooth development

PubMed Central

Kettunen, Päivi; Kivimäe, Saul; Keshari, Pankaj; Klein, Ophir D.; Cheyette, Benjamin N.R.; Luukko, Keijo

2010-01-01

Wnt signaling is essential for tooth formation. Dact proteins modulate Wnt signaling by binding to the intracellular protein Dishevelled (Dvl). Comparison of all known mouse Dact genes, Dact1-3, from the morphological initiation of mandibular first molar development after the onset of the root formation using sectional in situ hybridization showed distinct, complementary and overlapping expression patterns for the studied genes. While Dact2 expression was restricted to the dental epithelium including the enamel knot signaling centers and tooth specific preameloblasts, Dact1 and Dact3 showed developmentally regulated expression in the dental mesenchyme. Both mRNAs were first detected in the presumptive dental mesenchyme. After being downregulated from the condensed dental mesenchyme of the bud stage tooth germ, Dact1 was upregulated in the dental follicle masenchyme at the cap stage and subsequently also in the dental papilla at the bell stage where the expression persisted to the postnatal stages. In contrast, Dact3 transcripts persisted throughout the dental mesenchymal tissue components including the tooth-specific cells, preodontoblasts before transcripts were largely downregulated from the tooth germ postnatally. Collectively these results suggest that Dact1 and -3 may contribute to early tooth formation by modulation of Wnt signaling pathways in the mesenchyme, including preodontoblasts, whereas Dact2 may play important signal-modulating roles in the adjacent epithelial cells including the enamel knot signaling centers and preameloblasts. Future loss-of-function studies will help elucidate whether any of these functions are redundant, particularly for Dact1 and Dact3. PMID:20170752

One-Dimensional Singlet Exciton Diffusion in Poly(3-hexylthiophene) Crystalline Domains.

PubMed

Tamai, Yasunari; Matsuura, Yuu; Ohkita, Hideo; Benten, Hiroaki; Ito, Shinzaburo

2014-01-16

Singlet exciton dynamics in crystalline domains of regioregular poly(3-hexylthiophene) (P3HT) films was studied by transient absorption spectroscopy. Upon the selective excitation of crystalline P3HT at the absorption edge, no red shift of the singlet exciton band was observed with an elapse of time, suggesting singlet exciton dynamics in relatively homogeneous P3HT crystalline domains without downhill relaxation in the energetic disorder. Even under such selective excitation conditions, the annihilation rate coefficient γ(t) was still dependent on time, γ(t) ∝ t(-1/2), which is attributed to anisotropic exciton diffusion in P3HT crystalline domains. From the annihilation rate coefficient, the singlet exciton diffusion coefficient D and exciton diffusion length LD in the crystalline domains were evaluated to be 7.9 × 10(-3) cm(2) s(-1) and 20 nm, respectively. The origin of the time-dependent exciton dynamics is discussed in terms of dimensionality.

Mass production of polyhedral occlusion bodies of NPV of Helicoverpa armigera in relation to dose, age and larval weight.

PubMed

Narayanan, K; Jayaraj, S

2002-07-01

A significant difference was noticed in the yield of polyhedral occlusion bodies (POBs) in various larval instars of H. armigera when three different doses of the nuclear polyhedrosis virus (NPV) were administered. The yield of POBs from a single larva ranged from 0.35 x 10(6) to 25033.33 x 10(6) with a mean of 18422.33 x 10(6) for fourth instar inoculated. Positive correlation existed between larval weight and number of POBs recovered. The regression analysis indicated POBs recovered responded with predictable manner to the weight of different larval instars and the various concentration of virus administered. The medium lethal time increased in the instars of the larva advanced with a minimum of 3.5 and maximum of 8 days in the first and fifth instars respectively.

Roles for N-terminal Extracellular Domains of Nicotinic Acetylcholine Receptor (nAChR) β3 Subunits in Enhanced Functional Expression of Mouse α6β2β3- and α6β4β3-nAChRs*

PubMed Central

Dash, Bhagirathi; Li, Ming D.; Lukas, Ronald J.

2014-01-01

Functional heterologous expression of naturally expressed mouse α6*-nicotinic acetylcholine receptors (mα6*-nAChRs; where “*” indicates the presence of additional subunits) has been difficult. Here we expressed and characterized wild-type (WT), gain-of-function, chimeric, or gain-of-function chimeric nAChR subunits, sometimes as hybrid nAChRs containing both human (h) and mouse (m) subunits, in Xenopus oocytes. Hybrid mα6mβ4hβ3- (∼5–8-fold) or WT mα6mβ4mβ3-nAChRs (∼2-fold) yielded higher function than mα6mβ4-nAChRs. Function was not detected when mα6 and mβ2 subunits were expressed together or in the additional presence of hβ3 or mβ3 subunits. However, function emerged upon expression of mα6mβ2mβ3V9′S-nAChRs containing β3 subunits having gain-of-function V9′S (valine to serine at the 9′-position) mutations in transmembrane domain II and was further elevated 9-fold when hβ3V9′S subunits were substituted for mβ3V9′S subunits. Studies involving WT or gain-of-function chimeric mouse/human β3 subunits narrowed the search for domains that influence functional expression of mα6*-nAChRs. Using hβ3 subunits as templates for site-directed mutagenesis studies, substitution with mβ3 subunit residues in extracellular N-terminal domain loops “C” (Glu221 and Phe223), “E” (Ser144 and Ser148), and “β2-β3” (Gln94 and Glu101) increased function of mα6mβ2*- (∼2–3-fold) or mα6mβ4* (∼2–4-fold)-nAChRs. EC50 values for nicotine acting at mα6mβ4*-nAChR were unaffected by β3 subunit residue substitutions in loop C or E. Thus, amino acid residues located in primary (loop C) or complementary (loops β2-β3 and E) interfaces of β3 subunits are some of the molecular impediments for functional expression of mα6mβ2β3- or mα6mβ4β3-nAChRs. PMID:25028511

Molecular LEGO by domain-imprinting of cytochrome P450 BM3.

PubMed

Jetzschmann, K J; Yarman, A; Rustam, L; Kielb, P; Urlacher, V B; Fischer, A; Weidinger, I M; Wollenberger, U; Scheller, F W

2018-04-01

Electrosynthesis of the MIP nano-film after binding of the separated domains or holo-cytochrome BM3 via an engineered anchor should result in domain-specific cavities in the polymer layer. Both the two domains and the holo P450 BM3 have been bound prior polymer deposition via a N-terminal engineered his6-anchor to the electrode surface. Each step of MIP preparation was characterized by cyclic voltammetry of the redox-marker ferricyanide. Rebinding after template removal was evaluated by quantifying the suppression of the diffusive permeability of the signal for ferricyanide and by the NADH-dependent reduction of cytochrome c by the reductase domain (BMR). The working hypothesis is verified by the discrimination of the two domains by the respective MIPs: The holoenzyme P450 BM3 was ca. 5.5 times more effectively recognized by the film imprinted with the oxidase domain (BMO) as compared to the BMR-MIP or the non-imprinted polymer (NIP). Obviously, a cavity is formed during the imprinting process around the his 6 -tag-anchored BMR which cannot accommodate the broader BMO or the P450 BM3. The affinity of the MIP towards P450 BM3 is comparable with that to the monomer in solution. The his 6 -tagged P450 BM3 binds (30 percent) stronger which shows the additive effect of the interaction with the MIP and the binding to the electrode. Copyright © 2018. Published by Elsevier B.V.

75 FR 76520 - Designation of Three Individuals Pursuant to Executive Order 13224

Federal Register 2010, 2011, 2012, 2013, 2014

2010-12-08

.... ULLAH, Aman; a.k.a. URS, Amanullah; a.k.a. ``MUFTI ILYAS''), Frontier Region Kohat, Pakistan; DOB 1973... 1974; POB Bwawal Pur, Pakistan; alt. POB Bahawalpur, Pakistan (individual) [SDGT] 3. UR-REHMAN, Mati (a...; nationality Pakistan (individual) [SDGT] Dated: December 2, 2010. Adam J. Szubin, Director, Office of Foreign...

Interface Engineering of Domain Structures in BiFeO 3 Thin Films

DOE PAGES

Chen, Deyang; Chen, Zuhuang; He, Qian; ...

2016-12-07

A wealth of fascinating phenomena have been discovered at the BiFeO 3 domain walls, examples such as domain wall conductivity, photovoltaic effects, and magnetoelectric coupling. Thus, the ability to precisely control the domain structures and accurately study their switching behaviors is critical to realize the next generation of novel devices based on domain wall functionalities. In this work, the introduction of a dielectric layer leads to the tunability of the depolarization field both in the multilayers and superlattices, which provides a novel approach to control the domain patterns of BiFeO 3 films. Moreover, we are able to study the switchingmore » behavior of the first time obtained periodic 109° stripe domains with a thick bottom electrode. Besides, the precise controlling of pure 71° and 109° periodic stripe domain walls enable us to make a clear demonstration that the exchange bias in the ferromagnet/BiFeO 3 system originates from 109° domain walls. Lastly, our findings provide future directions to study the room temperature electric field control of exchange bias and open a new pathway to explore the room temperature multiferroic vortices in the BiFeO 3 system.« less

Identification of functional domains in Arabidopsis thaliana mRNA decapping enzyme (AtDcp2)

PubMed Central

Gunawardana, Dilantha; Cheng, Heung-Chin; Gayler, Kenwyn R.

2008-01-01

The Arabidopsis thaliana decapping enzyme (AtDcp2) was characterized by bioinformatics analysis and by biochemical studies of the enzyme and mutants produced by recombinant expression. Three functionally significant regions were detected: (i) a highly disordered C-terminal region with a putative PSD-95, Discs-large, ZO-1 (PDZ) domain-binding motif, (ii) a conserved Nudix box constituting the putative active site and (iii) a putative RNA binding domain consisting of the conserved Box B and a preceding loop region. Mutation of the putative PDZ domain-binding motif improved the stability of recombinant AtDcp2 and secondary mutants expressed in Escherichia coli. Such recombinant AtDcp2 specifically hydrolysed capped mRNA to produce 7-methyl GDP and decapped RNA. AtDcp2 activity was Mn2+- or Mg2+-dependent and was inhibited by the product 7-methyl GDP. Mutation of the conserved glutamate-154 and glutamate-158 in the Nudix box reduced AtDcp2 activity up to 400-fold and showed that AtDcp2 employs the catalytic mechanism conserved amongst Nudix hydrolases. Unlike many Nudix hydrolases, AtDcp2 is refractory to inhibition by fluoride ions. Decapping was dependent on binding to the mRNA moiety rather than to the 7-methyl diguanosine triphosphate cap of the substrate. Mutational analysis of the putative RNA-binding domain confirmed the functional significance of an 11-residue loop region and the conserved Box B. PMID:18025047

Wavelet-based 3-D inversion for frequency-domain airborne EM data

NASA Astrophysics Data System (ADS)

Liu, Yunhe; Farquharson, Colin G.; Yin, Changchun; Baranwal, Vikas C.

2018-04-01

In this paper, we propose a new wavelet-based 3-D inversion method for frequency-domain airborne electromagnetic (FDAEM) data. Instead of inverting the model in the space domain using a smoothing constraint, this new method recovers the model in the wavelet domain based on a sparsity constraint. In the wavelet domain, the model is represented by two types of coefficients, which contain both large- and fine-scale informations of the model, meaning the wavelet-domain inversion has inherent multiresolution. In order to accomplish a sparsity constraint, we minimize an L1-norm measure in the wavelet domain that mostly gives a sparse solution. The final inversion system is solved by an iteratively reweighted least-squares method. We investigate different orders of Daubechies wavelets to accomplish our inversion algorithm, and test them on synthetic frequency-domain AEM data set. The results show that higher order wavelets having larger vanishing moments and regularity can deliver a more stable inversion process and give better local resolution, while the lower order wavelets are simpler and less smooth, and thus capable of recovering sharp discontinuities if the model is simple. At last, we test this new inversion algorithm on a frequency-domain helicopter EM (HEM) field data set acquired in Byneset, Norway. Wavelet-based 3-D inversion of HEM data is compared to L2-norm-based 3-D inversion's result to further investigate the features of the new method.

Stabilization and cytoskeletal-association of LDL receptor mRNA are mediated by distinct domains in its 3' untranslated region.

PubMed

Wilson, G M; Vasa, M Z; Deeley, R G

1998-05-01

The mRNA encoding the human low density lipoprotein (LDL) receptor is transiently stabilized after phorbol ester treatment of HepG2 cells and has been shown to associate with components of the cytoskeleton in this cell line (G. M. Wilson, E. A. Roberts, and R. G. Deeley, J. Lipid Res. 1997. 38: 437-446). Using an episomal expression system, fragments of the 3' untranslated region (3'UTR) of LDL receptor mRNA were transcribed in fusion with the coding region of beta-globin mRNA in HepG2 cells. Analyses of the decay kinetics of these beta-globin-LDL receptor fusion mRNA deletion mutants showed that sequences in the proximal 3'UTR of LDL receptor mRNA including several AU-rich elements (AREs) were sufficient to confer short constitutive mRNA half-life in the heterologous system. Stabilization of LDL receptor mRNA in the presence of PMA required sequences in the distal 3'UTR, at or near three Alu-like repetitive elements. Furthermore, the 3'UTR of LDL receptor mRNA conferred cytoskeletal association on the otherwise unassociated beta-globin mRNA, by a mechanism involving at least two distinct RNA elements. Comparisons of decay kinetics and subcellular localization of endogenous LDL receptor mRNA and beta-globin-LDL receptor mRNA fusions in HepG2 cells have demonstrated that several cis-acting elements in the receptor 3'UTR contribute to post-transcriptional regulation of receptor expression, and provide further support for involvement of the cytoskeleton in the regulation of LDL receptor mRNA turnover.

Structural plasticity of the TDRD3 Tudor domain probed by a fragment screening hit.

PubMed

Liu, Jiuyang; Zhang, Shuya; Liu, Mingqing; Liu, Yaqian; Nshogoza, Gilbert; Gao, Jia; Ma, Rongsheng; Yang, Yang; Wu, Jihui; Zhang, Jiahai; Li, Fudong; Ruan, Ke

2018-04-12

As a reader of di-methylated arginine on various proteins, such as histone, RNA polymerase II, PIWI and Fragile X mental retardation protein, the Tudor domain of Tudor domain-containing protein 3 (TDRD3) mediates transcriptional activation in nucleus and formation of stress granules in the cytoplasm. Despite the TDRD3 implication in cancer cell proliferation and invasion, warheads to block the di-methylated arginine recognition pocket of the TDRD3 Tudor domain have not yet been uncovered. Here we identified 14 small molecule hits against the TDRD3 Tudor domain through NMR fragment-based screening. These hits were further cross-validated by using competitive fluorescence polarization and isothermal titration calorimetry experiments. The crystal structure of the TDRD3 Tudor domain in complex with hit 1 reveals a distinct binding mode from the nature substrate. Hit 1 protrudes into the aromatic cage of the TDRD3 Tudor domain, where the aromatic residues are tilted to accommodate a sandwich-like π-π interaction. The side chain of the conserved residue N596 swings away 3.1 Å to form a direct hydrogen bond with hit 1. Moreover, this compound shows a decreased affinity against the single Tudor domain of survival motor neuron protein, but no detectable binding to neither the tandem Tudor domain of TP53-binding protein 1 nor the extended Tudor domain of staphylococcal nuclease domain-containing protein 1. Our work depicts the structural plasticity of the TDRD3 Tudor domain and paves the way for the subsequent structure-guided discovery of selective inhibitors targeting Tudor domains. Structural data are available in the PDB under the accession number 5YJ8. © 2018 Federation of European Biochemical Societies.

Distinct Ubiquitin Binding Modes Exhibited by SH3 Domains: Molecular Determinants and Functional Implications

PubMed Central

Ortega Roldan, Jose L.; Casares, Salvador; Ringkjøbing Jensen, Malene; Cárdenes, Nayra; Bravo, Jerónimo; Blackledge, Martin; Azuaga, Ana I.; van Nuland, Nico A. J.

2013-01-01

SH3 domains constitute a new type of ubiquitin-binding domains. We previously showed that the third SH3 domain (SH3-C) of CD2AP binds ubiquitin in an alternative orientation. We have determined the structure of the complex between first CD2AP SH3 domain and ubiquitin and performed a structural and mutational analysis to decipher the determinants of the SH3-C binding mode to ubiquitin. We found that the Phe-to-Tyr mutation in CD2AP and in the homologous CIN85 SH3-C domain does not abrogate ubiquitin binding, in contrast to previous hypothesis and our findings for the first two CD2AP SH3 domains. The similar alternative binding mode of the SH3-C domains of these related adaptor proteins is characterised by a higher affinity to C-terminal extended ubiquitin molecules. We conclude that CD2AP/CIN85 SH3-C domain interaction with ubiquitin constitutes a new ubiquitin-binding mode involved in a different cellular function and thus changes the previously established mechanism of EGF-dependent CD2AP/CIN85 mono-ubiquitination. PMID:24039852

Dynamic interplay between the periplasmic and transmembrane domains of GspL and GspM in the type II secretion system.

PubMed

Lallemand, Mathilde; Login, Frédéric H; Guschinskaya, Natalia; Pineau, Camille; Effantin, Géraldine; Robert, Xavier; Shevchik, Vladimir E

2013-01-01

The type II secretion system (T2SS) is a multiprotein nanomachine that transports folded proteins across the outer membrane of gram-negative bacteria. The molecular mechanisms that govern the secretion process remain poorly understood. The inner membrane components GspC, GspL and GspM possess a single transmembrane segment (TMS) and a large periplasmic region and they are thought to form a platform of unknown function. Here, using two-hybrid and pull-down assays we performed a systematic mapping of the GspC/GspL/GspM interaction regions in the plant pathogen Dickeya dadantii. We found that the TMS of these components interact with each other, implying a complex interaction network within the inner membrane. We also showed that the periplasmic, ferredoxin-like, domains of GspL and GspM drive homo- and heterodimerizations of these proteins. Disulfide bonding analyses revealed that the respective domain interfaces include the equivalent secondary-structure elements, suggesting alternating interactions of the periplasmic domains, L/L and M/M versus L/M. Finally, we found that displacements of the periplasmic GspM domain mediate coordinated shifts or rotations of the cognate TMS. These data suggest a plausible mechanism for signal transmission between the periplasmic and the cytoplasmic portions of the T2SS machine.

Dynamic Interplay between the Periplasmic and Transmembrane Domains of GspL and GspM in the Type II Secretion System

PubMed Central

Guschinskaya, Natalia; Pineau, Camille; Effantin, Géraldine; Robert, Xavier; Shevchik, Vladimir E.

2013-01-01

The type II secretion system (T2SS) is a multiprotein nanomachine that transports folded proteins across the outer membrane of gram-negative bacteria. The molecular mechanisms that govern the secretion process remain poorly understood. The inner membrane components GspC, GspL and GspM possess a single transmembrane segment (TMS) and a large periplasmic region and they are thought to form a platform of unknown function. Here, using two-hybrid and pull-down assays we performed a systematic mapping of the GspC/GspL/GspM interaction regions in the plant pathogen Dickeya dadantii. We found that the TMS of these components interact with each other, implying a complex interaction network within the inner membrane. We also showed that the periplasmic, ferredoxin-like, domains of GspL and GspM drive homo- and heterodimerizations of these proteins. Disulfide bonding analyses revealed that the respective domain interfaces include the equivalent secondary-structure elements, suggesting alternating interactions of the periplasmic domains, L/L and M/M versus L/M. Finally, we found that displacements of the periplasmic GspM domain mediate coordinated shifts or rotations of the cognate TMS. These data suggest a plausible mechanism for signal transmission between the periplasmic and the cytoplasmic portions of the T2SS machine. PMID:24223969

Super-Enhancers and Broad H3K4me3 Domains Form Complex Gene Regulatory Circuits Involving Chromatin Interactions.

PubMed

Cao, Fan; Fang, Yiwen; Tan, Hong Kee; Goh, Yufen; Choy, Jocelyn Yeen Hui; Koh, Bryan Thean Howe; Hao Tan, Jiong; Bertin, Nicolas; Ramadass, Aroul; Hunter, Ewan; Green, Jayne; Salter, Matthew; Akoulitchev, Alexandre; Wang, Wilson; Chng, Wee Joo; Tenen, Daniel G; Fullwood, Melissa J

2017-05-19

Stretched histone regions, such as super-enhancers and broad H3K4me3 domains, are associated with maintenance of cell identity and cancer. We connected super-enhancers and broad H3K4me3 domains in the K562 chronic myelogenous leukemia cell line as well as the MCF-7 breast cancer cell line with chromatin interactions. Super-enhancers and broad H3K4me3 domains showed higher association with chromatin interactions than their typical counterparts. Interestingly, we identified a subset of super-enhancers that overlap with broad H3K4me3 domains and show high association with cancer-associated genes including tumor suppressor genes. Besides cell lines, we could observe chromatin interactions by a Chromosome Conformation Capture (3C)-based method, in primary human samples. Several chromatin interactions involving super-enhancers and broad H3K4me3 domains are constitutive and can be found in both cancer and normal samples. Taken together, these results reveal a new layer of complexity in gene regulation by super-enhancers and broad H3K4me3 domains.

PNPLA3 I148M polymorphism and progressive liver disease

PubMed Central

Dongiovanni, Paola; Donati, Benedetta; Fares, Roberta; Lombardi, Rosa; Mancina, Rosellina Margherita; Romeo, Stefano; Valenti, Luca

2013-01-01

The 148 Isoleucine to Methionine protein variant (I148M) of patatin-like phospholipase domain-containing 3 (PNPLA3), a protein is expressed in the liver and is involved in lipid metabolism, has recently been identified as a major determinant of liver fat content. Several studies confirmed that the I148M variant predisposes towards the full spectrum of liver damage associated with fatty liver: from simple steatosis to steatohepatitis and progressive fibrosis. Furthermore, the I148M variant represents a major determinant of progression of alcohol related steatohepatitis to cirrhosis, and to influence fibrogenesis and related clinical outcomes in chronic hepatitis C virus hepatitis, and possibly chronic hepatitis B virus hepatitis, hereditary hemochromatosis and primary sclerosing cholangitis. All in all, studies suggest that the I148M polymorphism may represent a general modifier of fibrogenesis in liver diseases. Remarkably, the effect of the I148M variant on fibrosis was independent of that on hepatic steatosis and inflammation, suggesting that it may affect both the quantity and quality of hepatic lipids and the biology of non-parenchymal liver cells besides hepatocytes, directly promoting fibrogenesis. Therefore, PNPLA3 is a key player in liver disease progression. Assessment of the I148M polymorphism will possibly inform clinical practice in the future, whereas the determination of the effect of the 148M variant will reveal mechanisms involved in hepatic fibrogenesis. PMID:24222941

Structure-Activity Relationships of Peptides Incorporating a Bioactive Reverse-Turn Heterocycle at the Melanocortin Receptors: Identification of a 5,800-fold Mouse Melanocortin-3 Receptor (mMC3R) Selective Antagonist/Partial Agonist versus the Mouse Melanocortin-4 Receptor (mMC4R)

PubMed Central

Singh, Anamika; Dirain, Marvin; Witek, Rachel; Rocca, James R.; Edison, Arthur S; Haskell-Luevano, Carrie

2013-01-01

The melanocortin-3 (MC3) and melanocortin-4 (MC4) receptors regulate energy homeostasis, food intake, and associated physiological conditions. The MC4R has been studied extensively. Less is known about specific physiological roles of the MC3R. A major obstacle to this lack of knowledge is attributed to a limited number of identified MC3R selective ligands. We previously reported a spatial scanning approach of a 10-membered thioether-heterocycle ring incorporated into a chimeric peptide template that identified a lead nM MC4R ligand. Based upon those results, 17 compounds were designed and synthesized that focused upon modification in the pharmacophore domain. Notable results include the identification of a 0.13 nM potent 5800-fold mMC3R selective antagonist/slight partial agonist versus a 760 nM mMC4R full agonist (ligand 11). Biophysical experiments (2D 1H NMR and computer assisted molecular modeling) of this ligand resulted in the identification of an inverse γ-turn secondary structure in the ligand pharmacophore domain. PMID:23432160

Cingulin Contains Globular and Coiled-Coil Domains and Interacts with Zo-1, Zo-2, Zo-3, and Myosin

PubMed Central

Cordenonsi, Michelangelo; D'Atri, Fabio; Hammar, Eva; Parry, David A.D.; Kendrick-Jones, John; Shore, David; Citi, Sandra

1999-01-01

We characterized the sequence and protein interactions of cingulin, an M r 140–160-kD phosphoprotein localized on the cytoplasmic surface of epithelial tight junctions (TJ). The derived amino acid sequence of a full-length Xenopus laevis cingulin cDNA shows globular head (residues 1–439) and tail (1,326–1,368) domains and a central α-helical rod domain (440–1,325). Sequence analysis, electron microscopy, and pull-down assays indicate that the cingulin rod is responsible for the formation of coiled-coil parallel dimers, which can further aggregate through intermolecular interactions. Pull-down assays from epithelial, insect cell, and reticulocyte lysates show that an NH2-terminal fragment of cingulin (1–378) interacts in vitro with ZO-1 (K d ∼5 nM), ZO-2, ZO-3, myosin, and AF-6, but not with symplekin, and a COOH-terminal fragment (377–1,368) interacts with myosin and ZO-3. ZO-1 and ZO-2 immunoprecipitates contain cingulin, suggesting in vivo interactions. Full-length cingulin, but not NH2-terminal and COOH-terminal fragments, colocalizes with endogenous cingulin in transfected MDCK cells, indicating that sequences within both head and rod domains are required for TJ localization. We propose that cingulin is a functionally important component of TJ, linking the submembrane plaque domain of TJ to the actomyosin cytoskeleton. PMID:10613913

Janus-faced Sestrin2 controls ROS and mTOR signalling through two separate functional domains

NASA Astrophysics Data System (ADS)

Kim, Hanseong; An, Sojin; Ro, Seung-Hyun; Teixeira, Filipa; Jin Park, Gyeong; Kim, Cheal; Cho, Chun-Seok; Kim, Jeong-Sig; Jakob, Ursula; Hee Lee, Jun; Cho, Uhn-Soo

2015-11-01

Sestrins are stress-inducible metabolic regulators with two seemingly unrelated but physiologically important functions: reduction of reactive oxygen species (ROS) and inhibition of the mechanistic target of rapamycin complex 1 (mTORC1). How Sestrins fulfil this dual role has remained elusive so far. Here we report the crystal structure of human Sestrin2 (hSesn2), and show that hSesn2 is twofold pseudo-symmetric with two globular subdomains, which are structurally similar but functionally distinct from each other. While the N-terminal domain (Sesn-A) reduces alkylhydroperoxide radicals through its helix-turn-helix oxidoreductase motif, the C-terminal domain (Sesn-C) modified this motif to accommodate physical interaction with GATOR2 and subsequent inhibition of mTORC1. These findings clarify the molecular mechanism of how Sestrins can attenuate degenerative processes such as aging and diabetes by acting as a simultaneous inhibitor of ROS accumulation and mTORC1 activation.

Wall touching kink mode calculations with the M3D code

NASA Astrophysics Data System (ADS)

Breslau, J. A.

2014-10-01

In recent years there have been a number of results published concerning the transient vessel currents and forces occurring during a tokamak VDE, as predicted by simulations with the nonlinear MHD code M3D. The nature of the simulations is such that these currents and forces occur at the boundary of the computational domain, making the proper choice of boundary conditions critical to the reliability of the results. The M3D boundary condition includes the prescription that the normal component of the velocity vanish at the wall. It has been argued that this prescription invalidates the calculations because it would seem to rule out the possibility of advection of plasma surface currents into the wall. This claim has been tested by applying M3D to an idealized case - a kink-unstable plasma column - in order to abstract the essential physics from the complications involved in the attempt to model real devices. While comparison of the results is complicated by effects arising from the higher dimensionality and complexity of M3D, we have verified that M3D is capable of reproducing both the correct saturation behavior of the free boundary kink and the ``Hiro'' currents arising when the kink interacts with a conducting tile surface interior to the ideal wall.

Computational Insights into the Interactions between Calmodulin and the c/nSH2 Domains of p85α Regulatory Subunit of PI3Kα: Implication for PI3Kα Activation by Calmodulin.

PubMed

Ni, Duan; Liu, Dingyu; Zhang, Jian; Lu, Shaoyong

2018-01-04

Calmodulin (CaM) and phosphatidylinositide-3 kinase (PI3Kα) are well known for their multiple roles in a series of intracellular signaling pathways and in the progression of several human cancers. Crosstalk between CaM and PI3Kα has been an area of intensive research. Recent experiments have shown that in adenocarcinoma, K-Ras4B is involved in the CaM-PI3Kα crosstalk. Based on experimental results, we have recently put forward a hypothesis that the coordination of CaM and PI3Kα with K-Ras4B forms a CaM-PI3Kα-K-Ras4B ternary complex, which leads to the formation of pancreatic ductal adenocarcinoma. However, the mechanism for the CaM-PI3Kα crosstalk is unresolved. Based on molecular modeling and molecular dynamics simulations, here we explored the potential interactions between CaM and the c/nSH2 domains of p85α subunit of PI3Kα. We demonstrated that CaM can interact with the c/nSH2 domains and the interaction details were unraveled. Moreover, the possible modes for the CaM-cSH2 and CaM-nSH2 interactions were uncovered and we used them to construct a complete CaM-PI3Kα complex model. The structural model of CaM-PI3Kα interaction not only offers a support for our previous ternary complex hypothesis, but also is useful for drug design targeted at CaM-PI3Kα protein-protein interactions.

Structural and Biochemical Basis for the Inhibitory Effect of Liprin-α3 on Mouse Diaphanous 1 (mDia1) Function*

PubMed Central

Brenig, Julian; de Boor, Susanne; Knyphausen, Philipp; Kuhlmann, Nora; Wroblowski, Sarah; Baldus, Linda; Scislowski, Lukas; Artz, Oliver; Trauschies, Philip; Baumann, Ulrich; Neundorf, Ines; Lammers, Michael

2015-01-01

Diaphanous-related formins are eukaryotic actin nucleation factors regulated by an autoinhibitory interaction between the N-terminal RhoGTPase-binding domain (mDiaN) and the C-terminal Diaphanous-autoregulatory domain (DAD). Although the activation of formins by Rho proteins is well characterized, its inactivation is only marginally understood. Recently, liprin-α3 was shown to interact with mDia1. Overexpression of liprin-α3 resulted in a reduction of the cellular actin filament content. The molecular mechanisms of how liprin-α3 exerts this effect and counteracts mDia1 activation by RhoA are unknown. Here, we functionally and structurally define a minimal liprin-α3 core region, sufficient to recapitulate the liprin-α3 determined mDia1-respective cellular functions. We show that liprin-α3 alters the interaction kinetics and thermodynamics of mDiaN with RhoA·GTP and DAD. RhoA displaces liprin-α3 allosterically, whereas DAD competes with liprin-α3 for a highly overlapping binding site on mDiaN. Liprin-α3 regulates actin polymerization by lowering the regulatory potency of RhoA and DAD on mDiaN. We present a model of a mechanistically unexplored and new aspect of mDiaN regulation by liprin-α3. PMID:25911102

Structure at 1.3 A resolution of Rhodothermus marinus caa(3) cytochrome c domain.

PubMed

Srinivasan, Vasundara; Rajendran, Chitra; Sousa, Filipa L; Melo, Ana M P; Saraiva, Lígia M; Pereira, Manuela M; Santana, Margarida; Teixeira, Miguel; Michel, Hartmut

2005-02-04

The cytochrome c domain of subunit II from the Rhodothermus marinus caa(3) HiPIP:oxygen oxidoreductase, a member of the superfamily of heme-copper-containing terminal oxidases, was produced in Escherichia coli and characterised. The recombinant protein, which shows the same optical absorption and redox properties as the corresponding domain in the holo enzyme, was crystallized and its structure was determined to a resolution of 1.3 A by the multiwavelength anomalous dispersion (MAD) technique using the anomalous dispersion of the heme iron atom. The model was refined to final R(cryst) and R(free) values of 13.9% and 16.7%, respectively. The structure reveals the insertion of two short antiparallel beta-strands forming a small beta-sheet, an interesting variation of the classical all alpha-helical cytochrome c fold. This modification appears to be common to all known caa(3)-type terminal oxidases, as judged by comparative modelling and by analyses of the available amino acid sequences for these enzymes. This is the first high-resolution crystal structure reported for a cytochrome c domain of a caa(3)-type terminal oxidase. The R.marinus caa(3) uses HiPIP as the redox partner. The calculation of the electrostatic potential at the molecular surface of this extra C-terminal domain provides insights into the binding to its redox partner on one side and its interaction with the remaining subunit II on the other side.

Optical second harmonic images of 90 deg domain structure in BaTiO3 and periodically inverted antiparallel domains in LiTaO3

NASA Astrophysics Data System (ADS)

Uesu, Y.; Kurimura, S.; Yamamoto, Y.

1995-04-01

Applied is a microscope to observations of 90 deg ferroelectric domain structure in BaTiO3 and inverted periodically are ferroelectric domains in LiTaO3. It is founded that the second harmonic generation microscope gives information which cannot be obtained by ordinary optical microscopes. The developed nonlinear optical microscope builds two dimensional second harmonic image of a specimen with inhomogenous distribution of d(sub ijk) and applied the microscope to observations of inhomogeneity in some nonlinear-optical organic microcrystals.

[Toxicity of Pythium oligandrum broth to animal and its control effect on rot diseases caused by Penicillium italicum and Penicillium digitatum in orange fruit storage].

PubMed

Tan, Yan; Peng, Liangzhi; Yuan, Ling; Wang, Shaobo

2015-11-04

In order to develop a safe, nontoxic and efficient biological antistaling agent and to decrease the incidence of rot diseases caused by the Penicillium italicum and Penicillium digitatum in orange fruit storage. the present experiment was carried out with Pythium oligandrum broth (POB) produced by our self-isolated strain (P. oligandrum CQ2010) to study the toxicity to animal. Thereafter, mycelium growth and spore germination of both P. digitatum and P. italicum and control effect of rot disease in orange storage were compared after treated by liquid culture medium (control), POB, prochloraz (PC) , and PC + POB. Gastric lavage with large amount POB did not influence mouse weight. The animals also showed no abnormality in appearance, behaviors and pathology changes in heart, liver, kidney, lung and intestine. POB decreased the hyphal growth by 70.24% - 93.74% and spore germination by 44.91% - 87.82% (24 h after POB addition) of these two pathogenic fungi. Disease incidence of orange fruit following P. italicum inoculation changed in the sequence: CK > POB > PC > PC + POB and the control efficacy behaved otherwise. In commercial simulation storage, the disease incidence of orange fruit caused by P. digitatum and P. italicum was above 50% of the total. The fruit rot rate was 26.40% (CK), 15.03% (POB), 16.61% (PC) and 4.21% (PC + POB). There were no significant differences in fruit quality under different treatments. POB was safe to animal and could decrease rot disease incidence caused by P. italicum and P. digitatum in orange storage whereby producing a positive interaction with prochloraz and controlling rot diseases caused by these two fungi.

An Msh3 ATPase domain mutation has no effect on MMR function.

PubMed

Edwards, Yasmin

2017-11-25

To demonstrate that the Msh3 ATPase domain is required for DNA mismatch repair and tumor suppression in a murine model. The DNA mismatch repair proteins are members of the ABC family of ATPases. ATP binding and hydrolysis regulates their mismatch repair function. In the current study, a mouse model was generated harboring a glycine to aspartic acid residue change in the Walker A motif of the ATPase domain of Msh3. Impaired ATP mediated release of the Msh2-Msh3 GD/GD complex from it's DNA substrate in vitro confirmed the presence of an ATPase defect. However, the mismatch repair function of the protein was not significantly affected. Therefore, mutation of a critical residue within the ATPase domain of Msh3 did not preclude mismatch repair at the genomic sequences tested. Indicating that Msh3 mediated mismatch function is retained the absence of a functional ATPase domain.

Field-Polarity-Dependent Domain Growth in Epitaxial BaTiO 3 Films

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

Roth, Robert; Guo, Er-Jia; Rafique, Mohsin

The growth of circular tip-induced domains has been studied in epitaxial BaTiO 3 (BTO) films as a function of writing voltage and time using piezoresponse force microscopy (PFM). While abundant for Pb(Zr,Ti)O 3 (PZT) films, such studies are rare for BTO. Strong relaxation of written domains is observed in the form of reduction of the PFM amplitude inside the area of written domains which occurs additionally to a decrease of the domain radius. The domain wall velocity observed for negative tip voltage is systematically smaller than that for positive tip voltage. Based on maps of the positive and negative switchingmore » fields, the effective driving field for both polarities has been estimated. The polarity-dependent effective field cannot account for the different velocities, indicating a polarity-dependent mobility of the domain walls.« less

Field-Polarity-Dependent Domain Growth in Epitaxial BaTiO 3 Films

DOE PAGES

Roth, Robert; Guo, Er-Jia; Rafique, Mohsin; ...

2018-03-23

The growth of circular tip-induced domains has been studied in epitaxial BaTiO 3 (BTO) films as a function of writing voltage and time using piezoresponse force microscopy (PFM). While abundant for Pb(Zr,Ti)O 3 (PZT) films, such studies are rare for BTO. Strong relaxation of written domains is observed in the form of reduction of the PFM amplitude inside the area of written domains which occurs additionally to a decrease of the domain radius. The domain wall velocity observed for negative tip voltage is systematically smaller than that for positive tip voltage. Based on maps of the positive and negative switchingmore » fields, the effective driving field for both polarities has been estimated. The polarity-dependent effective field cannot account for the different velocities, indicating a polarity-dependent mobility of the domain walls.« less

AQP3 is regulated by PPARγ and JNK in hepatic stellate cells carrying PNPLA3 I148M.

PubMed

Tardelli, Matteo; Bruschi, Francesca V; Claudel, Thierry; Moreno-Viedma, Veronica; Halilbasic, Emina; Marra, Fabio; Herac, Merima; Stulnig, Thomas M; Trauner, Michael

2017-11-07

Aquaglyceroporins (AQPs) allow the movement of glycerol that is required for triglyceride formation in hepatic stellate cells (HSC), as key cellular source of fibrogenesis in the liver. The genetic polymorphism I148M of the patatin-like phospholipase domain-containing 3 (PNPLA3) is associated with hepatic steatosis and its progression to steatohepatitis (NASH), fibrosis and cancer. We aimed to explore the role of AQP3 for HSC activation and unveil its potential interactions with PNPLA3. HSC were isolated from human liver, experiments were performed in primary HSC and human HSC line LX2. AQP3 was the only aquaglyceroporin present in HSC and its expression decreased during activation. The PPARγ agonist, rosiglitazone, recovered AQP3 expression also in PNPLA3 I148M carrying HSC. When PNPLA3 was silenced, AQP3 expression increased. In liver sections from patients with NASH, the decreased amount of AQP3 was proportional to the severity of fibrosis and presence of the PNPLA3 I148M variant. In PNPLA3 I148M cells, the blockade of JNK pathway upregulated AQP3 in synergism with PPARγ. In conclusion, we demonstrated profound reduction of AQP3 in HSC carrying the PNPLA3 I148M variant in parallel to decreased PPARγ activation, which could be rescued by rosiglitazone and blockade of JNK.

Identification of the gene transcription repressor domain of Gli3.

PubMed

Tsanev, Robert; Tiigimägi, Piret; Michelson, Piret; Metsis, Madis; Østerlund, Torben; Kogerman, Priit

2009-01-05

Gli transcription factors are downstream targets of the Hedgehog signaling pathway. Two of the three Gli proteins harbor gene transcription repressor function in the N-terminal half. We have analyzed the sequences and identified a potential repressor domain in Gli2 and Gli3 and have tested this experimentally. Overexpression studies confirm that the N-terminal parts harbor gene repression activity and we mapped the minimal repressor to residues 106 till 236 in Gli3. Unlike other mechanisms that inhibit Gli induced gene transcription, the repressor domain identified here does not utilize Histone deacetylases (HDACs) to achieve repression, as confirmed by HDAC inhibition studies and pull-down assays. This distinguishes the identified domain from other regulatory parts with negative influence on transcription.

Domain analysis of 3 Keto Acyl-CoA synthase for structural variations in Vitis vinifera and Oryza brachyantha using comparative modelling.

PubMed

Sagar, Mamta; Pandey, Neetesh; Qamar, Naseha; Singh, Brijendra; Shukla, Akanksha

2015-03-01

The long chain fatty acids incorporated into plant lipids are derived from the iterative addition of C2 units which is provided by malonyl-CoA to an acyl-CoA after interactions with 3-ketoacyl-CoA synthase (KCS), found in several plants. This study provides functional characterization of three 3 ketoacyl CoA synthase like proteins in Vitis vinifera (one) and Oryza brachyantha (two proteins). Sequence analysis reveals that protein of Oryza brachyantha shows 96% similarity to a hypothetical protein in Sorghum bicolor; total 11 homologs were predicted in Sorghum bicolor. Conserved domain prediction confirm the presence of FAE1/Type III polyketide synthase-like protein, Thiolase-like, subgroup; Thiolase-like and 3-Oxoacyl-ACP synthase III, C-terminal and chalcone synthase like domain but very long chain 3-keto acyl CoA domain is absent. All three proteins were found to have Chalcone and stilbene synthases C terminal domain which is similar to domain of thiolase and β keto acyl synthase. Its N terminal domain is absent in J3M9Z7 protein of Oryza brachyantha and F6HH63 protein of Vitis vinifera. Differences in N-terminal domain is responsible for distinguish activity. The J3MF16 protein of Oryza brachyantha contains N terminal domain and C terminal domain and characterized using annotation of these domains. Domains Gcs (streptomyces coelicolor) and Chalcone-stilbene synthases (KAS) in 2-pyrone synthase (Gerbera hybrid) and chalcone synthase 2 (Medicago sativa) were found to be present in three proteins. This similarity points toward anthocyanin biosynthetic process. Similarity to chalcone synthase 2 reveals its possible role in Naringenine and Chalcone synthase like activity. In 3 keto acyl CoA synthase of Oryza brachyantha. Active site residues C-240, H-407, N-447 are present in J3MF16 protein that are common in these three protein at different positions. Structural variations among dimer interface, product binding site, malonyl-CoA binding sites, were predicted in

Structure of Pseudoknot PK26 Shows 3D Domain Swapping in an RNA

NASA Technical Reports Server (NTRS)

Lietzke, Susan E; Barnes, Cindy L.

1998-01-01

3D domain swapping provides a facile pathway for the evolution of oligomeric proteins and allosteric mechanisms and a means for using monomer-oligomer equilibria to regulate biological activity. The term "3D domain swapping" describes the exchange of identical domains between two protein monomers to create an oligomer. 3D domain swapping has, so far, only been recognized in proteins. In this study, the structure of the pseudoknot PK26 is reported and it is a clear example of 3D domain swapping in RNA. PK26 was chosen for study because RNA pseudoknots are required structures in several biological processes and they arise frequently in in vitro selection experiments directed against protein targets. PK26 specifically inhibits HIV-1 reverse transcriptase with nanomolar affinity. We have now determined the 3.1 A resolution crystal structure of PK26 and find that it forms a 3D domain swapped dimer. PK26 shows extensive base pairing between and within strands. Formation of the dimer requires the linker region between the pseudoknot folds to adopt a unique conformation that allows a base within a helical stem to skip one base in the stacking register. Rearrangement of the linker would permit a monomeric pseudoknot to form. This structure shows how RNA can use 3D domain swapping to build large scale oligomers like the putative hexamer in the packaging RNA of bacteriophage Phi29.

Electric-field-induced domain intersection in BaTiO3 single crystal

NASA Astrophysics Data System (ADS)

He, Ming; Wang, Mengxia; Zhang, Zhihua

2017-03-01

Large-angle convergent beam electron diffraction was used to determine the directions of polarization vectors in a BaTiO3 single crystal. Domain intersections driven by an electric field were investigated by in situ transmission electron microscopy. The dark triangles observed in the domain intersection region can be accounted for by dislocations and the strain field. Domains nucleate at the domain tip depending on the dislocations and strain field to relieve the accumulated stress. Schematic representations of the intersecting domains and the microscopic structure are given, clarifying the special electric-field-induced domain structure.

Modulation of superconducting transition temperature in LaAlO3/SrTiO3 by SrTiO3 structural domains

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

Noad, Hilary; Moler, Kathryn

2018-01-01

The tetragonal domain structure in SrTiO3 (STO) is known to modulate the normal-state carrier density in LaAlO3/SrTiO3 (LAO/STO) heterostructures, among other electronic properties, but the effect of STO domains on the superconductivity in LAO/STO has not been fully explored. Using a scanning SQUID susceptometer microscope to map the superconducting response as a function of temperature in LAO/STO, we find that the superconducting transition temperature is spatially inhomogeneous and modulated in a pattern that is characteristic of structural domains in the STO.

Single-domain multiferroic BiFeO 3 films

DOE PAGES

Kuo, Chang -Yang; Hu, Z.; Yang, J. C.; ...

2016-09-01

The strong coupling between antiferromagnetism and ferroelectricity at room temperature found in BiFeO 3 generates high expectations for the design and development of technological devices with novel functionalities. However, the multi-domain nature of the material tends to nullify the properties of interest and complicates the thorough understanding of the mechanisms that are responsible for those properties. Here we report the realization of a BiFeO 3 material in thin film form with single-domain behaviour in both its magnetism and ferroelectricity: the entire film shows its antiferromagnetic axis aligned along the crystallographic b axis and its ferroelectric polarization along the c axis.more » With this we are able to reveal that the canted ferromagnetic moment due to the Dzyaloshinskii–Moriya interaction is parallel to the a axis. Moreover, by fabricating a Co/BiFeO 3 heterostructure, we demonstrate that the ferromagnetic moment of the Co film does couple directly to the canted moment of BiFeO 3.« less

Domain characterization of Pb(Zn1/3Nb2/3)O3-(6%-7%)PbTiO3 single crystals using scanning electron acoustic microscopy

NASA Astrophysics Data System (ADS)

Wong, Meng Fei; Heng, Xiangxin; Zeng, Kaiyang

2008-10-01

Domain structures of [001]T and [011]T-cut Pb(Zn1/3Nb2/3)O3-(6%-7%)PbTiO3 (PZN-PT) single crystals are studied using scanning electron acoustic microscope (SEAM) technique. The observation of the orientation of domain walls agree reasonably well with the trigonometric projection of rhombohedral and orthorhombic dipoles on the (001) and (011) surfaces, respectively. After mechanical loading with microindentation, domain switching is also observed to form a hyperbolic butterfly shape and extend preferentially along four diagonal directions, i.e., ⟨110⟩ on (001) surface and ⟨111¯⟩ on (011) surface. The critical shear stress to cause domain switching for PZN-PT crystal is estimated to be approximately 49 MPa for both {110} and {111¯} planes based on theoretical analysis. Generally, the SEAM technique has been successfully demonstrated to be a valid technique for observation of domain structures in single crystal PZN-PTs.

Pnpla3I148M knockin mice accumulate PNPLA3 on lipid droplets and develop hepatic steatosis

PubMed Central

Smagris, Eriks; BasuRay, Soumik; Li, John; Huang, Yongcheng; Lai, Ka-man V; Gromada, Jesper; Cohen, Jonathan C; Hobbs, Helen H

2015-01-01

A sequence polymorphism (rs738409, I148M) in patatin-like phospholipid domain containing protein 3 (PNPLA3) is strongly associated with nonalcoholic fatty liver disease (NAFLD), but the mechanistic basis for this association remains enigmatic. Neither ablation nor overexpression of wild-type PNPLA3 affects liver fat content in mice, whereas hepatic overexpression of the human 148M transgene causes steatosis. To determine whether the 148M allele causes fat accumulation in the liver when expressed at physiological levels, we introduced a methionine codon at position 148 of the mouse Pnpla3 gene. Knockin mice had normal levels of hepatic fat on a chow diet, but when challenged with a high-sucrose diet their liver fat levels increased 2 to 3-fold compared to wild-type littermates without any associated changes in glucose homeostasis. The increased liver fat in the knockin mice was accompanied by a 40-fold increase in PNPLA3 on hepatic lipid droplets, with no increase in hepatic PNPLA3 messenger RNA (mRNA). Similar results were obtained when the catalytic dyad of PNPLA3 was inactivated by substituting the catalytic serine with alanine (S47A). Conclusion: These data provide the first direct evidence that physiological expression of PNPLA3 148M variant causes NAFLD, and that the accumulation of catalytically inactive PNPLA3 on the surfaces of lipid droplets is associated with the accumulation of TG in the liver. (Hepatology 2015;61:108–118) PMID:24917523

Functional relationship between CABIT, SAM and 14-3-3 binding domains of GAREM1 that play a role in its subcellular localization

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

Nishino, Tasuku; Matsunaga, Ryota; Konishi, Hiroaki, E-mail: hkonishi@pu-hiroshima.ac.jp

2015-08-21

GAREM1 (Grb2-associated regulator of Erk/MAPK1) is an adaptor protein that is involved in the epidermal growth factor (EGF) pathway. The nuclear localization of GAREM1 depends on the nuclear localization sequence (NLS), which is located at the N-terminal CABIT (cysteine-containing, all in Themis) domain. Here, we identified 14-3-3ε as a GAREM-binding protein, and its binding site is closely located to the NLS. This 14-3-3 binding site was of the atypical type and independent of GAREM phosphorylation. Moreover, the binding of 14-3-3 had an effect on the nuclear localization of GAREM1. Unexpectedly, we observed that the CABIT domain had intramolecular association withmore » the C-terminal SAM (sterile alpha motif) domain. This association might be inhibited by binding of 14-3-3 at the CABIT domain. Our results demonstrate that the mechanism underlying the nuclear localization of GAREM1 depends on its NLS in the CABIT domain, which is controlled by the binding of 14-3-3 and the C-terminal SAM domain. We suggest that the interplay between 14-3-3, SAM domain and CABIT domain might be responsible for the distribution of GAREM1 in mammalian cells. - Highlights: • 14-3-3ε regulated the nuclear localization of GAREM1 as its binding partner. • The atypical 14-3-3 binding site of GAREM1 is located near the NLS in CABIT domain. • The CABIT domain had intramolecular association with the SAM domain in GAREM1. • Subcellular localization of GAREM1 is affected with its CABIT-SAM interaction.« less

Engineered domain based assays to identify individual antibodies in oligoclonal combinations targeting the same protein

PubMed Central

Meng, Q.; Garcia-Rodriguez, C.; Manzanarez, G.; Silberg, M.A.; Conrad, F.; Bettencourt, J.; Pan, X.; Breece, T.; To, R.; Li, M.; Lee, D.; Thorner, L.; Tomic, M.T.; Marks, J.D.

2014-01-01

Quantitation of individual mAbs within a combined antibody drug product is required for preclinical and clinical drug development. We have developed two antitoxins (XOMA 3B and XOMA 3E) each consisting of three monoclonal antibodies (mAbs) that neutralize type B and type E botulinum neurotoxin (BoNT/B and BoNT/E) to treat serotype B and E botulism. To develop mAb-specific binding assays for each antitoxin, we mapped the epitopes of the six mAbs. Each mAb bound an epitope on either the BoNT light chain (LC) or translocation domain (HN). Epitope mapping data was used to design LC-HN domains with orthogonal mutations to make them specific for only one mAb in either XOMA 3B or 3E. Mutant LC-HN domains were cloned, expressed, and purified from E. coli. Each mAb bound only to its specific domain with affinity comparable to the binding to holotoxin. Further engineering of domains allowed construction of ELISAs that could characterize the integrity, binding affinity, and identity of each of the six mAbs in XOMA 3B, and 3E without interference from the three BoNT/A mAbs in XOMA 3AB. Such antigen engineering is a general method allowing quantitation and characterization of individual mAbs in a mAb cocktail that bind the same protein. PMID:22922799

Probing conformational changes in the I-like domain and the cysteine-rich repeat of human beta 3 integrins following disulfide bond disruption by cysteine mutations: identification of cysteine 598 involved in alphaIIbbeta3 activation.

PubMed

Chen, P; Melchior, C; Brons, N H; Schlegel, N; Caen, J; Kieffer, N

2001-10-19

We have investigated receptor function and epitope expression of recombinant alpha(IIb)beta(3) mutated at Cys(177) or Cys(273) in the I-like domain as well as Cys(598), located in the fourth repeat of the membrane-proximal cysteine-rich region and mutated in a Glanzmann's thrombasthenia type II patient. The beta(3) mutants beta(3)C177A, beta(3)C273A, and beta(3)C598Y exhibited a decreased electrophoretic mobility in SDS-polyacrylamide gel electrophoresis under nonreducing conditions, confirming the disruption of the respective disulfide loops. Despite reduced surface expression, the alpha(IIb)beta(3)C177A, alpha(IIb)beta(3)C273A, and alpha(IIb)beta(3)C598Y receptors mediated cell adhesion to immobilized fibrinogen and translocated into focal adhesion plaques. The beta(3)C598Y mutation, but not the beta(3)C177A or beta(3)C273A mutations, induced spontaneous binding of the ligand mimetic monoclonal antibody PAC-1, while the beta(3)C177A and beta(3)C273A mutants exhibited reduced complex stability in the absence of Ca(2+). Epitope mapping of function-blocking monoclonal antibodies (mAbs) allowed the identification of two distinct subgroups; mAbs A2A9, pl2-46, 10E5, and P256 did not interact with alpha(IIb)beta(3)C273A and bound only weakly to alpha(IIb)beta(3)C177A, while mAbs AP2, LM609 and 7E3 bound normally to mutant alpha(IIb)beta(3)C273A, but interacted only weakly with mutant alpha(IIb)beta(3)C177A. Furthermore, a cryptic epitope recognized by mAb 4D10G3 and not exposed on wild type alpha(IIb)beta(3) became accessible only on mutant alpha(IIb)beta(3)C177A and was mapped to the 60-kDa chymotrypsin fragment of beta(3). Finally, the ligand-induced binding site (LIBS) epitopes AP5, D3, LIBS1, and LIBS2 were spontaneously expressed on all three mutants independent of RGDS or dithiothreitol treatment. Our results provide evidence that disruption of a single cysteine disulfide bond in the cysteine-rich repeat domain, but not in the I-like domain, activates integrin

Rapid autocatalytic activation of the M4 metalloprotease aureolysin is controlled by a conserved N-terminal fungalysin-thermolysin-propeptide domain.

PubMed

Nickerson, Nicholas N; Joag, Vineet; McGavin, Martin J

2008-09-01

The Staphylococcus aureus proteolytic cascade consists of a metalloprotease aureolysin (Aur), which activates a serine protease zymogen proSspA, which in turn activates the SspB cysteine protease. As with other M4 metalloproteases, including elastase of Pseudomonas aeruginosa, the propeptide of proAur contains an N-terminal fungalysin-thermolysin-propeptide (FTP) domain. Autocatalytic activation of proAur was initiated by processing at T85 downward arrowL(86) in the FTP domain. This differed from the mechanism described for proElastase, where the FTP domain has an RY motif in place of TL(86), and processing occurred at the junction of the propeptide and metalloprotease domains, which remained as an inactive complex during passage across the outer membrane. When TL(86) in the FTP domain was replaced with RY, an intact N-terminal propeptide was secreted, but the M4 metalloprotease domain was degraded. Consequently, this segment of the FTP domain promotes intramolecular processing of proAur while bestowing a chaperone function, but discourages processing within the FTP domain of proElastase, where activation must be co-ordinated with passage across a second membrane. We conclude that the FTP domain of proAur is adapted to facilitate a rapid autocatalytic activation mechanism, consistent with the role or proAur as initiator of the staphylococcal proteolytic cascade.

Artificial proteins as allosteric modulators of PDZ3 and SH3 in two-domain constructs: A computational characterization of novel chimeric proteins.

PubMed

Kirubakaran, Palani; Pfeiferová, Lucie; Boušová, Kristýna; Bednarova, Lucie; Obšilová, Veronika; Vondrášek, Jiří

2016-10-01

Artificial multidomain proteins with enhanced structural and functional properties can be utilized in a broad spectrum of applications. The design of chimeric fusion proteins utilizing protein domains or one-domain miniproteins as building blocks is an important advancement for the creation of new biomolecules for biotechnology and medical applications. However, computational studies to describe in detail the dynamics and geometry properties of two-domain constructs made from structurally and functionally different proteins are lacking. Here, we tested an in silico design strategy using all-atom explicit solvent molecular dynamics simulations. The well-characterized PDZ3 and SH3 domains of human zonula occludens (ZO-1) (3TSZ), along with 5 artificial domains and 2 types of molecular linkers, were selected to construct chimeric two-domain molecules. The influence of the artificial domains on the structure and dynamics of the PDZ3 and SH3 domains was determined using a range of analyses. We conclude that the artificial domains can function as allosteric modulators of the PDZ3 and SH3 domains. Proteins 2016; 84:1358-1374. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Identification and functional characterization of an Src homology domain 3 domain-binding site on Cbl.

PubMed

Sanjay, Archana; Miyazaki, Tsuyoshi; Itzstein, Cecile; Purev, Enkhtsetseg; Horne, William C; Baron, Roland

2006-12-01

Cbl is an adaptor protein and ubiquitin ligase that binds and is phosphorylated by the nonreceptor tyrosine kinase Src. We previously showed that the primary interaction between Src and Cbl is mediated by the Src homology domain 3 (SH3) of Src binding to proline-rich sequences of Cbl. The peptide Cbl RDLPPPPPPDRP(540-551), which corresponds to residues 540-551 of Cbl, inhibited the binding of a GST-Src SH3 fusion protein to Cbl, whereas RDLAPPAPPPDR(540-551) did not, suggesting that Src binds to this site on Cbl in a class I orientation. Mutating prolines 543-548 reduced Src binding to the Cbl 479-636 fragment significantly more than mutating the prolines in the PPVPPR(494-499) motif, which was previously reported to bind Src SH3. Mutating Cbl prolines 543-548 to alanines substantially reduced Src binding to Cbl, Src-induced phosphorylation of Cbl, and the inhibition of Src kinase activity by Cbl. Expressing the mutated Cbl in osteoclasts induced a moderate reduction in bone-resorbing activity and increased amounts of Src protein. In contrast, disabling the tyrosine kinase-binding domain of full-length Cbl by mutating glycine 306 to glutamic acid, and thereby preventing the previously described binding of the tyrosine kinase-binding domain to the Src phosphotyrosine 416, had no effect on Cbl phosphorylation, the inhibition of Src activity by full-length Cbl, or bone resorption. These data indicate that the Cbl RDLPPPP(540-546) sequence is a functionally important binding site for Src.

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

Su, M; Sura, S

Purpose: To evaluate dosimetric impact of two axillary nodes (AX) boost techniques: (1) posterior-oblique optimized field boost (POB), (2) traditional posterior-anterior boost (PAB) with field optimization (O-PAB), for a postmastectomy breast patient with positive axillary lymph nodes. Methods: Five patients, 3 left and 2 right chest walls, were included in this study. All patients were simulated in 5mm CT slice thickness. Supraclavicular (SC) and level I/II/III AX were contoured based on the RTOG atlas guideline. Five treatment plans, (1) tangential chest wall, (2) oblique SC including AX, (3) PAB, O-PAB and POB, were created for each patient. Three plan sumsmore » (PS) were generated by sum one of (3) plan with plan (1) and (2). The field optimization was done through PS dose distribution, which included a field adjustment, a fractional dose, a calculation location and a gantry angle selection for POB. A dosimetric impact was evaluated by comparing a SC and AX coverage, a PS maximum dose, an irradiated area percentage volume received dose over 105% prescription dose (V105), an ipsi-laterial mean lung dose (MLD), an ipsi-laterial mean humeral head dose (MHHD), a mean heart dose (MHD) (for left case only) and their DVH amount these three technique. Results: O-PAB, POB and PAB dosimetric results showed that there was no significant different on SC and AX coverage (p>0.43) and MHD (p>0.16). The benefit of sparing lung irradiation from PAB to O-PAB to POB was significant (p<0.004). PAB showed a highest PS maximum dose (p<0.005), V105 (p<0.023) and MLD (compared with OPAB, p=0.055). MHHD showed very sensitive to the patient arm positioning and anatomy. O-PAB convinced a lower MHHD than PAB (p=0.03). Conclusion: 3D CT contouring plays main role in accuracy radiotherapy. Dosimetric advantage of POB and O-PAB was observed for a better normal tissue irradiation sparing.« less

Electron backscatter diffraction as a domain analysis technique in BiFeO(3)-PbTiO(3) single crystals.

PubMed

Burnett, T L; Comyn, T P; Merson, E; Bell, A J; Mingard, K; Hegarty, T; Cain, M

2008-05-01

xBiFeO(3)-(1-x)PbTiO(3) single crystals were grown via a flux method for a range of compositions. Presented here is a study of the domain configuration in the 0.5BiFeO(3)-0.5PbTiO(3) composition using electron backscatter diffraction to demonstrate the ability of the technique to map ferroelastic domain structures at the micron and submicron scale. The micron-scale domains exhibit an angle of approximately 85 degrees between each variant, indicative of a ferroelastic domain wall in a tetragonal system with a spontaneous strain, c/a - 1 of 0.10, in excellent agreement with the lattice parameters derived from x-ray diffraction. Contrast seen in forescatter images is attributed to variations in the direction of the electrical polarization vector, providing images of ferroelectric domain patterns.

Hydroxychloroquine binding to cytoplasmic domain of Band 3 in human erythrocytes: Novel mechanistic insights into drug structure, efficacy and toxicity.

PubMed

Nakagawa, Mizuki; Sugawara, Kotomi; Goto, Tatsufumi; Wakui, Hideki; Nunomura, Wataru

2016-05-13

Hydroxychloroquine (HCQ) is a widely used drug in the treatment of autoimmune diseases, such as arthritis and systemic lupus erythematosus. It has also been prescribed for the treatment of malaria owing to its lower toxicity compared to its closely related compound chloroquine (CQ). However, the mechanisms of action of HCQ in erythrocytes (which bind preferentially this drug) have not been documented and the reasons underlying the lower side effects of HCQ compared to CQ remain unclear. Here we show that, although the activity of erythrocyte lactate dehydrogenase (LDH), but not GAPDH, was inhibited by both HCQ and CQ in vitro, LDH activity in erythrocytes incubated with 20 mM HCQ was not significantly reduced within 5 h in contrast to CQ did. Using HCQ coupled Sepharose chromatography (HCQ-Sepharose), we identified Band 3, spectrin, ankyrin, protein 4.1R and protein 4.2 as HCQ binding proteins in human erythrocyte plasma membrane. Recombinant cytoplasmic N-terminal 43 kDa domain of Band 3 bound to HCQ-Sepharose and was eluted with 40 mM (but not 20 mM) HCQ. Band 3 transport activity was reduced by only 23% in the presence of 20 mM HCQ. Taken together, these data demonstrate that HCQ binds to the cytoplasmic N-terminal domain of Band 3 in human erythrocytes but does not inhibit dramatically its transport activity. We hypothesize that the trapping of HCQ on Band 3 contributes to the lower side effects of the drug on energy production in erythrocytes. Copyright © 2016 Elsevier Inc. All rights reserved.

Investigations into the Membrane Interactions of m-Calpain Domain V

PubMed Central

Dennison, Sarah R.; Dante, Silvia; Hauß, Thomas; Brandenburg, Klaus; Harris, Frederick; Phoenix, David A.

2005-01-01

m-Calpain is a calcium-dependent heterodimeric protease implicated in a number of pathological conditions. The activation of m-calpain appears to be modulated by membrane interaction, which has been predicted to involve oblique-orientated α-helix formation by a GTAMRILGGVI segment located in domain V of the protein's small subunit. Here, we have investigated this prediction. Fourier transform infrared conformational analysis showed that VP1, a peptide homolog of this segment, exhibited α-helicity of ∼45% in the presence of dimyristoylphosphatidylcholine/dimyristoylphosphatidylserine (DMPS) vesicles. The level of helicity was unaffected over a 1- to 8-mM concentration range and did not alter when the anionic lipid composition of these vesicles was varied between 1% and 10% DMPS. Similar levels of α-helicity were observed in trifluoroethanol and the peptide appeared to adopt α-helical structure at an air/water interface with a molecular area of 164 Å2 at the monolayer collapse pressure. VP1 was found to penetrate dimyristoylphosphatidylcholine/DMPS monolayers, and at an initial surface pressure of 30 mN m−1, the peptide induced surface pressure changes in these monolayers that correlated strongly with their anionic lipid content (maximal at 4 mN m−1 in the presence of 10% DMPS). Neutron diffraction studies showed VP1 to be localized at the hydrophobic core of model palmitoyloleylphosphatidylcholine/palmitoyloleylphosphatidylserine (10:1 molar ratio) bilayer structures and, in combination, these results are consistent with the oblique membrane penetration predicted for the peptide. It would also appear that although not needed for structural stabilization anionic lipid was required for membrane penetration. PMID:15653743

Efficient T-cell receptor signaling requires a high-affinity interaction between the Gads C-SH3 domain and the SLP-76 RxxK motif.

PubMed

Seet, Bruce T; Berry, Donna M; Maltzman, Jonathan S; Shabason, Jacob; Raina, Monica; Koretzky, Gary A; McGlade, C Jane; Pawson, Tony

2007-02-07

The relationship between the binding affinity and specificity of modular interaction domains is potentially important in determining biological signaling responses. In signaling from the T-cell receptor (TCR), the Gads C-terminal SH3 domain binds a core RxxK sequence motif in the SLP-76 scaffold. We show that residues surrounding this motif are largely optimized for binding the Gads C-SH3 domain resulting in a high-affinity interaction (K(D)=8-20 nM) that is essential for efficient TCR signaling in Jurkat T cells, since Gads-mediated signaling declines with decreasing affinity. Furthermore, the SLP-76 RxxK motif has evolved a very high specificity for the Gads C-SH3 domain. However, TCR signaling in Jurkat cells is tolerant of potential SLP-76 crossreactivity, provided that very high-affinity binding to the Gads C-SH3 domain is maintained. These data provide a quantitative argument that the affinity of the Gads C-SH3 domain for SLP-76 is physiologically important and suggest that the integrity of TCR signaling in vivo is sustained both by strong selection of SLP-76 for the Gads C-SH3 domain and by a capacity to buffer intrinsic crossreactivity.

Engineered domain-based assays to identify individual antibodies in oligoclonal combinations targeting the same protein.

PubMed

Meng, Q; Garcia-Rodriguez, C; Manzanarez, G; Silberg, M A; Conrad, F; Bettencourt, J; Pan, X; Breece, T; To, R; Li, M; Lee, D; Thorner, L; Tomic, M T; Marks, J D

2012-11-15

Quantitation of individual monoclonal antibodies (mAbs) within a combined antibody drug product is required for preclinical and clinical drug development. We have developed two antitoxins, XOMA 3B and XOMA 3E, each consisting of three mAbs that neutralize type B and type E botulinum neurotoxin (BoNT/B and BoNT/E) to treat serotype B and E botulism. To develop mAb-specific binding assays for each antitoxin, we mapped the epitopes of the six mAbs. Each mAb bound an epitope on either the BoNT light chain (LC) or translocation domain (H(N)). Epitope mapping data were used to design LC-H(N) domains with orthogonal mutations to make them specific for only one mAb in either XOMA 3B or XOMA 3E. Mutant LC-H(N) domains were cloned, expressed, and purified from Escherichia coli. Each mAb bound only to its specific domain with affinity comparable to the binding to holotoxin. Further engineering of domains allowed construction of enzyme-linked immunosorbent assays (ELISAs) that could characterize the integrity, binding affinity, and identity of each of the six mAbs in XOMA 3B and 3E without interference from the three BoNT/A mAbs in XOMA 3AB. Such antigen engineering is a general method allowing quantitation and characterization of individual mAbs in a mAb cocktail that bind the same protein. Copyright © 2012 Elsevier Inc. All rights reserved.

The ShcA SH2 domain engages a 14-3-3/PI3'K signaling complex and promotes breast cancer cell survival.

PubMed

Ursini-Siegel, J; Hardy, W R; Zheng, Y; Ling, C; Zuo, D; Zhang, C; Podmore, L; Pawson, T; Muller, W J

2012-11-29

The ShcA adapter protein transmits activating signals downstream of receptor and cytoplasmic tyrosine kinases through the establishment of phosphotyrosine-dependent complexes. In this regard, ShcA possesses both a phosphotyrosine-binding domain (PTB) and Src homology 2 domain (SH2), which bind phosphotyrosine residues in a sequence-specific manner. Although the majority of receptor tyrosine kinases expressed in breast cancer cells bind the PTB domain, very little is known regarding the biological importance of SH2-driven ShcA signaling during mammary tumorigenesis. To address this, we employed transgenic mice expressing a mutant ShcA allele harboring a non-functional SH2 domain (ShcR397K) under the transcriptional control of the endogenous ShcA promoter. Using transplantation approaches, we demonstrate that SH2-dependent ShcA signaling within the mammary epithelial compartment is essential for breast tumor outgrowth, survival and the development of lung metastases. We further show that the ShcA SH2 domain activates the AKT pathway, potentially through a novel SH2-mediated complex between ShcA, 14-3-3ζ and the p85 regulatory subunit of phosphatidylinositol 3 (PI3') kinase. This study is the first to demonstrate that the SH2 domain of ShcA is critical for tumor survival during mammary tumorigenesis.

Ferroelectric domain structure of anisotropically strained NaNbO3 epitaxial thin films

NASA Astrophysics Data System (ADS)

Schwarzkopf, J.; Braun, D.; Schmidbauer, M.; Duk, A.; Wördenweber, R.

2014-05-01

NaNbO3 thin films have been grown under anisotropic biaxial strain on several oxide substrates by liquid-delivery spin metalorganic chemical vapor deposition. Compressive lattice strain of different magnitude, induced by the deposition of NaNbO3 films with varying film thickness on NdGaO3 single crystalline substrates, leads to modifications of film orientation and phase symmetry, which are similar to the phase transitions in Pb-containing oxides near the morphotropic phase boundary. Piezoresponse force microscopy measurements exhibit large out-of-plane polarization components, but no distinctive domain structure, while C-V measurements indicate relaxor properties in these films. When tensile strain is provoked by the epitaxial growth on DyScO3, TbScO3, and GdScO3 single crystalline substrates, NaNbO3 films behave rather like a normal ferroelectric. The application of these rare-earth scandate substrates yields well-ordered ferroelectric stripe domains of the type a1/a2 with coherent domain walls aligned along the [001] substrate direction as long as the films are fully strained. With increasing plastic lattice relaxation, initially, a 2D domain pattern with still exclusively in-plane electric polarization, and finally, domains with in-plane and out-of-plane polar components evolve.

Non-3D domain swapped crystal structure of truncated zebrafish alphaA crystallin

PubMed Central

Laganowsky, A; Eisenberg, D

2010-01-01

In previous work on truncated alpha crystallins (Laganowsky et al., Protein Sci 2010; 19:1031–1043), we determined crystal structures of the alpha crystallin core, a seven beta-stranded immunoglobulin-like domain, with its conserved C-terminal extension. These extensions swap into neighboring cores forming oligomeric assemblies. The extension is palindromic in sequence, binding in either of two directions. Here, we report the crystal structure of a truncated alphaA crystallin (AAC) from zebrafish (Danio rerio) revealing C-terminal extensions in a non three-dimensional (3D) domain swapped, “closed” state. The extension is quasi-palindromic, bound within its own zebrafish core domain, lying in the opposite direction to that of bovine AAC, which is bound within an adjacent core domain (Laganowsky et al., Protein Sci 2010; 19:1031–1043). Our findings establish that the C-terminal extension of alpha crystallin proteins can be either 3D domain swapped or non-3D domain swapped. This duality provides another molecular mechanism for alpha crystallin proteins to maintain the polydispersity that is crucial for eye lens transparency. PMID:20669149

Pnpla3I148M knockin mice accumulate PNPLA3 on lipid droplets and develop hepatic steatosis.

PubMed

Smagris, Eriks; BasuRay, Soumik; Li, John; Huang, Yongcheng; Lai, Ka-man V; Gromada, Jesper; Cohen, Jonathan C; Hobbs, Helen H

2015-01-01

A sequence polymorphism (rs738409, I148M) in patatin-like phospholipid domain containing protein 3 (PNPLA3) is strongly associated with nonalcoholic fatty liver disease (NAFLD), but the mechanistic basis for this association remains enigmatic. Neither ablation nor overexpression of wild-type PNPLA3 affects liver fat content in mice, whereas hepatic overexpression of the human 148M transgene causes steatosis. To determine whether the 148M allele causes fat accumulation in the liver when expressed at physiological levels, we introduced a methionine codon at position 148 of the mouse Pnpla3 gene. Knockin mice had normal levels of hepatic fat on a chow diet, but when challenged with a high-sucrose diet their liver fat levels increased 2 to 3-fold compared to wild-type littermates without any associated changes in glucose homeostasis. The increased liver fat in the knockin mice was accompanied by a 40-fold increase in PNPLA3 on hepatic lipid droplets, with no increase in hepatic PNPLA3 messenger RNA (mRNA). Similar results were obtained when the catalytic dyad of PNPLA3 was inactivated by substituting the catalytic serine with alanine (S47A). These data provide the first direct evidence that physiological expression of PNPLA3 148M variant causes NAFLD, and that the accumulation of catalytically inactive PNPLA3 on the surfaces of lipid droplets is associated with the accumulation of TG in the liver. © 2014 The Authors. Hepatology published by Wiley Periodicals, Inc., on behalf of the American Association for the Study of Liver Diseases.

Characterization of Novel Calmodulin Binding Domains within IQ Motifs of IQGAP1

PubMed Central

Jang, Deok-Jin; Ban, Byungkwan; Lee, Jin-A

2011-01-01

IQ motif-containing GTPase-activating protein 1 (IQGAP1), which is a well-known calmodulin (CaM) binding protein, is involved in a wide range of cellular processes including cell proliferation, tumorigenesis, adhesion, and migration. Interaction of IQGAP1 with CaM is important for its cellular functions. Although each IQ domain of IQGAP1 for CaM binding has been characterized in a Ca2+-dependent or -independent manner, it was not clear which IQ motifs are physiologically relevant for CaM binding in the cells. In this study, we performed immunoprecipitation using 3xFLAGhCaM in mammalian cell lines to characterize the domains of IQGAP1 that are key for CaM binding under physiological conditions. Interestingly, using this method, we identified two novel domains, IQ(2.7-3) and IQ(3.5-4.4), within IQGAP1 that were involved in Ca2+-independent or -dependent CaM binding, respectively. Mutant analysis clearly showed that the hydrophobic regions within IQ(2.7-3) were mainly involved in apoCaM binding, while the basic amino acids and hydrophobic region of IQ(3.5-4.4) were required for Ca2+/CaM binding. Finally, we showed that IQ(2.7-3) was the main apoCaM binding domain and both IQ(2.7-3) and IQ(3.5-4.4) were required for Ca2+/CaM binding within IQ(1- 2-3-4). Thus, we identified and characterized novel direct CaM binding motifs essential for IQGAP1. This finding indicates that IQGAP1 plays a dynamic role via direct interactions with CaM in a Ca2+-dependent or -independent manner. PMID:22080369

A graph kernel approach for alignment-free domain-peptide interaction prediction with an application to human SH3 domains.

PubMed

Kundu, Kousik; Costa, Fabrizio; Backofen, Rolf

2013-07-01

State-of-the-art experimental data for determining binding specificities of peptide recognition modules (PRMs) is obtained by high-throughput approaches like peptide arrays. Most prediction tools applicable to this kind of data are based on an initial multiple alignment of the peptide ligands. Building an initial alignment can be error-prone, especially in the case of the proline-rich peptides bound by the SH3 domains. Here, we present a machine-learning approach based on an efficient graph-kernel technique to predict the specificity of a large set of 70 human SH3 domains, which are an important class of PRMs. The graph-kernel strategy allows us to (i) integrate several types of physico-chemical information for each amino acid, (ii) consider high-order correlations between these features and (iii) eliminate the need for an initial peptide alignment. We build specialized models for each human SH3 domain and achieve competitive predictive performance of 0.73 area under precision-recall curve, compared with 0.27 area under precision-recall curve for state-of-the-art methods based on position weight matrices. We show that better models can be obtained when we use information on the noninteracting peptides (negative examples), which is currently not used by the state-of-the art approaches based on position weight matrices. To this end, we analyze two strategies to identify subsets of high confidence negative data. The techniques introduced here are more general and hence can also be used for any other protein domains, which interact with short peptides (i.e. other PRMs). The program with the predictive models can be found at http://www.bioinf.uni-freiburg.de/Software/SH3PepInt/SH3PepInt.tar.gz. We also provide a genome-wide prediction for all 70 human SH3 domains, which can be found under http://www.bioinf.uni-freiburg.de/Software/SH3PepInt/Genome-Wide-Predictions.tar.gz. Supplementary data are available at Bioinformatics online.

The Abl SH2-kinase linker naturally adopts a conformation competent for SH3 domain binding.

PubMed

Chen, Shugui; Brier, Sébastien; Smithgall, Thomas E; Engen, John R

2007-04-01

The core of the Abelson tyrosine kinase (c-Abl) is structurally similar to Src-family kinases where SH3 and SH2 domains pack against the backside of the kinase domain in the down-regulated conformation. Both kinase families depend upon intramolecular association of SH3 with the linker joining the SH2 and kinase domains for suppression of kinase activity. Hydrogen deuterium exchange (HX) and mass spectrometry (MS) were used to probe intramolecular interaction of the c-Abl SH3 domain with the linker in recombinant constructs lacking the kinase domain. Under physiological conditions, the c-Abl SH3 domain undergoes partial unfolding, which is stabilized by ligand binding, providing a unique assay for SH3:linker interaction in solution. Using this approach, we observed dynamic association of the SH3 domain with the linker in the absence of the kinase domain. Truncation of the linker before W254 completely prevented cis-interaction with SH3, while constructs containing amino acids past this point showed SH3:linker interactions. The observation that the Abl linker sequence exhibits SH3-binding activity in the absence of the kinase domain is unique to Abl and was not observed with Src-family kinases. These results suggest that SH3:linker interactions may have a more prominent role in Abl regulation than in Src kinases, where the down-regulated conformation is further stabilized by a second intramolecular interaction between the C-terminal tail and the SH2 domain.

Computational Identification of Genomic Features That Influence 3D Chromatin Domain Formation.

PubMed

Mourad, Raphaël; Cuvier, Olivier

2016-05-01

Recent advances in long-range Hi-C contact mapping have revealed the importance of the 3D structure of chromosomes in gene expression. A current challenge is to identify the key molecular drivers of this 3D structure. Several genomic features, such as architectural proteins and functional elements, were shown to be enriched at topological domain borders using classical enrichment tests. Here we propose multiple logistic regression to identify those genomic features that positively or negatively influence domain border establishment or maintenance. The model is flexible, and can account for statistical interactions among multiple genomic features. Using both simulated and real data, we show that our model outperforms enrichment test and non-parametric models, such as random forests, for the identification of genomic features that influence domain borders. Using Drosophila Hi-C data at a very high resolution of 1 kb, our model suggests that, among architectural proteins, BEAF-32 and CP190 are the main positive drivers of 3D domain borders. In humans, our model identifies well-known architectural proteins CTCF and cohesin, as well as ZNF143 and Polycomb group proteins as positive drivers of domain borders. The model also reveals the existence of several negative drivers that counteract the presence of domain borders including P300, RXRA, BCL11A and ELK1.

Computational Identification of Genomic Features That Influence 3D Chromatin Domain Formation

PubMed Central

Mourad, Raphaël; Cuvier, Olivier

2016-01-01

Recent advances in long-range Hi-C contact mapping have revealed the importance of the 3D structure of chromosomes in gene expression. A current challenge is to identify the key molecular drivers of this 3D structure. Several genomic features, such as architectural proteins and functional elements, were shown to be enriched at topological domain borders using classical enrichment tests. Here we propose multiple logistic regression to identify those genomic features that positively or negatively influence domain border establishment or maintenance. The model is flexible, and can account for statistical interactions among multiple genomic features. Using both simulated and real data, we show that our model outperforms enrichment test and non-parametric models, such as random forests, for the identification of genomic features that influence domain borders. Using Drosophila Hi-C data at a very high resolution of 1 kb, our model suggests that, among architectural proteins, BEAF-32 and CP190 are the main positive drivers of 3D domain borders. In humans, our model identifies well-known architectural proteins CTCF and cohesin, as well as ZNF143 and Polycomb group proteins as positive drivers of domain borders. The model also reveals the existence of several negative drivers that counteract the presence of domain borders including P300, RXRA, BCL11A and ELK1. PMID:27203237

The Effects of Organizational Justice on Positive Organizational Behavior: Evidence from a Large-Sample Survey and a Situational Experiment.

PubMed

Pan, Xiaofu; Chen, Mengyan; Hao, Zhichao; Bi, Wenfen

2017-01-01

Employees' positive organizational behavior (POB) is not only to promote organizational function but also improve individual and organizational performance. As an important concept in organizational research, organizational justice is thought to be a universal predictor of employee and organizational outcomes. The current set of two studies examined the effects of organizational justice (OJ) on POB of employees with two different studies, a large-sample survey and a situational experiment. In study 1, a total of 2,566 employees from 45 manufacturing enterprises completed paper-and-pencil questionnaires assessing organizational justice (OJ) and positive organizational behavior (POB) of employees. In study 2, 747 employees were randomly sampled to participate in the situational experiment with 2 × 2 between-subjects design. They were asked to read one of the four situational stories and to image that this situation happen to the person in the story or them, and then they were asked to imagine how the person in the story or they would have felt and what the person or they subsequently would have done. The results of study 1 suggested that OJ was correlated with POB of employees and OJ is a positive predictor of POB. The results of study 2 suggested that OJ had significant effects on POB and negative organizational behavior (NOB). Procedural justice accounted for significantly more variance than distributive justice in POB of employees. Distributive justice and procedural justice have different influences on POB and NOB in terms of effectiveness and direction. The effect of OJ on POB was greater than that of NOB. In addition, path analysis indicated that the direct effect of OJ on POB was smaller than its indirect effect. Thus, many intermediary effects could possibly be between them.

The Effects of Organizational Justice on Positive Organizational Behavior: Evidence from a Large-Sample Survey and a Situational Experiment

PubMed Central

Pan, Xiaofu; Chen, Mengyan; Hao, Zhichao; Bi, Wenfen

2018-01-01

Employees' positive organizational behavior (POB) is not only to promote organizational function but also improve individual and organizational performance. As an important concept in organizational research, organizational justice is thought to be a universal predictor of employee and organizational outcomes. The current set of two studies examined the effects of organizational justice (OJ) on POB of employees with two different studies, a large-sample survey and a situational experiment. In study 1, a total of 2,566 employees from 45 manufacturing enterprises completed paper-and-pencil questionnaires assessing organizational justice (OJ) and positive organizational behavior (POB) of employees. In study 2, 747 employees were randomly sampled to participate in the situational experiment with 2 × 2 between-subjects design. They were asked to read one of the four situational stories and to image that this situation happen to the person in the story or them, and then they were asked to imagine how the person in the story or they would have felt and what the person or they subsequently would have done. The results of study 1 suggested that OJ was correlated with POB of employees and OJ is a positive predictor of POB. The results of study 2 suggested that OJ had significant effects on POB and negative organizational behavior (NOB). Procedural justice accounted for significantly more variance than distributive justice in POB of employees. Distributive justice and procedural justice have different influences on POB and NOB in terms of effectiveness and direction. The effect of OJ on POB was greater than that of NOB. In addition, path analysis indicated that the direct effect of OJ on POB was smaller than its indirect effect. Thus, many intermediary effects could possibly be between them. PMID:29375434

Structural studies and SH3 domain binding properties of a human antiviral salivary proline-rich peptide.

PubMed

Righino, Benedetta; Pirolli, Davide; Radicioni, Giorgia; Marzano, Valeria; Longhi, Renato; Arcovito, Alessandro; Sanna, Maria Teresa; De Rosa, Maria Cristina; Paoluzi, Serena; Cesareni, Gianni; Messana, Irene; Castagnola, Massimo; Vitali, Alberto

2016-09-01

Human saliva contains hundreds of small proline-rich peptides originated by the proteolytic cleavage of the salivary basic Proline-Rich Proteins. Nevertheless only for few of them a specific biological activity has been assigned to date. Among them, the 1932 Da peptide (p1932) has been patented as an anti-HIV agent. In order to shed light on the possible mechanism of action of this peptide, we assessed in this study, by means of molecular dynamics calculations, circular dichroism and FTIR spectroscopic techniques, that p1932 has an intrinsic propensity to adopt a polyproline-II helix arrangement. This structural feature combined with the presence of PxxP motifs in its primary structure, represents an essential property for the exploitation of several biological activities. Next to these findings, we recently demonstrated the ability of this peptide to be internalized within cells of the oral mucosa, thus we focused onto a possible intracellular target, represented by the SH3 domains family. Its ability to interact with selected SH3 domains was finally assayed by Surface Plasmon Resonance spectroscopy. As a result, only Fyn, Hck, and c-Src SH3 domains gave positive results in terms of interaction, showing dissociation constants ranging from nanomolar to micromolar values having the best performer a KD of 148 nM. It is noteworthy that all the interacting domains belong to the Src kinases family, suggesting a role for p1932 as a modulator of the signal transduction pathways mediated by these kinases. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 714-725, 2016. © 2016 Wiley Periodicals, Inc.

Importance of the REM (Ras exchange) domain for membrane interactions by RasGRP3.

PubMed

Czikora, Agnes; Kedei, Noemi; Kalish, Heather; Blumberg, Peter M

2017-12-01

RasGRP comprises a family of guanine nucleotide exchange factors, regulating the dissociation of GDP from Ras GTPases to enhance the formation of the active GTP-bound form. RasGRP1 possesses REM (Ras exchange), GEF (catalytic), EF-hand, C1, SuPT (suppressor of PT), and PT (plasma membrane-targeting) domains, among which the C1 domain drives membrane localization in response to diacylglycerol or phorbol ester and the PT domain recognizes phosphoinositides. The homologous family member RasGRP3 shows less plasma membrane localization. The objective of this study was to explore the role of the different domains of RasGRP3 in membrane translocation in response to phorbol esters. The full-length RasGRP3 shows limited translocation to the plasma membrane in response to PMA, even when the basic hydrophobic cluster in the PT domain, reported to be critical for RasGRP1 translocation to endogenous activators, is mutated to resemble that of RasGRP1. Moreover, exchange of the C-termini (SuPT-PT domain) of the two proteins had little effect on their plasma membrane translocation. On the other hand, while the C1 domain of RasGRP3 alone showed partial plasma membrane translocation, truncated RasGRP3 constructs, which contain the PT domain and are missing the REM, showed stronger translocation, indicating that the REM of RasGRP3 was a suppressor of its membrane interaction. The REM of RasGRP1 failed to show comparable suppression of RasGRP3 translocation. The marked differences between RasGRP3 and RasGRP1 in membrane interaction necessarily will contribute to their different behavior in cells and are relevant to the design of selective ligands as potential therapeutic agents. Published by Elsevier B.V.

3DIANA: 3D Domain Interaction Analysis: A Toolbox for Quaternary Structure Modeling

PubMed Central

Segura, Joan; Sanchez-Garcia, Ruben; Tabas-Madrid, Daniel; Cuenca-Alba, Jesus; Sorzano, Carlos Oscar S.; Carazo, Jose Maria

2016-01-01

Electron microscopy (EM) is experiencing a revolution with the advent of a new generation of Direct Electron Detectors, enabling a broad range of large and flexible structures to be resolved well below 1 nm resolution. Although EM techniques are evolving to the point of directly obtaining structural data at near-atomic resolution, for many molecules the attainable resolution might not be enough to propose high-resolution structural models. However, accessing information on atomic coordinates is a necessary step toward a deeper understanding of the molecular mechanisms that allow proteins to perform specific tasks. For that reason, methods for the integration of EM three-dimensional maps with x-ray and NMR structural data are being developed, a modeling task that is normally referred to as fitting, resulting in the so called hybrid models. In this work, we present a novel application—3DIANA—specially targeted to those cases in which the EM map resolution is medium or low and additional experimental structural information is scarce or even lacking. In this way, 3DIANA statistically evaluates proposed/potential contacts between protein domains, presents a complete catalog of both structurally resolved and predicted interacting regions involving these domains and, finally, suggests structural templates to model the interaction between them. The evaluation of the proposed interactions is computed with DIMERO, a new method that scores physical binding sites based on the topology of protein interaction networks, which has recently shown the capability to increase by 200% the number of domain-domain interactions predicted in interactomes as compared to previous approaches. The new application displays the information at a sequence and structural level and is accessible through a web browser or as a Chimera plugin at http://3diana.cnb.csic.es. PMID:26772592

Quantitation of pyridyloxobutyl-DNA adducts in tissues of rats treated chronically with (R)- or (S)-N’-nitrosonornicotine (NNN) in a carcinogenicity study

PubMed Central

Zhao, Lijiao; Balbo, Silvia; Wang, Mingyao; Upadhyaya, Pramod; Khariwala, Samir S.; Villalta, Peter W.; Hecht, Stephen S.

2013-01-01

We quantified DNA adducts resulting from 2’-hydroxylation of enantiomers of the tobacco-specific nitrosamine N’-nitrosonornicotine (NNN) in tissues of male F-344 rats after 10, 30, 50, and 70 weeks of treatment with 14 ppm in the drinking water. These rats were in subgroups of a carcinogenicity study in which (S)-NNN was highly tumorigenic in the oral cavity and esophagus while (R)-NNN was relatively weakly active. DNA adducts were quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry in six tissues – oral mucosa, esophageal mucosa, nasal respiratory mucosa, nasal olfactory mucosa, liver, and lung. O2-[4-(3-Pyridyl)-4-oxobut-1-yl]thymidine (O2-POB-dThd, 7) and 7-[4-(3-pyridyl)-4-oxobut-1-yl]-2′-deoxyguanosine (7-POB-dGuo, 8), the latter as 7-[4-(3-pyridyl)-4-oxobut-1-yl]guanine (7-POB-Gua, 11), were detected at each time point in each tissue. In the target tissues for carcinogenicity, oral mucosa and esophageal mucosa, levels of 7-POB-Gua (11) and O2-POB-dThd (7) were similar, or 11 predominated, while in all other tissues at all time points for both enantiomers, 7 was clearly present in greater amounts than 11. Total measured DNA adduct levels in esophageal mucosa and oral mucosa were higher in rats treated with (S)-NNN than (R)-NNN. The highest adduct levels were found in the nasal respiratory mucosa. DNA adducts generally persisted in all tissues without any sign of substantial decreases throughout the 70 week time course. The results of this study suggest that inefficient repair of 7-POB-dGuo (8) in the rat oral cavity and esophagus may be important in carcinogenesis by NNN and support the development of these DNA adducts as potential biomarkers of NNN metabolic activation in people who use tobacco products. PMID:24001146

Domain alignment within ferroelectric/dielectric PbTiO 3 /SrTiO 3 superlattice nanostructures

DOE PAGES

Park, Joonkyu; Mangeri, John; Zhang, Qingteng; ...

2018-01-01

The ferroelectric domain pattern within lithographically defined PbTiO 3/SrTiO 3ferroelectric/dielectric heteroepitaxial superlattice nanostructures is strongly influenced by the edges of the structures.

Akt substrate TBC1D1 regulates GLUT1 expression through the mTOR pathway in 3T3-L1 adipocytes

PubMed Central

Zhou, Qiong L.; Jiang, Zhen Y.; Holik, John; Chawla, Anil; Hagan, G. Nana; Leszyk, John; Czech, Michael P.

2010-01-01

Multiple studies have suggested that the protein kinase Akt/PKB (protein kinase B) is required for insulin-stimulated glucose transport in skeletal muscle and adipose cells. In an attempt to understand links between Akt activation and glucose transport regulation, we applied mass spectrometry-based proteomics and bioinformatics approaches to identify potential Akt substrates containing the phospho-Akt substrate motif RXRXXpS/T. The present study describes the identification of the Rab GAP (GTPase-activating protein)-domain containing protein TBC1D1 [TBC (Tre-2/Bub2/Cdc16) domain family, member 1], which is closely related to TBC1D4 [TBC domain family, member 4, also denoted AS160 (Akt substrate of 160 kDa)], as an Akt substrate that is phosphorylated at Thr590. RNAi (RNA interference)-me-diated silencing of TBC1D1 elevated basal deoxyglucose uptake by approx. 61% in 3T3-L1 mouse embryo adipocytes, while the suppression of TBC1D4 and RapGAP220 under the same conditions had little effect on basal and insulin-stimulated deoxy-glucose uptake. Silencing of TBC1D1 strongly increased expression of the GLUT1 glucose transporter but not GLUT4 in cultured adipocytes, whereas the decrease in TBC1D4 had no effect. Remarkably, loss of TBC1D1 in 3T3-L1 adipocytes activated the mTOR (mammalian target of rapamycin)-p70 S6 protein kinase pathway, and the increase in GLUT1 expression in the cells treated with TBC1D1 siRNA (small interfering RNA) was blocked by the mTOR inhibitor rapamycin. Furthermore, overexpression of the mutant TBC1D1-T590A, lacking the putative Akt/PKB phosphorylation site, inhibited insulin stimulation of p70 S6 kinase phosphorylation at Thr389, a phosphorylation induced by mTOR. Taken together, our data suggest that TBC1D1 may be involved in controlling GLUT1 glucose transporter expression through the mTOR-p70 S6 kinase pathway. PMID:18215134

SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins.

PubMed

Koch, C A; Anderson, D; Moran, M F; Ellis, C; Pawson, T

1991-05-03

Src homology (SH) regions 2 and 3 are noncatalytic domains that are conserved among a series of cytoplasmic signaling proteins regulated by receptor protein-tyrosine kinases, including phospholipase C-gamma, Ras GTPase (guanosine triphosphatase)-activating protein, and Src-like tyrosine kinases. The SH2 domains of these signaling proteins bind tyrosine phosphorylated polypeptides, implicated in normal signaling and cellular transformation. Tyrosine phosphorylation acts as a switch to induce the binding of SH2 domains, thereby mediating the formation of heteromeric protein complexes at or near the plasma membrane. The formation of these complexes is likely to control the activation of signal transduction pathways by tyrosine kinases. The SH3 domain is a distinct motif that, together with SH2, may modulate interactions with the cytoskeleton and membrane. Some signaling and transforming proteins contain SH2 and SH3 domains unattached to any known catalytic element. These noncatalytic proteins may serve as adaptors to link tyrosine kinases to specific target proteins. These observations suggest that SH2 and SH3 domains participate in the control of intracellular responses to growth factor stimulation.

78 FR 23827 - Designation of Eighteen Individuals Pursuant to the Sergei Magnitsky Rule of Law Accountability...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-04-22

...; POB Moscow Region, Russia (individual) [MAGNIT]. 2. KUZNETSOV, Artem (a.k.a. KUZNETSOV, Artyom); DOB... Samarkand, Uzbekistan (individual) [MAGNIT]. 4. STEPANOVA, Olga G.; DOB 29 Jul 1962; POB Moscow, Russia... Region, Russia (individual) [MAGNIT]. 6. KARPOV, Pavel; DOB 27 Aug 1977; POB Moscow, Russia (individual...

77 FR 76624 - Unblocking of Specially Designated Nationals and Blocked Persons Pursuant to the Foreign...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-12-28

..., Fraccionamiento Quintas Del Sol, Chihuahua, Chihuahua, Mexico; DOB 10 Apr 1962; POB Culiacan, Sinaloa, Mexico... Sol, Chihuahua, Chihuahua, Mexico; Calle Ohio 3200, Chihuahua, Chihuahua, Mexico; DOB 19 Jan 1972; POB... Michigan, Fraccionamiento Quintas del Sol, Chihuahua, Chihuahua, Mexico; DOB 05 Oct 1966; POB Chihuahua...

Fresnel Lorentz Microscopy Imaging of Domains in Fe3O4 Nanoparticle Arrays

NASA Astrophysics Data System (ADS)

Majetich, S. A.; Evarts, E. R.; Hogg, C.; Yamamoto, K.; Hirayama, T.

2009-03-01

Fresnel Lorentz microscopy was used to study the magnetic domain structures of self-assembled nanoparticle arrays as a function of temperature, from 24 to 605 C. 11 nm diameter Fe3O4 nanoparticles with an edge-to-edge spacing of 2.5 nm form magnetic domains through magnetostatic interactions alone. At room temperature stripe domains were evident in monolayer arrays. The average domain size in monolayer regions is larger than that in bilayers. Mean field theories predict a reduced stabilization energy for bilayers, relative to that for monolayers. The domain wall positions were fairly stable up to 500 C, though the contrast in the walls diminished, indicating reduced magnetic order. Above 500 C there were large temperature-dependent changes. The walls surrounding the smaller domains disappeared at lower temperatures than those of the larger domains. Some magnetic contrast was visible up to 575 C, close to the Curie temperature of Fe3O4 (585 C). Transmission electron microscopy after cooling showed that the particle shape and position in the ordered arrays had been preserved during the high temperature imaging experiments.

Direct observation of interlocked domain walls and topological four-state vortex-like domain patterns in multiferroic YMnO{sub 3} single crystal

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

Tian, Lei; School of Materials Science and Engineering, Dalian Jiaotong University, Dalian, Liaoning 116028; Wang, Yumei, E-mail: wangym@iphy.ac.cn

2015-03-16

Using the advanced spherical aberration-corrected high angle annular dark field scanning transmission electron microscope imaging techniques, we investigated atomic-scale structural features of domain walls and domain patterns in YMnO{sub 3} single crystal. Three different types of interlocked ferroelectric-antiphase domain walls and two abnormal topological four-state vortex-like domain patterns are identified. Each ferroelectric domain wall is accompanied by a translation vector, i.e., 1/6[210] or −1/6[210], demonstrating its interlocked nature. Different from the four-state vortex domain patterns caused by a partial edge dislocation, two four-state vortex-like domain configurations have been obtained at atomic level. These observed phenomena can further extend our understandingmore » of the fascinating vortex domain patterns in multiferroic hexagonal rare-earth manganites.« less

Domain switching kinetics in ferroelectric-resistive BiFeO3 thin film memories

NASA Astrophysics Data System (ADS)

Meng, Jianwei; Jiang, Jun; Geng, Wenping; Chen, Zhihui; Zhang, Wei; Jiang, Anquan

2015-02-01

We fabricated (00l) BiFeO3 (BFO) thin films in different growth modes on SrRuO3/SrTiO3 substrates using a pulsed laser deposition technique. X-ray diffraction patterns show an out-of-plane lattice constant of 4.03 Å and ferroelectric polarization of 82 µC/cm2 for the BFO thin film in a layer-by-layer growth mode (2D-BFO), larger than 3.96 Å and 51 µC/cm2 for the thin film in the 3D-island formation growth mode (3D-BFO). The 2D-BFO thin film at 300 K shows switchable on/off diode currents upon polarization flipping near a negative coercive voltage, which is nevertheless absent from the above 3D-BFO thin film. From a positive-up-negative-down pulse characterization technique, we measured domain switching current transients as well as polarization-voltage (Pf-Vf) hysteresis loops in both semiconducting thin films. Pf-Vf hysteresis loops after 1 µs-retention time show the preferred domain orientation pointing to bottom electrodes in a 3D-BFO thin film. The poor retention of the domains pointing to top electrodes can be improved considerably in a 2D-BFO thin film. From these measurements, we extracted domain switching time dependence of coercive voltage at temperatures of 78-300 K. From these dependences, we found coercive voltages in semiconducting ferroelectric thin films much higher than those in insulating thin films, disobeying the traditional Merz equation. Finally, an equivalent resistance model in description of free-carrier compensation of the front domain boundary charge is developed to interpret this difference. This equivalent resistance can be coincidently extracted either from domain switching time dependence of coercive voltage or from applied voltage dependence of domain switching current, which drops almost linearly with the temperature until down to 0 in a ferroelectric insulator at 78 K.

Stability of M 3S 3 complexes on fcc M(111) surfaces: M = Au, Ag, Cu, and Ni

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

Liu, Da-Jiang; Lee, Jiyoung; Windus, Theresa L.

Density Functional Theory is utilized to assess the stability of metal (M)-sulfur (S) complexes adsorbed on fcc M(111) surfaces, specifically considering S-decorated planar M trimers, M 3S 3. Scanning Tunneling Microscopy studies have identified structures proposed to be Ni 3S 3 on Ni(111), and Au 3S 3 on Au(111). In addition, Cu 3S 3 on Cu(111) has been suggested to facilitate enhanced Cu surface mass transport. Our analysis considers M 3S 3 complexes for M = Au, Ag, Cu, and Ni, assessing key measures of stability on surfaces, and also comparing behavior with trends in gas-phase stability. These surface andmore » gas-phase analyses are systematically related within the framework of Hess’s law, which allows elucidation of various contributions to the overall energetics. In all cases, the adsorbed complex is stable relative to its separated constituents adsorbed on the terrace. However, only for Ag does one find a negative energy of formation from excess S on terraces and M extracted from kink sites along step edges, implying spontaneous complex formation for this pathway. We interpret various experimental observations in the context of our results for energetics.« less

Stability of M 3S 3 complexes on fcc M(111) surfaces: M = Au, Ag, Cu, and Ni

DOE PAGES

Liu, Da-Jiang; Lee, Jiyoung; Windus, Theresa L.; ...

2018-02-08

Density Functional Theory is utilized to assess the stability of metal (M)-sulfur (S) complexes adsorbed on fcc M(111) surfaces, specifically considering S-decorated planar M trimers, M 3S 3. Scanning Tunneling Microscopy studies have identified structures proposed to be Ni 3S 3 on Ni(111), and Au 3S 3 on Au(111). In addition, Cu 3S 3 on Cu(111) has been suggested to facilitate enhanced Cu surface mass transport. Our analysis considers M 3S 3 complexes for M = Au, Ag, Cu, and Ni, assessing key measures of stability on surfaces, and also comparing behavior with trends in gas-phase stability. These surface andmore » gas-phase analyses are systematically related within the framework of Hess’s law, which allows elucidation of various contributions to the overall energetics. In all cases, the adsorbed complex is stable relative to its separated constituents adsorbed on the terrace. However, only for Ag does one find a negative energy of formation from excess S on terraces and M extracted from kink sites along step edges, implying spontaneous complex formation for this pathway. We interpret various experimental observations in the context of our results for energetics.« less

The Smad3 linker region contains a transcriptional activation domain

PubMed Central

2004-01-01

Transforming growth factor-β (TGF-β)/Smads regulate a wide variety of biological responses through transcriptional regulation of target genes. Smad3 plays a key role in TGF-β/Smad-mediated transcriptional responses. Here, we show that the proline-rich linker region of Smad3 contains a transcriptional activation domain. When the linker region is fused to a heterologous DNA-binding domain, it activates transcription. We show that the linker region physically interacts with p300. The adenovirus E1a protein, which binds to p300, inhibits the transcriptional activity of the linker region, and overexpression of p300 can rescue the linker-mediated transcriptional activation. In contrast, an adenovirus E1a mutant, which cannot bind to p300, does not inhibit the linker-mediated transcription. The native Smad3 protein lacking the linker region is unable to mediate TGF-β transcriptional activation responses, although it can be phosphorylated by the TGF-β receptor at the C-terminal tail and has a significantly increased ability to form a heteromeric complex with Smad4. We show further that the linker region and the C-terminal domain of Smad3 synergize for transcriptional activation in the presence of TGF-β. Thus our findings uncover an important function of the Smad3 linker region in Smad-mediated transcriptional control. PMID:15588252

The Smad3 linker region contains a transcriptional activation domain.

PubMed

Wang, Guannan; Long, Jianyin; Matsuura, Isao; He, Dongming; Liu, Fang

2005-02-15

Transforming growth factor-beta (TGF-beta)/Smads regulate a wide variety of biological responses through transcriptional regulation of target genes. Smad3 plays a key role in TGF-beta/Smad-mediated transcriptional responses. Here, we show that the proline-rich linker region of Smad3 contains a transcriptional activation domain. When the linker region is fused to a heterologous DNA-binding domain, it activates transcription. We show that the linker region physically interacts with p300. The adenovirus E1a protein, which binds to p300, inhibits the transcriptional activity of the linker region, and overexpression of p300 can rescue the linker-mediated transcriptional activation. In contrast, an adenovirus E1a mutant, which cannot bind to p300, does not inhibit the linker-mediated transcription. The native Smad3 protein lacking the linker region is unable to mediate TGF-beta transcriptional activation responses, although it can be phosphorylated by the TGF-beta receptor at the C-terminal tail and has a significantly increased ability to form a heteromeric complex with Smad4. We show further that the linker region and the C-terminal domain of Smad3 synergize for transcriptional activation in the presence of TGF-beta. Thus our findings uncover an important function of the Smad3 linker region in Smad-mediated transcriptional control.

Domain alignment within ferroelectric/dielectric PbTiO 3 /SrTiO 3 superlattice nanostructures

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

Park, Joonkyu; Mangeri, John; Zhang, Qingteng

The ferroelectric domain pattern within lithographically defined PbTiO 3/SrTiO 3 ferroelectric/dielectric heteroepitaxial superlattice nanostructures is strongly influenced by the edges of the structures. Synchrotron X-ray nanobeam diffraction reveals that the spontaneously formed 180° ferroelectric stripe domains exhibited by such superlattices adopt a configuration in rectangular nanostructures in which domain walls are aligned with long patterned edges. The angular distribution of X-ray diffuse scattering intensity from nanodomains indicates that domains are aligned within an angular range of approximately 20° with respect to the edges. Computational studies based on a time-dependent Landau–Ginzburg–Devonshire model show that the preferred direction of the alignment resultsmore » from lowering of the bulk and electrostrictive contributions to the free energy of the system due to the release of the lateral mechanical constraint. This unexpected alignment appears to be intrinsic and not a result of distortions or defects caused by the patterning process. Thus, our work demonstrates how nanostructuring and patterning of heteroepitaxial superlattices allow for pathways to create and control ferroelectric structures that may appear counterintuitive.« less

Domain alignment within ferroelectric/dielectric PbTiO 3 /SrTiO 3 superlattice nanostructures

DOE PAGES

Park, Joonkyu; Mangeri, John; Zhang, Qingteng; ...

2018-01-22

The ferroelectric domain pattern within lithographically defined PbTiO 3/SrTiO 3 ferroelectric/dielectric heteroepitaxial superlattice nanostructures is strongly influenced by the edges of the structures. Synchrotron X-ray nanobeam diffraction reveals that the spontaneously formed 180° ferroelectric stripe domains exhibited by such superlattices adopt a configuration in rectangular nanostructures in which domain walls are aligned with long patterned edges. The angular distribution of X-ray diffuse scattering intensity from nanodomains indicates that domains are aligned within an angular range of approximately 20° with respect to the edges. Computational studies based on a time-dependent Landau–Ginzburg–Devonshire model show that the preferred direction of the alignment resultsmore » from lowering of the bulk and electrostrictive contributions to the free energy of the system due to the release of the lateral mechanical constraint. This unexpected alignment appears to be intrinsic and not a result of distortions or defects caused by the patterning process. Thus, our work demonstrates how nanostructuring and patterning of heteroepitaxial superlattices allow for pathways to create and control ferroelectric structures that may appear counterintuitive.« less

Domain mapping of the Rad51 paralog protein complexes

PubMed Central

Miller, Kristi A.; Sawicka, Dorota; Barsky, Daniel; Albala, Joanna S.

2004-01-01

The five human Rad51 paralogs are suggested to play an important role in the maintenance of genome stability through their function in DNA double-strand break repair. These proteins have been found to form two distinct complexes in vivo, Rad51B–Rad51C–Rad51D–Xrcc2 (BCDX2) and Rad51C–Xrcc3 (CX3). Based on the recent Pyrococcus furiosus Rad51 structure, we have used homology modeling to design deletion mutants of the Rad51 paralogs. The models of the human Rad51B, Rad51C, Xrcc3 and murine Rad51D (mRad51D) proteins reveal distinct N-terminal and C-terminal domains connected by a linker region. Using yeast two-hybrid and co-immunoprecipitation techniques, we have demonstrated that a fragment of Rad51B containing amino acid residues 1–75 interacts with the C-terminus and linker of Rad51C, residues 79–376, and this region of Rad51C also interacts with mRad51D and Xrcc3. We have also determined that the N-terminal domain of mRad51D, residues 4–77, binds to Xrcc2 while the C-terminal domain of mRad51D, residues 77–328, binds Rad51C. By this, we have identified the binding domains of the BCDX2 and CX3 complexes to further characterize the interaction of these proteins and propose a scheme for the three-dimensional architecture of the BCDX2 and CX3 paralog complexes. PMID:14704354

77 FR 28670 - Additional Designations, Foreign Narcotics Kingpin Designation Act

Federal Register 2010, 2011, 2012, 2013, 2014

2012-05-15

... 1968; Alt. DOB 24 Jun 1968; Alt. DOB 16 Oct 1962; POB Badiraguato, Sinaloa, Mexico; Citizen Mexico... SALAZAR, Ivan Archibaldo); Mexico; DOB 02 Oct 1980; POB Sinaloa, Mexico; Citizen Mexico; Nationality Mexico; (INDIVIDUAL) [SDNTK] 2. GUZMAN LOPEZ, Ovidio, Mexico; DOB 29 Mar 1990; POB Sinaloa, Mexico...

3D time-domain airborne EM modeling for an arbitrarily anisotropic earth

NASA Astrophysics Data System (ADS)

Yin, Changchun; Qi, Yanfu; Liu, Yunhe

2016-08-01

Time-domain airborne EM data is currently interpreted based on an isotropic model. Sometimes, it can be problematic when working in the region with distinct dipping stratifications. In this paper, we simulate the 3D time-domain airborne EM responses over an arbitrarily anisotropic earth with topography by edge-based finite-element method. Tetrahedral meshes are used to describe the abnormal bodies with complicated shapes. We further adopt the Backward Euler scheme to discretize the time-domain diffusion equation for electric field, obtaining an unconditionally stable linear equations system. We verify the accuracy of our 3D algorithm by comparing with 1D solutions for an anisotropic half-space. Then, we switch attentions to effects of anisotropic media on the strengths and the diffusion patterns of time-domain airborne EM responses. For numerical experiments, we adopt three typical anisotropic models: 1) an anisotropic anomalous body embedded in an isotropic half-space; 2) an isotropic anomalous body embedded in an anisotropic half-space; 3) an anisotropic half-space with topography. The modeling results show that the electric anisotropy of the subsurface media has big effects on both the strengths and the distribution patterns of time-domain airborne EM responses; this effect needs to be taken into account when interpreting ATEM data in areas with distinct anisotropy.

Eukaryotic ribonucleases P/MRP: the crystal structure of the P3 domain.

PubMed

Perederina, Anna; Esakova, Olga; Quan, Chao; Khanova, Elena; Krasilnikov, Andrey S

2010-02-17

Ribonuclease (RNase) P is a site-specific endoribonuclease found in all kingdoms of life. Typical RNase P consists of a catalytic RNA component and a protein moiety. In the eukaryotes, the RNase P lineage has split into two, giving rise to a closely related enzyme, RNase MRP, which has similar components but has evolved to have different specificities. The eukaryotic RNases P/MRP have acquired an essential helix-loop-helix protein-binding RNA domain P3 that has an important function in eukaryotic enzymes and distinguishes them from bacterial and archaeal RNases P. Here, we present a crystal structure of the P3 RNA domain from Saccharomyces cerevisiae RNase MRP in a complex with RNase P/MRP proteins Pop6 and Pop7 solved to 2.7 A. The structure suggests similar structural organization of the P3 RNA domains in RNases P/MRP and possible functions of the P3 domains and proteins bound to them in the stabilization of the holoenzymes' structures as well as in interactions with substrates. It provides the first insight into the structural organization of the eukaryotic enzymes of the RNase P/MRP family.

Mouse Noxa uses only the C-terminal BH3-domain to inactivate Mcl-1.

PubMed

Weber, Arnim; Ausländer, David; Häcker, Georg

2013-09-01

Noxa is a member of the pro-apoptotic BH3-only group of Bcl-2 proteins that is known to bind specifically to anti-apoptotic Mcl-1 and A1, antagonizing their function. Mcl-1 has been reported to have a short half-life, and Noxa up-regulation accelerates Mcl-1 degradation by the proteasome. Unlike human Noxa, mouse Noxa has two BH3-domains, which both have affinity for Mcl-1. We here investigate two aspects of the molecular function of Noxa, namely the requirements for the two BH3-domains in mouse Noxa and the role of Noxa in Mcl-1-degradation. We found that only the C-terminal BH3-domain of mouse Noxa is active in neutralizing Mcl-1. This was the result of the targeting of Noxa to the outer mitochondrial membrane through its C-terminal alpha-helix, which allowed Mcl-1-neutralization only when the BH3-domain was immediately N-terminal of the membrane anchor. However, the N-terminal BH3-domain enhanced interaction with Mcl-1 and A1. The Noxa-dependent degradation of Mcl-1 was independent of the kinase GSK3 and the deubiquitinase Usp9x in mouse embryonic fibroblasts. These data show that Noxa is targeted to the mitochondrial membrane where it neutralises Mcl-1 via its C-terminal BH3-domain and suggest that Noxa is co-degraded with Noxa, in a way independent of ubiquitin-modifying enzymes described for Mcl-1.

Shear at Twin Domain Boundaries in YBa2Cu3O7-x

NASA Astrophysics Data System (ADS)

Caldwell, W. A.; Tamura, N.; Celestre, R. S.; MacDowell, A. A.; Padmore, H. A.; Geballe, T. H.; Koster, G.; Batterman, B. W.; Patel, J. R.

2004-05-01

The microstructure and strain state of twin domains in YBa2Cu3O7-x are discussed based upon synchrotron white-beam x-ray microdiffraction measurements. Intensity variations of the fourfold twin splitting of Laue diffraction peaks are used to determine the twin domain structure. Strain analysis shows that interfaces between neighboring twin domains are strained in shear, whereas the interior of these domains are regions of low strain. These measurements are consistent with the orientation relationships of twin boundaries within and across domains and show that basal plane shear stresses can exceed 100MPa where twin domains meet. Our results support stress field pinning of magnetic flux vortices by twin domain boundaries.

Quantitative relation between intermolecular and intramolecular binding of pro-rich peptides to SH3 domains.

PubMed

Zhou, Huan-Xiang

2006-11-01

Flexible linkers are often found to tether binding sequence motifs or connect protein domains. Here we analyze three usages of flexible linkers: 1), intramolecular binding of proline-rich peptides (PRPs) to SH3 domains for kinase regulation; 2), intramolecular binding of PRP for increasing the folding stability of SH3 domains; and 3), covalent linking of PRPs and other ligands for high-affinity bivalent binding. The basis of these analyses is a quantitative relation between intermolecular and intramolecular binding constants. This relation has the form K(i) = K(e0)p for intramolecular binding and K(e) = K(e01)K(e02)p for bivalent binding. The effective concentration p depends on the length of the linker and the distance between the linker attachment points in the bound state. Several applications illustrate the usefulness of the quantitative relation. These include intramolecular binding to the Itk SH3 domain by an internal PRP and to a circular permutant of the alpha-spectrin SH3 domain by a designed PRP, and bivalent binding to the two SH3 domains of Grb2 by two linked PRPs. These and other examples suggest that flexible linkers and sequence motifs tethered to them, like folded protein domains, are also subject to tight control during evolution.

Ultraviolet-infrared laser-induced domain inversion in MgO-doped congruent LiNbO3 and near stoichiometric LiTaO3 crystals

NASA Astrophysics Data System (ADS)

Zhi, Ya'nan; Qu, Weijuan; Liu, De'an; Sun, Jianfeng; Yan, Aimin; Liu, Liren

2008-08-01

Laser-induced domain inversion is a promising technique for domain engineering in LiNbO3 and LiTaO3. The ultraviolet-infrared laser induced domain inversions in MgO-doped congruent LiNbO3 and near stoichiometric LiTaO3 crystals are investigated for the first time here. Within the wavelength range from 351 to 799 nm, the different reductions of nucleation field induced by the focused continuous laser irradiation are systematically investigated in the MgO-doped congruent LiNbO3 crystals. The investigation of ultrashort-pulse laser-induced domain inversion in MgO-doped congruent LiNbO3 is performed with 800 nm wavelength irradiation. The focused continuous ultraviolet laser-induced ferroelectric domain inversion in the near stoichiometric LiTaO3 is also investigated. The different physical explanations, based on space charge field and defect formation, are presented for the laser-induced domain inversion, and the solid experimental proofs are also presented. The results provide the solid experimental proofs and feasible schemes for the further investigation of laser-induced domain engineering in MgO-doped LiNbO3 and near stoichiometric LiTaO3 crystals. The important characteristics of domain inversion, including domain wall and internal field, in LiNbO3 crystals are also investigated by the digital holographic interferometry with an improved reconstruction method, and some creative experimental results and conclusions are achieved.

MDC9, a widely expressed cellular disintegrin containing cytoplasmic SH3 ligand domains

PubMed Central

1996-01-01

Cellular disintegrins are a family of proteins that are related to snake venom integrin ligands and metalloproteases. We have cloned and sequenced the mouse and human homologue of a widely expressed cellular disintegrin, which we have termed MDC9 (for metalloprotease/disintegrin/cysteine-rich protein 9). The deduced mouse and human protein sequences are 82% identical. MDC9 contains several distinct protein domains: a signal sequence is followed by a prodomain and a domain with sequence similarity to snake venom metalloproteases, a disintegrin domain, a cysteine-rich region, an EGF repeat, a membrane anchor, and a cytoplasmic tail. The cytoplasmic tail of MDC9 has two proline-rich sequences which can bind the SH3 domain of Src, and may therefore function as SH3 ligand domains. Western blot analysis shows that MDC9 is an approximately 84-kD glycoprotein in all mouse tissues examined, and in NIH 3T3 fibroblast and C2C12 myoblast mouse cell lines. MDC9 can be both cell surface biotinylated and 125I-labeled in NIH 3T3 mouse fibroblasts, indicating that the protein is present on the plasma membrane. Expression of MDC9 in COS-7 cells yields an 84-kD protein, and immunofluorescence analysis of COS-7 cells expressing MDC9 shows a staining pattern that is consistent with a plasma membrane localization. The apparent molecular mass of 84 kD suggests that MDC9 contains a membrane-anchored metalloprotease and disintegrin domain. We propose that MDC9 might function as a membrane-anchored integrin ligand or metalloprotease, or that MDC9 may combine both activities in one protein. PMID:8647900

Time domain simulation of Gd3+-Gd3+ distance measurements by EPR

NASA Astrophysics Data System (ADS)

Manukovsky, Nurit; Feintuch, Akiva; Kuprov, Ilya; Goldfarb, Daniella

2017-07-01

Gd3+-based spin labels are useful as an alternative to nitroxides for intramolecular distance measurements at high fields in biological systems. However, double electron-electron resonance (DEER) measurements using model Gd3+ complexes featured a low modulation depth and an unexpected broadening of the distance distribution for short Gd3+-Gd3+ distances, when analysed using the software designed for S = 1/2 pairs. It appears that these effects result from the different spectroscopic characteristics of Gd3+—the high spin, the zero field splitting (ZFS), and the flip-flop terms in the dipolar Hamiltonian that are often ignored for spin-1/2 systems. An understanding of the factors affecting the modulation frequency and amplitude is essential for the correct analysis of Gd3+-Gd3+ DEER data and for the educated choice of experimental settings, such as Gd3+ spin label type and the pulse parameters. This work uses time-domain simulations of Gd3+-Gd3+ DEER by explicit density matrix propagation to elucidate the factors shaping Gd3+ DEER traces. The simulations show that mixing between the |+½, -½> and |-½, +½> states of the two spins, caused by the flip-flop term in the dipolar Hamiltonian, leads to dampening of the dipolar modulation. This effect may be mitigated by a large ZFS or by pulse frequency settings allowing for a decreased contribution of the central transition and the one adjacent to it. The simulations reproduce both the experimental line shapes of the Fourier-transforms of the DEER time domain traces and the trends in the behaviour of the modulation depth, thus enabling a more systematic design and analysis of Gd3+ DEER experiments.

Virus-like Particles Containing Multiple M2 Extracellular Domains Confer Improved Cross-protection Against Various Subtypes of Influenza Virus

PubMed Central

Kim, Min-Chul; Song, Jae-Min; O, Eunju; Kwon, Young-Man; Lee, Youn-Jeong; Compans, Richard W; Kang, Sang-Moo

2013-01-01

The extracellular domain of M2 (M2e), a small ion channel membrane protein, is well conserved among different human influenza A virus strains. To improve the protective efficacy of M2e vaccines, we genetically engineered a tandem repeat of M2e epitope sequences (M2e5x) of human, swine, and avian origin influenza A viruses, which was expressed in a membrane-anchored form and incorporated in virus-like particles (VLPs). The M2e5x protein with the transmembrane domain of hemagglutinin (HA) was effectively incorporated into VLPs at a several 100-fold higher level than that on influenza virions. Intramuscular immunization with M2e5x VLP vaccines was highly effective in inducing M2e-specific antibodies reactive to different influenza viruses, mucosal and systemic immune responses, and cross-protection regardless of influenza virus subtypes in the absence of adjuvant. Importantly, immune sera were found to be sufficient for conferring protection in naive mice, which was long-lived and cross-protective. Thus, molecular designing and presenting M2e immunogens on VLPs provide a promising platform for developing universal influenza vaccines without using adjuvants. PMID:23247101

Theoretical Insights Reveal Novel Motions in Csk’s SH3 Domain That Control Kinase Activation

PubMed Central

Barkho, Sulyman; Pierce, Levi C. T.; Li, Sheng; Adams, Joseph A.; Jennings, Patricia A.

2015-01-01

The Src family of tyrosine kinases (SFKs) regulate numerous aspects of cell growth and differentiation and are under the principal control of the C-terminal Src Kinase (Csk). Although Csk and SFKs share conserved kinase, SH2 and SH3 domains, they differ considerably in three-dimensional structure, regulatory mechanism, and the intrinsic kinase activities. Although the SH2 and SH3 domains are known to up- or down-regulate tyrosine kinase function, little is known about the global motions in the full-length kinase that govern these catalytic variations. We use a combination of accelerated Molecular Dynamics (aMD) simulations and experimental methods to provide a new view of functional motions in the Csk scaffold. These computational studies suggest that high frequency vibrations in the SH2 domain are coupled through the N-terminal lobe of the kinase domain to motions in the SH3 domain. The effects of these reflexive movements on the kinase domain can be viewed using both Deuterium Exchange Mass Spectrometry (DXMS) and steady-state kinetic methods. Removal of several contacts, including a crystallographically unobserved N-terminal segment, between the SH3 and kinase domains short-circuit these coupled motions leading to reduced catalytic efficiency and stability of N-lobe motifs within the kinase domain. The data expands the model of Csk’s activation whereby separate domains productively interact with two diametrically opposed surfaces of the kinase domain. Such reversible transitions may organize the active structure of the tyrosine kinase domain of Csk. PMID:26030592

Raptor binds the SAIN (Shc and IRS-1 NPXY binding) domain of insulin receptor substrate-1 (IRS-1) and regulates the phosphorylation of IRS-1 at Ser-636/639 by mTOR.

PubMed

Tzatsos, Alexandros

2009-08-21

In normal physiological states mTOR phosphorylates and activates Akt. However, under diabetic-mimicking conditions mTOR inhibits phosphatidylinositol (PI) 3-kinase/Akt signaling by phosphorylating insulin receptor substrate-1 (IRS-1) at Ser-636/639. The molecular basis for the differential effect of mTOR signaling on Akt is poorly understood. Here, it has been shown that knockdown of mTOR, Raptor, and mLST8, but not Rictor and mSin1, suppresses insulin-stimulated phosphorylation of IRS-1 at Ser-636/639 and stabilizes IRS-1 after long term insulin stimulation. This phosphorylation depends on the PI 3-kinase/PDK1 axis but is Akt-independent. At the molecular level, Raptor binds the SAIN (Shc and IRS-1 NPXY binding) domain of IRS-1 and regulates the phosphorylation of IRS-1 at Ser-636/639 by mTOR. IRS-1 lacking the SAIN domain does not interact with Raptor, is not phosphorylated at Ser-636/639, and favorably interacts with PI 3-kinase. Overall, these data provide new insights in the molecular mechanisms by which mTORC1 inhibits PI 3-kinase/Akt signaling at the level of IRS-1 and suggest that mTOR signaling toward Akt is scaffold-dependent.

Pairwise domain adaptation module for CNN-based 2-D/3-D registration.

PubMed

Zheng, Jiannan; Miao, Shun; Jane Wang, Z; Liao, Rui

2018-04-01

Accurate two-dimensional to three-dimensional (2-D/3-D) registration of preoperative 3-D data and intraoperative 2-D x-ray images is a key enabler for image-guided therapy. Recent advances in 2-D/3-D registration formulate the problem as a learning-based approach and exploit the modeling power of convolutional neural networks (CNN) to significantly improve the accuracy and efficiency of 2-D/3-D registration. However, for surgery-related applications, collecting a large clinical dataset with accurate annotations for training can be very challenging or impractical. Therefore, deep learning-based 2-D/3-D registration methods are often trained with synthetically generated data, and a performance gap is often observed when testing the trained model on clinical data. We propose a pairwise domain adaptation (PDA) module to adapt the model trained on source domain (i.e., synthetic data) to target domain (i.e., clinical data) by learning domain invariant features with only a few paired real and synthetic data. The PDA module is designed to be flexible for different deep learning-based 2-D/3-D registration frameworks, and it can be plugged into any pretrained CNN model such as a simple Batch-Norm layer. The proposed PDA module has been quantitatively evaluated on two clinical applications using different frameworks of deep networks, demonstrating its significant advantages of generalizability and flexibility for 2-D/3-D medical image registration when a small number of paired real-synthetic data can be obtained.

Theoretical descriptions of novel triplet germylenes M1-Ge-M2-M3 (M1 = H, Li, Na, K; M2 = Be, Mg, Ca; M3 = H, F, Cl, Br).

PubMed

Kassaee, Mohamad Zaman; Ashenagar, Samaneh

2018-02-06

In a quest to identify new ground-state triplet germylenes, the stabilities (singlet-triplet energy differences, ΔE S-T ) of 96 singlet (s) and triplet (t) M 1 -Ge-M 2 -M 3 species were compared and contrasted at the B3LYP/6-311++G**, QCISD(T)/6-311++G**, and CCSD(T)/6-311++G** levels of theory (M 1  = H, Li, Na, K; M 2  = Be, Mg, Ca; M 3  = H, F, Cl, Br). Interestingly, F-substituent triplet germylenes (M 3  = F) appear to be more stable and linear than the corresponding Cl- or Br-substituent triplet germylenes (M 3  = Cl or Br). Triplets with M 1  = K (i.e., the K-Ge-M 2 -M 3 series) seem to be more stable than the corresponding triplets with M 1  = H, Li, or Na. This can be attributed to the higher electropositivity of potassium. Triplet species with M 3  = Cl behave similarly to those with M 3  = Br. Conversely, triplets with M 3  = H show similar stabilities and linearities to those with M 3  = F. Singlet species of formulae K-Ge-Ca-Cl and K-Ge-Ca-Br form unexpected cyclic structures. Finally, the triplet germylenes M 1 -Ge-M 2 -M 3 become more stable as the electropositivities of the α-substituents (M 1 and M 2 ) and the electronegativity of the β-substituent (M 3 ) increase.

Magnetoelectric domain wall dynamics and its implications for magnetoelectric memory

DOE PAGES

Belashchenko, K. D.; Tchernyshyov, O.; Kovalev, Alexey A.; ...

2016-03-30

Domain wall dynamics in a magnetoelectric antiferromagnet is analyzed, and its implications for magnetoelectric memory applications are discussed. Cr 2O 3 is used in the estimates of the materials parameters. It is found that the domain wall mobility has a maximum as a function of the electric field due to the gyrotropic coupling induced by it. In Cr 2O 3, the maximal mobility of 0.1 m/(s Oe) is reached at E≈0.06 V/nm. Fields of this order may be too weak to overcome the intrinsic depinning field, which is estimated for B-doped Cr 2O 3. These major drawbacks for device implementationmore » can be overcome by applying a small in-plane shear strain, which blocks the domain wall precession. Domain wall mobility of about 0.7 m/(s Oe) can then be achieved at E = 0.2 V/nm. Furthermore, a split-gate scheme is proposed for the domain-wall controlled bit element; its extension to multiple-gate linear arrays can offer advantages in memory density, programmability, and logic functionality.« less

The Caenorhabditis elegans gene unc-89, required fpr muscle M-line assembly, encodes a giant modular protein composed of Ig and signal transduction domains

PubMed Central

1996-01-01

Mutations in the Caenorhabditis elegans gene unc-89 result in nematodes having disorganized muscle structure in which thick filaments are not organized into A-bands, and there are no M-lines. Beginning with a partial cDNA from the C. elegans sequencing project, we have cloned and sequenced the unc-89 gene. An unc-89 allele, st515, was found to contain an 84-bp deletion and a 10-bp duplication, resulting in an in- frame stop codon within predicted unc-89 coding sequence. Analysis of the complete coding sequence for unc-89 predicts a novel 6,632 amino acid polypeptide consisting of sequence motifs which have been implicated in protein-protein interactions. UNC-89 begins with 67 residues of unique sequences, SH3, dbl/CDC24, and PH domains, 7 immunoglobulins (Ig) domains, a putative KSP-containing multiphosphorylation domain, and ends with 46 Ig domains. A polyclonal antiserum raised to a portion of unc-89 encoded sequence reacts to a twitchin-sized polypeptide from wild type, but truncated polypeptides from st515 and from the amber allele e2338. By immunofluorescent microscopy, this antiserum localizes to the middle of A-bands, consistent with UNC-89 being a structural component of the M-line. Previous studies indicate that myofilament lattice assembly begins with positional cues laid down in the basement membrane and muscle cell membrane. We propose that the intracellular protein UNC-89 responds to these signals, localizes, and then participates in assembling an M-line. PMID:8603916

Chromatin interaction networks revealed unique connectivity patterns of broad H3K4me3 domains and super enhancers in 3D chromatin.

PubMed

Thibodeau, Asa; Márquez, Eladio J; Shin, Dong-Guk; Vera-Licona, Paola; Ucar, Duygu

2017-10-31

Broad domain promoters and super enhancers are regulatory elements that govern cell-specific functions and harbor disease-associated sequence variants. These elements are characterized by distinct epigenomic profiles, such as expanded deposition of histone marks H3K27ac for super enhancers and H3K4me3 for broad domains, however little is known about how they interact with each other and the rest of the genome in three-dimensional chromatin space. Using network theory methods, we studied chromatin interactions between broad domains and super enhancers in three ENCODE cell lines (K562, MCF7, GM12878) obtained via ChIA-PET, Hi-C, and Hi-CHIP assays. In these networks, broad domains and super enhancers interact more frequently with each other compared to their typical counterparts. Network measures and graphlets revealed distinct connectivity patterns associated with these regulatory elements that are robust across cell types and alternative assays. Machine learning models showed that these connectivity patterns could effectively discriminate broad domains from typical promoters and super enhancers from typical enhancers. Finally, targets of broad domains in these networks were enriched in disease-causing SNPs of cognate cell types. Taken together these results suggest a robust and unique organization of the chromatin around broad domains and super enhancers: loci critical for pathologies and cell-specific functions.

Disparity of secondary electron emission in ferroelectric domains of YMnO{sub 3}

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

Cheng, Shaobo; Deng, S. Q.; Yuan, Wenjuan

2015-07-20

The applications of multiferroic materials require our understanding about the behaviors of domains with different polarization directions. Taking advantage of the scanning electron microscope, we investigate the polar surface of single crystal YMnO{sub 3} sample in secondary electron (SE) mode. By slowing down the scanning speed of electron beam, the negative surface potential of YMnO{sub 3} can be realized, and the domain contrast can be correspondingly changed. Under this experimental condition, with the help of a homemade Faraday cup, the difference of intrinsic SE emission coefficients of antiparallel domains is measured to be 0.12 and the downward polarization domains showmore » a larger SE emission ability. Our results indicate that the total SE emission of this material can be altered by changing the ratio of the antiparallel domains, which provide an avenue for device design with this kind of materials.« less

Chemical shift assignments of the partially deuterated Fyn SH2-SH3 domain.

PubMed

Kieken, Fabien; Loth, Karine; van Nuland, Nico; Tompa, Peter; Lenaerts, Tom

2018-04-01

Src Homology 2 and 3 (SH2 and SH3) are two key protein interaction modules involved in regulating the activity of many proteins such as tyrosine kinases and phosphatases by respective recognition of phosphotyrosine and proline-rich regions. In the Src family kinases, the inactive state of the protein is the direct result of the interaction of the SH2 and the SH3 domain with intra-molecular regions, leading to a closed structure incompetent with substrate modification. Here, we report the 1 H, 15 N and 13 C backbone- and side-chain chemical shift assignments of the partially deuterated Fyn SH3-SH2 domain and structural differences between tandem and single domains. The BMRB accession number is 27165.

Amyloid-like assembly of the low complexity domain of yeast Nab3.

PubMed

O'Rourke, Thomas W; Loya, Travis J; Head, PamelaSara E; Horton, John R; Reines, Daniel

2015-01-01

Termination of transcription of short non-coding RNAs is carried out in yeast by the Nab3-Nrd1-Sen1 complex. Nab3 and Nrd1 are hnRNP-like proteins that dimerize and bind RNA with sequence specificity. We show here that an essential region of Nab3 that is predicted to be prion-like based upon its sequence bias, formed amyloid-like filaments. A similar region from Nrd1 also assembled into filaments in vitro. The purified Nab3 domain formed a macroscopic gel whose lattice organization was observed by X-ray fiber diffraction. Filaments were resistant to dissociation in anionic detergent, bound the fluorescent dye thioflavin T, and showed a β-sheet rich structure by circular dichroism spectroscopy, similar to human amyloid β which served as a reference amyloid. A version of the Nab3 domain with a mutation that impairs its termination function, also formed fibers as observed by electron microscopy. Using a protein fragment interaction assay, the purified Nab3 domain was seen to interact with itself in living yeast. A similar observation was made for full length Nab3. These results suggest that the Nab3 and Nrd1 RNA-binding proteins can attain a complex polymeric form and raise the possibility that this property is important for organizing their functional state during termination. These findings are congruent with recent work showing that RNA binding proteins with low complexity domains form a dynamic subcellular matrix in which RNA metabolism takes place but can also aberrantly yield pathological aggregated particles.

PREFACE: Domain wall dynamics in nanostructures Domain wall dynamics in nanostructures

NASA Astrophysics Data System (ADS)

Marrows, C. H.; Meier, G.

2012-01-01

forms of ordered phases such as antiferromagnetism and ferroelectricity. We would like to thank the scientists from all over the world who happily agreed to contribute their latest results to this special issue, and the Journal of Physics: Condensed Matter staff for their help, patience and professionalism. In such a fast-moving field it is not possible to give a definitive account, and this special issue can be no more than a snapshot of the current state of knowledge regarding this topic. Nevertheless, we hope that this collection of papers is a useful resource for experienced workers in the field, forms a useful introduction to researchers early in their careers and inspires others in related areas of nanotechnology to enter into the study of domain dynamics in nanostructures. Domain wall dynamics in nanostructures contents Temperature estimation in a ferromagnetic Fe-Ni nanowire involving a current-driven domain wall motionA Yamaguchi, A Hirohata, T Ono and H Miyajima Magnetization reversal in magnetic nanostripes via Bloch wall formation M Zeisberger and R Mattheis Magnetic soft x-ray microscopy of the domain wall depinning process in permalloy magnetic nanowiresMi-Young Im, Lars Bocklage, Guido Meier and Peter Fischer Domain wall propagation in meso- and nanoscale ferroelectrics R G P McQuaid, M McMillen, L-W Chang, A Gruverman and J M Gregg Transverse and vortex domain wall structure in magnetic nanowires with uniaxial in-plane anisotropyM T Bryan, S Bance, J Dean, T Schrefl and D A Allwood The stochastic nature of the domain wall motion along high perpendicular anisotropy strips with surface roughness Eduardo Martinez Temperature-dependent dynamics of stochastic domain-wall depinning in nanowiresClemens Wuth, Peter Lendecke and Guido Meier Controlled pinning and depinning of domain walls in nanowires with perpendicular magnetic anisotropyTheo Gerhardt, André Drews and Guido Meier The interaction of transverse domain wallsBenjamin Krüger The increase of the

The C-Terminal Domain of the Virulence Factor MgtC Is a Divergent ACT Domain

PubMed Central

Yang, Yinshan; Labesse, Gilles; Carrère-Kremer, Séverine; Esteves, Kevin; Kremer, Laurent

2012-01-01

MgtC is a virulence factor of unknown function important for survival inside macrophages in several intracellular bacterial pathogens, including Mycobacterium tuberculosis. It is also involved in adaptation to Mg2+ deprivation, but previous work suggested that MgtC is not a Mg2+ transporter. In this study, we demonstrated that the amount of the M. tuberculosis MgtC protein is not significantly increased by Mg2+ deprivation. Members of the MgtC protein family share a conserved membrane N-terminal domain and a more divergent cytoplasmic C-terminal domain. To get insights into MgtC functional and structural organization, we have determined the nuclear magnetic resonance (NMR) structure of the C-terminal domain of M. tuberculosis MgtC. This structure is not affected by the Mg2+ concentration, indicating that it does not bind Mg2+. The structure of the C-terminal domain forms a βαββαβ fold found in small molecule binding domains called ACT domains. However, the M. tuberculosis MgtC ACT domain differs from canonical ACT domains because it appears to lack the ability to dimerize and to bind small molecules. We have shown, using a bacterial two-hybrid system, that the M. tuberculosis MgtC protein can dimerize and that the C-terminal domain somehow facilitates this dimerization. Taken together, these results indicate that M. tuberculosis MgtC does not have an intrinsic function related to Mg2+ uptake or binding but could act as a regulatory factor based on protein-protein interaction that could be facilitated by its ACT domain. PMID:22984256

von Willebrand factor (VWF) propeptide binding to VWF D'D3 domain attenuates platelet activation and adhesion.

PubMed

Madabhushi, Sri R; Shang, Chengwei; Dayananda, Kannayakanahalli M; Rittenhouse-Olson, Kate; Murphy, Mary; Ryan, Thomas E; Montgomery, Robert R; Neelamegham, Sriram

2012-05-17

Noncovalent association between the von Willebrand factor (VWF) propeptide (VWFpp) and mature VWF aids N-terminal multimerization and protein compartmentalization in storage granules. This association is currently thought to dissipate after secretion into blood. In the present study, we examined this proposition by quantifying the affinity and kinetics of VWFpp binding to mature VWF using surface plasmon resonance and by developing novel anti-VWF D'D3 mAbs. Our results show that the only binding site for VWFpp in mature VWF is in its D'D3 domain. At pH 6.2 and 10mM Ca(2+), conditions mimicking intracellular compartments, VWFpp-VWF binding occurs with high affinity (K(D) = 0.2nM, k(off) = 8 × 10(-5) s(-1)). Significant, albeit weaker, binding (K(D) = 25nM, k(off) = 4 × 10(-3) s(-1)) occurs under physiologic conditions of pH 7.4 and 2.5mM Ca(2+). This interaction was also observed in human plasma (K(D) = 50nM). The addition of recombinant VWFpp in both flow-chamber-based platelet adhesion assays and viscometer-based shear-induced platelet aggregation and activation studies reduced platelet adhesion and activation partially. Anti-D'D3 mAb DD3.1, which blocks VWFpp binding to VWF-D'D3, also abrogated platelet adhesion, as shown by shear-induced platelet aggregation and activation studies. Our data demonstrate that VWFpp binding to mature VWF occurs in the circulation, which can regulate the hemostatic potential of VWF by reducing VWF binding to platelet GpIbα.

Potent and Selective Phosphopeptide Mimetic Prodrugs Targeted to the Src Homology 2 (SH2) Domain of Signal Transducer and Activator of Transcription 3

PubMed Central

Mandal, Pijus K.; Gao, Fengqin; Lu, Zhen; Ren, Zhiyong; Ramesh, Rajagopal; Birtwistle, J. Sanderson; Kaluarachchi, Kumaralal K.; Chen, Xiaomin; Bast, Robert C.; Liao, Warren S.; McMurray, John S.

2011-01-01

Signal transducer and activator of transcription 3 (Stat3), a target for anticancer drug design, is activated by recruitment to phosphotyrosine residues on growth factor and cytokine receptors via its SH2 domain. We report here structure-activity relationship studies on phosphopeptide mimics targeted to the SH2 domain of Stat3. Inclusion of a methyl group on the β-position of the pTyr mimic, 4-phosphocinfnamide, enhanced affinity 2–3 fold. Bis-pivaloyloxymethyl prodrugs containing β-methyl cinnamide, dipeptide scaffolds Haic and Nle-cis-3,4-methanoproline, and glutamine surrogates were highly potent, completely inhibiting phosphorylation of Stat3 Tyr705 at 0.5–1 µM in a variety of cancer cell lines. The inhibitors were selective for Stat3 over Stat1, Stat5, Src, and p85 of PI3K, indicating ability to discriminate individual SH2 domains in intact cells. At concentrations that completely inhibited Stat3 phosphorylation, the prodrugs were not cytotoxic to a panel of tumor cells, thereby showing clear distinction between cytotoxicity and effects downstream of activated Stat3. PMID:21486047

The Minimum M3-M4 Loop Length of Neurotransmitter-activated Pentameric Receptors Is Critical for the Structural Integrity of Cytoplasmic Portals*

PubMed Central

Baptista-Hon, Daniel T.; Deeb, Tarek Z.; Lambert, Jeremy J.; Peters, John A.; Hales, Tim G.

2013-01-01

The 5-HT3A receptor homology model, based on the partial structure of the nicotinic acetylcholine receptor from Torpedo marmorata, reveals an asymmetric ion channel with five portals framed by adjacent helical amphipathic (HA) stretches within the 114-residue loop between the M3 and M4 membrane-spanning domains. The positive charge of Arg-436, located within the HA stretch, is a rate-limiting determinant of single channel conductance (γ). Further analysis reveals that positive charge and volume of residue 436 are determinants of 5-HT3A receptor inward rectification, exposing an additional role for portals. A structurally unresolved stretch of 85 residues constitutes the bulk of the M3-M4 loop, leaving a >45-Å gap in the model between M3 and the HA stretch. There are no additional structural data for this loop, which is vestigial in bacterial pentameric ligand-gated ion channels and was largely removed for crystallization of the Caenorhabditis elegans glutamate-activated pentameric ligand-gated ion channels. We created 5-HT3A subunit loop truncation mutants, in which sequences framing the putative portals were retained, to determine the minimum number of residues required to maintain their functional integrity. Truncation to between 90 and 75 amino acids produced 5-HT3A receptors with unaltered rectification. Truncation to 70 residues abolished rectification and increased γ. These findings reveal a critical M3-M4 loop length required for functions attributable to cytoplasmic portals. Examination of all 44 subunits of the human neurotransmitter-activated Cys-loop receptors reveals that, despite considerable variability in their sequences and lengths, all M3-M4 loops exceed 70 residues, suggesting a fundamental requirement for portal integrity. PMID:23740249

IRS-1 activates phosphatidylinositol 3'-kinase by associating with src homology 2 domains of p85.

PubMed Central

Myers, M G; Backer, J M; Sun, X J; Shoelson, S; Hu, P; Schlessinger, J; Yoakim, M; Schaffhausen, B; White, M F

1992-01-01

IRS-1 is an insulin receptor substrate that undergoes tyrosine phosphorylation and associates with the phosphatidylinositol (PtdIns) 3'-kinase immediately after insulin stimulation. Recombinant IRS-1 protein was tyrosine phosphorylated by the insulin receptor in vitro and associated with the PtdIns 3'-kinase from lysates of quiescent 3T3 fibroblasts. Bacterial fusion proteins containing the src homology 2 domains (SH2 domains) of the 85-kDa subunit (p85) of the PtdIns 3'-kinase bound quantitatively to tyrosine phosphorylated, but not unphosphorylated, IRS-1, and this association was blocked by phosphotyrosine-containing synthetic peptides. Moreover, the phosphorylated peptides and the SH2 domains each inhibited binding of PtdIns 3'-kinase to IRS-1. Phosphorylated IRS-1 activated PtdIns 3'-kinase in anti-p85 immunoprecipitates in vitro, and this activation was blocked by SH2 domain fusion proteins. These data suggest that the interaction between PtdIns 3'-kinase and IRS-1 is mediated by tyrosine phosphorylated motifs on IRS-1 and the SH2 domains of p85, and IRS-1 activates PtdIns 3'-kinase by binding to the SH2 domains of p85. Thus, IRS-1 likely serves to transmit the insulin signal by binding and regulating intracellular enzymes containing SH2 domains. Images PMID:1332046

Fabrication of Large Domain YBa2Cu3O(x) for Magnetic Suspension Applications

NASA Technical Reports Server (NTRS)

Sengupta, S.; Corpus, J.; Gaines, J. R., Jr.; Todt, V. R.; Zhang, X.; Miller, D. J.

1996-01-01

Large domain YBa2Cu3O(x) levitators have been fabricated using a seeded melt processing technique. Depending upon the seed, either a single or five domained sample can be obtained. The grain boundaries separating each domains in the five domain levitator are found to be 90 degrees. Similar levitation forces can be observed for single and five domained samples. After thermal cycling, however, a small decrease in the levitation force of the five domain levitator was observed as a function of thermal cycles while nearly no change in force was observed in the single domain levitator. Finally, it is shown that both, single and five domain YBCO, behave similarly as a function of sample thickness.

Local conductivity enhancement due to the tetragonal domain structure in LaAlO3- SrTiO3 heterointerfaces

NASA Astrophysics Data System (ADS)

Moler, Kathryn

2014-03-01

Progress in the difficult task of growing oxide heterostructures has enabled the field of oxide interface engineering. The ability to control materials properties through interface engineering is demonstrated by the appearance of conductivity at the interface of certain insulators, most famously the {001}interface of the band insulators LaAlO3 (LAO) and TiO2-terminated SrTiO3 (STO). The prevailing explanation of conduction at the interface is electronic reconstruction due to a `polar catastrophe' in which charge migrates from the top LAO layer to the interface. Transport and other measurements in this system display a plethora of diverse physical phenomena. To better understand the interface conductivity, we used scanning superconducting quantum interference device (SQUID) microscopy to image the magnetic field locally generated by current in an interface. At low temperature, we found that the current flowed in conductive narrow paths oriented along the crystallographic axes, embedded in a less conductive background. The configuration of these paths changed upon thermal cycling above the STO cubic to tetragonal structural transition temperature, implying that the local conductivity is strongly modified by the STO tetragonal domain structure. In this talk, I will summarize these results and also report on measurements of conductivity and diamagnetism in related materials that firmly establish the influence of the STO tetragonal domains on electronic properties. Coauthors C. Bell, H.K. Sato, M. Hosoda, Y. Xie, Y. Hikita, & H.Y. Hwang (SIMES); R. Jany & C. Richter (Augsburg); C. Woltmann, G. Pfanzelt, & J. Mannhart (MP Stuttgart); B. Kalisky, E.M. Spanton, H. Noad, K.C. Nowack, A. Rosenberg, & J.R. Kirtley.

Magnetic domain configuration of (111)-oriented LaFeO 3 epitaxial thin films

DOE PAGES

Hallsteinsen, I.; Moreau, M.; Chopdekar, R. V.; ...

2017-08-22

In antiferromagnetic spintronics control of the domains and corresponding spin axis orientation is crucial for devices. Here we investigate the antiferromagnetic axis in (111)-oriented LaFeO 3 SrTiO 3 , which is coupled to structural twin domains. The structural domains have either the orthorhombic a- or b-axis along the in-plane <1more » $$\\bar{1}$$0> cubic directions of the substrate, and the corresponding magnetic domains have the antiferromagnetic axis in the sample plane. Six degenerate antiferromagnetic axes are found corresponding to the <1$$\\bar{1}$$0> and <11$$\\bar{2}$$> in-plane directions. This is in contrast to the biaxial anisotropy in (001)-oriented films and reflects how crystal orientation can be used to control magnetic anisotropy in antiferromagnets.« less

Single-domain angiotensin I converting enzyme (kininase II): characterization and properties.

PubMed

Deddish, P A; Wang, L X; Jackman, H L; Michel, B; Wang, J; Skidgel, R A; Erdös, E G

1996-12-01

Somatic angiotensin I converting enzyme (ACE; kininase II) has two active sites, in two (N and C) domains. We studied the active centers with separate N-domain ACE (N-ACE), testicular C-domain ACE (germinal ACE) and, as control, renal somatic ACE. Germinal ACE cleaved the nonapeptide bradykinin about two times faster than N-ACE in 20 mM Cl-. Bradykinin1-7 was hydrolyzed further to bradykinin1-5 by N-ACE four times faster in the absence of Cl-, but at 300 mM Cl- the C-domain hydrolyzed it twice as fast. The hematopoietic system regulatory peptide acetyl-Ser-Asp-Lys-Pro was split to two dipeptides by N-ACE, depending on the chloride concentration, 8 to 24 times faster than by germinal ACE; at 100 mM Cl-, the Kcat with N-ACE was eight times higher. One millimolar 1-fluoro-2,4-dinitrobenzene inhibited germinal ACE 96% but it inhibited N-ACE by only 31%. [3H]Ramiprilat was displaced by other unlabeled ACE inhibitors to establish their relative affinities. Captopril had the lowest IC50 (0.5 nM) with N-ACE and the highest IC50 (8.3 nM) with the germinal ACE. The IC50 values of ramiprilat and quinaprilat were about the same with both active sites. The association and dissociation constants of [3H]ramiprilat indicated faster association with and faster dissociation from N-ACE than from germinal ACE. After exposure to alkali or moderate heat, somatic ACE was cleaved by plasmin and kallikrein, releasing N-ACE and apparently inactivating the C-domain. These studies affirm the differences in the activity, stability and inhibition of the two active sites of ACE.

M-Learning: Implications in Learning Domain Specificities, Adaptive Learning, Feedback, Augmented Reality, and the Future of Online Learning

ERIC Educational Resources Information Center

Squires, David R.

2014-01-01

The aim of this paper is to examine the potential and effectiveness of m-learning in the field of Education and Learning domains. The purpose of this research is to illustrate how mobile technology can and is affecting novel change in instruction, from m-learning and the link to adaptive learning, to the uninitiated learner and capacities of…

Probing the interaction between cHAVc3 peptide and the EC1 domain of E-cadherin using NMR and molecular dynamics simulations.

PubMed

Alaofi, Ahmed; Farokhi, Elinaz; Prasasty, Vivitri D; Anbanandam, Asokan; Kuczera, Krzysztof; Siahaan, Teruna J

2017-01-01

The goal of this work is to probe the interaction between cyclic cHAVc3 peptide and the EC1 domain of human E-cadherin protein. Cyclic cHAVc3 peptide (cyclo(1,6)Ac-CSHAVC-NH 2 ) binds to the EC1 domain as shown by chemical shift perturbations in the 2D 1 H,- 15 N-HSQC NMR spectrum. The molecular dynamics (MD) simulations of the EC1 domain showed folding of the C-terminal tail region into the main head region of the EC1 domain. For cHAVc3 peptide, replica exchange molecular dynamics (REMD) simulations generated five structural clusters of cHAVc3 peptide. Representative structures of cHAVc3 and the EC1 structure from MD simulations were used in molecular docking experiments with NMR constraints to determine the binding site of the peptide on EC1. The results suggest that cHAVc3 binds to EC1 around residues Y36, S37, I38, I53, F77, S78, H79, and I94. The dissociation constants (K d values) of cHAVc3 peptide to EC1 were estimated using the NMR chemical shifts data and the estimated K d s are in the range of .5 × 10 -5 -7.0 × 10 -5  M.

PNPLA3 variant I148M is associated with altered hepatic lipid composition in humans.

PubMed

Peter, Andreas; Kovarova, Marketa; Nadalin, Silvio; Cermak, Tomas; Königsrainer, Alfred; Machicao, Fausto; Stefan, Norbert; Häring, Hans-Ulrich; Schleicher, Erwin

2014-10-01

The common sequence variant I148M of the patatin-like phospholipase domain-containing protein 3 gene (PNPLA3) is associated with increased hepatic triacylglycerol (TAG) content, but not with insulin resistance, in humans. The PNPLA3 (I148M) variant was previously reported to alter the specificity of the encoded enzyme and subsequently affect lipid composition. We analysed the fatty acid composition of five lipid fractions from liver tissue samples from 52 individuals, including 19 carriers of the minor PNPLA3 (I148M) variant. PNPLA3 (I148M) was associated with a strong increase (1.75-fold) in liver TAGs, but with no change in other lipid fractions. PNPLA3 (I148M) minor allele carriers had an increased n-3 polyunsaturated fatty acid (PUFA) α-linolenic acid content and reductions in several n-6 PUFAs in the liver TAG fraction. Furthermore, there was a strong inverse correlation between n-6 PUFA and TAG content independent of PNPLA3 genotype. In a multivariate model including liver fat content, PNPLA3 genotype and fatty acid composition, two significant differences could be exclusively attributed to the PNPLA3 (I148M) minor allele: reduced stearic acid and increased α-linolenic acid content in the hepatic TAG fraction. These changes therefore suggest a mechanism to explain the PNPLA3 (I148M)-dependent increase in liver fat content without causing insulin resistance. Stearic acid can induce insulin resistance, whereas α-linolenic acid may protect against it.

Lipid binding by the Unique and SH3 domains of c-Src suggests a new regulatory mechanism

PubMed Central

Pérez, Yolanda; Maffei, Mariano; Igea, Ana; Amata, Irene; Gairí, Margarida; Nebreda, Angel R.; Bernadó, Pau; Pons, Miquel

2013-01-01

c-Src is a non-receptor tyrosine kinase involved in numerous signal transduction pathways. The kinase, SH3 and SH2 domains of c-Src are attached to the membrane-anchoring SH4 domain through the flexible Unique domain. Here we show intra- and intermolecular interactions involving the Unique and SH3 domains suggesting the presence of a previously unrecognized additional regulation layer in c-Src. We have characterized lipid binding by the Unique and SH3 domains, their intramolecular interaction and its allosteric modulation by a SH3-binding peptide or by Calcium-loaded calmodulin binding to the Unique domain. We also show reduced lipid binding following phosphorylation at conserved sites of the Unique domain. Finally, we show that injection of full-length c-Src with mutations that abolish lipid binding by the Unique domain causes a strong in vivo phenotype distinct from that of wild-type c-Src in a Xenopus oocyte model system, confirming the functional role of the Unique domain in c-Src regulation. PMID:23416516

Phase-field simulations of thickness-dependent domain stability in PbTiO3 thin films

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

Sheng, Guang; Hu, Jia-Mian; Zhang, Jinxian

Phase-field approach is used to predict the thickness effect on the domain stability in ferroelectric thin films. The strain relaxation mechanism and critical thickness for dislocation formation from both Matthews-Blakeslee (MB) and People-Bean (PB) models are employed. Thickness - strain domain stability diagrams are obtained for PbTiO3 thin films under different strain relaxation models. The relative domain fractions as a function of film thickness are also calculated and compared with experiment measurements in PbTiO3 thin films grown on SrTiO3 and KTaO3 substrates.

Dielectric and domain studies on Fe doped KNbO3 single crystal

NASA Astrophysics Data System (ADS)

Shamkuwar, Sanjaykumar H.; Patil, Naresh M.; Korde, Vivek B.; Pradnyakar, Namrata V.

2018-05-01

Synthesis of Fe doped KNbO3 single crystals by flux method is reported here. The effect of Fe-doping on phase transition temperatures of KNbO3 single crystals was investigated using dielectric studies. The phase transition temperatures were found to be 225°C and 425°C which almost same as reported by others. The domain studies were carried out using metallurgical microscope and it shows the presence of 60° and 90° domains in the grown crystals.

Positive and Negative Allosteric Modulation of an α1β3γ2 γ-Aminobutyric Acid Type A (GABAA) Receptor by Binding to a Site in the Transmembrane Domain at the γ+-β− Interface*

PubMed Central

Jayakar, Selwyn S.; Zhou, Xiaojuan; Savechenkov, Pavel Y.; Chiara, David C.; Desai, Rooma; Bruzik, Karol S.; Miller, Keith W.; Cohen, Jonathan B.

2015-01-01

In the process of developing safer general anesthetics, isomers of anesthetic ethers and barbiturates have been discovered that act as convulsants and inhibitors of γ-aminobutyric acid type A receptors (GABAARs) rather than potentiators. It is unknown whether these convulsants act as negative allosteric modulators by binding to the intersubunit anesthetic-binding sites in the GABAAR transmembrane domain (Chiara, D. C., Jayakar, S. S., Zhou, X., Zhang, X., Savechenkov, P. Y., Bruzik, K. S., Miller, K. W., and Cohen, J. B. (2013) J. Biol. Chem. 288, 19343–19357) or to known convulsant sites in the ion channel or extracellular domains. Here, we show that S-1-methyl-5-propyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid (S-mTFD-MPPB), a photoreactive analog of the convulsant barbiturate S-MPPB, inhibits α1β3γ2 but potentiates α1β3 GABAAR responses. In the α1β3γ2 GABAAR, S-mTFD-MPPB binds in the transmembrane domain with high affinity to the γ+-β− subunit interface site with negative energetic coupling to GABA binding in the extracellular domain at the β+-α− subunit interfaces. GABA inhibits S-[3H]mTFD-MPPB photolabeling of γ2Ser-280 (γM2–15′) in this site. In contrast, within the same site GABA enhances photolabeling of β3Met-227 in βM1 by an anesthetic barbiturate, R-[3H]methyl-5-allyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (mTFD-MPAB), which differs from S-mTFD-MPPB in structure only by chirality and two hydrogens (propyl versus allyl). S-mTFD-MPPB and R-mTFD-MPAB are predicted to bind in different orientations at the γ+-β− site, based upon the distance in GABAAR homology models between γ2Ser-280 and β3Met-227. These results provide an explanation for S-mTFD-MPPB inhibition of α1β3γ2 GABAAR function and provide a first demonstration that an intersubunit-binding site in the GABAAR transmembrane domain binds negative and positive allosteric modulators. PMID:26229099

Ferroelectric Self-Poling, Switching, and Monoclinic Domain Configuration in BiFeO 3 Thin Films

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

Beekman, C.; Siemons, W.; Chi, M.

2016-05-23

Self-poling of ferroelectric films, i.e., a preferred, uniform direction of the ferroelectric polarization in as-grown samples is often observed yet poorly understood despite its importance for device applications. The multiferroic perovskite BiFeO 3, which crystallizes in two distinct structural polymorphs depending on applied epitaxial strain, is well known to exhibit self-poling. This study investigates the effect of self-poling on the monoclinic domain configuration and the switching properties of the two polymorphs of BiFeO 3 (R' and T') in thin films grown on LaAlO 3 substrates with slightly different La 0.3Sr 0.7MnO 3 buffer layers. Our study shows that the polarizationmore » state formed during the growth acts as “imprint” on the polarization and that switching the polarization away from this self-poled direction can only be done at the expense of the sample's monoclinic domain configuration. We observed reduction of the monoclinic domain size and found that it was largely reversible; hence, the domain size is restored when the polarization is switched back to its original orientation. This is a direct consequence of the growth taking place in the polar phase (below T c). Finally, switching the polarization away from the preferred configuration, in which defects and domain patterns synergistically minimize the system's energy, leads to a domain state with smaller (and more highly strained and distorted) monoclinic domains.« less

YY1 as a controlling factor for the Peg3 and Gnas imprinted domains

PubMed Central

Kim, Jeong Do; Hinz, Angela K.; Choo, Jung Ha; Stubbs, Lisa; Kim, Joomyeong

2007-01-01

Imprinting Control Regions (ICRs) often harbor tandem arrays of transcription factor binding sites, as demonstrated by the identification of multiple YY1 binding sites within the ICRs of Peg3, Nespas, and Xist/Tsix domains. In the current study, we have sought to characterize possible roles of YY1 in transcriptional control and epigenetic modification of these imprinted domains. RNA interference-based knockdown experiments in Neuro2A cells resulted in overall transcriptional up-regulation of most of the imprinted genes within the Peg3 domain and also, concomitantly, caused significant loss in the DNA methylation of Peg3-DMR (Differentially Methylated Regions). A similar overall and coordinated expression change was also observed for the imprinted genes of the Gnas domain: up-regulation of Nespas and down-regulation of Nesp and Gnasxl. YY1 knockdown also resulted in changes in the expression levels of Xist and Snrpn. These results support the idea that YY1 plays a major role, as a trans factor, for the control of these imprinted domains. PMID:17067777

Keap1/Cullin3 Modulates p62/SQSTM1 Activity via UBA domain Ubiquitination

PubMed Central

Lee, YouJin; Chou, Tsui-Fen; Pittman, Sara K.; Keith, Amy L.; Razani, Babak; Weihl, Conrad C.

2017-01-01

Summary p62/SQSTM1 (p62) is a scaffolding protein that facilitates the formation and degradation of ubiquitinated aggregates via its self-interaction and ubiquitin binding domains. The regulation of this process is unclear but may relate to the post-translational modification of p62. In the present study, we find that Keap1/Cullin3 ubiquitinates p62 at lysine 420 within its UBA domain. Substitution of lysine 420 with an arginine diminishes p62 sequestration and degradation activity similar to that seen when the UBA domain is deleted. Overexpression of Keap1/Cullin3 in p62-WT expressing cells increases ubiquitinated inclusion formation, p62’s association with LC3 and rescues proteotoxicity. This effect is not seen in cells expressing a mutant p62 that fails to interact with Keap1. Interestingly, p62 disease mutants have diminished or absent UBA domain ubiquitination. These data suggest that the ubiquitination of p62’s UBA domain at lysine 420 may regulate p62’s function and be disrupted in p62 associated disease. PMID:28380357

Single domain YBa2Cu3Oy thick films on metallic substrates

NASA Astrophysics Data System (ADS)

Reddy, E. S.; Noudem, J. G.; Goodilin, E. A.; Tarka, M.; Schmitz, G. J.

2003-03-01

The fabrication of single domain YBa2Cu3Oy (123) thick films (10-100 mum) on metallic substrates is reported. The process involves the formation of the 123 phase by a peritectic reaction between an air-brushed dense Y2BaCuO5 (211) layer on a Ag12Pd substrate and infiltrated liquid phases containing barium cuprates and copper oxides. Single domain growth is achieved by seeding the green films with a c-axis oriented NdBa2Cu3Oy crystal prior to processing. The maximum processing temperatures are lowered to 970 °C by modifying the characteristics of the liquid phases meant for infiltration by addition of Ag powder. The fabrication technique, processing conditions for single domain growth and the resulting microstructures are discussed.

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

Jha, Ramesh K.; Kern, Teresa L.; Kim, Youngchang

A whole-cell biosensor utilizing a transcription factor (TF) is an effective tool for sensitive and selective detection of specialty chemicals or anthropogenic molecules, but requires access to an expanded repertoire of TFs. Using homology modeling and ligand docking for binding pocket identification, assisted by conservative mutations in the pocket, we engineered a novel specificity in an Acinetobacter TF, PobR, to ‘sense’ a chemical p-nitrophenol (pNP) and measured the response via a fluorescent protein reporter expressed from a PobR promoter. Out of 107 variants of PobR, four were active when dosed with pNP, with two mutants showing a specificity switch frommore » the native effector 4-hydroxybenzoate (4HB). One of the mutants, pNPmut1 was then used to create a smart microbial cell responding to pNP production from hydrolysis of an insecticide, paraoxon, in a coupled assay involving phosphotriesterase (PTE) enzyme expressed from a separate promoter. We show the fluorescence of the cells correlated with the catalytic efficiency of the PTE variant expressed in each cell. High selectivity between similar molecules (4HB versus pNP), high sensitivity for pNP detection (~2 μM) and agreement of apo- and holo-structures of PobR scaffold with predetermined computational models are other significant results presented in this work.« less

A microbial sensor for organophosphate hydrolysis exploiting an engineered specificity switch in a transcription factor

DOE PAGES

Jha, Ramesh K.; Kern, Theresa L.; Kim, Youngchang; ...

2016-08-30

A whole-cell biosensor utilizing a transcription factor (TF) is an effective tool for sensitive and selective detection of specialty chemicals or anthropogenic molecules, but requires an access to an expanded repertoire of TFs. Using ligand docked homology models for binding pocket identification, assisted by conservative mutations in the pocket, we engineered a novel specificity in an Acinetobacter TF, PobR, to ‘sense’ a chemical p-nitrophenol (pNP) and measured the response via a fluorescent protein reporter expressed from a PobR promoter. Out of 10 7 variants of PobR, four were active when pNP was added as an inducer, with two mutants showingmore » a specificity switch from the native effector 4-hydroxybenzoate (4HB). One of the mutants, pNPmut1 was then used to create a smart microbial cell responding to pNP production and detect hydrolysis of an insecticide, paraoxon, in a coupled assay involving phosphotriesterase (PTE) enzyme expressed from a separate promoter. We show that the fluorescence of the cells correlated with the catalytic efficiency of PTE variants, each cell expressed. High selectivity for similar molecules (4HB vs pNP), high sensitivity for pNP detection (~2 μM) and agreement of apo- and holo- structures of PobR scaffold with computational models are notable successes presented in this work.« less

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

Jha, Ramesh K.; Kern, Theresa L.; Kim, Youngchang

A whole-cell biosensor utilizing a transcription factor (TF) is an effective tool for sensitive and selective detection of specialty chemicals or anthropogenic molecules, but requires an access to an expanded repertoire of TFs. Using ligand docked homology models for binding pocket identification, assisted by conservative mutations in the pocket, we engineered a novel specificity in an Acinetobacter TF, PobR, to ‘sense’ a chemical p-nitrophenol (pNP) and measured the response via a fluorescent protein reporter expressed from a PobR promoter. Out of 10 7 variants of PobR, four were active when pNP was added as an inducer, with two mutants showingmore » a specificity switch from the native effector 4-hydroxybenzoate (4HB). One of the mutants, pNPmut1 was then used to create a smart microbial cell responding to pNP production and detect hydrolysis of an insecticide, paraoxon, in a coupled assay involving phosphotriesterase (PTE) enzyme expressed from a separate promoter. We show that the fluorescence of the cells correlated with the catalytic efficiency of PTE variants, each cell expressed. High selectivity for similar molecules (4HB vs pNP), high sensitivity for pNP detection (~2 μM) and agreement of apo- and holo- structures of PobR scaffold with computational models are notable successes presented in this work.« less

Enhanced strain effect of aged acceptor-doped BaTiO3 ceramics with clamping domain structures

NASA Astrophysics Data System (ADS)

Wang, Lei; Zhou, Zhiyong; Zhao, Xiaobo; Liu, Zhen; Liang, Ruihong; Dong, Xianlin

2017-03-01

A clamping domain structure is proposed to improve the amount of non-180° domain switching in BaTiO3 based piezoelectric ceramics. Experimental results show a large unipolar strain of 0.23% at 5 kV/mm in aged 0.5 mol. % Mn doped BaTiO3 ceramics with clamping domain structures, and the normalized strain (d33*= Smax/Emax) reaches 600 pm/V at low electric fields of 2 or 3 kV/mm. In contrast, pure BaTiO3 ceramics with clamping domain structures exhibit no clear polarization constriction or strain enhancement at 3 kV/mm. Electron paramagnetic resonance spectra verify the existence of titanium vacancies, Mn2+ and Mn4+, in 0.5 mol. % Mn doped BaTiO3 ceramics. These results indicate that the enhanced strain effect can be attributed to the combined effect of the clamping domain structure and stabilization of defect dipoles. This method provides a general way to obtain large strain in ferroelectrics.

Interaction of the S100A6 mutant (C3S) with the V domain of the receptor for advanced glycation end products (RAGE)

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

Mohan, Sepuru K., E-mail: mohansepuri@gmail.com; Gupta, Arun A., E-mail: ninja14gupta@gmail.com; Yu, Chin, E-mail: cyu.nthu@gmail.com

2013-05-03

Highlights: •The halo human S100A6 (C3S) NMR chemical shifts were assigned. •The interactions between S100A6m and RAGE V domain was investigated by ITC. •The residues involved in the S100A6m–RAGE V domain binding were mapped by {sup 1}H–{sup 15}N HSQC titration. •S100A6–RAGE V domain tetrameric complex model was generated from NMR studies. •The S100A6–RAGE V domain interface regions were elucidated based on HADDOCK model. -- Abstract: S100A6 is involved in several vital biological functions, such as calcium sensing and cell proliferation. It is a homodimeric protein that belongs to the S100 protein family. The receptor for advanced glycation end products (RAGE)more » has been shown to play a role in the progression of various disease conditions, such as diabetes and immune/inflammatory disorders. Information regarding the association of RAGE with S100 proteins at a molecular level is useful to understand the diversity of the RAGE signaling pathways. In this report, biomolecular NMR techniques were utilized for the resonance assignment of the C3S mutation in human S100A6 and characterizing its interaction with the RAGE V domain. Further binding affinity between S100A6m and the RAGE V domain was determined by isothermal titration calorimetric studies. HADDOCK was used to generate a heterotetramer model of the S100A6m–RAGE V domain complex. This model provides an important insights into the S100–RAGE cellular signaling pathway.« less

Folding of a LysM Domain: Entropy-Enthalpy Compensation in the Transition State of an Ideal Two-state Folder

PubMed Central

Nickson, Adrian A.; Stoll, Kate E.; Clarke, Jane

2008-01-01

Protein-engineering methods (Φ-values) were used to investigate the folding transition state of a lysin motif (LysM) domain from Escherichia coli membrane-bound lytic murein transglycosylase D. This domain consists of just 48 structured residues in a symmetrical βααβ arrangement and is the smallest αβ protein yet investigated using these methods. An extensive mutational analysis revealed a highly robust folding pathway with no detectable transition state plasticity, indicating that LysM is an example of an ideal two-state folder. The pattern of Φ-values denotes a highly polarised transition state, with significant formation of the helices but no structure within the β-sheet. Remarkably, this transition state remains polarised after circularisation of the domain, and exhibits an identical Φ-value pattern; however, the interactions within the transition state are uniformly weaker in the circular variant. This observation is supported by results from an Eyring analysis of the folding rates of the two proteins. We propose that the folding pathway of LysM is dominated by enthalpic rather than entropic considerations, and suggest that the lower entropy cost of formation of the circular transition state is balanced, to some extent, by the lower enthalpy of contacts within this structure. PMID:18538343

N-terminal functional domain of Gasdermin A3 regulates mitochondrial homeostasis via mitochondrial targeting.

PubMed

Lin, Pei-Hsuan; Lin, Hsien-Yi; Kuo, Cheng-Chin; Yang, Liang-Tung

2015-06-24

The epidermis forms a critical barrier that is maintained by orchestrated programs of proliferation, differentiation, and cell death. Gene mutations that disturb this turnover process may cause skin diseases. Human GASDERMIN A (GSDMA) is frequently silenced in gastric cancer cell lines and its overexpression has been reported to induce apoptosis. GSDMA has also been linked with airway hyperresponsiveness in genetic association studies. The function of GSDMA in the skin was deduced by dominant mutations in mouse gasdermin A3 (Gsdma3), which caused skin inflammation and hair loss. However, the mechanism for the autosomal dominance of Gsdma3 mutations and the mode of Gsdma3's action remain unanswered. We demonstrated a novel function of Gsdma3 in modulating mitochondrial oxidative stress. We showed that Gsdma3 is regulated by intramolecular fold-back inhibition, which is disrupted by dominant mutations in the C-terminal domain. The unmasked N-terminal domain of Gsdma3 associates with Hsp90 and is delivered to mitochondrial via mitochondrial importer receptor Tom70, where it interacts with the mitochondrial chaperone Trap1 and causes increased production of mitochondrial reactive oxygen species (ROS), dissipation of mitochondrial membrane potential, and mitochondrial permeability transition (MPT). Overexpression of the C-terminal domain of Gsdma3 as well as pharmacological interventions of mitochondrial translocation, ROS production, and MPT pore opening alleviate the cell death induced by Gsdma3 mutants. Our results indicate that the genetic mutations in the C-terminal domain of Gsdma3 are gain-of-function mutations which unmask the N-terminal functional domain of Gsdma3. Gsdma3 regulates mitochondrial oxidative stress through mitochondrial targeting. Since mitochondrial ROS has been shown to promote epidermal differentiation, we hypothesize that Gsdma3 regulates context-dependent response of keratinocytes to differentiation and cell death signals by impinging on

Decoy peptide targeted to Toll-IL-1R domain inhibits LPS and TLR4-active metabolite morphine-3 glucuronide sensitization of sensory neurons.

PubMed

Allette, Yohance M; Kim, Youngsook; Randolph, Aaron L; Smith, Jared A; Ripsch, Matthew S; White, Fletcher A

2017-06-16

Accumulating evidence indicates that Toll-like receptor (TLR) signaling adapter protein interactions with Toll/Interleukin-1 Receptor (TIR) domains present in sensory neurons may modulate neuropathic pain states. Following ligand interaction with TLRs, TIR serves to both initiate intracellular signaling and facilitate recruitment of signaling adapter proteins to the intracytoplasmic domain. Although TLR TIR is central to a number of TLR signaling cascades, its role in sensory neurons is poorly understood. In this study we investigated the degree to which TLR TIR decoy peptide modified to include a TAT sequence (Trans-Activator of Transcription gene in HIV; TAT-4BB) affected LPS-induced intracellular calcium flux and excitation in sensory neurons, and behavioral changes due to TLR4 active metabolite, morphine-3-glucuronide (M3G) exposure in vivo. TAT-4BB inhibited LPS-induced calcium changes in a majority of sensory neurons and decreased LPS-dependent neuronal excitability in small diameter neurons. Acute systemic administration of the TAT-4BB reversed M3G-induced tactile allodynia in a dose-dependent manner but did not affect motor activity, anxiety or responses to noxious thermal stimulus. These data suggest that targeting TLR TIR domains may provide novel pharmacological targets to reduce or reverse TLR4-dependent pain behavior in the rodent.

Mutations in a novel gene with transmembrane domains underlie Usher syndrome type 3.

PubMed

Joensuu, T; Hämäläinen, R; Yuan, B; Johnson, C; Tegelberg, S; Gasparini, P; Zelante, L; Pirvola, U; Pakarinen, L; Lehesjoki, A E; de la Chapelle, A; Sankila, E M

2001-10-01

Usher syndrome type 3 (USH3) is an autosomal recessive disorder characterized by progressive hearing loss, severe retinal degeneration, and variably present vestibular dysfunction, assigned to 3q21-q25. Here, we report on the positional cloning of the USH3 gene. By haplotype and linkage-disequilibrium analyses in Finnish carriers of a putative founder mutation, the critical region was narrowed to 250 kb, of which we sequenced, assembled, and annotated 207 kb. Two novel genes-NOPAR and UCRP-and one previously identified gene-H963-were excluded as USH3, on the basis of mutational analysis. USH3, the candidate gene that we identified, encodes a 120-amino-acid protein. Fifty-two Finnish patients were homozygous for a termination mutation, Y100X; patients in two Finnish families were compound heterozygous for Y100X and for a missense mutation, M44K, whereas patients in an Italian family were homozygous for a 3-bp deletion leading to an amino acid deletion and substitution. USH3 has two predicted transmembrane domains, and it shows no homology to known genes. As revealed by northern blotting and reverse-transcriptase PCR, it is expressed in many tissues, including the retina.

Symmetrical refolding of protein domains and subunits: example of the dimeric two-domain 3-isopropylmalate dehydrogenases.

PubMed

Gráczer, Eva; Varga, Andrea; Melnik, Bogdan; Semisotnov, Gennady; Závodszky, Péter; Vas, Mária

2009-02-10

The refolding mechanism of the homodimeric two-domain 3-isopropylmalate dehydrogenase (IPMDH) from the organisms adapted to different temperatures, Thermus thermophilus (Tt), Escherichia coli (Ec), and Vibrio sp. I5 (Vib), is described. In all three cases, instead of a self-template mechanism, the high extent of symmetry and cooperativity in folding of subunits and domains have been concluded from the following experimental findings: The complex time course of refolding, monitored by Trp fluorescence, consists of a fast (the rate constant varies as 16.5, 25.0, and 11.7 min-1 in the order of Tt, Ec, and Vib IPMDHs) and a slow (the rate constants are 0.11, 0.80, and 0.23 min-1 for the three different species) first-order process. However, a burst increase of Trp fluorescence anisotropy to the value of the native states indicates that in all three cases the association of the two polypeptide chains occurs at the beginning of refolding. This dimeric species binds the substrate IPM, but the native-like interactions of the tertiary and quaternary structures are only formed during the slow phase of refolding, accompanied by further increase of protein fluorescence and appearance of FRET between Trp side chain(s) and the bound NADH. Joining the contacting arms of each subunit also takes place exclusively during this slow phase. To monitor refolding of each domain within the intact molecule of T. thermophilus IPMDH, Trp's (located in separate domains) were systematically replaced with Phe's. The refolding processes of the mutants were followed by measuring changes in Trp fluorescence and in FRET between the particular Trp and NADH. The high similarity of time courses (both in biphasicity and in their rates) strongly suggests cooperative folding of the domains during formation of the native three-dimensional structure of IPMDH.

Imaging and tuning polarity at SrTiO3 domain walls

NASA Astrophysics Data System (ADS)

Frenkel, Yiftach; Haham, Noam; Shperber, Yishai; Bell, Christopher; Xie, Yanwu; Chen, Zhuoyu; Hikita, Yasuyuki; Hwang, Harold Y.; Salje, Ekhard K. H.; Kalisky, Beena

2017-12-01

Electrostatic fields tune the ground state of interfaces between complex oxide materials. Electronic properties, such as conductivity and superconductivity, can be tuned and then used to create and control circuit elements and gate-defined devices. Here we show that naturally occurring twin boundaries, with properties that are different from their surrounding bulk, can tune the LaAlO3/SrTiO3 interface 2DEG at the nanoscale. In particular, SrTiO3 domain boundaries have the unusual distinction of remaining highly mobile down to low temperatures, and were recently suggested to be polar. Here we apply localized pressure to an individual SrTiO3 twin boundary and detect a change in LaAlO3/SrTiO3 interface current distribution. Our data directly confirm the existence of polarity at the twin boundaries, and demonstrate that they can serve as effective tunable gates. As the location of SrTiO3 domain walls can be controlled using external field stimuli, our findings suggest a novel approach to manipulate SrTiO3-based devices on the nanoscale.

Zc3h13/Flacc is required for adenosine methylation by bridging the mRNA-binding factor Rbm15/Spenito to the m6A machinery component Wtap/Fl(2)d

PubMed Central

Knuckles, Philip; Lence, Tina; Haussmann, Irmgard U.; Jacob, Dominik; Kreim, Nastasja; Carl, Sarah H.; Masiello, Irene; Hares, Tina; Villaseñor, Rodrigo; Hess, Daniel; Andrade-Navarro, Miguel A.; Biggiogera, Marco; Helm, Mark; Soller, Matthias; Bühler, Marc; Roignant, Jean-Yves

2018-01-01

N6-methyladenosine (m6A) is the most abundant mRNA modification in eukaryotes, playing crucial roles in multiple biological processes. m6A is catalyzed by the activity of methyltransferase-like 3 (Mettl3), which depends on additional proteins whose precise functions remain poorly understood. Here we identified Zc3h13 (zinc finger CCCH domain-containing protein 13)/Flacc [Fl(2)d-associated complex component] as a novel interactor of m6A methyltransferase complex components in Drosophila and mice. Like other components of this complex, Flacc controls m6A levels and is involved in sex determination in Drosophila. We demonstrate that Flacc promotes m6A deposition by bridging Fl(2)d to the mRNA-binding factor Nito. Altogether, our work advances the molecular understanding of conservation and regulation of the m6A machinery. PMID:29535189

Structural and biophysical characterisation of agrin laminin G3 domain constructs.

PubMed

Tidow, Henning; Mattle, Daniel; Nissen, Poul

2011-01-01

Agrin mediates accumulation of acetylcholine receptors (AChRs) at the developing neuromuscular junction, but has also been implicated as a regulator of central nervous system (CNS) synapses. A C-terminal region of agrin (Ag-C20) binds to the α3 subunit of the sodium-potassium ATPase (NKA) in CNS neurons suggesting that α3NKA is a neuronal agrin receptor, whereas a shorter agrin fragment (Ag-C15) was shown to act as a competitive antagonist. Here, we show that the agrin C22 construct, which represents the naturally occurring neurotrypsin cleavage product, constitutes a well-folded, stable domain, while the deletion of 48 residues that correspond to strands β1-β4 of the agrin laminin G3 domain imposed by the agrin C15 construct leads to a misfolded protein.

The decapping activator Edc3 and the Q/N-rich domain of Lsm4 function together to enhance mRNA stability and alter mRNA decay pathway dependence in Saccharomyces cerevisiae.

PubMed

Huch, Susanne; Müller, Maren; Muppavarapu, Mridula; Gommlich, Jessie; Balagopal, Vidya; Nissan, Tracy

2016-10-15

The rate and regulation of mRNA decay are major elements in the proper control of gene expression. Edc3 and Lsm4 are two decapping activator proteins that have previously been shown to function in the assembly of RNA granules termed P bodies. Here, we show that deletion of edc3, when combined with a removal of the glutamine/asparagine rich region of Lsm4 (edc3Δ lsm4ΔC) reduces mRNA stability and alters pathways of mRNA degradation. Multiple tested mRNAs exhibited reduced stability in the edc3Δ lsm4ΔC mutant. The destabilization was linked to an increased dependence on Ccr4-mediated deadenylation and mRNA decapping. Unlike characterized mutations in decapping factors that either are neutral or are able to stabilize mRNA, the combined edc3Δ lsm4ΔC mutant reduced mRNA stability. We characterized the growth and activity of the major mRNA decay systems and translation in double mutant and wild-type yeast. In the edc3Δ lsm4ΔC mutant, we observed alterations in the levels of specific mRNA decay factors as well as nuclear accumulation of the catalytic subunit of the decapping enzyme Dcp2. Hence, we suggest that the effects on mRNA stability in the edc3Δ lsm4ΔC mutant may originate from mRNA decay protein abundance or changes in mRNPs, or alternatively may imply a role for P bodies in mRNA stabilization. © 2016. Published by The Company of Biologists Ltd.

The decapping activator Edc3 and the Q/N-rich domain of Lsm4 function together to enhance mRNA stability and alter mRNA decay pathway dependence in Saccharomyces cerevisiae

PubMed Central

Huch, Susanne; Müller, Maren; Muppavarapu, Mridula; Gommlich, Jessie; Balagopal, Vidya; Nissan, Tracy

2016-01-01

ABSTRACT The rate and regulation of mRNA decay are major elements in the proper control of gene expression. Edc3 and Lsm4 are two decapping activator proteins that have previously been shown to function in the assembly of RNA granules termed P bodies. Here, we show that deletion of edc3, when combined with a removal of the glutamine/asparagine rich region of Lsm4 (edc3Δ lsm4ΔC) reduces mRNA stability and alters pathways of mRNA degradation. Multiple tested mRNAs exhibited reduced stability in the edc3Δ lsm4ΔC mutant. The destabilization was linked to an increased dependence on Ccr4-mediated deadenylation and mRNA decapping. Unlike characterized mutations in decapping factors that either are neutral or are able to stabilize mRNA, the combined edc3Δ lsm4ΔC mutant reduced mRNA stability. We characterized the growth and activity of the major mRNA decay systems and translation in double mutant and wild-type yeast. In the edc3Δ lsm4ΔC mutant, we observed alterations in the levels of specific mRNA decay factors as well as nuclear accumulation of the catalytic subunit of the decapping enzyme Dcp2. Hence, we suggest that the effects on mRNA stability in the edc3Δ lsm4ΔC mutant may originate from mRNA decay protein abundance or changes in mRNPs, or alternatively may imply a role for P bodies in mRNA stabilization. PMID:27543059

A histone H3K9M mutation traps histone methyltransferase Clr4 to prevent heterochromatin spreading

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

Shan, Chun-Min; Wang, Jiyong; Xu, Ke

2016-09-20

Histone lysine-to-methionine (K-to-M) mutations are associated with multiple cancers, and they function in a dominant fashion to block the methylation of corresponding lysines on wild type histones. However, their mechanisms of function are controversial. Here we show that in fission yeast, introducing the K9M mutation into one of the three histone H3 genes dominantly blocks H3K9 methylation on wild type H3 across the genome. In addition, H3K9M enhances the interaction of histone H3 tail with the H3K9 methyltransferase Clr4 in a SAM (S-adenosyl-methionine)-dependent manner, and Clr4 is trapped at nucleation sites to prevent its spreading and the formation of largemore » heterochromatin domains. We further determined the crystal structure of an H3K9M peptide in complex with human H3K9 methyltransferase G9a and SAM, which reveales that the methionine side chain had enhanced van der Waals interactions with G9a. Therefore, our results provide a detailed mechanism by which H3K9M regulates H3K9 methylation.« less

Keap1/Cullin3 Modulates p62/SQSTM1 Activity via UBA Domain Ubiquitination.

PubMed

Lee, YouJin; Chou, Tsui-Fen; Pittman, Sara K; Keith, Amy L; Razani, Babak; Weihl, Conrad C

2017-04-04

p62/SQSTM1 (p62) is a scaffolding protein that facilitates the formation and degradation of ubiquitinated aggregates via its self-interaction and ubiquitin binding domains. The regulation of this process is unclear but may relate to the post-translational modification of p62. In the present study, we find that Keap1/Cullin3 ubiquitinates p62 at lysine 420 within its UBA domain. Substitution of lysine 420 with an arginine diminishes p62 sequestration and degradation activity similar what is seen when the UBA domain is deleted. Overexpression of Keap1/Cullin3 in p62-WT-expressing cells increases ubiquitinated inclusion formation and p62's association with LC3 and rescues proteotoxicity. This effect is not seen in cells expressing a mutant p62 that fails to interact with Keap1. Interestingly, p62 disease mutants have diminished or absent UBA domain ubiquitination. These data suggest that the ubiquitination of p62's UBA domain at lysine 420 may regulate p62's function and be disrupted in p62-associated disease. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Autonomous Frequency Domain Identification: Theory and Experiment

DTIC Science & Technology

1989-04-15

4,4,3)). This approach is particularly well suited to provide accurate estimation using sampled-data -3 DO 2 ^ UJ H « > x 2 ^ ui M (d 5 -P m...criteria for resonance requires a unimodal search. Search strategies such as golden search, Fibonacci search etc. are well known and can be found for...identified nonparametrically and a frequency domain de - scription is available, a parametric representation of the transfer function can be found by

Identification of the NC1 domain of {alpha}3 chain as critical for {alpha}3{alpha}4{alpha}5 type IV collagen network assembly.

PubMed

LeBleu, Valerie; Sund, Malin; Sugimoto, Hikaru; Birrane, Gabriel; Kanasaki, Keizo; Finan, Elizabeth; Miller, Caroline A; Gattone, Vincent H; McLaughlin, Heather; Shield, Charles F; Kalluri, Raghu

2010-12-31

The network organization of type IV collagen consisting of α3, α4, and α5 chains in the glomerular basement membrane (GBM) is speculated to involve interactions of the triple helical and NC1 domain of individual α-chains, but in vivo evidence is lacking. To specifically address the contribution of the NC1 domain in the GBM collagen network organization, we generated a mouse with specific loss of α3NC1 domain while keeping the triple helical α3 chain intact by connecting it to the human α5NC1 domain. The absence of α3NC1 domain leads to the complete loss of the α4 chain. The α3 collagenous domain is incapable of incorporating the α5 chain, resulting in the impaired organization of the α3α4α5 chain-containing network. Although the α5 chain can assemble with the α1, α2, and α6 chains, such assembly is incapable of functionally replacing the α3α4α5 protomer. This novel approach to explore the assembly type IV collagen in vivo offers novel insights in the specific role of the NC1 domain in the assembly and function of GBM during health and disease.

The mechano-sensing role of the unique SH3 insertion in plakin domains revealed by Molecular Dynamics simulations.

PubMed

Daday, Csaba; Kolšek, Katra; Gräter, Frauke

2017-09-15

The plakin family of proteins, important actors in cross-linking force-bearing structures in the cell, contain a curious SH3 domain insertion in their chain of spectrin repeats (SRs). While SH3 domains are known to mediate protein-protein interactions, here, its canonical binding site is autoinhibited by the preceding SR. Under force, however, this SH3 domain could be released, and possibly launch a signaling cascade. We performed large-scale force-probe molecular dynamics simulations, across two orders of magnitude of loading rates, to test this hypothesis, on two prominent members of the plakin family: desmoplakin and plectin, obligate proteins at desmosomes and hemidesmosomes, respectively. Our simulations show that force unravels the SRs and abolishes the autoinhibition of the SH3 domain, an event well separated from the unfolding of this domain. The SH3 domain is free and fully functional for a significant portion of the unfolding trajectories. The rupture forces required for the two proteins significantly decrease when the SH3 domain is removed, which implies that the SH3 domain also stabilizes this junction. Our results persist across all simulations, and support a force-sensing as well as a stabilizing role of the unique SH3 insertion, putting forward this protein family as a new class of mechano-sensors.

Experimental investigation of in-situ transformations of the M 7C3 carbide in the cast Fe-Cr-Ni alloy

NASA Astrophysics Data System (ADS)

Kraposhin, V. S.; Kondrat'ev, S. Yu.; Talis, A. L.; Anastasiadi, G. P.

2017-03-01

The microstructure and the phase composition of a heat-resistant Fe-Cr-Ni alloy (0. 45C-25Cr-35Ni) has been investigated in the cast state and after annealing at 1150°C for 2-100 h. After a 2-h high-temperature annealing, the fragmentation of the crystal structure of the eutectic M 7C3 carbides into domains of 500 nm in size with a partial transition into M 23C6 carbides is observed. After a 100-h holding, the complete transition of the hexagonal M 7C3 carbides into M 23C6 with a face-centered cubic structure occurs. The carbide transition M 7C3 → M 23 can be considered to be an in situ transformation.

New approaches to high-throughput structure characterization of SH3 complexes: the example of Myosin-3 and Myosin-5 SH3 domains from S. cerevisiae.

PubMed

Musi, Valeria; Birdsall, Berry; Fernandez-Ballester, Gregorio; Guerrini, Remo; Salvatori, Severo; Serrano, Luis; Pastore, Annalisa

2006-04-01

SH3 domains are small protein modules that are involved in protein-protein interactions in several essential metabolic pathways. The availability of the complete genome and the limited number of clearly identifiable SH3 domains make the yeast Saccharomyces cerevisae an ideal proteomic-based model system to investigate the structural rules dictating the SH3-mediated protein interactions and to develop new tools to assist these studies. In the present work, we have determined the solution structure of the SH3 domain from Myo3 and modeled by homology that of the highly homologous Myo5, two myosins implicated in actin polymerization. We have then implemented an integrated approach that makes use of experimental and computational methods to characterize their binding properties. While accommodating their targets in the classical groove, the two domains have selectivity in both orientation and sequence specificity of the target peptides. From our study, we propose a consensus sequence that may provide a useful guideline to identify new natural partners and suggest a strategy of more general applicability that may be of use in other structural proteomic studies.

Recognition of β-calcineurin by the domains of calmodulin: thermodynamic and structural evidence for distinct roles.

PubMed

O'Donnell, Susan E; Yu, Liping; Fowler, C Andrew; Shea, Madeline A

2011-03-01

Calcineurin (CaN, PP2B, PPP3), a heterodimeric Ca(2+)-calmodulin-dependent Ser/Thr phosphatase, regulates swimming in Paramecia, stress responses in yeast, and T-cell activation and cardiac hypertrophy in humans. Calcium binding to CaN(B) (the regulatory subunit) triggers conformational change in CaN(A) (the catalytic subunit). Two isoforms of CaN(A) (α, β) are both abundant in brain and heart and activated by calcium-saturated calmodulin (CaM). The individual contribution of each domain of CaM to regulation of calcineurin is not known. Hydrodynamic analyses of (Ca(2+))₄-CaM(1-148) bound to βCaNp, a peptide representing its CaM-binding domain, indicated a 1:1 stoichiometry. βCaNp binding to CaM increased the affinity of calcium for the N- and C-domains equally, thus preserving intrinsic domain differences, and the preference of calcium for sites III and IV. The equilibrium constants for individual calcium-saturated CaM domains dissociating from βCaNp were ∼1 μM. A limiting K(d) ≤ 1 nM was measured directly for full-length CaM, while thermodynamic linkage analysis indicated that it was approximately 1 pM. βCaNp binding to ¹⁵N-(Ca(2+))₄-CaM(1-148) monitored by ¹⁵N/¹HN HSQC NMR showed that association perturbed the N-domain of CaM more than its C-domain. NMR resonance assignments of CaM and βCaNp, and interpretation of intermolecular NOEs observed in the ¹³C-edited and ¹²C-¹⁴N-filtered 3D NOESY spectrum indicated anti-parallel binding. The sole aromatic residue (Phe) located near the βCaNp C-terminus was in close contact with several residues of the N-domain of CaM outside the hydrophobic cleft. These structural and thermodynamic properties would permit the domains of CaM to have distinct physiological roles in regulating activation of βCaN. Copyright © 2010 Wiley-Liss, Inc.

Interactions between the S-domain receptor kinases and AtPUB-ARM E3 ubiquitin ligases suggest a conserved signaling pathway in Arabidopsis.

PubMed

Samuel, Marcus A; Mudgil, Yashwanti; Salt, Jennifer N; Delmas, Frédéric; Ramachandran, Shaliny; Chilelli, Andrea; Goring, Daphne R

2008-08-01

The Arabidopsis (Arabidopsis thaliana) genome encompasses multiple receptor kinase families with highly variable extracellular domains. Despite their large numbers, the various ligands and the downstream interacting partners for these kinases have been deciphered only for a few members. One such member, the S-receptor kinase, is known to mediate the self-incompatibility (SI) response in Brassica. S-receptor kinase has been shown to interact and phosphorylate a U-box/ARM-repeat-containing E3 ligase, ARC1, which, in turn, acts as a positive regulator of the SI response. In an effort to identify conserved signaling pathways in Arabidopsis, we performed yeast two-hybrid analyses of various S-domain receptor kinase family members with representative Arabidopsis plant U-box/ARM-repeat (AtPUB-ARM) E3 ligases. The kinase domains from S-domain receptor kinases were found to interact with ARM-repeat domains from AtPUB-ARM proteins. These kinase domains, along with M-locus protein kinase, a positive regulator of SI response, were also able to phosphorylate the ARM-repeat domains in in vitro phosphorylation assays. Subcellular localization patterns were investigated using transient expression assays in tobacco (Nicotiana tabacum) BY-2 cells and changes were detected in the presence of interacting kinases. Finally, potential links to the involvement of these interacting modules to the hormone abscisic acid (ABA) were investigated. Interestingly, AtPUB9 displayed redistribution to the plasma membrane of BY-2 cells when either treated with ABA or coexpressed with the active kinase domain of ARK1. As well, T-DNA insertion mutants for ARK1 and AtPUB9 lines were altered in their ABA sensitivity during germination and acted at or upstream of ABI3, indicating potential involvement of these proteins in ABA responses.

WW domain of BAG3 is required for the induction of autophagy in glioma cells.

PubMed

Merabova, Nana; Sariyer, Ilker Kudret; Saribas, A Sami; Knezevic, Tijana; Gordon, Jennifer; Turco, M Caterina; Rosati, Alessandra; Weaver, Michael; Landry, Jacques; Khalili, Kamel

2015-04-01

Autophagy is an evolutionarily conserved, selective degradation pathway of cellular components that is important for cell homeostasis under healthy and pathologic conditions. Here we demonstrate that an increase in the level of BAG3 results in stimulation of autophagy in glioblastoma cells. BAG3 is a member of a co-chaperone family of proteins that associates with Hsp70 through a conserved BAG domain positioned near the C-terminus of the protein. Expression of BAG3 is induced by a variety of environmental changes that cause stress to cells. Our results show that BAG3 overexpression induces autophagy in glioma cells. Interestingly, inhibition of the proteasome caused an increase in BAG3 levels and induced autophagy. Further analysis using specific siRNA against BAG3 suggests that autophagic activation due to proteosomal inhibition is mediated by BAG3. Analyses of BAG3 domain mutants suggest that the WW domain of BAG3 is crucial for the induction of autophagy. BAG3 overexpression also increased the interaction between Bcl2 and Beclin-1, instead of disrupting them, suggesting that BAG3 induced autophagy is Beclin-1 independent. These observations reveal a novel role for the WW domain of BAG3 in the regulation of autophagy. © 2014 Wiley Periodicals, Inc.

WW domain of BAG3 is required for the induction of autophagy in glioma cells

PubMed Central

Merabova, Nana; Sariyer, Ilker Kudret; Saribas, A Sami; Knezevic, Tijana; Gordon, Jennifer; Weaver, Michael; Landry, Jacques; Khalili, Kamel

2015-01-01

Autophagy is an evolutionarily conserved, selective degradation pathway of cellular components that is important for cell homeostasis under healthy and pathologic conditions. Here we demonstrate that an increase in the level of BAG3 results in stimulation of autophagy in glioblastoma cells. BAG3 is a member of a co-chaperone family of proteins that associate with Hsp70 through a conserved BAG domain positioned near the C-terminus of the protein. Expression of BAG3 is induced by a variety of environmental changes that cause stress to cells. Our results show that BAG3 overexpression induces autophagy in glioma cells. Interestingly, inhibition of the proteasome caused an increase in BAG3 levels and induced autophagy. Further analysis using specific siRNA against BAG3 suggests that autophagic activation due to proteosomal inhibition is mediated by BAG3. Analyses of BAG3 domain mutants suggest that the WW domain of BAG3 is crucial for the induction of autophagy. BAG3 overexpression also increased the interaction between Bcl2 and Beclin-1, instead of disrupting them, suggesting that BAG3 induced autophagy is Beclin-1 independent. These observations reveal a novel role for the WW domain of BAG3 in the regulation of autophagy. PMID:25204229

Applying Frequency-Domain Equalization to Code-Division Multiple Access and Transform-Domain Communications Systems

DTIC Science & Technology

2008-03-01

terms the last time we spoke, I can say without a doubt that he was my favorite cousin. You are both missed, always.... I want to thank my wife for her...IEEE Communications Magazine, 50:S11–S15, September 2005. 3. Haker , M. E. Hardware Realization of a Transform Domain Communication Sys- tem. Master’s

Sequence-Specific Interaction between the Disintegrin Domain of Mouse ADAM 3 and Murine Eggs: Role of β1 Integrin-associated Proteins CD9, CD81, and CD98

PubMed Central

Takahashi, Yuji; Bigler, Dora; Ito, Yasuhiko; White, Judith M.

2001-01-01

ADAM 3 is a sperm surface glycoprotein that has been implicated in sperm-egg adhesion. Because little is known about the adhesive activity of ADAMs, we investigated the interaction of ADAM 3 disintegrin domains, made in bacteria and in insect cells, with murine eggs. Both recombinant proteins inhibited sperm-egg binding and fusion with potencies similar to that which we recently reported for the ADAM 2 disintegrin domain. Alanine scanning mutagenesis revealed a critical importance for the glutamine at position 7 of the disintegrin loop. Fluorescent beads coated with the ADAM 3 disintegrin domain bound to the egg surface. Bead binding was inhibited by an authentic, but not by a scrambled, peptide analog of the disintegrin loop. Bead binding was also inhibited by the function-blocking anti-α6 monoclonal antibody (mAb) GoH3, but not by a nonfunction blocking anti-α6 mAb, or by mAbs against either the αv or β3 integrin subunits. We also present evidence that in addition to the tetraspanin CD9, two other β1-integrin-associated proteins, the tetraspanin CD81 as well as the single pass transmembrane protein CD98 are expressed on murine eggs. Antibodies to CD9 and CD98 inhibited in vitro fertilization and binding of the ADAM 3 disintegrin domain. Our findings are discussed in terms of the involvement of multiple sperm ADAMs and multiple egg β1 integrin-associated proteins in sperm-egg binding and fusion. We propose that an egg surface “tetraspan web” facilitates fertilization and that it may do so by fostering ADAM–integrin interactions. PMID:11294888

Sequence-specific interaction between the disintegrin domain of mouse ADAM 3 and murine eggs: role of beta1 integrin-associated proteins CD9, CD81, and CD98.

PubMed

Takahashi, Y; Bigler, D; Ito, Y; White, J M

2001-04-01

ADAM 3 is a sperm surface glycoprotein that has been implicated in sperm-egg adhesion. Because little is known about the adhesive activity of ADAMs, we investigated the interaction of ADAM 3 disintegrin domains, made in bacteria and in insect cells, with murine eggs. Both recombinant proteins inhibited sperm-egg binding and fusion with potencies similar to that which we recently reported for the ADAM 2 disintegrin domain. Alanine scanning mutagenesis revealed a critical importance for the glutamine at position 7 of the disintegrin loop. Fluorescent beads coated with the ADAM 3 disintegrin domain bound to the egg surface. Bead binding was inhibited by an authentic, but not by a scrambled, peptide analog of the disintegrin loop. Bead binding was also inhibited by the function-blocking anti-alpha6 monoclonal antibody (mAb) GoH3, but not by a nonfunction blocking anti-alpha6 mAb, or by mAbs against either the alphav or beta3 integrin subunits. We also present evidence that in addition to the tetraspanin CD9, two other beta1-integrin-associated proteins, the tetraspanin CD81 as well as the single pass transmembrane protein CD98 are expressed on murine eggs. Antibodies to CD9 and CD98 inhibited in vitro fertilization and binding of the ADAM 3 disintegrin domain. Our findings are discussed in terms of the involvement of multiple sperm ADAMs and multiple egg beta1 integrin-associated proteins in sperm-egg binding and fusion. We propose that an egg surface "tetraspan web" facilitates fertilization and that it may do so by fostering ADAM-integrin interactions.

An examination of dynamics crosstalk between SH2 and SH3 domains by hydrogen/deuterium exchange and mass spectrometry

PubMed Central

Hochrein, James M.; Lerner, Edwina C.; Schiavone, Anthony P.; Smithgall, Thomas E.; Engen, John R.

2006-01-01

The ability of proteins to regulate their own enzymatic activity can be facilitated by changes in structure or protein dynamics in response to external regulators. Because many proteins contain SH2 and SH3 domains, transmission of information between the domains is a potential method of allosteric regulation. To determine if ligand binding to one modular domain may alter structural dynamics in an adjacent domain, allowing potential transmission of information through the protein, we used hydrogen exchange and mass spectrometry to measure changes in protein dynamics in the SH3 and SH2 domains of hematopoietic cell kinase (Hck). Ligand binding to either domain had little or no effect on hydrogen exchange in the adjacent domain, suggesting that changes in protein structure or dynamics are not a means of SH2/SH3 crosstalk. Furthermore, ligands of varying affinity covalently attached to SH3/SH2 altered dynamics only in the domain to which they bind. Such results demonstrate that ligand binding may not structurally alter adjacent SH3/SH2 domains and implies that other aspects of protein architecture contribute to the multiple levels of regulation in proteins containing SH3 and SH2 domains. PMID:16322569

Strong ground motion from the November 12, 2017, M 7.3 Kermanshah earthquake in western Iran

NASA Astrophysics Data System (ADS)

Babaie Mahani, Alireza; Kazemian, Javad

2018-05-01

In this paper, we analyzed the strong ground motion from the November 12, 2017, Kermanshah earthquake in western Iran with moment magnitude (M) of 7.3. Nonlinear and linear amplification of ground motion amplitudes were observed at stations with soft soil condition at hypocentral distances below and above 100 km, respectively. Observation of large ground motion amplitudes dominated with long-period pulses on the strike-normal component of the velocity time series suggests a right-lateral component of movement and propagation of rupture towards southeast. Comparison of the horizontal peak ground acceleration (PGA) from the M 7.3 earthquake with global PGA values showed a similar decay in ground motion amplitudes, although it seems that PGA from the M 7.3 Kermanshah earthquake is higher than global values for NEHRP site class B. We also found that the bracketed duration (D b) was higher in the velocity domain than in the acceleration domain for the same modified Mercalli intensity (MMI) threshold. For example, D b reached 30 s at the maximum PGA while it was 50 s at the maximum peak ground velocity above the threshold of MMI = 5. Although the standard design spectrum from Iranian Code of Practice for Seismic Resistant Design of Buildings (standard No. 2800) seems to include appropriate values for the design of structures with fundamental period of 1 s and higher, it is underestimated for near-field ground motions at lower periods.

Functional and physical interaction between Bcl-XL and a BH3-like domain in Beclin-1

PubMed Central

Maiuri, M Chiara; Le Toumelin, Gaëtane; Criollo, Alfredo; Rain, Jean-Christophe; Gautier, Fabien; Juin, Philippe; Tasdemir, Ezgi; Pierron, Gérard; Troulinaki, Kostoula; Tavernarakis, Nektarios; Hickman, John A; Geneste, Olivier; Kroemer, Guido

2007-01-01

The anti-apoptotic proteins Bcl-2 and Bcl-XL bind and inhibit Beclin-1, an essential mediator of autophagy. Here, we demonstrate that this interaction involves a BH3 domain within Beclin-1 (residues 114–123). The physical interaction between Beclin-1 and Bcl-XL is lost when the BH3 domain of Beclin-1 or the BH3 receptor domain of Bcl-XL is mutated. Mutation of the BH3 domain of Beclin-1 or of the BH3 receptor domain of Bcl-XL abolishes the Bcl-XL-mediated inhibition of autophagy triggered by Beclin-1. The pharmacological BH3 mimetic ABT737 competitively inhibits the interaction between Beclin-1 and Bcl-2/Bcl-XL, antagonizes autophagy inhibition by Bcl-2/Bcl-XL and hence stimulates autophagy. Knockout or knockdown of the BH3-only protein Bad reduces starvation-induced autophagy, whereas Bad overexpression induces autophagy in human cells. Gain-of-function mutation of the sole BH3-only protein from Caenorhabditis elegans, EGL-1, induces autophagy, while deletion of EGL-1 compromises starvation-induced autophagy. These results reveal a novel autophagy-stimulatory function of BH3-only proteins beyond their established role as apoptosis inducers. BH3-only proteins and pharmacological BH3 mimetics induce autophagy by competitively disrupting the interaction between Beclin-1 and Bcl-2 or Bcl-XL. PMID:17446862

Crystallography of in-situ transformations of the M 7C3 carbide in the cast Fe-Cr-Ni alloy

NASA Astrophysics Data System (ADS)

Kraposhin, V. S.; Kondrat'ev, S. Yu.; Talis, A. L.; Anastasiadi, G. P.

2017-03-01

In the process of holding of the cast heat-resistant Fe-Cr-Ni (0.45C-25Cr-35Ni) alloy at 1150°C, the eutectic chromium carbide present in its structure undergoes a gradual transition M 7C3 → M 23C6. The gradual formation of domains of the M 23C6 carbide inside the particles of the M 7C3 carbide makes it possible to assume that the observed phase transition is the well-known carbide transformation of the in situ type. The mechanism of the in situ transformation of the crystal structure of the carbide from M 7C3 into M 23C6 with a change in the number of nearest metal neighbors of carbon atoms is explained within the previously developed combinatory model of polymorphic transitions in the metals.

Identification of YTH Domain-Containing Proteins as the Readers for N1-Methyladenosine in RNA.

PubMed

Dai, Xiaoxia; Wang, Tianlu; Gonzalez, Gwendolyn; Wang, Yinsheng

2018-06-05

N1-methyladenosine (m 1 A) is an important post-transcriptional modification in RNA; however, the exact biological role of m 1 A remains to be determined. By employing a quantitative proteomics method, we identified multiple putative protein readers of m 1 A in RNA, including several YTH domain family proteins. We showed that YTHDF1-3 and YTHDC1, but not YTHDC2, could bind directly to m 1 A in RNA. We also found that Trp 432 in YTHDF2, a conserved residue in the hydrophobic pocket of the YTH domain that is necessary for its binding to N 6 -methyladenosine (m 6 A), is required for its recognition of m 1 A. An analysis of previously published data revealed transcriptome-wide colocalization of YTH domain-containing proteins and m 1 A sites in HeLa cells, suggesting that YTH domain-containing proteins can bind to m 1 A in cells. Together, our results uncovered YTH domain-containing proteins as readers for m 1 A in RNA and provided new insight into the functions of m 1 A in RNA biology.

The coiled-coil domain of MURC/cavin-4 is involved in membrane trafficking of caveolin-3 in cardiomyocytes.

PubMed

Naito, Daisuke; Ogata, Takehiro; Hamaoka, Tetsuro; Nakanishi, Naohiko; Miyagawa, Kotaro; Maruyama, Naoki; Kasahara, Takeru; Taniguchi, Takuya; Nishi, Masahiro; Matoba, Satoaki; Ueyama, Tomomi

2015-12-15

Muscle-restricted coiled-coil protein (MURC), also referred to as cavin-4, is a member of the cavin family that works cooperatively with caveolins in caveola formation and function. Cavins are cytoplasmic proteins with coiled-coil domains and form heteromeric complexes, which are recruited to caveolae in cells expressing caveolins. Among caveolins, caveolin-3 (Cav3) is exclusively expressed in muscle cells, similar to MURC/cavin-4. In the heart, Cav3 overexpression contributes to cardiac protection, and its deficiency leads to progressive cardiomyopathy. Mutations in the MURC/cavin-4 gene have been identified in patients with dilated cardiomyopathy. In the present study, we show the role of MURC/cavin-4 as a caveolar component in the heart. In H9c2 cells, MURC/cavin-4 was localized at the plasma membrane, whereas a MURC/cavin-4 mutant lacking the coiled-coil domain (ΔCC) was primarily localized to the cytoplasm. ΔCC bound to Cav3 and impaired membrane localization of Cav3 in cardiomyocytes. Additionally, although ΔCC did not alter Cav3 mRNA expression, ΔCC decreased the Cav3 protein level. MURC/cavin-4 and ΔCC similarly induced cardiomyocyte hypertrophy; however, ΔCC showed higher hypertrophy-related fetal gene expression than MURC/cavin-4. ΔCC induced ERK activation in cardiomyocytes. Transgenic mice expressing ΔCC in the heart (ΔCC-Tg mice) showed impaired cardiac function accompanied by cardiomyocyte hypertrophy and marked interstitial fibrosis. Hearts from ΔCC-Tg mice showed a reduction of the Cav3 protein level and activation of ERK. These results suggest that MURC/cavin-4 requires its coiled-coil domain to target the plasma membrane and to stabilize Cav3 at the plasma membrane of cardiomyocytes and that MURC/cavin-4 functions as a crucial caveolar component to regulate cardiac function. Copyright © 2015 the American Physiological Society.

Distinct peptide binding specificities of Src homology 3 (SH3) protein domains can be determined by modulation of local energetics across the binding interface.

PubMed

Gorelik, Maryna; Davidson, Alan R

2012-03-16

The yeast Nbp2p SH3 and Bem1p SH3b domains bind certain target peptides with similar high affinities, yet display vastly different affinities for other targets. To investigate this unusual behavior, we have solved the structure of the Nbp2p SH3-Ste20 peptide complex and compared it with the previously determined structure of the Bem1p SH3b bound to the same peptide. Although the Ste20 peptide interacts with both domains in a structurally similar manner, extensive in vitro studies with domain and peptide mutants revealed large variations in interaction strength across the binding interface of the two complexes. Whereas the Nbp2p SH3 made stronger contacts with the peptide core RXXPXXP motif, the Bem1p SH3b domain made stronger contacts with residues flanking the core motif. Remarkably, this modulation of local binding energetics can explain the distinct and highly nuanced binding specificities of these two domains.

Voltage control of magnetic single domains in Ni discs on ferroelectric BaTiO3

NASA Astrophysics Data System (ADS)

Ghidini, M.; Zhu, B.; Mansell, R.; Pellicelli, R.; Lesaine, A.; Moya, X.; Crossley, S.; Nair, B.; Maccherozzi, F.; Barnes, C. H. W.; Cowburn, R. P.; Dhesi, S. S.; Mathur, N. D.

2018-06-01

For 1 µm-diameter Ni discs on a BaTiO3 substrate, the local magnetization direction is determined by ferroelectric domain orientation as a consequence of growth strain, such that single-domain discs lie on single ferroelectric domains. On applying a voltage across the substrate, ferroelectric domain switching yields non-volatile magnetization rotations of 90°, while piezoelectric effects that are small and continuous yield non-volatile magnetization reversals that are non-deterministic. This demonstration of magnetization reversal without ferroelectric domain switching implies reduced fatigue, and therefore represents a step towards applications.

The RPAP3-Cterminal domain identifies R2TP-like quaternary chaperones.

PubMed

Maurizy, Chloé; Quinternet, Marc; Abel, Yoann; Verheggen, Céline; Santo, Paulo E; Bourguet, Maxime; C F Paiva, Ana; Bragantini, Benoît; Chagot, Marie-Eve; Robert, Marie-Cécile; Abeza, Claire; Fabre, Philippe; Fort, Philippe; Vandermoere, Franck; M F Sousa, Pedro; Rain, Jean-Christophe; Charpentier, Bruno; Cianférani, Sarah; Bandeiras, Tiago M; Pradet-Balade, Bérengère; Manival, Xavier; Bertrand, Edouard

2018-05-29

R2TP is an HSP90 co-chaperone that assembles important macro-molecular machineries. It is composed of an RPAP3-PIH1D1 heterodimer, which binds the two essential AAA+ATPases RUVBL1/RUVBL2. Here, we resolve the structure of the conserved C-terminal domain of RPAP3, and we show that it directly binds RUVBL1/RUVBL2 hexamers. The human genome encodes two other proteins bearing RPAP3-C-terminal-like domains and three containing PIH-like domains. Systematic interaction analyses show that one RPAP3-like protein, SPAG1, binds PIH1D2 and RUVBL1/2 to form an R2TP-like complex termed R2SP. This co-chaperone is enriched in testis and among 68 of the potential clients identified, some are expressed in testis and others are ubiquitous. One substrate is liprin-α2, which organizes large signaling complexes. Remarkably, R2SP is required for liprin-α2 expression and for the assembly of liprin-α2 complexes, indicating that R2SP functions in quaternary protein folding. Effects are stronger at 32 °C, suggesting that R2SP could help compensating the lower temperate of testis.

Comparison of SH3 and SH2 domain dynamics when expressed alone or in an SH(3+2) construct: the role of protein dynamics in functional regulation.

PubMed

Engen, J R; Smithgall, T E; Gmeiner, W H; Smith, D L

1999-04-02

Protein dynamics play an important role in protein function and regulation of enzymatic activity. To determine how additional interactions with surrounding structure affects local protein dynamics, we have used hydrogen exchange and mass spectrometry to investigate the SH2 and SH3 domains of the protein tyrosine kinase Hck. Exchange rates of isolated Hck SH3 and SH2 domains were compared with rates for the same domains when part of a larger SH(3+2) construct. Increased deuterium incorporation was observed for the SH3 domain in the joint construct, particularly near the SH2 interface and the short sequence that connects SH3 to SH2, implying greater flexibility of SH3 when it is part of SH(3+2). Slow cooperative unfolding of the SH3 domain occurred at the same rate in isolated SH3 as in the SH(3+2) construct, suggesting a functional significance for this unfolding. The SH2 domain displayed relatively smaller changes in flexibility when part of the SH(3+2) construct. These results suggest that the domains influence each other. Further, our results imply a link between functional regulation and structural dynamics of SH3 and SH2 domains. Copyright 1999 Academic Press.

Characterization of the SAM domain of the PKD-related protein ANKS6 and its interaction with ANKS3.

PubMed

Leettola, Catherine N; Knight, Mary Jane; Cascio, Duilio; Hoffman, Sigrid; Bowie, James U

2014-07-07

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic disorder leading to end-stage renal failure in humans. In the PKD/Mhm(cy/+) rat model of ADPKD, the point mutation R823W in the sterile alpha motif (SAM) domain of the protein ANKS6 is responsible for disease. SAM domains are known protein-protein interaction domains, capable of binding each other to form polymers and heterodimers. Despite its physiological importance, little is known about the function of ANKS6 and how the R823W point mutation leads to PKD. Recent work has revealed that ANKS6 interacts with a related protein called ANKS3. Both ANKS6 and ANKS3 have a similar domain structure, with ankyrin repeats at the N-terminus and a SAM domain at the C-terminus. The SAM domain of ANKS3 is identified as a direct binding partner of the ANKS6 SAM domain. We find that ANKS3-SAM polymerizes and ANKS6-SAM can bind to one end of the polymer. We present crystal structures of both the ANKS3-SAM polymer and the ANKS3-SAM/ANKS6-SAM complex, revealing the molecular details of their association. We also learn how the R823W mutation disrupts ANKS6 function by dramatically destabilizing the SAM domain such that the interaction with ANKS3-SAM is lost. ANKS3 is a direct interacting partner of ANKS6. By structurally and biochemically characterizing the interaction between the ANKS3 and ANKS6 SAM domains, our work provides a basis for future investigation of how the interaction between these proteins mediates kidney function.

Characterization of the SAM domain of the PKD-related protein ANKS6 and its interaction with ANKS3

PubMed Central

2014-01-01

Background Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic disorder leading to end-stage renal failure in humans. In the PKD/Mhm(cy/+) rat model of ADPKD, the point mutation R823W in the sterile alpha motif (SAM) domain of the protein ANKS6 is responsible for disease. SAM domains are known protein-protein interaction domains, capable of binding each other to form polymers and heterodimers. Despite its physiological importance, little is known about the function of ANKS6 and how the R823W point mutation leads to PKD. Recent work has revealed that ANKS6 interacts with a related protein called ANKS3. Both ANKS6 and ANKS3 have a similar domain structure, with ankyrin repeats at the N-terminus and a SAM domain at the C-terminus. Results The SAM domain of ANKS3 is identified as a direct binding partner of the ANKS6 SAM domain. We find that ANKS3-SAM polymerizes and ANKS6-SAM can bind to one end of the polymer. We present crystal structures of both the ANKS3-SAM polymer and the ANKS3-SAM/ANKS6-SAM complex, revealing the molecular details of their association. We also learn how the R823W mutation disrupts ANKS6 function by dramatically destabilizing the SAM domain such that the interaction with ANKS3-SAM is lost. Conclusions ANKS3 is a direct interacting partner of ANKS6. By structurally and biochemically characterizing the interaction between the ANKS3 and ANKS6 SAM domains, our work provides a basis for future investigation of how the interaction between these proteins mediates kidney function. PMID:24998259

Improving the Effectiveness of Speaker Verification Domain Adaptation With Inadequate In-Domain Data

DTIC Science & Technology

2017-08-20

Improving the Effectiveness of Speaker Verification Domain Adaptation With Inadequate In-Domain Data Bengt J. Borgström1, Elliot Singer1, Douglas...ll.mit.edu.edu, dar@ll.mit.edu, es@ll.mit.edu, omid.sadjadi@nist.gov Abstract This paper addresses speaker verification domain adaptation with...contain speakers with low channel diversity. Existing domain adaptation methods are reviewed, and their shortcomings are discussed. We derive an

H3K9me2/3 Binding of the MBT Domain Protein LIN-61 Is Essential for Caenorhabditis elegans Vulva Development

PubMed Central

Koester-Eiserfunke, Nora; Fischle, Wolfgang

2011-01-01

MBT domain proteins are involved in developmental processes and tumorigenesis. In vitro binding and mutagenesis studies have shown that individual MBT domains within clustered MBT repeat regions bind mono- and dimethylated histone lysine residues with little to no sequence specificity but discriminate against the tri- and unmethylated states. However, the exact function of promiscuous histone methyl-lysine binding in the biology of MBT domain proteins has not been elucidated. Here, we show that the Caenorhabditis elegans four MBT domain protein LIN-61, in contrast to other MBT repeat factors, specifically interacts with histone H3 when methylated on lysine 9, displaying a strong preference for di- and trimethylated states (H3K9me2/3). Although the fourth MBT repeat is implicated in this interaction, H3K9me2/3 binding minimally requires MBT repeats two to four. Further, mutagenesis of residues conserved with other methyl-lysine binding MBT regions in the fourth MBT repeat does not abolish interaction, implicating a distinct binding mode. In vivo, H3K9me2/3 interaction of LIN-61 is required for C. elegans vulva development within the synMuvB pathway. Mutant LIN-61 proteins deficient in H3K9me2/3 binding fail to rescue lin-61 synMuvB function. Also, previously identified point mutant synMuvB alleles are deficient in H3K9me2/3 interaction although these target residues that are outside of the fourth MBT repeat. Interestingly, lin-61 genetically interacts with two other synMuvB genes, hpl-2, an HP1 homologous H3K9me2/3 binding factor, and met-2, a SETDB1 homologous H3K9 methyl transferase (H3K9MT), in determining C. elegans vulva development and fertility. Besides identifying the first sequence specific and di-/trimethylation binding MBT domain protein, our studies imply complex multi-domain regulation of ligand interaction of MBT domains. Our results also introduce a mechanistic link between LIN-61 function and biology, and they establish interplay of the H3K9me2/3

Elucidation of the binding preferences of peptide recognition modules: SH3 and PDZ domains.

PubMed

Teyra, Joan; Sidhu, Sachdev S; Kim, Philip M

2012-08-14

Peptide-binding domains play a critical role in regulation of cellular processes by mediating protein interactions involved in signalling. In recent years, the development of large-scale technologies has enabled exhaustive studies on the peptide recognition preferences for a number of peptide-binding domain families. These efforts have provided significant insights into the binding specificities of these modular domains. Many research groups have taken advantage of this unprecedented volume of specificity data and have developed a variety of new algorithms for the prediction of binding specificities of peptide-binding domains and for the prediction of their natural binding targets. This knowledge has also been applied to the design of synthetic peptide-binding domains in order to rewire protein-protein interaction networks. Here, we describe how these experimental technologies have impacted on our understanding of peptide-binding domain specificities and on the elucidation of their natural ligands. We discuss SH3 and PDZ domains as well characterized examples, and we explore the feasibility of expanding high-throughput experiments to other peptide-binding domains. Copyright © 2012. Published by Elsevier B.V.

Interkinase domain of kit contains the binding site for phosphatidylinositol 3' kinase.

PubMed Central

Lev, S; Givol, D; Yarden, Y

1992-01-01

Our previous analysis of the signal transduction pathway used by the c-kit-encoded receptor for the stem cell factor (SCF) indicated efficient coupling to the type I phosphatidylinositol 3' kinase (PI3K). In an attempt to localize the receptor's site of interaction with PI3K, we separately deleted either the noncatalytic 68-amino-acid-long interkinase domain or the carboxyl-terminal portion distal to the catalytic sequences. Loss of ligand-induced association of PI3K with the former deletion mutant and retention of the PI3K association by the carboxyl-terminally deleted receptor implied interactions of PI3K with the kinase insert. This was further supported by partial inhibition of the association by an anti-peptide antibody directed against the kinase insert and lack of effect of an antibody directed to the carboxyl tail of the SCF receptor. A bacterially expressed kinase insert domain was used as a fusion protein to directly test its presumed function as a PI3K association site. This protein bound PI3K from cell lysate as demonstrated by PI3K activity and by an associated phosphoprotein of 85 kDa. The association was dependent on phosphorylation of the tyrosine residues on the expressed kinase insert. On the basis of these observations, we conclude that the kinase insert domain of the SCF receptor selectively interacts with the p85 regulatory subunit of PI3K and that this association requires phosphorylation of tyrosine residues in the kinase insert region, with apparently no involvement of the bulk cytoplasmic structure or tyrosine kinase function of the receptor. Images PMID:1370584

The SARS Coronavirus 3a protein binds calcium in its cytoplasmic domain.

PubMed

Minakshi, Rinki; Padhan, Kartika; Rehman, Safikur; Hassan, Md Imtaiyaz; Ahmad, Faizan

2014-10-13

The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is a positive stranded RNA virus with ∼30kb genome. Among all open reading frames (orfs) of this virus, the orf3a is the largest, and encodes a protein of 274 amino acids, named as 3a protein. Sequence analysis suggests that the orf3a aligned to one calcium pump present in Plasmodium falciparum and the enzyme glutamine synthetase found in Leptospira interrogans. This sequence similarity was found to be limited only to amino acid residues 209-264 which form the cytoplasmic domain of the orf3a. Furthermore, this region was predicted to be involved in the calcium binding. Owing to this hypothesis, we were driven to establish its calcium binding property in vitro. Here, we expressed and purified the cytoplasmic domain of the 3a protein, called Cyto3a, as a recombinant His-tagged protein in the E. coli. The calcium binding nature was established by performing various staining methods such as ruthenium red and stains-all. (45)Ca overlay method was also done to further support the data. Since the 3a protein forms ion channels, we were interested to see any conformational changes occurring in the Cyot3a upon calcium binding, using fluorescence spectroscopy and circular dichroism. These studies clearly indicate a significant change in the conformation of the Cyto3a protein after binding with calcium. Our results strongly suggest that the cytoplasmic domain of the 3a protein of SARS-CoV binds calcium in vitro, causing a change in protein conformation. Copyright © 2014 Elsevier B.V. All rights reserved.

Crystal structure of the Candida albicans Kar3 kinesin motor domain fused to maltose-binding protein

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

Delorme, Caroline; Joshi, Monika; Allingham, John S., E-mail: allinghj@queensu.ca

2012-11-30

Highlights: Black-Right-Pointing-Pointer The Candida albicans Kar3 motor domain structure was solved as a maltose-binding protein fusion. Black-Right-Pointing-Pointer The electrostatic surface and part of the ATPase pocket of the motor domain differs markedly from other kinesins. Black-Right-Pointing-Pointer The MBP-Kar3 interface highlights a new site for intramolecular or intermolecular interactions. -- Abstract: In the human fungal pathogen Candida albicans, the Kinesin-14 motor protein Kar3 (CaKar3) is critical for normal mitotic division, nuclear fusion during mating, and morphogenic transition from the commensal yeast form to the virulent hyphal form. As a first step towards detailed characterization of this motor of potential medical significance,more » we have crystallized and determined the X-ray structure of the motor domain of CaKar3 as a maltose-binding protein (MBP) fusion. The structure shows strong conservation of overall motor domain topology to other Kar3 kinesins, but with some prominent differences in one of the motifs that compose the nucleotide-binding pocket and the surface charge distribution. The MBP and Kar3 modules are arranged such that MBP interacts with the Kar3 motor domain core at the same site where the neck linker of conventional kinesins docks during the 'ATP state' of the mechanochemical cycle. This site differs from the Kar3 neck-core interface in the recent structure of the ScKar3Vik1 heterodimer. The position of MBP is also completely distinct from the Vik1 subunit in this complex. This may suggest that the site of MBP interaction on the CaKar3 motor domain provides an interface for the neck, or perhaps a partner subunit, at an intermediate state of its motile cycle that has not yet been observed for Kinesin-14 motors.« less

Free-electron gas at charged domain walls in insulating BaTiO3

PubMed Central

Sluka, Tomas; Tagantsev, Alexander K.; Bednyakov, Petr; Setter, Nava

2013-01-01

Hetero interfaces between metal-oxides display pronounced phenomena such as semiconductor-metal transitions, magnetoresistance, the quantum hall effect and superconductivity. Similar effects at compositionally homogeneous interfaces including ferroic domain walls are expected. Unlike hetero interfaces, domain walls can be created, displaced, annihilated and recreated inside a functioning device. Theory predicts the existence of 'strongly' charged domain walls that break polarization continuity, but are stable and conduct steadily through a quasi-two-dimensional electron gas. Here we show this phenomenon experimentally in charged domain walls of the prototypical ferroelectric BaTiO3. Their steady metallic-type conductivity, 109 times that of the parent matrix, evidence the presence of stable degenerate electron gas, thus adding mobility to functional interfaces. PMID:23651996

Different domains of the murine RNA polymerase I-specific termination factor mTTF-I serve distinct functions in transcription termination.

PubMed

Evers, R; Smid, A; Rudloff, U; Lottspeich, F; Grummt, I

1995-03-15

Termination of mouse ribosomal gene transcription by RNA polymerase I (Pol I) requires the specific interaction of a DNA binding protein, mTTF-I, with an 18 bp sequence element located downstream of the rRNA coding region. Here we describe the molecular cloning and functional characterization of the cDNA encoding this transcription termination factor. Recombinant mTTF-I binds specifically to the murine terminator elements and terminates Pol I transcription in a reconstituted in vitro system. Deletion analysis has defined a modular structure of mTTF-I comprising a dispensable N-terminal half, a large C-terminal DNA binding region and an internal domain which is required for transcription termination. Significantly, the C-terminal region of mTTF-I reveals striking homology to the DNA binding domains of the proto-oncogene c-Myb and the yeast transcription factor Reb1p. Site-directed mutagenesis of one of the tryptophan residues that is conserved in the homology region of c-Myb, Reb1p and mTTF-I abolishes specific DNA binding, a finding which underscores the functional relevance of these residues in DNA-protein interactions.

Different domains of the murine RNA polymerase I-specific termination factor mTTF-I serve distinct functions in transcription termination.

PubMed Central

Evers, R; Smid, A; Rudloff, U; Lottspeich, F; Grummt, I

1995-01-01

Termination of mouse ribosomal gene transcription by RNA polymerase I (Pol I) requires the specific interaction of a DNA binding protein, mTTF-I, with an 18 bp sequence element located downstream of the rRNA coding region. Here we describe the molecular cloning and functional characterization of the cDNA encoding this transcription termination factor. Recombinant mTTF-I binds specifically to the murine terminator elements and terminates Pol I transcription in a reconstituted in vitro system. Deletion analysis has defined a modular structure of mTTF-I comprising a dispensable N-terminal half, a large C-terminal DNA binding region and an internal domain which is required for transcription termination. Significantly, the C-terminal region of mTTF-I reveals striking homology to the DNA binding domains of the proto-oncogene c-Myb and the yeast transcription factor Reb1p. Site-directed mutagenesis of one of the tryptophan residues that is conserved in the homology region of c-Myb, Reb1p and mTTF-I abolishes specific DNA binding, a finding which underscores the functional relevance of these residues in DNA-protein interactions. Images PMID:7720715

Drosophila CTCF tandemly aligns with other insulator proteins at the borders of H3K27me3 domains.

PubMed

Van Bortle, Kevin; Ramos, Edward; Takenaka, Naomi; Yang, Jingping; Wahi, Jessica E; Corces, Victor G

2012-11-01

Several multiprotein DNA complexes capable of insulator activity have been identified in Drosophila melanogaster, yet only CTCF, a highly conserved zinc finger protein, and the transcription factor TFIIIC have been shown to function in mammals. CTCF is involved in diverse nuclear activities, and recent studies suggest that the proteins with which it associates and the DNA sequences that it targets may underlie these various roles. Here we show that the Drosophila homolog of CTCF (dCTCF) aligns in the genome with other Drosophila insulator proteins such as Suppressor of Hairy wing [SU(HW)] and Boundary Element Associated Factor of 32 kDa (BEAF-32) at the borders of H3K27me3 domains, which are also enriched for associated insulator proteins and additional cofactors. RNAi depletion of dCTCF and combinatorial knockdown of gene expression for other Drosophila insulator proteins leads to a reduction in H3K27me3 levels within repressed domains, suggesting that insulators are important for the maintenance of appropriate repressive chromatin structure in Polycomb (Pc) domains. These results shed new insights into the roles of insulators in chromatin domain organization and support recent models suggesting that insulators underlie interactions important for Pc-mediated repression. We reveal an important relationship between dCTCF and other Drosophila insulator proteins and speculate that vertebrate CTCF may also align with other nuclear proteins to accomplish similar functions.

PNPLA3 p.I148M variant is associated with greater reduction of liver fat content after bariatric surgery.

PubMed

Krawczyk, Marcin; Jiménez-Agüero, Raúl; Alustiza, José M; Emparanza, José I; Perugorria, María J; Bujanda, Luis; Lammert, Frank; Banales, Jesús M

2016-12-01

Obesity is the major trigger of nonalcoholic fatty liver disease (NAFLD). NAFLD is further favored by the patatin-like phospholipase domain-containing 3 (PNPLA3) p.I148M, transmembrane 6 superfamily member 2 (TM6SF2) p.E167K, and membrane-bound O-acyltransferase domain containing 7 (MBOAT7) rs641738 variants. To investigate the relationship between the PNPLA3, TM6SF2, and MBOAT7 genotypes and the outcomes of bariatric surgery. University hospital. Prospectively we monitored 84 obese individuals (body mass index 35-64 kg/m 2 ) scheduled for bariatric surgery. The PNPLA3 p.I148M, TM6SF2 p.E167K, and MBOAT7 rs641738 variants were genotyped using restriction fragment length polymorphism analysis and TaqMan assays. Hepatic steatosis was determined before surgery using analysis of liver biopsy samples and a novel magnetic resonance imaging-based equation. One year later, steatosis was reevaluated by magnetic resonance imaging. The presence of the PNPLA3 allle [M] was associated with increased hepatic triglyceride content (P = .03), steatosis detected by magnetic resonance imaging (P = 0.04), and decreased serum glucose concentrations (P = .04). Neither variant TM6SF2 nor MBOAT7 increased hepatic steatosis (all P>.05); however, the MBOAT7 polymorphism was associated with increased triglyceride, total cholesterol, low density lipoprotein, and serum glucose levels (all P<.05). Patients carrying the prosteatotic PNPLA3 allele [M] lost more weight (P<.01) and liver fat (P = .04) one year after surgery, as compared to individuals having the common genotype. The PNPLA3 genotype and initial grade of steatosis, but not the TM6SF2 or MBOAT7 variants, were independent predictors of NAFLD improvement (P = .03 and P<.01, respectively). In obese patients, the presence of the PNPLA3 p.I148M allele might be associated with greater improvement of hepatic steatosis after bariatric surgery in comparison to carriers of PNPLA3 wild-type alleles. Copyright © 2016 American Society for

Striatal Transcriptome and Interactome Analysis of Shank3-overexpressing Mice Reveals the Connectivity between Shank3 and mTORC1 Signaling

PubMed Central

Lee, Yeunkum; Kim, Sun Gyun; Lee, Bokyoung; Zhang, Yinhua; Kim, Yoonhee; Kim, Shinhyun; Kim, Eunjoon; Kang, Hyojin; Han, Kihoon

2017-01-01

Mania causes symptoms of hyperactivity, impulsivity, elevated mood, reduced anxiety and decreased need for sleep, which suggests that the dysfunction of the striatum, a critical component of the brain motor and reward system, can be causally associated with mania. However, detailed molecular pathophysiology underlying the striatal dysfunction in mania remains largely unknown. In this study, we aimed to identify the molecular pathways showing alterations in the striatum of SH3 and multiple ankyrin repeat domains 3 (Shank3)-overexpressing transgenic (TG) mice that display manic-like behaviors. The results of transcriptome analysis suggested that mammalian target of rapamycin complex 1 (mTORC1) signaling may be the primary molecular signature altered in the Shank3 TG striatum. Indeed, we found that striatal mTORC1 activity, as measured by mTOR S2448 phosphorylation, was significantly decreased in the Shank3 TG mice compared to wild-type (WT) mice. To elucidate the potential underlying mechanism, we re-analyzed previously reported protein interactomes, and detected a high connectivity between Shank3 and several upstream regulators of mTORC1, such as tuberous sclerosis 1 (TSC1), TSC2 and Ras homolog enriched in striatum (Rhes), via 94 common interactors that we denominated “Shank3-mTORC1 interactome”. We noticed that, among the 94 common interactors, 11 proteins were related to actin filaments, the level of which was increased in the dorsal striatum of Shank3 TG mice. Furthermore, we could co-immunoprecipitate Shank3, Rhes and Wiskott-Aldrich syndrome protein family verprolin-homologous protein 1 (WAVE1) proteins from the striatal lysate of Shank3 TG mice. By comparing with the gene sets of psychiatric disorders, we also observed that the 94 proteins of Shank3-mTORC1 interactome were significantly associated with bipolar disorder (BD). Altogether, our results suggest a protein interaction-mediated connectivity between Shank3 and certain upstream regulators of mTORC1

Effects of mutations in the Arabidopsis Cold Shock Domain Protein 3 (AtCSP3) gene on leaf cell expansion.

PubMed

Yang, Yongil; Karlson, Dale

2012-08-01

The cold shock domain is among the most evolutionarily conserved nucleic acid binding domains from prokaryotes to higher eukaryotes, including plants. Although eukaryotic cold shock domain proteins have been extensively studied as transcriptional and post-transcriptional regulators during various developmental processes, their functional roles in plants remains poorly understood. In this study, AtCSP3 (At2g17870), which is one of four Arabidopsis thaliana c old s hock domain proteins (AtCSPs), was functionally characterized. Quantitative RT-PCR analysis confirmed high expression of AtCSP3 in reproductive and meristematic tissues. A homozygous atcsp3 loss-of-function mutant exhibits an overall reduced seedling size, stunted and orbicular rosette leaves, reduced petiole length, and curled leaf blades. Palisade mesophyll cells are smaller and more circular in atcsp3 leaves. Cell size analysis indicated that the reduced size of the circular mesophyll cells appears to be generated by a reduction of cell length along the leaf-length axis, resulting in an orbicular leaf shape. It was also determined that leaf cell expansion is impaired for lateral leaf development in the atcsp3 loss-of-function mutant, but leaf cell proliferation is not affected. AtCSP3 loss-of-function resulted in a dramatic reduction of LNG1 transcript, a gene that is involved in two-dimensional leaf polarity regulation. Transient subcellular localization of AtCSP3 in onion epidermal cells confirmed a nucleocytoplasmic localization pattern. Collectively, these data suggest that AtCSP3 is functionally linked to the regulation of leaf length by affecting LNG1 transcript accumulation during leaf development. A putative function of AtCSP3 as an RNA binding protein is also discussed in relation to leaf development.

Differential Recognition Preferences of the Three Src Homology 3 (SH3) Domains from the Adaptor CD2-associated Protein (CD2AP) and Direct Association with Ras and Rab Interactor 3 (RIN3)*

PubMed Central

Rouka, Evgenia; Simister, Philip C.; Janning, Melanie; Kumbrink, Joerg; Konstantinou, Tassos; Muniz, João R. C.; Joshi, Dhira; O'Reilly, Nicola; Volkmer, Rudolf; Ritter, Brigitte; Knapp, Stefan; von Delft, Frank; Kirsch, Kathrin H.; Feller, Stephan M.

2015-01-01

CD2AP is an adaptor protein involved in membrane trafficking, with essential roles in maintaining podocyte function within the kidney glomerulus. CD2AP contains three Src homology 3 (SH3) domains that mediate multiple protein-protein interactions. However, a detailed comparison of the molecular binding preferences of each SH3 remained unexplored, as well as the discovery of novel interactors. Thus, we studied the binding properties of each SH3 domain to the known interactor Casitas B-lineage lymphoma protein (c-CBL), conducted a peptide array screen based on the recognition motif PxPxPR and identified 40 known or novel candidate binding proteins, such as RIN3, a RAB5-activating guanine nucleotide exchange factor. CD2AP SH3 domains 1 and 2 generally bound with similar characteristics and specificities, whereas the SH3-3 domain bound more weakly to most peptide ligands tested yet recognized an unusually extended sequence in ALG-2-interacting protein X (ALIX). RIN3 peptide scanning arrays revealed two CD2AP binding sites, recognized by all three SH3 domains, but SH3-3 appeared non-functional in precipitation experiments. RIN3 recruited CD2AP to RAB5a-positive early endosomes via these interaction sites. Permutation arrays and isothermal titration calorimetry data showed that the preferred binding motif is Px(P/A)xPR. Two high-resolution crystal structures (1.65 and 1.11 Å) of CD2AP SH3-1 and SH3-2 solved in complex with RIN3 epitopes 1 and 2, respectively, indicated that another extended motif is relevant in epitope 2. In conclusion, we have discovered novel interaction candidates for CD2AP and characterized subtle yet significant differences in the recognition preferences of its three SH3 domains for c-CBL, ALIX, and RIN3. PMID:26296892

Metabolic roles of the M3 muscarinic acetylcholine receptor studied with M3 receptor mutant mice: a review.

PubMed

Gautam, Dinesh; Jeon, Jongrye; Li, Jian Hua; Han, Sung-Jun; Hamdan, Fadi F; Cui, Yinghong; Lu, Huiyan; Deng, Chuxia; Gavrilova, Oksana; Wess, Jürgen

2008-01-01

The M(3) muscarinic acetylcholine (ACh) receptor (M(3) mAChR) is expressed in many central and peripheral tissues. It is a prototypic member of the superfamily of G protein-coupled receptors and preferentially activates G proteins of the G(q) family. Recent studies involving the use of newly generated mAChR mutant mice have revealed that the M(3) mAChR plays a key role in regulating many important metabolic functions. Phenotypic analyses of mutant mice that either selectively lacked or overexpressed M(3) receptors in pancreatic beta -cells indicated that beta -cell M(3) mAChRs are essential for maintaining proper insulin release and glucose homeostasis. The experimental data also suggested that strategies aimed at enhancing signaling through beta -cell M(3) mAChRs might be beneficial for the treatment of type 2 diabetes. Recent studies with whole body M(3) mAChR knockout mice showed that the absence of M(3) receptors protected mice against various forms of experimentally or genetically induced obesity and obesity-associated metabolic deficits. Under all experimental conditions tested, M(3) receptor-deficient mice showed greatly ameliorated impairments in glucose homeostasis and insulin sensitivity, reduced food intake, and a significant elevation in basal and total energy expenditure, most likely due to increased central sympathetic outflow and increased rate of fatty acid oxidation. These findings are of potential interest for the development of novel therapeutic approaches for the treatment of obesity and associated metabolic disorders.

Ferroelectric domain switching dynamics and memristive behaviors in BiFeO3-based magnetoelectric heterojunctions

NASA Astrophysics Data System (ADS)

Huang, Weichuan; Liu, Yukuai; Luo, Zhen; Hou, Chuangming; Zhao, Wenbo; Yin, Yuewei; Li, Xiaoguang

2018-06-01

The ferroelectric domain reversal dynamics and the corresponding resistance switching as well as the memristive behaviors in epitaxial BiFeO3 (BFO, ~150 nm) based multiferroic heterojunctions were systematically investigated. The ferroelectric domain reversal dynamics could be described by the nucleation-limited-switching model with the Lorentzian distribution of logarithmic domain-switching times. By engineering the domain states, multi and even continuously tunable resistances states, i.e. memristive states, could be non-volatilely achieved. The resistance switching speed can be as fast as 30 ns in the BFO-based multiferroic heterojunctions with a write voltage of ~20 V. By reducing the thickness of BFO, the La0.6Sr0.4MnO3/BFO (~5 nm)/La0.6Sr0.4MnO3 multiferroic tunnel junction (MFTJ) shows an even a quicker switching speed (20 ns) with a much lower operation voltage (~4 V). Importantly, the MFTJ exhibits a tunable interfacial magnetoelectric coupling related to the ferroelectric domain switching dynamics. These findings enrich the potential applications of multiferroic BFO based devices in high-speed, low-power, and high-density memories as well as future neuromorphic computational architectures.

The Chemical Composition Contrast between M3 and M13 Revisited: New Abundances for 28 Giant Stars in M3

NASA Astrophysics Data System (ADS)

Sneden, Christopher; Kraft, Robert P.; Guhathakurta, Puragra; Peterson, Ruth C.; Fulbright, Jon P.

2004-04-01

We report new chemical abundances of 23 bright red giant members of the globular cluster M3, based on high-resolution (R~45,000) spectra obtained with the Keck I telescope. The observations, which involve the use of multislits in the HIRES Keck I spectrograph, are described in detail. Combining these data with a previously reported small sample of M3 giants obtained with the Lick 3 m telescope, we compare metallicities and [X/Fe] ratios for 28 M3 giants with a 35-star sample in the similar-metallicity cluster M13, and with Galactic halo field stars having [Fe/H]<-1. For elements having atomic number A>=A(Si), we derive little difference in [X/Fe] ratios in the M3, M13, or halo field samples. All three groups exhibit C depletion with advancing evolutionary state beginning at the level of the red giant branch ``bump,'' but the overall depletion of about 0.7-0.9 dex seen in the clusters is larger than that associated with the field stars. The behaviors of O, Na, Mg, and Al are distinctively different among the three stellar samples. Field halo giants and subdwarfs have a positive correlation of Na with Mg, as predicted from explosive or hydrostatic carbon burning in Type II supernova sites. Both M3 and M13 show evidence of high-temperature proton-capture synthesis from the ON, NeNa, and MgAl cycles, while there is no evidence for such synthesis among halo field stars. But the degree of such extreme proton-capture synthesis in M3 is smaller than it is in M13: the M3 giants exhibit only modest deficiencies of O and corresponding enhancements of Na, less extreme overabundances of Al, fewer stars with low Mg and correspondingly high Na, and no indication that O depletions are a function of advancing evolutionary state, as has been claimed for M13. We have also considered NGC 6752, for which Mg isotopic abundances have been reported by Yong et al. Giants in NGC 6752 and M13 satisfy the same anticorrelation of O abundances with the ratio (25Mg+26Mg)/24Mg, which measures the

Mutations in the HECT domain of NEDD4L lead to AKT-mTOR pathway deregulation and cause periventricular nodular heterotopia.

PubMed

Broix, Loïc; Jagline, Hélène; Ivanova, Ekaterina; Schmucker, Stéphane; Drouot, Nathalie; Clayton-Smith, Jill; Pagnamenta, Alistair T; Metcalfe, Kay A; Isidor, Bertrand; Louvier, Ulrike Walther; Poduri, Annapurna; Taylor, Jenny C; Tilly, Peggy; Poirier, Karine; Saillour, Yoann; Lebrun, Nicolas; Stemmelen, Tristan; Rudolf, Gabrielle; Muraca, Giuseppe; Saintpierre, Benjamin; Elmorjani, Adrienne; Moïse, Martin; Weirauch, Nathalie Bednarek; Guerrini, Renzo; Boland, Anne; Olaso, Robert; Masson, Cecile; Tripathy, Ratna; Keays, David; Beldjord, Cherif; Nguyen, Laurent; Godin, Juliette; Kini, Usha; Nischké, Patrick; Deleuze, Jean-François; Bahi-Buisson, Nadia; Sumara, Izabela; Hinckelmann, Maria-Victoria; Chelly, Jamel

2016-11-01

Neurodevelopmental disorders with periventricular nodular heterotopia (PNH) are etiologically heterogeneous, and their genetic causes remain in many cases unknown. Here we show that missense mutations in NEDD4L mapping to the HECT domain of the encoded E3 ubiquitin ligase lead to PNH associated with toe syndactyly, cleft palate and neurodevelopmental delay. Cellular and expression data showed sensitivity of PNH-associated mutants to proteasome degradation. Moreover, an in utero electroporation approach showed that PNH-related mutants and excess wild-type NEDD4L affect neurogenesis, neuronal positioning and terminal translocation. Further investigations, including rapamycin-based experiments, found differential deregulation of pathways involved. Excess wild-type NEDD4L leads to disruption of Dab1 and mTORC1 pathways, while PNH-related mutations are associated with deregulation of mTORC1 and AKT activities. Altogether, these data provide insights into the critical role of NEDD4L in the regulation of mTOR pathways and their contributions in cortical development.

Cold Shock Domain Protein 3 Regulates Freezing Tolerance in Arabidopsis thaliana*

PubMed Central

Kim, Myung-Hee; Sasaki, Kentaro; Imai, Ryozo

2009-01-01

In response to cold, Escherichia coli produces cold shock proteins (CSPs) that have essential roles in cold adaptation as RNA chaperones. Here, we demonstrate that Arabidopsis cold shock domain protein 3 (AtCSP3), which shares a cold shock domain with bacterial CSPs, is involved in the acquisition of freezing tolerance in plants. AtCSP3 complemented a cold-sensitive phenotype of the E. coli CSP quadruple mutant and displayed nucleic acid duplex melting activity, suggesting that AtCSP3 also functions as an RNA chaperone. Promoter-GUS transgenic plants revealed tissue-specific expression of AtCSP3 in shoot and root apical regions. When exposed to low temperature, GUS activity was extensively induced in a broader region of the roots. In transgenic plants expressing an AtCSP3-GFP fusion, GFP signals were detected in both the nucleus and cytoplasm. An AtCSP3 knock-out mutant (atcsp3-2) was sensitive to freezing compared with wild-type plants under non-acclimated and cold-acclimated conditions, whereas expression of C-repeat-binding factors and their downstream genes during cold acclimation was not altered in the atcsp3-2 mutant. Overexpression of AtCSP3 in transgenic plants conferred enhanced freezing tolerance over wild-type plants. Together, the data demonstrated an essential role of RNA chaperones for cold adaptation in higher plants. PMID:19556243

LRAT-specific domain facilitates vitamin A metabolism by domain swapping in HRASLS3

DOE PAGES

Golczak, Marcin; Sears, Avery E.; Kiser, Philip D.; ...

2014-11-10

Cellular uptake of vitamin A, production of visual chromophore and triglyceride homeostasis in adipocytes depend on two representatives of the vertebrate N1pC/P60 protein family, lecithin:retinol acyltransferase (LRAT) and HRAS-like tumor suppressor 3 (HRASLS3). Both proteins function as lipid-metabolizing enzymes but differ in their substrate preferences and dominant catalytic activity. The mechanism of this catalytic diversity is not understood. In this paper, by using a gain-of-function approach, we identified a specific sequence responsible for the substrate specificity of N1pC/P60 proteins. A 2.2-Å crystal structure of the HRASLS3-LRAT chimeric enzyme in a thioester catalytic intermediate state revealed a major structural rearrangement accompaniedmore » by three-dimensional domain swapping dimerization not observed in native HRASLS proteins. Structural changes affecting the active site environment contributed to slower hydrolysis of the catalytic intermediate, supporting efficient acyl transfer. Finally, these findings reveal structural adaptation that facilitates selective catalysis and mechanism responsible for diverse substrate specificity within the LRAT-like enzyme family.« less

77 FR 58912 - Designation of One (1) Individual Pursuant to Executive Order 13582 of August 17, 2011, “Blocking...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-09-24

...; alt. DOB 31 Aug 1955; alt. DOB 31 Aug 1958; POB Al Basuriyah, Lebanon; alt. POB Beirut, Lebanon; Passport 042833 (Lebanon); Secretary General of HIZBALLAH (individual) [SDT] [SYRIA]. Dated: September 13...

Identification of a Novel TGF-β-Binding Site in the Zona Pellucida C-terminal (ZP-C) Domain of TGF-β-Receptor-3 (TGFR-3)

PubMed Central

Diestel, Uschi; Resch, Marcus; Meinhardt, Kathrin; Weiler, Sigrid; Hellmann, Tina V.; Mueller, Thomas D.; Nickel, Joachim; Eichler, Jutta; Muller, Yves A.

2013-01-01

The zona pellucida (ZP) domain is present in extracellular proteins such as the zona pellucida proteins and tectorins and participates in the formation of polymeric protein networks. However, the ZP domain also occurs in the cytokine signaling co-receptor transforming growth factor β (TGF-β) receptor type 3 (TGFR-3, also known as betaglycan) where it contributes to cytokine ligand recognition. Currently it is unclear how the ZP domain architecture enables this dual functionality. Here, we identify a novel major TGF-β-binding site in the FG loop of the C-terminal subdomain of the murine TGFR-3 ZP domain (ZP-C) using protein crystallography, limited proteolysis experiments, surface plasmon resonance measurements and synthetic peptides. In the murine 2.7 Å crystal structure that we are presenting here, the FG-loop is disordered, however, well-ordered in a recently reported homologous rat ZP-C structure. Surprisingly, the adjacent external hydrophobic patch (EHP) segment is registered differently in the rat and murine structures suggesting that this segment only loosely associates with the remaining ZP-C fold. Such a flexible and temporarily-modulated association of the EHP segment with the ZP domain has been proposed to control the polymerization of ZP domain-containing proteins. Our findings suggest that this flexibility also extends to the ZP domain of TGFR-3 and might facilitate co-receptor ligand interaction and presentation via the adjacent FG-loop. This hints that a similar C-terminal region of the ZP domain architecture possibly regulates both the polymerization of extracellular matrix proteins and cytokine ligand recognition of TGFR-3. PMID:23826237

Structural and biochemical analysis of nuclease domain of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 3 (Cas3).

PubMed

Mulepati, Sabin; Bailey, Scott

2011-09-09

RNA transcribed from clustered regularly interspaced short palindromic repeats (CRISPRs) protects many prokaryotes from invasion by foreign DNA such as viruses, conjugative plasmids, and transposable elements. Cas3 (CRISPR-associated protein 3) is essential for this CRISPR protection and is thought to mediate cleavage of the foreign DNA through its N-terminal histidine-aspartate (HD) domain. We report here the 1.8 Å crystal structure of the HD domain of Cas3 from Thermus thermophilus HB8. Structural and biochemical studies predict that this enzyme binds two metal ions at its active site. We also demonstrate that the single-stranded DNA endonuclease activity of this T. thermophilus domain is activated not by magnesium but by transition metal ions such as manganese and nickel. Structure-guided mutagenesis confirms the importance of the metal-binding residues for the nuclease activity and identifies other active site residues. Overall, these results provide a framework for understanding the role of Cas3 in the CRISPR system.

1H, 15N and 13C assignments of domain 5 of Dictyostelium discoideum gelation factor (ABP-120) in its native and 8M urea-denatured states.

PubMed

Hsu, Shang-Te Danny; Cabrita, Lisa D; Christodoulou, John; Dobson, Christopher M

2009-06-01

The gelation factor from Dictyostelium discoideum (ABP-120) is an actin binding protein consisting of six immunoglobulin (Ig) domains in the C-terminal rod domain. We have recently used the pair of domains 5 and 6 of ABP-120 as a model system for studying multi-domain nascent chain folding on the ribosome. Here we present the NMR assignments of domain 5 in its native and 8M urea-denatured states.

Myosin 3A kinase activity is regulated by phosphorylation of the kinase domain activation loop.

PubMed

Quintero, Omar A; Unrath, William C; Stevens, Stanley M; Manor, Uri; Kachar, Bechara; Yengo, Christopher M

2013-12-27

Class III myosins are unique members of the myosin superfamily in that they contain both a motor and kinase domain. We have found that motor activity is decreased by autophosphorylation, although little is known about the regulation of the kinase domain. We demonstrate by mass spectrometry that Thr-178 and Thr-184 in the kinase domain activation loop and two threonines in the loop 2 region of the motor domain are autophosphorylated (Thr-908 and Thr-919). The kinase activity of MYO3A 2IQ with the phosphomimic (T184E) or phosphoblock (T184A) mutations demonstrates that kinase activity is reduced 30-fold as a result of the T184A mutation, although the Thr-178 site only had a minor impact on kinase activity. Interestingly, the actin-activated ATPase activity of MYO3A 2IQ is slightly reduced as a result of the T178A and T184A mutations suggesting coupling between motor and kinase domains. Full-length GFP-tagged T184A and T184E MYO3A constructs transfected into COS7 cells do not disrupt the ability of MYO3A to localize to filopodia structures. In addition, we demonstrate that T184E MYO3A reduces filopodia elongation in the presence of espin-1, whereas T184A enhances filopodia elongation in a similar fashion to kinase-dead MYO3A. Our results suggest that as MYO3A accumulates at the tips of actin protrusions, autophosphorylation of Thr-184 enhances kinase activity resulting in phosphorylation of the MYO3A motor and reducing motor activity. The differential regulation of the kinase and motor activities allows for MYO3A to precisely self-regulate its concentration in the actin bundle-based structures of cells.

Myosin 3A Kinase Activity Is Regulated by Phosphorylation of the Kinase Domain Activation Loop*

PubMed Central

Quintero, Omar A.; Unrath, William C.; Stevens, Stanley M.; Manor, Uri; Kachar, Bechara; Yengo, Christopher M.

2013-01-01

Class III myosins are unique members of the myosin superfamily in that they contain both a motor and kinase domain. We have found that motor activity is decreased by autophosphorylation, although little is known about the regulation of the kinase domain. We demonstrate by mass spectrometry that Thr-178 and Thr-184 in the kinase domain activation loop and two threonines in the loop 2 region of the motor domain are autophosphorylated (Thr-908 and Thr-919). The kinase activity of MYO3A 2IQ with the phosphomimic (T184E) or phosphoblock (T184A) mutations demonstrates that kinase activity is reduced 30-fold as a result of the T184A mutation, although the Thr-178 site only had a minor impact on kinase activity. Interestingly, the actin-activated ATPase activity of MYO3A 2IQ is slightly reduced as a result of the T178A and T184A mutations suggesting coupling between motor and kinase domains. Full-length GFP-tagged T184A and T184E MYO3A constructs transfected into COS7 cells do not disrupt the ability of MYO3A to localize to filopodia structures. In addition, we demonstrate that T184E MYO3A reduces filopodia elongation in the presence of espin-1, whereas T184A enhances filopodia elongation in a similar fashion to kinase-dead MYO3A. Our results suggest that as MYO3A accumulates at the tips of actin protrusions, autophosphorylation of Thr-184 enhances kinase activity resulting in phosphorylation of the MYO3A motor and reducing motor activity. The differential regulation of the kinase and motor activities allows for MYO3A to precisely self-regulate its concentration in the actin bundle-based structures of cells. PMID:24214986

C-Terminal Helical Domains of Dengue Virus Type 4 E Protein Affect the Expression/Stability of prM Protein and Conformation of prM and E Proteins

PubMed Central

Tsai, Wen-Yang; Hsieh, Szu-Chia; Lai, Chih-Yun; Lin, Hong-En; Nerurkar, Vivek R.; Wang, Wei-Kung

2012-01-01

Background The envelope (E) protein of dengue virus (DENV) is the major immunogen for dengue vaccine development. At the C-terminus are two α-helices (EH1 and EH2) and two transmembrane domains (ET1 and ET2). After synthesis, E protein forms a heterodimer with the precursor membrane (prM) protein, which has been shown as a chaperone for E protein and could prevent premature fusion of E protein during maturation. Recent reports of enhancement of DENV infectivity by anti-prM monoclonal antibodies (mAbs) suggest the presence of prM protein in dengue vaccine is potentially harmful. A better understanding of prM-E interaction and its effect on recognition of E and prM proteins by different antibodies would provide important information for future design of safe and effective subunit dengue vaccines. Methodology/Principal Findings In this study, we examined a series of C-terminal truncation constructs of DENV4 prME, E and prM. In the absence of E protein, prM protein expressed poorly. In the presence of E protein, the expression of prM protein increased in a dose-dependent manner. Radioimmunoprecipitation, sucrose gradient sedimentation and pulse-chase experiments revealed ET1 and EH2 were involved in prM-E interaction and EH2 in maintaining the stability of prM protein. Dot blot assay revealed E protein affected the recognition of prM protein by an anti-prM mAb; truncation of EH2 or EH1 affected the recognition of E protein by several anti-E mAbs, which was further verified by capture ELISA. The E protein ectodomain alone can be recognized well by all anti-E mAbs tested. Conclusions/Significance A C-terminal domain (EH2) of DENV E protein can affect the expression and stability of its chaperone prM protein. These findings not only add to our understanding of the interaction between prM and E proteins, but also suggest the ectodomain of E protein alone could be a potential subunit immunogen without inducing anti-prM response. PMID:23300717

Enhancement of Local Photovoltaic Current at Ferroelectric Domain Walls in BiFeO3.

PubMed

Yang, Ming-Min; Bhatnagar, Akash; Luo, Zheng-Dong; Alexe, Marin

2017-02-20

Domain walls, which are intrinsically two dimensional nano-objects exhibiting nontrivial electronic and magnetic behaviours, have been proven to play a crucial role in photovoltaic properties of ferroelectrics. Despite this recognition, the electronic properties of domain walls under illumination until now have been accessible only to macroscopic studies and their effects upon the conduction of photovoltaic current still remain elusive. The lack of understanding hinders the developing of nanoscale devices based on ferroelectric domain walls. Here, we directly characterize the local photovoltaic and photoconductive properties of 71° domain walls on BiFeO 3 thin films with a nanoscale resolution. Local photovoltaic current, proven to be driven by the bulk photovoltaic effect, has been probed over the whole illuminated surface by using a specially designed photoelectric atomic force microscopy and found to be significantly enhanced at domain walls. Additionally, spatially resolved photoconductive current distribution reveals a higher density of excited carriers at domain walls in comparison with domains. Our measurements demonstrate that domain wall enhanced photovoltaic current originates from its high conduction rather than the internal electric field. This photoconduction facilitated local photovoltaic current is likely to be a universal property of topological defects in ferroelectric semiconductors.

Stress induced modulation of magnetic domain diffraction of single crystalline yttrium iron garnet

NASA Astrophysics Data System (ADS)

Mito, Shinichiro; Yoshihara, Yuki; Takagi, Hiroyuki; Inoue, Mitsuteru

2018-05-01

Stress induced modulation of the diffraction angle and efficiency of the light reflected from a stripe-domain magnetic garnet was demonstrated. The spacing of the magnetic domain was changed using the inverse magnetostriction effect. The sample structure was a piezo actuator/Al reflection layer/magnetic garnet substrate. A diffraction angle between the 0th and 1st ordered light was changed from 9.12 deg. to 10.20 deg. This result indicates that the domain spacing was changed from 3.3 μm to 3.0 μm. The change of the diffraction angle was irreversible for the voltage. However, reversible, linear and continuous change of the diffraction efficiency was observed. These results could be applicable for a voltage-driven optical solid state light deflector with low power consumption and high switching speed.

A test of the domain-specific acculturation strategy hypothesis.

PubMed

Miller, Matthew J; Yang, Minji; Lim, Robert H; Hui, Kayi; Choi, Na-Yeun; Fan, Xiaoyan; Lin, Li-Ling; Grome, Rebekah E; Farrell, Jerome A; Blackmon, Sha'kema

2013-01-01

Acculturation literature has evolved over the past several decades and has highlighted the dynamic ways in which individuals negotiate experiences in multiple cultural contexts. The present study extends this literature by testing M. J. Miller and R. H. Lim's (2010) domain-specific acculturation strategy hypothesis-that individuals might use different acculturation strategies (i.e., assimilated, bicultural, separated, and marginalized strategies; J. W. Berry, 2003) across behavioral and values domains-in 3 independent cluster analyses with Asian American participants. Present findings supported the domain-specific acculturation strategy hypothesis as 67% to 72% of participants from 3 independent samples using different strategies across behavioral and values domains. Consistent with theory, a number of acculturation strategy cluster group differences emerged across generational status, acculturative stress, mental health symptoms, and attitudes toward seeking professional psychological help. Study limitations and future directions for research are discussed.

Transduction of Recombinant M3-p53-R12 Protein Enhances Human Leukemia Cell Apoptosis

PubMed Central

Lu, Tsung Chi; Zhao, Guan- Hao; Chen, Yao Yun; Chien, Chia-Ying; Huang, Chi-Hung; Lin, Kwang Hui; Chen, Shen Liang

2016-01-01

Tumor suppressor protein p53 plays important roles in initiating cell cycle arrest and promoting tumor cell apoptosis. Previous studies have shown that p53 is either mutated or defective in approximately 50% of human cancers; therefore restoring normal p53 activity in cancer cells might be an effective anticancer therapeutic approach. Herein, we designed a chimeric p53 protein flanked with the MyoD N-terminal transcriptional activation domain (amino acids 1-62, called M3) and a poly-arginine (R12) cell penetrating signal in its N-and C-termini respectively. This chimeric protein, M3-p53-R12, can be expressed in E. coli and purified using immobilized metal ion chromatography followed by serial refolding dialysis. The purified M3-p53-R12 protein retains DNA-binding activity and gains of cell penetrating ability. Using MTT assay, we demonstrated that M3-p53-R12 inhibited the growth of K562, Jurkat as well as HL-60 leukemia cells carrying mutant p53 genes. Results from FACS analysis also demonstrated that transduction of M3-p53-R12 protein induced cell cycle arrest of these leukemia cells. Of special note, M3-p53-R12 has no apoptotic effect on normal mesenchymal stem cells (MSC) and leukocytes, highlighting its differential effects on normal and tumor cells. To sum up, our results reveal that purified recombinant M3-p53-R12 protein has functions of suppressing the leukemia cell lines' proliferation and launching cell apoptosis, suggesting the feasibility of using M3-p53-R12 protein as an anticancer drug. In the future we will test whether this chimeric protein can preferentially trigger the death of malignant cancer cells without affecting normal cells in animals carrying endogenous or xenographic tumors. PMID:27390612

The D0 immunoglobulin-like domain plays a central role for the stronger binding of KIR3DL2 to B27 free heavy chain dimers

PubMed Central

Hatano, Hiroko; Shaw, Jacqueline; Marquardt, Kaitlin; Zhang, Zhiyong; Gauthier, Laurent; Chanteux, Stephanie; Rossi, Benjamin; Li, Demin; Mitchell, Julie; Kollnberger, Simon

2015-01-01

We have proposed that the killer cell immunoglobulin-like receptor KIR3DL2 binding more strongly to HLA-B27 (B27) β2m-free heavy chain (FHC) dimers regulates lymphocyte function in arthritis and infection. We compared the function of B27 FHC dimers with other class I heavy chains and identified contact residues in KIR3DL2. B27 FHC dimers interacted functionally with KIR3DL2 on NK and reporter cells more strongly than other class I FHC. Mutagenesis identified key residues in the D0 and other immunoglobulin-like domains which were shared and distinct from KIR3DL1, for KIR3DL2 binding to B27 and other class I FHC. We modeled B27 dimer binding to KIR3DL2 and compared experimental mutagenesis data with computational “hot spot” predictions. Modelling predicts the stronger binding of B27 dimers to KIR3DL2 is mediated by non-symmetrical complementary contacts of the D0 and D1 domains with the α1, α2 and α3 domains of both B27 heavy chains. By contrast, the D2 domain primarily contacts residues in the α2 domain of one B27 heavy chain. These findings both provide novel insights about the molecular basis of KIR3DL2 binding to HLA-B27 and other ligands and suggest an important role for KIR3DL2 HLA-B27 interactions in controlling the function of NK cells in HLA-B27+ individuals. PMID:25582852

Effect of the SH3-SH2 domain linker sequence on the structure of Hck kinase.

PubMed

Meiselbach, Heike; Sticht, Heinrich

2011-08-01

The coordination of activity in biological systems requires the existence of different signal transduction pathways that interact with one another and must be precisely regulated. The Src-family tyrosine kinases, which are found in many signaling pathways, differ in their physiological function despite their high overall structural similarity. In this context, the differences in the SH3-SH2 domain linkers might play a role for differential regulation, but the structural consequences of linker sequence remain poorly understood. We have therefore performed comparative molecular dynamics simulations of wildtype Hck and of a mutant Hck in which the SH3-SH2 domain linker is replaced by the corresponding sequence from the homologous kinase Lck. These simulations reveal that linker replacement not only affects the orientation of the SH3 domain itself, but also leads to an alternative conformation of the activation segment in the Hck kinase domain. The sequence of the SH3-SH2 domain linker thus exerts a remote effect on the active site geometry and might therefore play a role in modulating the structure of the inactive kinase or in fine-tuning the activation process itself.

Structural insights into the specific binding of huntingtin proline-rich region with the SH3 and WW domains.

PubMed

Gao, Yong-Guang; Yan, Xian-Zhong; Song, Ai-Xin; Chang, Yong-Gang; Gao, Xue-Chao; Jiang, Nan; Zhang, Qi; Hu, Hong-Yu

2006-12-01

The interactions of huntingtin (Htt) with the SH3 domain- or WW domain-containing proteins have been implicated in the pathogenesis of Huntington's disease (HD). We report the specific interactions of Htt proline-rich region (PRR) with the SH3GL3-SH3 domain and HYPA-WW1-2 domain pair by NMR. The results show that Htt PRR binds with the SH3 domain through nearly its entire chain, and that the binding region on the domain includes the canonical PxxP-binding site and the specificity pocket. The C terminus of PRR orients to the specificity pocket, whereas the N terminus orients to the PxxP-binding site. Htt PRR can also specifically bind to WW1-2; the N-terminal portion preferentially binds to WW1, while the C-terminal portion binds to WW2. This study provides structural insights into the specific interactions between Htt PRR and its binding partners as well as the alteration of these interactions that involve PRR, which may have implications for the understanding of HD.

Drosophila CTCF tandemly aligns with other insulator proteins at the borders of H3K27me3 domains

PubMed Central

Van Bortle, Kevin; Ramos, Edward; Takenaka, Naomi; Yang, Jingping; Wahi, Jessica E.; Corces, Victor G.

2012-01-01

Several multiprotein DNA complexes capable of insulator activity have been identified in Drosophila melanogaster, yet only CTCF, a highly conserved zinc finger protein, and the transcription factor TFIIIC have been shown to function in mammals. CTCF is involved in diverse nuclear activities, and recent studies suggest that the proteins with which it associates and the DNA sequences that it targets may underlie these various roles. Here we show that the Drosophila homolog of CTCF (dCTCF) aligns in the genome with other Drosophila insulator proteins such as Suppressor of Hairy wing [SU(HW)] and Boundary Element Associated Factor of 32 kDa (BEAF-32) at the borders of H3K27me3 domains, which are also enriched for associated insulator proteins and additional cofactors. RNAi depletion of dCTCF and combinatorial knockdown of gene expression for other Drosophila insulator proteins leads to a reduction in H3K27me3 levels within repressed domains, suggesting that insulators are important for the maintenance of appropriate repressive chromatin structure in Polycomb (Pc) domains. These results shed new insights into the roles of insulators in chromatin domain organization and support recent models suggesting that insulators underlie interactions important for Pc-mediated repression. We reveal an important relationship between dCTCF and other Drosophila insulator proteins and speculate that vertebrate CTCF may also align with other nuclear proteins to accomplish similar functions. PMID:22722341

Up-regulation of Hyperpolarization-activated Cyclic Nucleotide-gated Channel 3 (HCN3) by Specific Interaction with K+ Channel Tetramerization Domain-containing Protein 3 (KCTD3)*

PubMed Central

Cao-Ehlker, Xiaochun; Zong, Xiangang; Hammelmann, Verena; Gruner, Christian; Fenske, Stefanie; Michalakis, Stylianos; Wahl-Schott, Christian; Biel, Martin

2013-01-01

Most ion channels consist of the principal ion-permeating core subunit(s) and accessory proteins that are assembled with the channel core. The biological functions of the latter proteins are diverse and include the regulation of the biophysical properties of the ion channel, its connection to signaling pathways and the control of its cell surface expression. There is recent evidence that native hyperpolarization-activated cyclic nucleotide-gated channel complexes (HCN1–4) also contain accessory subunits, among which TRIP8b (tetratricopeptide repeat-containing Rab8b-interacting protein) has been most extensively studied. Here, we identify KCTD3, a so far uncharacterized member of the potassium channel tetramerization-domain containing (KCTD) protein family as an HCN3-interacting protein. KCTD3 is widely expressed in brain and some non-neuronal tissues and colocalizes with HCN3 in specific regions of the brain including hypothalamus. Within the HCN channel family, KCTD3 specifically binds to HCN3 and leads to a profound up-regulation of cell surface expression and current density of this channel. HCN3 can also functionally interact with TRIP8b; however, we found no evidence for channel complexes containing both TRIP8b and KCTD3. The C terminus of HCN3 is crucially required for functional interaction with KCTD3. Replacement of the cytosolic C terminus of HCN2 by the corresponding domain of HCN3 renders HCN2 sensitive to regulation by KCTD3. The C-terminal-half of KCTD3 is sufficient for binding to HCN3. However, the complete protein including the N-terminal tetramerization domain is needed for HCN3 current up-regulation. Together, our experiments indicate that KCTD3 is an accessory subunit of native HCN3 complexes. PMID:23382386

M2-F3 on lakebed

NASA Image and Video Library

1970-06-19

The M2-F3 Lifting Body is seen here on the lakebed next to the NASA Flight Research Center (later the Dryden Flight Research Center), Edwards, California. Redesigned and rebuilt from the M2-F2, the M2-F3 featured as its most visible change a center fin for greater stability. While the M2-F3 was still demanding to fly, the center fin eliminated the high risk of pilot induced oscillation (PIO) that was characteristic of the M2-F2.

Chromatoid Body Protein TDRD6 Supports Long 3’ UTR Triggered Nonsense Mediated mRNA Decay

PubMed Central

Fanourgakis, Grigorios; Akpinar, Müge; Dahl, Andreas; Jessberger, Rolf

2016-01-01

Chromatoid bodies (CBs) are spermiogenesis-specific organelles of largely unknown function. CBs harbor various RNA species, RNA-associated proteins and proteins of the tudor domain family like TDRD6, which is required for a proper CB architecture. Proteome analysis of purified CBs revealed components of the nonsense-mediated mRNA decay (NMD) machinery including UPF1. TDRD6 is essential for UPF1 localization to CBs, for UPF1-UPF2 and UPF1-MVH interactions. Upon removal of TDRD6, the association of several mRNAs with UPF1 and UPF2 is disturbed, and the long 3’ UTR-stimulated but not the downstream exon-exon junction triggered pathway of NMD is impaired. Reduced association of the long 3’ UTR mRNAs with UPF1 and UPF2 correlates with increased stability and enhanced translational activity. Thus, we identified TDRD6 within CBs as required for mRNA degradation, specifically the extended 3’ UTR-triggered NMD pathway, and provide evidence for the requirement of NMD in spermiogenesis. This function depends on TDRD6-promoted assembly of mRNA and decay enzymes in CBs. PMID:27149095

Domain wall magnetoresistance in BiFeO3 thin films measured by scanning probe microscopy

NASA Astrophysics Data System (ADS)

Domingo, N.; Farokhipoor, S.; Santiso, J.; Noheda, B.; Catalan, G.

2017-08-01

We measure the magnetotransport properties of individual 71° domain walls in multiferroic BiFeO3 by means of conductive—atomic force microscopy (C-AFM) in the presence of magnetic fields up to one Tesla. The results suggest anisotropic magnetoresistance at room temperature, with the sign of the magnetoresistance depending on the relative orientation between the magnetic field and the domain wall plane. A consequence of this finding is that macroscopically averaged magnetoresistance measurements for domain wall bunches are likely to underestimate the magnetoresistance of each individual domain wall.

Molecular dynamic simulation of mGluR5 amino terminal domain: essential dynamics analysis captures the agonist or antagonist behaviour of ligands.

PubMed

Casoni, Alessandro; Clerici, Francesca; Contini, Alessandro

2013-04-01

We describe the application of molecular dynamics followed by principal component analysis to study the inter-domain movements of the ligand binding domain (LBD) of mGluR5 in response to the binding of selected agonists or antagonists. Our results suggest that the method is an attractive alternative to current approaches to predict the agonist-induced or antagonist-blocked LBD responses. The ratio between the eigenvalues of the first and second eigenvectors (R1,2) is also proposed as a numerical descriptor for discriminating the ligand behavior as a mGluR5 agonist or antagonist. Copyright © 2013 Elsevier Inc. All rights reserved.

Phospholipase C-independent effects of 3M3FBS in murine colon.

PubMed

Dwyer, Laura; Kim, Hyun Jin; Koh, Byoung Ho; Koh, Sang Don

2010-02-25

The muscarinic receptor subtype M(3) is coupled to Gq/11 proteins. Muscarinic receptor agonists such as carbachol stimulate these receptors that result in activation of phospholipase C (PLC) which hydrolyzes phosphatidylinositol 4,5-bisphosphate into diacylglycerol and Ins(1,4,5)P(3). This pathway leads to excitation and smooth muscle contraction. In this study the PLC agonist, 2, 4, 6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benezenesulfonamide (m-3M3FBS), was used to investigate whether direct PLC activation mimics carbachol-induced excitation. We examined the effects of m-3M3FBS and 2, 4, 6-trimethyl-N-(ortho-3-trifluoromethyl-phenyl)-benzenesulfonamide (o-3M3FBS), on murine colonic smooth muscle tissue and cells by performing conventional microelectrode recordings, isometric force measurements and patch clamp experiments. Application of m-3M3FBS decreased spontaneous contractility in murine colonic smooth muscle without affecting the resting membrane potential. Patch clamp studies revealed that delayed rectifier K(+) channels were reversibly inhibited by m-3M3FBS and o-3M3FBS. The PLC inhibitor, 1-(6-((17b-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), did not prevent this inhibition by m-3M3FBS. Both m-3M3FBS and o-3M3FBS decreased two components of delayed rectifier K(+) currents in the presence of tetraethylammonium chloride or 4-aminopyridine. Ca(2+) currents were significantly suppressed by m-3M3FBS and o-3M3FBS with a simultaneous increase in intracellular Ca(2+). Pretreatment with U73122 did not prevent the decrease in Ca(2+) currents upon m-3M3FBS application. In conclusion, both m-3M3FBS and o-3M3FBS inhibit inward and outward currents via mechanisms independent of PLC acting in an antagonistic manner. In contrast, both compounds also caused an increase in [Ca(2+)](i) in an agonistic manner. Therefore caution must be employed when interpreting their effects at the tissue and cellular level.

2-μm optical time domain reflectometry measurements from novel Al-, Ge-, CaAlSi- doped and standard single-mode fibers

NASA Astrophysics Data System (ADS)

Rodriguez-Novelo, J. C.; Sanchez-Nieves, J. A.; Sierra-Calderon, A.; Sanchez-Lara, R.; Alvarez-Chavez, J. A.

2017-08-01

The development of novel Al-, Ge- doped and un-doped standard single mode fibers for future optical communication at 2μm requires the integration of, among other pieces of equipment, an optical time domain reflectometry (OTDR) technique for precise spectral attenuation characterization, including the well-known cut-back method. The integration of a state of the art OTDR at 2μm could provide valuable attenuation information from the aforementioned novel fibers. The proposed setup consists of a 1.7 mW, 1960nm pump source, a 30 dB gain Thulium doped fibre amplifier at 2μm, an 0.8mm focal length lens with a 0.5 NA, a 30 MHz acusto-optic modulator, a 3.1 focal length lens with a 0.68NA, an optical circulator at 2μm, an InGaAs photodetector for 1.2 nm-2.6 nm range, a voltage amplifier and an oscilloscope. The propagated pulse rate is 50 KHz, with 500 ns, 200 ns, 100 ns and 50 ns pulse widths. Attenuation versus novel fibers types for lengths ranging from 400- to 1000- meter samples were obtained using the proposed setup.

Chemical composition of stars in globular clusters: M5, M13, and M3

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

Pilachowski, C.A.; Wallerstein, G.; Myckky Leep, E.

The composition of giant stars in the globular clusters M3, M5, and M13 have been determined by detailed model atmosphere analysis; the average iron deficiencies relative to the Sun are -1.55, -1.33, and -1.42, respectively. Oxygen is overabundant relative to iron in M3 and M5 but not in M13. Differences in the oxygen abundance can account for variation in horizontal-branch morphology; clusters of intermediate metallicity with high oxygen have red horizontal-branch stars, and those with low oxygen have only blue horizontal-branch stars Z/sub cno/ is the second abundance parameter.

Laplace-domain waveform modeling and inversion for the 3D acoustic-elastic coupled media