Developing EAL Learners' Intercultural Sensitivity through a Digital Literacy Project

ERIC Educational Resources Information Center

Galante, Angelica

2014-01-01

Language and culture are informally integrated in many English as an Additional Language (EAL) programs, but cultural discussions are often regarded from the perspective of a particular dominant culture. Although this integration is crucial for the development of communicative competence, practical applications are still challenging as language…

Developing EAL Learners' Science Conceptual Understanding through Visualisation

ERIC Educational Resources Information Center

Hainsworth, Mark

2017-01-01

Science can be a difficult subject for EAL (English as an Additional Language) learners to master, mainly because of the prominent role that language plays in the acquisition of scientific concepts. Language is essential to science because it is the means by which we envisage and communicate new ideas. Teaching this new language to pupils involves…

How Does the Use of Role-Play Affect the Learning of Year 4 Children in a Predominately EAL Class?

ERIC Educational Resources Information Center

Grant, Katrina; Mistry, Malini Tina

2010-01-01

This paper investigates role-play activities and how they can be used within learning for English as an Additional Language (EAL) pupils in a Key Stage 2 setting in England. Through observations, results showed effective role-play activities can be beneficial to EAL pupils, allowing them to practice words and phrases in a relaxed atmosphere…

Comparative analysis of diguanylate cyclase and phosphodiesterase genes in Klebsiella pneumoniae.

PubMed

Cruz, Diana P; Huertas, Mónica G; Lozano, Marcela; Zárate, Lina; Zambrano, María Mercedes

2012-07-09

Klebsiella pneumoniae can be found in environmental habitats as well as in hospital settings where it is commonly associated with nosocomial infections. One of the factors that contribute to virulence is its capacity to form biofilms on diverse biotic and abiotic surfaces. The second messenger Bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) is a ubiquitous signal in bacteria that controls biofilm formation as well as several other cellular processes. The cellular levels of this messenger are controlled by c-di-GMP synthesis and degradation catalyzed by diguanylate cyclase (DGC) and phophodiesterase (PDE) enzymes, respectively. Many bacteria contain multiple copies of these proteins with diverse organizational structure that highlight the complex regulatory mechanisms of this signaling network. This work was undertaken to identify DGCs and PDEs and analyze the domain structure of these proteins in K. pneumoniae. A search for conserved GGDEF and EAL domains in three sequenced K. pneumoniae genomes showed that there were multiple copies of GGDEF and EAL containing proteins. Both single domain and hybrid GGDEF proteins were identified: 21 in K. pneumoniae Kp342, 18 in K. pneumoniae MGH 78578 and 17 in K. pneumoniae NTUH-K2044. The majority had only the GGDEF domain, most with the GGEEF motif, and hybrid proteins containing both GGDEF and EAL domains were also found. The I site for allosteric control was identified only in single GGDEF domain proteins and not in hybrid proteins. EAL-only proteins, containing either intact or degenerate domains, were also identified: 15 in Kp342, 15 in MGH 78578 and 10 in NTUH-K2044. Several input sensory domains and transmembrane segments were identified, which together indicate complex regulatory circuits that in many cases can be membrane associated. The comparative analysis of proteins containing GGDEF/EAL domains in K. pneumoniae showed that most copies were shared among the three strains and that some were unique to a particular strain

Complex regulatory network encompassing the Csr, c-di-GMP and motility systems of Salmonella Typhimurium.

PubMed

Jonas, Kristina; Edwards, Adrianne N; Ahmad, Irfan; Romeo, Tony; Römling, Ute; Melefors, Ojar

2010-02-01

Bacterial survival depends on the ability to switch between sessile and motile lifestyles in response to changing environmental conditions. In many species, this switch is governed by (3'-5')-cyclic-diguanosine monophosphate (c-di-GMP), a signalling molecule, which is metabolized by proteins containing GGDEF and/or EAL domains. Salmonella Typhimurium contains 20 such proteins. Here, we show that the RNA-binding protein CsrA regulates the expression of eight genes encoding GGDEF, GGDEF-EAL and EAL domain proteins. CsrA bound directly to the mRNA leaders of five of these genes, suggesting that it may regulate these genes post-transcriptionally. The c-di-GMP-specific phosphodiesterase STM3611, which reciprocally controls flagella function and production of biofilm matrix components, was regulated by CsrA binding to the mRNA, but was also indirectly regulated by CsrA through the FlhDC/FliA flagella cascade and STM1344. STM1344 is an unconventional (c-di-GMP-inactive) EAL domain protein, recently identified as a negative regulator of flagella gene expression. Here, we demonstrate that CsrA directly downregulates expression of STM1344, which in turn regulates STM3611 through fliA and thus reciprocally controls motility and biofilm factors. Altogether, our data reveal that the concerted and complex regulation of several genes encoding GGDEF/EAL domain proteins allows CsrA to control the motility-sessility switch in S. Typhimurium at multiple levels.

Documentation for a Structural Optimization Procedure Developed Using the Engineering Analysis Language (EAL)

NASA Technical Reports Server (NTRS)

Martin, Carl J., Jr.

1996-01-01

This report describes a structural optimization procedure developed for use with the Engineering Analysis Language (EAL) finite element analysis system. The procedure is written primarily in the EAL command language. Three external processors which are written in FORTRAN generate equivalent stiffnesses and evaluate stress and local buckling constraints for the sections. Several built-up structural sections were coded into the design procedures. These structural sections were selected for use in aircraft design, but are suitable for other applications. Sensitivity calculations use the semi-analytic method, and an extensive effort has been made to increase the execution speed and reduce the storage requirements. There is also an approximate sensitivity update method included which can significantly reduce computational time. The optimization is performed by an implementation of the MINOS V5.4 linear programming routine in a sequential liner programming procedure.

Negotiating Contradictions in Developing Teacher Identity during the EAL Practicum in Australia

ERIC Educational Resources Information Center

Nguyen, Minh Hue

2017-01-01

This paper reports on how Maria (pseudonym), a non-native English speaker (NNES) and preservice teacher (PST) of English as an additional language (EAL), developed her professional identity during the practicum in an Australian secondary school. Drawing on activity theory, the study identified contradictions in Maria's practicum activity and…

Creating Inclusive EAL Classrooms: How Language Instruction for Newcomers to Canada (LINC) Instructors Understand and Mitigate Barriers for Students Who Have Experienced Trauma

ERIC Educational Resources Information Center

Wilbur, Amea

2016-01-01

This article draws on my dissertation, "Creating Inclusive EAL Classrooms: How LINC Instructors Understand and Mitigate Barriers for Students Who Have Experienced Trauma." The article explores some assumptions and understandings that English as an Additional Language (EAL) teachers bring to teaching students believed to have experienced…

Genome-Based Comparison of Cyclic Di-GMP Signaling in Pathogenic and Commensal Escherichia coli Strains

PubMed Central

Povolotsky, Tatyana L.

2015-01-01

ABSTRACT The ubiquitous bacterial second messenger cyclic di-GMP (c-di-GMP) has recently become prominent as a trigger for biofilm formation in many bacteria. It is generated by diguanylate cyclases (DGCs; with GGDEF domains) and degraded by specific phosphodiesterases (PDEs; containing either EAL or HD-GYP domains). Most bacterial species contain multiples of these proteins with some having specific functions that are based on direct molecular interactions in addition to their enzymatic activities. Escherichia coli K-12 laboratory strains feature 29 genes encoding GGDEF and/or EAL domains, resulting in a set of 12 DGCs, 13 PDEs, and four enzymatically inactive “degenerate” proteins that act by direct macromolecular interactions. We present here a comparative analysis of GGDEF/EAL domain-encoding genes in 61 genomes of pathogenic, commensal, and probiotic E. coli strains (including enteric pathogens such as enteroaggregative, enterohemorrhagic, enteropathogenic, enterotoxigenic, and adherent and invasive Escherichia coli and the 2011 German outbreak O104:H4 strain, as well as extraintestinal pathogenic E. coli, such as uropathogenic and meningitis-associated E. coli). We describe additional genes for two membrane-associated DGCs (DgcX and DgcY) and four PDEs (the membrane-associated PdeT, as well as the EAL domain-only proteins PdeW, PdeX, and PdeY), thus showing the pangenome of E. coli to contain at least 35 GGDEF/EAL domain proteins. A core set of only eight proteins is absolutely conserved in all 61 strains: DgcC (YaiC), DgcI (YliF), PdeB (YlaB), PdeH (YhjH), PdeK (YhjK), PdeN (Rtn), and the degenerate proteins CsrD and CdgI (YeaI). In all other GGDEF/EAL domain genes, diverse point and frameshift mutations, as well as small or large deletions, were discovered in various strains. IMPORTANCE Our analysis reveals interesting trends in pathogenic Escherichia coli that could reflect different host cell adherence mechanisms. These may either benefit from or be

Genome-Based Comparison of Cyclic Di-GMP Signaling in Pathogenic and Commensal Escherichia coli Strains.

PubMed

Povolotsky, Tatyana L; Hengge, Regine

2016-01-01

The ubiquitous bacterial second messenger cyclic di-GMP (c-di-GMP) has recently become prominent as a trigger for biofilm formation in many bacteria. It is generated by diguanylate cyclases (DGCs; with GGDEF domains) and degraded by specific phosphodiesterases (PDEs; containing either EAL or HD-GYP domains). Most bacterial species contain multiples of these proteins with some having specific functions that are based on direct molecular interactions in addition to their enzymatic activities. Escherichia coli K-12 laboratory strains feature 29 genes encoding GGDEF and/or EAL domains, resulting in a set of 12 DGCs, 13 PDEs, and four enzymatically inactive "degenerate" proteins that act by direct macromolecular interactions. We present here a comparative analysis of GGDEF/EAL domain-encoding genes in 61 genomes of pathogenic, commensal, and probiotic E. coli strains (including enteric pathogens such as enteroaggregative, enterohemorrhagic, enteropathogenic, enterotoxigenic, and adherent and invasive Escherichia coli and the 2011 German outbreak O104:H4 strain, as well as extraintestinal pathogenic E. coli, such as uropathogenic and meningitis-associated E. coli). We describe additional genes for two membrane-associated DGCs (DgcX and DgcY) and four PDEs (the membrane-associated PdeT, as well as the EAL domain-only proteins PdeW, PdeX, and PdeY), thus showing the pangenome of E. coli to contain at least 35 GGDEF/EAL domain proteins. A core set of only eight proteins is absolutely conserved in all 61 strains: DgcC (YaiC), DgcI (YliF), PdeB (YlaB), PdeH (YhjH), PdeK (YhjK), PdeN (Rtn), and the degenerate proteins CsrD and CdgI (YeaI). In all other GGDEF/EAL domain genes, diverse point and frameshift mutations, as well as small or large deletions, were discovered in various strains. Our analysis reveals interesting trends in pathogenic Escherichia coli that could reflect different host cell adherence mechanisms. These may either benefit from or be counteracted by the c

Mesodomain and Protein-Associated Solvent Phases with Temperature-Tunable (200-265 K) Dynamics Surround Ethanolamine Ammonia-Lyase in Globally Polycrystalline Aqueous Solution Containing Dimethyl Sulfoxide.

PubMed

Nforneh, Benjamen; Warncke, Kurt

2017-12-14

Electron paramagnetic resonance spectroscopy of the spin probe, TEMPOL, is used to resolve solvent phases that surround the ethanolamine ammonia-lyase (EAL) protein from Salmonella typhimurium at low temperature (T) in frozen, globally polycrystalline aqueous solution and to report on the T dependence of their detectably rigid and fluid states. EAL plays a role in human gut microbiome-based disease conditions, and physicochemical studies provide insight into protein structure and mechanism, toward potential therapeutics. Temperature dependences of the rotational correlation times (τ c ; detection range, 10 -11 ≤ τ c ≤ 10 -7 s) and the corresponding weights of TEMPOL tumbling components from 200 to 265 K in the presence of EAL are measured in two frozen systems: (1) water-only and (2) 1% v/v dimethyl sulfoxide (DMSO). In the water-only condition, a protein-vicinal solvent component detectably fluidizes at 230 K and melts the surrounding ice-crystalline region with increasing T, creating a bounded, relatively high-viscosity aqueous solvent domain, up to 265 K. In the EAL, 1% v/v DMSO condition, two distinct concentric solvent phases are resolved around EAL: protein-associated domain (PAD) and mesodomain. The DMSO aqueous mesodomain fluidizes at 200 K, followed by PAD fluidization at 210 K. The interphase dynamical coupling is consistent with the spatial arrangement and significant contact areas of the phases, indicated by the experimentally determined mean volume ratio, V(mesodomain)/V(PAD)/V(protein) = 0.5:0.3:1.0. The results provide a rationale for native chemical reactions of EAL at T < 250 K and an advance toward precise control of solvent dynamics as a tunable parameter for quantifying the coupling between solvent and protein fluctuations and chemical reaction steps in EAL and other enzymes.

The Use of Makaton for Supporting Talk, through Play, for Pupils Who have English as an Additional Language (EAL) in the Foundation Stage

ERIC Educational Resources Information Center

Mistry, Malini; Barnes, Danielle

2013-01-01

This study examines the use of Makaton® , a language programme based on the use of signing, symbols and speech, as a pedagogic tool to support the development of talk for pupils learning English as an Additional Language (EAL). The research setting was a Reception class with a high percentage of pupils who have EAL in the initial stages of…

2015 EAL evidence-based nutrition practice guildeline for the management of hypertension in adults

USDA-ARS?s Scientific Manuscript database

Hypertension (HTN) or high blood pressure (BP) is one of the most prevalent forms of cardiovascular disease and occurs in approximately one of every three adults in the United States. Nutrition therapy is an important component of treatment. The purpose of this Evidence Analysis Library (EAL) gui...

Poly(A) polymerase contains multiple functional domains.

PubMed Central

Raabe, T; Murthy, K G; Manley, J L

1994-01-01

Poly(A) polymerase (PAP) contains regions of similarity with several known protein domains. Through site-directed mutagenesis, we provide evidence that PAP contains a functional ribonucleoprotein-type RNA binding domain (RBD) that is responsible for primer binding, making it the only known polymerase to contain such a domain. The RBD is adjacent to, and probably overlaps with, an apparent catalytic region responsible for polymerization. Despite the presence of sequence similarities, this catalytic domain appears to be distinct from the conserved polymerase module found in a large number of RNA-dependent polymerases. PAP contains two nuclear localization signals (NLSs) in its C terminus, each by itself similar to the consensus bipartite NLS found in many nuclear proteins. Mutagenesis experiments indicate that both signals, which are separated by nearly 140 residues, play important roles in directing PAP exclusively to the nucleus. Surprisingly, basic amino acids in the N-terminal-most NLS are also essential for AAUAAA-dependent polyadenylation but not for nonspecific poly(A) synthesis, suggesting that this region of PAP is involved in interactions both with nuclear targeting proteins and with nuclear polyadenylation factors. The serine/threonine-rich C terminus is multiply phosphorylated, including at sites affected by mutations in either NLS. Images PMID:8164653

Applied Linguistics Project: Student-Led Computer Assisted Research in High School EAL/EAP

ERIC Educational Resources Information Center

Bohát, Róbert; Rödlingová, Beata; Horáková, Nina

2015-01-01

The Applied Linguistics Project (ALP) started at the International School of Prague (ISP) in 2013. Every year, Grade 9 English as an Additional Language (EAL) students identify an area of learning in need of improvement and design a research method followed by data collection and analysis using basic computer software tools or online corpora.…

Scaffolding during the Formal Assessment of Young EAL Learners: A New Zealand Case Study

ERIC Educational Resources Information Center

Booth, Dawn

2012-01-01

Despite growing interest in the theorisation of teacher-based assessment (TBA), very little research has paid close attention to how teachers practice assessment embedded in real classroom contexts. This longitudinal study over one school term reports on the TBA of young learners with English as an additional language (EAL) in New Zealand primary…

[Family of ribosomal proteins S1 contains unique conservative domain].

PubMed

Deriusheva, E I; Machulin, A V; Selivanova, O M; Serdiuk, I N

2010-01-01

Different representatives of bacteria have different number of amino acid residues in the ribosomal proteins S1. This number varies from 111 (Spiroplasma kunkelii) to 863 a.a. (Treponema pallidum). Traditionally and for lack of this protein three-dimensional structure, its architecture is represented as repeating S1 domains. Number of these domains depends on the protein's length. Domain's quantity and its boundaries data are contained in the specialized databases, such as SMART, Pfam and PROSITE. However, for the same object these data may be very different. For search of domain's quantity and its boundaries, new approach, based on the analysis of dicted secondary structure (PsiPred), was used. This approach allowed us to reveal structural domains in amino acid sequences of S1 proteins and at that number varied from one to six. Alignment of S1 proteins, containing different domain's number, with the S1 RNAbinding domain of Escherichia coli PNPase elicited a fact that in family of ribosomal proteins SI one domain has maximal homology with S1 domain from PNPase. This conservative domain migrates along polypeptide chain and locates in proteins, containing different domain's number, according to specified pattern. In this domain as well in the S1 domain from PNPase, residues Phe-19, Phe-22, His-34, Asp-64 and Arg-68 are clustered on the surface and formed RNA binding site.

A Healing Space: The Experiences of First Nations and Inuit Youth with Equine-Assisted Learning (EAL)

ERIC Educational Resources Information Center

Dell, Colleen Anne; Chalmers, Darlene; Bresette, Nora; Swain, Sue; Rankin, Deb; Hopkins, Carol

2011-01-01

The Nimkee NupiGawagan Healing Centre (NNHC) in Muncey, ON provides residential treatment to First Nations and Inuit youth who abuse solvents. As a complement to its culture-based programming, in 2008 the centre began offering weekly equine-assisted learning (EAL) curriculum to its clients in partnership with the Keystone Equine Centre and the…

An Exploration of School Communication Approaches for Newly Arrived EAL Students: Applying Three Dimensions of Organisational Communication Theory

ERIC Educational Resources Information Center

Schneider, Claudia; Arnot, Madeleine

2018-01-01

This article explores the modes of school communication associated with language and cultural diversity, demonstrating how organisational communication theory can be applied to the analysis of schools' communication responses to the presence of pupils who have English as an additional language (EAL). The article highlights three analytical…

In silico analysis of Schmidtea mediterranea TIR domain-containing proteins.

PubMed

Tsoumtsa, Landry Laure; Sougoufora, Seynabou; Torre, Cedric; Lemichez, Emmanuel; Pontarotti, Pierre; Ghigo, Eric

2018-09-01

While genetic evidence points towards an absence of Toll-Like Receptors (TLRs) in Platyhelminthes, the Toll/IL-1 Receptor (TIR)-domains that drive the assembly of signalling complexes downstream TLR are present in these organisms. Here, we undertook the characterisation of the repertoire of TIR-domain containing proteins in Schmidtea mediterranea in order to gain valuable information on TLR evolution in metazoan. We report the presence of twenty proteins containing between one and two TIR domains. In addition, our phylogenetic-based reconstruction approach identified Smed-SARM and Smed-MyD88 as conserved TLR adaptors. Copyright © 2018 Elsevier Ltd. All rights reserved.

Holographic imaging based on time-domain data of natural-fiber-containing materials

DOEpatents

Bunch, Kyle J.; McMakin, Douglas L.

2012-09-04

Methods and apparatuses for imaging material properties in natural-fiber-containing materials can utilize time-domain data. In particular, images can be constructed that provide quantified measures of localized moisture content. For example, one or more antennas and at least one transceiver can be configured to collect time-domain data from radiation interacting with the natural-fiber-containing materials. The antennas and the transceivers are configured to transmit and receive electromagnetic radiation at one or more frequencies, which are between 50 MHz and 1 THz, according to a time-domain impulse function. A computing device is configured to transform the time-domain data to frequency-domain data, to apply a synthetic imaging algorithm for constructing a three-dimensional image of the natural-fiber-containing materials, and to provide a quantified measure of localized moisture content based on a pre-determined correlation of moisture content to frequency-domain data.

Cloning and characterization of a novel human STAR domain containing cDNA KHDRBS2.

PubMed

Wang, Liu; Xu, Jian; Zeng, Li; Ye, Xin; Wu, Qihan; Dai, Jianfeng; Ji, Chaoneng; Gu, Shaohua; Zhao, Chunhua; Xie, Yi; Mao, Yumin

2002-12-01

KHDRBS2, KH domain containing, RNA binding, signal transduction associated 2, is an RNA-binding protein that is tyrosine phosphorylated by Src during mitosis. It contains a KH domain,which is embedded in a larger conserved domain called the STAR domain. This protein has a 99% sequence identity with rat SLM-1 (the Sam68-like mammalian protein 1) and 98% sequence identity with mouse SLM-1 in its STAR domain. KHDRBS2 has the characteristic Sam68 SH2 and SH3 domain binding sites. RT-PCR analysis showed its transcript is ubiquitously expressed. The characterization of KHDRBS2 indicates it may link tyrosine kinase signaling cascades with some aspect of RNA metabolism.

Ermelin, an endoplasmic reticulum transmembrane protein, contains the novel HELP domain conserved in eukaryotes.

PubMed

Suzuki, Akiko; Endo, Takeshi

2002-02-06

We have cloned a cDNA encoding a novel protein referred to as ermelin from mouse C2 skeletal muscle cells. This protein contained six hydrophobic amino acid stretches corresponding to transmembrane domains, two histidine-rich sequences, and a sequence homologous to the fusion peptides of certain fusion proteins. Ermelin also contained a novel modular sequence, designated as HELP domain, which was highly conserved among eukaryotes, from yeast to higher plants and animals. All these HELP domain-containing proteins, including mouse KE4, Drosophila Catsup, and Arabidopsis IAR1, possessed multipass transmembrane domains and histidine-rich sequences. Ermelin was predominantly expressed in brain and testis, and induced during neuronal differentiation of N1E-115 neuroblastoma cells but downregulated during myogenic differentiation of C2 cells. The mRNA was accumulated in hippocampus and cerebellum of brain and central areas of seminiferous tubules in testis. Epitope-tagging experiments located ermelin and KE4 to a network structure throughout the cytoplasm. Staining with the fluorescent dye DiOC(6)(3) identified this structure as the endoplasmic reticulum. These results suggest that at least some, if not all, of the HELP domain-containing proteins are multipass endoplasmic reticulum membrane proteins with functions conserved among eukaryotes.

Characterization of a double WAP domain-containing protein from the red swamp crayfish Procambarus clarkii

USDA-ARS?s Scientific Manuscript database

Crustaceans express multiple whey acidic protein (WAP) domain containing proteins which are components of host immunity. In the present study, a new double WAP domain containing protein was identified from red swamp crayfish Procambarus clarkii, designated Pc-DWD. The ORF is 387 bp, encoding 128 ami...

OnpA, an Unusual Flavin-Dependent Monooxygenase Containing a Cytochrome b5 Domain

PubMed Central

Xiao, Yi; Liu, Ting-Ting; Dai, Hui; Zhang, Jun-Jie; Liu, Hong; Tang, Huiru; Leak, David J.

2012-01-01

ortho-Nitrophenol 2-monooxygenase (EC 1.14.13.31) from Alcaligenes sp. strain NyZ215 catalyzes monooxygenation of ortho-nitrophenol to form catechol via ortho-benzoquinone. Sequence analysis of this onpA-encoded enzyme revealed that it contained a flavin-binding monooxygenase domain and a heme-binding cytochrome b5 domain. OnpA was purified to homogeneity as a His-tagged protein and was considered a monomer, as determined by gel filtration. FAD and heme were identified by high-performance liquid chromatography (HPLC) and HPLC-mass spectrometry (HPLC-MS) as cofactors in this enzyme, and quantitative analysis indicated that 1 mol of the purified recombinant OnpA contained 0.66 mol of FAD and 0.20 mol of heme. However, the enzyme activity of OnpA was increased by 60% and 450% after addition of FAD and hemin, respectively, suggesting that the optimal stoichiometry was 1:1:1. In addition, site-directed mutagenesis experiments confirmed that two highly conserved histidines located in the cytochrome b5 domain were associated with binding of the heme, and the cytochrome b5 domain was involved in the OnpA activity. These results indicate that OnpA is an unusual FAD-dependent monooxygenase containing a fused cytochrome b5 domain that is essential for its activity. Therefore, we here demonstrate a link between cytochrome b5 and flavin-dependent monooxygenases. PMID:22267507

Structure of PA1221, a nonribosomal peptide synthetase containing adenylation and peptidyl carrier protein domains.

PubMed

Mitchell, Carter A; Shi, Ce; Aldrich, Courtney C; Gulick, Andrew M

2012-04-17

Many bacteria use large modular enzymes for the synthesis of polyketide and peptide natural products. These multidomain enzymes contain integrated carrier domains that deliver bound substrates to multiple catalytic domains, requiring coordination of these chemical steps. Nonribosomal peptide synthetases (NRPSs) load amino acids onto carrier domains through the activity of an upstream adenylation domain. Our lab recently determined the structure of an engineered two-domain NRPS containing fused adenylation and carrier domains. This structure adopted a domain-swapped dimer that illustrated the interface between these two domains. To continue our investigation, we now examine PA1221, a natural two-domain protein from Pseudomonas aeruginosa. We have determined the amino acid specificity of this new enzyme and used domain specific mutations to demonstrate that loading the downstream carrier domain within a single protein molecule occurs more quickly than loading of a nonfused carrier domain intermolecularly. Finally, we have determined crystal structures of both apo- and holo-PA1221 proteins, the latter using a valine-adenosine vinylsulfonamide inhibitor that traps the adenylation domain-carrier domain interaction. The protein adopts an interface similar to that seen with the prior adenylation domain-carrier protein construct. A comparison of these structures with previous structures of multidomain NRPSs suggests that a large conformational change within the NRPS adenylation domains guides the carrier domain into the active site for thioester formation.

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.

Cystathionine β-Synthase (CBS) Domain-containing Pyrophosphatase as a Target for Diadenosine Polyphosphates in Bacteria*

PubMed Central

Anashkin, Viktor A.; Salminen, Anu; Tuominen, Heidi K.; Orlov, Victor N.; Lahti, Reijo; Baykov, Alexander A.

2015-01-01

Among numerous proteins containing pairs of regulatory cystathionine β-synthase (CBS) domains, family II pyrophosphatases (CBS-PPases) are unique in that they generally contain an additional DRTGG domain between the CBS domains. Adenine nucleotides bind to the CBS domains in CBS-PPases in a positively cooperative manner, resulting in enzyme inhibition (AMP or ADP) or activation (ATP). Here we show that linear P1,Pn-diadenosine 5′-polyphosphates (ApnAs, where n is the number of phosphate residues) bind with nanomolar affinity to DRTGG domain-containing CBS-PPases of Desulfitobacterium hafniense, Clostridium novyi, and Clostridium perfringens and increase their activity up to 30-, 5-, and 7-fold, respectively. Ap4A, Ap5A, and Ap6A bound noncooperatively and with similarly high affinities to CBS-PPases, whereas Ap3A bound in a positively cooperative manner and with lower affinity, like mononucleotides. All ApnAs abolished kinetic cooperativity (non-Michaelian behavior) of CBS-PPases. The enthalpy change and binding stoichiometry, as determined by isothermal calorimetry, were ∼10 kcal/mol nucleotide and 1 mol/mol enzyme dimer for Ap4A and Ap5A but 5.5 kcal/mol and 2 mol/mol for Ap3A, AMP, ADP, and ATP, suggesting different binding modes for the two nucleotide groups. In contrast, Eggerthella lenta and Moorella thermoacetica CBS-PPases, which contain no DRTGG domain, were not affected by ApnAs and showed no enthalpy change, indicating the importance of the DTRGG domain for ApnA binding. These findings suggest that ApnAs can control CBS-PPase activity and hence affect pyrophosphate level and biosynthetic activity in bacteria. PMID:26400082

Static and dynamic structural-sensitivity derivative calculations in the finite-element-based Engineering Analysis Language (EAL) system

NASA Technical Reports Server (NTRS)

Camarda, C. J.; Adelman, H. M.

1984-01-01

The implementation of static and dynamic structural-sensitivity derivative calculations in a general purpose, finite-element computer program denoted the Engineering Analysis Language (EAL) System is described. Derivatives are calculated with respect to structural parameters, specifically, member sectional properties including thicknesses, cross-sectional areas, and moments of inertia. Derivatives are obtained for displacements, stresses, vibration frequencies and mode shapes, and buckling loads and mode shapes. Three methods for calculating derivatives are implemented (analytical, semianalytical, and finite differences), and comparisons of computer time and accuracy are made. Results are presented for four examples: a swept wing, a box beam, a stiffened cylinder with a cutout, and a space radiometer-antenna truss.

Characterization of a novel MIIA domain-containing protein (MdcE) in Bradyrhizobium spp.

PubMed

Durán, David; Imperial, Juan; Palacios, José; Ruiz-Argüeso, Tomás; Göttfert, Michael; Zehner, Susanne; Rey, Luis

2018-03-01

Several genes coding for proteins with metal ion-inducible autocleavage (MIIA) domains were identified in type III secretion system tts gene clusters from draft genomes of recently isolated Bradyrhizobium spp. MIIA domains have been first described in the effectors NopE1 and NopE2 of Bradyrhizobium diazoefficiens USDA 110. All identified genes are preceded by tts box promoter motifs. The identified proteins contain one or two MIIA domains. A phylogenetic analysis of 35 MIIA domain sequences from 16 Bradyrhizobium strains revealed four groups. The protein from Bradyrhizobium sp. LmjC strain contains a single MIIA domain and was designated MdcE (MdcELmjC). It was expressed as a fusion to maltose-binding protein (MalE) in Escherichia coli and subsequently purified by affinity chromatography. Recombinant MalE-MdcELmjC-Strep protein exhibited autocleavage in the presence of Ca2+, Cu2+, Cd2+ and Mn2+, but not in the presence of Mg2+, Ni2+ or Co2+. Site-directed mutagenesis at the predicted cleavage site abolished autocleavage activity of MdcELmjC. An LmjC mdcE- mutant was impaired in the ability to nodulate Lupinus angustifolius and Macroptilium atropurpureum. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Taxonomic distribution, repeats, and functions of the S1 domain-containing proteins as members of the OB-fold family.

PubMed

Deryusheva, Evgeniia I; Machulin, Andrey V; Selivanova, Olga M; Galzitskaya, Oxana V

2017-04-01

Proteins of the nucleic acid-binding proteins superfamily perform such functions as processing, transport, storage, stretching, translation, and degradation of RNA. It is one of the 16 superfamilies containing the OB-fold in protein structures. Here, we have analyzed the superfamily of nucleic acid-binding proteins (the number of sequences exceeds 200,000) and obtained that this superfamily prevalently consists of proteins containing the cold shock DNA-binding domain (ca. 131,000 protein sequences). Proteins containing the S1 domain compose 57% from the cold shock DNA-binding domain family. Furthermore, we have found that the S1 domain was identified mainly in the bacterial proteins (ca. 83%) compared to the eukaryotic and archaeal proteins, which are available in the UniProt database. We have found that the number of multiple repeats of S1 domain in the S1 domain-containing proteins depends on the taxonomic affiliation. All archaeal proteins contain one copy of the S1 domain, while the number of repeats in the eukaryotic proteins varies between 1 and 15 and correlates with the protein size. In the bacterial proteins, the number of repeats is no more than 6, regardless of the protein size. The large variation of the repeat number of S1 domain as one of the structural variants of the OB-fold is a distinctive feature of S1 domain-containing proteins. Proteins from the other families and superfamilies have either one OB-fold or change slightly the repeat numbers. On the whole, it can be supposed that the repeat number is a vital for multifunctional activity of the S1 domain-containing proteins. Proteins 2017; 85:602-613. © 2016 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Thrombospondin Type-1 Repeat Domain-Containing Proteins Are Strongly Expressed in the Head Region of Hydra.

PubMed

Hamaguchi-Hamada, Kayoko; Kurumata-Shigeto, Mami; Minobe, Sumiko; Fukuoka, Nozomi; Sato, Manami; Matsufuji, Miyuki; Koizumi, Osamu; Hamada, Shun

2016-01-01

The head region of Hydra, the hypostome, is a key body part for developmental control and the nervous system. We herein examined genes specifically expressed in the head region of Hydra oligactis using suppression subtractive hybridization (SSH) cloning. A total of 1414 subtracted clones were sequenced and found to be derived from at least 540 different genes by BLASTN analyses. Approximately 25% of the subtracted clones had sequences encoding thrombospondin type-1 repeat (TSR) domains, and were derived from 17 genes. We identified 11 TSR domain-containing genes among the top 36 genes that were the most frequently detected in our SSH library. Whole-mount in situ hybridization analyses confirmed that at least 13 out of 17 TSR domain-containing genes were expressed in the hypostome of Hydra oligactis. The prominent expression of TSR domain-containing genes suggests that these genes play significant roles in the hypostome of Hydra oligactis.

Evidence that the tandem-pleckstrin-homology-domain-containing protein TAPP1 interacts with Ptd(3,4)P2 and the multi-PDZ-domain-containing protein MUPP1 in vivo.

PubMed Central

Kimber, Wendy A; Trinkle-Mulcahy, Laura; Cheung, Peter C F; Deak, Maria; Marsden, Louisa J; Kieloch, Agnieszka; Watt, Stephen; Javier, Ronald T; Gray, Alex; Downes, C Peter; Lucocq, John M; Alessi, Dario R

2002-01-01

PtdIns(3,4,5)P3 is an established second messenger of growth-factor and insulin-induced signalling pathways. There is increasing evidence that one of the immediate breakdown products of PtdIns(3,4,5)P3, namely PtdIns(3,4)P2, whose levels are elevated by numerous extracellular agonists, might also function as a signalling molecule. Recently, we identified two related pleckstrin-homology (PH)-domain-containing proteins, termed 'tandem-PH-domain-containing protein-1' (TAPP1) and TAPP2, which interacted in vitro with high affinity with PtdIns(3,4)P2, but did not bind PtdIns(3,4,5)P3 or other phosphoinositides. In the present study we demonstrate that stimulation of Swiss 3T3 or 293 cells with agonists that stimulate PtdIns(3,4)P2 production results in the marked translocation of TAPP1 to the plasma membrane. This recruitment is dependent on a functional PtdIns(3,4)P2-binding PH domain and is inhibited by wortmannin, a phosphoinositide 3-kinase inhibitor that prevents PtdIns(3,4)P2 generation. A search for proteins that interact with TAPP1 identified the multi-PDZ-containing protein termed 'MUPP1', a protein possessing 13 PDZ domains and no other known modular or catalytic domains [PDZ is postsynaptic density protein (PSD-95)/Drosophila disc large tumour suppressor (dlg)/tight junction protein (ZO1)]. We demonstrate that immunoprecipitation of endogenously expressed TAPP1 from 293-cell lysates results in the co-immunoprecipitation of endogenous MUPP1, indicating that these proteins are likely to interact with each other physiologically. We show that TAPP1 and TAPP2 interact with the 10th and 13th PDZ domain of MUPP1 through their C-terminal amino acids. The results of the present study suggest that TAPP1 and TAPP2 could function in cells as adapter proteins to recruit MUPP1, or other proteins that they may interact with, to the plasma membrane in response to signals that elevate PtdIns(3,4)P2. PMID:11802782

Ovarian Tumor (OTU)-domain Containing Viral Proteases Evade Ubiquitin- and ISG15-dependent Innate Immune Responses

PubMed Central

Frias-Staheli, Natalia; Giannakopoulos, Nadia V.; Kikkert, Marjolein; Taylor, Shannon L.; Bridgen, Anne; Paragas, Jason J.; Richt, Juergen A.; Rowland, Raymond R.; Schmaljohn, Connie S.; Lenschow, Deborah J.; Snijder, Eric J.; García-Sastre, Adolfo; Virgin, Herbert Whiting

2007-01-01

Summary Ubiquitin (Ub) and interferon stimulated gene product 15 (ISG15) reversibly conjugate to proteins via a conserved LRLRGG C-terminal motif, mediating important innate antiviral responses. The ovarian tumor (OTU) domain represents a superfamily of predicted proteases found in eukaryotic, bacterial and viral proteins, some of which have Ub-deconjugating activity. We show that the OTU domain-containing proteases of nairoviruses and arteriviruses hydrolyze Ub and ISG15 from cellular target proteins. This broad activity contrasts with the target specificity of known mammalian OTU domain-containing proteins. The biological significance of this activity of viral OTU domain-containing proteases was evidenced by their capacity to inhibit NF-κB dependent signaling and to antagonize the antiviral effects of ISG15 during Sindbis virus infection in vivo. The deconjugating activity of viral OTU proteases represents a novel viral immune evasion mechanism that inhibits Ub-and ISG15-dependent antiviral pathways. PMID:18078692

Thrombospondin Type-1 Repeat Domain-Containing Proteins Are Strongly Expressed in the Head Region of Hydra

PubMed Central

Hamaguchi-Hamada, Kayoko; Kurumata-Shigeto, Mami; Minobe, Sumiko; Fukuoka, Nozomi; Sato, Manami; Matsufuji, Miyuki; Koizumi, Osamu; Hamada, Shun

2016-01-01

The head region of Hydra, the hypostome, is a key body part for developmental control and the nervous system. We herein examined genes specifically expressed in the head region of Hydra oligactis using suppression subtractive hybridization (SSH) cloning. A total of 1414 subtracted clones were sequenced and found to be derived from at least 540 different genes by BLASTN analyses. Approximately 25% of the subtracted clones had sequences encoding thrombospondin type-1 repeat (TSR) domains, and were derived from 17 genes. We identified 11 TSR domain-containing genes among the top 36 genes that were the most frequently detected in our SSH library. Whole-mount in situ hybridization analyses confirmed that at least 13 out of 17 TSR domain-containing genes were expressed in the hypostome of Hydra oligactis. The prominent expression of TSR domain-containing genes suggests that these genes play significant roles in the hypostome of Hydra oligactis. PMID:27043211

Are South African Speech-Language Therapists adequately equipped to assess English Additional Language (EAL) speakers who are from an indigenous linguistic and cultural background? A profile and exploration of the current situation

PubMed Central

Mdlalo, Thandeka; Flack, Penelope

2016-01-01

This article presents the results of a survey conducted on Speech-Language Therapists (SLTs) regarding current practices in the assessment of English Additional Language (EAL) speakers in South Africa. It forms part of the rationale for a broader (PhD) study that critiques the use of assessment instruments on EAL speakers from an indigenous linguistic and cultural background. This article discusses an aspect of the broader research and presents the background, method, findings, discussion and implications of the survey. The results of this survey highlight the challenges of human and material resources to, and the dominance of English in, the profession in South Africa. The findings contribute to understanding critical factors for acquiring reliable and valid assessment results with diverse populations, particularly the implications from a cultural and linguistic perspective. PMID:27247254

The GDP-switched GAF domain of DcpA modulates the concerted synthesis/hydrolysis of c-di-GMP in Mycobacterium smegmatis.

PubMed

Chen, Hui-Jie; Li, Na; Luo, Ye; Jiang, Yong-Liang; Zhou, Cong-Zhao; Chen, Yuxing; Li, Qiong

2018-04-09

The second messenger c-di-GMP [bis-(3'-5')-cyclic dimeric guanosine monophosphate] plays a key role in bacterial growth, survival and pathogenesis, and thus its intracellular homeostasis should be finely maintained. Mycobacterium smegmatis encodes a GAF (mammalian c G MP-regulated phosphodiesterases, Anabaena a denylyl cyclases and Escherichia coli transcription activator F hlA) domain containing bifunctional enzyme DcpA ( d iguanylate c yclase and p hosphodiesterase A ) that catalyzes the synthesis and hydrolysis of c-di-GMP . Here, we found that M. smegmatis DcpA catalyzes the hydrolysis of c-di-GMP at a higher velocity, compared with synthetic activity, resulting in a sum reaction from the ultimate substrate GTP to the final product pGpG [5'-phosphoguanylyl-(3'-5')-guanosine]. Fusion with the N-terminal GAF domain enables the GGDEF (Gly-Gly-Asp-Glu-Phe) domain of DcpA to dimerize and accordingly gain synthetic activity. Screening of putative metabolites revealed that GDP is the ligand of the GAF domain. Binding of GDP to the GAF domain down-regulates synthetic activity, but up-regulates hydrolytic activity, which, in consequence, might enable a timely response to the transient accumulation of c-di-GMP at the stationary phase or under stresses. Combined with the crystal structure of the EAL (Glu-Ala-Leu) domain and the small-angle X-ray scattering data, we propose a putative regulatory model of the GAF domain finely tuned by the intracellular GTP/GDP ratio. These findings help us to better understand the concerted control of the synthesis and hydrolysis of c-di-GMP in M. smegmatis in various microenvironments. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Structural insights into the functional versatility of WW domain-containing oxidoreductase tumor suppressor

PubMed Central

2015-01-01

Recent work on WW domain-containing oxidoreductase (WWOX) tumor suppressor is beginning to shed new light on both the molecular mechanism of action of its WW domains as well as the contiguous catalytic domain. Herein, the structural basis underlying the ability of WW1 domain to bind to various physiological ligands and how the orphan WW2 tandem partner synergizes its ligand binding in the context of WW1–WW2 tandem module of WWOX is discussed. Notably, the WW domains within the WW1–WW2 tandem module physically associate so as to adopt a fixed spatial orientation relative to each other. In this manner, the association of WW2 domain with WW1 hinders ligand binding to the latter. Consequently, ligand binding to WW1 domain not only results in the displacement of WW2 lid but also disrupts the fixed orientation of WW domains in the liganded conformation. Equally importantly, structure-guided functional approach suggests that the catalytic domain of WWOX likely serves as a retinal oxidoreductase that catalyzes the reversible oxidation and reduction of all-trans-retinal. Collectively, this review provides structural insights into the functional versatility of a key signaling protein with important implications on its biology. PMID:25662954

Structural insights into the functional versatility of WW domain-containing oxidoreductase tumor suppressor.

PubMed

Farooq, Amjad

2015-03-01

Recent work on WW domain-containing oxidoreductase (WWOX) tumor suppressor is beginning to shed new light on both the molecular mechanism of action of its WW domains as well as the contiguous catalytic domain. Herein, the structural basis underlying the ability of WW1 domain to bind to various physiological ligands and how the orphan WW2 tandem partner synergizes its ligand binding in the context of WW1-WW2 tandem module of WWOX is discussed. Notably, the WW domains within the WW1-WW2 tandem module physically associate so as to adopt a fixed spatial orientation relative to each other. In this manner, the association of WW2 domain with WW1 hinders ligand binding to the latter. Consequently, ligand binding to WW1 domain not only results in the displacement of WW2 lid but also disrupts the fixed orientation of WW domains in the liganded conformation. Equally importantly, structure-guided functional approach suggests that the catalytic domain of WWOX likely serves as a retinal oxidoreductase that catalyzes the reversible oxidation and reduction of all-trans-retinal. Collectively, this review provides structural insights into the functional versatility of a key signaling protein with important implications on its biology. © 2015 by the Society for Experimental Biology and Medicine.

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

Improving fMRI reliability in presurgical mapping for brain tumours.

PubMed

Stevens, M Tynan R; Clarke, David B; Stroink, Gerhard; Beyea, Steven D; D'Arcy, Ryan Cn

2016-03-01

Functional MRI (fMRI) is becoming increasingly integrated into clinical practice for presurgical mapping. Current efforts are focused on validating data quality, with reliability being a major factor. In this paper, we demonstrate the utility of a recently developed approach that uses receiver operating characteristic-reliability (ROC-r) to: (1) identify reliable versus unreliable data sets; (2) automatically select processing options to enhance data quality; and (3) automatically select individualised thresholds for activation maps. Presurgical fMRI was conducted in 16 patients undergoing surgical treatment for brain tumours. Within-session test-retest fMRI was conducted, and ROC-reliability of the patient group was compared to a previous healthy control cohort. Individually optimised preprocessing pipelines were determined to improve reliability. Spatial correspondence was assessed by comparing the fMRI results to intraoperative cortical stimulation mapping, in terms of the distance to the nearest active fMRI voxel. The average ROC-r reliability for the patients was 0.58±0.03, as compared to 0.72±0.02 in healthy controls. For the patient group, this increased significantly to 0.65±0.02 by adopting optimised preprocessing pipelines. Co-localisation of the fMRI maps with cortical stimulation was significantly better for more reliable versus less reliable data sets (8.3±0.9 vs 29±3 mm, respectively). We demonstrated ROC-r analysis for identifying reliable fMRI data sets, choosing optimal postprocessing pipelines, and selecting patient-specific thresholds. Data sets with higher reliability also showed closer spatial correspondence to cortical stimulation. ROC-r can thus identify poor fMRI data at time of scanning, allowing for repeat scans when necessary. ROC-r analysis provides optimised and automated fMRI processing for improved presurgical mapping. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence

Differential Occurrence of Interactions and Interaction Domains in Proteins Containing Homopolymeric Amino Acid Repeats

PubMed Central

Pelassa, Ilaria; Fiumara, Ferdinando

2015-01-01

Homopolymeric amino acids repeats (AARs), which are widespread in proteomes, have often been viewed simply as spacers between protein domains, or even as “junk” sequences with no obvious function but with a potential to cause harm upon expansion as in genetic diseases associated with polyglutamine or polyalanine expansions, including Huntington disease and cleidocranial dysplasia. A growing body of evidence indicates however that at least some AARs can form organized, functional protein structures, and can regulate protein function. In particular, certain AARs can mediate protein-protein interactions, either through homotypic AAR-AAR contacts or through heterotypic contacts with other protein domains. It is still unclear however, whether AARs may have a generalized, proteome-wide role in shaping protein-protein interaction networks. Therefore, we have undertaken here a bioinformatics screening of the human proteome and interactome in search of quantitative evidence of such a role. We first identified the sets of proteins that contain repeats of any one of the 20 amino acids, as well as control sets of proteins chosen at random in the proteome. We then analyzed the connectivity between the proteins of the AAR-containing protein sets and we compared it with that observed in the corresponding control networks. We find evidence for different degrees of connectivity in the different AAR-containing protein networks. Indeed, networks of proteins containing polyglutamine, polyglutamate, polyproline, and other AARs show significantly increased levels of connectivity, whereas networks containing polyleucine and other hydrophobic repeats show lower degrees of connectivity. Furthermore, we observed that numerous protein-protein, -nucleic acid, and -lipid interaction domains are significantly enriched in specific AAR protein groups. These findings support the notion of a generalized, combinatorial role of AARs, together with conventional protein interaction domains, in

Phyletic Distribution and Lineage-Specific Domain Architectures of Archaeal Two-Component Signal Transduction Systems

PubMed Central

Makarova, Kira S.; Wolf, Yuri I.

2017-01-01

ABSTRACT The two-component signal transduction (TCS) machinery is a key mechanism of sensing environmental changes in the prokaryotic world. TCS systems have been characterized thoroughly in bacteria but to a much lesser extent in archaea. Here, we provide an updated census of more than 2,000 histidine kinases and response regulators encoded in 218 complete archaeal genomes, as well as unfinished genomes available from metagenomic data. We describe the domain architectures of the archaeal TCS components, including several novel output domains, and discuss the evolution of the archaeal TCS machinery. The distribution of TCS systems in archaea is strongly biased, with high levels of abundance in haloarchaea and thaumarchaea but none detected in the sequenced genomes from the phyla Crenarchaeota, Nanoarchaeota, and Korarchaeota. The archaeal sensor histidine kinases are generally similar to their well-studied bacterial counterparts but are often located in the cytoplasm and carry multiple PAS and/or GAF domains. In contrast, archaeal response regulators differ dramatically from the bacterial ones. Most archaeal genomes do not encode any of the major classes of bacterial response regulators, such as the DNA-binding transcriptional regulators of the OmpR/PhoB, NarL/FixJ, NtrC, AgrA/LytR, and ActR/PrrA families and the response regulators with GGDEF and/or EAL output domains. Instead, archaea encode multiple copies of response regulators containing either the stand-alone receiver (REC) domain or combinations of REC with PAS and/or GAF domains. Therefore, the prevailing mechanism of archaeal TCS signaling appears to be via a variety of protein-protein interactions, rather than direct transcriptional regulation. IMPORTANCE Although the Archaea represent a separate domain of life, their signaling systems have been assumed to be closely similar to the bacterial ones. A study of the domain architectures of the archaeal two-component signal transduction (TCS) machinery

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.

Yield Scaling of Frequency Domain Moment Tensors from Contained Chemical Explosions Detonated in Granite

NASA Astrophysics Data System (ADS)

MacPhail, M. D.; Stump, B. W.; Zhou, R.

2017-12-01

The Source Phenomenology Experiment (SPE - Arizona) was a series of nine, contained and partially contained chemical explosions within the porphyry granite at the Morenci Copper mine in Arizona. Its purpose was to detonate, record and analyze seismic waveforms from these single-fired explosions. Ground motion data from the SPE is analyzed in this study to assess the uniqueness of the time domain moment tensor source representation and its ability to quantify containment and yield scaling. Green's functions were computed for each of the explosions based on a 1D velocity model developed for the SPE. The Green's functions for the sixteen, near-source stations focused on observations from 37 to 680 m. This study analyzes the three deepest, fully contained explosions with a depth of burial of 30 m and yields of 0.77e-3, 3.08e-3 and 6.17e-3 kt. Inversions are conducted within the frequency domain and moment tensors are decomposed into deviatoric and isotropic components to evaluate the effects of containment and yield on the resulting source representation. Isotropic moments are compared to those for other contained explosions as reported by Denny and Johnson, 1991, and are in good agreement with their scaling results. The explosions in this study have isotropic moments of 1.2e12, 3.1e12 and 6.1e13 n*m. Isotropic and Mzz moment tensor spectra are compared to Mueller-Murphy, Denny-Johnson and revised Heard-Ackerman (HA) models and suggest that the larger explosions fit the HA model better. Secondary source effects resulting from free surface interactions including the effects of spallation contribute to the resulting moment tensors which include a CLVD component. Hudson diagrams, using frequency domain moment tensor data, are computed as a tool to assess how these containment scenarios affect the source representation. Our analysis suggests that, within our band of interest (2-20 Hz), as the frequency increases, the source representation becomes more explosion like

Possible Domain Formation In PE/PC Bilayers Containing High Cholesterol

NASA Astrophysics Data System (ADS)

Hein, Matthew; Hussain, Fazle; Huang, Juyang

2015-03-01

Cholesterol is a significant component of animal cell membranes, and its presence has the effects of not only adding rigidity to the lipid bilayer, but also leading to the formation of lipid domains. Two other lipids of interest are phosphatidylethanolamine (PE), which constitutes about 45 percent of the phospholipids found in human nervous tissues, and phosphatidylcholine (PC), which is found in every cell of the human body. The maximum solubility of cholesterol is the highest mole fraction of cholesterol that the lipid bilayer can retain, at which point cholesterol begins to precipitate out to form cholesterol monohydrate crystals. We have measured the maximum solubility of cholesterol in mixtures of 16:0-18:1PE and 16:0-18:1PC using a new light scattering technique, which utilizes the anisotropic nature of light scattering by cholesterol crystals. This new method is highly accurate and reproducible. Our results show that the maximum solubility of cholesterol increases linearly as a function of the molar ratio POPC/(POPE+POPC), which suggests possible domain formation in mixtures of PE and PC containing maximum amount of cholesterol.

Roots of angiosperm formins: The evolutionary history of plant FH2 domain-containing proteins

PubMed Central

2008-01-01

Background Shuffling of modular protein domains is an important source of evolutionary innovation. Formins are a family of actin-organizing proteins that share a conserved FH2 domain but their overall domain architecture differs dramatically between opisthokonts (metazoans and fungi) and plants. We performed a phylogenomic analysis of formins in most eukaryotic kingdoms, aiming to reconstruct an evolutionary scenario that may have produced the current diversity of domain combinations with focus on the origin of the angiosperm formin architectures. Results The Rho GTPase-binding domain (GBD/FH3) reported from opisthokont and Dictyostelium formins was found in all lineages except plants, suggesting its ancestral character. Instead, mosses and vascular plants possess the two formin classes known from angiosperms: membrane-anchored Class I formins and Class II formins carrying a PTEN-like domain. PTEN-related domains were found also in stramenopile formins, where they have been probably acquired independently rather than by horizontal transfer, following a burst of domain rearrangements in the chromalveolate lineage. A novel RhoGAP-related domain was identified in some algal, moss and lycophyte (but not angiosperm) formins that define a specific branch (Class III) of the formin family. Conclusion We propose a scenario where formins underwent multiple domain rearrangements in several eukaryotic lineages, especially plants and chromalveolates. In plants this replaced GBD/FH3 by a probably inactive RhoGAP-like domain, preserving a formin-mediated association between (membrane-anchored) Rho GTPases and the actin cytoskeleton. Subsequent amplification of formin genes, possibly coincident with the expansion of plants to dry land, was followed by acquisition of alternative membrane attachment mechanisms present in extant Class I and Class II formins, allowing later loss of the RhoGAP-like domain-containing formins in angiosperms. PMID:18430232

Effect of Dialkyl Ammonium Cationic Surfactants on the Microfluidity of Membranes Containing Raft Domains.

PubMed

Uyama, Makoto; Inoue, Kaori; Kinoshita, Koichi; Miyahara, Reiji; Yokoyama, Hirokazu; Nakano, Minoru

2018-01-01

It has been reported that a lot of receptors localize in lipid raft domains and that the microfluidity of these domains regulates the activation of these receptors. In this study, we focused on the lipid raft and in order to evaluate the physicochemical effects of surfactants on microfluidity of lipid membranes, we used liposomes comprising of egg-yolk L-α-phosphatidylcholine, egg-yolk sphingomyelin, and cholesterol as a model of cell membranes containing raft domains. The microfluidity of the domains was characterized by fluorescence spectrometry using 1,6-diphenyl-1,3,5-hexatriene and 2-dimethylamino-6-lauroylnaphthalene. Among several surfactants, dialkylammonium-type cationic surfactants most efficiently increased the microfluidity. It is therefore concluded that (1) the electrostatic interaction between the cationic surfactant and eggPC/eggSM/cholesterol liposome could be important, (2) surfactants with alkyl chains more effectively inserted into membranes than those with acyl chains, and (3) cationic surfactants with lower T m values have a greater ability to increase the fluidity.

The Popeye Domain Containing Genes and Their Function as cAMP Effector Proteins in Striated Muscle.

PubMed

Brand, Thomas

2018-03-13

The Popeye domain containing (POPDC) genes encode transmembrane proteins, which are abundantly expressed in striated muscle cells. Hallmarks of the POPDC proteins are the presence of three transmembrane domains and the Popeye domain, which makes up a large part of the cytoplasmic portion of the protein and functions as a cAMP-binding domain. Interestingly, despite the prediction of structural similarity between the Popeye domain and other cAMP binding domains, at the protein sequence level they strongly differ from each other suggesting an independent evolutionary origin of POPDC proteins. Loss-of-function experiments in zebrafish and mouse established an important role of POPDC proteins for cardiac conduction and heart rate adaptation after stress. Loss-of function mutations in patients have been associated with limb-girdle muscular dystrophy and AV-block. These data suggest an important role of these proteins in the maintenance of structure and function of striated muscle cells.

Phyletic Distribution and Lineage-Specific Domain Architectures of Archaeal Two-Component Signal Transduction Systems.

PubMed

Galperin, Michael Y; Makarova, Kira S; Wolf, Yuri I; Koonin, Eugene V

2018-04-01

The two-component signal transduction (TCS) machinery is a key mechanism of sensing environmental changes in the prokaryotic world. TCS systems have been characterized thoroughly in bacteria but to a much lesser extent in archaea. Here, we provide an updated census of more than 2,000 histidine kinases and response regulators encoded in 218 complete archaeal genomes, as well as unfinished genomes available from metagenomic data. We describe the domain architectures of the archaeal TCS components, including several novel output domains, and discuss the evolution of the archaeal TCS machinery. The distribution of TCS systems in archaea is strongly biased, with high levels of abundance in haloarchaea and thaumarchaea but none detected in the sequenced genomes from the phyla Crenarchaeota , Nanoarchaeota , and Korarchaeota The archaeal sensor histidine kinases are generally similar to their well-studied bacterial counterparts but are often located in the cytoplasm and carry multiple PAS and/or GAF domains. In contrast, archaeal response regulators differ dramatically from the bacterial ones. Most archaeal genomes do not encode any of the major classes of bacterial response regulators, such as the DNA-binding transcriptional regulators of the OmpR/PhoB, NarL/FixJ, NtrC, AgrA/LytR, and ActR/PrrA families and the response regulators with GGDEF and/or EAL output domains. Instead, archaea encode multiple copies of response regulators containing either the stand-alone receiver (REC) domain or combinations of REC with PAS and/or GAF domains. Therefore, the prevailing mechanism of archaeal TCS signaling appears to be via a variety of protein-protein interactions, rather than direct transcriptional regulation. IMPORTANCE Although the Archaea represent a separate domain of life, their signaling systems have been assumed to be closely similar to the bacterial ones. A study of the domain architectures of the archaeal two-component signal transduction (TCS) machinery revealed an

Adverse childhood experiences are associated with irritable bowel syndrome and gastrointestinal symptom severity.

PubMed

Park, S H; Videlock, E J; Shih, W; Presson, A P; Mayer, E A; Chang, L

2016-08-01

Early adverse life events (EALs) are associated with irritable bowel syndrome (IBS). Exposure to EALs as assessed by the Adverse Childhood Experiences (ACE) questionnaire is associated with greater disease prevalence, but ACE has not been studied in gastrointestinal disorders. Study aims were to: (i) Estimate the prevalence of EALs in the IBS patients using the ACE questionnaire; (ii) Determine correlations between ACE and Early Trauma Inventory Self Report-Short Form (ETI-SR) scores to confirm its validity in IBS; and (iii) Correlate ACE scores with IBS symptom severity. A total of 148 IBS (73% women, mean age = 31 years) and 154 HCs (59% women, mean age = 30 years) completed the ACE and ETI-SR between June 2010 and April 2015. These surveys measured EALs before age 18 in the domains of physical, sexual, and emotional abuse, and general trauma. IBS and abdominal pain severity was measured by a 20-point scale (0 = none, 20 = worst symptoms). The ACE score increased the odds of having IBS (odds ratio [OR] = 2.05, 95% confidence interval [CI]: 1.21-3.48, p = 0.008). Household mental illness (p < 0.001), emotional abuse (p = 0.004), and incarcerated household member (p = 0.019) were significant predictors of IBS. Adverse childhood experiences and ETI-SR scores were strongly correlated (r = 0.59, p < 0.001). ACE, but not ETI-SR, modestly correlated with IBS severity (r = 0.17, p = 0.036) and abdominal pain (r = 0.20, p = 0.015). The ACE questionnaire is a useful instrument to measure EALs in IBS based on its use in large studies, its ability to measure prevalence across different EAL domains, and its correlation with symptom severity. © 2016 John Wiley & Sons Ltd.

Phosphate homeostasis in the yeast Saccharomyces cerevisiae, the key role of the SPX domain-containing proteins.

PubMed

Secco, David; Wang, Chuang; Shou, Huixia; Whelan, James

2012-02-17

In the yeast Saccharomyces cerevisiae, a working model for nutrient homeostasis in eukaryotes, inorganic phosphate (Pi) homeostasis is regulated by the PHO pathway, a set of phosphate starvation induced genes, acting to optimize Pi uptake and utilization. Among these, a subset of proteins containing the SPX domain has been shown to be key regulators of Pi homeostasis. In this review, we summarize the recent progresses in elucidating the mechanisms controlling Pi homeostasis in yeast, focusing on the key roles of the SPX domain-containing proteins in these processes, as well as describing the future challenges and opportunities in this fast-moving field. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Structural insight into the interaction of proteins containing NPF, DPF, and GPF motifs with the C-terminal EH-domain of EHD1

PubMed Central

Kieken, Fabien; Jović, Marko; Tonelli, Marco; Naslavsky, Naava; Caplan, Steve; Sorgen, Paul L

2009-01-01

Eps15 homology (EH)-domain containing proteins are regulators of endocytic membrane trafficking. EH-domain binding to proteins containing the tripeptide NPF has been well characterized, but recent studies have shown that EH-domains are also able to interact with ligands containing DPF or GPF motifs. We demonstrate that the three motifs interact in a similar way with the EH-domain of EHD1, with the NPF motif having the highest affinity due to the presence of an intermolecular hydrogen bond. The weaker affinity for the DPF and GPF motifs suggests that if complex formation occurs in vivo, they may require high ligand concentrations, the presence of successive motifs and/or specific flanking residues. PMID:19798736

Multifunctional G-Rich and RRM-Containing Domains of TbRGG2 Perform Separate yet Essential Functions in Trypanosome RNA Editing

PubMed Central

Foda, Bardees M.; Downey, Kurtis M.; Fisk, John C.

2012-01-01

Efficient editing of Trypanosoma brucei mitochondrial RNAs involves the actions of multiple accessory factors. T. brucei RGG2 (TbRGG2) is an essential protein crucial for initiation and 3′-to-5′ progression of editing. TbRGG2 comprises an N-terminal G-rich region containing GWG and RG repeats and a C-terminal RNA recognition motif (RRM)-containing domain. Here, we perform in vitro and in vivo separation-of-function studies to interrogate the mechanism of TbRGG2 action in RNA editing. TbRGG2 preferentially binds preedited mRNA in vitro with high affinity attributable to its G-rich region. RNA-annealing and -melting activities are separable, carried out primarily by the G-rich and RRM domains, respectively. In vivo, the G-rich domain partially complements TbRGG2 knockdown, but the RRM domain is also required. Notably, TbRGG2's RNA-melting activity is dispensable for RNA editing in vivo. Interactions between TbRGG2 and MRB1 complex proteins are mediated by both G-rich and RRM-containing domains, depending on the binding partner. Overall, our results are consistent with a model in which the high-affinity RNA binding and RNA-annealing activities of the G-rich domain are essential for RNA editing in vivo. The RRM domain may have key functions involving interactions with the MRB1 complex and/or regulation of the activities of the G-rich domain. PMID:22798390

Decoy Plasminogen Receptor Containing a Selective Kunitz-Inhibitory Domain

PubMed Central

2015-01-01

Kunitz domain 1 (KD1) of tissue factor pathway inhibitor-2 in which P2′ residue Leu17 (bovine pancreatic trypsin inhibitor numbering) is mutated to Arg selectively inhibits the active site of plasmin with ∼5-fold improved affinity. Thrombin cleavage (24 h extended incubation at a 1:50 enzyme-to-substrate ratio) of the KD1 mutant (Leu17Arg) yielded a smaller molecule containing the intact Kunitz domain with no detectable change in the active-site inhibitory function. The N-terminal sequencing and MALDI-TOF/ESI data revealed that the starting molecule has a C-terminal valine (KD1L17R-VT), whereas the smaller molecule has a C-terminal lysine (KD1L17R-KT). Because KD1L17R-KT has C-terminal lysine, we examined whether it could serve as a decoy receptor for plasminogen/plasmin. Such a molecule might inhibit plasminogen activation as well as the active site of generated plasmin. In surface plasmon resonance experiments, tissue plasminogen activator (tPA) and Glu-plasminogen bound to KD1L17R-KT (Kd ∼ 0.2 to 0.3 μM) but not to KD1L17R-VT. Furthermore, KD1L17R-KT inhibited tPA-induced plasma clot fibrinolysis more efficiently than KD1L17R-VT. Additionally, compared to ε-aminocaproic acid KD1L17R-KT was more effective in reducing blood loss in a mouse liver-laceration injury model, where the fibrinolytic system is activated. In further experiments, the micro(μ)-plasmin–KD1L17R-KT complex inhibited urokinase-induced plasminogen activation on phorbol-12-myristate-13-acetate-stimulated U937 monocyte-like cells, whereas the μ-plasmin–KD1L17R-VT complex failed to inhibit this process. In conclusion, KD1L17R-KT inhibits the active site of plasmin as well as acts as a decoy receptor for the kringle domain(s) of plasminogen/plasmin; hence, it limits both plasmin generation and activity. With its dual function, KD1L17R-KT could serve as a preferred agent for controlling plasminogen activation in pathological processes. PMID:24383758

Decoy plasminogen receptor containing a selective Kunitz-inhibitory domain.

PubMed

Kumar, Yogesh; Vadivel, Kanagasabai; Schmidt, Amy E; Ogueli, Godwin I; Ponnuraj, Sathya M; Rannulu, Nalaka; Loo, Joseph A; Bajaj, Madhu S; Bajaj, S Paul

2014-01-28

Kunitz domain 1 (KD1) of tissue factor pathway inhibitor-2 in which P2' residue Leu17 (bovine pancreatic trypsin inhibitor numbering) is mutated to Arg selectively inhibits the active site of plasmin with ∼5-fold improved affinity. Thrombin cleavage (24 h extended incubation at a 1:50 enzyme-to-substrate ratio) of the KD1 mutant (Leu17Arg) yielded a smaller molecule containing the intact Kunitz domain with no detectable change in the active-site inhibitory function. The N-terminal sequencing and MALDI-TOF/ESI data revealed that the starting molecule has a C-terminal valine (KD1L17R-VT), whereas the smaller molecule has a C-terminal lysine (KD1L17R-KT). Because KD1L17R-KT has C-terminal lysine, we examined whether it could serve as a decoy receptor for plasminogen/plasmin. Such a molecule might inhibit plasminogen activation as well as the active site of generated plasmin. In surface plasmon resonance experiments, tissue plasminogen activator (tPA) and Glu-plasminogen bound to KD1L17R-KT (Kd ∼ 0.2 to 0.3 μM) but not to KD1L17R-VT. Furthermore, KD1L17R-KT inhibited tPA-induced plasma clot fibrinolysis more efficiently than KD1L17R-VT. Additionally, compared to ε-aminocaproic acid KD1L17R-KT was more effective in reducing blood loss in a mouse liver-laceration injury model, where the fibrinolytic system is activated. In further experiments, the micro(μ)-plasmin-KD1L17R-KT complex inhibited urokinase-induced plasminogen activation on phorbol-12-myristate-13-acetate-stimulated U937 monocyte-like cells, whereas the μ-plasmin-KD1L17R-VT complex failed to inhibit this process. In conclusion, KD1L17R-KT inhibits the active site of plasmin as well as acts as a decoy receptor for the kringle domain(s) of plasminogen/plasmin; hence, it limits both plasmin generation and activity. With its dual function, KD1L17R-KT could serve as a preferred agent for controlling plasminogen activation in pathological processes.

Alternative splicing for members of human mosaic domain superfamilies. I. The CH and LIM domains containing group of proteins.

PubMed

Friedberg, Felix

2009-05-01

In this paper we examine (restricted to homo sapiens) the products resulting from gene duplication and the subsequent alternative splicing for the members of a multidomain group of proteins which possess the evolutionary conserved calponin homology CH domain, i.e. an "actin binding domain", as a singlet and which, in addition, contain the conserved cysteine rich double Zn finger possessing Lim domain, also as a singlet. Seven genes, resulting from gene duplications, were identified that code for seven group members for which pre-mRNAs appear to have undergone multiple alternative splicing: Mical 1, 2 and 3 are located on chromosomes 6q21, 11p15 and 22q11, respectively. The LMO7 gene is present on chromosome 13q22 and the LIMCH1 gene on chromosome 4p13. Micall1 is mapped to chromosome 22q13 and Micall2 to chromosome 7p22. Translated Gen/Bank ESTs suggest the existence of multiple products alternatively spliced from the pre-mRNAs encoded by these genes. Characteristic indicators of such splicing among the proteins derived from one gene must include containment of some common extensive 100% identical regions. In some instances only one exon might be partly or completely eliminated. Sometimes alternative splicing is also associated with an increased frequency of creation of an exon or part of an exon from an intron. Not only coding regions for the body of the protein but also for its N- or -C ends could be affected by the splicing. If created forms are merely beginning at different starting points but remain identical in sequence thereafter, their existence as products of alternate splicing must be questioned. In the splicings, described in this paper, multiple isoforms rather than a single isoform appear as products during the gene expression.

Crystal Structure of the Human, FIC-Domain Containing Protein HYPE and Implications for Its Functions

PubMed Central

Bunney, Tom D.; Cole, Ambrose R.; Broncel, Malgorzata; Esposito, Diego; Tate, Edward W.; Katan, Matilda

2014-01-01

Summary Protein AMPylation, the transfer of AMP from ATP to protein targets, has been recognized as a new mechanism of host-cell disruption by some bacterial effectors that typically contain a FIC-domain. Eukaryotic genomes also encode one FIC-domain protein, HYPE, which has remained poorly characterized. Here we describe the structure of human HYPE, solved by X-ray crystallography, representing the first structure of a eukaryotic FIC-domain protein. We demonstrate that HYPE forms stable dimers with structurally and functionally integrated FIC-domains and with TPR-motifs exposed for protein-protein interactions. As HYPE also uniquely possesses a transmembrane helix, dimerization is likely to affect its positioning and function in the membrane vicinity. The low rate of autoAMPylation of the wild-type HYPE could be due to autoinhibition, consistent with the mechanism proposed for a number of putative FIC AMPylators. Our findings also provide a basis to further consider possible alternative cofactors of HYPE and distinct modes of target-recognition. PMID:25435325

Crystal structure of the human, FIC-domain containing protein HYPE and implications for its functions.

PubMed

Bunney, Tom D; Cole, Ambrose R; Broncel, Malgorzata; Esposito, Diego; Tate, Edward W; Katan, Matilda

2014-12-02

Protein AMPylation, the transfer of AMP from ATP to protein targets, has been recognized as a new mechanism of host-cell disruption by some bacterial effectors that typically contain a FIC-domain. Eukaryotic genomes also encode one FIC-domain protein,HYPE, which has remained poorly characterized.Here we describe the structure of human HYPE, solved by X-ray crystallography, representing the first structure of a eukaryotic FIC-domain protein. We demonstrate that HYPE forms stable dimers with structurally and functionally integrated FIC-domains and with TPR-motifs exposed for protein-protein interactions. As HYPE also uniquely possesses a transmembrane helix, dimerization is likely to affect its positioning and function in the membrane vicinity. The low rate of auto AMPylation of the wild-type HYPE could be due to autoinhibition, consistent with the mechanism proposed for a number of putative FIC AMPylators. Our findings also provide a basis to further consider possible alternative cofactors of HYPE and distinct modes of target-recognition.

The Popeye domain containing genes: essential elements in heart rate control

PubMed Central

Schindler, Roland F.; Poon, Kar Lai; Simrick, Subreena

2012-01-01

The Popeye domain containing (Popdc) gene family displays preferential expression in skeletal muscle and heart. Only recently a significant gain in the understanding of the function of Popdc genes in the heart has been obtained. The Popdc genes encode membrane proteins harboring an evolutionary conserved Popeye domain, which functions as a binding domain for cyclic adenosine monophosphate (cAMP). Popdc proteins interact with the two-pore channel TREK-1 and enhance its current. This protein interaction is modulated by cAMP. Null mutations of members of the Popdc gene family in zebrafish and mouse are associated with severe cardiac arrhythmia phenotypes. While in zebrafish an atrioventricular block was prevalent, in mouse a stress-induced sinus bradycardia was observed, which was due to the presence of sinus pauses. Moreover, the phenotype develops in an age-dependent manner, being absent in the young animal and becoming increasingly severe, as the animals grow older. This phenotype is reminiscent of the sick sinus syndrome (SSS), which affects mostly the elderly and is characterized by the poor ability of the cardiac pacemaker to adapt the heart rate to the physiological demand. While being a prevalent disease, which is responsible for a large fraction of pacemaker implantations in Western countries, SSS is poorly understood at the molecular level. It is therefore expected that the study of the molecular basis of the stress-induced bradycardia in Popdc mice will shed new light on the etiology of pacemaker disease. PMID:24282731

The Popeye domain containing genes: essential elements in heart rate control.

PubMed

Schindler, Roland F; Poon, Kar Lai; Simrick, Subreena; Brand, Thomas

2012-12-01

The Popeye domain containing (Popdc) gene family displays preferential expression in skeletal muscle and heart. Only recently a significant gain in the understanding of the function of Popdc genes in the heart has been obtained. The Popdc genes encode membrane proteins harboring an evolutionary conserved Popeye domain, which functions as a binding domain for cyclic adenosine monophosphate (cAMP). Popdc proteins interact with the two-pore channel TREK-1 and enhance its current. This protein interaction is modulated by cAMP. Null mutations of members of the Popdc gene family in zebrafish and mouse are associated with severe cardiac arrhythmia phenotypes. While in zebrafish an atrioventricular block was prevalent, in mouse a stress-induced sinus bradycardia was observed, which was due to the presence of sinus pauses. Moreover, the phenotype develops in an age-dependent manner, being absent in the young animal and becoming increasingly severe, as the animals grow older. This phenotype is reminiscent of the sick sinus syndrome (SSS), which affects mostly the elderly and is characterized by the poor ability of the cardiac pacemaker to adapt the heart rate to the physiological demand. While being a prevalent disease, which is responsible for a large fraction of pacemaker implantations in Western countries, SSS is poorly understood at the molecular level. It is therefore expected that the study of the molecular basis of the stress-induced bradycardia in Popdc mice will shed new light on the etiology of pacemaker disease.

c-di-GMP Turn-Over in Clostridium difficile Is Controlled by a Plethora of Diguanylate Cyclases and Phosphodiesterases

PubMed Central

Bordeleau, Eric; Fortier, Louis-Charles; Malouin, François; Burrus, Vincent

2011-01-01

Clostridium difficile infections have become a major healthcare concern in the last decade during which the emergence of new strains has underscored this bacterium's capacity to cause persistent epidemics. c-di-GMP is a bacterial second messenger regulating diverse bacterial phenotypes, notably motility and biofilm formation, in proteobacteria such as Vibrio cholerae, Pseudomonas aeruginosa, and Salmonella. c-di-GMP is synthesized by diguanylate cyclases (DGCs) that contain a conserved GGDEF domain. It is degraded by phosphodiesterases (PDEs) that contain either an EAL or an HD-GYP conserved domain. Very little is known about the role of c-di-GMP in the regulation of phenotypes of Gram-positive or fastidious bacteria. Herein, we exposed the main components of c-di-GMP signalling in 20 genomes of C. difficile, revealed their prevalence, and predicted their enzymatic activity. Ectopic expression of 31 of these conserved genes was carried out in V. cholerae to evaluate their effect on motility and biofilm formation, two well-characterized phenotype alterations associated with intracellular c-di-GMP variation in this bacterium. Most of the predicted DGCs and PDEs were found to be active in the V. cholerae model. Expression of truncated versions of CD0522, a protein with two GGDEF domains and one EAL domain, suggests that it can act alternatively as a DGC or a PDE. The activity of one purified DGC (CD1420) and one purified PDE (CD0757) was confirmed by in vitro enzymatic assays. GTP was shown to be important for the PDE activity of CD0757. Our results indicate that, in contrast to most Gram-positive bacteria including its closest relatives, C. difficile encodes a large assortment of functional DGCs and PDEs, revealing that c-di-GMP signalling is an important and well-conserved signal transduction system in this human pathogen. PMID:21483756

Serine protease inhibitors containing a Kunitz domain: their role in modulation of host inflammatory responses and parasite survival.

PubMed

de Magalhães, Mariana T Q; Mambelli, Fábio S; Santos, Bruno P O; Morais, Suellen B; Oliveira, Sergio C

2018-03-31

Proteins containing a Kunitz domain have the typical serine protease inhibition function ranging from sea anemone to man. Protease inhibitors play major roles in infection, inflammation disorders and cancer. This review discusses the role of serine proteases containing a Kunitz domain in immunomodulation induced by helminth parasites. Helminth parasites are associated with protection from inflammatory conditions. Therefore, interest has raised whether worm parasites or their products hold potential as drugs for treatment of immunological disorders. Finally, we also propose the use of recombinant SmKI-1 from Schistosoma mansoni as a potential therapeutic molecule to treat inflammatory diseases. Copyright © 2018 Institut Pasteur. Published by Elsevier Masson SAS. 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

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.

Change in single cystathionine β-synthase domain-containing protein from a bent to flat conformation upon adenosine monophosphate binding.

PubMed

Jeong, Byung-Cheon; Park, Si Hoon; Yoo, Kyoung Shin; Shin, Jeong Sheop; Song, Hyun Kyu

2013-07-01

Cystathionine β-synthase (CBS) domains are small intracellular modules that can act as binding domains for adenosine derivatives, and they may regulate the activity of associated enzymes or other functional domains. Among these, the single CBS domain-containing proteins, CBSXs, from Arabidopsis thaliana, have recently been identified as redox regulators of the thioredoxin system. Here, the crystal structure of CBSX2 in complex with adenosine monophosphate (AMP) is reported at 2.2Å resolution. The structure of dimeric CBSX2 with bound-AMP is shown to be approximately flat, which is in stark contrast to the bent form of apo-CBSXs. This conformational change in quaternary structure is triggered by a local structural change of the unique α5 helix, and by moving each loop P into an open conformation to accommodate incoming ligands. Furthermore, subtle rearrangement of the dimer interface triggers movement of all subunits, and consequently, the bent structure of the CBSX2 dimer becomes a flat structure. This reshaping of the structure upon complex formation with adenosine-containing ligand provides evidence that ligand-induced conformational reorganization of antiparallel CBS domains is an important regulatory mechanism. Copyright © 2013 Elsevier Inc. All rights reserved.

Fungal mediator tail subunits contain classical transcriptional activation domains.

PubMed

Liu, Zhongle; Myers, Lawrence C

2015-04-01

Classical activation domains within DNA-bound eukaryotic transcription factors make weak interactions with coactivator complexes, such as Mediator, to stimulate transcription. How these interactions stimulate transcription, however, is unknown. The activation of reporter genes by artificial fusion of Mediator subunits to DNA binding domains that bind to their promoters has been cited as evidence that the primary role of activators is simply to recruit Mediator. We have identified potent classical transcriptional activation domains in the C termini of several tail module subunits of Saccharomyces cerevisiae, Candida albicans, and Candida dubliniensis Mediator, while their N-terminal domains are necessary and sufficient for their incorporation into Mediator but do not possess the ability to activate transcription when fused to a DNA binding domain. This suggests that Mediator fusion proteins actually are functioning in a manner similar to that of a classical DNA-bound activator rather than just recruiting Mediator. Our finding that deletion of the activation domains of S. cerevisiae Med2 and Med3, as well as C. dubliniensis Tlo1 (a Med2 ortholog), impairs the induction of certain genes shows these domains function at native promoters. Activation domains within coactivators are likely an important feature of these complexes and one that may have been uniquely leveraged by a common fungal pathogen. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

WW domain-containing oxidoreductase promotes neuronal differentiation via negative regulation of glycogen synthase kinase 3β.

PubMed

Wang, H-Y; Juo, L-I; Lin, Y-T; Hsiao, M; Lin, J-T; Tsai, C-H; Tzeng, Y-H; Chuang, Y-C; Chang, N-S; Yang, C-N; Lu, P-J

2012-06-01

WW domain-containing oxidoreductase (WWOX), a putative tumour suppressor, is suggested to be involved in the hyperphosphorylation of Alzheimer's Tau. Tau is a microtubule-associated protein that has an important role in microtubule assembly and stability. Glycogen synthase kinase 3β (GSK3β) has a vital role in Tau hyperphosphorylation at its microtubule-binding domains. Hyperphosphorylated Tau has a low affinity for microtubules, thus disrupting microtubule stability. Bioinformatics analysis indicated that WWOX contains two potential GSK3β-binding FXXXLI/VXRLE motifs. Immunofluorescence, immunoprecipitation and molecular modelling showed that WWOX interacts physically with GSK3β. We demonstrated biochemically that WWOX can bind directly to GSK3β through its short-chain alcohol dehydrogenase/reductase domain. Moreover, the overexpression of WWOX inhibited GSK3β-stimulated S396 and S404 phosphorylation within the microtubule domains of Tau, indicating that WWOX is involved in regulating GSK3β activity in cells. WWOX repressed GSK3β activity, restored the microtubule assembly activity of Tau and promoted neurite outgrowth in SH-SY5Y cells. Conversely, RNAi-mediated knockdown of WWOX in retinoic acid (RA)-differentiated SH-SY5Y cells inhibited neurite outgrowth. These results suggest that WWOX is likely to be involved in regulating GSK3β activity, reducing the level of phosphorylated Tau, and subsequently promoting neurite outgrowth during neuron differentiation. In summary, our data reveal a novel mechanism by which WWOX promotes neuronal differentiation in response to RA.

Forkhead-associated (FHA) Domain Containing ABC Transporter Rv1747 Is Positively Regulated by Ser/Thr Phosphorylation in Mycobacterium tuberculosis*

PubMed Central

Spivey, Vicky L.; Molle, Virginie; Whalan, Rachael H.; Rodgers, Angela; Leiba, Jade; Stach, Lasse; Walker, K. Barry; Smerdon, Stephen J.; Buxton, Roger S.

2011-01-01

One major signaling method employed by Mycobacterium tuberculosis, the causative agent of tuberculosis, is through reversible phosphorylation of proteins mediated by protein kinases and phosphatases. This study concerns one of these enzymes, the serine/threonine protein kinase PknF, that is encoded in an operon with Rv1747, an ABC transporter that is necessary for growth of M. tuberculosis in vivo and contains two forkhead-associated (FHA) domains. FHA domains are phosphopeptide recognition motifs that specifically recognize phosphothreonine-containing epitopes. Experiments to determine how PknF regulates the function of Rv1747 demonstrated that phosphorylation occurs on two specific threonine residues, Thr-150 and Thr-208. To determine the in vivo consequences of phosphorylation, infection experiments were performed in bone marrow-derived macrophages and in mice using threonine-to-alanine mutants of Rv1747 that prevent specific phosphorylation and revealed that phosphorylation positively modulates Rv1747 function in vivo. The role of the FHA domains in this regulation was further demonstrated by isothermal titration calorimetry, using peptides containing both phosphothreonine residues. FHA-1 domain mutation resulted in attenuation in macrophages highlighting the critical role of this domain in Rv1747 function. A mutant deleted for pknF did not, however, have a growth phenotype in an infection, suggesting that other kinases can fulfill its role when it is absent. This study provides the first information on the molecular mechanism(s) regulating Rv1747 through PknF-dependent phosphorylation but also indicates that phosphorylation activates Rv1747, which may have important consequences in regulating growth of M. tuberculosis. PMID:21622570

Relative increase in Alzheimer's disease of soluble forms of cerebral Abeta amyloid protein precursor containing the Kunitz protease inhibitory domain.

PubMed

Moir, R D; Lynch, T; Bush, A I; Whyte, S; Henry, A; Portbury, S; Multhaup, G; Small, D H; Tanzi, R E; Beyreuther, K; Masters, C L

1998-02-27

Although a number of studies have examined amyloid precursor protein (APP) mRNA levels in Alzheimer's disease (AD), no clear consensus has emerged as to whether the levels of transcripts for isoforms containing a Kunitz protease inhibitory (KPI)-encoded region are increased or decreased in AD. Here we compare AD and control brain for the relative amounts of APP protein containing KPI to APP protein lacking this domain. APP protein was purified from the soluble subcellular fraction and Triton X-100 membrane pellet extract of one hemisphere of AD (n = 10), normal (n = 7), and neurological control (n = 5) brains. The amount of KPI-containing APP in the purified protein samples was determined using two independent assay methods. The first assay exploited the inhibitory action of KPI-containing APP on trypsin. The second assay employed reflectance analysis of Western blots. The proportion of KPI-containing forms of APP in the soluble subcellular fraction of AD brains is significantly elevated (p < 0.01) compared with controls. Species containing a KPI domain comprise 32-41 and 76-77% of purified soluble APP from control and AD brains, respectively. For purified membrane-associated APP, 72-77 and 65-82% of control and AD samples, respectively, contain a KPI domain. Since KPI-containing species of APP may be more amyloidogenic (Ho, L., Fukuchi, K., and Yonkin, S. G. (1996) J. Biol. Chem. 271, 30929-30934), our findings support an imbalance of isoforms as one possible mechanism for amyloid deposition in sporadic AD.

The Presentation of Animals in English as an Additional Language Coursebooks

ERIC Educational Resources Information Center

Jacobs, George M.; Jiexin, Teh; Michael, Joyce J.

2016-01-01

Education materials can be analysed in many ways. The current study analysed EAL (English as an Additional Language) coursebooks as to the presentation of nonhuman animals in the books. The study examined 22 EAL coursebooks. The research looked at the percentage of activities that contained animals in the coursebooks, what types of animals were…

The N-terminal Region of the Ubiquitin Regulatory X (UBX) Domain-containing Protein 1 (UBXD1) Modulates Interdomain Communication within the Valosin-containing Protein p97*

PubMed Central

Trusch, Franziska; Matena, Anja; Vuk, Maja; Koerver, Lisa; Knævelsrud, Helene; Freemont, Paul S.; Meyer, Hemmo; Bayer, Peter

2015-01-01

Valosin-containing protein/p97 is an ATP-driven protein segregase that cooperates with distinct protein cofactors to control various aspects of cellular homeostasis. Mutations at the interface between the regulatory N-domain and the first of two ATPase domains (D1 and D2) deregulate the ATPase activity and cause a multisystem degenerative disorder, inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia/amyotrophic lateral sclerosis. Intriguingly, the mutations affect only a subset of p97-mediated pathways correlating with unbalanced cofactor interactions and most prominently compromised binding of the ubiquitin regulatory X domain-containing protein 1 (UBXD1) cofactor during endolysosomal sorting of caveolin-1. However, how the mutations impinge on the p97-cofactor interplay is unclear so far. In cell-based endosomal localization studies, we identified a critical role of the N-terminal region of UBXD1 (UBXD1-N). Biophysical studies using NMR and CD spectroscopy revealed that UBXD1-N can be classified as intrinsically disordered. NMR titration experiments confirmed a valosin-containing protein/p97 interaction motif and identified a second binding site at helices 1 and 2 of UBXD1-N as binding interfaces for p97. In reverse titration experiments, we identified two distant epitopes on the p97 N-domain that include disease-associated residues and an additional interaction between UBXD1-N and the D1D2 barrel of p97 that was confirmed by fluorescence anisotropy. Functionally, binding of UBXD1-N to p97 led to a reduction of ATPase activity and partial protection from proteolysis. These findings indicate that UBXD1-N intercalates into the p97-ND1 interface, thereby modulating interdomain communication of p97 domains and its activity with relevance for disease pathogenesis. We propose that the polyvalent binding mode characterized for UBXD1-N is a more general principle that defines a subset of p97 cofactors. PMID:26475856

RHOMBOID DOMAIN CONTAINING 2 (RHBDD2)

PubMed Central

Abba, MC; Lacunza, E; Nunez, MI; Colussi, A; Isla-Larrain, M; Segal-Eiras, A; Croce, MV; Aldaz, CM

2009-01-01

In the course of breast cancer global gene expression studies, we identified an uncharacterized gene known as RHBDD2 (Rhomboid domain containing 2) to be markedly over-expressed in primary tumors from patients with recurrent disease. In this study, we identified RHBDD2 mRNA and protein expression significantly elevated in breast carcinomas compared with normal breast samples as analyzed by SAGE (n=46) and immunohistochemistry (n=213). Interestingly, specimens displaying RHBDD2 over-expression were predominantly advanced stage III breast carcinomas (p=0.001). Western-blot, RT-PCR and cDNA sequencing analyses allowed us to identify two RHBDD2 alternatively spliced mRNA isoforms expressed in breast cancer cell lines. We further investigated the occurrence and frequency of gene amplification and over-expression affecting RHBDD2 in 131 breast samples. RHBDD2 gene amplification was detected in 21% of 98 invasive breast carcinomas analyzed. However, no RHBDD2 amplification was detected in normal breast tissues (n=17) or breast benign lesions (n=16) (p=0.014). Interestingly, siRNA mediated silencing of RHBDD2 expression results in a decrease of MCF7 breast cancer cells proliferation compared with the corresponding controls (p=0.001). In addition, analysis of publicly available gene expression data showed a strong association between high RHBDD2 expression and decreased overall survival (p=0.0023), relapse-free survival (p= 0.0013), and metastasis-free interval (p=0.006) in patients with primary ER-negative breast carcinomas. In conclusion, our findings suggest that RHBDD2 over-expression behaves as an indicator of poor prognosis and may play a role facilitating breast cancer progression. PMID:19616622

Arabidopsis F-box protein containing a Nictaba-related lectin domain interacts with N-acetyllactosamine structures.

PubMed

Stefanowicz, Karolina; Lannoo, Nausicaä; Proost, Paul; Van Damme, Els J M

2012-01-01

The Arabidopsis thaliana genome contains a small group of bipartite F-box proteins, consisting of an N-terminal F-box domain and a C-terminal domain sharing sequence similarity with Nictaba, the jasmonate-induced glycan-binding protein (lectin) from tobacco. Based on the high sequence similarity between the C-terminal domain of these proteins and Nictaba, the hypothesis was put forward that the so-called F-box-Nictaba proteins possess carbohydrate-binding activity and accordingly can be considered functional homologs of the mammalian sugar-binding F-box or Fbs proteins which are involved in proteasomal degradation of glycoproteins. To obtain experimental evidence for the carbohydrate-binding activity and specificity of the A. thaliana F-box-Nictaba proteins, both the complete F-box-Nictaba sequence of one selected Arabidopsis F-box protein (in casu At2g02360) as well as the Nictaba-like domain only were expressed in Pichia pastoris and analyzed by affinity chromatography, agglutination assays and glycan micro-array binding assays. These results demonstrated that the C-terminal Nictaba-like domain provides the F-box-protein with a carbohydrate-binding activity that is specifically directed against N- and O-glycans containing N-acetyllactosamine (Galβ1-3GlcNAc and Galβ1-4GlcNAc) and poly-N-acetyllactosamine ([Galβ1-4GlcNAc]n) as well as Lewis A (Galβ1-3(Fucα1-4)GlcNAc), Lewis X (Galβ1-4(Fucα1-3)GlcNAc, Lewis Y (Fucα1-2Galβ1-4(Fucα1-3)GlcNAc) and blood type B (Galα1-3(Fucα1-2)Galβ1-3GlcNAc) motifs. Based on these findings one can reasonably conclude that at least the A. thaliana F-box-Nictaba protein encoded by At2g02360 can act as a carbohydrate-binding protein. The results from the glycan array assays revealed differences in sugar-binding specificity between the F-box protein and Nictaba, indicating that the same carbohydrate-binding motif can accommodate unrelated oligosaccharides.

The C-terminal extension of human RTEL1, mutated in Hoyeraal-Hreidarsson syndrome, contains harmonin-N-like domains.

PubMed

Faure, Guilhem; Revy, Patrick; Schertzer, Michael; Londono-Vallejo, Arturo; Callebaut, Isabelle

2014-06-01

Several studies have recently shown that germline mutations in RTEL1, an essential DNA helicase involved in telomere regulation and DNA repair, cause Hoyeraal-Hreidarsson syndrome (HHS), a severe form of dyskeratosis congenita. Using original new softwares, facilitating the delineation of the different domains of the protein and the identification of remote relationships for orphan domains, we outline here that the C-terminal extension of RTEL1, downstream of its catalytic domain and including several HHS-associated mutations, contains a yet unidentified tandem of harmonin-N-like domains, which may serve as a hub for partner interaction. This finding highlights the potential critical role of this region for the function of RTEL1 and gives insights into the impact that the identified mutations would have on the structure and function of these domains. © 2013 Wiley Periodicals, Inc.

The cyclic-di-GMP phosphodiesterase BinA negatively regulates cellulose-containing biofilms in Vibrio fischeri.

PubMed

Bassis, Christine M; Visick, Karen L

2010-03-01

Bacteria produce different types of biofilms under distinct environmental conditions. Vibrio fischeri has the capacity to produce at least two distinct types of biofilms, one that relies on the symbiosis polysaccharide Syp and another that depends upon cellulose. A key regulator of biofilm formation in bacteria is the intracellular signaling molecule cyclic diguanylate (c-di-GMP). In this study, we focused on a predicted c-di-GMP phosphodiesterase encoded by the gene binA, located directly downstream of syp, a cluster of 18 genes critical for biofilm formation and the initiation of symbiotic colonization of the squid Euprymna scolopes. Disruption or deletion of binA increased biofilm formation in culture and led to increased binding of Congo red and calcofluor, which are indicators of cellulose production. Using random transposon mutagenesis, we determined that the phenotypes of the DeltabinA mutant strain could be disrupted by insertions in genes in the bacterial cellulose biosynthesis cluster (bcs), suggesting that cellulose production is negatively regulated by BinA. Replacement of critical amino acids within the conserved EAL residues of the EAL domain disrupted BinA activity, and deletion of binA increased c-di-GMP levels in the cell. Together, these data support the hypotheses that BinA functions as a phosphodiesterase and that c-di-GMP activates cellulose biosynthesis. Finally, overexpression of the syp regulator sypG induced binA expression. Thus, this work reveals a mechanism by which V. fischeri inhibits cellulose-dependent biofilm formation and suggests that the production of two different polysaccharides may be coordinated through the action of the cellulose inhibitor BinA.

Biotin Attachment Domain-Containing Proteins Irreversibly Inhibit Acetyl CoA Carboxylase

DOE PAGES

Keereetaweep, Jantana; Liu, Hui; Zhai, Zhiyang; ...

2018-04-06

The first committed step in fatty acid synthesis is mediated by Acetyl-CoA carboxylase (ACCase), a biotin-dependent enzyme that carboxylates acetyl-CoA to produce malonyl-CoA. ACCase can be feedback-regulated by short-term (reversible) and longer-term (irreversible) inhibition upon oversupply of fatty acids (FA) provided by Tween80 (predominantly containing oleic acid; 18:1). Biotin-Attachment-Domain-Containing (BADC) proteins are inactive analogs of biotin carboxyl transfer protein (BCCP) that lack biotin and their incorporation into ACCase downregulates it by displacing active (biotin-containing) BCCP subunits. Individual T-DNA insertion lines of BADC1, BADC2, and BADC3 were used to generate badc1badc2 and badc1badc3. The badc1badc3 mutant and wild-type exhibited normal growthmore » and development, however ACCase activity was 26% higher in badc1badc3 relative to wild-type and its seeds contained 30.1 %DW more FA and 32.6 %DW more TAG than wild-type. Cell suspension cultures were generated from leaves of badc1badc3 and wild-type plants to test whether BADC contributes to the irreversible phase of ACCase inhibition resulting from culture in medium containing 10mM Tween80. While the reversible phase of ACCase inhibition after two days of Tween80 feeding was equivalent for badc1badc3 and wild-type, the irreversible phase of inhibition following four days of Tween80 feeding was reduced by 50% in badc1badc3 relative to wild-type. In this work we present evidence for two important homeostatic roles for BADC proteins in downregulating ACCase activity: during normal growth and development, and by contributing to its long-term irreversible feedback inhibition resulting from oversupply of fatty acids.« less

Biotin Attachment Domain-Containing Proteins Irreversibly Inhibit Acetyl CoA Carboxylase

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

Keereetaweep, Jantana; Liu, Hui; Zhai, Zhiyang

The first committed step in fatty acid synthesis is mediated by Acetyl-CoA carboxylase (ACCase), a biotin-dependent enzyme that carboxylates acetyl-CoA to produce malonyl-CoA. ACCase can be feedback-regulated by short-term (reversible) and longer-term (irreversible) inhibition upon oversupply of fatty acids (FA) provided by Tween80 (predominantly containing oleic acid; 18:1). Biotin-Attachment-Domain-Containing (BADC) proteins are inactive analogs of biotin carboxyl transfer protein (BCCP) that lack biotin and their incorporation into ACCase downregulates it by displacing active (biotin-containing) BCCP subunits. Individual T-DNA insertion lines of BADC1, BADC2, and BADC3 were used to generate badc1badc2 and badc1badc3. The badc1badc3 mutant and wild-type exhibited normal growthmore » and development, however ACCase activity was 26% higher in badc1badc3 relative to wild-type and its seeds contained 30.1 %DW more FA and 32.6 %DW more TAG than wild-type. Cell suspension cultures were generated from leaves of badc1badc3 and wild-type plants to test whether BADC contributes to the irreversible phase of ACCase inhibition resulting from culture in medium containing 10mM Tween80. While the reversible phase of ACCase inhibition after two days of Tween80 feeding was equivalent for badc1badc3 and wild-type, the irreversible phase of inhibition following four days of Tween80 feeding was reduced by 50% in badc1badc3 relative to wild-type. In this work we present evidence for two important homeostatic roles for BADC proteins in downregulating ACCase activity: during normal growth and development, and by contributing to its long-term irreversible feedback inhibition resulting from oversupply of fatty acids.« less

EAL Teacher Agency: Implications for Participation in Professional Development

ERIC Educational Resources Information Center

Gurney, Laura; Liyanage, Indika

2016-01-01

Teachers construct their practice, education and professional development within two domains of professionalism: sponsored and independent. The association between these two domains, however, is complex; it is overlapping, inseparable and sometimes uneasy. The complexity is further exacerbated by the codependent nature of association between the…

TGN38 is maintained in the trans-Golgi network by a tyrosine-containing motif in the cytoplasmic domain.

PubMed Central

Bos, K; Wraight, C; Stanley, K K

1993-01-01

Sorting of proteins destined for different plasma membrane domains, lysosomes and secretory pathways takes place in the trans-Golgi network (TGN). TGN38 is an integral membrane protein found in this intracellular compartment. We show that TGN38 contains an autonomous targeting signal within its cytoplasmic domain which determines its intracellular location. Deletion analysis and site-directed mutagenesis of this domain demonstrate that a tyrosine motif homologous to the internalization signal of surface receptors is necessary and sufficient for correct localization. These findings suggest that TGN38 is maintained in the TGN by retrieval from the plasma membrane and employs a different mechanism for retention from that of the transferase enzymes of the trans-Golgi. Images PMID:8491209

A single WAP domain (SWD)-containing protein with antiviral activity from Pacific white shrimp Litopenaeus vannamei.

PubMed

Yang, Linwei; Niu, Shengwen; Gao, Jiefeng; Zuo, Hongliang; Yuan, Jia; Weng, Shaoping; He, Jianguo; Xu, Xiaopeng

2018-02-01

The single whey acidic protein (WAP) domain (SWD)-containing proteins, also called type III crustins, are a group of antimicrobial peptides (AMPs) in crustaceans. At present, a number of SWDs have been identified in shrimp, which showed essential antibacterial activities. However, the roles of SWDs in antiviral immune responses have not been reported up to now. In this study, a novel SWD (LvSWD3) was identified from Pacific white shrimp, Litopenaeus vannamei, which contained a typical single WAP domain homologous to those of other crustacean SWDs. Although lacking the pro and arg-rich region between the signal peptide and the WAP domain, LvSWD3 was closely clustered with other shrimp SWDs in the phylogenetic tree. Similar to many shrimp SWDs, the highest expression of LvSWD3 was detected in hemocytes. The LvSWD3 expression exhibited only limited changes after challenges with Vibrio parahaemolyticus, Poly (I:C) and lipopolysaccharide, but was significantly up-regulated after white spot syndrome virus (WSSV) infection. Silencing of LvSWDs significantly accelerated the death of the WSSV-infected but not the V. parahaemolyticus-infected shrimp. The recombinant LvSWD3 protein did not show proteinase inhibitory and antibacterial activities but could significantly postpone the death of WSSV-infected shrimp and reduce the viral load in tissues. These suggested that LvSWD3 was a novel SWD with antiviral activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Genome-wide search and comparative genomic analysis of the trypsin inhibitor-like cysteine-rich domain-containing peptides.

PubMed

Zeng, Xian-Chun; Liu, Yichen; Shi, Wanxia; Zhang, Lei; Luo, Xuesong; Nie, Yao; Yang, Ye

2014-03-01

It was shown that peptides containing trypsin inhibitor-like cysteine-rich (TIL) domain are able to inhibit proteinase activities, and thus play important roles in various biological processes, such as immune response and anticoagulation. However, only a limited number of the TIL peptides have been identified and characterized so far; and little has been known about the evolutionary relationships of the genes encoding the TIL peptides. BmKAPi is a TIL domain-containing peptide that was identified from Mesobuthus martensii Karsch. Here, we conducted genome-wide searches for new peptides that are homologous to BmKAPi or possess a cysteine pattern similar to that of BmKAPi. As a result, we identified a total of 80 different TIL peptides from 34 species of arthropods. We found that these peptides can be classified into seven evolutionarily distinct groups. Furthermore, we cloned the genomic sequence of BmKAPi; the genomic sequences of the majority of other TIL peptides were also identified from the GenBank database using bioinformatical approaches. Through phylogenetic and comparative genomic analysis, we found 26 cases of intron gain events occurred in the genes of the TIL peptides; however, no instances of intron loss were observed. Moreover, we found that alternative splicing contributes to the diversification of the TIL peptides. It is interesting to see that four genes of the TIL domain-containing peptides overlap in a DNA region located on the chromosome LG B15 of Bombus terretris. These data suggest that the evolution of the TIL peptide genes are dynamic, which was dominated by intron gain. Copyright © 2013 Elsevier Inc. All rights reserved.

Ubiquitin Regulates Caspase Recruitment Domain-mediated Signaling by Nucleotide-binding Oligomerization Domain-containing Proteins NOD1 and NOD2*

PubMed Central

Ver Heul, Aaron M.; Fowler, C. Andrew; Ramaswamy, S.; Piper, Robert C.

2013-01-01

NOD1 and NOD2 (nucleotide-binding oligomerization domain-containing proteins) are intracellular pattern recognition receptors that activate inflammation and autophagy. These pathways rely on the caspase recruitment domains (CARDs) within the receptors, which serve as protein interaction platforms that coordinately regulate immune signaling. We show that NOD1 CARD binds ubiquitin (Ub), in addition to directly binding its downstream targets receptor-interacting protein kinase 2 (RIP2) and autophagy-related protein 16-1 (ATG16L1). NMR spectroscopy and structure-guided mutagenesis identified a small hydrophobic surface of NOD1 CARD that binds Ub. In vitro, Ub competes with RIP2 for association with NOD1 CARD. In vivo, we found that the ligand-stimulated activity of NOD1 with a mutant CARD lacking Ub binding but retaining ATG16L1 and RIP2 binding is increased relative to wild-type NOD1. Likewise, point mutations in the tandem NOD2 CARDs at positions analogous to the surface residues defining the Ub interface on NOD1 resulted in loss of Ub binding and increased ligand-stimulated NOD2 signaling. These data suggest that Ub binding provides a negative feedback loop upon NOD-dependent activation of RIP2. PMID:23300079

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

PubMed

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

2012-12-01

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

Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration

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

Simarro, Maria; Gimenez-Cassina, Alfredo; Kedersha, Nancy

2010-10-22

Research highlights: {yields} Five members of the FAST kinase domain-containing proteins are localized to mitochondria in mammalian cells. {yields} The FASTKD3 interactome includes proteins involved in various aspects of mitochondrial metabolism. {yields} Targeted knockdown of FASTKD3 significantly reduces basal and maximal mitochondrial oxygen consumption. -- Abstract: Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated withmore » reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.« less

Insights into jumonji c-domain containing protein 6 (JMJD6): a multifactorial role in FMDV replication in cells

USDA-ARS?s Scientific Manuscript database

The Jumonji C-domain containing protein 6 (JMJD6) has had a convoluted history. It was first identified as the phosphatidylserine receptor (PSR) on the cell surface responsible for recognizing phosphatidylserine on the surface of apoptotic cells resulting in their engulfment by phagocytic cells. Sub...

Therapeutic inhibition of prolyl hydroxylase domain-containing enzymes in surgery: putative applications and challenges

PubMed Central

Harnoss, Jonathan Michael; Strowitzki, Moritz Johannes; Radhakrishnan, Praveen; Platzer, Lisa Katharina; Harnoss, Julian Camill; Hank, Thomas; Cai, Jun; Ulrich, Alexis; Schneider, Martin

2015-01-01

Oxygen is essential for metazoans to generate energy. Upon oxygen deprivation adaptive and protective pathways are induced, mediated by hypoxia-inducible factors (HIFs) and prolyl hydroxylase domain-containing enzymes (PHDs). Both play a pivotal role in various conditions associated with prolonged ischemia and inflammation, and are promising targets for therapeutic intervention. This review focuses on aspects of therapeutic PHD modulation in surgically relevant disease conditions such as hepatic and intestinal disorders, wound healing, innate immune responses, and tumorigenesis, and discusses the therapeutic potential and challenges of PHD inhibition in surgical patients. PMID:27774478

Investigation of pharmaceutical drugs and caffeine-containing foods using Fourier and terahertz time-domain spectroscopy

NASA Astrophysics Data System (ADS)

KaraliÅ«nas, Mindaugas; Venckevičius, Rimvydas; Kašalynas, Irmantas; Puc, Uroš; Abina, Andreja; Jeglič, Anton; Zidanšek, Aleksander; Valušis, Gintaras

2015-08-01

Several pharmaceutical drugs, such as alprazolam, ibuprofen, acetaminophen, activated carbon and others, and caffeine-containing foods were tested using terahertz (THz) time domain spectroscopy in the range from 0.3 to 2 THz. The dry powder of pharmaceutical drugs was mixed with HDPE and pressed into the pellets using hydraulic press. The coffee grounds were also pressed into the pellets after ball-milling and mixing with HDPE. The caffeine containing liquid foods were dried out on the paper strips of various stacking. Experiments allow one to determine characteristic spectral signatures of the investigated substances within THz range caused by active pharmaceutical ingredients, like in the case of caffeine, as well as supporting pharmaceutical ingredients. Spectroscopic THz imaging approach is considered as a possible option to identify packaged pharmaceutical drugs. The caffeine spectral features in the tested caffeine containing foods are difficult to observed due to the low caffeine concentration and complex caffeine chemical surrounding.

Domain-to-domain coupling in voltage-sensing phosphatase.

PubMed

Sakata, Souhei; Matsuda, Makoto; Kawanabe, Akira; Okamura, Yasushi

2017-01-01

Voltage-sensing phosphatase (VSP) consists of a transmembrane voltage sensor and a cytoplasmic enzyme region. The enzyme region contains the phosphatase and C2 domains, is structurally similar to the tumor suppressor phosphatase PTEN, and catalyzes the dephosphorylation of phosphoinositides. The transmembrane voltage sensor is connected to the phosphatase through a short linker region, and phosphatase activity is induced upon membrane depolarization. Although the detailed molecular characteristics of the voltage sensor domain and the enzyme region have been revealed, little is known how these two regions are coupled. In addition, it is important to know whether mechanism for coupling between the voltage sensor domain and downstream effector function is shared among other voltage sensor domain-containing proteins. Recent studies in which specific amino acid sites were genetically labeled using a fluorescent unnatural amino acid have enabled detection of the local structural changes in the cytoplasmic region of Ciona intestinalis VSP that occur with a change in membrane potential. The results of those studies provide novel insight into how the enzyme activity of the cytoplasmic region of VSP is regulated by the voltage sensor domain.

Domain-to-domain coupling in voltage-sensing phosphatase

PubMed Central

Sakata, Souhei; Matsuda, Makoto; Kawanabe, Akira; Okamura, Yasushi

2017-01-01

Voltage-sensing phosphatase (VSP) consists of a transmembrane voltage sensor and a cytoplasmic enzyme region. The enzyme region contains the phosphatase and C2 domains, is structurally similar to the tumor suppressor phosphatase PTEN, and catalyzes the dephosphorylation of phosphoinositides. The transmembrane voltage sensor is connected to the phosphatase through a short linker region, and phosphatase activity is induced upon membrane depolarization. Although the detailed molecular characteristics of the voltage sensor domain and the enzyme region have been revealed, little is known how these two regions are coupled. In addition, it is important to know whether mechanism for coupling between the voltage sensor domain and downstream effector function is shared among other voltage sensor domain-containing proteins. Recent studies in which specific amino acid sites were genetically labeled using a fluorescent unnatural amino acid have enabled detection of the local structural changes in the cytoplasmic region of Ciona intestinalis VSP that occur with a change in membrane potential. The results of those studies provide novel insight into how the enzyme activity of the cytoplasmic region of VSP is regulated by the voltage sensor domain. PMID:28744425

The Surface Layer Homology Domain-Containing Proteins of Alkaliphilic Bacillus pseudofirmus OF4 Play an Important Role in Alkaline Adaptation via Peptidoglycan Synthesis.

PubMed

Fujinami, Shun; Ito, Masahiro

2018-01-01

It is well known that the Na + cycle and the cell wall are essential for alkaline adaptation of Na + -dependent alkaliphilic Bacillus species. In Bacillus pseudofirmus OF4, surface layer protein A (SlpA), the most abundant protein in the surface layer (S-layer) of the cell wall, is involved in alkaline adaptation, especially under low Na + concentrations. The presence of a large number of genes that encode S-layer homology (SLH) domain-containing proteins has been suggested from the genome sequence of B. pseudofirmus OF4. However, other than SlpA, the functions of SLH domain-containing proteins are not well known. Therefore, a deletion mutant of the csaB gene, required for the retention of SLH domain-containing proteins on the cell wall, was constructed to investigate its physiological properties. The csaB mutant strain of B. pseudofirmus OF4 had a chained morphology and alkaline sensitivity even under a 230 mM Na + concentration at which there is no growth difference between the parental strain and the slpA mutant strain. Ultra-thin section transmission electron microscopy showed that a csaB mutant strain lacked an S-layer part, and its peptidoglycan (PG) layer was disturbed. The slpA mutant strain also lacked an S-layer part, although its PG layer was not disturbed. These results suggested that the surface layer homology domain-containing proteins of B. pseudofirmus OF4 play an important role in alkaline adaptation via peptidoglycan synthesis.

Entry into the nuclear pore complex is controlled by a cytoplasmic exclusion zone containing dynamic GLFG-repeat nucleoporin domains.

PubMed

Fiserova, Jindriska; Spink, Matthew; Richards, Shane A; Saunter, Christopher; Goldberg, Martin W

2014-01-01

Nuclear pore complexes (NPCs) mediate nucleocytoplasmic movement. The central channel contains proteins with phenylalanine-glycine (FG) repeats, or variations (GLFG, glycine-leucine-phenylalanine-glycine). These are 'intrinsically disordered' and often represent weak interaction sites that become ordered upon interaction. We investigated this possibility during nuclear transport. Using electron microscopy of S. cerevisiae, we show that NPC cytoplasmic filaments form a dome-shaped structure enclosing GLFG domains. GLFG domains extend out of this structure and are part of an 'exclusion zone' that might act as a partial barrier to entry of transport-inert proteins. The anchor domain of a GLFG nucleoporin locates exclusively to the central channel. By contrast, the localisation of the GLFG domains varied between NPCs and could be cytoplasmic, central or nucleoplasmic and could stretch up to 80 nm. These results suggest a dynamic exchange between ordered and disordered states. In contrast to diffusion through the NPC, transport cargoes passed through the exclusion zone and accumulated near the central plane. We also show that movement of cargo through the NPC is accompanied by relocation of GLFG domains, suggesting that binding, restructuring and movement of these domains could be part of the translocation mechanism.

Research Advances in CKLFSF-like MARVEL Transmembrane Domain Containing Member 3.

PubMed

Hu, Feng-zhan; Sheng, Zheng-zuo; Qin, Cai-peng; Xu, Tao

2016-06-10

CKLF-like MARVEL transmembrane domain containing member/chemokine-like factor super family member (CKLFSF/CMTM) is a novel tumor suppressor gene. CMTM3 is broadly expressed in normal human tissues and evolutionary conserved,especially in testis,spleen,and some cells of peripheral blood mononuclear cells. However,its expression is undetectable or down-regulated in most carcinoma cell lines and tissues. Restoration of CMTM3 may inhibit the proliferation,migration,and invasion of carcinoma cells. Although the exact mechanism of its anti-tumor activity remains unclear,CKLFSF3/CMTM3 is closely connected with immune system and associated with sex during tumorigenesis. The study advances of CKLFSF3/CMTM3 are elaborated in this review as CMTM3 may be a new target in the gene therapies for tumors,especially genitourinary tumors,while further studies on CMTM3 and its anti-tumor mechanisms are warranted.

TAIL1: an isthmin-like gene, containing type 1 thrombospondin-repeat and AMOP domain, mapped to ARVD1 critical region.

PubMed

Rossi, Valeria; Beffagna, Giorgia; Rampazzo, Alessandra; Bauce, Barbara; Danieli, Gian Antonio

2004-06-23

Isthmins represent a novel family of vertebrate secreted proteins containing one copy of the thrombospondin type 1 repeat (TSR), which in mammals is shared by several proteins with diverse biological functions, including cell adhesion, angiogenesis, and patterning of developing nervous system. We have determined the genomic organization of human TAIL1 (thrombospondin and AMOP containing isthmin-like 1), a novel isthmin-like gene encoding a protein that contains a TSR and a C-terminal AMOP domain (adhesion-associated domain in MUC4 and other proteins), characteristic of extracellular proteins involved in adhesion processes. TAIL1 gene encompasses more than 24.4 kb. Analysis of the DNA sequence surrounding the putative transcriptional start region revealed a TATA-less promoter located in a CpG island. Several consensus binding sites for the transcription factors Sp1 and MZF-1 were identified in this promoter region. In humans, TAIL1 gene is located on chromosome 14q24.3 within ARVD1 (arrhythmogenic right ventricular dysplasia/cardiomyopathy, type 1) critical region; preliminary evidence suggests that it is expressed in several tissues, showing multiple alternative splicing.

Cohesin and related coiled-coil domain-containing complexes physically and functionally connect the dots across the genome

PubMed Central

Poon, Betty P.K

2011-01-01

Interactions between genetic regions located across the genome maintain its three-dimensional organization and function. Recent studies point to key roles for a set of coiled-coil domain-containing complexes (cohibin, cohesin, condensin and monopolin) and related factors in the regulation of DNA-DNA connections across the genome. These connections are critical to replication, recombination, gene expression as well as chromosome segregation. PMID:21822055

Human Topoisomerase I C-Terminal Domain Fragment Containing the Active Site Tyrosine is a Molten Globule: Implication for the Formation of Competent Productive Complex

PubMed Central

Punchihewa, Chandanamali; Dai, Jixun; Carver, Megan; Yang, Danzhou

2007-01-01

Human topoisomerase I (topo I) is an essential cellular enzyme that relaxes DNA supercoiling. The 6.3 kDa C-terminal domain of topo I contains the active site tyrosine (Tyr723) but lacks enzymatic activity by itself. Activity can be fully reconstituted when the C-terminal is associated with the 56 kDa core domain. Even though several crystal structures of topo I/DNA complexes are available, crystal structures of the free topo I protein or its individual domain fragments have been difficult to obtain. In this report we analyze the human topo I C-terminal domain structure using a variety of biophysical methods. Our results indicate that this fragment protein (topo6.3) appears to be in a molten globule state. It appears to have a native-like tertiary fold that contains a large population of α-helix secondary structure and extensive surface hydrophobic regions. Topo6.3 is known to be readily activated with the association of the topo I core domain, and the molten globule state of topo6.3 is likely to be an energy-favorable conformation for the free topo I C-terminal domain protein. The structural fluctuation and plasticity may represent an efficient mechanism in the topo I functional pathway, where the flexibility aids in the complementary association with the core domain and in the formation of a fully productive topo I complex. PMID:17434318

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

Histone Code Modulation by Oncogenic PWWP-Domain Protein in Breast Cancers

DTIC Science & Technology

2010-06-01

athanogene 4 * DDHD2 DDHD domain containing 2 * PPAPDC1B phosphatidic acid phosphatase type 2 domain containing 1B * WHSC1L1 Wolf-Hirschhorn syndrome...from alternative splicing of exon 10. The WHSC1L1 long isoform encodes a 1437 amino acid protein containing 2 PWWP domains, 2 PHD-type zinc finger...motifs, a TANG2 domain, an AWS domain and a SET domain. The short isoform encodes a 645 amino acid protein containing a PWWP domain only. Our western

Three-dimensional local ALE-FEM method for fluid flow in domains containing moving boundaries/objects interfaces

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

Carrington, David Bradley; Monayem, A. K. M.; Mazumder, H.

2015-03-05

A three-dimensional finite element method for the numerical simulations of fluid flow in domains containing moving rigid objects or boundaries is developed. The method falls into the general category of Arbitrary Lagrangian Eulerian methods; it is based on a fixed mesh that is locally adapted in the immediate vicinity of the moving interfaces and reverts to its original shape once the moving interfaces go past the elements. The moving interfaces are defined by separate sets of marker points so that the global mesh is independent of interface movement and the possibility of mesh entanglement is eliminated. The results is amore » fully robust formulation capable of calculating on domains of complex geometry with moving boundaries or devises that can also have a complex geometry without danger of the mesh becoming unsuitable due to its continuous deformation thus eliminating the need for repeated re-meshing and interpolation. Moreover, the boundary conditions on the interfaces are imposed exactly. This work is intended to support the internal combustion engines simulator KIVA developed at Los Alamos National Laboratories. The model's capabilities are illustrated through application to incompressible flows in different geometrical settings that show the robustness and flexibility of the technique to perform simulations involving moving boundaries in a three-dimensional domain.« less

The Popeye Domain Containing Genes and Their Function in Striated Muscle

PubMed Central

Schindler, Roland F. R.; Scotton, Chiara; French, Vanessa; Ferlini, Alessandra; Brand, Thomas

2016-01-01

The Popeye domain containing (POPDC) genes encode a novel class of cAMP effector proteins, which are abundantly expressed in heart and skeletal muscle. Here, we will review their role in striated muscle as deduced from work in cell and animal models and the recent analysis of patients carrying a missense mutation in POPDC1. Evidence suggests that POPDC proteins control membrane trafficking of interacting proteins. Furthermore, we will discuss the current catalogue of established protein-protein interactions. In recent years, the number of POPDC-interacting proteins has been rising and currently includes ion channels (TREK-1), sarcolemma-associated proteins serving functions in mechanical stability (dystrophin), compartmentalization (caveolin 3), scaffolding (ZO-1), trafficking (NDRG4, VAMP2/3) and repair (dysferlin) or acting as a guanine nucleotide exchange factor for Rho-family GTPases (GEFT). Recent evidence suggests that POPDC proteins might also control the cellular level of the nuclear proto-oncoprotein c-Myc. These data suggest that this family of cAMP-binding proteins probably serves multiple roles in striated muscle. PMID:27347491

A BEN-domain-containing protein associates with heterochromatin and represses transcription.

PubMed

Sathyan, Kizhakke M; Shen, Zhen; Tripathi, Vidisha; Prasanth, Kannanganattu V; Prasanth, Supriya G

2011-09-15

In eukaryotes, higher order chromatin structure governs crucial cellular processes including DNA replication, transcription and post-transcriptional gene regulation. Specific chromatin-interacting proteins play vital roles in the maintenance of chromatin structure. We have identified BEND3, a quadruple BEN domain-containing protein that is highly conserved amongst vertebrates. BEND3 colocalizes with HP1 and H3 trimethylated at K9 at heterochromatic regions in mammalian cells. Using an in vivo gene locus, we have been able to demonstrate that BEND3 associates with the locus only when it is heterochromatic and dissociates upon activation of transcription. Furthermore, tethering BEND3 inhibits transcription from the locus, indicating that BEND3 is involved in transcriptional repression through its interaction with histone deacetylases and Sall4, a transcription repressor. We further demonstrate that BEND3 is SUMOylated and that such modifications are essential for its role in transcriptional repression. Finally, overexpression of BEND3 causes premature chromatin condensation and extensive heterochromatinization, resulting in cell cycle arrest. Taken together, our data demonstrate the role of a novel heterochromatin-associated protein in transcriptional repression.

A BEN-domain-containing protein associates with heterochromatin and represses transcription

PubMed Central

Sathyan, Kizhakke M.; Shen, Zhen; Tripathi, Vidisha; Prasanth, Kannanganattu V.; Prasanth, Supriya G.

2011-01-01

In eukaryotes, higher order chromatin structure governs crucial cellular processes including DNA replication, transcription and post-transcriptional gene regulation. Specific chromatin-interacting proteins play vital roles in the maintenance of chromatin structure. We have identified BEND3, a quadruple BEN domain-containing protein that is highly conserved amongst vertebrates. BEND3 colocalizes with HP1 and H3 trimethylated at K9 at heterochromatic regions in mammalian cells. Using an in vivo gene locus, we have been able to demonstrate that BEND3 associates with the locus only when it is heterochromatic and dissociates upon activation of transcription. Furthermore, tethering BEND3 inhibits transcription from the locus, indicating that BEND3 is involved in transcriptional repression through its interaction with histone deacetylases and Sall4, a transcription repressor. We further demonstrate that BEND3 is SUMOylated and that such modifications are essential for its role in transcriptional repression. Finally, overexpression of BEND3 causes premature chromatin condensation and extensive heterochromatinization, resulting in cell cycle arrest. Taken together, our data demonstrate the role of a novel heterochromatin-associated protein in transcriptional repression. PMID:21914818

Cancer Research Advance in CKLF-like MARVEL Transmembrane Domain Containing Member Family (Review).

PubMed

Lu, Jia; Wu, Qian-Qian; Zhou, Ya-Bo; Zhang, Kai-Hua; Pang, Bing-Xin; Li, Liang; Sun, Nan; Wang, Heng-Shu; Zhang, Song; Li, Wen-Jian; Zheng, Wei; Liu, Wei

2016-01-01

CKLF-like MARVEL transmembrane domain-containing family (CMTM) is a novel family of genes first reported at international level by Peking University Human Disease Gene Research Center. The gene products are between chemokines and the transmembrane-4 superfamily. Loaceted in several human chromosomes, CMTMs, which are unregulated in kinds of tumors, are potential tumor suppressor genes consisting of CKLF and CMTM1 to CMTM8. CMTMs play important roles in immune, male reproductive and hematopoietic systems. Also, it has been approved that CMTM family has strong connection with diseases of autoimmunity, haematopoietic system and haematopoietic system. The in-depth study in recent years found the close relation between CMTMs and umorigenesis, tumor development and metastasis. CMTM family has a significant clinical value in diagnosis and treatment to the diseases linking to tumor and immune system.

Grb-IR: A SH2-Domain-Containing Protein that Binds to the Insulin Receptor and Inhibits Its Function

NASA Astrophysics Data System (ADS)

Liu, Feng; Roth, Richard A.

1995-10-01

To identify potential signaling molecules involved in mediating insulin-induced biological responses, a yeast two-hybrid screen was performed with the cytoplasmic domain of the human insulin receptor (IR) as bait to trap high-affinity interacting proteins encoded by human liver or HeLa cDNA libraries. A SH2-domain-containing protein was identified that binds with high affinity in vitro to the autophosphorylated IR. The mRNA for this protein was found by Northern blot analyses to be highest in skeletal muscle and was also detected in fat by PCR. To study the role of this protein in insulin signaling, a full-length cDNA encoding this protein (called Grb-IR) was isolated and stably expressed in Chinese hamster ovary cells overexpressing the human IR. Insulin treatment of these cells resulted in the in situ formation of a complex of the IR and the 60-kDa Grb-IR. Although almost 75% of the Grb-IR protein was bound to the IR, it was only weakly tyrosine-phosphorylated. The formation of this complex appeared to inhibit the insulin-induced increase in tyrosine phosphorylation of two endogenous substrates, a 60-kDa GTPase-activating-protein-associated protein and, to a lesser extent, IR substrate 1. The subsequent association of this latter protein with phosphatidylinositol 3-kinase also appeared to be inhibited. These findings raise the possibility that Grb-IR is a SH2-domain-containing protein that directly complexes with the IR and serves to inhibit signaling or redirect the IR signaling pathway.

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

Preprotein mature domains contain translocase targeting signals that are essential for secretion.

PubMed

Chatzi, Katerina E; Sardis, Marios Frantzeskos; Tsirigotaki, Alexandra; Koukaki, Marina; Šoštarić, Nikolina; Konijnenberg, Albert; Sobott, Frank; Kalodimos, Charalampos G; Karamanou, Spyridoula; Economou, Anastassios

2017-05-01

Secretory proteins are only temporary cytoplasmic residents. They are typically synthesized as pre proteins, carrying signal peptides N-terminally fused to their mature domains. In bacteria secretion largely occurs posttranslationally through the membrane-embedded SecA-SecYEG translocase. Upon crossing the plasma membrane, signal peptides are cleaved off and mature domains reach their destinations and fold. Targeting to the translocase is mediated by signal peptides. The role of mature domains in targeting and secretion is unclear. We now reveal that mature domains harbor their own independent targeting signals (mature domain targeting signals [MTSs]). These are multiple, degenerate, interchangeable, linear or 3D hydrophobic stretches that become available because of the unstructured states of targeting-competent preproteins. Their receptor site on the cytoplasmic face of the SecYEG-bound SecA is also of hydrophobic nature and is located adjacent to the signal peptide cleft. Both the preprotein MTSs and their receptor site on SecA are essential for protein secretion. Evidently, mature domains have their own previously unsuspected distinct roles in preprotein targeting and secretion. © 2017 Chatzi et al.

Preprotein mature domains contain translocase targeting signals that are essential for secretion

PubMed Central

Tsirigotaki, Alexandra; Koukaki, Marina; Šoštarić, Nikolina; Konijnenberg, Albert; Sobott, Frank; Kalodimos, Charalampos G.; Karamanou, Spyridoula

2017-01-01

Secretory proteins are only temporary cytoplasmic residents. They are typically synthesized as preproteins, carrying signal peptides N-terminally fused to their mature domains. In bacteria secretion largely occurs posttranslationally through the membrane-embedded SecA-SecYEG translocase. Upon crossing the plasma membrane, signal peptides are cleaved off and mature domains reach their destinations and fold. Targeting to the translocase is mediated by signal peptides. The role of mature domains in targeting and secretion is unclear. We now reveal that mature domains harbor their own independent targeting signals (mature domain targeting signals [MTSs]). These are multiple, degenerate, interchangeable, linear or 3D hydrophobic stretches that become available because of the unstructured states of targeting-competent preproteins. Their receptor site on the cytoplasmic face of the SecYEG-bound SecA is also of hydrophobic nature and is located adjacent to the signal peptide cleft. Both the preprotein MTSs and their receptor site on SecA are essential for protein secretion. Evidently, mature domains have their own previously unsuspected distinct roles in preprotein targeting and secretion. PMID:28404644

Programs for transferring data between a relational data base and a finite element structural analysis program

NASA Technical Reports Server (NTRS)

Johnson, S. C.

1982-01-01

An interface system for passing data between a relational information management (RIM) data base complex and engineering analysis language (EAL), a finite element structural analysis program is documented. The interface system, implemented on a CDC Cyber computer, is composed of two FORTRAN programs called RIM2EAL and EAL2RIM. The RIM2EAL reads model definition data from RIM and creates a file of EAL commands to define the model. The EAL2RIM reads model definition and EAL generated analysis data from EAL's data library and stores these data dirctly in a RIM data base. These two interface programs and the format for the RIM data complex are described.

An ice-binding and tandem beta-sandwich domain-containing protein in Shewanella frigidimarina is a potential new type of ice adhesin.

PubMed

Vance, Tyler D R; Graham, Laurie A; Davies, Peter L

2018-04-01

Out of the dozen different ice-binding protein (IBP) structures known, the DUF3494 domain is the most widespread, having been passed many times between prokaryotic and eukaryotic microorganisms by horizontal gene transfer. This ~25-kDa β-solenoid domain with an adjacent parallel α-helix is most commonly associated with an N-terminal secretory signal peptide. However, examples of the DUF3494 domain preceded by tandem Bacterial Immunoglobulin-like (BIg) domains are sometimes found, though uncharacterized. Here, we present one such protein (SfIBP_1) from the Antarctic bacterium Shewanella frigidimarina. We have confirmed and characterized the ice-binding activity of its ice-binding domain using thermal hysteresis measurements, fluorescent ice plane affinity analysis, and ice recrystallization inhibition assays. X-ray crystallography was used to solve the structure of the SfIBP_1 ice-binding domain, to further characterize its ice-binding surface and unique method of stabilizing or 'capping' the ends of the solenoid structure. The latter is formed from the interaction of two loops mediated by a combination of tandem prolines and electrostatic interactions. Furthermore, given their domain architecture and membrane association, we propose that these BIg-containing DUF3494 IBPs serve as ice-binding adhesion proteins that are capable of adsorbing their host bacterium onto ice. Submitted new structure to the Protein Data Bank (PDB: 6BG8). © 2018 Federation of European Biochemical Societies.

Concomitant Alterations of Desmosomes, Adhesiveness, and Diffusion through Gap Junction Channels in a Rat Ovarian Transformation Model System

DTIC Science & Technology

1993-01-01

while using a sterile glass slide, held parallel to the monolayer, as a MATERIALS AND METHODS straight edge. Cultures were photographed using phase...contrastmicrosoyo tonoa~r movng ofi I spheria mi rocr rie bed ntI h CEILL CELIL CONTACT AND DIFFU SION 23 and cytokeratin 8 antibodies (refer to Figs. 4a

Cold-dependent alternative splicing of a Jumonji C domain-containing gene MtJMJC5 in Medicago truncatula.

PubMed

Shen, Yingfang; Wu, Xiaopei; Liu, Demei; Song, Shengjing; Liu, Dengcai; Wang, Haiqing

2016-05-27

Histone methylation is an epigenetic modification mechanism that regulates gene expression in eukaryotic cells. Jumonji C domain-containing demethylases are involved in removal of methyl groups at lysine or arginine residues. The JmjC domain-only member, JMJ30/JMJD5 of Arabidopsis, is a component of the plant circadian clock. Although some plant circadian clock genes undergo alternative splicing in response to external cues, there is no evidence that JMJ30/JMJD5 is regulated by alternative splicing. In this study, the expression of an Arabidopsis JMJ30/JMJD5 ortholog in Medicago truncatula, MtJMJC5, in response to circadian clock and abiotic stresses were characterized. The results showed that MtJMJC5 oscillates with a circadian rhythm, and undergoes cold specifically induced alternative splicing. The cold-induced alternative splicing could be reversed after ambient temperature returning to the normal. Sequencing results revealed four alternative splicing RNA isoforms including a full-length authentic protein encoding variant, and three premature termination condon-containing variants due to alternative 3' splice sites at the first and second intron. Under cold treatment, the variants that share a common 3' alternative splicing site at the second intron were intensively up-regulated while the authentic protein encoding variant and the premature termination condon-containing variant only undergoing a 3' alternative splicing at the first intron were down regulated. Although all the premature termination condon-harboring alternative splicing variants were sensitive to nonsense-mediated decay, the premature termination codon-harboring alternative splicing variants sharing the 3' alternative splicing site at the second intron showed less sensitivity than the one only containing the 3' alternative slicing site at the first intron under cold treatment. These results suggest that the cold-dependent alternative splicing of MtJMJC5 is likely a species or genus

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

Mutations in type 3 reovirus that determine binding to sialic acid are contained in the fibrous tail domain of viral attachment protein sigma1.

PubMed

Chappell, J D; Gunn, V L; Wetzel, J D; Baer, G S; Dermody, T S

1997-03-01

The reovirus attachment protein, sigma1, determines numerous aspects of reovirus-induced disease, including viral virulence, pathways of spread, and tropism for certain types of cells in the central nervous system. The sigma1 protein projects from the virion surface and consists of two distinct morphologic domains, a virion-distal globular domain known as the head and an elongated fibrous domain, termed the tail, which is anchored into the virion capsid. To better understand structure-function relationships of sigma1 protein, we conducted experiments to identify sequences in sigma1 important for viral binding to sialic acid, a component of the receptor for type 3 reovirus. Three serotype 3 reovirus strains incapable of binding sialylated receptors were adapted to growth in murine erythroleukemia (MEL) cells, in which sialic acid is essential for reovirus infectivity. MEL-adapted (MA) mutant viruses isolated by serial passage in MEL cells acquired the capacity to bind sialic acid-containing receptors and demonstrated a dependence on sialic acid for infection of MEL cells. Analysis of reassortant viruses isolated from crosses of an MA mutant virus and a reovirus strain that does not bind sialic acid indicated that the sigma1 protein is solely responsible for efficient growth of MA mutant viruses in MEL cells. The deduced sigma1 amino acid sequences of the MA mutant viruses revealed that each strain contains a substitution within a short region of sequence in the sigma1 tail predicted to form beta-sheet. These studies identify specific sequences that determine the capacity of reovirus to bind sialylated receptors and suggest a location for a sialic acid-binding domain. Furthermore, the results support a model in which type 3 sigma1 protein contains discrete receptor binding domains, one in the head and another in the tail that binds sialic acid.

Viral Interactions with PDZ Domain-Containing Proteins-An Oncogenic Trait?

PubMed

James, Claire D; Roberts, Sally

2016-01-18

Many of the human viruses with oncogenic capabilities, either in their natural host or in experimental systems (hepatitis B and C, human T cell leukaemia virus type 1, Kaposi sarcoma herpesvirus, human immunodeficiency virus, high-risk human papillomaviruses and adenovirus type 9), encode in their limited genome the ability to target cellular proteins containing PSD95/ DLG/ZO-1 (PDZ) interaction modules. In many cases (but not always), the viruses have evolved to bind the PDZ domains using the same short linear peptide motifs found in host protein-PDZ interactions, and in some cases regulate the interactions in a similar fashion by phosphorylation. What is striking is that the diverse viruses target a common subset of PDZ proteins that are intimately involved in controlling cell polarity and the structure and function of intercellular junctions, including tight junctions. Cell polarity is fundamental to the control of cell proliferation and cell survival and disruption of polarity and the signal transduction pathways involved is a key event in tumourigenesis. This review focuses on the oncogenic viruses and the role of targeting PDZ proteins in the virus life cycle and the contribution of virus-PDZ protein interactions to virus-mediated oncogenesis. We highlight how many of the viral associations with PDZ proteins lead to deregulation of PI3K/AKT signalling, benefitting virus replication but as a consequence also contributing to oncogenesis.

Caspase-3 mediated release of SAC domain containing fragment from Par-4 is necessary for the sphingosine-induced apoptosis in Jurkat cells

PubMed Central

2013-01-01

Background Prostate apoptosis response-4 (Par-4) is a tumor-suppressor protein that selectively activates and induces apoptosis in cancer cells, but not in normal cells. The cancer specific pro-apoptotic function of Par-4 is encoded in its centrally located SAC (Selective for Apoptosis induction in Cancer cells) domain (amino acids 137–195). The SAC domain itself is capable of nuclear entry, caspase activation, inhibition of NF-κB activity, and induction of apoptosis in cancer cells. However, the precise mechanism(s) of how the SAC domain is released from Par-4, in response to apoptotic stimulation, is not well explored. Results In this study, we demonstrate for the first time that sphingosine (SPH), a member of the sphingolipid family, induces caspase-dependant cleavage of Par-4, leading to the release of SAC domain containing fragment from it. Par-4 is cleaved at the EEPD131G site on incubation with caspase-3 in vitro, and by treating cells with several anti-cancer agents. The caspase-3 mediated cleavage of Par-4 is blocked by addition of the pan-caspase inhibitor z-VAD-fmk, caspase-3 specific inhibitor Ac-DEVD-CHO, and by introduction of alanine substitution for D131 residue. Moreover, suppression of SPH-induced Akt dephosphorylation also abrogated the caspase dependant cleavage of Par-4. Conclusion Evidence provided here shows that Par-4 is cleaved by caspase-3 during SPH-induced apoptosis. Cleavage of Par-4 leads to the generation of SAC domain containing fragment which may possibly be essential and sufficient to induce or augment apoptosis in cancer cells. PMID:23442976

Supra-domains: evolutionary units larger than single protein domains.

PubMed

Vogel, Christine; Berzuini, Carlo; Bashton, Matthew; Gough, Julian; Teichmann, Sarah A

2004-02-20

Domains are the evolutionary units that comprise proteins, and most proteins are built from more than one domain. Domains can be shuffled by recombination to create proteins with new arrangements of domains. Using structural domain assignments, we examined the combinations of domains in the proteins of 131 completely sequenced organisms. We found two-domain and three-domain combinations that recur in different protein contexts with different partner domains. The domains within these combinations have a particular functional and spatial relationship. These units are larger than individual domains and we term them "supra-domains". Amongst the supra-domains, we identified some 1400 (1203 two-domain and 166 three-domain) combinations that are statistically significantly over-represented relative to the occurrence and versatility of the individual component domains. Over one-third of all structurally assigned multi-domain proteins contain these over-represented supra-domains. This means that investigation of the structural and functional relationships of the domains forming these popular combinations would be particularly useful for an understanding of multi-domain protein function and evolution as well as for genome annotation. These and other supra-domains were analysed for their versatility, duplication, their distribution across the three kingdoms of life and their functional classes. By examining the three-dimensional structures of several examples of supra-domains in different biological processes, we identify two basic types of spatial relationships between the component domains: the combined function of the two domains is such that either the geometry of the two domains is crucial and there is a tight constraint on the interface, or the precise orientation of the domains is less important and they are spatially separate. Frequently, the role of the supra-domain becomes clear only once the three-dimensional structure is known. Since this is the case for only a

Detergent/Nanodisc Screening for High-Resolution NMR Studies of an Integral Membrane Protein Containing a Cytoplasmic Domain

PubMed Central

Maslennikov, Innokentiy; Choe, Senyon; Riek, Roland

2013-01-01

Because membrane proteins need to be extracted from their natural environment and reconstituted in artificial milieus for the 3D structure determination by X-ray crystallography or NMR, the search for membrane mimetic that conserve the native structure and functional activities remains challenging. We demonstrate here a detergent/nanodisc screening study by NMR of the bacterial α-helical membrane protein YgaP containing a cytoplasmic rhodanese domain. The analysis of 2D [15N,1H]-TROSY spectra shows that only a careful usage of low amounts of mixed detergents did not perturb the cytoplasmic domain while solubilizing in parallel the transmembrane segments with good spectral quality. In contrast, the incorporation of YgaP into nanodiscs appeared to be straightforward and yielded a surprisingly high quality [15N,1H]-TROSY spectrum opening an avenue for the structural studies of a helical membrane protein in a bilayer system by solution state NMR. PMID:23349867

A TPR domain-containing N-terminal module of MPS1 is required for its kinetochore localization by Aurora B.

PubMed

Nijenhuis, Wilco; von Castelmur, Eleonore; Littler, Dene; De Marco, Valeria; Tromer, Eelco; Vleugel, Mathijs; van Osch, Maria H J; Snel, Berend; Perrakis, Anastassis; Kops, Geert J P L

2013-04-15

The mitotic checkpoint ensures correct chromosome segregation by delaying cell cycle progression until all kinetochores have attached to the mitotic spindle. In this paper, we show that the mitotic checkpoint kinase MPS1 contains an N-terminal localization module, organized in an N-terminal extension (NTE) and a tetratricopeptide repeat (TPR) domain, for which we have determined the crystal structure. Although the module was necessary for kinetochore localization of MPS1 and essential for the mitotic checkpoint, the predominant kinetochore binding activity resided within the NTE. MPS1 localization further required HEC1 and Aurora B activity. We show that MPS1 localization to kinetochores depended on the calponin homology domain of HEC1 but not on Aurora B-dependent phosphorylation of the HEC1 tail. Rather, the TPR domain was the critical mediator of Aurora B control over MPS1 localization, as its deletion rendered MPS1 localization insensitive to Aurora B inhibition. These data are consistent with a model in which Aurora B activity relieves a TPR-dependent inhibitory constraint on MPS1 localization.

A novel activity of HMG domains: promotion of the triple-stranded complex formation between DNA containing (GGA/TCC)11 and d(GGA)11 oligonucleotides.

PubMed Central

Suda, T; Mishima, Y; Takayanagi, K; Asakura, H; Odani, S; Kominami, R

1996-01-01

The high mobility group protein (HMG)-box is a DNA-binding domain found in many proteins that bind preferentially to DNA of irregular structures in a sequence-independent manner and can bend the DNA. We show here that GST-fusion proteins of HMG domains from HMG1 and HMG2 promote a triple-stranded complex formation between DNA containing the (GGA/TCC)11 repeat and oligonucleotides of d(GGA)11 probably due to G:G base pairing. The activity is to reduce association time and requirements of Mg2+ and oligonucleotide concentrations. The HMG box of SRY, the protein determining male-sex differentiation, also has the activity, suggesting that it is not restricted to the HMG-box domains derived from HMG1/2 but is common to those from other members of the HMG-box family of proteins. Interestingly, the box-AB and box-B of HMG1 bend DNA containing the repeat, but SRY fails to bend in a circularization assay. The difference suggests that the two activities of association-promotion and DNA bending are distinct. These results suggest that the HMG-box domain has a novel activity of promoting the association between GGA repeats which might be involved in higher-order architecture of chromatin. PMID:8972860

Molecular cloning, structural analysis, and expression of a human IRLB, MYC promoter-binding protein: new DENN domain-containing protein family emerges.

PubMed

Semova, Natalia; Kapanadze, Bagrat; Corcoran, Martin; Kutsenko, Alexei; Baranova, Ancha; Semov, Alexandre

2003-09-01

IRLB was originally identified as a partial cDNA clone, encoding a 191-aa protein binding the interferon-stimulated response element (ISRE) in the P2 promoter of human MYC. Here, we cloned the full-size IRLB using different bioinformatics tools and an RT-PCR approach. The full-size gene encompasses 131 kb within chromosome 15q22 and consists of 32 exons. IRLB is transcribed as a 6.6-kb mRNA encoding a protein of 1865 aa. IRLB is ubiquitously expressed and its expression is regulated in a growth- and cell cycle-dependent manner. In addition to the ISRE-binding domain IRLB contains a tripartite DENN domain, a nuclear localization signal, two PPRs, and a calmodulin-binding domain. The presence of DENN domains predicts possible interactions of IRLB with GTPases from the Rab family or regulation of growth-induced MAPKs. Strongly homologous proteins were identified in all available vertebrate genomes as well as in Caenorhabditis elegans and Drosophila melanogaster. In human and mouse a family of IRLB proteins exists, consisting of at least three members.

MIT domain of Vps4 is a Ca2+-dependent phosphoinositide-binding domain.

PubMed

Iwaya, Naoko; Takasu, Hirotoshi; Goda, Natsuko; Shirakawa, Masahiro; Tanaka, Toshiki; Hamada, Daizo; Hiroaki, Hidekazu

2013-05-01

The microtubule interacting and trafficking (MIT) domain is a small protein module that is conserved in proteins of diverged function, such as Vps4, spastin and sorting nexin 15 (SNX15). The molecular function of the MIT domain is protein-protein interaction, in which the domain recognizes peptides containing MIT-interacting motifs. Recently, we identified an evolutionarily related domain, 'variant' MIT domain at the N-terminal region of the microtubule severing enzyme katanin p60. We found that the domain was responsible for binding to microtubules and Ca(2+). Here, we have examined whether the authentic MIT domains also bind Ca(2+). We found that the loop between the first and second α-helices of the MIT domain binds a Ca(2+) ion. Furthermore, the MIT domains derived from Vps4b and SNX15a showed phosphoinositide-binding activities in a Ca(2+)-dependent manner. We propose that the MIT domain is a novel membrane-associating domain involved in endosomal trafficking.

Popeye domain containing proteins are essential for stress-mediated modulation of cardiac pacemaking in mice

PubMed Central

Froese, Alexander; Breher, Stephanie S.; Waldeyer, Christoph; Schindler, Roland F.R.; Nikolaev, Viacheslav O.; Rinné, Susanne; Wischmeyer, Erhard; Schlueter, Jan; Becher, Jan; Simrick, Subreena; Vauti, Franz; Kuhtz, Juliane; Meister, Patrick; Kreissl, Sonja; Torlopp, Angela; Liebig, Sonja K.; Laakmann, Sandra; Müller, Thomas D.; Neumann, Joachim; Stieber, Juliane; Ludwig, Andreas; Maier, Sebastian K.; Decher, Niels; Arnold, Hans-Henning; Kirchhof, Paulus; Fabritz, Larissa; Brand, Thomas

2012-01-01

Cardiac pacemaker cells create rhythmic pulses that control heart rate; pacemaker dysfunction is a prevalent disorder in the elderly, but little is known about the underlying molecular causes. Popeye domain containing (Popdc) genes encode membrane proteins with high expression levels in cardiac myocytes and specifically in the cardiac pacemaking and conduction system. Here, we report the phenotypic analysis of mice deficient in Popdc1 or Popdc2. ECG analysis revealed severe sinus node dysfunction when freely roaming mutant animals were subjected to physical or mental stress. In both mutants, bradyarrhythmia developed in an age-dependent manner. Furthermore, we found that the conserved Popeye domain functioned as a high-affinity cAMP-binding site. Popdc proteins interacted with the potassium channel TREK-1, which led to increased cell surface expression and enhanced current density, both of which were negatively modulated by cAMP. These data indicate that Popdc proteins have an important regulatory function in heart rate dynamics that is mediated, at least in part, through cAMP binding. Mice with mutant Popdc1 and Popdc2 alleles are therefore useful models for the dissection of the mechanisms causing pacemaker dysfunction and could aid in the development of strategies for therapeutic intervention. PMID:22354168

A thermophilic mini-chaperonin contains a conserved polypeptide-binding surface: combined crystallographic and NMR studies of the GroEL Apical Domain with implications for substrate interactions

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

Hua, Q. X. H.; Dementieva, I. S. D.; Walsh, M. A. W.

2001-02-23

A homologue of the Escherichia coli GroEL apical domain was obtained from thermophilic eubacterium Thermus thermophilus. The domains share 70 % sequence identity (101 out of 145 residues). The thermal stability of the T. thermophilus apical domain (T{sub m}>100{sup o}C as evaluated by circular dichroism) is at least 35{sup o}C greater than that of the E. coli apical domain (T{sub m}=65{sup o}C). The crystal structure of a selenomethione-substituted apical domain from T. thermophilus was determined to a resolution of 1.78 {angstrom} using multiwavelength-anomalous-diffraction phasing. The structure is similar to that of the E. coli apical domain (root-mean-square deviation 0.45 {angstrom}more » based on main-chain atoms). The thermophilic structure contains seven additional salt bridges of which four contain charge-stabilized hydrogen bonds. Only one of the additional salt bridges would face the 'Anfinsen cage' in GroEL. High temperatures were exploited to map sites of interactions between the apical domain and molten globules. NMR footprints of apical domain-protein complexes were obtained at elevated temperature using {sup 15}N-{sup 1}H correlation spectra of {sup 15}N-labeled apical domain. Footprints employing two polypeptides unrelated in sequence or structure (an insulin monomer and the SRY high-mobility-group box, each partially unfolded at 50{sup o}C) are essentially the same and consistent with the peptide-binding surface previously defined in E. coli GroEL and its apical domain-peptide complexes. An additional part of this surface comprising a short N-terminal {alpha}-helix is observed. The extended footprint rationalizes mutagenesis studies of intact GroEL in which point mutations affecting substrate binding were found outside the 'classical' peptide-binding site. Our results demonstrate structural conservation of the apical domain among GroEL homologues and conservation of an extended non-polar surface recognizing diverse polypeptides.« less

Tandem-repeat protein domains across the tree of life.

PubMed

Jernigan, Kristin K; Bordenstein, Seth R

2015-01-01

Tandem-repeat protein domains, composed of repeated units of conserved stretches of 20-40 amino acids, are required for a wide array of biological functions. Despite their diverse and fundamental functions, there has been no comprehensive assessment of their taxonomic distribution, incidence, and associations with organismal lifestyle and phylogeny. In this study, we assess for the first time the abundance of armadillo (ARM) and tetratricopeptide (TPR) repeat domains across all three domains in the tree of life and compare the results to our previous analysis on ankyrin (ANK) repeat domains in this journal. All eukaryotes and a majority of the bacterial and archaeal genomes analyzed have a minimum of one TPR and ARM repeat. In eukaryotes, the fraction of ARM-containing proteins is approximately double that of TPR and ANK-containing proteins, whereas bacteria and archaea are enriched in TPR-containing proteins relative to ARM- and ANK-containing proteins. We show in bacteria that phylogenetic history, rather than lifestyle or pathogenicity, is a predictor of TPR repeat domain abundance, while neither phylogenetic history nor lifestyle predicts ARM repeat domain abundance. Surprisingly, pathogenic bacteria were not enriched in TPR-containing proteins, which have been associated within virulence factors in certain species. Taken together, this comparative analysis provides a newly appreciated view of the prevalence and diversity of multiple types of tandem-repeat protein domains across the tree of life. A central finding of this analysis is that tandem repeat domain-containing proteins are prevalent not just in eukaryotes, but also in bacterial and archaeal species.

Tandem-repeat protein domains across the tree of life

PubMed Central

Jernigan, Kristin K.

2015-01-01

Tandem-repeat protein domains, composed of repeated units of conserved stretches of 20–40 amino acids, are required for a wide array of biological functions. Despite their diverse and fundamental functions, there has been no comprehensive assessment of their taxonomic distribution, incidence, and associations with organismal lifestyle and phylogeny. In this study, we assess for the first time the abundance of armadillo (ARM) and tetratricopeptide (TPR) repeat domains across all three domains in the tree of life and compare the results to our previous analysis on ankyrin (ANK) repeat domains in this journal. All eukaryotes and a majority of the bacterial and archaeal genomes analyzed have a minimum of one TPR and ARM repeat. In eukaryotes, the fraction of ARM-containing proteins is approximately double that of TPR and ANK-containing proteins, whereas bacteria and archaea are enriched in TPR-containing proteins relative to ARM- and ANK-containing proteins. We show in bacteria that phylogenetic history, rather than lifestyle or pathogenicity, is a predictor of TPR repeat domain abundance, while neither phylogenetic history nor lifestyle predicts ARM repeat domain abundance. Surprisingly, pathogenic bacteria were not enriched in TPR-containing proteins, which have been associated within virulence factors in certain species. Taken together, this comparative analysis provides a newly appreciated view of the prevalence and diversity of multiple types of tandem-repeat protein domains across the tree of life. A central finding of this analysis is that tandem repeat domain-containing proteins are prevalent not just in eukaryotes, but also in bacterial and archaeal species. PMID:25653910

Activation of nucleotide-binding domain-like receptor containing protein 3 inflammasome in dendritic cells and macrophages by Streptococcus sanguinis.

PubMed

Saeki, Ayumi; Suzuki, Toshihiko; Hasebe, Akira; Kamezaki, Ryousuke; Fujita, Mari; Nakazawa, Futoshi; Shibata, Ken-Ichiro

2017-03-01

Streptococcus sanguinis is frequently isolated from the blood of patients with infective endocarditis and contributes to the pathology of this disease through induction of interleukin (IL)-1β responsible for the development of the disease. However, the mechanism of IL-1β induction remains unknown. In this study, S. sanguinis activated a murine dendritic cell (DC) to induce IL-1β and this activity was attenuated by silencing the mRNAs of nucleotide-binding domain-like receptor containing protein 3 (NLRP3) and caspase-1. S. sanguinis induced IL-1β production in murine bone marrow-derived macrophage, but this activity was significantly reduced in bone marrow-derived macrophages from NLRP3-, apoptosis-associated speck-like protein containing a caspase-recruitment domain-, and caspase-1-deficient mice. DC phagocytosed S. sanguinis cells, followed by the release of adenosine triphosphate (ATP). The ATP-degradating enzyme attenuated the release of ATP and IL-1β. The inhibitors for ATP receptor reduced IL-1β release in DC. These results strongly suggest that S. sanguinis has the activity to induce IL-1β through the NLRP3 inflammasome in macrophage and DC and interaction of purinergic receptors with ATP released is involved in expression of the activity. © 2016 John Wiley & Sons Ltd.

Towards the identification of the common features of bacterial biofilm development.

PubMed

Lasa, Iñigo

2006-03-01

Microorganisms can live and proliferate as individual cells swimming freely in the environment, or they can grow as highly organized, multicellular communities encased in a self-produced polymeric matrix in close association with surfaces and interfaces. This microbial lifestyle is referred to as biofilms. The intense search over the last few years for factors involved in biofilm development has revealed that distantly related bacterial species recurrently make use of the same elements to produce biofilms. These common elements include a group of proteins containing GGDEF/EAL domains, surface proteins homologous to Bap of Staphylococcus aureus, and some types of exopolysaccharides, such as cellulose and the poly-beta-1,6-N-acetylglucosamine. This review summarizes current knowledge about these three common elements and their role in biofilm development.

Big domains are novel Ca²+-binding modules: evidences from big domains of Leptospira immunoglobulin-like (Lig) proteins.

PubMed

Raman, Rajeev; Rajanikanth, V; Palaniappan, Raghavan U M; Lin, Yi-Pin; He, Hongxuan; McDonough, Sean P; Sharma, Yogendra; Chang, Yung-Fu

2010-12-29

Many bacterial surface exposed proteins mediate the host-pathogen interaction more effectively in the presence of Ca²+. Leptospiral immunoglobulin-like (Lig) proteins, LigA and LigB, are surface exposed proteins containing Bacterial immunoglobulin like (Big) domains. The function of proteins which contain Big fold is not known. Based on the possible similarities of immunoglobulin and βγ-crystallin folds, we here explore the important question whether Ca²+ binds to a Big domains, which would provide a novel functional role of the proteins containing Big fold. We selected six individual Big domains for this study (three from the conserved part of LigA and LigB, denoted as Lig A3, Lig A4, and LigBCon5; two from the variable region of LigA, i.e., 9(th) (Lig A9) and 10(th) repeats (Lig A10); and one from the variable region of LigB, i.e., LigBCen2. We have also studied the conserved region covering the three and six repeats (LigBCon1-3 and LigCon). All these proteins bind the calcium-mimic dye Stains-all. All the selected four domains bind Ca²+ with dissociation constants of 2-4 µM. Lig A9 and Lig A10 domains fold well with moderate thermal stability, have β-sheet conformation and form homodimers. Fluorescence spectra of Big domains show a specific doublet (at 317 and 330 nm), probably due to Trp interaction with a Phe residue. Equilibrium unfolding of selected Big domains is similar and follows a two-state model, suggesting the similarity in their fold. We demonstrate that the Lig are Ca²+-binding proteins, with Big domains harbouring the binding motif. We conclude that despite differences in sequence, a Big motif binds Ca²+. This work thus sets up a strong possibility for classifying the proteins containing Big domains as a novel family of Ca²+-binding proteins. Since Big domain is a part of many proteins in bacterial kingdom, we suggest a possible function these proteins via Ca²+ binding.

The N-Terminal Domain of Human DNA Helicase Rtel1 Contains a Redox Active Iron-Sulfur Cluster

PubMed Central

Landry, Aaron P.

2014-01-01

Human telomere length regulator Rtel1 is a superfamily II DNA helicase and is essential for maintaining proper length of telomeres in chromosomes. Here we report that the N-terminal domain of human Rtel1 (RtelN) expressed in Escherichia coli cells produces a protein that contains a redox active iron-sulfur cluster with the redox midpoint potential of −248 ± 10 mV (pH 8.0). The iron-sulfur cluster in RtelN is sensitive to hydrogen peroxide and nitric oxide, indicating that reactive oxygen/nitrogen species may modulate the DNA helicase activity of Rtel1 via modification of its iron-sulfur cluster. Purified RtelN retains a weak binding affinity for the single-stranded (ss) and double-stranded (ds) DNA in vitro. However, modification of the iron-sulfur cluster by hydrogen peroxide or nitric oxide does not significantly affect the DNA binding activity of RtelN, suggesting that the iron-sulfur cluster is not directly involved in the DNA interaction in the N-terminal domain of Rtel1. PMID:25147792

The N-terminal domain of human DNA helicase Rtel1 contains a redox active iron-sulfur cluster.

PubMed

Landry, Aaron P; Ding, Huangen

2014-01-01

Human telomere length regulator Rtel1 is a superfamily II DNA helicase and is essential for maintaining proper length of telomeres in chromosomes. Here we report that the N-terminal domain of human Rtel1 (RtelN) expressed in Escherichia coli cells produces a protein that contains a redox active iron-sulfur cluster with the redox midpoint potential of -248 ± 10 mV (pH 8.0). The iron-sulfur cluster in RtelN is sensitive to hydrogen peroxide and nitric oxide, indicating that reactive oxygen/nitrogen species may modulate the DNA helicase activity of Rtel1 via modification of its iron-sulfur cluster. Purified RtelN retains a weak binding affinity for the single-stranded (ss) and double-stranded (ds) DNA in vitro. However, modification of the iron-sulfur cluster by hydrogen peroxide or nitric oxide does not significantly affect the DNA binding activity of RtelN, suggesting that the iron-sulfur cluster is not directly involved in the DNA interaction in the N-terminal domain of Rtel1.

Structural organization and chromosomal assignment of the mouse embryonic TEA domain-containing factor (ETF) gene

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

Suzuki, Kazuo; Yasunami, Michio; Matsuda, Yoichi

1996-09-01

Embryonic TEA domain-containing factor (ETF) belongs to the family of proteins structurally related to transcriptional enhancer factor-1 (TEF-1) and is implicated in neural development. Isolation and characterization of the cosmid clones encoding the mouse ETF gene (Etdf) revealed that Etdf spans approximately 17.9 kb and consists of 12 exons. The exon-intron structure of Etdf closely resembles that of the Drosophila scalloped gene, indicating that these genes may have evolved from a common ancestor. Then multiple transcription initiation sites revealed by S1 protection and primer extension analyses are consistent with the absence of the canonical TATA and CAAT boxes in themore » 5{prime}-flanking region, which contains many potential regulatory sequences, such as the E-box, N-box, Sp1 element, GATA-1 element, TAATGARAT element, and B2 short interspersed element (SINE) as well as several direct and inverted repeat sequences. The Etdf locus was assigned to the proximal region of mouse chromosome 7 using fluorescence in situ hybridization and linkage mapping analyses. These results provide the molecular basis for studying the regulation, in vivo function, and evolution of Etdf. 29 refs., 5 figs., 1 tab.« less

Structural organization and chromosomal assignment of the mouse embryonic TEA domain-containing factor (ETF) gene.

PubMed

Suzuki, K; Yasunami, M; Matsuda, Y; Maeda, T; Kobayashi, H; Terasaki, H; Ohkubo, H

1996-09-01

Embryonic TEA domain-containing factor (ETF) belongs to the family of proteins structurally related to transcriptional enhancer factor-1 (TEF-1) and is implicated in neural development. Isolation and characterization of the cosmid clones encoding the mouse ETF gene (Etdf) revealed that Etdf spans approximately 17.9 kb and consists of 12 exons. The exon-intron structure of Etdf closely resembles that of the Drosophila scalloped gene, indicating that these genes may have evolved from a common ancestor. The multiple transcription initiation sites revealed by S1 protection and primer extension analyses are consistent with the absence of the canonical TATA and CAAT boxes in the 5'-flanking region, which contains many potential regulatory sequences, such as the E-box, N-box, Sp1 element, GATA-1 element, TAATGARAT element, and B2 short interspersed element (SINE) as well as several direct and inverted repeat sequences. The Etdf locus was assigned to the proximal region of mouse chromosome 7 using fluorescence in situ hybridization and linkage mapping analyses. These results provide the molecular basis for studying the regulation, in vivo function, and evolution of Etdf.

Arctium lappa ameliorates endothelial dysfunction in rats fed with high fat/cholesterol diets.

PubMed

Lee, Yun Jung; Choi, Deok Ho; Cho, Guk Hyun; Kim, Jin Sook; Kang, Dae Gill; Lee, Ho Sub

2012-08-06

Arctium lappa L. (Asteraceae), burdock, is a medicinal plant that is popularly used for treating hypertension, gout, hepatitis, and other inflammatory disorders. This study was performed to test the effect of ethanol extract of Arctium lappa L. (EAL) seeds on vascular reactivity and inflammatory factors in rats fed a high fat/cholesterol diet (HFCD). EAL-I (100 mg·kg-1/day), EAL-II (200 mg·kg-1/day), and fluvastatin (3 mg·kg-1/day) groups initially received HFCD alone for 8 weeks, with EAL supplementation provided during the final 6 weeks. Treatment with low or high doses of EAL markedly attenuated plasma levels of triglycerides and augmented plasma levels of high-density lipoprotein (HDL) in HFCD-fed rats. Chronic treatment with EAL markedly reduced impairments of acetylcholine (ACh)-induced relaxation of aortic rings. Furthermore, chronic treatment with EAL significantly lowered systolic blood pressure (SBP) and maintained smooth and flexible intimal endothelial layers in HFCD-fed rats. Chronic treatment with EAL suppressed upregulation of intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and E-selectin in the aorta. Chronic treatment with EAL also suppressed increases in matrix metalloproteinase (MMP)-2 expression. These results suggested that EAL can inhibit HFCD-induced vascular inflammation in the rat model. The present study provides evidence that EAL ameliorates HFCD-induced vascular dysfunction through protection of vascular relaxation and suppression of vascular inflammation.

Arctium lappa ameliorates endothelial dysfunction in rats fed with high fat/cholesterol diets

PubMed Central

2012-01-01

Background Arctium lappa L. (Asteraceae), burdock, is a medicinal plant that is popularly used for treating hypertension, gout, hepatitis, and other inflammatory disorders. This study was performed to test the effect of ethanol extract of Arctium lappa L. (EAL) seeds on vascular reactivity and inflammatory factors in rats fed a high fat/cholesterol diet (HFCD). Method EAL-I (100 mg·kg−1/day), EAL-II (200 mg·kg−1/day), and fluvastatin (3 mg·kg−1/day) groups initially received HFCD alone for 8 weeks, with EAL supplementation provided during the final 6 weeks. Results Treatment with low or high doses of EAL markedly attenuated plasma levels of triglycerides and augmented plasma levels of high-density lipoprotein (HDL) in HFCD-fed rats. Chronic treatment with EAL markedly reduced impairments of acetylcholine (ACh)-induced relaxation of aortic rings. Furthermore, chronic treatment with EAL significantly lowered systolic blood pressure (SBP) and maintained smooth and flexible intimal endothelial layers in HFCD-fed rats. Chronic treatment with EAL suppressed upregulation of intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and E-selectin in the aorta. Chronic treatment with EAL also suppressed increases in matrix metalloproteinase (MMP)-2 expression. These results suggested that EAL can inhibit HFCD-induced vascular inflammation in the rat model. Conclusion The present study provides evidence that EAL ameliorates HFCD-induced vascular dysfunction through protection of vascular relaxation and suppression of vascular inflammation. PMID:22866890

Novel receptor-like kinases in cacao contain PR-1 extracellular domains.

PubMed

Teixeira, Paulo José Pereira Lima; Costa, Gustavo Gilson Lacerda; Fiorin, Gabriel Lorencini; Pereira, Gonçalo Amarante Guimarães; Mondego, Jorge Maurício Costa

2013-08-01

Members of the pathogenesis-related protein 1 (PR-1) family are well-known markers of plant defence responses, forming part of the arsenal of the secreted proteins produced on pathogen recognition. Here, we report the identification of two cacao (Theobroma cacao L.) PR-1s that are fused to transmembrane regions and serine/threonine kinase domains, in a manner characteristic of receptor-like kinases (RLKs). These proteins (TcPR-1f and TcPR-1g) were named PR-1 receptor kinases (PR-1RKs). Phylogenetic analysis of RLKs and PR-1 proteins from cacao indicated that PR-1RKs originated from a fusion between sequences encoding PR-1 and the kinase domain of a LecRLK (Lectin Receptor-Like Kinase). Retrotransposition marks surround TcPR-1f, suggesting that retrotransposition was involved in the origin of PR-1RKs. Genes with a similar domain architecture to cacao PR-1RKs were found in rice (Oryza sativa), barrel medic (Medicago truncatula) and a nonphototrophic bacterium (Herpetosiphon aurantiacus). However, their kinase domains differed from those found in LecRLKs, indicating the occurrence of convergent evolution. TcPR-1g expression was up-regulated in the biotrophic stage of witches' broom disease, suggesting a role for PR-1RKs during cacao defence responses. We hypothesize that PR-1RKs transduce a defence signal by interacting with a PR-1 ligand. © 2013 BSPP AND JOHN WILEY & SONS LTD.

Functional domains of plant chimeric calcium/calmodulin-dependent protein kinase: regulation by autoinhibitory and visinin-like domains

NASA Technical Reports Server (NTRS)

Ramachandiran, S.; Takezawa, D.; Wang, W.; Poovaiah, B. W.

1997-01-01

A novel calcium-binding calcium/calmodulin-dependent protein kinase (CCaMK) with a catalytic domain, calmodulin-binding domain, and a neural visinin-like domain was cloned and characterized from plants [Patil et al., (1995) Proc. Natl. Acad. Sci. USA 92, 4797-4801; Takezawa et al. (1996) J. Biol. Chem. 271, 8126-8132]. The mechanisms of CCaMK activation by calcium and calcium/calmodulin were investigated using various deletion mutants. The use of deletion mutants of CCaMK lacking either one, two, or all three calcium-binding EF hands indicated that all three calcium-binding sites in the visinin-like domain were crucial for the full calcium/calmodulin-dependent kinase activity. As each calcium-binding EF hand was deleted, there was a gradual reduction in calcium/calmodulin-dependent kinase activity from 100 to 4%. Another mutant (amino acids 1-322) which lacks both the visinin-like domain containing three EF hands and the calmodulin-binding domain was constitutively active, indicating the presence of an autoinhibitory domain around the calmodulin-binding domain. By using various synthetic peptides and the constitutively active mutant, we have shown that CCaMK contains an autoinhibitory domain within the residues 322-340 which overlaps its calmodulin-binding domain. Kinetic studies with both ATP and the GS peptide substrate suggest that the autoinhibitory domain of CCaMK interacts only with the peptide substrate binding motif of the catalytic domain, but not with the ATP-binding motif.

Tudor domain containing 12 (TDRD12) is essential for secondary PIWI interacting RNA biogenesis in mice.

PubMed

Pandey, Radha Raman; Tokuzawa, Yoshimi; Yang, Zhaolin; Hayashi, Eri; Ichisaka, Tomoko; Kajita, Shimpei; Asano, Yuka; Kunieda, Tetsuo; Sachidanandam, Ravi; Chuma, Shinichiro; Yamanaka, Shinya; Pillai, Ramesh S

2013-10-08

Piwi-interacting RNAs (piRNAs) are gonad-specific small RNAs that provide defense against transposable genetic elements called transposons. Our knowledge of piRNA biogenesis is sketchy, partly due to an incomplete inventory of the factors involved. Here, we identify Tudor domain-containing 12 (TDRD12; also known as ECAT8) as a unique piRNA biogenesis factor in mice. TDRD12 is detected in complexes containing Piwi protein MILI (PIWIL2), its associated primary piRNAs, and TDRD1, all of which are already implicated in secondary piRNA biogenesis. Male mice carrying either a nonsense point mutation (reproductive mutant 23 or repro23 mice) or a targeted deletion in the Tdrd12 locus are infertile and derepress retrotransposons. We find that TDRD12 is dispensable for primary piRNA biogenesis but essential for production of secondary piRNAs that enter Piwi protein MIWI2 (PIWIL4). Cell-culture studies with the insect ortholog of TDRD12 suggest a role for the multidomain protein in mediating complex formation with other participants during secondary piRNA biogenesis.

Z-disc-associated, Alternatively Spliced, PDZ Motif-containing Protein (ZASP) Mutations in the Actin-binding Domain Cause Disruption of Skeletal Muscle Actin Filaments in Myofibrillar Myopathy*

PubMed Central

Lin, Xiaoyan; Ruiz, Janelle; Bajraktari, Ilda; Ohman, Rachel; Banerjee, Soojay; Gribble, Katherine; Kaufman, Joshua D.; Wingfield, Paul T.; Griggs, Robert C.; Fischbeck, Kenneth H.; Mankodi, Ami

2014-01-01

The core of skeletal muscle Z-discs consists of actin filaments from adjacent sarcomeres that are cross-linked by α-actinin homodimers. Z-disc-associated, alternatively spliced, PDZ motif-containing protein (ZASP)/Cypher interacts with α-actinin, myotilin, and other Z-disc proteins via the PDZ domain. However, these interactions are not sufficient to maintain the Z-disc structure. We show that ZASP directly interacts with skeletal actin filaments. The actin-binding domain is between the modular PDZ and LIM domains. This ZASP region is alternatively spliced so that each isoform has unique actin-binding domains. All ZASP isoforms contain the exon 6-encoded ZASP-like motif that is mutated in zaspopathy, a myofibrillar myopathy (MFM), whereas the exon 8–11 junction-encoded peptide is exclusive to the postnatal long ZASP isoform (ZASP-LΔex10). MFM is characterized by disruption of skeletal muscle Z-discs and accumulation of myofibrillar degradation products. Wild-type and mutant ZASP interact with α-actin, α-actinin, and myotilin. Expression of mutant, but not wild-type, ZASP leads to Z-disc disruption and F-actin accumulation in mouse skeletal muscle, as in MFM. Mutations in the actin-binding domain of ZASP-LΔex10, but not other isoforms, cause disruption of the actin cytoskeleton in muscle cells. These isoform-specific mutation effects highlight the essential role of the ZASP-LΔex10 isoform in F-actin organization. Our results show that MFM-associated ZASP mutations in the actin-binding domain have deleterious effects on the core structure of the Z-discs in skeletal muscle. PMID:24668811

Translocation of the Catalytic Domain of Diphtheria Toxin across Planar Phospholipid Bilayers by Its Own T Domain

NASA Astrophysics Data System (ADS)

Oh, Kyoung Joon; Senzel, Lisa; Collier, R. John; Finkelstein, Alan

1999-07-01

The T domain of diphtheria toxin is known to participate in the pH-dependent translocation of the catalytic C domain of the toxin across the endosomal membrane, but how it does so, and whether cellular proteins are also required for this process, remain unknown. Here, we report results showing that the T domain alone is capable of translocating the entire C domain across model, planar phospholipid bilayers in the absence of other proteins. The T domain therefore contains the entire molecular machinery for mediating transfer of the catalytic domain of diphtheria toxin across membranes.

Adaptor protein containing PH domain, PTB domain and leucine zipper (APPL1) regulates the protein level of EGFR by modulating its trafficking

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

Lee, Jae-Rin; Hahn, Hwa-Sun; Kim, Young-Hoon

2011-11-11

Highlights: Black-Right-Pointing-Pointer APPL1 regulates the protein level of EGFR in response to EGF stimulation. Black-Right-Pointing-Pointer Depletion of APPL1 accelerates the movement of EGF/EGFR from the cell surface to the perinuclear region in response to EGF. Black-Right-Pointing-Pointer Knockdown of APPL1 enhances the activity of Rab5. -- Abstract: The EGFR-mediated signaling pathway regulates multiple biological processes such as cell proliferation, survival and differentiation. Previously APPL1 (adaptor protein containing PH domain, PTB domain and leucine zipper 1) has been reported to function as a downstream effector of EGF-initiated signaling. Here we demonstrate that APPL1 regulates EGFR protein levels in response to EGF stimulation.more » Overexpression of APPL1 enhances EGFR stabilization while APPL1 depletion by siRNA reduces EGFR protein levels. APPL1 depletion accelerates EGFR internalization and movement of EGF/EGFR from cell surface to the perinuclear region in response to EGF treatment. Conversely, overexpression of APPL1 decelerates EGFR internalization and translocation of EGF/EGFR to the perinuclear region. Furthermore, APPL1 depletion enhances the activity of Rab5 which is involved in internalization and trafficking of EGFR and inhibition of Rab5 in APPL1-depleted cells restored EGFR levels. Consistently, APPL1 depletion reduced activation of Akt, the downstream signaling effector of EGFR and this is restored by inhibition of Rab5. These findings suggest that APPL1 is required for EGFR signaling by regulation of EGFR stabilities through inhibition of Rab5.« less

Function of multiple Lis-Homology domain/WD-40 repeat-containing proteins in feed-forward transcriptional repression by silencing mediator for retinoic and thyroid receptor/nuclear receptor corepressor complexes.

PubMed

Choi, Hyo-Kyoung; Choi, Kyung-Chul; Kang, Hee-Bum; Kim, Han-Cheon; Lee, Yoo-Hyun; Haam, Seungjoo; Park, Hyoung-Gi; Yoon, Ho-Geun

2008-05-01

Lis-homology (LisH) motifs are involved in protein dimerization, and the discovery of the conserved N-terminal LisH domain in transducin beta-like protein 1 and its receptor (TBL1 and TBLR1) led us to examine the role of this domain in transcriptional repression. Here we show that multiple beta-transducin (WD-40) repeat-containing proteins interact to form oligomers in solution and that oligomerization depends on the presence of the LisH domain in each protein. Repression of transcription, as assayed using Gal4 fusion proteins, also depended on the presence of the LisH domain, suggesting that oligomerization is a prerequisite for efficient transcriptional repression. Furthermore, we show that the LisH domain is responsible for the binding to the hypoacetylated histone H4 tail and for stable chromatin targeting by the nuclear receptor corepressor complex. Mutations in conserved residues in the LisH motif of TBL1 and TBLR1 block histone binding, oligomerization, and transcriptional repression, supporting the functional importance of the LisH motif in transcriptional repression. Our results indicate that another WD-40 protein, TBL3, also preferentially binds to the N-terminal domain of TBL1 and TBLR1, and forms oligomers with other WD-40 proteins. Finally, we observed that the WD-40 proteins RbAp46 and RbAp48 of the sin3A corepressor complex failed to dimerize. We also found the specific interaction UbcH/E2 with TBL1, but not RbAp46/48. Altogether, our results thus indicate that the presence of multiple LisH/WD-40 repeat containing proteins is exclusive to nuclear receptor corepressor/ silencing mediator for retinoic and thyroid receptor complexes compared with other class 1 histone deacetylase-containing corepessor complexes.

Regulation of lipopolysaccharide-inducible genes by MyD88 and Toll/IL-1 domain containing adaptor inducing IFN-beta.

PubMed

Hirotani, Tomonori; Yamamoto, Masahiro; Kumagai, Yutaro; Uematsu, Satoshi; Kawase, Ichiro; Takeuchi, Osamu; Akira, Shizuo

2005-03-11

Macrophages recognize lipopolysaccharide (LPS) by Toll-like receptor 4 and activate inflammatory responses by inducing expression of various genes. TLR4 activates intracellular signaling pathways via TIR domain containing adaptor molecules, MyD88, and Toll/IL-1 domain containing adaptor inducing IFN-beta (TRIF). Although macrophages lacking MyD88 or TRIF showed impaired cytokine production, activation of intracellular signaling molecules still occurred in response to LPS in these cells. In the present study, we implemented cDNA microarrays to investigate the contribution of MyD88 and TRIF in gene expression induced by LPS stimulation. Whereas wild-type macrophages induced 148 genes in response to LPS, macrophages lacking both MyD88 and TRIF did not upregulate any genes in response to LPS. Surprisingly, 80 LPS-inducible genes were redundantly regulated by either MyD88 or TRIF. In contrast, proinflammatory cytokines and chemokines were critically regulated by MyD88 or TRIF alone. Genes critically regulated by MyD88 alone tend to be induced quickly after LPS stimulation and regulated by mRNA stability as well as transcription. Genes known to be induced by type I interferons were simply dependent on TRIF for their expression. Taken together, MyD88 and TRIF play both redundant and distinct roles in LPS-induced gene expression.

Inhibition of discoidin domain receptor 2-mediated lung cancer cells progression by gold nanoparticle-aptamer-assisted delivery of peptides containing transmembrane-juxtamembrane 1/2 domain

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

Kim, Daehwan; Yeom, Ji-Hyun; Lee, Boeun

The delivery of biologically functional peptides into mammalian cells can be a direct and effective method for cancer therapy and treatment of other diseases. Discoidin domain receptor 2 (DDR2) is a collagen-induced receptor tyrosine kinase recently identified as a novel therapeutic target in lung cancer. In this study, we report that peptides containing the functional domain of DDR2 can be efficiently delivered into lung malignant cancer cells via a gold nanoparticle-DNA aptamer conjugate (AuNP-Apt)-based system. Peptide delivery resulted in the abrogation of DDR2 activation triggered by collagen. Moreover, the peptide delivered by the AuNP-Apt system inhibited cancer cell proliferation andmore » invasion mediated by DDR2 activation. Thus, these results suggest that peptide loaded onto AuNP-Apt conjugates can be used for the development of peptide-based biomedical applications for the treatment of DDR2-positive cancer. - Highlights: • TM-JM1/2 peptides are efficiently delivered into cells by AuNP-Apt-conjugates. • TM-JM1/2 peptides loaded onto AuNP-Apt conjugates inhibit DDR2 activation. • Inhibition of DDR2 activation by TM-JM1/2 peptides decreases tumor progression.« less

Jumonji Domain Containing Protein 6: A Novel Oxygen Sensor in the Human Placenta.

PubMed

Alahari, Sruthi; Post, Martin; Caniggia, Isabella

2015-08-01

Persistent low oxygen is implicated in the pathogenesis of placental-associated pathologies such as preeclampsia, a serious disorder of pregnancy. Emerging evidence implicates a novel family of Jumonji C catalytic domain proteins as mediators of hypoxic gene expression. Here, we investigated the regulatory relationship between Jumonji C domain containing protein 6 (JMJD6) and hypoxia-inducible factor (HIF)1A in the human placenta at physiological and pathological conditions. JMJD6 expression inversely correlated with changes in oxygen tension during early placental development, ie, high at 7-9 weeks when-partial pressure of O2 is low and declining afterwards when-partial pressure of O2 increases. Moreover, JMJD6 protein was significantly elevated in early-onset preeclamptic placentae, localizing to the syncytiotrophoblast layer and syncytial knots. Exposure of primary isolated trophoblast cells, human villous explants, and JEG3 choriocarcinoma cells to low oxygen (3%) and sodium nitroprusside (inducer of oxidative stress) also resulted in elevated JMJD6 levels, which was abrogated by HIF1A knockdown. In normoxia, knockdown of JMJD6 in JEG3 cells stabilized HIF1A with a concomitant decrease in von Hippel-Lindau (VHL) tumor suppressor protein, a negative regulator of HIF1A stability. In contrast, overexpression of JMJD6 enhanced VHL expression and destabilized HIF1A. JMJD6 regulation of VHL stability did not involve the ubiquitin-proteasome system but likely occurred through lysyl hydroxylation and small ubiquitin-like modifier 1-dependent small ubiquitin-like modifierylation. In summary, our data signify a novel role for JMJD6 as an oxygen sensor in the human placenta, and alterations in the JMJD6-VHL-HIF1A feedback loop may indirectly contribute to elevated HIF1A found in preeclampsia.

Familial Blau syndrome without uveitis caused by a novel mutation in the nucleotide-binding oligomerization domain-containing protein 2 gene with good response to infliximab.

PubMed

Toral-López, Jaime; González-Huerta, Luz M; Martín-Del Campo, Mónica; Messina-Baas, Olga; Cuevas-Covarrubias, Sergio A

2018-05-01

The proband in this study was a 4-year-old Mexican girl with Blau syndrome. She and her affected family members had skin rash and arthritis but no uveitis. Exome sequencing and DNA direct sequencing from blood samples revealed a novel nucleotide-binding oligomerization domain-containing protein 2 gene mutation in the affected family members. This study is the first report of a Mexican family with Blau syndrome showing good infliximab treatment response. The novel mutation in the nucleotide-binding oligomerization domain-containing protein 2 gene (c.1808A>G) enriches the mutation spectrum in Blau syndrome. This family represents one of the few cases of autosomal Blau syndrome with no uveitis; because of phenotype variability, it is important to recognize Blau syndrome's clinical spectrum and recommend genetic consultation. © 2018 Wiley Periodicals, Inc.

Genome-wide identification and expression analysis of YTH domain-containing RNA-binding protein family in cucumber (Cucumis sativus).

PubMed

Zhou, Yong; Hu, Lifang; Jiang, Lunwei; Liu, Shiqiang

2018-06-01

YTH domain-containing RNA-binding proteins are involved in post-transcriptional regulation and play important roles in the growth and development as well as abiotic stress responses of plants. However, YTH genes have not been previously studied in cucumber (Cucumis sativus). In this study, a total of five YTH genes (CsYTH1-CsYTH5) were identified in cucumber, which could be mapped on three out of the seven cucumber chromosomes. All CsYTH proteins had highly conserved C-terminal YTH domains, and two of them (CsYTH1 and CsYTH4) harbored extra CCCH and P/Q/N-rich domains. The phylogenesis, conserved motifs and exon-intron structure of YTH genes from cucumber, Arabidopsis and rice were also analyzed. The phylogenetically closely clustered YTHs shared similar gene structures and conserved motifs. An analysis of the cis-acting regulatory elements in the upstream region of these genes resulted in the identification of many cis-elements related to stress, hormone and development. Expression analysis based on the transcriptome data showed that some CsYTHs had development- or tissue-specific expression. In addition, their expression levels were altered under various stresses such as salt, drought, cold, and abscisic acid (ABA) treatments. These findings lay the foundation for the functional analysis of CsYTHs in the future.

JFK, a Kelch domain-containing F-box protein, links the SCF complex to p53 regulation

PubMed Central

Sun, Luyang; Shi, Lei; Li, Wenqian; Yu, Wenhua; Liang, Jing; Zhang, Hua; Yang, Xiaohan; Wang, Yan; Li, Ruifang; Yao, Xingrong; Yi, Xia; Shang, Yongfeng

2009-01-01

The p53 tumor suppressor plays a central role in integrating cellular responses to various stresses. Tight regulation of p53 is thus essential for the maintenance of genome integrity and normal cell proliferation. Currently, several ubiquitin ligases, including the single-subunit RING-finger types—MDM2, Pirh2, and COP1—and the HECT-domain type—ARF-BP1—have been reported to target p53 for degradation. Here, we report the identification of a human Kelch domain-containing F-box protein, JFK. We showed that JFK promotes ubiquitination and degradation of p53. But unlike MDM2, Pirh2, COP1, and ARF-BP1, all of which possess an intrinsic ubiquitin ligase activity, JFK destabilizes p53 through the assembly of a Skp1-Cul1-F-box complex. Significantly, JFK inhibits p53-dependent transcription, and depletion of JFK stabilizes p53, promotes cell apoptosis, arrests cells in the G1 phase, and sensitizes cells to ionizing radiation-induced cell death. These data indicate that JFK is a critical negative regulator of p53 and represents a pathway for the maintenance of p53 levels in unstressed cells. Our experiments link the Skp1-Cul1-F-box system to p53 regulation. PMID:19509332

JFK, a Kelch domain-containing F-box protein, links the SCF complex to p53 regulation.

PubMed

Sun, Luyang; Shi, Lei; Li, Wenqian; Yu, Wenhua; Liang, Jing; Zhang, Hua; Yang, Xiaohan; Wang, Yan; Li, Ruifang; Yao, Xingrong; Yi, Xia; Shang, Yongfeng

2009-06-23

The p53 tumor suppressor plays a central role in integrating cellular responses to various stresses. Tight regulation of p53 is thus essential for the maintenance of genome integrity and normal cell proliferation. Currently, several ubiquitin ligases, including the single-subunit RING-finger types--MDM2, Pirh2, and COP1--and the HECT-domain type--ARF-BP1--have been reported to target p53 for degradation. Here, we report the identification of a human Kelch domain-containing F-box protein, JFK. We showed that JFK promotes ubiquitination and degradation of p53. But unlike MDM2, Pirh2, COP1, and ARF-BP1, all of which possess an intrinsic ubiquitin ligase activity, JFK destabilizes p53 through the assembly of a Skp1-Cul1-F-box complex. Significantly, JFK inhibits p53-dependent transcription, and depletion of JFK stabilizes p53, promotes cell apoptosis, arrests cells in the G(1) phase, and sensitizes cells to ionizing radiation-induced cell death. These data indicate that JFK is a critical negative regulator of p53 and represents a pathway for the maintenance of p53 levels in unstressed cells. Our experiments link the Skp1-Cul1-F-box system to p53 regulation.

Big Domains Are Novel Ca2+-Binding Modules: Evidences from Big Domains of Leptospira Immunoglobulin-Like (Lig) Proteins

PubMed Central

Palaniappan, Raghavan U. M.; Lin, Yi-Pin; He, Hongxuan; McDonough, Sean P.; Sharma, Yogendra; Chang, Yung-Fu

2010-01-01

Background Many bacterial surface exposed proteins mediate the host-pathogen interaction more effectively in the presence of Ca2+. Leptospiral immunoglobulin-like (Lig) proteins, LigA and LigB, are surface exposed proteins containing Bacterial immunoglobulin like (Big) domains. The function of proteins which contain Big fold is not known. Based on the possible similarities of immunoglobulin and βγ-crystallin folds, we here explore the important question whether Ca2+ binds to a Big domains, which would provide a novel functional role of the proteins containing Big fold. Principal Findings We selected six individual Big domains for this study (three from the conserved part of LigA and LigB, denoted as Lig A3, Lig A4, and LigBCon5; two from the variable region of LigA, i.e., 9th (Lig A9) and 10th repeats (Lig A10); and one from the variable region of LigB, i.e., LigBCen2. We have also studied the conserved region covering the three and six repeats (LigBCon1-3 and LigCon). All these proteins bind the calcium-mimic dye Stains-all. All the selected four domains bind Ca2+ with dissociation constants of 2–4 µM. Lig A9 and Lig A10 domains fold well with moderate thermal stability, have β-sheet conformation and form homodimers. Fluorescence spectra of Big domains show a specific doublet (at 317 and 330 nm), probably due to Trp interaction with a Phe residue. Equilibrium unfolding of selected Big domains is similar and follows a two-state model, suggesting the similarity in their fold. Conclusions We demonstrate that the Lig are Ca2+-binding proteins, with Big domains harbouring the binding motif. We conclude that despite differences in sequence, a Big motif binds Ca2+. This work thus sets up a strong possibility for classifying the proteins containing Big domains as a novel family of Ca2+-binding proteins. Since Big domain is a part of many proteins in bacterial kingdom, we suggest a possible function these proteins via Ca2+ binding. PMID:21206924

Cas13d Is a Compact RNA-Targeting Type VI CRISPR Effector Positively Modulated by a WYL-Domain-Containing Accessory Protein.

PubMed

Yan, Winston X; Chong, Shaorong; Zhang, Huaibin; Makarova, Kira S; Koonin, Eugene V; Cheng, David R; Scott, David A

2018-04-19

Bacterial class 2 CRISPR-Cas systems utilize a single RNA-guided protein effector to mitigate viral infection. We aggregated genomic data from multiple sources and constructed an expanded database of predicted class 2 CRISPR-Cas systems. A search for novel RNA-targeting systems identified subtype VI-D, encoding dual HEPN domain-containing Cas13d effectors and putative WYL-domain-containing accessory proteins (WYL1 and WYL-b1 through WYL-b5). The median size of Cas13d proteins is 190 to 300 aa smaller than that of Cas13a-Cas13c. Despite their small size, Cas13d orthologs from Eubacterium siraeum (Es) and Ruminococcus sp. (Rsp) are active in both CRISPR RNA processing and targeting, as well as collateral RNA cleavage, with no target-flanking sequence requirements. The RspWYL1 protein stimulates RNA cleavage by both EsCas13d and RspCas13d, demonstrating a common regulatory mechanism for divergent Cas13d orthologs. The small size, minimal targeting constraints, and modular regulation of Cas13d effectors further expands the CRISPR toolkit for RNA manipulation and detection. Copyright © 2018 Elsevier Inc. All rights reserved.

Comparison of structure, function and regulation of plant cold shock domain proteins to bacterial and animal cold shock domain proteins.

PubMed

Chaikam, Vijay; Karlson, Dale T

2010-01-01

The cold shock domain (CSD) is among the most ancient and well conserved nucleic acid binding domains from bacteria to higher animals and plants. The CSD facilitates binding to RNA, ssDNA and dsDNA and most functions attributed to cold shock domain proteins are mediated by this nucleic acid binding activity. In prokaryotes, cold shock domain proteins only contain a single CSD and are termed cold shock proteins (Csps). In animal model systems, various auxiliary domains are present in addition to the CSD and are commonly named Y-box proteins. Similar to animal CSPs, plant CSPs contain auxiliary C-terminal domains in addition to their N-terminal CSD. Cold shock domain proteins have been shown to play important roles in development and stress adaptation in wide variety of organisms. In this review, the structure, function and regulation of plant CSPs are compared and contrasted to the characteristics of bacterial and animal CSPs. [BMB reports 2010; 43(1): 1-8].

The S-layer homology domain-containing protein SlhA from Paenibacillus alvei CCM 2051(T) is important for swarming and biofilm formation.

PubMed

Janesch, Bettina; Koerdt, Andrea; Messner, Paul; Schäffer, Christina

2013-01-01

Swarming and biofilm formation have been studied for a variety of bacteria. While this is well investigated for Gram-negative bacteria, less is known about Gram-positive bacteria, including Paenibacillus alvei, a secondary invader of diseased honeybee colonies infected with Melissococcus pluton, the causative agent of European foulbrood (EFB). Paenibacillus alvei CCM 2051(T) is a Gram-positive bacterium which was recently shown to employ S-layer homology (SLH) domains as cell wall targeting modules to display proteins on its cell surface. This study deals with the newly identified 1335-amino acid protein SlhA from P. alvei which carries at the C‑terminus three consecutive SLH-motifs containing the predicted binding sequences SRGE, VRQD, and LRGD instead of the common TRAE motif. Based on the proof of cell surface location of SlhA by fluorescence microscopy using a SlhA-GFP chimera, the binding mechanism was investigated in an in vitro assay. To unravel a putative function of the SlhA protein, a knockout mutant was constructed. Experimental data indicated that one SLH domain is sufficient for anchoring of SlhA to the cell surface, and the SLH domains of SlhA recognize both the peptidoglycan and the secondary cell wall polymer in vitro. This is in agreement with previous data from the S-layer protein SpaA, pinpointing a wider utilization of that mechanism for cell surface display of proteins in P. alvei. Compared to the wild-type bacterium ΔslhA revealed changed colony morphology, loss of swarming motility and impaired biofilm formation. The phenotype was similar to that of the flagella knockout Δhag, possibly due to reduced EPS production influencing the functionality of the flagella of ΔslhA. This study demonstrates the involvement of the SLH domain-containing protein SlhA in swarming and biofilm formation of P. alvei CCM 2051(T).

The SARS-Unique Domain (SUD) of SARS Coronavirus Contains Two Macrodomains That Bind G-Quadruplexes

PubMed Central

Tan, Jinzhi; Vonrhein, Clemens; Smart, Oliver S.; Bricogne, Gerard; Bollati, Michela; Kusov, Yuri; Hansen, Guido; Mesters, Jeroen R.; Schmidt, Christian L.; Hilgenfeld, Rolf

2009-01-01

Since the outbreak of severe acute respiratory syndrome (SARS) in 2003, the three-dimensional structures of several of the replicase/transcriptase components of SARS coronavirus (SARS-CoV), the non-structural proteins (Nsps), have been determined. However, within the large Nsp3 (1922 amino-acid residues), the structure and function of the so-called SARS-unique domain (SUD) have remained elusive. SUD occurs only in SARS-CoV and the highly related viruses found in certain bats, but is absent from all other coronaviruses. Therefore, it has been speculated that it may be involved in the extreme pathogenicity of SARS-CoV, compared to other coronaviruses, most of which cause only mild infections in humans. In order to help elucidate the function of the SUD, we have determined crystal structures of fragment 389–652 (“SUDcore”) of Nsp3, which comprises 264 of the 338 residues of the domain. Both the monoclinic and triclinic crystal forms (2.2 and 2.8 Å resolution, respectively) revealed that SUDcore forms a homodimer. Each monomer consists of two subdomains, SUD-N and SUD-M, with a macrodomain fold similar to the SARS-CoV X-domain. However, in contrast to the latter, SUD fails to bind ADP-ribose, as determined by zone-interference gel electrophoresis. Instead, the entire SUDcore as well as its individual subdomains interact with oligonucleotides known to form G-quadruplexes. This includes oligodeoxy- as well as oligoribonucleotides. Mutations of selected lysine residues on the surface of the SUD-N subdomain lead to reduction of G-quadruplex binding, whereas mutations in the SUD-M subdomain abolish it. As there is no evidence for Nsp3 entering the nucleus of the host cell, the SARS-CoV genomic RNA or host-cell mRNA containing long G-stretches may be targets of SUD. The SARS-CoV genome is devoid of G-stretches longer than 5–6 nucleotides, but more extended G-stretches are found in the 3′-nontranslated regions of mRNAs coding for certain host-cell proteins involved

Deletion of genes involved in glutamate metabolism to improve poly-gamma-glutamic acid production in B. amyloliquefaciens LL3.

PubMed

Zhang, Wei; He, Yulian; Gao, Weixia; Feng, Jun; Cao, Mingfeng; Yang, Chao; Song, Cunjiang; Wang, Shufang

2015-02-01

Here, we attempted to elevate poly-gamma-glutamic acid (γ-PGA) production by modifying genes involved in glutamate metabolism in Bacillus amyloliquefaciens LL3. Products of rocR, rocG and gudB facilitate the conversion from glutamate to 2-oxoglutarate in Bacillus subtillis. The gene odhA is responsible for the synthesis of a component of the 2-oxoglutarate dehydrogenase complex that catalyzes the oxidative decarboxylation of 2-oxoglutarate to succinyl coenzyme A. In-frame deletions of these four genes were performed. In shake flask experiments the gudB/rocG double mutant presented enhanced production of γ-PGA, a 38 % increase compared with wild type. When fermented in a 5-L fermenter with pH control, the γ-PGA yield of the rocR mutant was increased to 5.83 g/L from 4.55 g/L for shake flask experiments. The gudB/rocG double mutant produced 5.68 g/L γ-PGA compared with that of 4.03 g/L for the wild type, a 40 % increase. Those results indicated the possibility of improving γ-PGA production by modifying glutamate metabolism, and identified potential genetic targets to improve γ-PGA production.

Same but not alike: Structure, flexibility and energetics of domains in multi-domain proteins are influenced by the presence of other domains

PubMed Central

Vishwanath, Sneha

2018-01-01

The majority of the proteins encoded in the genomes of eukaryotes contain more than one domain. Reasons for high prevalence of multi-domain proteins in various organisms have been attributed to higher stability and functional and folding advantages over single-domain proteins. Despite these advantages, many proteins are composed of only one domain while their homologous domains are part of multi-domain proteins. In the study presented here, differences in the properties of protein domains in single-domain and multi-domain systems and their influence on functions are discussed. We studied 20 pairs of identical protein domains, which were crystallized in two forms (a) tethered to other proteins domains and (b) tethered to fewer protein domains than (a) or not tethered to any protein domain. Results suggest that tethering of domains in multi-domain proteins influences the structural, dynamic and energetic properties of the constituent protein domains. 50% of the protein domain pairs show significant structural deviations while 90% of the protein domain pairs show differences in dynamics and 12% of the residues show differences in the energetics. To gain further insights on the influence of tethering on the function of the domains, 4 pairs of homologous protein domains, where one of them is a full-length single-domain protein and the other protein domain is a part of a multi-domain protein, were studied. Analyses showed that identical and structurally equivalent functional residues show differential dynamics in homologous protein domains; though comparable dynamics between in-silico generated chimera protein and multi-domain proteins were observed. From these observations, the differences observed in the functions of homologous proteins could be attributed to the presence of tethered domain. Overall, we conclude that tethered domains in multi-domain proteins not only provide stability or folding advantages but also influence pathways resulting in differences in

Same but not alike: Structure, flexibility and energetics of domains in multi-domain proteins are influenced by the presence of other domains.

PubMed

Vishwanath, Sneha; de Brevern, Alexandre G; Srinivasan, Narayanaswamy

2018-02-01

The majority of the proteins encoded in the genomes of eukaryotes contain more than one domain. Reasons for high prevalence of multi-domain proteins in various organisms have been attributed to higher stability and functional and folding advantages over single-domain proteins. Despite these advantages, many proteins are composed of only one domain while their homologous domains are part of multi-domain proteins. In the study presented here, differences in the properties of protein domains in single-domain and multi-domain systems and their influence on functions are discussed. We studied 20 pairs of identical protein domains, which were crystallized in two forms (a) tethered to other proteins domains and (b) tethered to fewer protein domains than (a) or not tethered to any protein domain. Results suggest that tethering of domains in multi-domain proteins influences the structural, dynamic and energetic properties of the constituent protein domains. 50% of the protein domain pairs show significant structural deviations while 90% of the protein domain pairs show differences in dynamics and 12% of the residues show differences in the energetics. To gain further insights on the influence of tethering on the function of the domains, 4 pairs of homologous protein domains, where one of them is a full-length single-domain protein and the other protein domain is a part of a multi-domain protein, were studied. Analyses showed that identical and structurally equivalent functional residues show differential dynamics in homologous protein domains; though comparable dynamics between in-silico generated chimera protein and multi-domain proteins were observed. From these observations, the differences observed in the functions of homologous proteins could be attributed to the presence of tethered domain. Overall, we conclude that tethered domains in multi-domain proteins not only provide stability or folding advantages but also influence pathways resulting in differences in

Interactions of the EGFR juxtamembrane domain with PIP2-containing lipid bilayers: Insights from multiscale molecular dynamics simulations☆

PubMed Central

Abd Halim, Khairul Bariyyah; Koldsø, Heidi; Sansom, Mark S.P.

2015-01-01

Background The epidermal growth factor receptor (EGFR) is the best characterised member of the receptor tyrosine kinases, which play an important role in signalling across mammalian cell membranes. The EGFR juxtamembrane (JM) domain is involved in the mechanism of activation of the receptor, interacting with the anionic lipid phosphatidylinositol 4,5-bisphosphate (PIP2) in the intracellular leaflet of the cell membrane. Methods Multiscale MD simulations were used to characterize PIP2–JM interactions. Simulations of the transmembrane helix plus JM region (TM–JM) dimer (PDB:2M20) in both PIP2-containing and PIP2-depleted lipid bilayer membranes revealed the interactions of the JM with PIP2 and other lipids. Results PIP2 forms strong interactions with the basic residues in the R645–R647 motif of the JM domain resulting in clustering of PIP2 around the protein. This association of PIP2 and the JM domain aids stabilization of JM-A dimer away from the membrane. Mutation (R645N/R646N/R647N) or PIP2-depletion results in deformation of the JM-A dimer and changes in JM–membrane interactions. Conclusions These simulations support the proposal that the positively charged residues at the start of the JM-A domain stabilize the JM-A helices in an orientation away from the membrane surface through binding to PIP2, thus promoting a conformation corresponding to an asymmetric (i.e. activated) kinase. General significance This study indicates that MD simulations may be used to characterise JM/lipid interactions, thus helping to define their role in the mechanisms of receptor tyrosine kinases. This article is part of a Special Issue entitled Recent developments of molecular dynamics. PMID:25219456

Interactions of the EGFR juxtamembrane domain with PIP2-containing lipid bilayers: Insights from multiscale molecular dynamics simulations.

PubMed

Abd Halim, Khairul Bariyyah; Koldsø, Heidi; Sansom, Mark S P

2015-05-01

The epidermal growth factor receptor (EGFR) is the best characterised member of the receptor tyrosine kinases, which play an important role in signalling across mammalian cell membranes. The EGFR juxtamembrane (JM) domain is involved in the mechanism of activation of the receptor, interacting with the anionic lipid phosphatidylinositol 4,5-bisphosphate (PIP2) in the intracellular leaflet of the cell membrane. Multiscale MD simulations were used to characterize PIP2-JM interactions. Simulations of the transmembrane helix plus JM region (TM-JM) dimer (PDB:2M20) in both PIP2-containing and PIP2-depleted lipid bilayer membranes revealed the interactions of the JM with PIP2 and other lipids. PIP2 forms strong interactions with the basic residues in the R645-R647 motif of the JM domain resulting in clustering of PIP2 around the protein. This association of PIP2 and the JM domain aids stabilization of JM-A dimer away from the membrane. Mutation (R645N/R646N/R647N) or PIP2-depletion results in deformation of the JM-A dimer and changes in JM-membrane interactions. These simulations support the proposal that the positively charged residues at the start of the JM-A domain stabilize the JM-A helices in an orientation away from the membrane surface through binding to PIP2, thus promoting a conformation corresponding to an asymmetric (i.e. activated) kinase. This study indicates that MD simulations may be used to characterise JM/lipid interactions, thus helping to define their role in the mechanisms of receptor tyrosine kinases. This article is part of a Special Issue entitled Recent developments of molecular dynamics. Copyright © 2014. Published by Elsevier B.V.

TIR-domain-containing adapter-inducing interferon-β (TRIF) forms filamentous structures, whose pro-apoptotic signalling is terminated by autophagy.

PubMed

Gentle, Ian E; McHenry, Kevin T; Weber, Arnim; Metz, Arlena; Kretz, Oliver; Porter, Dale; Häcker, Georg

2017-07-01

The formation of amyloid-like protein structures has recently emerged as a feature in signal transduction, particularly in innate immunity. These structures appear to depend on defined domains for their formation but likely also require dedicated ways to terminate signalling. We, here, define the innate immunity protein/Toll-like receptor adaptor TIR-domain-containing adapter-inducing interferon-β (TRIF) as a novel platform of fibril formation and probe signal initiation through TRIF as well as its termination in Toll-like receptor 3 (TLR3)-stimulated melanoma cells. A main signalling pathway triggered by TLR3 caused apoptosis, which was controlled by inhibitor of apoptosis proteins and was dependent on RIPK1 and independent of TNF. Using correlative electron/fluorescence microscopy, we visualised fibrillar structures formed through both Toll/interleukin-1 receptor and RIP homotypic interacting motif regions of TRIF. We provide evidence that these fibrillary structures are active signalling platforms whose activity is terminated by autophagy. TRIF-signalling enhanced autophagy, and fibrillary structures were partly contained within autophagosomes. Inhibition of autophagy increased levels of pro-apoptotic TRIF complexes, leading to the accumulation of active caspase-8 and enhanced apoptosis while stimulation of autophagy reduced TRIF-dependent death. We conclude that pro-death signals through TRIF are regulated by autophagy and propose that pro-apoptotic signalling through TRIF/RIPK1/caspase-8 occurs in fibrillary platforms. © 2017 Federation of European Biochemical Societies.

WW Domains of the Yes-Kinase-Associated-Protein (YAP) Transcriptional Regulator Behave as Independent Units with Different Binding Preferences for PPxY Motif-Containing Ligands

PubMed Central

Iglesias-Bexiga, Manuel; Castillo, Francisco; Cobos, Eva S.; Oka, Tsutomu; Sudol, Marius; Luque, Irene

2015-01-01

YAP is a WW domain-containing effector of the Hippo tumor suppressor pathway, and the object of heightened interest as a potent oncogene and stemness factor. YAP has two major isoforms that differ in the number of WW domains they harbor. Elucidating the degree of co-operation between these WW domains is important for a full understanding of the molecular function of YAP. We present here a detailed biophysical study of the structural stability and binding properties of the two YAP WW domains aimed at investigating the relationship between both domains in terms of structural stability and partner recognition. We have carried out a calorimetric study of the structural stability of the two YAP WW domains, both isolated and in a tandem configuration, and their interaction with a set of functionally relevant ligands derived from PTCH1 and LATS kinases. We find that the two YAP WW domains behave as independent units with different binding preferences, suggesting that the presence of the second WW domain might contribute to modulate target recognition between the two YAP isoforms. Analysis of structural models and phage-display studies indicate that electrostatic interactions play a critical role in binding specificity. Together, these results are relevant to understand of YAP function and open the door to the design of highly specific ligands of interest to delineate the functional role of each WW domain in YAP signaling. PMID:25607641

Crystal Structure of the Bovine lactadherin C2 Domain, a Membrane Binding Motif, Shows Similarity of the C2 Domains of Factor V and Factor VIII

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

Lin,L.; Huai, Q.; Huang, M.

2007-01-01

Lactadherin, a glycoprotein secreted by a variety of cell types, contains two EGF domains and two C domains with sequence homology to the C domains of blood coagulation proteins factor V and factor VIII. Like these proteins, lactadherin binds to phosphatidylserine (PS)-containing membranes with high affinity. We determined the crystal structure of the bovine lactadherin C2 domain (residues 1 to 158) at 2.4 Angstroms. The lactadherin C2 structure is similar to the C2 domains of factors V and VIII (rmsd of C? atoms of 0.9 Angstroms and 1.2 Angstroms, and sequence identities of 43% and 38%, respectively). The lactadherin C2more » domain has a discoidin-like fold containing two ?-sheets of five and three antiparallel ?-strands packed against one another. The N and C termini are linked by a disulfide bridge between Cys1 and Cys158. One ?-turn and two loops containing solvent-exposed hydrophobic residues extend from the C2 domain ?-sandwich core. In analogy with the C2 domains of factors V and VIII, some or all of these solvent-exposed hydrophobic residues, Trp26, Leu28, Phe31, and Phe81, likely participate in membrane binding. The C2 domain of lactadherin may serve as a marker of cell surface phosphatidylserine exposure and may have potential as a unique anti-thrombotic agent.« less

The Cysteine-rich Domain of the DHHC3 Palmitoyltransferase Is Palmitoylated and Contains Tightly Bound Zinc*

PubMed Central

Gottlieb, Colin D.; Zhang, Sheng; Linder, Maurine E.

2015-01-01

DHHC palmitoyltransferases catalyze the addition of the fatty acid palmitate to proteins on the cytoplasmic leaflet of cell membranes. There are 23 members of the highly diverse mammalian DHHC protein family, all of which contain a conserved catalytic domain called the cysteine-rich domain (CRD). DHHC proteins transfer palmitate via a two-step catalytic mechanism in which the enzyme first modifies itself with palmitate in a process termed autoacylation. The enzyme then transfers palmitate from itself onto substrate proteins. The number and location of palmitoylated cysteines in the autoacylated intermediate is unknown. In this study, we present evidence using mass spectrometry that DHHC3 is palmitoylated at the cysteine in the DHHC motif. Mutation of highly conserved CRD cysteines outside the DHHC motif resulted in activity deficits and a structural perturbation revealed by limited proteolysis. Treatment of DHHC3 with chelating agents in vitro replicated both the specific structural perturbations and activity deficits observed in conserved cysteine mutants, suggesting metal ion-binding in the CRD. Using the fluorescent indicator mag-fura-2, the metal released from DHHC3 was identified as zinc. The stoichiometry of zinc binding was measured as 2 mol of zinc/mol of DHHC3 protein. Taken together, our data demonstrate that coordination of zinc ions by cysteine residues within the CRD is required for the structural integrity of DHHC proteins. PMID:26487721

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

The Structure of an Unconventional HD-GYP Protein from Bdellovibrio Reveals the Roles of Conserved Residues in this Class of Cyclic-di-GMP Phosphodiesterases

PubMed Central

Lovering, Andrew L.; Capeness, Michael J.; Lambert, Carey; Hobley, Laura; Sockett, R. Elizabeth

2011-01-01

ABSTRACT Cyclic-di-GMP is a near-ubiquitous bacterial second messenger that is important in localized signal transmission during the control of various processes, including virulence and switching between planktonic and biofilm-based lifestyles. Cyclic-di-GMP is synthesized by GGDEF diguanylate cyclases and hydrolyzed by EAL or HD-GYP phosphodiesterases, with each functional domain often appended to distinct sensory modules. HD-GYP domain proteins have resisted structural analysis, but here we present the first structural representative of this family (1.28 Å), obtained using the unusual Bd1817 HD-GYP protein from the predatory bacterium Bdellovibrio bacteriovorus. Bd1817 lacks the active-site tyrosine present in most HD-GYP family members yet remains an excellent model of their features, sharing 48% sequence similarity with the archetype RpfG. The protein structure is highly modular and thus provides a basis for delineating domain boundaries in other stimulus-dependent homologues. Conserved residues in the HD-GYP family cluster around a binuclear metal center, which is observed complexed to a molecule of phosphate, providing information on the mode of hydroxide ion attack on substrate. The fold and active site of the HD-GYP domain are different from those of EAL proteins, and restricted access to the active-site cleft is indicative of a different mode of activity regulation. The region encompassing the GYP motif has a novel conformation and is surface exposed and available for complexation with binding partners, including GGDEF proteins. PMID:21990613

FBI-1 can stimulate HIV-1 Tat activity and is targeted to a novel subnuclear domain that includes the Tat-P-TEFb-containing nuclear speckles.

PubMed

Pendergrast, P Shannon; Wang, Chen; Hernandez, Nouria; Huang, Sui

2002-03-01

FBI-1 is a cellular POZ-domain-containing protein that binds to the HIV-1 LTR and associates with the HIV-1 transactivator protein Tat. Here we show that elevated levels of FBI-1 specifically stimulate Tat activity and that this effect is dependent on the same domain of FBI-1 that mediates Tat-FBI-1 association in vivo. FBI-1 also partially colocalizes with Tat and Tat's cellular cofactor, P-TEFb (Cdk9 and cyclin T1), at the splicing-factor-rich nuclear speckle domain. Further, a less-soluble population of FBI-1 distributes in a novel peripheral-speckle pattern of localization as well as in other nuclear regions. This distribution pattern is dependent on the FBI-1 DNA binding domain, on the presence of cellular DNA, and on active transcription. Taken together, these results suggest that FBI-1 is a cellular factor that preferentially associates with active chromatin and that can specifically stimulate Tat-activated HIV-1 transcription.

Peptides containing antigenic and cationic domains have enhanced, multivalent immunogenicity when bound to DNA vaccines.

PubMed

Riedl, Petra; Reimann, Jörg; Schirmbeck, Reinhold

2004-02-01

We explored strategies to codeliver DNA- and peptide-based vaccines in a way that enhances the immunogenicity of both components of the combination vaccine for T cells. Specific CD8(+) T cell responses to an antigenic peptide are primed when the peptide is fused to a cationic peptide domain that is bound to plasmid DNA or oligonucleotides (ODN; with or without CpG motifs). Plasmid DNA mixed with antigenic/cationic peptides or histones forms large complexes with different biological properties depending on the molar ratios of peptide/protein and polynucleotide. Complexes containing high (but not low) molar ratios of cationic peptide to DNA facilitate transfection (DNA uptake and expression of the plasmid-encoded product) of cells. In contrast, complexes containing low (but not high) molar ratios of cationic peptide to DNA prime potent multispecific T cell responses after a single intramuscular injection of the complexes. The general validity of this observation was confirmed mixing different antigenic/cationic peptides with different DNA vaccines. In these vaccine formulations, multispecific CD8(+) T cell responses specific for epitopes of the peptide- as well as the DNA-based vaccine were efficiently coprimed, together with humoral antibody responses to conformational determinants of large viral antigens encoded by the DNA vaccine. The data indicate that mixtures of DNA vaccines with antigenic, cationic peptides are immunogenic vaccine formulations particularly suited for the induction of multispecific T cell responses.

Human alpha beta hydrolase domain containing protein 11 and its yeast homolog are lipid hydrolases

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

Arya, Madhuri; Srinivasan, Malathi; Rajasekharan, Ram

Mammalian alpha/beta hydrolase domain (ABHD) family of proteins have emerged as key regulators of lipid metabolism and are found to be associated with human diseases. Human α/β-hydrolase domain containing protein 11 (ABHD11) has recently been predicted as a potential biomarker for human lung adenocarcinoma. In silico analyses of the ABHD11 protein sequence revealed the presence of a conserved lipase motif GXSXG. However, the role of ABHD11 in lipid metabolism is not known. To understand the biological function of ABHD11, we heterologously expressed the human ABHD11 in budding yeast, Saccharomyces cerevisiae. In vivo [{sup 14}C]acetate labeling of cellular lipids in yeast cellsmore » overexpressing ABHD11 showed a decrease in triacylglycerol content. Overexpression of ABHD11 also alters the molecular species of triacylglycerol in yeast. Similar activity was observed in its yeast homolog, Ygr031w. The role of the conserved lipase motif in the hydrolase activity was proven by the mutation of all conserved amino acid residues of GXSXG motif. Collectively, our results demonstrate that human ABHD11 and its yeast homolog YGR031W have a pivotal role in the lipid metabolism. - Highlights: • Overexpression of ABHD11 protein and its yeast homolog Ygr031w cause a reduction in triacylglycerol levels in yeast. • The reduction in triacylglycerol is due to the presence of lipase motif GXSXG. • Overexpression of ABHD11 and Ygr031w alters the molecular species of triacylglycerol.« less

A localized interaction surface for voltage-sensing domains on the pore domain of a K+ channel.

PubMed

Li-Smerin, Y; Hackos, D H; Swartz, K J

2000-02-01

Voltage-gated K+ channels contain a central pore domain and four surrounding voltage-sensing domains. How and where changes in the structure of the voltage-sensing domains couple to the pore domain so as to gate ion conduction is not understood. The crystal structure of KcsA, a bacterial K+ channel homologous to the pore domain of voltage-gated K+ channels, provides a starting point for addressing this question. Guided by this structure, we used tryptophan-scanning mutagenesis on the transmembrane shell of the pore domain in the Shaker voltage-gated K+ channel to localize potential protein-protein and protein-lipid interfaces. Some mutants cause only minor changes in gating and when mapped onto the KcsA structure cluster away from the interface between pore domain subunits. In contrast, mutants producing large changes in gating tend to cluster near this interface. These results imply that voltage-sensing domains interact with localized regions near the interface between adjacent pore domain subunits.

Crystal Structure of the Bovine lactadherin C2 Domain, a Membrane Binding Motif, Shows Similarity to the C2 Domains of Factor V and Factor VIII

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

Lin,L.

2007-01-01

Lactadherin, a glycoprotein secreted by a variety of cell types, contains two EGF domains and two C domains with sequence homology to the C domains of blood coagulation proteins factor V and factor VIII. Like these proteins, lactadherin binds to phosphatidylserine (PS)-containing membranes with high affinity. We determined the crystal structure of the bovine lactadherin C2 domain (residues 1 to 158) at 2.4 {angstrom}. The lactadherin C2 structure is similar to the C2 domains of factors V and VIII (rmsd of C{sub {alpha}} atoms of 0.9 {angstrom} and 1.2 {angstrom}, and sequence identities of 43% and 38%, respectively). The lactadherinmore » C2 domain has a discoidin-like fold containing two {beta}-sheets of five and three antiparallel {beta}-strands packed against one another. The N and C termini are linked by a disulfide bridge between Cys1 and Cys158. One {beta}-turn and two loops containing solvent-exposed hydrophobic residues extend from the C2 domain {beta}-sandwich core. In analogy with the C2 domains of factors V and VIII, some or all of these solvent-exposed hydrophobic residues, Trp26, Leu28, Phe31, and Phe81, likely participate in membrane binding. The C2 domain of lactadherin may serve as a marker of cell surface phosphatidylserine exposure and may have potential as a unique anti-thrombotic agent.« less

Estimation of exciton reverse transfer for variable spectra and high efficiency in interlayer-based organic light-emitting devices

NASA Astrophysics Data System (ADS)

Liu, Shengqiang; Zhao, Juan; Huang, Jiang; Yu, Junsheng

2016-12-01

Organic light-emitting devices (OLEDs) with three different exciton adjusting interlayers (EALs), which are inserted between two complementary blue and yellow emitting layers, are fabricated to demonstrate the relationship between the EAL and device performance. The results show that the variations of type and thickness of EAL have different adjusting capability and distribution control on excitons. However, we also find that the reverse Dexter transfer of triplet exciton from the light-emitting layer to the EAL is an energy loss path, which detrimentally affects electroluminescent (EL) spectral performance and device efficiency in different EAL-based devices. Based on exciton distribution and integration, an estimation of exciton reverse transfer is developed through a triplet energy level barrier to simulate the exciton behavior. Meanwhile, the estimation results also demonstrate the relationship between the EAL and device efficiency by a parameter of exciton reverse transfer probability. The estimation of exciton reverse transfer discloses a crucial role of the EALs in the interlayer-based OLEDs to achieve variable EL spectra and high efficiency.

Nucleo-cytoplasmic shuttling of the endonuclease ankyrin repeats and LEM domain-containing protein 1 (Ankle1) is mediated by canonical nuclear export- and nuclear import signals.

PubMed

Zlopasa, Livija; Brachner, Andreas; Foisner, Roland

2016-06-01

Ankyrin repeats and LEM domain containing protein 1 (Ankle1) belongs to the LEM protein family, whose members share a chromatin-interacting LEM motif. Unlike most other LEM proteins, Ankle1 is not an integral protein of the inner nuclear membrane but shuttles between the nucleus and the cytoplasm. It contains a GIY-YIG-type nuclease domain, but its function is unknown. The mammalian genome encodes only one other GIY-YIG domain protein, termed Slx1. Slx1 has been described as a resolvase that processes Holliday junctions during homologous recombination-mediated DNA double strand break repair. Resolvase activity is regulated in a spatial and temporal manner during the cell cycle. We hypothesized that Ankle1 may have a similar function and its nucleo-cytoplasmic shuttling may contribute to the regulation of Ankle1 activity. Hence, we aimed at identifying the domains mediating Ankle1 shuttling and investigating whether cellular localization is affected during DNA damage response. Sequence analysis predicts the presence of two canonical nuclear import and export signals in Ankle1. Immunofluorescence microscopy of cells expressing wild-type and various mutated Ankle1-fusion proteins revealed a C-terminally located classical monopartite nuclear localization signal and a centrally located CRM1-dependent nuclear export signal that mediate nucleo-cytoplasmic shuttling of Ankle1. These sequences are also functional in heterologous proteins. The predominant localization of Ankle1 in the cytoplasm, however, does not change upon induction of several DNA damage response pathways throughout the cell cycle. We identified the domains mediating nuclear import and export of Ankle1. Ankle1's cellular localization was not affected following DNA damage.

Transformation-specific interaction of the bovine papillomavirus E5 oncoprotein with the platelet-derived growth factor receptor transmembrane domain and the epidermal growth factor receptor cytoplasmic domain.

PubMed Central

Cohen, B D; Goldstein, D J; Rutledge, L; Vass, W C; Lowy, D R; Schlegel, R; Schiller, J T

1993-01-01

The bovine papillomavirus E5 transforming protein appears to activate both the epidermal growth factor receptor (EGF-R) and the platelet-derived growth factor receptor (PDGF-R) by a ligand-independent mechanism. To further investigate the ability of E5 to activate receptors of different classes and to determine whether this stimulation occurs through the extracellular domain required for ligand activation, we constructed chimeric genes encoding PDGF-R and EGF-R by interchanging the extracellular, membrane, and cytoplasmic coding domains. Chimeras were transfected into NIH 3T3 and CHO(LR73) cells. All chimeras expressed stable protein which, upon addition of the appropriate ligand, could be activated as assayed by tyrosine autophosphorylation and biological transformation. Cotransfection of E5 with the wild-type and chimeric receptors resulted in the ligand-independent activation of receptors, provided that a receptor contained either the transmembrane domain of the PDGF-R or the cytoplasmic domain of the EGF-R. Chimeric receptors that contained both of these domains exhibited the highest level of E5-induced biochemical and biological stimulation. These results imply that E5 activates the PDGF-R and EGR-R by two distinct mechanisms, neither of which specifically involves the extracellular domain of the receptor. Consistent with the biochemical and biological activation data, coimmunoprecipitation studies demonstrated that E5 formed a complex with any chimera that contained a PDGF-R transmembrane domain or an EGF-R cytoplasmic domain, with those chimeras containing both domains demonstrating the greatest efficiency of complex formation. These results suggest that although different domains of the PDGF-R and EGF-R are required for E5 activation, both receptors are activated directly by formation of an E5-containing complex. Images PMID:8394451

Reductive evolution and the loss of PDC/PAS domains from the genus Staphylococcus

PubMed Central

2013-01-01

Background The Per-Arnt-Sim (PAS) domain represents a ubiquitous structural fold that is involved in bacterial sensing and adaptation systems, including several virulence related functions. Although PAS domains and the subclass of PhoQ-DcuS-CitA (PDC) domains have a common structure, there is limited amino acid sequence similarity. To gain greater insight into the evolution of PDC/PAS domains present in the bacterial kingdom and staphylococci in specific, the PDC/PAS domains from the genomic sequences of 48 bacteria, representing 5 phyla, were identified using the sensitive search method based on HMM-to-HMM comparisons (HHblits). Results A total of 1,007 PAS domains and 686 PDC domains distributed over 1,174 proteins were identified. For 28 Gram-positive bacteria, the distribution, organization, and molecular evolution of PDC/PAS domains were analyzed in greater detail, with a special emphasis on the genus Staphylococcus. Compared to other bacteria the staphylococci have relatively fewer proteins (6–9) containing PDC/PAS domains. As a general rule, the staphylococcal genomes examined in this study contain a core group of seven PDC/PAS domain-containing proteins consisting of WalK, SrrB, PhoR, ArlS, HssS, NreB, and GdpP. The exceptions to this rule are: 1) S. saprophyticus lacks the core NreB protein; 2) S. carnosus has two additional PAS domain containing proteins; 3) S. epidermidis, S. aureus, and S. pseudintermedius have an additional protein with two PDC domains that is predicted to code for a sensor histidine kinase; 4) S. lugdunensis has an additional PDC containing protein predicted to be a sensor histidine kinase. Conclusions This comprehensive analysis demonstrates that variation in PDC/PAS domains among bacteria has limited correlations to the genome size or pathogenicity; however, our analysis established that bacteria having a motile phase in their life cycle have significantly more PDC/PAS-containing proteins. In addition, our analysis revealed a

Reductive evolution and the loss of PDC/PAS domains from the genus Staphylococcus.

PubMed

Shah, Neethu; Gaupp, Rosmarie; Moriyama, Hideaki; Eskridge, Kent M; Moriyama, Etsuko N; Somerville, Greg A

2013-07-31

The Per-Arnt-Sim (PAS) domain represents a ubiquitous structural fold that is involved in bacterial sensing and adaptation systems, including several virulence related functions. Although PAS domains and the subclass of PhoQ-DcuS-CitA (PDC) domains have a common structure, there is limited amino acid sequence similarity. To gain greater insight into the evolution of PDC/PAS domains present in the bacterial kingdom and staphylococci in specific, the PDC/PAS domains from the genomic sequences of 48 bacteria, representing 5 phyla, were identified using the sensitive search method based on HMM-to-HMM comparisons (HHblits). A total of 1,007 PAS domains and 686 PDC domains distributed over 1,174 proteins were identified. For 28 Gram-positive bacteria, the distribution, organization, and molecular evolution of PDC/PAS domains were analyzed in greater detail, with a special emphasis on the genus Staphylococcus. Compared to other bacteria the staphylococci have relatively fewer proteins (6-9) containing PDC/PAS domains. As a general rule, the staphylococcal genomes examined in this study contain a core group of seven PDC/PAS domain-containing proteins consisting of WalK, SrrB, PhoR, ArlS, HssS, NreB, and GdpP. The exceptions to this rule are: 1) S. saprophyticus lacks the core NreB protein; 2) S. carnosus has two additional PAS domain containing proteins; 3) S. epidermidis, S. aureus, and S. pseudintermedius have an additional protein with two PDC domains that is predicted to code for a sensor histidine kinase; 4) S. lugdunensis has an additional PDC containing protein predicted to be a sensor histidine kinase. This comprehensive analysis demonstrates that variation in PDC/PAS domains among bacteria has limited correlations to the genome size or pathogenicity; however, our analysis established that bacteria having a motile phase in their life cycle have significantly more PDC/PAS-containing proteins. In addition, our analysis revealed a tremendous amount of variation in the

Involvement of oxidative and nitrosative stress in promoting retinal vasculitis in patients with Eales' disease.

PubMed

Rajesh, Mohanraj; Sulochana, Konerirajapuram N; Punitham, Ranganathan; Biswas, Jyotirmay; Lakshmi, Soundarajan; Ramakrishnan, Sivaramakrishnan

2003-07-01

Eales' disease (ED) is an idiopathic retinal vasculitis condition, which affects retina of young adult males. The histopathological hallmark in ED is the adhesion of leukocytes to the endothelium and the infiltration of these cells into the retinal parenchyma. Phagocyte generated free radicals have been implicated in mediating tissue damage associated with various inflammatory vasculopathies. In the present study, we have investigated the possible role of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in causing retinal tissue damage in ED. 35 patients with ED and 20 healthy control subjects were included in the study. Monocytes (MC) were separated from peripheral blood of the respective study participants. Inducible nitric oxide synthase (iNOS) protein expression was assessed using Western blot and 3 nitrotyrosine (3NTYR) by reversed phase high performance liquid chromatography (RP HPLC). Thiobarbituric acid reactive substances (TBARS) were determined by measuring malondialdehyde (MDA) formed. Superoxide dismutase (SOD) activity was assayed based on the ability of SOD to inhibit auto-oxidation of epinephrine. Iron, copper and zinc content were determined using Atomic Absorption Spectrophotometer. Immunolocalization of iNOS and 3NTYR was performed on the surgically excised epiretinal membranes (ERM) from patients with ED. There was a significant increase in the expression of iNOS, as well as 3NTYR accumulation, diminished SOD activity, elevated lipid peroxides, iron, copper and decreased zinc content in the MC of patients with ED when compared with healthy control subjects. The elevated levels of ROS and RNS products correlated with diminished antioxidant status in patients with ED. Strong immunoreactivity for iNOS and 3NTYR was observed in inflammatory cells and endothelial cells in ERM obtained from patients with ED. Our findings from this study clearly reveal the involvement of RNS and ROS in the development of retinal vasculitis in ED. Based

Formation and release of arrestin domain-containing protein 1-mediated microvesicles (ARMMs) at plasma membrane by recruitment of TSG101 protein.

PubMed

Nabhan, Joseph F; Hu, Ruoxi; Oh, Raymond S; Cohen, Stanley N; Lu, Quan

2012-03-13

Mammalian cells are capable of delivering multiple types of membrane capsules extracellularly. The limiting membrane of late endosomes can fuse with the plasma membrane, leading to the extracellular release of multivesicular bodies (MVBs), initially contained within the endosomes, as exosomes. Budding viruses exploit the TSG101 protein and endosomal sorting complex required for transport (ESCRT) machinery used for MVB formation to mediate the egress of viral particles from host cells. Here we report the discovery of a virus-independent cellular process that generates microvesicles that are distinct from exosomes and which, like budding viruses, are produced by direct plasma membrane budding. Such budding is driven by a specific interaction of TSG101 with a tetrapeptide PSAP motif of an accessory protein, arrestin domain-containing protein 1 (ARRDC1), which we show is localized to the plasma membrane through its arrestin domain. This interaction results in relocation of TSG101 from endosomes to the plasma membrane and mediates the release of microvesicles that contain TSG101, ARRDC1, and other cellular proteins. Unlike exosomes, which are derived from MVBs, ARRDC1-mediated microvesicles (ARMMs) lack known late endosomal markers. ARMMs formation requires VPS4 ATPase and is enhanced by the E3 ligase WWP2, which interacts with and ubiquitinates ARRDC1. ARRDC1 protein discharged into ARMMs was observed in co-cultured cells, suggesting a role for ARMMs in intercellular communication. Our findings reveal an intrinsic cellular mechanism that results in direct budding of microvesicles from the plasma membrane, providing a formal paradigm for the evolutionary recruitment of ESCRT proteins in the release of budding viruses.

Comparing domain interactions within antibody Fabs with kappa and lambda light chains.

PubMed

Toughiri, Raheleh; Wu, Xiufeng; Ruiz, Diana; Huang, Flora; Crissman, John W; Dickey, Mark; Froning, Karen; Conner, Elaine M; Cujec, Thomas P; Demarest, Stephen J

2016-10-01

IgG antibodies are multi-domain proteins with complex inter-domain interactions. Human IgG heavy chains (HCs) associate with light chains (LCs) of the κ or λ isotype to form mature antibodies capable of binding antigen. The HC/LC interaction involves 4 domains: VH and CH1 from the HC and VL and CL from the LC. Human Fabs with κ LCs have been well characterized for their unfolding behaviors and demonstrate a significant level of cooperativity and stabilization when all 4 domains are intact. Very little is known regarding the thermodynamic properties of human Fabs with λ LCs. Here, we dissect the domain contributions to Fab stability for both κ and λ LC-containing Fabs. We find the cooperativity of unfolding between the constant domains, CH1/Cλ, and variable domains, VH/Vλ, within λ LC-containing Fabs is significantly weaker than that of κ LC-containing Fabs. The data suggests there may not be an evolutionary necessity for strong variable/constant domain cooperativity within λ LC-containing Fabs. After investigating the biophysical properties of Fabs with mismatched variable and constant domain subunits (e.g., VH/Vκ paired with CH1/Cλ or T cell receptor Cα/Cβ), the major role of the constant domains for both κ- and λ-containing Fabs may be to reduce the hydrophobic exposure at the VH/VL interface. Even though Fabs with these non-native pairings were thermodynamically less stable, they secreted well from mammalian cells as well behaved monodisperse proteins, which was in contrast to what was observed with the VH/Vκ and VH/Vλ scFvs that secreted as a mixture of monomer and aggregates.

APETALA 2-domain-containing transcription factors: focusing on abscisic acid and gibberellins antagonism.

PubMed

Shu, Kai; Zhou, Wenguan; Yang, Wenyu

2018-02-01

The phytohormones abscisic acid (ABA) and gibberellin (GA) antagonistically mediate diverse plant developmental processes including seed dormancy and germination, root development, and flowering time control, and thus the optimal balance between ABA and GA is essential for plant growth and development. Although more than a half and one century have passed since the initial discoveries of ABA and GA, respectively, the precise mechanisms underlying ABA-GA antagonism still need further investigation. Emerging evidence indicates that two APETALA 2 (AP2)-domain-containing transcription factors (ATFs), ABI4 in Arabidopsis and OsAP2-39 in rice, play key roles in ABA and GA antagonism. These two transcription factors precisely regulate the transcription pattern of ABA and GA biosynthesis or inactivation genes, mediating ABA and GA levels. In this Viewpoint article, we try to shed light on the effects of ATFs on ABA-GA antagonism, and summarize the overlapping but distinct biological functions of these ATFs in the antagonism between ABA and GA. Finally, we strongly propose that further research is needed into the detailed roles of additional numerous ATFs in ABA and GA crosstalk, which will improve our understanding of the antagonism between these two phytohormones. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Protective effects of Arctium lappa L. roots against hydrogen peroxide-induced cell injury and potential mechanisms in SH-SY5Y cells.

PubMed

Tian, Xing; Guo, Li-Ping; Hu, Xiao-Long; Huang, Jin; Fan, Yan-Hua; Ren, Tian-Shu; Zhao, Qing-Chun

2015-04-01

Accumulated evidence has shown that excessive reactive oxygen species (ROS) have been implicated in neuronal cell death related with various chronic neurodegenerative disorders. This study was designed to explore neuroprotective effects of ethyl acetate extract of Arctium lappa L. roots (EAL) on hydrogen peroxide (H2O2)-induced cell injury in human SH-SY5Y neuroblastoma cells. The cell viability was significantly decreased after exposure to 200 μM H2O2, whereas pretreatment with different concentrations of EAL attenuated the H2O2-induced cytotoxicity. Hoechst 33342 staining indicated that EAL reversed nuclear condensation in H2O2-treated cells. Meanwhile, TUNEL assay with DAPI staining showed that EAL attenuated apoptosis was induced by H2O2. Pretreatment with EAL also markedly elevated activities of antioxidant enzyme (GSH-Px and SOD), reduced lipid peroxidation (MDA) production, prevented ROS formation, and the decrease of mitochondrial membrane potential. In addition, EAL showed strong radical scavenging ability in 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) assays. Furthermore, EAL inhibited H2O2-induced apoptosis by increases in the Bcl-2/Bax ratio, decreases in cytochrome c release, and attenuation of caspase-3, caspase-9 activities, and expressions. These findings suggest that EAL may be regarded as a potential antioxidant agent and possess potent neuroprotective activity against H2O2-induced injury.

The comprehension skills of children learning English as an additional language.

PubMed

Burgoyne, K; Kelly, J M; Whiteley, H E; Spooner, A

2009-12-01

Data from national test results suggests that children who are learning English as an additional language (EAL) experience relatively lower levels of educational attainment in comparison to their monolingual, English-speaking peers. The relative underachievement of children who are learning EAL demands that the literacy needs of this group are identified. To this end, this study aimed to explore the reading- and comprehension-related skills of a group of EAL learners. Data are reported from 92 Year 3 pupils, of whom 46 children are learning EAL. Children completed standardized measures of reading accuracy and comprehension, listening comprehension, and receptive and expressive vocabulary. Results indicate that many EAL learners experience difficulties in understanding written and spoken text. These comprehension difficulties are not related to decoding problems but are related to significantly lower levels of vocabulary knowledge experienced by this group. Many EAL learners experience significantly lower levels of English vocabulary knowledge which has a significant impact on their ability to understand written and spoken text. Greater emphasis on language development is therefore needed in the school curriculum to attempt to address the limited language skills of children learning EAL.

Discussion summary: Fictitious domain methods

NASA Technical Reports Server (NTRS)

Glowinski, Rowland; Rodrigue, Garry

1991-01-01

Fictitious Domain methods are constructed in the following manner: Suppose a partial differential equation is to be solved on an open bounded set, Omega, in 2-D or 3-D. Let R be a rectangle domain containing the closure of Omega. The partial differential equation is first solved on R. Using the solution on R, the solution of the equation on Omega is then recovered by some procedure. The advantage of the fictitious domain method is that in many cases the solution of a partial differential equation on a rectangular region is easier to compute than on a nonrectangular region. Fictitious domain methods for solving elliptic PDEs on general regions are also very efficient when used on a parallel computer. The reason is that one can use the many domain decomposition methods that are available for solving the PDE on the fictitious rectangular region. The discussion on fictitious domain methods began with a talk by R. Glowinski in which he gave some examples of a variational approach to ficititious domain methods for solving the Helmholtz and Navier-Stokes equations.

Identification of DEP domain-containing proteins by a machine learning method and experimental analysis of their expression in human HCC tissues

NASA Astrophysics Data System (ADS)

Liao, Zhijun; Wang, Xinrui; Zeng, Yeting; Zou, Quan

2016-12-01

The Dishevelled/EGL-10/Pleckstrin (DEP) domain-containing (DEPDC) proteins have seven members. However, whether this superfamily can be distinguished from other proteins based only on the amino acid sequences, remains unknown. Here, we describe a computational method to segregate DEPDCs and non-DEPDCs. First, we examined the Pfam numbers of the known DEPDCs and used the longest sequences for each Pfam to construct a phylogenetic tree. Subsequently, we extracted 188-dimensional (188D) and 20D features of DEPDCs and non-DEPDCs and classified them with random forest classifier. We also mined the motifs of human DEPDCs to find the related domains. Finally, we designed experimental verification methods of human DEPDC expression at the mRNA level in hepatocellular carcinoma (HCC) and adjacent normal tissues. The phylogenetic analysis showed that the DEPDCs superfamily can be divided into three clusters. Moreover, the 188D and 20D features can both be used to effectively distinguish the two protein types. Motif analysis revealed that the DEP and RhoGAP domain was common in human DEPDCs, human HCC and the adjacent tissues that widely expressed DEPDCs. However, their regulation was not identical. In conclusion, we successfully constructed a binary classifier for DEPDCs and experimentally verified their expression in human HCC tissues.

Low energy electron attenuation lengths in core–shell nanoparticles

DOE PAGES

Jacobs, Michael I.; Kostko, Oleg; Ahmed, Musahid; ...

2017-05-05

Here, a velocity map imaging spectrometer is used to measure photoemission from free core–shell nanoparticles, where a salt core is coated with a liquid hydrocarbon shell (i.e. squalane). By varying the radial thickness of the hydrocarbon shell, electron attenuation lengths (EALs) are determined by measuring the decay in photoemission intensity from the salt core. In squalane, electrons with kinetic energy (KE) above 2 eV are found to have EALs of 3–5 nm, whereas electrons with smaller KE (<2 eV) have significantly larger EALs of >15 nm. These results (in the context of other energy-resolved EAL measurements) suggest that the energymore » dependent behavior of low energy electrons is similar in dielectrics when KE > 2 eV. At this energy the EALs do not appear to exhibit strong energy dependence. However, at very low KE (<2 eV), the EALs diverge and appear to be extremely material dependent.« less

Determinant representation of the domain-wall boundary condition partition function of a Richardson-Gaudin model containing one arbitrary spin

NASA Astrophysics Data System (ADS)

Faribault, Alexandre; Tschirhart, Hugo; Muller, Nicolas

2016-05-01

In this work we present a determinant expression for the domain-wall boundary condition partition function of rational (XXX) Richardson-Gaudin models which, in addition to N-1 spins \\frac{1}{2}, contains one arbitrarily large spin S. The proposed determinant representation is written in terms of a set of variables which, from previous work, are known to define eigenstates of the quantum integrable models belonging to this class as solutions to quadratic Bethe equations. Such a determinant can be useful numerically since systems of quadratic equations are much simpler to solve than the usual highly nonlinear Bethe equations. It can therefore offer significant gains in stability and computation speed.

A DEK Domain-Containing Protein Modulates Chromatin Structure and Function in Arabidopsis[W][OPEN

PubMed Central

Waidmann, Sascha; Kusenda, Branislav; Mayerhofer, Juliane; Mechtler, Karl; Jonak, Claudia

2014-01-01

Chromatin is a major determinant in the regulation of virtually all DNA-dependent processes. Chromatin architectural proteins interact with nucleosomes to modulate chromatin accessibility and higher-order chromatin structure. The evolutionarily conserved DEK domain-containing protein is implicated in important chromatin-related processes in animals, but little is known about its DNA targets and protein interaction partners. In plants, the role of DEK has remained elusive. In this work, we identified DEK3 as a chromatin-associated protein in Arabidopsis thaliana. DEK3 specifically binds histones H3 and H4. Purification of other proteins associated with nuclear DEK3 also established DNA topoisomerase 1α and proteins of the cohesion complex as in vivo interaction partners. Genome-wide mapping of DEK3 binding sites by chromatin immunoprecipitation followed by deep sequencing revealed enrichment of DEK3 at protein-coding genes throughout the genome. Using DEK3 knockout and overexpressor lines, we show that DEK3 affects nucleosome occupancy and chromatin accessibility and modulates the expression of DEK3 target genes. Furthermore, functional levels of DEK3 are crucial for stress tolerance. Overall, data indicate that DEK3 contributes to modulation of Arabidopsis chromatin structure and function. PMID:25387881

Internet Domain Names: Background and Policy Issues

DTIC Science & Technology

2010-04-05

was enacted to create a “ kids -friendly top level domain name” that would contain only age- appropriate content. The Dot Kids Implementation and...were introduced to require the Department of Commerce to compel ICANN to establish a mandatory top level domain name (such as . ) for material that...ICANN announced that it had entered into commercial and technical negotiations with a registry company (ICM Registry) to operate a new “. ” domain

The Pleckstrin Homology Domain of Phospholipase Cβ Transmits Enzymatic Activation through Modulation of Membrane - Domain Orientation§

PubMed Central

Drin, Guillaume; Douguet, Dominique; Scarlata, Suzanne

2008-01-01

Phospholipase C-beta (PLCβ) enzymes are activated by Gαq and Gβγ subunits and catalyze the hydrolysis of the minor membrane lipid phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2). Activation of PLCβ2 by Gβγ subunits has been shown to be conferred through its N-terminal pleckstrin homology (PH) domain although the underlying mechanism is unclear. Also unclear are observations that the extent of Gβγ activation differs on different membrane surfaces. In this study, we have identified a unique region of the PH domain of PLCβ2 domain (residues 71-88) which, when added to the enzyme as a peptide, causes enzyme activation similar to Gβγ subunits. This PH domain segment interacts strongly with membranes composed of lipid mixtures but not those containing lipids with electrically neutral zwitterionic head groups. Moreso, addition of this segment perturbs interaction of the catalytic domain, but not the PH domain, with membrane surfaces. We monitored the orientation of the PH and catalytic domains of PLC by intermolecular fluorescence resonance energy transfer (FRET) using the Gβγ activatable mutant, PLCβ2/δ1(C193S). We find an increase in FRET with binding to membranes with mixed lipids but not to those containing only lipids with electrically neutral head groups. These results suggest that enzymatic activation can be conferred through optimal association of the PHβ71-88 region to specific membrane surfaces. These studies allow us to understand the basis of variations of Gβγ activation on different membrane surfaces. PMID:16669615

New findings on phosphodiesterases, MoPdeH and MoPdeL, in Magnaporthe oryzae revealed by structural analysis.

PubMed

Yang, Li-Na; Yin, Ziyi; Zhang, Xi; Feng, Wanzhen; Xiao, Yuhan; Zhang, Haifeng; Zheng, Xiaobo; Zhang, Zhengguang

2018-05-01

The cyclic adenosine monophosphate (cAMP) signalling pathway mediates signal communication and sensing during infection-related morphogenesis in eukaryotes. Many studies have implicated cAMP as a critical mediator of appressorium development in the rice blast fungus, Magnaporthe oryzae. The cAMP phosphodiesterases, MoPdeH and MoPdeL, as key regulators of intracellular cAMP levels, play pleiotropic roles in cell wall integrity, cellular morphology, appressorium formation and infectious growth in M. oryzae. Here, we analysed the roles of domains of MoPdeH and MoPdeL separately or in chimeras. The results indicated that the HD and EAL domains of MoPdeH are indispensable for its phosphodiesterase activity and function. Replacement of the MoPdeH HD domain with the L1 and L2 domains of MoPdeL, either singly or together, resulted in decreased cAMP hydrolysis activity of MoPdeH. All of the transformants exhibited phenotypes similar to that of the ΔMopdeH mutant, but also revealed that EAL and L1 play additional roles in conidiation, and that L1 is involved in infectious growth. We further found that the intracellular cAMP level is important for surface signal recognition and hyphal autolysis. The intracellular cAMP level negatively regulates Mps1-MAPK and positively regulates Pmk1-MAPK in the rice blast fungus. Our results provide new information to better understand the cAMP signalling pathway in the development, differentiation and plant infection of the fungus. © 2017 BSPP AND JOHN WILEY & SONS LTD.

Sequence Analysis of Scaffolding Protein CipC and ORFXp, a New Cohesin-Containing Protein in Clostridium cellulolyticum: Comparison of Various Cohesin Domains and Subcellular Localization of ORFXp

PubMed Central

Pagès, Sandrine; Bélaïch, Anne; Fierobe, Henri-Pierre; Tardif, Chantal; Gaudin, Christian; Bélaïch, Jean-Pierre

1999-01-01

The gene encoding the scaffolding protein of the cellulosome from Clostridium cellulolyticum, whose partial sequence was published earlier (S. Pagès, A. Bélaïch, C. Tardif, C. Reverbel-Leroy, C. Gaudin, and J.-P. Bélaïch, J. Bacteriol. 178:2279–2286, 1996; C. Reverbel-Leroy, A. Bélaïch, A. Bernadac, C. Gaudin, J. P. Bélaïch, and C. Tardif, Microbiology 142:1013–1023, 1996), was completely sequenced. The corresponding protein, CipC, is composed of a cellulose binding domain at the N terminus followed by one hydrophilic domain (HD1), seven highly homologous cohesin domains (cohesin domains 1 to 7), a second hydrophilic domain, and a final cohesin domain (cohesin domain 8) which is only 57 to 60% identical to the seven other cohesin domains. In addition, a second gene located 8.89 kb downstream of cipC was found to encode a three-domain protein, called ORFXp, which includes a cohesin domain. By using antiserum raised against the latter, it was observed that ORFXp is associated with the membrane of C. cellulolyticum and is not detected in the cellulosome fraction. Western blot and BIAcore experiments indicate that cohesin domains 1 and 8 from CipC recognize the same dockerins and have similar affinity for CelA (Ka = 4.8 × 109 M−1) whereas the cohesin from ORFXp, although it is also able to bind all cellulosome components containing a dockerin, has a 19-fold lower Ka for CelA (2.6 × 108 M−1). Taken together, these data suggest that ORFXp may play a role in cellulosome assembly. PMID:10074072

Sequence analysis of scaffolding protein CipC and ORFXp, a new cohesin-containing protein in Clostridium cellulolyticum: comparison of various cohesin domains and subcellular localization of ORFXp.

PubMed

Pagès, S; Bélaïch, A; Fierobe, H P; Tardif, C; Gaudin, C; Bélaïch, J P

1999-03-01

The gene encoding the scaffolding protein of the cellulosome from Clostridium cellulolyticum, whose partial sequence was published earlier (S. Pagès, A. Bélaïch, C. Tardif, C. Reverbel-Leroy, C. Gaudin, and J.-P. Bélaïch, J. Bacteriol. 178:2279-2286, 1996; C. Reverbel-Leroy, A. Bélaïch, A. Bernadac, C. Gaudin, J. P. Bélaïch, and C. Tardif, Microbiology 142:1013-1023, 1996), was completely sequenced. The corresponding protein, CipC, is composed of a cellulose binding domain at the N terminus followed by one hydrophilic domain (HD1), seven highly homologous cohesin domains (cohesin domains 1 to 7), a second hydrophilic domain, and a final cohesin domain (cohesin domain 8) which is only 57 to 60% identical to the seven other cohesin domains. In addition, a second gene located 8.89 kb downstream of cipC was found to encode a three-domain protein, called ORFXp, which includes a cohesin domain. By using antiserum raised against the latter, it was observed that ORFXp is associated with the membrane of C. cellulolyticum and is not detected in the cellulosome fraction. Western blot and BIAcore experiments indicate that cohesin domains 1 and 8 from CipC recognize the same dockerins and have similar affinity for CelA (Ka = 4.8 x 10(9) M-1) whereas the cohesin from ORFXp, although it is also able to bind all cellulosome components containing a dockerin, has a 19-fold lower Ka for CelA (2.6 x 10(8) M-1). Taken together, these data suggest that ORFXp may play a role in cellulosome assembly.

Epilepsy in cerebrovascular diseases: Review of experimental and clinical data with meta-analysis of risk factors.

PubMed

Ferlazzo, Edoardo; Gasparini, Sara; Beghi, Ettore; Sueri, Chiara; Russo, Emilio; Leo, Antonio; Labate, Angelo; Gambardella, Antonio; Belcastro, Vincenzo; Striano, Pasquale; Paciaroni, Maurizio; Pisani, Laura Rosa; Aguglia, Umberto

2016-08-01

Seizures may occur in close temporal association with a stroke or after a variable interval. Moreover, epilepsy is often encountered in patients with leukoaraiosis. Although early post-stroke seizures have been studied extensively, less attention has been paid to post-stroke epilepsy (PSE) and to epilepsy associated with leukoaraiosis (EAL). The aim of this paper is to review data concerning pathophysiology, prognosis, and treatment of PSE and EAL. We performed an extensive literature search to identify experimental and clinical articles on PSE and EAL. We also conducted a systematic review of risk factors for PSE and EAL among eligible studies. PSE is caused by enhanced neuronal excitability within and near the scar. The role played by white matter changes in EAL remains to be elucidated. Meta-analysis showed that cortical involvement (odds ratio [OR] 3.71, 95% confidence interval [CI] 2.34-5.90, p < 0.001), cerebral hemorrhage (OR 2.41, 95% CI 1.57-3.70, p < 0.001), and early seizures (OR 4.43, 95% CI 2.36-8.32, p < 0.001) are associated with an increased risk of PSE. As regards EAL, no prospective, population-based studies evaluated the role of different variables on seizure risk. Studies about the management of PSE are limited. PSE is generally well controlled by drugs. Data about risk factors, prognosis, and treatment of EAL are lacking. Pathophysiology and risk factors are well defined for PSE but need to be elucidated for EAL. Management of PSE and EAL relies on the clinician's judgment and should be tailored on an individual basis. Wiley Periodicals, Inc. © 2016 International League Against Epilepsy.

English as an Additional Language and Initial Teacher Education: Views and Experiences from Northern Ireland

ERIC Educational Resources Information Center

Skinner, Barbara

2010-01-01

This paper addresses training for teaching English as an Additional Language (EAL) at initial teacher education (ITE) level in Northern Ireland. This small-scale qualitative study describes 15 primary and post-primary teachers' perspectives on their preparation for teaching EAL in Northern Ireland. It explores reflections on EAL content in ITE…

endodermal-amyloplast less 1 is a novel allele of SHORT-ROOT

NASA Astrophysics Data System (ADS)

Morita, Miyo T.; Saito, Chieko; Nakano, Akihiko; Tasaka, Masao

Plants can sense the direction of gravity and change the growth orientation of their organs. Arabidopsis mutants have been isolated and characterized in order to elucidate the molecular mechanisms of gravitropism. endodermal-amyloplast less 1 ( eal1) is a unique mutant that completely lacks gravitropism in inflorescence stems and exhibits reduced gravitropism in hypocotyls, whereas its roots showed normal gravitropism. Previously, it was suggested that differentiation or development of amyloplasts in shoot statocytes (endodermal cells) is affected by the eal1 mutation. Here, we have identified EAL1 as a SHORT-ROOT ( SHR) allele based on map position. Three nucleotides in the SHR coding region were deleted in the eal1 mutant, resulting in the deletion of just one amino acid. The protein encoded by the novel allele of SHR appears to have retained its function as a transcription factor since the endodermal cell layer was formed both in roots and in shoots of eal1. SCARECROW (SCR) promoter activity monitored by reporter protein expression was significantly decreased in eal1, suggesting that the activity of SHR lacking one amino acid is reduced. In addition, transcription levels of SHOOT GRAVITROPISM 5 (SGR5), which is mainly expressed in the endodermis of inflorescence stems, was markedly decreased. Together with the presence of abnormal endodermal amyloplasts in eal1, these results strongly suggest that the endodermis observed in eal1 is not sufficiently differentiated to execute shoot gravitropism.

Domain requirements for the Dock adapter protein in growth- cone signaling

PubMed Central

Rao, Yong; Zipursky, S. Lawrence

1998-01-01

Tyrosine phosphorylation has been implicated in growth-cone guidance through genetic, biochemical, and pharmacological studies. Adapter proteins containing src homology 2 (SH2) domains and src homology 3 (SH3) domains provide a means of linking guidance signaling through phosphotyrosine to downstream effectors regulating growth-cone motility. The Drosophila adapter, Dreadlocks (Dock), the homolog of mammalian Nck containing three N-terminal SH3 domains and a single SH2 domain, is highly specialized for growth-cone guidance. In this paper, we demonstrate that Dock can couple signals in either an SH2-dependent or an SH2-independent fashion in photoreceptor (R cell) growth cones, and that Dock displays different domain requirements in different neurons. PMID:9482841

Domain requirements for the Dock adapter protein in growth- cone signaling.

PubMed

Rao, Y; Zipursky, S L

1998-03-03

Tyrosine phosphorylation has been implicated in growth-cone guidance through genetic, biochemical, and pharmacological studies. Adapter proteins containing src homology 2 (SH2) domains and src homology 3 (SH3) domains provide a means of linking guidance signaling through phosphotyrosine to downstream effectors regulating growth-cone motility. The Drosophila adapter, Dreadlocks (Dock), the homolog of mammalian Nck containing three N-terminal SH3 domains and a single SH2 domain, is highly specialized for growth-cone guidance. In this paper, we demonstrate that Dock can couple signals in either an SH2-dependent or an SH2-independent fashion in photoreceptor (R cell) growth cones, and that Dock displays different domain requirements in different neurons.

Haem Recognition By a Staphylococcus Aureus NEAT Domain

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

Grigg, J.C.; Vermeiren, C.; Heinrichs, D.E.

2009-06-01

Successful pathogenic organisms have developed mechanisms to thrive under extreme levels of iron restriction. Haem-iron represents the largest iron reservoir in the human body and is a significant source of iron for some bacterial pathogens. NEAT (NEAr Transporter) domains are found exclusively in a family of cell surface proteins in Gram-positive bacteria. Many NEAT domain-containing proteins, including IsdA in Staphylococcus aureus, are implicated in haem binding. Here, we show that overexpression of IsdA in S. aureus enhances growth and an inactivation mutant of IsdA has a growth defect, compared with wild type, when grown in media containing haem as themore » sole iron source. Furthermore, the haem-binding property of IsdA is contained within the NEAT domain. Crystal structures of the apo-IsdA NEAT domain and in complex with haem were solved and reveal a clathrin adapter-like beta-sandwich fold with a large hydrophobic haem-binding pocket. Haem is bound with the propionate groups directed at the molecular surface and the iron is co-ordinated solely by Tyr(166). The phenol groups of Tyr(166) and Tyr(170) form an H-bond that may function in regulating haem binding and release. An analysis of IsdA structure-sequence alignments indicate that conservation of Tyr(166) is a predictor of haem binding by NEAT domains.« less

Formation of supported lipid bilayers containing phase-segregated domains and their interaction with gold nanoparticles

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

Melby, Eric S.; Mensch, Arielle C.; Lohse, Samuel E.

2016-01-01

The cell membrane represents an important biological interface that nanoparticles may encounter after being released into the environment. Interaction of nanoparticles with cellular membranes may alter membrane structure and function, lead to their uptake into cells, and elicit adverse biological responses. Supported lipid bilayers have proven to be valuable ex vivo models for biological membranes, allowing investigation of their mechanisms of interaction with nanoparticles with a degree of control impossible in living cells. To date, the majority of research on nanoparticle interaction with supported lipid bilayers has employed membranes composed of single or binary mixtures of phospholipids. Cellular membranes containmore » a wide variety of lipids and exhibit lateral organization. Ordered membrane domains enriched in specific membrane components are referred to as lipid rafts and have not been explored with respect to their interaction with nanoparticles. Here we develop model lipid raft-containing membranes amenable to investigation by a variety of surface-sensitive analytical techniques and demonstrate that lipid rafts influence the extent of nanoparticle attachment to model membranes. We determined conditions that allow reliable formation of bilayers containing rafts enriched in sphingomyelin and cholesterol and confirmed their morphology by structured illumination and atomic force microscopies. We demonstrate that lipid rafts increase attachment of cationic gold nanoparticles to model membranes under near physiological ionic strength conditions (0.1 M NaCl) at pH 7.4. We anticipate that these results will serve as the foundation for and motivate further study of nanoparticle interaction with compositionally varied lipid rafts.« less

SH2 domain-containing inositol 5-phosphatase (SHIP2) regulates de-novo lipogenesis and secretion of apoB100 containing lipoproteins in HepG2 cells.

PubMed

Gorgani-Firuzjaee, Sattar; Khatami, Shohreh; Adeli, Khosrow; Meshkani, Reza

2015-09-04

Hepatic de-novo lipogenesis and production of triglyceride rich VLDL are regulated via the phosphoinositide 3-kinase cascade, however, the role of a negative regulator of this pathway, the SH2 domain-containing inositol 5-phosphatase (SHIP2) in this process, remains unknown. In the present study, we investigated the molecular link between SHIP2 expression and metabolic dyslipidemia using overexpression or suppression of SHIP2 gene in HepG2 cells. The results showed that overexpression of the wild type SHIP2 gene (SHIP2-WT) led to a higher total lipid content (28%) compared to control, whereas overexpression of the dominant negative SHIP2 gene (SHIP2-DN) reduced total lipid content in oleate treated cells by 40%. Overexpression of SHIP2-WT also led to a significant increase in both secretion of apoB100 containing lipoproteins and de-novo lipogenesis, as demonstrated by an enhancement in secreted apoB100 and MTP expression, increased intra and extracellular triglyceride levels and enhanced expression of lipogenic genes such as SREBP1c, FAS and ACC. On the other hand, overexpression of the SHIP2-DN gene prevented oleate-induced de-novo lipogenesis and secretion of apoB100 containing lipoproteins in HepG2 cells. Collectively, these findings suggest that SHIP2 expression level is a key determinant of hepatic lipogenesis and lipoprotein secretion, and its inhibition could be considered as a potential target for treatment of dyslipidemia. Copyright © 2015 Elsevier Inc. All rights reserved.

The small phytoplasma virulence effector SAP11 contains distinct domains required for nuclear targeting and CIN-TCP binding and destabilization.

PubMed

Sugio, Akiko; MacLean, Allyson M; Hogenhout, Saskia A

2014-05-01

Phytoplasmas are insect-transmitted bacterial phytopathogens that secrete virulence effectors and induce changes in the architecture and defense response of their plant hosts. We previously demonstrated that the small (± 10 kDa) virulence effector SAP11 of Aster Yellows phytoplasma strain Witches' Broom (AY-WB) binds and destabilizes Arabidopsis CIN (CINCINNATA) TCP (TEOSINTE-BRANCHED, CYCLOIDEA, PROLIFERATION FACTOR 1 AND 2) transcription factors, resulting in dramatic changes in leaf morphogenesis and increased susceptibility to phytoplasma insect vectors. SAP11 contains a bipartite nuclear localization signal (NLS) that targets this effector to plant cell nuclei. To further understand how SAP11 functions, we assessed the involvement of SAP11 regions in TCP binding and destabilization using a series of mutants. SAP11 mutants lacking the entire N-terminal domain, including the NLS, interacted with TCPs but did not destabilize them. SAP11 mutants lacking the C-terminal domain were impaired in both binding and destabilization of TCPs. These SAP11 mutants did not alter leaf morphogenesis. A SAP11 mutant that did not accumulate in plant nuclei (SAP11ΔNLS-NES) was able to bind and destabilize TCP transcription factors, but instigated weaker changes in leaf morphogenesis than wild-type SAP11. Overall the results suggest that phytoplasma effector SAP11 has a modular organization in which at least three domains are required for efficient CIN-TCP destabilization in plants. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

Characterization of big bang, a novel gene encoding for PDZ domain-containing proteins that are dynamically expressed throughout Drosophila development.

PubMed

Kim, Sabrina Y; Renihan, Maia K; Boulianne, Gabrielle L

2006-06-01

PDZ (PSD-95, Discs-large, ZO-1) domain proteins often function as scaffolding proteins and have been shown to play important roles in diverse cellular processes such as the establishment and maintenance of cell polarity, and signal transduction. Here, we report the identification and cloning of a novel Drosophila melanogaster gene that is predicted to produce several different PDZ domain-containing proteins through alternative promoter usage and alternative splicing. This gene, that we have named big bang (bbg), was first identified as C96-GAL4, a GAL4 enhancer trap line that was generated in our lab. To further characterize bbg, its expression pattern was examined in ovaries, embryos, and late third instar larvae using UAS reporter gene constructs, in situ hybridization, or immunocytochemistry. In addition, the expression of alternatively spliced transcripts was examined in more detail using in situ hybridization. We find that during embryogenesis bbg is predominantly expressed in the developing gut, but it is also expressed in external sensory organs found in the epidermis. In the late third instar larva, bbg is expressed along the presumptive wing margin in the wing disc, broadly in the eye disc, and in other imaginal discs as well as in the brain. The expression patterns observed are dynamic and specific during development, suggesting that like other genes that encode for several different PDZ domain protein isoforms, bbg likely plays important roles in multiple developmental processes.

The SPOR Domain, a Widely Conserved Peptidoglycan Binding Domain That Targets Proteins to the Site of Cell Division.

PubMed

Yahashiri, Atsushi; Jorgenson, Matthew A; Weiss, David S

2017-07-15

Sporulation-related repeat (SPOR) domains are small peptidoglycan (PG) binding domains found in thousands of bacterial proteins. The name "SPOR domain" stems from the fact that several early examples came from proteins involved in sporulation, but SPOR domain proteins are quite diverse and contribute to a variety of processes that involve remodeling of the PG sacculus, especially with respect to cell division. SPOR domains target proteins to the division site by binding to regions of PG devoid of stem peptides ("denuded" glycans), which in turn are enriched in septal PG by the intense, localized activity of cell wall amidases involved in daughter cell separation. This targeting mechanism sets SPOR domain proteins apart from most other septal ring proteins, which localize via protein-protein interactions. In addition to SPOR domains, bacteria contain several other PG-binding domains that can exploit features of the cell wall to target proteins to specific subcellular sites. Copyright © 2017 American Society for Microbiology.

Generalized vector calculus on convex domain

NASA Astrophysics Data System (ADS)

Agrawal, Om P.; Xu, Yufeng

2015-06-01

In this paper, we apply recently proposed generalized integral and differential operators to develop generalized vector calculus and generalized variational calculus for problems defined over a convex domain. In particular, we present some generalization of Green's and Gauss divergence theorems involving some new operators, and apply these theorems to generalized variational calculus. For fractional power kernels, the formulation leads to fractional vector calculus and fractional variational calculus for problems defined over a convex domain. In special cases, when certain parameters take integer values, we obtain formulations for integer order problems. Two examples are presented to demonstrate applications of the generalized variational calculus which utilize the generalized vector calculus developed in the paper. The first example leads to a generalized partial differential equation and the second example leads to a generalized eigenvalue problem, both in two dimensional convex domains. We solve the generalized partial differential equation by using polynomial approximation. A special case of the second example is a generalized isoperimetric problem. We find an approximate solution to this problem. Many physical problems containing integer order integrals and derivatives are defined over arbitrary domains. We speculate that future problems containing fractional and generalized integrals and derivatives in fractional mechanics will be defined over arbitrary domains, and therefore, a general variational calculus incorporating a general vector calculus will be needed for these problems. This research is our first attempt in that direction.

The evolution of filamin-a protein domain repeat perspective.

PubMed

Light, Sara; Sagit, Rauan; Ithychanda, Sujay S; Qin, Jun; Elofsson, Arne

2012-09-01

Particularly in higher eukaryotes, some protein domains are found in tandem repeats, performing broad functions often related to cellular organization. For instance, the eukaryotic protein filamin interacts with many proteins and is crucial for the cytoskeleton. The functional properties of long repeat domains are governed by the specific properties of each individual domain as well as by the repeat copy number. To provide better understanding of the evolutionary and functional history of repeating domains, we investigated the mode of evolution of the filamin domain in some detail. Among the domains that are common in long repeat proteins, sushi and spectrin domains evolve primarily through cassette tandem duplications while scavenger and immunoglobulin repeats appear to evolve through clustered tandem duplications. Additionally, immunoglobulin and filamin repeats exhibit a unique pattern where every other domain shows high sequence similarity. This pattern may be the result of tandem duplications, serve to avert aggregation between adjacent domains or it is the result of functional constraints. In filamin, our studies confirm the presence of interspersed integrin binding domains in vertebrates, while invertebrates exhibit more varied patterns, including more clustered integrin binding domains. The most notable case is leech filamin, which contains a 20 repeat expansion and exhibits unique dimerization topology. Clearly, invertebrate filamins are varied and contain examples of similar adjacent integrin-binding domains. Given that invertebrate integrin shows more similarity to the weaker filamin binder, integrin β3, it is possible that the distance between integrin-binding domains is not as crucial for invertebrate filamins as for vertebrates. Copyright © 2012 Elsevier Inc. All rights reserved.

FBI-1 Can Stimulate HIV-1 Tat Activity and Is Targeted to a Novel Subnuclear Domain that Includes the Tat-P-TEFb—containing Nuclear Speckles

PubMed Central

Pendergrast, P. Shannon; Wang, Chen; Hernandez, Nouria; Huang, Sui

2002-01-01

FBI-1 is a cellular POZ-domain–containing protein that binds to the HIV-1 LTR and associates with the HIV-1 transactivator protein Tat. Here we show that elevated levels of FBI-1 specifically stimulate Tat activity and that this effect is dependent on the same domain of FBI-1 that mediates Tat-FBI-1 association in vivo. FBI-1 also partially colocalizes with Tat and Tat's cellular cofactor, P-TEFb (Cdk9 and cyclin T1), at the splicing-factor–rich nuclear speckle domain. Further, a less-soluble population of FBI-1 distributes in a novel peripheral-speckle pattern of localization as well as in other nuclear regions. This distribution pattern is dependent on the FBI-1 DNA binding domain, on the presence of cellular DNA, and on active transcription. Taken together, these results suggest that FBI-1 is a cellular factor that preferentially associates with active chromatin and that can specifically stimulate Tat-activated HIV-1 transcription. PMID:11907272

Molecular Mechanisms of SH2- and PTB-Domain-Containing Proteins in Receptor Tyrosine Kinase Signaling

PubMed Central

Wagner, Melany J.; Stacey, Melissa M.; Liu, Bernard A.; Pawson, Tony

2013-01-01

Intracellular signaling is mediated by reversible posttranslational modifications (PTMs) that include phosphorylation, ubiquitination, and acetylation, among others. In response to extracellular stimuli such as growth factors, receptor tyrosine kinases (RTKs) typically dimerize and initiate signaling through phosphorylation of their cytoplasmic tails and downstream scaffolds. Signaling effectors are recruited to these phosphotyrosine (pTyr) sites primarily through Src homology 2 (SH2) domains and pTyr-binding (PTB) domains. This review describes how these conserved domains specifically recognize pTyr residues and play a major role in mediating precise downstream signaling events. PMID:24296166

Molecular mechanisms of SH2- and PTB-domain-containing proteins in receptor tyrosine kinase signaling.

PubMed

Wagner, Melany J; Stacey, Melissa M; Liu, Bernard A; Pawson, Tony

2013-12-01

Intracellular signaling is mediated by reversible posttranslational modifications (PTMs) that include phosphorylation, ubiquitination, and acetylation, among others. In response to extracellular stimuli such as growth factors, receptor tyrosine kinases (RTKs) typically dimerize and initiate signaling through phosphorylation of their cytoplasmic tails and downstream scaffolds. Signaling effectors are recruited to these phosphotyrosine (pTyr) sites primarily through Src homology 2 (SH2) domains and pTyr-binding (PTB) domains. This review describes how these conserved domains specifically recognize pTyr residues and play a major role in mediating precise downstream signaling events.

CHPA, a Cysteine- and Histidine-Rich-Domain-Containing Protein, Contributes to Maintenance of the Diploid State in Aspergillus nidulans

PubMed Central

Sadanandom, Ari; Findlay, Kim; Doonan, John H.; Schulze-Lefert, Paul; Shirasu, Ken

2004-01-01

The alternation of eukaryotic life cycles between haploid and diploid phases is crucial for maintaining genetic diversity. In some organisms, the growth and development of haploid and diploid phases are nearly identical, and one might suppose that all genes required for one phase are likely to be critical for the other phase. Here, we show that targeted disruption of the chpA (cysteine- and histidine-rich-domain- [CHORD]-containing protein A) gene in haploid Aspergillus nidulans strains gives rise to chpA knockout haploids and heterozygous diploids but no chpA knockout diploids. A. nidulans chpA heterozygous diploids showed impaired conidiophore development and reduced conidiation. Deletion of chpA from diploid A. nidulans resulted in genome instability and reversion to a haploid state. Thus, our data suggest a vital role for chpA in maintenance of the diploid phase in A. nidulans. Furthermore, the human chpA homolog, Chp-1, was able to complement haploinsufficiency in A. nidulans chpA heterozygotes, suggesting that the function of CHORD-containing proteins is highly conserved in eukaryotes. PMID:15302831

Protein domain assignment from the recurrence of locally similar structures

PubMed Central

Tai, Chin-Hsien; Sam, Vichetra; Gibrat, Jean-Francois; Garnier, Jean; Munson, Peter J.

2010-01-01

Domains are basic units of protein structure and essential for exploring protein fold space and structure evolution. With the structural genomics initiative, the number of protein structures in the Protein Databank (PDB) is increasing dramatically and domain assignments need to be done automatically. Most existing structural domain assignment programs define domains using the compactness of the domains and/or the number and strength of intra-domain versus inter-domain contacts. Here we present a different approach based on the recurrence of locally similar structural pieces (LSSPs) found by one-against-all structure comparisons with a dataset of 6,373 protein chains from the PDB. Residues of the query protein are clustered using LSSPs via three different procedures to define domains. This approach gives results that are comparable to several existing programs that use geometrical and other structural information explicitly. Remarkably, most of the proteins that contribute the LSSPs defining a domain do not themselves contain the domain of interest. This study shows that domains can be defined by a collection of relatively small locally similar structural pieces containing, on average, four secondary structure elements. In addition, it indicates that domains are indeed made of recurrent small structural pieces that are used to build protein structures of many different folds as suggested by recent studies. PMID:21287617

English as an Additional Language and Attainment in Primary Schools in England

ERIC Educational Resources Information Center

Demie, Feyisa

2018-01-01

English as an additional language (EAL) and language diversity attract much interest amongst policymakers and educationists; yet little is known about the performance in English schools of EAL pupils who are not fluent in English and speak different languages at home. The findings of the aggregated data confirm that EAL pupils achieved less well…

HCV NS5A protein containing potential ligands for both Src homology 2 and 3 domains enhances autophosphorylation of Src family kinase Fyn in B cells.

PubMed

Nakashima, Kenji; Takeuchi, Kenji; Chihara, Kazuyasu; Horiguchi, Tomoko; Sun, Xuedong; Deng, Lin; Shoji, Ikuo; Hotta, Hak; Sada, Kiyonao

2012-01-01

Hepatitis C virus (HCV) infects B lymphocytes and induces mixed cryoglobulinemia and B cell non-Hodgkin's lymphoma. The molecular mechanism for the pathogenesis of HCV infection-mediated B cell disorders remains obscure. To identify the possible role for HCV nonstructural 5A (NS5A) protein in B cells, we generated the stable B cell lines expressing Myc-His tagged NS5A. Immunoprecipitation study in the presence or absence of pervanadate (PV) implied that NS5A was tyrosine phosphorylated by pervanadate (PV) treatment of the cells. Therefore we examined pull-down assay by using glutathione S-transferase (GST)-fusion proteins of various Src homology 2 (SH2) domains, which associates with phosphotyrosine within a specific amino acid sequence. The results showed that NS5A specifically bound to SH2 domain of Fyn from PV-treated B cells in addition to Src homology 3 (SH3) domain. Substitution of Arg(176) to Lys in the SH2 domain of Fyn abrogated this interaction. Deletion mutational analysis demonstrated that N-terminal region of NS5A was not required for the interaction with the SH2 domain of Fyn. Tyr(334) was identified as a tyrosine phosphorylation site in NS5A. Far-western analysis revealed that SH2 domain of Fyn directly bound to NS5A. Fyn and NS5A were colocalized in the lipid raft. These results suggest that NS5A directly binds to the SH2 domain of Fyn in a tyrosine phosphorylation-dependent manner. Lastly, we showed that the expression of NS5A in B cells increased phosphorylation of activation loop tyrosine in the kinase domain of Fyn. NS5A containing ligand for both SH2 and SH3 domains enhances an aberrant autophosphorylation and kinase activity of Fyn in B cells.

Cache domains that are homologous to, but different from PAS domains comprise the largest superfamily of extracellular sensors in prokaryotes

DOE PAGES

Upadhyay, Amit A.; Fleetwood, Aaron D.; Adebali, Ogun; ...

2016-04-06

Cellular receptors usually contain a designated sensory domain that recognizes the signal. Per/Arnt/Sim (PAS) domains are ubiquitous sensors in thousands of species ranging from bacteria to humans. Although PAS domains were described as intracellular sensors, recent structural studies revealed PAS-like domains in extracytoplasmic regions in several transmembrane receptors. However, these structurally defined extracellular PAS-like domains do not match sequence-derived PAS domain models, and thus their distribution across the genomic landscape remains largely unknown. Here we show that structurally defined extracellular PAS-like domains belong to the Cache superfamily, which is homologous to, but distinct from the PAS superfamily. Our newly builtmore » computational models enabled identification of Cache domains in tens of thousands of signal transduction proteins including those from important pathogens and model organisms.Moreover, we show that Cache domains comprise the dominant mode of extracellular sensing in prokaryotes.« less

Cache domains that are homologous to, but different from PAS domains comprise the largest superfamily of extracellular sensors in prokaryotes

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

Upadhyay, Amit A.; Fleetwood, Aaron D.; Adebali, Ogun

Cellular receptors usually contain a designated sensory domain that recognizes the signal. Per/Arnt/Sim (PAS) domains are ubiquitous sensors in thousands of species ranging from bacteria to humans. Although PAS domains were described as intracellular sensors, recent structural studies revealed PAS-like domains in extracytoplasmic regions in several transmembrane receptors. However, these structurally defined extracellular PAS-like domains do not match sequence-derived PAS domain models, and thus their distribution across the genomic landscape remains largely unknown. Here we show that structurally defined extracellular PAS-like domains belong to the Cache superfamily, which is homologous to, but distinct from the PAS superfamily. Our newly builtmore » computational models enabled identification of Cache domains in tens of thousands of signal transduction proteins including those from important pathogens and model organisms.Moreover, we show that Cache domains comprise the dominant mode of extracellular sensing in prokaryotes.« less

Src binds cortactin through an SH2 domain cystine-mediated linkage.

PubMed

Evans, Jason V; Ammer, Amanda G; Jett, John E; Bolcato, Chris A; Breaux, Jason C; Martin, Karen H; Culp, Mark V; Gannett, Peter M; Weed, Scott A

2012-12-15

Tyrosine-kinase-based signal transduction mediated by modular protein domains is critical for cellular function. The Src homology (SH)2 domain is an important conductor of intracellular signaling that binds to phosphorylated tyrosines on acceptor proteins, producing molecular complexes responsible for signal relay. Cortactin is a cytoskeletal protein and tyrosine kinase substrate that regulates actin-based motility through interactions with SH2-domain-containing proteins. The Src kinase SH2 domain mediates cortactin binding and tyrosine phosphorylation, but how Src interacts with cortactin is unknown. Here we demonstrate that Src binds cortactin through cystine bonding between Src C185 in the SH2 domain within the phosphotyrosine binding pocket and cortactin C112/246 in the cortactin repeats domain, independent of tyrosine phosphorylation. Interaction studies show that the presence of reducing agents ablates Src-cortactin binding, eliminates cortactin phosphorylation by Src, and prevents Src SH2 domain binding to cortactin. Tandem MS/MS sequencing demonstrates cystine bond formation between Src C185 and cortactin C112/246. Mutational studies indicate that an intact cystine binding interface is required for Src-mediated cortactin phosphorylation, cell migration, and pre-invadopodia formation. Our results identify a novel phosphotyrosine-independent binding mode between the Src SH2 domain and cortactin. Besides Src, one quarter of all SH2 domains contain cysteines at or near the analogous Src C185 position. This provides a potential alternative mechanism to tyrosine phosphorylation for cysteine-containing SH2 domains to bind cognate ligands that may be widespread in propagating signals regulating diverse cellular functions.

Src binds cortactin through an SH2 domain cystine-mediated linkage

PubMed Central

Evans, Jason V.; Ammer, Amanda G.; Jett, John E.; Bolcato, Chris A.; Breaux, Jason C.; Martin, Karen H.; Culp, Mark V.; Gannett, Peter M.; Weed, Scott A.

2012-01-01

Summary Tyrosine-kinase-based signal transduction mediated by modular protein domains is critical for cellular function. The Src homology (SH)2 domain is an important conductor of intracellular signaling that binds to phosphorylated tyrosines on acceptor proteins, producing molecular complexes responsible for signal relay. Cortactin is a cytoskeletal protein and tyrosine kinase substrate that regulates actin-based motility through interactions with SH2-domain-containing proteins. The Src kinase SH2 domain mediates cortactin binding and tyrosine phosphorylation, but how Src interacts with cortactin is unknown. Here we demonstrate that Src binds cortactin through cystine bonding between Src C185 in the SH2 domain within the phosphotyrosine binding pocket and cortactin C112/246 in the cortactin repeats domain, independent of tyrosine phosphorylation. Interaction studies show that the presence of reducing agents ablates Src-cortactin binding, eliminates cortactin phosphorylation by Src, and prevents Src SH2 domain binding to cortactin. Tandem MS/MS sequencing demonstrates cystine bond formation between Src C185 and cortactin C112/246. Mutational studies indicate that an intact cystine binding interface is required for Src-mediated cortactin phosphorylation, cell migration, and pre-invadopodia formation. Our results identify a novel phosphotyrosine-independent binding mode between the Src SH2 domain and cortactin. Besides Src, one quarter of all SH2 domains contain cysteines at or near the analogous Src C185 position. This provides a potential alternative mechanism to tyrosine phosphorylation for cysteine-containing SH2 domains to bind cognate ligands that may be widespread in propagating signals regulating diverse cellular functions. PMID:23097045

Deletion and site-specific mutagenesis of nucleolin's carboxy GAR domain.

PubMed

Pellar, Gregory J; DiMario, Patrick J

2003-04-01

Vertebrate nucleolin is an abundant RNA-binding protein in the dense fibrillar component of active nucleoli. Nucleolin is modular in composition. Its amino-terminal third contains alternating acidic and basic domains, its middle section contains four consensus RNA-binding domains (cRBDs), and its carboxy-terminus contains a distinctive glycine/arginine-rich (GAR) domain with several RGG motifs. The arginines within these motifs are asymmetrically dimethylated. Several laboratories have shown that the GAR domain is necessary but not sufficient for the efficient localization of nucleolin to nucleoli. We examined the distribution of endogenous fibrillarin, Nopp140, and B23 when full-length and DeltaGAR nucleolin were expressed exogenously as enhanced green fluorescent protein (EGFP)-tagged fusions. Only B23 redistributed when DeltaGAR-EGFP was expressed at moderate to high levels, suggesting an in vivo interaction between nucleolin and B23. Next we substituted all ten arginines within the GAR domain of Chinese hamster ovary (CHO) nucleolin with lysines to test the hypothesis that methylation of the carboxy GAR domain is necessary for the nucleolar association of nucleolin. The lysine-substituted mutant was not an in vitro substrate for the yeast protein methyltransferase, Hmt1p/Rmt1. It was, however, able to associate properly with interphase nucleoli and with interphase pre-nucleolar bodies upon recovery from hypotonic shock. We conclude, therefore, that although the GAR domain is necessary for the efficient localization of nucleolin to nucleoli, methylation of this domain is not required for proper nucleolar localization.

Label-free logic modules and two-layer cascade based on stem-loop probes containing a G-quadruplex domain.

PubMed

Guo, Yahui; Cheng, Junjie; Wang, Jine; Zhou, Xiaodong; Hu, Jiming; Pei, Renjun

2014-09-01

A simple, versatile, and label-free DNA computing strategy was designed by using toehold-mediated strand displacement and stem-loop probes. A full set of logic gates (YES, NOT, OR, NAND, AND, INHIBIT, NOR, XOR, XNOR) and a two-layer logic cascade were constructed. The probes contain a G-quadruplex domain, which was blocked or unfolded through inputs initiating strand displacement and the obviously distinguishable light-up fluorescent signal of G-quadruplex/NMM complex was used as the output readout. The inputs are the disease-specific nucleotide sequences with potential for clinic diagnosis. The developed versatile computing system based on our label-free and modular strategy might be adapted in multi-target diagnosis through DNA hybridization and aptamer-target interaction. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

hLARP7 C-terminal domain contains an xRRM that binds the 3' hairpin of 7SK RNA

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

Eichhorn, Catherine D.; Chug, Rahul; Feigon, Juli

The 7SK small nuclear ribonucleoprotein (snRNP) sequesters and inactivates the positive transcription elongation factor b (P-TEFb), an essential eukaryotic mRNA transcription factor. The human La-related protein group 7 (hLARP7) is a constitutive component of the 7SK snRNP and localizes to the 3' terminus of the 7SK long noncoding RNA. hLARP7, and in particular its C-terminal domain (CTD), is essential for 7SK RNA stability and assembly with P-TEFb. The hLARP7 N-terminal Lamodule binds and protects the 3' end from degradation, but the structural and functional role of its CTD is unclear.We report the solution NMR structure of the hLARP7 CTD andmore » show that this domain contains an xRRM, a class of atypical RRM first identified in the Tetrahymena thermophila telomerase LARP7 protein p65. The xRRM binds the 3' end of 7SK RNA at the top of stem-loop 4 (SL4) and interacts with both unpaired and base-paired nucleotides. This study thus confirms that the xRRM is general to the LARP7 family of proteins and defines the binding site for hLARP7 on the 7SK RNA, providing insight into function.« less

hLARP7 C-terminal domain contains an xRRM that binds the 3' hairpin of 7SK RNA

DOE PAGES

Eichhorn, Catherine D.; Chug, Rahul; Feigon, Juli

2016-09-26

The 7SK small nuclear ribonucleoprotein (snRNP) sequesters and inactivates the positive transcription elongation factor b (P-TEFb), an essential eukaryotic mRNA transcription factor. The human La-related protein group 7 (hLARP7) is a constitutive component of the 7SK snRNP and localizes to the 3' terminus of the 7SK long noncoding RNA. hLARP7, and in particular its C-terminal domain (CTD), is essential for 7SK RNA stability and assembly with P-TEFb. The hLARP7 N-terminal Lamodule binds and protects the 3' end from degradation, but the structural and functional role of its CTD is unclear.We report the solution NMR structure of the hLARP7 CTD andmore » show that this domain contains an xRRM, a class of atypical RRM first identified in the Tetrahymena thermophila telomerase LARP7 protein p65. The xRRM binds the 3' end of 7SK RNA at the top of stem-loop 4 (SL4) and interacts with both unpaired and base-paired nucleotides. This study thus confirms that the xRRM is general to the LARP7 family of proteins and defines the binding site for hLARP7 on the 7SK RNA, providing insight into function.« less

Contributions of individual domains to function of the HIV-1 Rev response element.

PubMed

O'Carroll, Ina P; Thappeta, Yashna; Fan, Lixin; Ramirez-Valdez, Edric A; Smith, Sean; Wang, Yun-Xing; Rein, Alan

2017-08-16

The HIV-1 Rev response element (RRE) is a 351-base element in unspliced and partially spliced viral RNA; binding of the RRE by the viral Rev protein induces nuclear export of RRE-containing RNAs, as required for virus replication. It contains one long, imperfect double helix (domain I), one branched domain (domain II) containing a high-affinity Rev-binding site, and two or three additional domains. We previously reported that the RRE assumes an "A" shape in solution and suggested that the location of the Rev binding sites in domains I and II, opposite each other on the two legs of the A, is optimal for Rev binding and explains Rev's specificity for RRE-containing RNAs. Using SAXS and a quantitative functional assay, we have now analyzed a panel of RRE mutants. All the results support the essential role of the A shape for RRE function. Moreover, they suggest that the distal portion of domain I and the three crowning domains all contribute to the maintenance of the A shape. Domains I and II are necessary and sufficient for substantial RRE function, provided they are joined by a flexible linker that allows the two domains to face each other. IMPORTANCE Retroviral replication requires that some of the viral RNAs transcribed in the cell nucleus be exported to the cytoplasm without being spliced. To achieve this, HIV-1 encodes a protein, Rev, which binds to a complex, highly structured element within viral RNA, the Rev Response Element (RRE), and escorts RRE-containing RNAs from the nucleus. We previously reported that the RRE is "A"-shaped and suggested that this architecture, with the 2 legs opposite one another, can explain the specificity of Rev for the RRE. We have analyzed the functional contributions of individual RRE domains, and now report that several domains contribute, with some redundancy, to maintenance of the overall RRE shape. The data strongly support the hypothesis that the opposed placement of the 2 legs is essential for RRE function. Copyright © 2017

Contributions of Individual Domains to Function of the HIV-1 Rev Response Element

PubMed Central

O'Carroll, Ina P.; Thappeta, Yashna; Fan, Lixin; Ramirez-Valdez, Edric A.; Smith, Sean; Wang, Yun-Xing

2017-01-01

ABSTRACT The HIV-1 Rev response element (RRE) is a 351-base element in unspliced and partially spliced viral RNA; binding of the RRE by the viral Rev protein induces nuclear export of RRE-containing RNAs, as required for virus replication. It contains one long, imperfect double helix (domain I), one branched domain (domain II) containing a high-affinity Rev-binding site, and two or three additional domains. We previously reported that the RRE assumes an “A” shape in solution and suggested that the location of the Rev binding sites in domains I and II, opposite each other on the two legs of the A, is optimal for Rev binding and explains Rev's specificity for RRE-containing RNAs. Using small-angle X-ray scattering (SAXS) and a quantitative functional assay, we have now analyzed a panel of RRE mutants. All the results support the essential role of the A shape for RRE function. Moreover, they suggest that the distal portion of domain I and the three crowning domains all contribute to the maintenance of the A shape. Domains I and II are necessary and sufficient for substantial RRE function, provided they are joined by a flexible linker that allows the two domains to face each other. IMPORTANCE Retroviral replication requires that some of the viral RNAs transcribed in the cell nucleus be exported to the cytoplasm without being spliced. To achieve this, HIV-1 encodes a protein, Rev, which binds to a complex, highly structured element within viral RNA, the Rev response element (RRE), and escorts RRE-containing RNAs from the nucleus. We previously reported that the RRE is “A” shaped and suggested that this architecture, with the 2 legs opposite one another, can explain the specificity of Rev for the RRE. We have analyzed the functional contributions of individual RRE domains and now report that several domains contribute, with some redundancy, to maintenance of the overall RRE shape. The data strongly support the hypothesis that the opposed placement of the 2 legs

SH3 Domain-Containing Protein 2 Plays a Crucial Role at the Step of Membrane Tubulation during Cell Plate Formation

PubMed Central

Ahn, Gyeongik; Kim, Hyeran; Kim, Dae Heon; Hanh, Hong; Yoon, Youngdae; Singaram, Indira; Wijesinghe, Kaveesha J.; Johnson, Kristen A.; Liang, Zizhen; Stahelin, Robert V.; Jiang, Liwen; Cho, Wonhwa; Kang, Byung-Ho

2017-01-01

During cytokinesis in plants, trans-Golgi network-derived vesicles accumulate at the center of dividing cells and undergo various structural changes to give rise to the planar cell plate. However, how this conversion occurs at the molecular level remains elusive. In this study, we report that SH3 Domain-Containing Protein 2 (SH3P2) in Arabidopsis thaliana plays a crucial role in converting vesicles to the planar cell plate. SH3P2 RNAi plants showed cytokinesis-defective phenotypes and produced aggregations of vesicles at the leading edge of the cell plate. SH3P2 localized to the leading edge of the cell plate, particularly the constricted or curved regions of the cell plate. The BAR domain of SH3P2 induced tubulation of vesicles. SH3P2 formed a complex with dynamin-related protein 1A (DRP1A) and affected DRP1A accumulation to the cell plate. Based on these results, we propose that SH3P2 functions together with DRP1A to convert the fused vesicles to tubular structures during cytokinesis. PMID:28584166

Crystal Structure of a Two-domain Fragment of Hepatocyte Growth Factor Activator Inhibitor-1: FUNCTIONAL INTERACTIONS BETWEEN THE KUNITZ-TYPE INHIBITOR DOMAIN-1 AND THE NEIGHBORING POLYCYSTIC KIDNEY DISEASE-LIKE DOMAIN.

PubMed

Hong, Zebin; De Meulemeester, Laura; Jacobi, Annemarie; Pedersen, Jan Skov; Morth, J Preben; Andreasen, Peter A; Jensen, Jan K

2016-07-01

Hepatocyte growth factor activator inhibitor-1 (HAI-1) is a type I transmembrane protein and inhibitor of several serine proteases, including hepatocyte growth factor activator and matriptase. The protein is essential for development as knock-out mice die in utero due to placental defects caused by misregulated extracellular proteolysis. HAI-1 contains two Kunitz-type inhibitor domains (Kunitz), which are generally thought of as a functionally self-contained protease inhibitor unit. This is not the case for HAI-1, where our results reveal how interdomain interactions have evolved to stimulate the inhibitory activity of an integrated Kunitz. Here we present an x-ray crystal structure of an HAI-1 fragment covering the internal domain and Kunitz-1. The structure reveals not only that the previously uncharacterized internal domain is a member of the polycystic kidney disease domain family but also how the two domains engage in interdomain interactions. Supported by solution small angle x-ray scattering and a combination of site-directed mutagenesis and functional assays, we show that interdomain interactions not only stabilize the fold of the internal domain but also stimulate the inhibitory activity of Kunitz-1. By completing our structural characterization of the previously unknown N-terminal region of HAI-1, we provide new insight into the interplay between tertiary structure and the inhibitory activity of a multidomain protease inhibitor. We propose a previously unseen mechanism by which the association of an auxiliary domain stimulates the inhibitory activity of a Kunitz-type inhibitor (i.e. the first structure of an intramolecular interaction between a Kunitz and another domain). © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The DEP domain-containing protein TOE-2 promotes apoptosis in the Q lineage of C. elegans through two distinct mechanisms

PubMed Central

Gurling, Mark; Talavera, Karla; Garriga, Gian

2014-01-01

Neuroblast divisions in the nematode Caenorhabditis elegans often give rise to a larger neuron and a smaller cell that dies. We have previously identified genes that, when mutated, result in neuroblast divisions that generate daughter cells that are more equivalent in size. This effect correlates with the survival of daughter cells that would normally die. We now describe a role for the DEP domain-containing protein TOE-2 in promoting the apoptotic fate in the Q lineage. TOE-2 localized at the plasma membrane and accumulated in the cleavage furrow of the Q.a and Q.p neuroblasts, suggesting that TOE-2 might position the cleavage furrow asymmetrically to generate daughter cells of different sizes. This appears to be the case for Q.a divisions where loss of TOE-2 led to a more symmetric division and to survival of the smaller Q.a daughter. Localization of TOE-2 to the membrane is required for this asymmetry, but, surprisingly, the DEP domain is dispensable. By contrast, loss of TOE-2 led to loss of the apoptotic fate in the smaller Q.p daughter but did not affect the size asymmetry of the Q.p daughters. This function of TOE-2 required the DEP domain but not localization to the membrane. We propose that TOE-2 ensures an apoptotic fate for the small Q.a daughter by promoting asymmetry in the daughter cell sizes of the Q.a neuroblast division but by a mechanism that is independent of cell size in the Q.p division. PMID:24961802

Generation of a Maize B Centromere Minimal Map Containing the Central Core Domain.

PubMed

Ellis, Nathanael A; Douglas, Ryan N; Jackson, Caroline E; Birchler, James A; Dawe, R Kelly

2015-10-28

The maize B centromere has been used as a model for centromere epigenetics and as the basis for building artificial chromosomes. However, there are no sequence resources for this important centromere. Here we used transposon display for the centromere-specific retroelement CRM2 to identify a collection of 40 sequence tags that flank CRM2 insertion points on the B chromosome. These were confirmed to lie within the centromere by assaying deletion breakpoints from centromere misdivision derivatives (intracentromere breakages caused by centromere fission). Markers were grouped together on the basis of their association with other markers in the misdivision series and assembled into a pseudocontig containing 10.1 kb of sequence. To identify sequences that interact directly with centromere proteins, we carried out chromatin immunoprecipitation using antibodies to centromeric histone H3 (CENH3), a defining feature of functional centromeric sequences. The CENH3 chromatin immunoprecipitation map was interpreted relative to the known transmission rates of centromere misdivision derivatives to identify a centromere core domain spanning 33 markers. A subset of seven markers was mapped in additional B centromere misdivision derivatives with the use of unique primer pairs. A derivative previously shown to have no canonical centromere sequences (Telo3-3) lacks these core markers. Our results provide a molecular map of the B chromosome centromere and identify key sequences within the map that interact directly with centromeric histone H3. Copyright © 2015 Ellis et al.

Starch Binding Domain-containing Protein 1 Plays a Dominant Role in Glycogen Transport to Lysosomes in Liver*

PubMed Central

Sun, Tao; Yi, Haiqing; Yang, Chunyu; Kishnani, Priya S.; Sun, Baodong

2016-01-01

A small portion of cellular glycogen is transported to and degraded in lysosomes by acid α-glucosidase (GAA) in mammals, but it is unclear why and how glycogen is transported to the lysosomes. Stbd1 has recently been proposed to participate in glycogen trafficking to lysosomes. However, our previous study demonstrated that knockdown of Stbd1 in GAA knock-out mice did not alter lysosomal glycogen storage in skeletal muscles. To further determine whether Stbd1 participates in glycogen transport to lysosomes, we generated GAA/Stbd1 double knock-out mice. In fasted double knock-out mice, glycogen accumulation in skeletal and cardiac muscles was not affected, but glycogen content in liver was reduced by nearly 73% at 3 months of age and by 60% at 13 months as compared with GAA knock-out mice, indicating that the transport of glycogen to lysosomes was suppressed in liver by the loss of Stbd1. Exogenous expression of human Stbd1 in double knock-out mice restored the liver lysosomal glycogen content to the level of GAA knock-out mice, as did a mutant lacking the Atg8 family interacting motif (AIM) and another mutant that contains only the N-terminal 24 hydrophobic segment and the C-terminal starch binding domain (CBM20) interlinked by an HA tag. Our results demonstrate that Stbd1 plays a dominant role in glycogen transport to lysosomes in liver and that the N-terminal transmembrane region and the C-terminal CBM20 domain are critical for this function. PMID:27358407

Domain structure of the ribozyme from eubacterial ribonuclease P.

PubMed Central

Loria, A; Pan, T

1996-01-01

Large RNAs can be composed of discrete domains that fold independently. One such "folding domain" has been identified previously in the ribozyme from Bacillus subtilis ribonuclease P (denoted P RNA). This domain contains roughly one-third of all residues. Folding of an RNA construct consisting of the remaining two-thirds of B. subtilis P RNA was examined by Fe(II)-EDTA hydroxyl radical protection. This molecule folds into the proper higher-order structure under identical conditions as the full-length P RNA, suggesting the presence of a second folding domain in B. subtilis P RNA. Folding analysis of the Escherichia coli P RNA by hydroxyl radical protection shows that this P RNA is completely folded at 5-6 mM Mg2+. In order to analyze the structural organization of folding domains in E. coli P RNA, constructs were designed based on the domain structure of B. subtilis P RNA. Fe(II)-EDTA protection indicates that E. coli P RNA also contains two folding domains. Despite the significant differences at the secondary structure level, both P RNAs appear to converge structurally at the folding domain level. The pre-tRNA substrate, localized in previous studies, may bind across the folding domains with the acceptor stem/3'CCA contacting the domain including the active site and the T stem-loop contacting the other. Because all eubacterial P RNAs share considerable homology in secondary structure to either B. subtilis or E. coli P RNA, these results suggest that this domain structure may be applicable for most, if not all, eubacterial P RNAs. Identification of folding domains should be valuable in dissecting structure-function relationship of large RNAs. PMID:8718684

Bacillus anthracis TIR Domain-Containing Protein Localises to Cellular Microtubule Structures and Induces Autophagy.

PubMed

Carlsson, Emil; Thwaite, Joanne E; Jenner, Dominic C; Spear, Abigail M; Flick-Smith, Helen; Atkins, Helen S; Byrne, Bernadette; Ding, Jeak Ling

2016-01-01

Toll-like receptors (TLRs) recognise invading pathogens and mediate downstream immune signalling via Toll/IL-1 receptor (TIR) domains. TIR domain proteins (Tdps) have been identified in multiple pathogenic bacteria and have recently been implicated as negative regulators of host innate immune activation. A Tdp has been identified in Bacillus anthracis, the causative agent of anthrax. Here we present the first study of this protein, designated BaTdp. Recombinantly expressed and purified BaTdp TIR domain interacted with several human TIR domains, including that of the key TLR adaptor MyD88, although BaTdp expression in cultured HEK293 cells had no effect on TLR4- or TLR2- mediated immune activation. During expression in mammalian cells, BaTdp localised to microtubular networks and caused an increase in lipidated cytosolic microtubule-associated protein 1A/1B-light chain 3 (LC3), indicative of autophagosome formation. In vivo intra-nasal infection experiments in mice showed that a BaTdp knockout strain colonised host tissue faster with higher bacterial load within 4 days post-infection compared to the wild type B. anthracis. Taken together, these findings indicate that BaTdp does not play an immune suppressive role, but rather, its absence increases virulence. BaTdp present in wild type B. anthracis plausibly interact with the infected host cell, which undergoes autophagy in self-defence.

Tudor-domain containing proteins act to make the piRNA pathways more robust in Drosophila.

PubMed

Sato, Kaoru; Iwasaki, Yuka W; Siomi, Haruhiko; Siomi, Mikiko C

2015-01-01

PIWI-interacting RNAs (piRNAs), a subset of small non-coding RNAs enriched in animal gonads, repress transposons by assembling with PIWI proteins to form potent gene-silencing RNP complexes, piRISCs. Accumulating evidence suggests that piRNAs are produced through three interdependent pathways; the de novo primary pathway, the ping-pong pathway, and the phased primary pathway. The de novo primary pathway in Drosophila ovaries produces primary piRNAs for two PIWI members, Piwi and Aub. Aub then initiates the ping-pong pathway to produce secondary piRNAs for AGO3. AGO3-slicer dependent cleavage subsequently produces secondary piRNAs for Aub. Trailer products of AGO3-slicer activity are consumed by the phased primary pathway to increase the Piwi-bound piRNA population. All these pathways are regulated by a number of piRNA factors in a highly coordinated fashion. Recent studies show that two Tudor-domain containing piRNA factors, Krimper (Krimp) and Qin/Kumo, play crucial roles in making Aub-AGO3 heterotypic ping-pong robust. This maintains the levels of piRNAs loaded onto Piwi and Aub to efficiently repress transposons at transcriptional and post-transcriptional levels, respectively.

The structure of an unconventional HD-GYP protein from Bdellovibrio reveals the roles of conserved residues in this class of cyclic-di-GMP phosphodiesterases.

PubMed

Lovering, Andrew L; Capeness, Michael J; Lambert, Carey; Hobley, Laura; Sockett, R Elizabeth

2011-01-01

Cyclic-di-GMP is a near-ubiquitous bacterial second messenger that is important in localized signal transmission during the control of various processes, including virulence and switching between planktonic and biofilm-based lifestyles. Cyclic-di-GMP is synthesized by GGDEF diguanylate cyclases and hydrolyzed by EAL or HD-GYP phosphodiesterases, with each functional domain often appended to distinct sensory modules. HD-GYP domain proteins have resisted structural analysis, but here we present the first structural representative of this family (1.28 Å), obtained using the unusual Bd1817 HD-GYP protein from the predatory bacterium Bdellovibrio bacteriovorus. Bd1817 lacks the active-site tyrosine present in most HD-GYP family members yet remains an excellent model of their features, sharing 48% sequence similarity with the archetype RpfG. The protein structure is highly modular and thus provides a basis for delineating domain boundaries in other stimulus-dependent homologues. Conserved residues in the HD-GYP family cluster around a binuclear metal center, which is observed complexed to a molecule of phosphate, providing information on the mode of hydroxide ion attack on substrate. The fold and active site of the HD-GYP domain are different from those of EAL proteins, and restricted access to the active-site cleft is indicative of a different mode of activity regulation. The region encompassing the GYP motif has a novel conformation and is surface exposed and available for complexation with binding partners, including GGDEF proteins. It is becoming apparent that many bacteria use the signaling molecule cyclic-di-GMP to regulate a variety of processes, most notably, transitions between motility and sessility. Importantly, this regulation is central to several traits implicated in chronic disease (adhesion, biofilm formation, and virulence gene expression). The mechanisms of cyclic-di-GMP synthesis via GGDEF enzymes and hydrolysis via EAL enzymes have been suggested by

The evolution of filamin – A protein domain repeat perspective

PubMed Central

Light, Sara; Sagit, Rauan; Ithychanda, Sujay S.; Qin, Jun; Elofsson, Arne

2013-01-01

Particularly in higher eukaryotes, some protein domains are found in tandem repeats, performing broad functions often related to cellular organization. For instance, the eukaryotic protein filamin interacts with many proteins and is crucial for the cytoskeleton. The functional properties of long repeat domains are governed by the specific properties of each individual domain as well as by the repeat copy number. To provide better understanding of the evolutionary and functional history of repeating domains, we investigated the mode of evolution of the filamin domain in some detail. Among the domains that are common in long repeat proteins, sushi and spectrin domains evolve primarily through cassette tandem duplications while scavenger and immunoglobulin repeats appear to evolve through clustered tandem duplications. Additionally, immunoglobulin and filamin repeats exhibit a unique pattern where every other domain shows high sequence similarity. This pattern may be the result of tandem duplications, serve to avert aggregation between adjacent domains or it is the result of functional constraints. In filamin, our studies confirm the presence of interspersed integrin binding domains in vertebrates, while invertebrates exhibit more varied patterns, including more clustered integrin binding domains. The most notable case is leech filamin, which contains a 20 repeat expansion and exhibits unique dimerization topology. Clearly, invertebrate filamins are varied and contain examples of similar adjacent integrin-binding domains. Given that invertebrate integrin shows more similarity to the weaker filamin binder, integrin β3, it is possible that the distance between integrin-binding domains is not as crucial for invertebrate filamins as for vertebrates. PMID:22414427

Elevated Aerosol Layers and Their Radiative Impact over Kanpur During Monsoon Onset Period

NASA Technical Reports Server (NTRS)

Sarangi, Chandan; Tripathi, S. N.; Mishra, A. K.; Welton, E. J.

2016-01-01

Accurate information about aerosol vertical distribution is needed to reduce uncertainties in aerosol radiative forcing and its effect on atmospheric dynamics. The present study deals with synergistic analyses of aerosol vertical distribution and aerosol optical depth (AOD) with meteorological variables using multisatellite and ground-based remote sensors over Kanpur in central Indo-Gangetic Plain (IGP). Micro-Pulse Lidar Network-derived aerosol vertical extinction (sigma) profiles are analyzed to quantify the interannual and daytime variations during monsoon onset period (May-June) for 2009-2011. The mean aerosol profile is broadly categorized into two layers viz., a surface layer (SL) extending up to 1.5 km (where sigma decreased exponentially with height) and an elevated aerosol layer (EAL) extending between 1.5 and 5.5 km. The increase in total columnar aerosol loading is associated with relatively higher increase in contribution from EAL loading than that from SL. The mean contributions of EALs are about 60%, 51%, and 50% to total columnar AOD during 2009, 2010, and 2011, respectively. We observe distinct parabolic EALs during early morning and late evening but uniformly mixed EALs during midday. The interannual and daytime variations of EALs are mainly influenced by long-range transport and convective capacity of the local emissions, respectively. Radiative flux analysis shows that clear-sky incoming solar radiation at surface is reduced with increase in AOD, which indicates significant cooling at surface. Collocated analysis of atmospheric temperature and aerosol loading reveals that increase in AOD not only resulted in surface dimming but also reduced the temperature (approximately 2-3 C) of lower troposphere (below 3 km altitude). Radiative transfer simulations indicate that the reduction of incoming solar radiation at surface is mainly due to increased absorption by EALs (with increase in total AOD). The observed cooling in lower troposphere in high

The Nucleotide-Binding Oligomerization Domain-Like Receptor Family Pyrin Domain-Containing 3 Inflammasome Regulates Bronchial Epithelial Cell Injury and Proapoptosis after Exposure to Biomass Fuel Smoke.

PubMed

Li, Chen; Zhihong, Huang; Wenlong, Li; Xiaoyan, Liu; Qing, Chen; Wenzhi, Luo; Siming, Xie; Shengming, Liu

2016-12-01

The number of individuals in the population exposed to biomass fuel smoke (BS) is far greater than the number of cigarette smokers. About 20% of cigarette smokers develop chronic obstructive pulmonary disease (COPD) due to smoke-induced irreversible damage and sustained inflammation of the airway epithelium. However, the role of BS in COPD pathogenesis remains to be elucidated. In this study, we investigated the expression of nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing (NLRP) 3 and caspase-1 in the bronchial epithelium from patients with COPD, and further determined the specific role of the NLRP3 inflammasome in bronchial epithelium injury using two in vitro models (BS and cigarette smoke [CS]) in the human bronchial epithelial (HBE) cell line (16HBE). After exposure to BS and CS, the release of damage-associated molecular patterns, the transcriptional and translational up-regulation of NLRP3, and the activation of caspase-1 were observed in cells at different time points. Because IL-1β secretion was dependent on the NLRP3 inflammasome, we assessed CXCL-8 production in response to smoke. Using a transwell migration assay in which 16HBE cells and human alveolar macrophages were cocultured, we showed that smoke-induced NLRP3 activation in 16HBE cells increased the migration of human alveolar macrophages. When the NLRP3 expression was silenced, the average migration distance of 16HBE was increased in scratch assay, because the activation of NLRP3 induced apoptosis by the p53-Bax mitochondrial pathway in the smoke-induced response. These results demonstrate the importance of the NLRP3 inflammasome in mediating BS- and CS-induced HBE cell damage and proapoptosis.

Cross-β Polymerization of Low Complexity Sequence Domains.

PubMed

Kato, Masato; McKnight, Steven L

2017-03-01

Most transcription factors and RNA regulatory proteins encoded by eukaryotic genomes ranging from yeast to humans contain polypeptide domains variously described as intrinsically disordered, prion-like, or of low complexity (LC). These LC domains exist in an unfolded state when DNA and RNA regulatory proteins are studied in biochemical isolation from cells. Upon incubation in the purified state, many of these LC domains polymerize into homogeneous, labile amyloid-like fibers. Here, we consider several lines of evidence that may favor biologic utility for LC domain polymers. Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved.

Thrombospondin Type-1 Domain-Containing 7A in Idiopathic Membranous Nephropathy

PubMed Central

Meyer-Schwesinger, Catherine; Seitz-Polski, Barbara; Ma, Hong; Zahner, Gunther; Dolla, Guillaume; Hoxha, Elion; Helmchen, Udo; Dabert-Gay, Anne-Sophie; Debayle, Delphine; Merchant, Michael; Klein, Jon; Salant, David J.; Stahl, Rolf A.K.; Lambeau, Gérard

2014-01-01

BACKGROUND Idiopathic membranous nephropathy is an autoimmune disease. In approximately 70% of patients, it is associated with autoantibodies against the phospholipase A2 receptor 1 (PLA2R1). Antigenic targets in the remaining patients are unknown. METHODS Using Western blotting, we screened serum samples from patients with idiopathic membranous nephropathy, patients with other glomerular diseases, and healthy controls for antibodies against human native glomerular proteins. We partially purified a putative new antigen, identified this protein by means of mass spectrometry of digested peptides, and validated the results by analysis of recombinant protein expression, immunoprecipitation, and immunohistochemical analysis. RESULTS Serum samples from 6 of 44 patients in a European cohort and 9 of 110 patients in a Boston cohort with anti-PLA2R1–negative idiopathic membranous nephropathy recognized a glomerular protein that was 250 kD in size. None of the serum samples from the 74 patients with idiopathic membranous nephropathy who were sero-positive for anti-PLA2R1 antibodies, from the 76 patients with other glomerular diseases, and from the 44 healthy controls reacted against this antigen. Although this newly identified antigen is clearly different from PLA2R1, it shares some biochemical features, such as N-glycosylation, membranous location, and reactivity with serum only under nonreducing conditions. Mass spectrometry identified this antigen as thrombospondin type-1 domain-containing 7A (THSD7A). All reactive serum samples recognized recombinant THSD7A and immunoprecipitated THSD7A from glomerular lysates. Moreover, immunohistochemical analyses of biopsy samples from patients revealed localization of THSD7A to podocytes, and IgG eluted from one of these samples was specific for THSD7A. CONCLUSIONS In our cohort, 15 of 154 patients with idiopathic membranous nephropathy had circulating autoantibodies to THSD7A but not to PLA2R1, a finding that suggests a distinct

The Popeye domain containing 2 (popdc2) gene in zebrafish is required for heart and skeletal muscle development

PubMed Central

Kirchmaier, Bettina C.; Poon, Kar Lai; Schwerte, Thorsten; Huisken, Jan; Winkler, Christoph; Jungblut, Benno; Stainier, Didier Y.; Brand, Thomas

2013-01-01

The Popeye domain containing (Popdc) genes encode a family of transmembrane proteins with an evolutionary conserved Popeye domain. These genes are abundantly expressed in striated muscle tissue, however their function is not well understood. In this study we have investigated the role of the popdc2 gene in zebrafish. Popdc2 transcripts were detected in the embryonic myocardium and transiently in the craniofacial and tail musculature. Morpholino oligonucleotide-mediated knockdown of popdc2 resulted in aberrant development of skeletal muscle and heart. Muscle segments in the trunk were irregularly shaped and craniofacial muscles were severely reduced or even missing. In the heart, pericardial edema was prevalent in the morphants and heart chambers were elongated and looping was abnormal. These pathologies in muscle and heart were alleviated after reducing the morpholino concentration. However the heart still was abnormal displaying cardiac arrhythmia at later stages of development. Optical recordings of cardiac contractility revealed irregular ventricular contractions with a 2:1, or 3:1 atrial/ventricular conduction ratio, which caused a significant reduction in heart frequency. Recordings of calcium transients with high spatiotemporal resolution using a transgenic calcium indicator line (Tg(cmlc2:gCaMP)s878) and SPIM microscopy confirmed the presence of a severe arrhythmia phenotype. Our results identify popdc2 as a gene important for striated muscle differentiation and cardiac morphogenesis. In addition it is required for the development of the cardiac conduction system. PMID:22290329

BRICHOS domain-containing leukocyte cell-derived chemotaxin 1-like cDNA from disk abalone Haliotis discus discus.

PubMed

Kim, Yucheol; De Zoysa, Mahanama; Lee, Youngdeuk; Whang, Ilson; Lee, Jehee

2010-11-01

A BRICHOS domain-containing leukocyte cell-derived chemotaxin 1-like cDNA was cloned from the disk abalone (Haliotis discus discus) and designated as AbLECT-1. A full-length (705 bp) of AbLECT-1 cDNA was composed of a 576 bp open reading frame that translates into a putative peptide of 192 amino acids. Deduced amino acid sequence of AbLECT-1 had 15.5- and 27.8% identity and similarity to human LECT-1, respectively. Quantitative real-time PCR analysis results showed that the mRNA of AbLECT-1 was constitutively expressed in abalone hemocytes, gills, mantle, muscle, digestive tract and hepatopancreas in a tissue-specific manner. Moreover, the AbLECT-1 transcription level was induced in hemocytes after challenge with Vibrio alginolyticus, Vibrio parahemolyticus, and Listeria monocytogenes suggesting that it may be involved in immune response reactions in abalone. Copyright 2010 Elsevier Ltd. All rights reserved.

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

Coiled-Coil Antagonism Regulates Activity of Venus Flytrap-Domain-Containing Sensor Kinases of the BvgS Family

PubMed Central

Lesne, Elodie; Dupré, Elian; Lensink, Marc F.; Locht, Camille

2018-01-01

ABSTRACT Bordetella pertussis controls the expression of its virulence regulon through the two-component system BvgAS. BvgS is a prototype for a family of multidomain sensor kinases. In BvgS, helical linkers connect periplasmic Venus flytrap (VFT) perception domains to a cytoplasmic Per-Arnt-Sim (PAS) domain and the PAS domain to the dimerization/histidine phosphotransfer (DHp) domain of the kinase. The two linkers can adopt coiled-coil structures but cannot do so simultaneously. The first linker forms a coiled coil in the kinase mode and the second in the phosphatase mode, with the other linker in both cases showing an increase in dynamic behavior. The intervening PAS domain changes its quaternary structure between the two modes. In BvgS homologues without a PAS domain, a helical “X” linker directly connects the VFT and DHp domains. Here, we used BvgS as a platform to characterize regulation in members of the PAS-less subfamily. BvgS chimeras of homologues with natural X linkers displayed various regulation phenotypes. We identified two distinct coiled-coil registers in the N- and C-terminal portions of the X linkers. Stable coil formation in the C-terminal moiety determines the phosphatase mode, similarly to BvgS; in contrast, coil formation in the N-terminal moiety along the other register leads to the kinase mode. Thus, antagonism between two registers in the VFT-DHp linker forms the basis for activity regulation in the absence of the PAS domain. The N and C moieties of the X linker play roles similar to those played by the two independent linkers in sensor kinases with a PAS domain, providing a unified mechanism of regulation for the entire family. PMID:29487240

Characterization of thin films on the nanometer scale by Auger electron spectroscopy and X-ray photoelectron spectroscopy

NASA Astrophysics Data System (ADS)

Powell, C. J.; Jablonski, A.; Werner, W. S. M.; Smekal, W.

2005-01-01

We describe two NIST databases that can be used to characterize thin films from Auger electron spectroscopy (AES) and X-ray photoelectron spectroscopy (XPS) measurements. First, the NIST Electron Effective-Attenuation-Length Database provides values of effective attenuation lengths (EALs) for user-specified materials and measurement conditions. The EALs differ from the corresponding inelastic mean free paths on account of elastic-scattering of the signal electrons. The database supplies "practical" EALs that can be used to determine overlayer-film thicknesses. Practical EALs are plotted as a function of film thickness, and an average value is shown for a user-selected thickness. The average practical EAL can be utilized as the "lambda parameter" to obtain film thicknesses from simple equations in which the effects of elastic-scattering are neglected. A single average practical EAL can generally be employed for a useful range of film thicknesses and for electron emission angles of up to about 60°. For larger emission angles, the practical EAL should be found for the particular conditions. Second, we describe a new NIST database for the Simulation of Electron Spectra for Surface Analysis (SESSA) to be released in 2004. This database provides data for many parameters needed in quantitative AES and XPS (e.g., excitation cross-sections, electron-scattering cross-sections, lineshapes, fluorescence yields, and backscattering factors). Relevant data for a user-specified experiment are automatically retrieved by a small expert system. In addition, Auger electron and photoelectron spectra can be simulated for layered samples. The simulated spectra, for layer compositions and thicknesses specified by the user, can be compared with measured spectra. The layer compositions and thicknesses can then be adjusted to find maximum consistency between simulated and measured spectra, and thus, provide more detailed characterizations of multilayer thin-film materials. SESSA can also

Further insight into BRUTUS domain composition and functionality

PubMed Central

Matthiadis, Anna; Long, Terri A.

2016-01-01

ABSTRACT BRUTUS (BTS) is a hemerythrin (HHE) domain containing E3 ligase that facilitates the degradation of POPEYE-like (PYEL) proteins in a proteasomal-dependent manner. Deletion of BTS HHE domains enhances BTS stability in the presence of iron and also complements loss of BTS function, suggesting that the HHE domains are critical for protein stability but not for enzymatic function. The RING E3 domain plays an essential role in BTS' capacity to both interact with PYEL proteins and to act as an E3 ligase. Here we show that removal of the RING domain does not complement loss of BTS function. We conclude that enzymatic activity of BTS via the RING domain is essential for response to iron deficiency in plants. Further, we analyze possible BTS domain structure evolution and predict that the combination of domains found in BTS is specific to photosynthetic organisms, potentially indicative of a role for BTS and its orthologs in mitigating the iron-related challenges presented by photosynthesis. PMID:27359166

Further insight into BRUTUS domain composition and functionality.

PubMed

Matthiadis, Anna; Long, Terri A

2016-08-02

BRUTUS (BTS) is a hemerythrin (HHE) domain containing E3 ligase that facilitates the degradation of POPEYE-like (PYEL) proteins in a proteasomal-dependent manner. Deletion of BTS HHE domains enhances BTS stability in the presence of iron and also complements loss of BTS function, suggesting that the HHE domains are critical for protein stability but not for enzymatic function. The RING E3 domain plays an essential role in BTS' capacity to both interact with PYEL proteins and to act as an E3 ligase. Here we show that removal of the RING domain does not complement loss of BTS function. We conclude that enzymatic activity of BTS via the RING domain is essential for response to iron deficiency in plants. Further, we analyze possible BTS domain structure evolution and predict that the combination of domains found in BTS is specific to photosynthetic organisms, potentially indicative of a role for BTS and its orthologs in mitigating the iron-related challenges presented by photosynthesis.

The CW domain, a structural module shared amongst vertebrates, vertebrate-infecting parasites and higher plants.

PubMed

Perry, Jason; Zhao, Yunde

2003-11-01

A previously undetected domain, named CW for its conserved cysteine and tryptophan residues, appears to be a four-cysteine zinc-finger motif found exclusively in vertebrates, vertebrate-infecting parasites and higher plants. Of the twelve distinct nuclear protein families that comprise the CW domain-containing superfamily, only the microrchida (MORC) family has begun to be characterized. However, several families contain other domains suggesting a relationship between the CW domain and either chromatin methylation status or early embryonic development.

Expression of Zinc Finger and BTB Domain-containing 7A in Colorectal Carcinoma.

PubMed

Joo, Jin Woo; Kim, Hyun-Soo; Do, Sung-Im; Sung, Ji-Youn

2018-05-01

Previous studies have revealed that zinc finger and BTB domain-containing 7A (ZBTB7A), an important proto-oncogene, plays multiple roles in carcinogenesis and is up-regulated in several human malignancies. However, the expression of ZBTB7A in colorectal carcinoma (CRC) has seldom been documented. In this study, we investigated the differential expression of ZBTB7A in CRC cell lines and tissues. Expression levels of ZBTB7A mRNA and protein were examined in CRC cell lines. ZBTB7A protein expression was also evaluated in tissue samples of normal colonic mucosa, high-grade dysplasia, and CRC using immunohistochemical staining. All CRC cell lines exhibited significantly higher ZBTB7A mRNA expression levels than did normal colonic epithelial cells. The ZBTB7A protein expression levels were clearly higher in the CRC cell lines than in the normal colonic epithelial cells. Consistent with the cell line data, immunostaining revealed that there were significant differences in ZBTB7A protein expression between tissue samples of CRC and normal colonic mucosa (p=0.048) and high-grade dysplasia (p=0.015). In addition, metastatic CRC exhibited significantly higher ZBTB7A protein expression levels than primary CRC (p=0.027). We demonstrated that ZBTB7A expression is up-regulated in CRC cell lines and tissues. Our data suggest that ZBTB7A is involved in the development and progression of CRC. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Starch Binding Domain-containing Protein 1 Plays a Dominant Role in Glycogen Transport to Lysosomes in Liver.

PubMed

Sun, Tao; Yi, Haiqing; Yang, Chunyu; Kishnani, Priya S; Sun, Baodong

2016-08-05

A small portion of cellular glycogen is transported to and degraded in lysosomes by acid α-glucosidase (GAA) in mammals, but it is unclear why and how glycogen is transported to the lysosomes. Stbd1 has recently been proposed to participate in glycogen trafficking to lysosomes. However, our previous study demonstrated that knockdown of Stbd1 in GAA knock-out mice did not alter lysosomal glycogen storage in skeletal muscles. To further determine whether Stbd1 participates in glycogen transport to lysosomes, we generated GAA/Stbd1 double knock-out mice. In fasted double knock-out mice, glycogen accumulation in skeletal and cardiac muscles was not affected, but glycogen content in liver was reduced by nearly 73% at 3 months of age and by 60% at 13 months as compared with GAA knock-out mice, indicating that the transport of glycogen to lysosomes was suppressed in liver by the loss of Stbd1. Exogenous expression of human Stbd1 in double knock-out mice restored the liver lysosomal glycogen content to the level of GAA knock-out mice, as did a mutant lacking the Atg8 family interacting motif (AIM) and another mutant that contains only the N-terminal 24 hydrophobic segment and the C-terminal starch binding domain (CBM20) interlinked by an HA tag. Our results demonstrate that Stbd1 plays a dominant role in glycogen transport to lysosomes in liver and that the N-terminal transmembrane region and the C-terminal CBM20 domain are critical for this function. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

TaUBA, a UBA domain-containing protein in wheat (Triticum aestivum L.), is a negative regulator of salt and drought stress response in transgenic Arabidopsis.

PubMed

Li, Xiao; Zhang, Shuang-shuang; Ma, Jun-xia; Guo, Guang-yan; Zhang, Xue-yong; Liu, Xu; Bi, Cai-li

2015-05-01

TaUBA functions as a negative regulator of salt and drought stress response in transgenic Arabidopsis, either the UBA domain or the zinc finger domain is crucial for TaUBA's function. TaUBA (DQ211935), which is a UBA domain-containing protein in wheat, was cloned and functionally characterized. Southern blot suggested that TaUBA is a low copy gene in common wheat. qRT-PCR assay showed that the expression of TaUBA was strongly induced by salt and drought stress. When suffering from drought and salt stresses, lower proline content and much higher MDA content in the TaUBA overexpressors were observed than those of the wild-type control, suggesting TaUBA may function as a negative regulator of salt and drought stress response in plants. To study whether the UBA domain or the zinc finger domain affects the function of TaUBA, TaUBAΔUBA (deletion of UBA domain) and TaUBA-M (Cys464Gly and Cys467Gly) overexpression vectors were constructed and transformed into Arabidopsis. Upon drought and salt stresses, the TaUBAΔUBA-and TaUBA-M-overexpressed plants accumulated much more proline and lower MDA than the wild-type control, the TaUBA-overexpressors lost water more quickly than TaUBAΔUBA-and TaUBA-M-overexpressed plants as well as the wild-type control, suggesting that overexpression of TaUBAΔUBA or TaUBA-M improved the drought and salt tolerance of transgenic Arabidopsis plants and the possibility of ubiquitination role in the regulation of osmolyte synthesis and oxidative stress responses in mediating stress tolerance. qRT-PCR assay of stress-related genes in transgenic plants upon drought and salt stresses suggested that TaUBA may function through down-regulating some stress related-transcription factors and by regulating P5CSs to cope with osmotic stress.

Flares In Time-Domain Surveys

NASA Astrophysics Data System (ADS)

Kowalski, Adam; Hawley, Suzanne; Davenport, James; Berlicki, Arkadiusz; Cauzzi, Gianna; Fletcher, Lyndsay; Heinzel, Petr; Notsu, Yuta; Loyd, Parke; Martinez Oliveros, Juan Carlos; Pugh, Chloe; Schmidt, Sarah Jane; Karmakar, Subhajeet; Pye, John; Flaccomio, Ettore

2016-07-01

Proceedings for the splinter session "Flares in Time-Domain Surveys" convened at Cool Stars 19 on June 07, 2016 in Uppsala, Sweden. Contains a two page summary of the splinter session, links to YouTube talks, and a PDF copy of the slides from the presenters.

Early-age Acute Leukemia: Revisiting Two Decades of the Brazilian Collaborative Study Group.

PubMed

Pombo-de-Oliveira, Maria S; Andrade, Francianne Gomes

2016-11-01

The understanding of leukemogenesis in early-age acute leukemia (EAL) has improved remarkably. Initiating somatic mutations detected in dried neonatal blood spots (DNBS) and in cord blood samples of affected children with leukemia have been proven to be acquired prenatally. However, to date, few epidemiological studies have been carried out exploring EAL that include infants and children 13-24 months of age at the diagnosis. Maternal exposure to transplacental DNA-damaging substances during pregnancy has been suggested to be a risk factor for EAL. Most cases of infants with acute lymphoblastic (i-ALL) or myeloid leukemia (i-AML) have KMT2A gene rearrangements (KMT2A-r), which disturb its essential role as an epigenetic regulator of hematopoiesis. Due to the short latency period for EAL and the fact that KMT2A-r resembles those found in secondary AML, exposure to topoisomerase II inhibitors has been associated with transplacental risk as proxi for causality. EAL studies have been conducted in Brazil for over two decades, combining observational epidemiology, leukemia biology, and clinical data. EAL was investigated considering (i) age strata (infants vs. 13-24 months-old); (ii) somatic mutations associated with i-ALL and i-AML; (iii) ethnic-geographic variations; (iv) contribution of maternal genotypes; and (v) time latency of exposures and mutations in DNBS. Interactions of acquired and constitutive gene mutations are challenging tools to test risk factor associations for EAL. In this review we summarize the EAL scenario (including B-cell precursor-ALL, T-ALL, and AML) results combining environmental and genetic susceptibility risk factors and we raise questions that should be considered for further action. Copyright © 2016 IMSS. Published by Elsevier Inc. All rights reserved.

WRNIP1 accumulates at laser light irradiated sites rapidly via its ubiquitin-binding zinc finger domain and independently from its ATPase domain

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

Nomura, Hironoshin; Yoshimura, Akari, E-mail: akari_yo@musashino-u.ac.jp; Edo, Takato

2012-01-27

Highlights: Black-Right-Pointing-Pointer WRNIP1 accumulates in laser light irradiated sites very rapidly via UBZ domain. Black-Right-Pointing-Pointer The ATPase domain of WRNIP1 is dispensable for its accumulation. Black-Right-Pointing-Pointer The accumulation of WRNIP1 seems not to be dependent on the interaction with WRN. -- Abstract: WRNIP1 (Werner helicase-interacting protein 1) was originally identified as a protein that interacts with the Werner syndrome responsible gene product. WRNIP1 contains a ubiquitin-binding zinc-finger (UBZ) domain in the N-terminal region and two leucine zipper motifs in the C-terminal region. In addition, it possesses an ATPase domain in the middle of the molecule and the lysine residues servingmore » as ubiquitin acceptors in the entire of the molecule. Here, we report that WRNIP1 accumulates in laser light irradiated sites very rapidly via its ubiquitin-binding zinc finger domain, which is known to bind polyubiquitin and to be involved in ubiquitination of WRNIP1 itself. The accumulation of WRNIP1 in laser light irradiated sites also required the C-terminal region containing two leucine zippers, which is reportedly involved in the oligomerization of WRNIP1. Mutated WRNIP1 with a deleted ATPase domain or with mutations in lysine residues, which serve as ubiquitin acceptors, accumulated in laser light irradiated sites, suggesting that the ATPase domain of WRNIP1 and ubiquitination of WRNIP1 are dispensable for the accumulation.« less

Elucidating nitric oxide synthase domain interactions by molecular dynamics.

PubMed

Hollingsworth, Scott A; Holden, Jeffrey K; Li, Huiying; Poulos, Thomas L

2016-02-01

Nitric oxide synthase (NOS) is a multidomain enzyme that catalyzes the production of nitric oxide (NO) by oxidizing L-Arg to NO and L-citrulline. NO production requires multiple interdomain electron transfer steps between the flavin mononucleotide (FMN) and heme domain. Specifically, NADPH-derived electrons are transferred to the heme-containing oxygenase domain via the flavin adenine dinucleotide (FAD) and FMN containing reductase domains. While crystal structures are available for both the reductase and oxygenase domains of NOS, to date there is no atomic level structural information on domain interactions required for the final FMN-to-heme electron transfer step. Here, we evaluate a model of this final electron transfer step for the heme-FMN-calmodulin NOS complex based on the recent biophysical studies using a 105-ns molecular dynamics trajectory. The resulting equilibrated complex structure is very stable and provides a detailed prediction of interdomain contacts required for stabilizing the NOS output state. The resulting equilibrated complex model agrees well with previous experimental work and provides a detailed working model of the final NOS electron transfer step required for NO biosynthesis. © 2015 The Protein Society.

CUB domain-containing protein 1 and the epidermal growth factor receptor cooperate to induce cell detachment.

PubMed

Law, Mary E; Ferreira, Renan B; Davis, Bradley J; Higgins, Paul J; Kim, Jae-Sung; Castellano, Ronald K; Chen, Sixue; Luesch, Hendrik; Law, Brian K

2016-08-05

While localized malignancies often respond to available therapies, most disseminated cancers are refractory. Novel approaches, therefore, are needed for the treatment of metastatic disease. CUB domain-containing protein1 (CDCP1) plays an important role in metastasis and drug resistance; the mechanism however, is poorly understood. Breast cancer cell lines were engineered to stably express EGFR, CDCP1 or phosphorylation site mutants of CDCP1. These cell lines were used for immunoblot analysis or affinity purification followed by immunoblot analysis to assess protein phosphorylation and/or protein complex formation with CDCP1. Kinase activity was evaluated using phosphorylation site-specific antibodies and immunoblot analysis in in vitro kinase assays. Protein band excision and mass spectrometry was utilized to further identify proteins complexed with CDCP1 or ΔCDCP1, which is a mimetic of the cleaved form of CDCP1. Cell detachment was assessed using cell counting. This paper reports that CDCP1 forms ternary protein complexes with Src and EGFR, facilitating Src activation and Src-dependent EGFR transactivation. Importantly, we have discovered that a class of compounds termed Disulfide bond Disrupting Agents (DDAs) blocks CDCP1/EGFR/Src ternary complex formation and downstream signaling. CDCP1 and EGFR cooperate to induce detachment of breast cancer cells from the substratum and to disrupt adherens junctions. Analysis of CDCP1-containing complexes using proteomics techniques reveals that CDCP1 associates with several proteins involved in cell adhesion, including adherens junction and desmosomal cadherins, and cytoskeletal elements. Together, these results suggest that CDCP1 may facilitate loss of adhesion by promoting activation of EGFR and Src at sites of cell-cell and cell-substratum contact.

Structural Basis for Endosomal Targeting by the Bro1 Domain

PubMed Central

Kim, Jaewon; Sitaraman, Sujatha; Hierro, Aitor; Beach, Bridgette M.; Odorizzi, Greg; Hurley, James H.

2010-01-01

Summary Proteins delivered to the lysosome or the yeast vacuole via late endosomes are sorted by the ESCRT complexes and by associated proteins, including Alix and its yeast homolog Bro1. Alix, Bro1, and several other late endosomal proteins share a conserved 160 residue Bro1 domain whose boundaries, structure, and function have not been characterized. The crystal structure of the Bro1 domain of Bro1 reveals a folded core of 367 residues. The extended Bro1 domain is necessary and sufficient for binding to the ESCRT-III subunit Snf7 and for the recruitment of Bro1 to late endosomes. The structure resembles a boomerang with its concave face filled in and contains a triple tetratricopeptide repeat domain as a substructure. Snf7 binds to a conserved hydrophobic patch on Bro1 that is required for protein complex formation and for the protein-sorting function of Bro1. These results define a conserved mechanism whereby Bro1 domain-containing proteins are targeted to endosomes by Snf7 and its orthologs. PMID:15935782

The macromolecular aromatic domain in suberized tissue: a changing paradigm

NASA Technical Reports Server (NTRS)

Bernards, M. A.; Lewis, N. G.

1998-01-01

As a structural feature of specialized cell walls, suberization remains an enigma, despite its obvious importance both during normal growth and development and as a stress response in plants. While it is clear that suberized tissues contain both polyaromatic and polyaliphatic domains, and that each of these has its own unique characteristics, whether there is a contiguous macromolecule that can be called suberin is an open question. From a structural perspective, the aromatic domain is unique and distinct from lignin, and is apparently comprised primarily of (poly)hydroxycinnamates, such as amides (e.g., feruloyltyramine). The aliphatic domain is also unique, being quite distinct from cutin in terms of both its chemical composition and cellular location. In the present paper, histochemical, structural and biochemical data, particularly, regarding the polyaromatic domain of suberized tissues, are critically reviewed. A revised description of the polyaromatic domain of suberized tissues, based on the consensus that is emerging from the current data, is presented and especially includes a spatially distinct (poly)hydroxycinnamoyl-containing macromolecule.

Possible role of the Nipah virus V protein in the regulation of the interferon beta induction by interacting with UBX domain-containing protein1.

PubMed

Uchida, Shotaro; Horie, Ryo; Sato, Hiroki; Kai, Chieko; Yoneda, Misako

2018-05-16

Nipah virus (NiV) is a highly pathogenic paramyxovirus that causes lethal encephalitis in humans. We previously reported that the V protein, one of the three accessory proteins encoded by the P gene, is one of the key determinants of the pathogenesis of NiV in a hamster infection model. Satterfield B.A. et al. have also revealed that V protein is required for the pathogenicity of henipavirus in a ferret infection model. However, the complete functions of NiV V have not been clarified. In this study, we identified UBX domain-containing protein 1 (UBXN1), a negative regulator of RIG-I-like receptor signaling, as a host protein that interacts with NiV V. NiV V interacted with the UBX domain of UBXN1 via its proximal zinc-finger motif in the C-terminal domain. NiV V increased the level of UBXN1 protein by suppressing its proteolysis. Furthermore, NiV V suppressed RIG-I and MDA5-dependent interferon signaling by stabilizing UBXN1 and increasing the interaction between MAVS and UBXN1 in addition to directly interrupting the activation of MDA5. Our results suggest a novel molecular mechanism by which the induction of interferon is potentially suppressed by NiV V protein via UBXN1.

Ribosomal small subunit domains radiate from a central core

NASA Astrophysics Data System (ADS)

Gulen, Burak; Petrov, Anton S.; Okafor, C. Denise; Vander Wood, Drew; O'Neill, Eric B.; Hud, Nicholas V.; Williams, Loren Dean

2016-02-01

The domain architecture of a large RNA can help explain and/or predict folding, function, biogenesis and evolution. We offer a formal and general definition of an RNA domain and use that definition to experimentally characterize the rRNA of the ribosomal small subunit. Here the rRNA comprising a domain is compact, with a self-contained system of molecular interactions. A given rRNA helix or stem-loop must be allocated uniquely to a single domain. Local changes such as mutations can give domain-wide effects. Helices within a domain have interdependent orientations, stabilities and interactions. With these criteria we identify a core domain (domain A) of small subunit rRNA. Domain A acts as a hub, linking the four peripheral domains and imposing orientational and positional restraints on the other domains. Experimental characterization of isolated domain A, and mutations and truncations of it, by methods including selective 2‧OH acylation analyzed by primer extension and circular dichroism spectroscopy are consistent with our architectural model. The results support the utility of the concept of an RNA domain. Domain A, which exhibits structural similarity to tRNA, appears to be an essential core of the small ribosomal subunit.

The SH2 domain interaction landscape.

PubMed

Tinti, Michele; Kiemer, Lars; Costa, Stefano; Miller, Martin L; Sacco, Francesca; Olsen, Jesper V; Carducci, Martina; Paoluzi, Serena; Langone, Francesca; Workman, Christopher T; Blom, Nikolaj; Machida, Kazuya; Thompson, Christopher M; Schutkowski, Mike; Brunak, Søren; Mann, Matthias; Mayer, Bruce J; Castagnoli, Luisa; Cesareni, Gianni

2013-04-25

Members of the SH2 domain family modulate signal transduction by binding to short peptides containing phosphorylated tyrosines. Each domain displays a distinct preference for the sequence context of the phosphorylated residue. We have developed a high-density peptide chip technology that allows for probing of the affinity of most SH2 domains for a large fraction of the entire complement of tyrosine phosphopeptides in the human proteome. Using this technique, we have experimentally identified thousands of putative SH2-peptide interactions for more than 70 different SH2 domains. By integrating this rich data set with orthogonal context-specific information, we have assembled an SH2-mediated probabilistic interaction network, which we make available as a community resource in the PepspotDB database. A predicted dynamic interaction between the SH2 domains of the tyrosine phosphatase SHP2 and the phosphorylated tyrosine in the extracellular signal-regulated kinase activation loop was validated by experiments in living cells. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Rsp5 WW domains interact directly with the carboxyl-terminal domain of RNA polymerase II.

PubMed

Chang, A; Cheang, S; Espanel, X; Sudol, M

2000-07-07

RSP5 is an essential gene in Saccharomyces cerevisiae and was recently shown to form a physical and functional complex with RNA polymerase II (RNA pol II). The amino-terminal half of Rsp5 consists of four domains: a C2 domain, which binds membrane phospholipids; and three WW domains, which are protein interaction modules that bind proline-rich ligands. The carboxyl-terminal half of Rsp5 contains a HECT (homologous to E6-AP carboxyl terminus) domain that catalytically ligates ubiquitin to proteins and functionally classifies Rsp5 as an E3 ubiquitin-protein ligase. The C2 and WW domains are presumed to act as membrane localization and substrate recognition modules, respectively. We report that the second (and possibly third) Rsp5 WW domain mediates binding to the carboxyl-terminal domain (CTD) of the RNA pol II large subunit. The CTD comprises a heptamer (YSPTSPS) repeated 26 times and a PXY core that is critical for interaction with a specific group of WW domains. An analysis of synthetic peptides revealed a minimal CTD sequence that is sufficient to bind to the second Rsp5 WW domain (Rsp5 WW2) in vitro and in yeast two-hybrid assays. Furthermore, we found that specific "imperfect" CTD repeats can form a complex with Rsp5 WW2. In addition, we have shown that phosphorylation of this minimal CTD sequence on serine, threonine and tyrosine residues acts as a negative regulator of the Rsp5 WW2-CTD interaction. In view of the recent data pertaining to phosphorylation-driven interactions between the RNA pol II CTD and the WW domain of Ess1/Pin1, we suggest that CTD dephosphorylation may be a prerequisite for targeted RNA pol II degradation.

NIPA-like domain containing 1 is a novel tumor-promoting factor in oral squamous cell carcinoma.

PubMed

Sasahira, Tomonori; Nishiguchi, Yukiko; Kurihara-Shimomura, Miyako; Nakashima, Chie; Kuniyasu, Hiroki; Kirita, Tadaaki

2018-05-01

In our previous global gene expression analysis, we identified NIPA-like domain containing 1 (NIPAL1), which encodes a magnesium transporter, as one of the most overexpressed genes in recurrent oral squamous cell carcinoma (OSCC). Although has been NIPAL1 linked with gout pathogenesis, little is known about its expression and function in human malignancies. In this study, we examined NIPAL1 expression in 192 cases of OSCC by immunohistochemistry and performed a functional analysis of human OSCC cells. NIPAL1 immunostaining was observed in 39 of 192 OSCC patients (20.3%). NIPAL1 expression correlated significantly with cancer cell intravsation (P = 0.0062), as well as with poorer disease-free survival in a Kaplan-Meier analysis (P < 0.0001). Moreover, a multivariate Cox proportional hazards model analysis revealed that NIPAL1 expression was an independent predictor of disease-free survival in OSCC (P < 0.0001). In a functional analysis, NIPAL1 regulated the growth and adhesion of OSCC tumor cells and endothelial cells. Our findings suggest that NIPAL1 might be a novel factor promoting OSCC tumorigenesis, as well as a useful molecular marker of OSCC.

TFIID TAF6-TAF9 Complex Formation Involves the HEAT Repeat-containing C-terminal Domain of TAF6 and Is Modulated by TAF5 Protein*

PubMed Central

Scheer, Elisabeth; Delbac, Frédéric; Tora, Laszlo; Moras, Dino; Romier, Christophe

2012-01-01

The general transcription factor TFIID recognizes specifically the core promoter of genes transcribed by eukaryotic RNA polymerase II, nucleating the assembly of the preinitiation complex at the transcription start site. However, the understanding in molecular terms of TFIID assembly and function remains poorly understood. Histone fold motifs have been shown to be extremely important for the heterodimerization of many TFIID subunits. However, these subunits display several evolutionary conserved noncanonical features when compared with histones, including additional regions whose role is unknown. Here we show that the conserved additional C-terminal region of TFIID subunit TAF6 can be divided into two domains: a small middle domain (TAF6M) and a large C-terminal domain (TAF6C). Our crystal structure of the TAF6C domain from Antonospora locustae at 1.9 Å resolution reveals the presence of five conserved HEAT repeats. Based on these data, we designed several mutants that were introduced into full-length human TAF6. Surprisingly, the mutants affect the interaction between TAF6 and TAF9, suggesting that the formation of the complex between these two TFIID subunits do not only depend on their histone fold motifs. In addition, the same mutants affect even more strongly the interaction between TAF6 and TAF9 in the context of a TAF5-TAF6-TAF9 complex. Expression of these mutants in HeLa cells reveals that most of them are unstable, suggesting their poor incorporation within endogenous TFIID. Taken together, our results suggest that the conserved additional domains in histone fold-containing subunits of TFIID and of co-activator SAGA are important for the assembly of these complexes. PMID:22696218

ATP Binding to p97/VCP D1 Domain Regulates Selective Recruitment of Adaptors to Its Proximal N-Domain

PubMed Central

Chia, Wei Sheng; Chia, Diana Xueqi; Rao, Feng; Bar Nun, Shoshana; Geifman Shochat, Susana

2012-01-01

p97/Valosin-containing protein (VCP) is a member of the AAA-ATPase family involved in many cellular processes including cell division, intracellular trafficking and extraction of misfolded proteins in endoplasmic reticulum-associated degradation (ERAD). It is a homohexamer with each subunit containing two tandem D1 and D2 ATPase domains and N- and C-terminal regions that function as adaptor protein binding domains. p97/VCP is directed to its many different functional pathways by associating with various adaptor proteins. The regulation of the recruitment of the adaptor proteins remains unclear. Two adaptor proteins, Ufd1/Npl4 and p47, which bind exclusively to the p97/VCP N-domain and direct p97/VCP to either ERAD-related processes or homotypic fusion of Golgi fragments, were studied here. Surface plasmon resonance biosensor-based assays allowed the study of binding kinetics in real time. In competition experiments, it was observed that in the presence of ATP, Ufd1/Npl4 was able to compete more effectively with p47 for binding to p97/VCP. By using non-hydrolysable ATP analogues and the hexameric truncated p97/N-D1 fragment, it was shown that binding rather than hydrolysis of ATP to the proximal D1 domain strengthened the Ufd1/Npl4 association with the N-domain, thus regulating the recruitment of either Ufd1/Npl4 or p47. This novel role of ATP and an assigned function to the D1 AAA-ATPase domain link the multiple functions of p97/VCP to the metabolic status of the cell. PMID:23226521

ATP binding to p97/VCP D1 domain regulates selective recruitment of adaptors to its proximal N-domain.

PubMed

Chia, Wei Sheng; Chia, Diana Xueqi; Rao, Feng; Bar Nun, Shoshana; Geifman Shochat, Susana

2012-01-01

p97/Valosin-containing protein (VCP) is a member of the AAA-ATPase family involved in many cellular processes including cell division, intracellular trafficking and extraction of misfolded proteins in endoplasmic reticulum-associated degradation (ERAD). It is a homohexamer with each subunit containing two tandem D1 and D2 ATPase domains and N- and C-terminal regions that function as adaptor protein binding domains. p97/VCP is directed to its many different functional pathways by associating with various adaptor proteins. The regulation of the recruitment of the adaptor proteins remains unclear. Two adaptor proteins, Ufd1/Npl4 and p47, which bind exclusively to the p97/VCP N-domain and direct p97/VCP to either ERAD-related processes or homotypic fusion of Golgi fragments, were studied here. Surface plasmon resonance biosensor-based assays allowed the study of binding kinetics in real time. In competition experiments, it was observed that in the presence of ATP, Ufd1/Npl4 was able to compete more effectively with p47 for binding to p97/VCP. By using non-hydrolysable ATP analogues and the hexameric truncated p97/N-D1 fragment, it was shown that binding rather than hydrolysis of ATP to the proximal D1 domain strengthened the Ufd1/Npl4 association with the N-domain, thus regulating the recruitment of either Ufd1/Npl4 or p47. This novel role of ATP and an assigned function to the D1 AAA-ATPase domain link the multiple functions of p97/VCP to the metabolic status of the cell.

Domain Nucleation Rates and Interfacial Line Tensions in Supported Bilayers of Ternary Mixtures Containing Galactosylceramide

PubMed Central

Blanchette, Craig D.; Lin, Wan-Chen; Orme, Christine A.; Ratto, Timothy V.; Longo, Marjorie L.

2008-01-01

Domains within the plane of the plasma membrane, referred to as membrane rafts, have been a topic of considerable interest in the field of membrane biophysics. Although model membrane systems have been used extensively to study lipid phase behavior as it relates to the existence of rafts, very little work has focused on either the initial stage of lipid domain nucleation, or the relevant physical parameters such as temperature and interfacial line tension which control nucleation. In this work, we utilize a method in which the kinetic process of lipid domain nucleation is imaged by atomic force microscopy and modeled using classical theory of nucleation to map interfacial line tension in ternary lipid mixtures. These mixtures consist of a fluid phase lipid component (1,2-dilauroyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, or 1,2-dioleoyl-sn-glycero-3-phosphocholine), a solid phase component (galactosylceramide), and cholesterol. Interfacial line tension measurements of galactosylceramide-rich domains track with our previously measured area/perimeter ratios and height mismatches measured here. Line tension also follows known trends in cholesterol interactions and partitioning, as we observed previously with area/perimeter ratios. Our line tension measurements are discussed in combination with recent line tension measurements to address line tension regulation by cholesterol and the dynamic nature of membrane rafts. PMID:18065459

Impact of Virtual Work Environment on Traditional Team Domains.

ERIC Educational Resources Information Center

Geroy, Gary D.; Olson, Joel; Hartman, Jackie

2002-01-01

Examines a virtual work team to determine the domains of the team and the effect the virtual work environment had on the domains. Discusses results of a literature review and a phenomenological heuristic case study, including the effects of post-modern philosophy and postindustrial society on changes in the marketplace. (Contains 79 references.)…

Method for identification of rigid domains and hinge residues in proteins based on exhaustive enumeration.

PubMed

Sim, Jaehyun; Sim, Jun; Park, Eunsung; Lee, Julian

2015-06-01

Many proteins undergo large-scale motions where relatively rigid domains move against each other. The identification of rigid domains, as well as the hinge residues important for their relative movements, is important for various applications including flexible docking simulations. In this work, we develop a method for protein rigid domain identification based on an exhaustive enumeration of maximal rigid domains, the rigid domains not fully contained within other domains. The computation is performed by mapping the problem to that of finding maximal cliques in a graph. A minimal set of rigid domains are then selected, which cover most of the protein with minimal overlap. In contrast to the results of existing methods that partition a protein into non-overlapping domains using approximate algorithms, the rigid domains obtained from exact enumeration naturally contain overlapping regions, which correspond to the hinges of the inter-domain bending motion. The performance of the algorithm is demonstrated on several proteins. © 2015 Wiley Periodicals, Inc.

LY6/PLAUR domain containing 3 has a role in the maintenance of colorectal cancer stem-like cells

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

Wang, Liming; Department of Surgery, Sapporo Higashi Tokusyukai Hospital, North-33 East-14, Higashi-Ku, Sapporo 065-0033; Hirohashi, Yoshihiko

Colorectal carcinoma (CRC) is one of the most frequently diagnosed cancers and the leading cause of cancer-related death for both men and women. Recent studies have revealed that a small sub-population of cancer cells, termed cancer stem-like cells (CSCs)/cancer-initiating cells (CICs), are endowed with tumor-initiating ability, self-renewal ability and differentiation ability. CSCs/CICs are resistant to current therapies including chemotherapy and radiotherapy. Thus, CSCs/CICs are responsible for recurrence and metastasis, and eradication of CSCs/CICs is essential to cure cancer. In this study, we isolated CR-CSCs/CICs as sphere-cultured cells and found that a product derived from LY6/PLAUR domain containing 3 (LYPD3) ismore » preferentially expressed in CSCs/CICs. Gene overexpression and gene knockdown experiments revealed that LYPD3 has a role in the maintenance of CR-CSCs/CICs. The findings provide a novel molecular insight into CR-CSCs/CICs.« less

Stability domain of alumina thermally grown on Fe-Cr-Al-based model alloys and modified surface layers exposed to oxygen-containing molten Pb

NASA Astrophysics Data System (ADS)

Jianu, A.; Fetzer, R.; Weisenburger, A.; Doyle, S.; Bruns, M.; Heinzel, A.; Hosemann, P.; Mueller, G.

2016-03-01

The paper gives experimental results concerning the morphology, composition, structure and thickness of the oxide scales grown on Fe-Cr-Al-based bulk alloys during exposure to oxygen-containing molten lead. The results are discussed and compared with former results obtained on Al-containing surface layers, modified by melting with intense pulsed electron beam and exposed to similar conditions. The present and previous results provide the alumina stability domain and also the criterion of the Al/Cr ratio for the formation of a highly protective alumina layer on the surface of Fe-Cr-Al-based alloys and on modified surface layers exposed to molten lead with 10-6 wt.% oxygen at 400-600 °C. The protective oxide scales, grown on alumina-forming Fe-Cr-Al alloys under the given experimental conditions, were transient aluminas, namely, kappa-Al2O3 and theta-Al2O3.

Chimeric antigen receptor containing ICOS signaling domain mediates specific and efficient antitumor effect of T cells against EGFRvIII expressing glioma

PubMed Central

2013-01-01

Background Adoptive transfer of chimeric antigen receptor (CAR)-modified T cells appears to be a promising immunotherapeutic strategy. CAR combines the specificity of antibody and cytotoxicity of cytotoxic T lymphocytes, enhancing T cells’ ability to specifically target antigens and to effectively kill cancer cells. Recent efforts have been made to integrate the costimulatory signals in the CAR to improve the antitumor efficacy. Epidermal growth factor receptor variant III (EGFRvIII) is an attractive therapeutic target as it frequently expresses in glioma and many other types of cancers. Our current study aimed to investigate the specific and efficient antitumor effect of T cells modified with CAR containing inducible costimulator (ICOS) signaling domain. Methods A second generation of EGFRvIII/CAR was generated and it contained the EGFRvIII single chain variable fragment, ICOS signaling domain and CD3ζ chain. Lentiviral EGFRvIII/CAR was prepared and human CD3+ T cells were infected by lentivirus encoding EGFRvIII/CAR. The expression of EGFRvIII/CAR on CD3+ T cells was confirmed by flow cytometry and Western blot. The functions of EGFRvIII/CAR+ T cells were evaluated using in vitro and in vivo methods including cytotoxicity assay, cytokine release assay and xenograft tumor mouse model. Results Chimeric EGFRvIIIscFv-ICOS-CD3ζ (EGFRvIII/CAR) was constructed and lentiviral EGFRvIII/CAR were made to titer of 106 TU/ml. The transduction efficiency of lentiviral EGFRvIII/CAR on T cells reached around 70% and expression of EGFRvIII/CAR protein was verified by immunoblotting as a band of about 57 kDa. Four hour 51Cr release assays demonstrated specific and efficient cytotoxicity of EGFRvIII/CAR+ T cells against EGFRvIII expressing U87 cells. A robust increase in the IFN-γ secretion was detected in the co-culture supernatant of the EGFRvIII/CAR+ T cells and the EGFRvIII expressing U87 cells. Intravenous and intratumor injection of EGFRvIII/CAR+ T cells inhibited the in

Chimeric antigen receptor containing ICOS signaling domain mediates specific and efficient antitumor effect of T cells against EGFRvIII expressing glioma.

PubMed

Shen, Chan-Juan; Yang, Yu-Xiu; Han, Ethan Q; Cao, Na; Wang, Yun-Fei; Wang, Yi; Zhao, Ying-Ying; Zhao, Li-Ming; Cui, Jian; Gupta, Puja; Wong, Albert J; Han, Shuang-Yin

2013-05-09

Adoptive transfer of chimeric antigen receptor (CAR)-modified T cells appears to be a promising immunotherapeutic strategy. CAR combines the specificity of antibody and cytotoxicity of cytotoxic T lymphocytes, enhancing T cells' ability to specifically target antigens and to effectively kill cancer cells. Recent efforts have been made to integrate the costimulatory signals in the CAR to improve the antitumor efficacy. Epidermal growth factor receptor variant III (EGFRvIII) is an attractive therapeutic target as it frequently expresses in glioma and many other types of cancers. Our current study aimed to investigate the specific and efficient antitumor effect of T cells modified with CAR containing inducible costimulator (ICOS) signaling domain. A second generation of EGFRvIII/CAR was generated and it contained the EGFRvIII single chain variable fragment, ICOS signaling domain and CD3ζ chain. Lentiviral EGFRvIII/CAR was prepared and human CD3+ T cells were infected by lentivirus encoding EGFRvIII/CAR. The expression of EGFRvIII/CAR on CD3+ T cells was confirmed by flow cytometry and Western blot. The functions of EGFRvIII/CAR+ T cells were evaluated using in vitro and in vivo methods including cytotoxicity assay, cytokine release assay and xenograft tumor mouse model. Chimeric EGFRvIIIscFv-ICOS-CD3ζ (EGFRvIII/CAR) was constructed and lentiviral EGFRvIII/CAR were made to titer of 106 TU/ml. The transduction efficiency of lentiviral EGFRvIII/CAR on T cells reached around 70% and expression of EGFRvIII/CAR protein was verified by immunoblotting as a band of about 57 kDa. Four hour 51Cr release assays demonstrated specific and efficient cytotoxicity of EGFRvIII/CAR+ T cells against EGFRvIII expressing U87 cells. A robust increase in the IFN-γ secretion was detected in the co-culture supernatant of the EGFRvIII/CAR+ T cells and the EGFRvIII expressing U87 cells. Intravenous and intratumor injection of EGFRvIII/CAR+ T cells inhibited the in vivo growth of the EGFRv

Remote-sensing image encryption in hybrid domains

NASA Astrophysics Data System (ADS)

Zhang, Xiaoqiang; Zhu, Guiliang; Ma, Shilong

2012-04-01

Remote-sensing technology plays an important role in military and industrial fields. Remote-sensing image is the main means of acquiring information from satellites, which always contain some confidential information. To securely transmit and store remote-sensing images, we propose a new image encryption algorithm in hybrid domains. This algorithm makes full use of the advantages of image encryption in both spatial domain and transform domain. First, the low-pass subband coefficients of image DWT (discrete wavelet transform) decomposition are sorted by a PWLCM system in transform domain. Second, the image after IDWT (inverse discrete wavelet transform) reconstruction is diffused with 2D (two-dimensional) Logistic map and XOR operation in spatial domain. The experiment results and algorithm analyses show that the new algorithm possesses a large key space and can resist brute-force, statistical and differential attacks. Meanwhile, the proposed algorithm has the desirable encryption efficiency to satisfy requirements in practice.

Insights into substrate specificity of NlpC/P60 cell wall hydrolases containing bacterial SH3 domains

DOE PAGES

Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.; ...

2015-09-15

Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. In addition, these enzymes all have γ-d-Glu-A 2pm (A 2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structuremore » consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.Peptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes

Insights into substrate specificity of NlpC/P60 cell wall hydrolases containing bacterial SH3 domains

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

Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.

Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. In addition, these enzymes all have γ-d-Glu-A 2pm (A 2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structuremore » consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.Peptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes

Insights into Substrate Specificity of NlpC/P60 Cell Wall Hydrolases Containing Bacterial SH3 Domains

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

Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.

ABSTRACT Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have γ-d-Glu-A 2pm (A 2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structure consistingmore » of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation. IMPORTANCEPeptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling

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

Over-Expression of Rififylin, a New RING Finger and FYVE-like Domain-containing Protein, Inhibits Recycling from the Endocytic Recycling Compartment

PubMed Central

Coumailleau, Franck; Das, Vincent; Alcover, Andres; Raposo, Graça; Vandormael-Pournin, Sandrine; Le Bras, Stéphanie; Baldacci, Patricia; Dautry-Varsat, Alice; Babinet, Charles; Cohen-Tannoudji, Michel

2004-01-01

Endocytosed membrane components are recycled to the cell surface either directly from early/sorting endosomes or after going through the endocytic recycling compartment (ERC). Studying recycling mechanisms is difficult, in part due to the fact that specific tools to inhibit this process are scarce. In this study, we have characterized a novel widely expressed protein, named Rififylin (Rffl) for RING Finger and FYVE-like domain-containing protein, that, when overexpressed in HeLa cells, induced the condensation of transferrin receptor-, Rab5-, and Rab11-positive recycling tubulovesicular membranes in the perinuclear region. Internalized transferrin was able to access these condensed endosomes but its exit from this compartment was delayed. Using deletion mutants, we show that the carboxy-terminal RING finger of Rffl is dispensable for its action. In contrast, the amino-terminal domain of Rffl, which shows similarities with the phosphatidylinositol-3-phosphate–binding FYVE finger, is critical for the recruitment of Rffl to recycling endocytic membranes and for the inhibition of recycling, albeit in a manner that is independent of PtdIns(3)-kinase activity. Rffl overexpression represents a novel means to inhibit recycling that will help to understand the mechanisms involved in recycling from the ERC to the plasma membrane. PMID:15229288

Over-expression of Rififylin, a new RING finger and FYVE-like domain-containing protein, inhibits recycling from the endocytic recycling compartment.

PubMed

Coumailleau, Franck; Das, Vincent; Alcover, Andres; Raposo, Graça; Vandormael-Pournin, Sandrine; Le Bras, Stéphanie; Baldacci, Patricia; Dautry-Varsat, Alice; Babinet, Charles; Cohen-Tannoudji, Michel

2004-10-01

Endocytosed membrane components are recycled to the cell surface either directly from early/sorting endosomes or after going through the endocytic recycling compartment (ERC). Studying recycling mechanisms is difficult, in part due to the fact that specific tools to inhibit this process are scarce. In this study, we have characterized a novel widely expressed protein, named Rififylin (Rffl) for RING Finger and FYVE-like domain-containing protein, that, when overexpressed in HeLa cells, induced the condensation of transferrin receptor-, Rab5-, and Rab11-positive recycling tubulovesicular membranes in the perinuclear region. Internalized transferrin was able to access these condensed endosomes but its exit from this compartment was delayed. Using deletion mutants, we show that the carboxy-terminal RING finger of Rffl is dispensable for its action. In contrast, the amino-terminal domain of Rffl, which shows similarities with the phosphatidylinositol-3-phosphate-binding FYVE finger, is critical for the recruitment of Rffl to recycling endocytic membranes and for the inhibition of recycling, albeit in a manner that is independent of PtdIns(3)-kinase activity. Rffl overexpression represents a novel means to inhibit recycling that will help to understand the mechanisms involved in recycling from the ERC to the plasma membrane.

Fas Apoptosis Inhibitory Molecule (FAIM) Contains a Novel Beta Sandwich in Contact with a Partially Ordered Domain

PubMed Central

Hemond, Michael; Rothstein, Thomas L.; Wagner, Gerhard

2009-01-01

Summary Fas apoptosis inhibitory molecule (FAIM) is a soluble cytosolic protein inhibitor of programmed cell death and is found in organisms throughout the animal kingdom. A short isoform (FAIM-S) is expressed in all tissue types, while an alternatively spliced long isoform (FAIM-L) is specifically expressed in the brain. Here FAIM-S is shown to consist of two independently folding domains in contact with one another. The NMR solution structure of the C-terminal domain of murine FAIM is solved in isolation and revealed to be a novel protein fold, a noninterleaved seven-stranded beta sandwich. The structure and sequence reveal several residues that are likely to be involved in functionally significant interactions with the N-terminal domain or other binding partners. Chemical shift perturbation is used to elucidate contacts made between the N- and C-terminal domains. PMID:19168072

A thermodynamic definition of protein domains.

PubMed

Porter, Lauren L; Rose, George D

2012-06-12

Protein domains are conspicuous structural units in globular proteins, and their identification has been a topic of intense biochemical interest dating back to the earliest crystal structures. Numerous disparate domain identification algorithms have been proposed, all involving some combination of visual intuition and/or structure-based decomposition. Instead, we present a rigorous, thermodynamically-based approach that redefines domains as cooperative chain segments. In greater detail, most small proteins fold with high cooperativity, meaning that the equilibrium population is dominated by completely folded and completely unfolded molecules, with a negligible subpopulation of partially folded intermediates. Here, we redefine structural domains in thermodynamic terms as cooperative folding units, based on m-values, which measure the cooperativity of a protein or its substructures. In our analysis, a domain is equated to a contiguous segment of the folded protein whose m-value is largely unaffected when that segment is excised from its parent structure. Defined in this way, a domain is a self-contained cooperative unit; i.e., its cooperativity depends primarily upon intrasegment interactions, not intersegment interactions. Implementing this concept computationally, the domains in a large representative set of proteins were identified; all exhibit consistency with experimental findings. Specifically, our domain divisions correspond to the experimentally determined equilibrium folding intermediates in a set of nine proteins. The approach was also proofed against a representative set of 71 additional proteins, again with confirmatory results. Our reframed interpretation of a protein domain transforms an indeterminate structural phenomenon into a quantifiable molecular property grounded in solution thermodynamics.

Cooperative interactions between paired domain and homeodomain.

PubMed

Jun, S; Desplan, C

1996-09-01

The Pax proteins are a family of transcriptional regulators involved in many developmental processes in all higher eukaryotes. They are characterized by the presence of a paired domain (PD), a bipartite DNA binding domain composed of two helix-turn-helix (HTH) motifs,the PAI and RED domains. The PD is also often associated with a homeodomain (HD) which is itself able to form homo- and hetero-dimers on DNA. Many of these proteins therefore contain three HTH motifs each able to recognize DNA. However, all PDs recognize highly related DNA sequences, and most HDs also recognize almost identical sites. We show here that different Pax proteins use multiple combinations of their HTHs to recognize several types of target sites. For instance, the Drosophila Paired protein can bind, in vitro, exclusively through its PAI domain, or through a dimer of its HD, or through cooperative interaction between PAI domain and HD. However, prd function in vivo requires the synergistic action of both the PAI domain and the HD. Pax proteins with only a PD appear to require both PAI and RED domains, while a Pax-6 isoform and a new Pax protein, Lune, may rely on the RED domain and HD. We propose a model by which Pax proteins recognize different target genes in vivo through various combinations of their DNA binding domains, thus expanding their recognition repertoire.

A Cysteine-Rich CCG Domain Contains a Novel [4Fe-4S] Cluster Binding Motif As Deduced From Studies With Subunit B of Heterodisulfide Reductase From Methanothermobacter Marburgensis

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

Hamann, N.; Mander, G.J.; Shokes, J.E.

Heterodisulfide reductase (HDR) of methanogenic archaea with its active-site [4Fe-4S] cluster catalyzes the reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic coenzyme M (CoM-SH) and coenzyme B (CoB-SH). CoM-HDR, a mechanistic-based paramagnetic intermediate generated upon half-reaction of the oxidized enzyme with CoM-SH, is a novel type of [4Fe-4S]{sup 3+} cluster with CoM-SH as a ligand. Subunit HdrB of the Methanothermobacter marburgensis HdrABC holoenzyme contains two cysteine-rich sequence motifs (CX{sub 31-39}CCX{sub 35-36}CXXC), designated as CCG domain in the Pfam database and conserved in many proteins. Here we present experimental evidence that the C-terminal CCG domain of HdrB binds this unusualmore » [4Fe-4S] cluster. HdrB was produced in Escherichia coli, and an iron-sulfur cluster was subsequently inserted by in vitro reconstitution. In the oxidized state the cluster without the substrate exhibited a rhombic EPR signal (g{sub zyx} = 2.015, 1.995, and 1.950) reminiscent of the CoM-HDR signal. {sup 57}Fe ENDOR spectroscopy revealed that this paramagnetic species is a [4Fe-4S] cluster with {sup 57}Fe hyperfine couplings very similar to that of CoM-HDR. CoM-{sup 33}SH resulted in a broadening of the EPR signal, and upon addition of CoM-SH the midpoint potential of the cluster was shifted to values observed for CoM-HDR, both indicating binding of CoM-SH to the cluster. Site-directed mutagenesis of all 12 cysteine residues in HdrB identified four cysteines of the C-terminal CCG domain as cluster ligands. Combined with the previous detection of CoM-HDR-like EPR signals in other CCG domain-containing proteins our data indicate a general role of the C-terminal CCG domain in coordination of this novel [4Fe-4S] cluster. In addition, Zn K-edge X-ray absorption spectroscopy identified an isolated Zn site with an S{sub 3}(O/N){sub 1} geometry in HdrB and the HDR holoenzyme. The N-terminal CCG domain is suggested to provide ligands to

Mechanical Properties of Nanoscopic Lipid Domains

DOE PAGES

Nickels, Jonathan D.; Cheng, Xiaolin; Mostofian, Barmak; ...

2015-09-28

We found that the lipid raft hypothesis presents insight into how the cell membrane organizes proteins and lipids to accomplish its many vital functions. Yet basic questions remain about the physical mechanisms that lead to the formation, stability, and size of lipid rafts. Thus, much interest has been generated in the study of systems that contain similar lateral heterogeneities, or domains. In the current work we present an experimental approach that is capable of isolating the bending moduli of lipid domains. This is accomplished using neutron scattering and its unique sensitivity to the isotopes of hydrogen. Combining contrast matching approachesmore » with inelastic neutron scattering, we isolate the bending modulus of ~13 nm diameter domains residing in 60 nm unilamellar vesicles, whose lipid composition mimics the mammalian plasma membrane outer leaflet. Importantly, the bending modulus of the nanoscopic domains differs from the modulus of the continuous phase surrounding them. Moreover, from additional structural measurements and all-atom simulations, we also determine that nanoscopic domains are in-register across the bilayer leaflets. Taken together, these results inform a number of theoretical models of domain/raft formation and highlight the fact that mismatches in bending modulus must be accounted for when explaining the emergence of lateral heterogeneities in lipid systems and biological membranes.« less

Structure-function analysis of the auxilin J-domain reveals an extended Hsc70 interaction interface.

PubMed

Jiang, Jianwen; Taylor, Alexander B; Prasad, Kondury; Ishikawa-Brush, Yumiko; Hart, P John; Lafer, Eileen M; Sousa, Rui

2003-05-20

J-domains are widespread protein interaction modules involved in recruiting and stimulating the activity of Hsp70 family chaperones. We have determined the crystal structure of the J-domain of auxilin, a protein which is involved in uncoating clathrin-coated vesicles. Comparison to the known structures of J-domains from four other proteins reveals that the auxilin J-domain is the most divergent of all J-domain structures described to date. In addition to the canonical J-domain features described previously, the auxilin J-domain contains an extra N-terminal helix and a long loop inserted between helices I and II. The latter loop extends the positively charged surface which forms the Hsc70 binding site, and is shown by directed mutagenesis and surface plasmon resonance to contain side chains important for binding to Hsc70.

77 FR 63355 - Proposed Revision to Emergency Action Level Development Guidance Document

Federal Register 2010, 2011, 2012, 2013, 2014

2012-10-16

... action level (EAL) scheme. The NRC is publishing this proposed revision to inform the public and solicit... EAL scheme using site-specific information. Dated at Rockville, Maryland, this 10th day of October...

Generalization of the Schwarz-Christoffel mapping to multiply connected polygonal domains.

PubMed

Vasconcelos, Giovani L

2014-06-08

A generalization of the Schwarz-Christoffel mapping to multiply connected polygonal domains is obtained by making a combined use of two preimage domains, namely, a rectilinear slit domain and a bounded circular domain. The conformal mapping from the circular domain to the polygonal region is written as an indefinite integral whose integrand consists of a product of powers of the Schottky-Klein prime functions, which is the same irrespective of the preimage slit domain, and a prefactor function that depends on the choice of the rectilinear slit domain. A detailed derivation of the mapping formula is given for the case where the preimage slit domain is the upper half-plane with radial slits. Representation formulae for other canonical slit domains are also obtained but they are more cumbersome in that the prefactor function contains arbitrary parameters in the interior of the circular domain.

Characterization of the microtubule binding domain of microtubule actin crosslinking factor (MACF): identification of a novel group of microtubule associated proteins.

PubMed

Sun, D; Leung, C L; Liem, R K

2001-01-01

MACF (microtubule actin cross-linking factor) is a large, 608-kDa protein that can associate with both actin microfilaments and microtubules (MTs). Structurally, MACF can be divided into 3 domains: an N-terminal domain that contains both a calponin type actin-binding domain and a plakin domain; a rod domain that is composed of 23 dystrophin-like spectrin repeats; and a C-terminal domain that includes two EF-hand calcium-binding motifs, as well as a region that is homologous to two related proteins, GAR22 and Gas2. We have previously demonstrated that the C-terminal domain of MACF binds to MTs, although no homology was observed between this domain and other known microtubule-binding proteins. In this report, we describe the characterization of this microtubule-binding domain of MACF by transient transfection studies and in vitro binding assays. We found that the C-terminus of MACF contains at least two microtubule-binding regions, a GAR domain and a domain containing glycine-serine-arginine (GSR) repeats. In transfected cells, the GAR domain bound to and partially stabilized MTs to depolymerization by nocodazole. The GSR-containing domain caused MTs to form bundles that are still sensitive to nocodazole-induced depolymerization. When present together, these two domains acted in concert to bundle MTs and render them stable to nocodazole treatment. Recently, a study has shown that the N-terminal half of the plakin domain (called the M1 domain) of MACF also binds MTs. We therefore examined the microtubule binding ability of the M1 domain in the context of the entire plakin domain with and without the remaining N-terminal regions of two different MACF isoforms. Interestingly, in the presence of the surrounding sequences, the M1 domain did not bind MTs. In addition to MACF, cDNA sequences encoding the GAR and GSR-containing domains are also found in the partial human EST clone KIAA0728, which has high sequence homology to the 3' end of the MACF cDNA; hence, we refer to

Improved toughness of silicon carbide

NASA Technical Reports Server (NTRS)

Palm, J. A.

1976-01-01

Impact energy absorbing layers (EALs) comprised of partially densified silicon carbide were formed in situ on fully sinterable silicon carbide substrates. After final sintering, duplex silicon carbide structures resulted which were comprised of a fully sintered, high density silicon carbide substrate or core, overlayed with an EAL of partially sintered silicon carbide integrally bonded to its core member. Thermal cycling tests proved such structures to be moderately resistant to oxidation and highly resistant to thermal shock stresses. The strength of the developed structures in some cases exceeded but essentially it remained the same as the fully sintered silicon carbide without the EAL. Ballistic impact tests indicated that substantial improvements in the toughness of sintered silicon carbide were achieved by the use of the partially densified silicon carbide EALs.

Transcriptional machinery of TNF-α-inducible YTH domain containing 2 (YTHDC2) gene.

PubMed

Tanabe, Atsushi; Konno, Junpei; Tanikawa, Kenya; Sahara, Hiroeki

2014-02-01

We previously demonstrated that a cellular factor, cyclosporin A (CsA) associated helicase-like protein (CAHL) that is identical to YTH domain containing 2 (YTHDC2), forms trimer complex with cyclophilin B and NS5B of hepatitis C virus (HCV) and facilitates HCV genome replication. Gene expression of YTHDC2 was shown in tumor cell lines and tumor necrosis factor (TNF)-α-treated hepatocytes, but not in untreated. However, the function of YTHDC2 in the tumor cells and the mechanism by which the YTHDC2 gene is transcribed in these cells is largely unknown. We first evaluated that the role of YTHDC2 in the proliferation of hepatocellular carcinoma (HCC) cell line Huh7 using RNA interference and found that YTHDC2-downregulated Huh7 were significantly decreased cell growth as compared to control. We next demonstrated that the cAMP response element (CRE) site in the promoter region of the YTHDC2 gene is critical for YTHDC2 transcription. To further investigate the transcription factors bound to the CRE site, we performed chromatin immunoprecipitation assays. Our findings demonstrate that c-Jun and ATF-2 bind to the CRE site in Huh7, and that TNF-α induces the biological activity of these transcription factors in hepatocytes as well as Huh7. Moreover, treatment with the HDAC inhibitor, trichostatin A (TSA), reduces YTHDC2 expression in Huh7 and in TNF-α-stimulated hepatocytes. Collectively, these data show that YTHDC2 plays an important role in tumor cells growth and activation/recruitment of c-Jun and ATF-2 to the YTHDC2 promoter is necessary for the transcription of YTHDC2, and that HDAC activity is required for the efficient expression of YTHDC2 in both of hepatocyte and HCC cells. Copyright © 2013 Elsevier B.V. All rights reserved.

Hybrid Sterility in Rice (Oryza sativa L.) Involves the Tetratricopeptide Repeat Domain Containing Protein.

PubMed

Yu, Yang; Zhao, Zhigang; Shi, Yanrong; Tian, Hua; Liu, Linglong; Bian, Xiaofeng; Xu, Yang; Zheng, Xiaoming; Gan, Lu; Shen, Yumin; Wang, Chaolong; Yu, Xiaowen; Wang, Chunming; Zhang, Xin; Guo, Xiuping; Wang, Jiulin; Ikehashi, Hiroshi; Jiang, Ling; Wan, Jianmin

2016-07-01

Intersubspecific hybrid sterility is a common form of reproductive isolation in rice (Oryza sativa L.), which significantly hampers the utilization of heterosis between indica and japonica varieties. Here, we elucidated the mechanism of S7, which specially causes Aus-japonica/indica hybrid female sterility, through cytological and genetic analysis, map-based cloning, and transformation experiments. Abnormal positioning of polar nuclei and smaller embryo sac were observed in F1 compared with male and female parents. Female gametes carrying S7(cp) and S7(i) were aborted in S7(ai)/S7(cp) and S7(ai)/S7(i), respectively, whereas they were normal in both N22 and Dular possessing a neutral allele, S7(n) S7 was fine mapped to a 139-kb region in the centromere region on chromosome 7, where the recombination was remarkably suppressed due to aggregation of retrotransposons. Among 16 putative open reading frames (ORFs) localized in the mapping region, ORF3 encoding a tetratricopeptide repeat domain containing protein was highly expressed in the pistil. Transformation experiments demonstrated that ORF3 is the candidate gene: downregulated expression of ORF3 restored spikelet fertility and eliminated absolutely preferential transmission of S7(ai) in heterozygote S7(ai)/S7(cp); sterility occurred in the transformants Cpslo17-S7(ai) Our results may provide implications for overcoming hybrid embryo sac sterility in intersubspecific hybrid rice and utilization of hybrid heterosis for cultivated rice improvement. Copyright © 2016 by the Genetics Society of America.

δ-COP contains a helix C-terminal to its longin domain key to COPI dynamics and function

PubMed Central

Arakel, Eric C.; Richter, Kora P.; Clancy, Anne; Schwappach, Blanche

2016-01-01

Membrane recruitment of coatomer and formation of coat protein I (COPI)-coated vesicles is crucial to homeostasis in the early secretory pathway. The conformational dynamics of COPI during cargo capture and vesicle formation is incompletely understood. By scanning the length of δ-COP via functional complementation in yeast, we dissect the domains of the δ-COP subunit. We show that the μ-homology domain is dispensable for COPI function in the early secretory pathway, whereas the N-terminal longin domain is essential. We map a previously uncharacterized helix, C-terminal to the longin domain, that is specifically required for the retrieval of HDEL-bearing endoplasmic reticulum-luminal residents. It is positionally analogous to an unstructured linker that becomes helical and membrane-facing in the open form of the AP2 clathrin adaptor complex. Based on the amphipathic nature of the critical helix it may probe the membrane for lipid packing defects or mediate interaction with cargo and thus contribute to stabilizing membrane-associated coatomer. PMID:27298352

Ubiquitin-like and ubiquitin-associated domain proteins: significance in proteasomal degradation

PubMed Central

Lau, Alan F.

2009-01-01

The ubiquitin–proteasome pathway of protein degradation is one of the major mechanisms that are involved in the maintenance of the proper levels of cellular proteins. The regulation of proteasomal degradation thus ensures proper cell functions. The family of proteins containing ubiquitin-like (UbL) and ubiquitin-associated (UBA) domains has been implicated in proteasomal degradation. UbL–UBA domain containing proteins associate with substrates destined for degradation as well as with subunits of the proteasome, thus regulating the proper turnover of proteins. PMID:19468686

Voltage-sensing domain of voltage-gated proton channel Hv1 shares mechanism of block with pore domains.

PubMed

Hong, Liang; Pathak, Medha M; Kim, Iris H; Ta, Dennis; Tombola, Francesco

2013-01-23

Voltage-gated sodium, potassium, and calcium channels are made of a pore domain (PD) controlled by four voltage-sensing domains (VSDs). The PD contains the ion permeation pathway and the activation gate located on the intracellular side of the membrane. A large number of small molecules are known to inhibit the PD by acting as open channel blockers. The voltage-gated proton channel Hv1 is made of two VSDs and lacks the PD. The location of the activation gate in the VSD is unknown and open channel blockers for VSDs have not yet been identified. Here, we describe a class of small molecules which act as open channel blockers on the Hv1 VSD and find that a highly conserved phenylalanine in the charge transfer center of the VSD plays a key role in blocker binding. We then use one of the blockers to show that Hv1 contains two intracellular and allosterically coupled gates. Copyright © 2013 Elsevier Inc. All rights reserved.

Extracytoplasmic function σ factors of the widely distributed group ECF41 contain a fused regulatory domain

PubMed Central

Wecke, Tina; Halang, Petra; Staroń, Anna; Dufour, Yann S; Donohue, Timothy J; Mascher, Thorsten

2012-01-01

Bacteria need signal transducing systems to respond to environmental changes. Next to one- and two-component systems, alternative σ factors of the extra-cytoplasmic function (ECF) protein family represent the third fundamental mechanism of bacterial signal transduction. A comprehensive classification of these proteins identified more than 40 phylogenetically distinct groups, most of which are not experimentally investigated. Here, we present the characterization of such a group with unique features, termed ECF41. Among analyzed bacterial genomes, ECF41 σ factors are widely distributed with about 400 proteins from 10 different phyla. They lack obvious anti-σ factors that typically control activity of other ECF σ factors, but their structural genes are often predicted to be cotranscribed with carboxymuconolactone decarboxylases, oxidoreductases, or epimerases based on genomic context conservation. We demonstrate for Bacillus licheniformis and Rhodobacter sphaeroides that the corresponding genes are preceded by a highly conserved promoter motif and are the only detectable targets of ECF41-dependent gene regulation. In contrast to other ECF σ factors, proteins of group ECF41 contain a large C-terminal extension, which is crucial for σ factor activity. Our data demonstrate that ECF41 σ factors are regulated by a novel mechanism based on the presence of a fused regulatory domain. PMID:22950025

Patatin-like phospholipase domain-containing protein 3 (PNPLA3): A potential role in the association between liver disease and bipolar disorder.

PubMed

Kenneson, Aileen; Funderburk, Jennifer S

2017-02-01

Due to the increased prevalence of liver disease in patients with bipolar disorder, we examined the potential role of the patatin-like phospholipase domain-containing protein 3 (PNPLA3) variant among individuals with bipolar disorder and those with no mood disorder. We used the National Health and Nutrition Examination Survey (NHANES) database (aged 15-39 years) to identify a group of individuals with a bipolar diagnosis and a control group of individuals with no mood disorder. A total of 1931 individuals were randomly selected, one from each family containing information on the PNPLA3 genotype to be used in the analysis. Analyses revealed individuals with the recessive variant genotype (MM) had an adjusted odds ratio for bipolar disorder of about 4.6 compared to individuals with either IM or II genotypes of the PNPLA3 variant. Limitations of this study include the use of a lay-administered survey in for diagnosis of bipolar disorder in NHANES. The association between the PNPLA3 variant and bipolar disorder may help guide further work on medication effectiveness, treatment options, prevention approaches, and understanding potential medication side effects among specific subgroups of individuals with the MM genotype. Published by Elsevier B.V.

Characterization of substrate binding of the WW domains in human WWP2 protein.

PubMed

Jiang, Jiahong; Wang, Nan; Jiang, Yafei; Tan, Hongwei; Zheng, Jimin; Chen, Guangju; Jia, Zongchao

2015-07-08

WW domains harbor substrates containing proline-rich motifs, but the substrate specificity and binding mechanism remain elusive for those WW domains less amenable for structural studies, such as human WWP2 (hWWP2). Herein we have employed multiple techniques to investigate the second WW domain (WW2) in hWWP2. Our results show that hWWP2 is a specialized E3 for PPxY motif-containing substrates only and does not recognize other amino acids and phospho-residues. The strongest binding affinity of WW2, and the incompatibility between each WW domain, imply a novel relationship, and our SPR experiment reveals a dynamic binding mode in Class-I WW domains for the first time. The results from alanine-scanning mutagenesis and modeling further point to functionally conserved residues in WW2. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Generalization of the Schwarz–Christoffel mapping to multiply connected polygonal domains

PubMed Central

Vasconcelos, Giovani L.

2014-01-01

A generalization of the Schwarz–Christoffel mapping to multiply connected polygonal domains is obtained by making a combined use of two preimage domains, namely, a rectilinear slit domain and a bounded circular domain. The conformal mapping from the circular domain to the polygonal region is written as an indefinite integral whose integrand consists of a product of powers of the Schottky-Klein prime functions, which is the same irrespective of the preimage slit domain, and a prefactor function that depends on the choice of the rectilinear slit domain. A detailed derivation of the mapping formula is given for the case where the preimage slit domain is the upper half-plane with radial slits. Representation formulae for other canonical slit domains are also obtained but they are more cumbersome in that the prefactor function contains arbitrary parameters in the interior of the circular domain. PMID:24910523

Structural and functional properties of the N transcriptional activation domain of thyroid transcription factor-1: similarities with the acidic activation domains.

PubMed Central

Tell, G; Perrone, L; Fabbro, D; Pellizzari, L; Pucillo, C; De Felice, M; Acquaviva, R; Formisano, S; Damante, G

1998-01-01

The thyroid transcription factor 1 (TTF-1) is a tissue-specific transcription factor involved in the development of thyroid and lung. TTF-1 contains two transcriptional activation domains (N and C domain). The primary amino acid sequence of the N domain does not show any typical characteristic of known transcriptional activation domains. In aqueous solution the N domain exists in a random-coil conformation. The increase of the milieu hydrophobicity, by the addition of trifluoroethanol, induces a considerable gain of alpha-helical structure. Acidic transcriptional activation domains are largely unstructured in solution, but, under hydrophobic conditions, folding into alpha-helices or beta-strands can be induced. Therefore our data indicate that the inducibility of alpha-helix by hydrophobic conditions is a property not restricted to acidic domains. Co-transfections experiments indicate that the acidic domain of herpes simplex virus protein VP16 (VP16) and the TTF-1 N domain are interchangeable and that a chimaeric protein, which combines VP16 linked to the DNA-binding domain of TTF-1, undergoes the same regulatory constraints that operate for the wild-type TTF-1. In addition, we demonstrate that the TTF-1 N domain possesses two typical properties of acidic activation domains: TBP (TATA-binding protein) binding and ability to activate transcription in yeast. Accordingly, the TTF-1 N domain is able to squelch the activity of the p65 acidic domain. Altogether, these structural and functional data suggest that a non-acidic transcriptional activation domain (TTF-1 N domain) activates transcription by using molecular mechanisms similar to those used by acidic domains. TTF-1 N domain and acidic domains define a family of proteins whose common property is to activate transcription through the use of mechanisms largely conserved during evolutionary development. PMID:9425125

The PpaA/AerR regulators of photosynthesis gene expression from anoxygenic phototrophic proteobacteria contain heme-binding SCHIC domains.

PubMed

Moskvin, Oleg V; Gilles-Gonzalez, Marie-Alda; Gomelsky, Mark

2010-10-01

The SCHIC domain of the B12-binding domain family present in the Rhodobacter sphaeroides AppA protein binds heme and senses oxygen. Here we show that the predicted SCHIC domain PpaA/AerR regulators also bind heme and respond to oxygen in vitro, despite their low sequence identity with AppA.

Homoallylglycine residues are superior precursors to orthogonally modified thioether containing polypeptides.

PubMed

Perlin, Pesach; Gharakhanian, Eric G; Deming, Timothy J

2018-06-12

Homoallylglycine N-carboxyanhydride, Hag NCA, monomers were synthesized and used to prepare polypeptides containing Hag segments with controllable lengths of up to 245 repeats. Poly(l-homoallylglycine), GHA, was found to adopt an α-helical conformation, which provided good solubility in organic solvents and allowed high yield functionalization of its alkene side-chains via radical promoted addition of thiols. The conformations of these derivatives were shown to be switchable between α-helical and disordered states in aqueous media using thioether alkylation or oxidation reactions. Incorporation of GHA segments into block copolymers with poly(l-methionine), M, segments provided a means to orthogonally modify thioether side-chains different ways in separate copolypeptide domains. This approach allows preparation of functional polypeptides containing discrete domains of oxidized and alkylated thioether containing residues, where chain conformation and functionality of each domain can be independently modified.

The TIR domain of TIR-NB-LRR resistance proteins is a signaling domain involved in cell death induction.

PubMed

Swiderski, Michal R; Birker, Doris; Jones, Jonathan D G

2009-02-01

In plants, the TIR (toll interleukin 1 receptor) domain is found almost exclusively in nucleotide-binding (NB) leucine-rich repeat resistance proteins and their truncated homologs, and has been proposed to play a signaling role during resistance responses mediated by TIR containing R proteins. Transient expression in Nicotiana benthamiana leaves of "TIR + 80", the RPS4 truncation without the NB-ARC domain, leads to EDS1-, SGT1-, and HSP90-dependent cell death. Transgenic Arabidopsis plants expressing the RPS4 TIR+80 from either dexamethasone or estradiol-inducible promoters display inducer-dependent cell death. Cell death is also elicited by transient expression of similarly truncated constructs from two other R proteins, RPP1A and At4g19530, but is not elicited by similar constructs representing RPP2A and RPP2B proteins. Site-directed mutagenesis of the RPS4 TIR domain identified many loss-of-function mutations but also revealed several gain-of function substitutions. Lack of cell death induction by the E160A substitution suggests that amino acids outside of the TIR domain contribute to cell death signaling in addition to the TIR domain itself. This is consistent with previous observations that the TIR domain itself is insufficient to induce cell death upon transient expression.

Identification of Staphylococcal Nuclease Domain-containing 1 (SND1) as a Metadherin-interacting Protein with Metastasis-promoting Functions*

PubMed Central

Blanco, Mario Andres; Alečković, Maša; Hua, Yuling; Li, Tuo; Wei, Yong; Xu, Zhen; Cristea, Ileana M.; Kang, Yibin

2011-01-01

Metastasis is the deadliest and most poorly understood feature of malignant diseases. Recent work has shown that Metadherin (MTDH) is overexpressed in over 40% of breast cancer patients and promotes metastasis and chemoresistance in experimental models of breast cancer progression. Here we applied mass spectrometry-based screen to identify staphylococcal nuclease domain-containing 1 (SND1) as a candidate MTDH-interacting protein. After confirming the interaction between SND1 and MTDH, we tested the role of SND1 in breast cancer and found that it strongly promotes lung metastasis. SND1 was further shown to promote resistance to apoptosis and to regulate the expression of genes associated with metastasis and chemoresistance. Analyses of breast cancer clinical microarray data indicated that high expression of SND1 in primary tumors is strongly associated with reduced metastasis-free survival in multiple large scale data sets. Thus, we have uncovered SND1 as a novel MTDH-interacting protein and shown that it is a functionally and clinically significant mediator of metastasis. PMID:21478147

Role of ELA region in auto-activation of mutant KIT receptor: a molecular dynamics simulation insight.

PubMed

Purohit, Rituraj

2014-01-01

KIT receptor is the prime target in gastrointestinal stromal tumor (GISTs) therapy. Second generation inhibitor, Sunitinib, binds to an inactivated conformation of KIT receptor and stabilizes it in order to prevent tumor formation. Here, we investigated the dynamic behavior of wild type and mutant D816H KIT receptor, and emphasized the extended A-loop (EAL) region (805-850) by conducting molecular dynamics simulation (∼100 ns). We analyzed different properties such as root mean square cutoff or deviation, root mean square fluctuation, radius of gyration, solvent-accessible surface area, hydrogen bonding network analysis, and essential dynamics. Apart from this, clustering and cross-correlation matrix approach was used to explore the conformational space of the wild type and mutant EAL region of KIT receptor. Molecular dynamics analysis indicated that mutation (D816H) was able to alter intramolecular hydrogen bonding pattern and affected the structural flexibility of EAL region. Moreover, flexible secondary elements, specially, coil and turns were dominated in EAL region of mutant KIT receptor during simulation. This phenomenon increased the movement of EAL region which in turn helped in shifting the equilibrium towards the active kinase conformation. Our atomic investigation of mutant KIT receptor which emphasized on EAL region provided a better insight into the understanding of Sunitinib resistance mechanism of KIT receptor and would help to discover new therapeutics for KIT-based resistant tumor cells in GIST therapy.

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

Liu, Shengqiang; Li, Jie; Yu, Junsheng, E-mail: jsyu@uestc.edu.cn

A color tuning index (I{sub CT}) parameter for evaluating the color change capability of color-tunable organic light-emitting diodes (CT-OLEDs) was proposed and formulated. And a series of CT-OLEDs, consisting of five different carrier/exciton adjusting interlayers (C/EALs) inserted between two complementary emitting layers, were fabricated and applied to disclose the relationship between I{sub CT} and C/EALs. The result showed that the trend of electroluminescence spectra behavior in CT-OLEDs has good accordance with I{sub CT} values, indicating that the I{sub CT} parameter is feasible for the evaluation of color variation. Meanwhile, by changing energy level and C/EAL thickness, the optimized device withmore » the widest color tuning range was based on N,N′-dicarbazolyl-3,5-benzene C/EAL, exhibiting the highest I{sub CT} value of 41.2%. Based on carrier quadratic hopping theory and exciton transfer model, two fitting I{sub CT} formulas derived from the highest occupied molecular orbital (HOMO) energy level and triplet energy level were simulated. Finally, a color tuning prediction (CTP) model was developed to deduce the I{sub CT} via C/EAL HOMO and triplet energy levels, and verified by the fabricated OLEDs with five different C/EALs. We believe that the CTP model assisted with I{sub CT} parameter will be helpful for fabricating high performance CT-OLEDs with a broad range of color tuning.« less

Internet Protocol Handbook. Volume 4. The Domain Name System (DNS) handbook

DTIC Science & Technology

1989-08-01

Mockapetris [Page 1] 4-11 INTERNET PROTOCOL HA TDBOOK - Voue Four 1989 RFC 1034 Domain Concepts and Facilities November 1987 bandwidth consumed in distributing...Domain Names- Concepts and Facilities KFC 1034 RFC 1034 Domain Concepts and Facilities November 1’)87 - Queries contain a bit called recursion desired...during periodic sweeps to reclaim the memory consumed by old RRS. Mockapetris [Page 33] 4-43 INTERNET PROTOCOL HANDBOOK - Volume Four 1989 RFC 1034

Embedding international benchmarks of proficiency in English in undergraduate nursing programmes: challenges and strategies in equipping culturally and linguistically diverse students with English as an additional language for nursing in Australia.

PubMed

Glew, Paul J

2013-01-01

To meet the expected shortfalls in the number of registered nurses throughout the coming decade Australian universities have been recruiting an increasing number of students from culturally and linguistically diverse (CaLD) backgrounds. Given that international and domestic students who use English as an additional language (EAL) complement the number of native English speaking nursing students, they represent a valuable nurse education investment. Although university programmes are in a position to meet the education and learning needs of native English speaking nursing students, they can experience considerable challenges in effectively equipping EAL students with the English and academic language skills for nursing studies and registration in Australia. However, success in a nursing programme and in preparing for nurse registration can require EAL students to achieve substantial literacy skills in English and academic language through their engagement with these tertiary learning contexts. This paper discusses the education implications for nursing programmes and EAL students of developing literacy skills through pre-registration nursing studies to meet the English language skills standard for nurse registration and presents intervention strategies for nursing programmes that aim to build EAL student capacity in using academic English.

English Language Proficiency and Early School Attainment Among Children Learning English as an Additional Language.

PubMed

Whiteside, Katie E; Gooch, Debbie; Norbury, Courtenay F

2017-05-01

Children learning English as an additional language (EAL) often experience lower academic attainment than monolingual peers. In this study, teachers provided ratings of English language proficiency and social, emotional, and behavioral functioning for 782 children with EAL and 6,485 monolingual children in reception year (ages 4-5). Academic attainment was assessed in reception and Year 2 (ages 6-7). Relative to monolingual peers with comparable English language proficiency, children with EAL displayed fewer social, emotional, and behavioral difficulties in reception, were equally likely to meet curriculum targets in reception, and were more likely to meet targets in Year 2. Academic attainment and social, emotional, and behavioral functioning in children with EAL are associated with English language proficiency at school entry. © 2016 The Authors. Child Development published by Wiley Periodicals, Inc. on behalf of Society for Research in Child Development.

The insulin receptor substrate (IRS)-1 pleckstrin homology domain functions in downstream signaling.

PubMed

Vainshtein, I; Kovacina, K S; Roth, R A

2001-03-16

The pleckstrin homology (PH) domain of the insulin receptor substrate-1 (IRS-1) plays a role in directing this molecule to the insulin receptor, thereby regulating its tyrosine phosphorylation. In this work, the role of the PH domain in subsequent signaling was studied by constructing constitutively active forms of IRS-1 in which the inter-SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase was fused to portions of the IRS-1 molecule. Chimeric molecules containing the PH domain were found to activate the downstream response of stimulating the Ser/Thr kinase Akt. A chimera containing point mutations in the PH domain that abolished the ability of this domain to bind phosphatidylinositol 4,5-bisphosphate prevented these molecules from activating Akt. These mutations also decreased by about 70% the amount of the constructs present in a particulate fraction of the cells. These results indicate that the PH domain of IRS-1, in addition to directing this protein to the receptor for tyrosine phosphorylation, functions in the ability of this molecule to stimulate subsequent responses. Thus, compromising the function of the PH domain, e.g. in insulin-resistant states, could decrease both the ability of IRS-1 to be tyrosine phosphorylated by the insulin receptor and to link to subsequent downstream targets.

The Prp19 WD40 Domain Contains a Conserved Protein Interaction Region Essential for its Function

PubMed Central

Vander Kooi, Craig W.; Ren, Liping; Xu, Ping; Ohi, Melanie D.; Gould, Kathleen L.; Chazin, Walter J.

2010-01-01

Summary Prp19 is a member of the WD40-repeat family of E3 ubiquitin ligases and a conserved eukaryotic RNA splicing factor essential for activation and stabilization of the spliceosome. To understand the role of the WD40 repeat domain of Prp19 we have determined its structure using X-ray crystallography. The domain has a distorted seven bladed WD40 architecture with significant asymmetry due to irregular packing of blades one and seven into the core of the WD40 domain. Structure-based mutagenesis identified a highly conserved surface centered around blade five that is required for the physical interaction between Prp19 and Cwc2, another essential splicing factor. This region is found to be required for Prp19 function and yeast viability. Experiments in vitro and in vivo demonstrate that two molecules of Cwc2 bind to the Prp19 tetramer. These coupled structural and functional studies provide a model for the functional architecture of Prp19. PMID:20462492

Genetic reductionist approach for dissecting individual roles of GGDEF proteins within the c-di-GMP signaling network in Salmonella

PubMed Central

Solano, Cristina; García, Begoña; Latasa, Cristina; Toledo-Arana, Alejandro; Zorraquino, Violeta; Valle, Jaione; Casals, Joan; Pedroso, Enrique; Lasa, Iñigo

2009-01-01

Bacteria have developed an exclusive signal transduction system involving multiple diguanylate cyclase and phosphodiesterase domain-containing proteins (GGDEF and EAL/HD-GYP, respectively) that modulate the levels of the same diffusible molecule, 3′-5′-cyclic diguanylic acid (c-di-GMP), to transmit signals and obtain specific cellular responses. Current knowledge about c-di-GMP signaling has been inferred mainly from the analysis of recombinant bacteria that either lack or overproduce individual members of the pathway, without addressing potential compensatory effects or interferences between them. Here, we dissected c-di-GMP signaling by constructing a Salmonella strain lacking all GGDEF-domain proteins and then producing derivatives, each restoring 1 protein. Our analysis showed that most GGDEF proteins are constitutively expressed and that their expression levels are not interdependent. Complete deletion of genes encoding GGDEF-domain proteins abrogated virulence, motility, long-term survival, and cellulose and fimbriae synthesis. Separate restoration revealed that 4 proteins from Salmonella and 1 from Yersinia pestis exclusively restored cellulose synthesis in a c-di-GMP–dependent manner, indicating that c-di-GMP produced by different GGDEF proteins can activate the same target. However, the restored strain containing the STM4551-encoding gene recovered all other phenotypes by means of gene expression modulation independently of c-di-GMP. Specifically, fimbriae synthesis and virulence were recovered through regulation of csgD and the plasmid-encoded spvAB mRNA levels, respectively. This study provides evidence that the regulation of the GGDEF-domain proteins network occurs at 2 levels: a level that strictly requires c-di-GMP to control enzymatic activities directly, restricted to cellulose synthesis in our experimental conditions, and another that involves gene regulation for which c-di-GMP synthesis can be dispensable. PMID:19416883

Control of plant phosphate homeostasis by inositol pyrophosphates and the SPX domain.

PubMed

Jung, Ji-Yul; Ried, Martina K; Hothorn, Michael; Poirier, Yves

2018-02-01

Proteins containing a SPX domain are involved in phosphate (Pi) homeostasis, including Pi transport and adaptation to Pi deficiency. The SPX domain harbors a basic surface binding Pi at low affinity and inositol pyrophosphates (PP-InsPs) at high affinity. Genetic and biochemical studies revealed that PP-InsPs serve as ligands for the SPX domain. Residues in the PHO1 SPX domain involved in PP-InsPs binding are critical for its Pi export activity, and the interaction between SPX proteins and the PHR1 transcription factor, which results in PHR1 inactivation, is promoted by PP-InsPs. Changes in PP-InsPs levels in response to Pi deficiency may thus contribute to the adaptation of plants to stress via the modulation of the activity of SPX-containing proteins and their interactors. Modulating PP-InsP levels or the affinity/specificity of the SPX domain for PP-InsP could potentially be used to engineer crops to maintain high yield under reduced Pi fertilizer input. Copyright © 2017 Elsevier Ltd. All rights reserved.

Modelling protein functional domains in signal transduction using Maude

NASA Technical Reports Server (NTRS)

Sriram, M. G.

2003-01-01

Modelling of protein-protein interactions in signal transduction is receiving increased attention in computational biology. This paper describes recent research in the application of Maude, a symbolic language founded on rewriting logic, to the modelling of functional domains within signalling proteins. Protein functional domains (PFDs) are a critical focus of modern signal transduction research. In general, Maude models can simulate biological signalling networks and produce specific testable hypotheses at various levels of abstraction. Developing symbolic models of signalling proteins containing functional domains is important because of the potential to generate analyses of complex signalling networks based on structure-function relationships.

Structure and stability of the ankyrin domain of the Drosophila Notch receptor.

PubMed

Zweifel, Mark E; Leahy, Daniel J; Hughson, Frederick M; Barrick, Doug

2003-11-01

The Notch receptor contains a conserved ankyrin repeat domain that is required for Notch-mediated signal transduction. The ankyrin domain of Drosophila Notch contains six ankyrin sequence repeats previously identified as closely matching the ankyrin repeat consensus sequence, and a putative seventh C-terminal sequence repeat that exhibits lower similarity to the consensus sequence. To better understand the role of the Notch ankyrin domain in Notch-mediated signaling and to examine how structure is distributed among the seven ankyrin sequence repeats, we have determined the crystal structure of this domain to 2.0 angstroms resolution. The seventh, C-terminal, ankyrin sequence repeat adopts a regular ankyrin fold, but the first, N-terminal ankyrin repeat, which contains a 15-residue insertion, appears to be largely disordered. The structure reveals a substantial interface between ankyrin polypeptides, showing a high degree of shape and charge complementarity, which may be related to homotypic interactions suggested from indirect studies. However, the Notch ankyrin domain remains largely monomeric in solution, demonstrating that this interface alone is not sufficient to promote tight association. Using the structure, we have classified reported mutations within the Notch ankyrin domain that are known to disrupt signaling into those that affect buried residues and those restricted to surface residues. We show that the buried substitutions greatly decrease protein stability, whereas the surface substitutions have only a marginal affect on stability. The surface substitutions are thus likely to interfere with Notch signaling by disrupting specific Notch-effector interactions and map the sites of these interactions.

The SLP-76 SH2 domain is required for T cell development and activation

PubMed Central

Burns, Jeremy C.; Corbo, Evann; Degen, Janine; Gohil, Mercy; Anterasian, Christine; Schraven, Burkart; Koretzky, Gary A.; Kliche, Stefanie; Jordan, Martha S.

2011-01-01

The adaptor protein Src homology 2 (SH2) domain containing leukocyte protein of 76 kDa (SLP-76) is critical for multiple aspects of T cell development and function. Through its protein-binding domains, SLP-76 serves as a platform for the assembly of multiple enzymes and adaptor proteins that function together to activate second messengers required for TCR signal propagation. The N-terminus of SLP-76, which contains three tyrosines that serve as docking sites for SH2 domain-containing proteins, and the central proline-rich region of SLP-76 have been well studied and are known to be important for both thymocyte selection and activation of peripheral T cells. Less is known about the function of the C-terminal SH2 domain of SLP-76. This region inducibly associates with the adhesion- and degranulation-promoting adaptor protein (ADAP) and hematopoietic progenitor kinase 1 (HPK1). Combining regulated deletion of endogenous SLP-76 with transgenic expression of a SLP-76 SH2 domain mutant, we demonstrate that the SLP-76 SH2 domain is required for peripheral T cell activation and positive selection of thymocytes, a function not previously attributed to this region. This domain is also important for T cell proliferation, IL-2 production and phosphorylation of protein kinase D (PKD) and IκB. ADAP-deficient T cells display similar, but in some cases less severe, defects despite phosphorylation of a negative regulatory site on SLP-76 by HPK1, a function that is lost in SLP-76 SH2 domain mutant T cells. PMID:21949020

An integrated genomic analysis of Tudor domain-containing proteins identifies PHD finger protein 20-like 1 (PHF20L1) as a candidate oncogene in breast cancer.

PubMed

Jiang, Yuanyuan; Liu, Lanxin; Shan, Wenqi; Yang, Zeng-Quan

2016-02-01

Tudor domain-containing proteins (TDRDs), which recognize and bind to methyl-lysine/arginine residues on histones and non-histone proteins, play critical roles in regulating chromatin architecture, transcription, genomic stability, and RNA metabolism. Dysregulation of several TDRDs have been observed in various types of cancer. However, neither the genomic landscape nor clinical significance of TDRDs in breast cancer has been explored comprehensively. Here, we performed an integrated genomic and transcriptomic analysis of 41 TDRD genes in breast cancer (TCGA and METABRIC datasets) and identified associations among recurrent copy number alterations, gene expressions, clinicopathological features, and survival of patients. Among seven TDRDs that had the highest frequency (>10%) of gene amplification, the plant homeodomain finger protein 20-like 1 (PHF20L1) was the most commonly amplified (17.62%) TDRD gene in TCGA breast cancers. Different subtypes of breast cancer had different patterns of copy number and expression for each TDRD. Notably, amplification and overexpression of PHF20L1 were more prevalent in aggressive basal-like and Luminal B subtypes and were significantly associated with shorter survival of breast cancer patients. Furthermore, knockdown of PHF20L1 inhibited cell proliferation in PHF20L1-amplified breast cancer cell lines. PHF20L1 protein contains N-terminal Tudor and C-terminal plant homeodomain domains. Detailed characterization of PHF20L1 in breast cancer revealed that the Tudor domain likely plays a critical role in promoting cancer. Mechanistically, PHF20L1 might participate in regulating DNA methylation by stabilizing DNA methyltransferase 1 (DNMT1) protein in breast cancer. Thus, our results demonstrated the oncogenic potential of PHF20L1 and its association with poor prognostic parameters in breast cancer. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Distribution of PASTA domains in penicillin-binding proteins and serine/threonine kinases of Actinobacteria.

PubMed

Ogawara, Hiroshi

2016-09-01

PASTA domains (penicillin-binding protein and serine/threonine kinase-associated domains) have been identified in penicillin-binding proteins and serine/threonine kinases of Gram-positive Firmicutes and Actinobacteria. They are believed to bind β-lactam antibiotics, and be involved in peptidoglycan metabolism, although their biological function is not definitively clarified. Actinobacteria, especially Streptomyces species, are distinct in that they undergo complex cellular differentiation and produce various antibiotics including β-lactams. This review focuses on the distribution of PASTA domains in penicillin-binding proteins and serine/threonine kinases in Actinobacteria. In Actinobacteria, PASTA domains are detectable exclusively in class A but not in class B penicillin-binding proteins, in sharp contrast to the cases in other bacteria. In penicillin-binding proteins, PASTA domains distribute independently from taxonomy with some distribution bias. Particularly interesting thing is that no Streptomyces species have penicillin-binding protein with PASTA domains. Protein kinases in Actinobacteria possess 0 to 5 PASTA domains in their molecules. Protein kinases in Streptomyces can be classified into three groups: no PASTA domain, 1 PASTA domain and 4 PASTA domain-containing groups. The 4 PASTA domain-containing groups can be further divided into two subgroups. The serine/threonine kinases in different groups may perform different functions. The pocket region in one of these subgroup is more dense and extended, thus it may be involved in binding of ligands like β-lactams more efficiently.

Solution structure of leptospiral LigA4 Big domain

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

Mei, Song; Zhang, Jiahai; Zhang, Xuecheng

Pathogenic Leptospiraspecies express immunoglobulin-like proteins which serve as adhesins to bind to the extracellular matrices of host cells. Leptospiral immunoglobulin-like protein A (LigA), a surface exposed protein containing tandem repeats of bacterial immunoglobulin-like (Big) domains, has been proved to be involved in the interaction of pathogenic Leptospira with mammalian host. In this study, the solution structure of the fourth Big domain of LigA (LigA4 Big domain) from Leptospira interrogans was solved by nuclear magnetic resonance (NMR). The structure of LigA4 Big domain displays a similar bacterial immunoglobulin-like fold compared with other Big domains, implying some common structural aspects of Bigmore » domain family. On the other hand, it displays some structural characteristics significantly different from classic Ig-like domain. Furthermore, Stains-all assay and NMR chemical shift perturbation revealed the Ca{sup 2+} binding property of LigA4 Big domain. - Highlights: • Determining the solution structure of a bacterial immunoglobulin-like domain from a surface protein of Leptospira. • The solution structure shows some structural characteristics significantly different from the classic Ig-like domains. • A potential Ca{sup 2+}-binding site was identified by strains-all and NMR chemical shift perturbation.« less

Pathogenesis and micro-anatomic characterization of a cell-adapted mutant foot-and-mouth disease virus in cattle: impact of the Jumonji C-domain containing protein 6 (JMJD6) and route of innoculation

USDA-ARS?s Scientific Manuscript database

In a companion study, we reported that the cellular Jumonji-C Domain containing Protein 6 (JMJD6) protein is involved in an alternate integrin- and HS-independent pathway of FMDV infection in CHO cells. Here, we investigated the JMJD6 localization in animal tissues from cattle infected with either ...

Effect of obesity and exercise on the expression of the novel myokines, Myonectin and Fibronectin type III domain containing 5

PubMed Central

Mart, Ryan; Bond, Cherie E.

2014-01-01

Metabolic dysfunction in skeletal muscle is a major contributor to the development of type 2 diabetes. Endurance exercise training has long been established as an effective means to directly restore skeletal muscle glucose and lipid uptake and metabolism. However, in addition to the direct effects of skeletal muscle on glucose and lipids, there is renewed interest in the ability of skeletal muscle to coordinate metabolic activity of other tissues, such as adipose tissue and liver. The purpose of this study was to examine the effects of endurance exercise on the expression level of two novel muscle-derived secreted factors, or myokines, Myonectin and Fibronectin type III domain containing 5 (FNDC5), the precursor for Irisin. Methods. We performed immunoblot analysis and quantitative real-time PCR analysis of Myonectin and FNDC5 in the diaphragm muscles of obese Zucker rat (OZR) and lean Zucker rat (LZR) with 9 weeks of aerobic training on a motorized treadmill. Results. We show that myonectin gene expression is increased in the OZR model of obesity and decreases with exercise in both lean and obese Zucker rats. Conversely, myonectin protein concentration was elevated with exercise. Similarly, FNDC5 mRNA levels are significantly higher in the OZR, however exercise training had no effect on the expression level of FNDC5 in either the LZR or OZR. We did not observe any difference in muscle protein content of Irisin with obesity or exercise. Conclusion. Our data shows that exercise training does not increase either FNDC5 or myonectin gene expression, indicating that increased transcriptional regulation of these myokines is not induced by exercise. However, our data also indicates a yet to be explored disconnect between myonectin gene expression and protein content. Further, this report highlights the importance of verifying reference genes when completing gene expression analysis. We found that many commonly used reference genes varied significantly by obesity and

The C Terminus of Formin FMNL3 Accelerates Actin Polymerization and Contains a WH2 Domain-like Sequence That Binds Both Monomers and Filament Barbed Ends*

PubMed Central

Heimsath, Ernest G.; Higgs, Henry N.

2012-01-01

Formin proteins are actin assembly factors that accelerate filament nucleation then remain on the elongating barbed end and modulate filament elongation. The formin homology 2 (FH2) domain is central to these activities, but recent work has suggested that additional sequences enhance FH2 domain function. Here we show that the C-terminal 76 amino acids of the formin FMNL3 have a dramatic effect on the ability of the FH2 domain to accelerate actin assembly. This C-terminal region contains a WASp homology 2 (WH2)-like sequence that binds actin monomers in a manner that is competitive with other WH2 domains and with profilin. In addition, the C terminus binds filament barbed ends. As a monomer, the FMNL3 C terminus inhibits actin polymerization and slows barbed end elongation with moderate affinity. As a dimer, the C terminus accelerates actin polymerization from monomers and displays high affinity inhibition of barbed end elongation. These properties are not common to all formin C termini, as those of mDia1 and INF2 do not behave similarly. Interestingly, mutation of two aliphatic residues, which blocks high affinity actin binding by the WH2-like sequence, has no effect on the ability of the C terminus to enhance FH2-mediated polymerization. However, mutation of three successive basic residues at the C terminus of the WH2-like sequence compromises polymerization enhancement. These results illustrate that the C termini of formins are highly diverse in their interactions with actin. PMID:22094460

The SLP-76 Src homology 2 domain is required for T cell development and activation.

PubMed

Burns, Jeremy C; Corbo, Evann; Degen, Janine; Gohil, Mercy; Anterasian, Christine; Schraven, Burkart; Koretzky, Gary A; Kliche, Stefanie; Jordan, Martha S

2011-11-01

The adapter protein Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP-76) is critical for multiple aspects of T cell development and function. Through its protein-binding domains, SLP-76 serves as a platform for the assembly of multiple enzymes and adapter proteins that function together to activate second messengers required for TCR signal propagation. The N terminus of SLP-76, which contains three tyrosines that serve as docking sites for SH2 domain-containing proteins, and the central proline-rich region of SLP-76 have been well studied and are known to be important for both thymocyte selection and activation of peripheral T cells. Less is known about the function of the C-terminal SH2 domain of SLP-76. This region inducibly associates with ADAP and HPK1. Combining regulated deletion of endogenous SLP-76 with transgenic expression of a SLP-76 SH2 domain mutant, we demonstrate that the SLP-76 SH2 domain is required for peripheral T cell activation and positive selection of thymocytes, a function not previously attributed to this region. This domain is also important for T cell proliferation, IL-2 production, and phosphorylation of protein kinase D and IκB. ADAP-deficient T cells display similar, but in some cases less severe, defects despite phosphorylation of a negative regulatory site on SLP-76 by HPK1, a function that is lost in SLP-76 SH2 domain mutant T cells.

Three-dimensional structure of the NLRP7 pyrin domain: insight into pyrin-pyrin-mediated effector domain signaling in innate immunity.

PubMed

Pinheiro, Anderson S; Proell, Martina; Eibl, Clarissa; Page, Rebecca; Schwarzenbacher, Robert; Peti, Wolfgang

2010-08-27

The innate immune system provides an initial line of defense against infection. Nucleotide-binding domain- and leucine-rich repeat-containing protein (NLR or (NOD-like)) receptors play a critical role in the innate immune response by surveying the cytoplasm for traces of intracellular invaders and endogenous stress signals. NLRs themselves are multi-domain proteins. Their N-terminal effector domains (typically a pyrin or caspase activation and recruitment domain) are responsible for driving downstream signaling and initiating the formation of inflammasomes, multi-component complexes necessary for cytokine activation. However, the currently available structures of NLR effector domains have not yet revealed the mechanism of their differential modes of interaction. Here, we report the structure and dynamics of the N-terminal pyrin domain of NLRP7 (NLRP7 PYD) obtained by NMR spectroscopy. The NLRP7 PYD adopts a six-alpha-helix bundle death domain fold. A comparison of conformational and dynamics features of the NLRP7 PYD with other PYDs showed distinct differences for helix alpha3 and loop alpha2-alpha3, which, in NLRP7, is stabilized by a strong hydrophobic cluster. Moreover, the NLRP7 and NLRP1 PYDs have different electrostatic surfaces. This is significant, because death domain signaling is driven by electrostatic contacts and stabilized by hydrophobic interactions. Thus, these results provide new insights into NLRP signaling and provide a first molecular understanding of inflammasome formation.

Recombinant spider silk genetically functionalized with affinity domains.

PubMed

Jansson, Ronnie; Thatikonda, Naresh; Lindberg, Diana; Rising, Anna; Johansson, Jan; Nygren, Per-Åke; Hedhammar, My

2014-05-12

Functionalization of biocompatible materials for presentation of active protein domains is an area of growing interest. Herein, we describe a strategy for functionalization of recombinant spider silk via gene fusion to affinity domains of broad biotechnological use. Four affinity domains of different origin and structure; the IgG-binding domains Z and C2, the albumin-binding domain ABD, and the biotin-binding domain M4, were all successfully produced as soluble silk fusion proteins under nondenaturing purification conditions. Silk films and fibers produced from the fusion proteins were demonstrated to be chemically and thermally stable. Still, the bioactive domains are concluded to be folded and accessible, since their respective targets could be selectively captured from complex samples, including rabbit serum and human plasma. Interestingly, materials produced from mixtures of two different silk fusion proteins displayed combined binding properties, suggesting that tailor-made materials with desired stoichiometry and surface distributions of several binding domains can be produced. Further, use of the IgG binding ability as a general mean for presentation of desired biomolecules could be demonstrated for a human vascular endothelial growth factor (hVEGF) model system, via a first capture of anti-VEGF IgG to silk containing the Z-domain, followed by incubation with hVEGF. Taken together, this study demonstrates the potential of recombinant silk, genetically functionalized with affinity domains, for construction of biomaterials capable of presentation of almost any desired biomolecule.

The B-Box Domain Protein BBX21 Promotes Photomorphogenesis.

PubMed

Xu, Dongqing; Jiang, Yan; Li, Jian; Holm, Magnus; Deng, Xing Wang

2018-03-01

B-box-containing (BBX) proteins play critical roles in a variety of cellular and developmental processes in plants. BBX21 (also known as SALT TOLERANCE HOMOLOG2), which contains two B-box domains in tandem at the N terminus, has been previously demonstrated as a key component involved in the COP1-HY5 signaling hub. However, the exact molecular and physiological roles of B-box domains in BBX21 are largely unclear. Here, we found that structurally disruption of the second B-box domain, but not the first one, in BBX21 completely abolishes its biological and physiological activity in conferring hyperphotomorphogenetic phenotype in Arabidopsis ( Arabidopsis thaliana ). Intact B-box domains in BBX21 are not required for interaction with COP1 and its degradation by COP1 via the 26S proteasome system. However, disruption of the second B-box of BBX21 nearly impairs its ability for binding of T/G-box within the HY5 promoter both in vitro and in vivo, as well as controlling HY5 and HY5-regulated gene expression in Arabidopsis seedlings. Taken together, this study provides a mechanistic framework in which BBX21 directly binds to the T/G-box present in the HY5 promoter possibly through its second B-box domain, which in turn controls HY5 and HY5-regulated gene expression to promote photomorphogenesis. © 2018 American Society of Plant Biologists. All Rights Reserved.

NovelFam3000 – Uncharacterized human protein domains conserved across model organisms

PubMed Central

Kemmer, Danielle; Podowski, Raf M; Arenillas, David; Lim, Jonathan; Hodges, Emily; Roth, Peggy; Sonnhammer, Erik LL; Höög, Christer; Wasserman, Wyeth W

2006-01-01

Background Despite significant efforts from the research community, an extensive portion of the proteins encoded by human genes lack an assigned cellular function. Most metazoan proteins are composed of structural and/or functional domains, of which many appear in multiple proteins. Once a domain is characterized in one protein, the presence of a similar sequence in an uncharacterized protein serves as a basis for inference of function. Thus knowledge of a domain's function, or the protein within which it arises, can facilitate the analysis of an entire set of proteins. Description From the Pfam domain database, we extracted uncharacterized protein domains represented in proteins from humans, worms, and flies. A data centre was created to facilitate the analysis of the uncharacterized domain-containing proteins. The centre both provides researchers with links to dispersed internet resources containing gene-specific experimental data and enables them to post relevant experimental results or comments. For each human gene in the system, a characterization score is posted, allowing users to track the progress of characterization over time or to identify for study uncharacterized domains in well-characterized genes. As a test of the system, a subset of 39 domains was selected for analysis and the experimental results posted to the NovelFam3000 system. For 25 human protein members of these 39 domain families, detailed sub-cellular localizations were determined. Specific observations are presented based on the analysis of the integrated information provided through the online NovelFam3000 system. Conclusion Consistent experimental results between multiple members of a domain family allow for inferences of the domain's functional role. We unite bioinformatics resources and experimental data in order to accelerate the functional characterization of scarcely annotated domain families. PMID:16533400

Jumonji domain-containing protein 3 regulates the early inflammatory response epigenetically in human periodontal ligament cells.

PubMed

Wang, Puyu; Yue, Junli; Xu, Weizhe; Chen, Xi; Yi, Xiaowei; Ye, Ling; Zhang, Lan; Huang, Dingming

2018-05-30

To investigate the role of the histone 3 lysine 27 trimethylation (H3K27me3) demethylase Jumonji domain-containing protein 3 (Jmjd3) in the epigenetic regulation of the inflammatory response in human periodontal ligament cells (HPDLs). HPDLs were stimulated with lipopolysaccharide from E. coli. The expression of Jmjd3 in HPDLs was examined by quantitative real-time polymerase chain reaction (Q-PCR), Western Blot and immunofluorescent staining. Potential target genes were selected by silencing Jmjd3 and were confirmed by Chromatin Immunoprecipitation (ChIP). Q-PCR, Western Blot and immunofluorescent staining revealed that the expression of Jmjd3 was increased in inflamed HPDLs. Knockdown of Jmjd3 led to the suppression of inflammation-induced up-regulation of interleukin-6 and interleukin-12. Moreover, ChIP assays demonstrated that Jmjd3 was recruited to the promoters of interleukin-6 and interleukin-12b and this recruitment was associated with decreased levels of trimethylated histone 3 lysine 27 (H3K27). It was concluded that Jmjd3 regulated the activation of interleukin-6 and interleukin-12b in the early inflammatory response of HPDLs via demethylation of H3K27me3 at promoters. This molecular event may play an important role in the regulation of the inflammatory response in HPDLs. Copyright © 2018. Published by Elsevier Ltd.

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.

Lune/eye gone, a Pax-like protein, uses a partial paired domain and a homeodomain for DNA recognition.

PubMed

Jun, S; Wallen, R V; Goriely, A; Kalionis, B; Desplan, C

1998-11-10

Pax proteins, characterized by the presence of a paired domain, play key regulatory roles during development. The paired domain is a bipartite DNA-binding domain that contains two helix-turn-helix domains joined by a linker region. Each of the subdomains, the PAI and RED domains, has been shown to be a distinct DNA-binding domain. The PAI domain is the most critical, but in specific circumstances, the RED domain is involved in DNA recognition. We describe a Pax protein, originally called Lune, that is the product of the Drosophila eye gone gene (eyg). It is unique among Pax proteins, because it contains only the RED domain. eyg seems to play a role both in the organogenesis of the salivary gland during embryogenesis and in the development of the eye. A high-affinity binding site for the Eyg RED domain was identified by using systematic evolution of ligands by exponential enrichment techniques. This binding site is related to a binding site previously identified for the RED domain of the Pax-6 5a isoform. Eyg also contains another DNA-binding domain, a Prd-class homeodomain (HD), whose palindromic binding site is similar to other Prd-class HDs. The ability of Pax proteins to use the PAI, RED, and HD, or combinations thereof, may be one mechanism that allows them to be used at different stages of development to regulate various developmental processes through the activation of specific target genes.

Lune/eye gone, a Pax-like protein, uses a partial paired domain and a homeodomain for DNA recognition

PubMed Central

Jun, Susie; Wallen, Robert V.; Goriely, Anne; Kalionis, Bill; Desplan, Claude

1998-01-01

Pax proteins, characterized by the presence of a paired domain, play key regulatory roles during development. The paired domain is a bipartite DNA-binding domain that contains two helix–turn–helix domains joined by a linker region. Each of the subdomains, the PAI and RED domains, has been shown to be a distinct DNA-binding domain. The PAI domain is the most critical, but in specific circumstances, the RED domain is involved in DNA recognition. We describe a Pax protein, originally called Lune, that is the product of the Drosophila eye gone gene (eyg). It is unique among Pax proteins, because it contains only the RED domain. eyg seems to play a role both in the organogenesis of the salivary gland during embryogenesis and in the development of the eye. A high-affinity binding site for the Eyg RED domain was identified by using systematic evolution of ligands by exponential enrichment techniques. This binding site is related to a binding site previously identified for the RED domain of the Pax-6 5a isoform. Eyg also contains another DNA-binding domain, a Prd-class homeodomain (HD), whose palindromic binding site is similar to other Prd-class HDs. The ability of Pax proteins to use the PAI, RED, and HD, or combinations thereof, may be one mechanism that allows them to be used at different stages of development to regulate various developmental processes through the activation of specific target genes. PMID:9811867

Functional domains of the Drosophila Engrailed protein.

PubMed Central

Han, K; Manley, J L

1993-01-01

We have studied the transcriptional activity of the Drosophila homeodomain protein Engrailed (En) by using a transient expression assay employing Schneider L2 cells. En was found to very strongly repress promoters activated by a variety of different activator proteins. However, unlike another Drosophila homeodomain-containing repressor, Even-skipped (Eve), En was unable to repress the activity of several basal promoters in the absence of activator expression. These findings indicate that En is a specific repressor of activated transcription, and suggest that En may repress transcription by a different mechanism than Eve, perhaps by interfering with interactions between transcriptional activators and the general transcription machinery. By analyzing the properties of a variety of En mutants, we identified a minimal repression domain composed of 55 residues, which can function when fused to a heterologous DNA binding domain. Like repression domains identified in the Drosophila repressors Eve and Krüppel, the En repression domain is rich in alanine residues (26%), but unlike these other domains, is moderately charged (six arginine and three glutamic acid residues). Separate regions of En that may in some circumstances function in transcriptional activation were also identified. Images PMID:8334991

Deficiency of a Retinal Dystrophy Protein, Acyl-CoA Binding Domain-containing 5 (ACBD5), Impairs Peroxisomal β-Oxidation of Very-long-chain Fatty Acids*

PubMed Central

Yagita, Yuichi; Shinohara, Kyoko; Abe, Yuichi; Nakagawa, Keiko; Al-Owain, Mohammed; Alkuraya, Fowzan S.; Fujiki, Yukio

2017-01-01

Acyl-CoA binding domain-containing 5 (ACBD5) is a peroxisomal protein that carries an acyl-CoA binding domain (ACBD) at its N-terminal region. The recent identification of a mutation in the ACBD5 gene in patients with a syndromic form of retinal dystrophy highlights the physiological importance of ACBD5 in humans. However, the underlying pathogenic mechanisms and the precise function of ACBD5 remain unclear. We herein report that ACBD5 is a peroxisomal tail-anchored membrane protein exposing its ACBD to the cytosol. Using patient-derived fibroblasts and ACBD5 knock-out HeLa cells generated via genome editing, we demonstrate that ACBD5 deficiency causes a moderate but significant defect in peroxisomal β-oxidation of very-long-chain fatty acids (VLCFAs) and elevates the level of cellular phospholipids containing VLCFAs without affecting peroxisome biogenesis, including the import of membrane and matrix proteins. Both the N-terminal ACBD and peroxisomal localization of ACBD5 are prerequisite for efficient VLCFA β-oxidation in peroxisomes. Furthermore, ACBD5 preferentially binds very-long-chain fatty acyl-CoAs (VLC-CoAs). Together, these results suggest a direct role of ACBD5 in peroxisomal VLCFA β-oxidation. Based on our findings, we propose that ACBD5 captures VLC-CoAs on the cytosolic side of the peroxisomal membrane so that the transport of VLC-CoAs into peroxisomes and subsequent β-oxidation thereof can proceed efficiently. Our study reclassifies ACBD5-related phenotype as a novel peroxisomal disorder. PMID:27899449

Substrate specificity of human metallocarboxypeptidase D: Comparison of the two active carboxypeptidase domains

PubMed Central

Tanco, Sebastian; Díaz, Lucía; Dasgupta, Sayani; Fernandez-Recio, Juan; Lorenzo, Julia; Aviles, Francesc X.; Fricker, Lloyd D.

2017-01-01

Metallocarboxypeptidase D (CPD) is a membrane-bound component of the trans-Golgi network that cycles to the cell surface through exocytic and endocytic pathways. Unlike other members of the metallocarboxypeptidase family, CPD is a multicatalytic enzyme with three carboxypeptidase-like domains, although only the first two domains are predicted to be enzymatically active. To investigate the enzymatic properties of each domain in human CPD, a critical active site Glu in domain I and/or II was mutated to Gln and the protein expressed, purified, and assayed with a wide variety of peptide substrates. CPD with all three domains intact displays >50% activity from pH 5.0 to 7.5 with a maximum at pH 6.5, as does CPD with mutation of domain I. In contrast, the domain II mutant displayed >50% activity from pH 6.5–7.5. CPD with mutations in both domains I and II was completely inactive towards all substrates and at all pH values. A quantitative peptidomics approach was used to compare the activities of CPD domains I and II towards a large number of peptides. CPD cleaved C-terminal Lys or Arg from a subset of the peptides. Most of the identified substrates of domain I contained C-terminal Arg, whereas comparable numbers of Lys- and Arg-containing peptides were substrates of domain II. We also report that some peptides with C-terminal basic residues were not cleaved by either domain I or II, showing the importance of the P1 position for CPD activity. Finally, the preference of domain I for C-terminal Arg was validated through molecular docking experiments. Together with the differences in pH optima, the different substrate specificities of CPD domains I and II allow the enzyme to perform distinct functions in the various locations within the cell. PMID:29131831

Application of Wavelet Transform for PDZ Domain Classification

PubMed Central

Daqrouq, Khaled; Alhmouz, Rami; Balamesh, Ahmed; Memic, Adnan

2015-01-01

PDZ domains have been identified as part of an array of signaling proteins that are often unrelated, except for the well-conserved structural PDZ domain they contain. These domains have been linked to many disease processes including common Avian influenza, as well as very rare conditions such as Fraser and Usher syndromes. Historically, based on the interactions and the nature of bonds they form, PDZ domains have most often been classified into one of three classes (class I, class II and others - class III), that is directly dependent on their binding partner. In this study, we report on three unique feature extraction approaches based on the bigram and trigram occurrence and existence rearrangements within the domain's primary amino acid sequences in assisting PDZ domain classification. Wavelet packet transform (WPT) and Shannon entropy denoted by wavelet entropy (WE) feature extraction methods were proposed. Using 115 unique human and mouse PDZ domains, the existence rearrangement approach yielded a high recognition rate (78.34%), which outperformed our occurrence rearrangements based method. The recognition rate was (81.41%) with validation technique. The method reported for PDZ domain classification from primary sequences proved to be an encouraging approach for obtaining consistent classification results. We anticipate that by increasing the database size, we can further improve feature extraction and correct classification. PMID:25860375

Towards optimal intraoperative conditions in esophageal surgery: A review of literature for the prevention of esophageal anastomotic leakage.

PubMed

Bootsma, Boukje Titia; Huisman, Daitlin Esmee; Plat, Victor Dirk; Schoonmade, Linda Jeanne; Stens, Jurre; Hubens, Guy; van der Peet, Donald Leonard; Daams, Freek

2018-04-30

Esophageal anastomotic leakage (EAL) is a severe complication following gastric and esophageal surgery for cancer. Several non-modifiable, patient or surgery related risk factors for EAL have been identified, however, the contribution of modifiable intraoperative parameters remains undetermined. This review provides an overview of current literature on potentially modifiable intraoperative risk factors for EAL. The PubMed, EMBASE and Cochrane databases were searched by two researchers independently. Clinical studies published in English between 1970 and January 2017 that evaluated the effect of intraoperative parameters on the development of EAL were included. Levels of evidence as defined by the Centre of Evidence Based Medicine (CEBM) were assigned to the studies. A total of 25 articles were included in the final analysis. These articles show evidence that anemia, increased amount of blood loss, low pH and high pCO2 values, prolonged duration of procedure and lack of surgical experience independently increase the risk of EAL. Supplemental oxygen therapy, epidural analgesia and selective digestive decontamination seem to have a beneficial effect. Potential risk factors include blood pressure, requirement of blood products, vasopressor use and glucocorticoid administration, however the results are ambiguous. Apart from fixed surgical and patient related factors, several intraoperative factors that can be modified in clinical practice can influence the risk of developing EAL. More prospective, observational studies are necessary focusing on modifiable intraoperative parameters to assess more evidence and to elucidate optimal values of these factors. Copyright © 2018 IJS Publishing Group Ltd. Published by Elsevier Ltd. All rights reserved.

Unravelling the differences in attrition and academic performance of international and domestic nursing students with English as an additional language.

PubMed

Zheng, Roy Xun; Everett, Bronwyn; Glew, Paul; Salamonson, Yenna

2014-12-01

High attrition and academic underperformance have been highlighted among students who speak English as an additional language (EAL) in higher education, and a lack of language skills is often cited as a key explanatory factor. Although the relationship between English-language skills and academic performance among EAL students has been established, group differences between international and domestic EAL nursing students is not known. The aim of this study was to compare attrition rates and academic performance of international and domestic EAL nursing students, taking into consideration levels of English-language usage and socio-demographic characteristics of these groups. A prospective correlational study. From 2010 to 2012, nursing students at a large Australian university, who attended an orientation session before course commencement, were invited to complete a survey to assess their English-language usage. Data collected included students' enrolment status and GPA at 12months. Compared with their domestic counterparts, the attrition rate of international EAL students was significantly lower (7.9% versus 13.3%, p=0.018). Similarly, international students also had higher GPAs (4.1 versus 4.0, p=0.011). Although the levels of English-language usage were not related to academic performance, recent arrivals in both international (p=0.047) and domestic (p=0.001) student groups had higher GPAs. This study suggests that language acculturation, indicated by English-language usage and the length of stay in the host country, was not sufficient to ensure successful transition into the academic environment for either international or domestic EAL nursing students. Copyright © 2014 Elsevier Ltd. All rights reserved.

PDZ affinity chromatography: a general method for affinity purification of proteins based on PDZ domains and their ligands.

PubMed

Walkup, Ward G; Kennedy, Mary B

2014-06-01

PDZ (PSD-95, DiscsLarge, ZO1) domains function in nature as protein binding domains within scaffold and membrane-associated proteins. They comprise ∼90 residues and make specific, high affinity interactions with complementary C-terminal peptide sequences, with other PDZ domains, and with phospholipids. We hypothesized that the specific, strong interactions of PDZ domains with their ligands would make them well suited for use in affinity chromatography. Here we describe a novel affinity chromatography method applicable for the purification of proteins that contain PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We created a series of affinity resins comprised of PDZ domains from the scaffold protein PSD-95, or from neuronal nitric oxide synthase (nNOS), coupled to solid supports. We used them to purify heterologously expressed neuronal proteins or protein domains containing endogenous PDZ domain ligands, eluting the proteins with free PDZ domain peptide ligands. We show that Proteins of Interest (POIs) lacking endogenous PDZ domain ligands can be engineered as fusion products containing C-terminal PDZ domain ligand peptides or internal, N- or C-terminal PDZ domains and then can be purified by the same method. Using this method, we recovered recombinant GFP fused to a PDZ domain ligand in active form as verified by fluorescence yield. Similarly, chloramphenicol acetyltransferase (CAT) and β-Galactosidase (LacZ) fused to a C-terminal PDZ domain ligand or an N-terminal PDZ domain were purified in active form as assessed by enzymatic assay. In general, PDZ domains and ligands derived from PSD-95 were superior to those from nNOS for this method. PDZ Domain Affinity Chromatography promises to be a versatile and effective method for purification of a wide variety of natural and recombinant proteins. Copyright © 2014 Elsevier Inc. All rights reserved.

Identification and Characterization of the Novel LysM Domain-Containing Surface Protein Sep from Lactobacillus fermentum BR11 and Its Use as a Peptide Fusion Partner in Lactobacillus and Lactococcus

PubMed Central

Turner, Mark S.; Hafner, Louise M.; Walsh, Terry; Giffard, Philip M.

2004-01-01

Examination of supernatant fractions from broth cultures of Lactobacillus fermentum BR11 revealed the presence of a number of proteins, including a 27-kDa protein termed Sep. The amino-terminal sequence of Sep was determined, and the gene encoding it was cloned and sequenced. Sep is a 205-amino-acid protein and contains a 30-amino-acid secretion signal and has overall homology (between 39 and 92% identity) with similarly sized proteins of Lactobacillus reuteri, Enterococcus faecium, Streptococcus pneumoniae, Streptococcus agalactiae, and Lactobacillus plantarum. The carboxy-terminal 81 amino acids of Sep also have strong homology (86% identity) to the carboxy termini of the aggregation-promoting factor (APF) surface proteins of Lactobacillus gasseri and Lactobacillus johnsonii. The mature amino terminus of Sep contains a putative peptidoglycan-binding LysM domain, thereby making it distinct from APF proteins. We have identified a common motif within LysM domains that is shared with carbohydrate binding YG motifs which are found in streptococcal glucan-binding proteins and glucosyltransferases. Sep was investigated as a heterologous peptide expression vector in L. fermentum, Lactobacillus rhamnosus GG and Lactococcus lactis MG1363. Modified Sep containing an amino-terminal six-histidine epitope was found associated with the cells but was largely present in the supernatant in the L. fermentum, L. rhamnosus, and L. lactis hosts. Sep as well as the previously described surface protein BspA were used to express and secrete in L. fermentum or L. rhamnosus a fragment of human E-cadherin, which contains the receptor region for Listeria monocytogenes. This study demonstrates that Sep has potential for heterologous protein expression and export in lactic acid bacteria. PMID:15184172

Identification of a human src homology 2-containing protein-tyrosine-phosphatase: a putative homolog of Drosophila corkscrew.

PubMed Central

Freeman, R M; Plutzky, J; Neel, B G

1992-01-01

src homology 2 (SH2) domains direct binding to specific phosphotyrosyl proteins. Recently, SH2-containing protein-tyrosine-phosphatases (PTPs) were identified. Using degenerate oligonucleotides and the PCR, we have cloned a cDNA for an additional PTP, SH-PTP2, which contains two SH2 domains and is expressed ubiquitously. When expressed in Escherichia coli, SH-PTP2 displays tyrosine-specific phosphatase activity. Strong sequence similarity between SH-PTP2 and the Drosophila gene corkscrew (csw) and their similar patterns of expression suggest that SH-PTP2 is the human corkscrew homolog. Sequence comparisons between SH-PTP2, SH-PTP1, corkscrew, and other SH2-containing proteins suggest the existence of a subfamily of SH2 domains found specifically in PTPs, whereas comparison of the PTP domains of the SH2-containing PTPs with other tyrosine phosphatases suggests the existence of a subfamily of PTPs containing SH2 domains. Since corkscrew, a member of the terminal class signal transduction pathway, acts in concert with D-raf to positively transduce the signal generated by the receptor tyrosine kinase torso, these findings suggest several mechanisms by which SH-PTP2 may participate in mammalian signal transduction. Images PMID:1280823

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

Non-contact fluid characterization in containers using ultrasonic waves

DOEpatents

Sinha, Dipen N [Los Alamos, NM

2012-05-15

Apparatus and method for non-contact (stand-off) ultrasonic determination of certain characteristics of fluids in containers or pipes are described. A combination of swept frequency acoustic interferometry (SFAI), wide-bandwidth, air-coupled acoustic transducers, narrowband frequency data acquisition, and data conversion from the frequency domain to the time domain, if required, permits meaningful information to be extracted from such fluids.

Nucleotide-Binding Domain Leucine-Rich Repeat Containing Proteins and Intestinal Microbiota: Pivotal Players in Colitis and Colitis-Associated Cancer Development.

PubMed

Prossomariti, Anna; Sokol, Harry; Ricciardiello, Luigi

2018-01-01

The nucleotide-binding domain leucine-rich repeat containing (NLR) proteins play a fundamental role in innate immunity and intestinal tissue repair. A dysbiotic intestinal microbiota, developed as a consequence of alterations in NLR proteins, has recently emerged as a crucial hit for the development of ulcerative colitis (UC) and colitis-associated cancer (CAC). The concept of the existence of functional axes interconnecting bacteria with NLR proteins in a causal role in intestinal inflammation and CAC aroused a great interest for the potential development of preventive and therapeutic strategies against UC and CAC. However, the most recent scientific evidence, which highlights many confounding factors in studies based on microbiota characterization, underlines the need for an in-depth reconsideration of the data obtained until now. The purpose of this review is to discuss the recent findings concerning the cross talk between the NLR signaling and the intestinal microbiota in UC and CAC development, and to highlight the open issues that should be explored and addressed in future studies.

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

Prioritisation of associations between protein domains and complex diseases using domain-domain interaction networks.

PubMed

Wang, W; Zhang, W; Jiang, R; Luan, Y

2010-05-01

It is of vital importance to find genetic variants that underlie human complex diseases and locate genes that are responsible for these diseases. Since proteins are typically composed of several structural domains, it is reasonable to assume that harmful genetic variants may alter structures of protein domains, affect functions of proteins and eventually cause disorders. With this understanding, the authors explore the possibility of recovering associations between protein domains and complex diseases. The authors define associations between protein domains and disease families on the basis of associations between non-synonymous single nucleotide polymorphisms (nsSNPs) and complex diseases, similarities between diseases, and relations between proteins and domains. Based on a domain-domain interaction network, the authors propose a 'guilt-by-proximity' principle to rank candidate domains according to their average distance to a set of seed domains in the domain-domain interaction network. The authors validate the method through large-scale cross-validation experiments on simulated linkage intervals, random controls and the whole genome. Results show that areas under receiver operating characteristic curves (AUC scores) can be as high as 77.90%, and the mean rank ratios can be as low as 21.82%. The authors further offer a freely accessible web interface for a genome-wide landscape of associations between domains and disease families.

The Popeye domain containing protein family--A novel class of cAMP effectors with important functions in multiple tissues.

PubMed

Schindler, Roland F R; Brand, Thomas

2016-01-01

Popeye domain containing (Popdc) proteins are a unique family, which combine several different properties and functions in a surprisingly complex fashion. They are expressed in multiple tissues and cell types, present in several subcellular compartments, interact with different classes of proteins, and are associated with a variety of physiological and pathophysiological processes. Moreover, Popdc proteins bind the second messenger cAMP with high affinity and it is thought that they act as a novel class of cAMP effector proteins. Here, we will review the most important findings about the Popdc family, which accumulated since its discovery about 15 years ago. We will be focussing on Popdc protein interaction and function in striated muscle tissue. However, as a full picture only emerges if all aspects are taken into account, we will also describe what is currently known about the role of Popdc proteins in epithelial cells and in various types of cancer, and discuss these findings with regard to their relevance for cardiac and skeletal muscle. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

A BAR domain in the N terminus of the Arf GAP ASAP1 affects membrane structure and trafficking of epidermal growth factor receptor.

PubMed

Nie, Zhongzhen; Hirsch, Dianne S; Luo, Ruibai; Jian, Xiaoying; Stauffer, Stacey; Cremesti, Aida; Andrade, Josefa; Lebowitz, Jacob; Marino, Michael; Ahvazi, Bijan; Hinshaw, Jenny E; Randazzo, Paul A

2006-01-24

Arf GAPs are multidomain proteins that function in membrane traffic by inactivating the GTP binding protein Arf1. Numerous Arf GAPs contain a BAR domain, a protein structural element that contributes to membrane traffic by either inducing or sensing membrane curvature. We have examined the role of a putative BAR domain in the function of the Arf GAP ASAP1. ASAP1's N terminus, containing the putative BAR domain together with a PH domain, dimerized to form an extended structure that bound to large unilamellar vesicles containing acidic phospholipids, properties that define a BAR domain. A recombinant protein containing the BAR domain of ASAP1, together with the PH and Arf GAP domains, efficiently bent the surface of large unilamellar vesicles, resulting in the formation of tubular structures. This activity was regulated by Arf1*GTP binding to the Arf GAP domain. In vivo, the tubular structures induced by ASAP1 mutants contained epidermal growth factor receptor (EGFR) and Rab11, and ASAP1 colocalized in tubular structures with EGFR during recycling of receptor. Expression of ASAP1 accelerated EGFR trafficking and slowed cell spreading. An ASAP1 mutant lacking the BAR domain had no effect. The N-terminal BAR domain of ASAP1 mediates membrane bending and is necessary for ASAP1 function. The Arf dependence of the bending activity is consistent with ASAP1 functioning as an Arf effector.

Identification of novel protein domains required for the expression of an active dehydratase fragment from a polyunsaturated fatty acid synthase

PubMed Central

Oyola-Robles, Delise; Gay, Darren C; Trujillo, Uldaeliz; Sánchez-Parés, John M; Bermúdez, Mei-Ling; Rivera-Díaz, Mónica; Carballeira, Néstor M; Baerga-Ortiz, Abel

2013-01-01

Polyunsaturated fatty acids (PUFAs) are made in some strains of deep-sea bacteria by multidomain proteins that catalyze condensation, ketoreduction, dehydration, and enoyl-reduction. In this work, we have used the Udwary-Merski Algorithm sequence analysis tool to define the boundaries that enclose the dehydratase (DH) domains in a PUFA multienzyme. Sequence analysis revealed the presence of four areas of high structure in a region that was previously thought to contain only two DH domains as defined by FabA-homology. The expression of the protein fragment containing all four protein domains resulted in an active enzyme, while shorter protein fragments were not soluble. The tetradomain fragment was capable of catalyzing the conversion of crotonyl-CoA to β-hydroxybutyryl-CoA efficiently, as shown by UV absorbance change as well as by chromatographic retention of reaction products. Sequence alignments showed that the two novel domains contain as much sequence conservation as the FabA-homology domains, suggesting that they too may play a functional role in the overall reaction. Structure predictions revealed that all domains belong to the hotdog protein family: two of them contain the active site His70 residue present in FabA-like DHs, while the remaining two do not. Replacing the active site His residues in both FabA domains for Ala abolished the activity of the tetradomain fragment, indicating that the DH activity is contained within the FabA-homology regions. Taken together, these results provide a first glimpse into a rare arrangement of DH domains which constitute a defining feature of the PUFA synthases. PMID:23696301

The Persistence and Functional Impact of English Language Difficulties Experienced by Children Learning English as an Additional Language and Monolingual Peers

ERIC Educational Resources Information Center

Whiteside, Katie E.; Norbury, Courtenay Frazier

2017-01-01

Purpose: This study explored whether a monolingual-normed English language battery could identify children with English as an additional language (EAL) who have persistent English language learning difficulties that affect functional academic attainment. Method: Children with EAL (n = 43) and monolingual English-speaking children (n = 46)…

Teachers' Voices: Teachers' Reflections on Learning and Teaching through the Medium of English as an Additional Language in South Africa.

ERIC Educational Resources Information Center

Probyn, Margaret

2001-01-01

Explores perceptions and practice of teachers teaching through English as an additional language (EAL) in township schools in South Africa. Lessons of five excellent teachers teaching mathematics, accounting, science, business economics, and history through EAL were videotaped, and teachers were interviewed about their perceptions of teaching…

Structure of a double ubiquitin-like domain in the talin head: a role in integrin activation

PubMed Central

Goult, Benjamin T; Bouaouina, Mohamed; Elliott, Paul R; Bate, Neil; Patel, Bipin; Gingras, Alexandre R; Grossmann, J Günter; Roberts, Gordon C K; Calderwood, David A; Critchley, David R; Barsukov, Igor L

2010-01-01

Talin is a 270-kDa protein that activates integrins and couples them to cytoskeletal actin. Talin contains an N-terminal FERM domain comprised of F1, F2 and F3 domains, but it is atypical in that F1 contains a large insert and is preceded by an extra domain F0. Although F3 contains the binding site for β-integrin tails, F0 and F1 are also required for activation of β1-integrins. Here, we report the solution structures of F0, F1 and of the F0F1 double domain. Both F0 and F1 have ubiquitin-like folds joined in a novel fixed orientation by an extensive charged interface. The F1 insert forms a loop with helical propensity, and basic residues predicted to reside on one surface of the helix are required for binding to acidic phospholipids and for talin-mediated activation of β1-integrins. This and the fact that basic residues on F2 and F3 are also essential for integrin activation suggest that extensive interactions between the talin FERM domain and acidic membrane phospholipids are required to orientate the FERM domain such that it can activate integrins. PMID:20150896

Slicing-independent RISC activation requires the argonaute PAZ domain.

PubMed

Gu, Shuo; Jin, Lan; Huang, Yong; Zhang, Feijie; Kay, Mark A

2012-08-21

Small RNAs regulate genetic networks through a ribonucleoprotein complex called the RNA-induced silencing complex (RISC), which, in mammals, contains at its center one of four Argonaute proteins (Ago1-Ago4). A key regulatory event in the RNA interference (RNAi) and microRNA (miRNA) pathways is Ago loading, wherein double-stranded small-RNA duplexes are incorporated into RISC (pre-RISC) and then become single-stranded (mature RISC), a process that is not well understood. The Agos contain an evolutionarily conserved PAZ (Piwi/Argonaute/Zwille) domain whose primary function is to bind the 3' end of small RNAs. We created multiple PAZ-domain-disrupted mutant Ago proteins and studied their biochemical properties and biological functionality in cells. We found that the PAZ domain is dispensable for Ago loading of slicing-competent RISC. In contrast, in the absence of slicer activity or slicer-substrate duplex RNAs, PAZ-disrupted Agos bound duplex small interfering RNAs, but were unable to unwind or eject the passenger strand and form functional RISC complexes. We have discovered that the highly conserved PAZ domain plays an important role in RISC activation, providing new mechanistic insights into how miRNAs regulate genes, as well as new insights for future design of miRNA- and RNAi-based therapeutics. Copyright © 2012 Elsevier Ltd. All rights reserved.

An improved model for teaching use of electronic apex locators.

PubMed

Tchorz, J P; Hellwig, E; Altenburger, M J

2012-04-01

To develop a simple, practical and inexpensive model, which enables the use of electronic apex locators (EALs) during pre-clinical and continuing education. Extracted teeth were placed in a mould and embedded in acrylic resin. The resin was applied in two consecutive steps to form a cavity around the root apices. A closable plastic tube serves as a valve, and a steel wire connects to the EAL. With its semi-closed reservoir for conductive fluids surrounding the root apices, the new model enables working length measurements of root canals using EALs. The model simulates the clinical situation for endodontic teaching purposes, as it allows working length determination of root canals as recommended. The measuring results of the EAL can be verified by radiography. At the same time, the roots are not directly visible and accessible to the user, allowing a precise evaluation and grading of the treatment. © 2011 International Endodontic Journal.

The large terminase DNA packaging motor grips DNA with its ATPase domain for cleavage by the flexible nuclease domain

PubMed Central

Hilbert, Brendan J.; Hayes, Janelle A.; Stone, Nicholas P.; Xu, Rui-Gang

2017-01-01

Abstract Many viruses use a powerful terminase motor to pump their genome inside an empty procapsid shell during virus maturation. The large terminase (TerL) protein contains both enzymatic activities necessary for packaging in such viruses: the adenosine triphosphatase (ATPase) that powers DNA translocation and an endonuclease that cleaves the concatemeric genome at both initiation and completion of genome packaging. However, how TerL binds DNA during translocation and cleavage remains mysterious. Here we investigate DNA binding and cleavage using TerL from the thermophilic phage P74-26. We report the structure of the P74-26 TerL nuclease domain, which allows us to model DNA binding in the nuclease active site. We screened a large panel of TerL variants for defects in binding and DNA cleavage, revealing that the ATPase domain is the primary site for DNA binding, and is required for nuclease activity. The nuclease domain is dispensable for DNA binding but residues lining the active site guide DNA for cleavage. Kinetic analysis of DNA cleavage suggests flexible tethering of the nuclease domains during DNA cleavage. We propose that interactions with the procapsid during DNA translocation conformationally restrict the nuclease domain, inhibiting cleavage; TerL release from the capsid upon completion of packaging unlocks the nuclease domains to cleave DNA. PMID:28082398

Adjoint-based optimization of PDEs in moving domains

NASA Astrophysics Data System (ADS)

Protas, Bartosz; Liao, Wenyuan

2008-02-01

In this investigation we address the problem of adjoint-based optimization of PDE systems in moving domains. As an example we consider the one-dimensional heat equation with prescribed boundary temperatures and heat fluxes. We discuss two methods of deriving an adjoint system necessary to obtain a gradient of a cost functional. In the first approach we derive the adjoint system after mapping the problem to a fixed domain, whereas in the second approach we derive the adjoint directly in the moving domain by employing methods of the noncylindrical calculus. We show that the operations of transforming the system from a variable to a fixed domain and deriving the adjoint do not commute and that, while the gradient information contained in both systems is the same, the second approach results in an adjoint problem with a simpler structure which is therefore easier to implement numerically. This approach is then used to solve a moving boundary optimization problem for our model system.

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

PubMed

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

2017-10-15

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

Exploring the Roles of Proline in Three-Dimensional Domain Swapping from Structure Analysis and Molecular Dynamics Simulations.

PubMed

Huang, Yongqi; Gao, Meng; Su, Zhengding

2018-02-01

Three-dimensional (3D) domain swapping is a mechanism to form protein oligomers. It has been proposed that several factors, including proline residues in the hinge region, may affect the occurrence of 3D domain swapping. Although introducing prolines into the hinge region has been found to promote domain swapping for some proteins, the opposite effect has also been observed in several studies. So far, how proline affects 3D domain swapping remains elusive. In this work, based on a large set of 3D domain-swapped structures, we performed a systematic analysis to explore the correlation between the presence of proline in the hinge region and the occurrence of 3D domain swapping. We further analyzed the conformations of proline and pre-proline residues to investigate the roles of proline in 3D domain swapping. We found that more than 40% of the domain-swapped structures contained proline residues in the hinge region. Unexpectedly, conformational transitions of proline residues were rarely observed upon domain swapping. Our analyses showed that hinge regions containing proline residues preferred more extended conformations, which may be beneficial for the occurrence of domain swapping by facilitating opening of the exchanged segments.

HrpW of Erwinia amylovora, a New Harpin That Contains a Domain Homologous to Pectate Lyases of a Distinct Class

PubMed Central

Kim, Jihyun F.; Beer, Steven V.

1998-01-01

Harpins, such as HrpN of Erwinia amylovora, are extracellular glycine-rich proteins that elicit the hypersensitive reaction (HR). We identified hrpW of E. amylovora, which encodes a protein similar to known harpins in that it is acidic, rich in glycine and serine, and lacks cysteine. A putative HrpL-dependent promoter was identified upstream of hrpW, and Western blot analysis of hrpL mutants indicated that the production of HrpW is regulated by hrpL. HrpW is secreted via the Hrp (type III) pathway based on analysis of wild-type strains and hrp secretion mutants. When infiltrated into plants, HrpW induced rapid tissue collapse, which required active plant metabolism. The HR-eliciting activity was heat stable and protease sensitive. Thus, we concluded that HrpW is a new harpin. HrpW of E. amylovora consists of two domains connected by a Pro and Ser-rich sequence. A fragment containing the N-terminal domain was sufficient to elicit the HR. Although no pectate lyase activity was detected, the C-terminal region of HrpW is homologous to pectate lyases of a unique class, suggesting that HrpW may be targeted to the plant cell wall. Southern analysis indicated that hrpW is conserved among several Erwinia species, and hrpW, provided in trans, enhanced the HR-inducing ability of a hrpN mutant. However, HrpW did not increase the virulence of a hrpN mutant in host tissue, and hrpW mutants retained the wild-type ability to elicit the HR in nonhosts and to cause disease in hosts. PMID:9748455

Using llama derived single domain antibodies to target botulinum neurotoxins

NASA Astrophysics Data System (ADS)

Swain, Marla D.; Anderson, George P.; Bernstein, Rachael D.; Liu, Jinny L.; Goldman, Ellen R.

2010-04-01

Llama serum contains both conventional IgG as well as unique forms of antibody that contain only heavy chains where antigen binding is mediated through a single variable domain. These variable domains can be expressed recombinantly and are referred to as single domain antibodies (sdAb). SdAb are among the smallest known naturally derived antigen binding fragments, possess good solubility, thermal stability, and can refold after heat and chemical denaturation. Llamas were immunized with either BoNT A or B toxoid and phage display libraries prepared. Single domain antibodies (sdAb) that were able to detect botulinum neurotoxin (BoNT) serotypes A and B were selected from their respective libraries. Here, the binders obtained by panning the BoNT B library on either BoNT B toxoid or BoNT B complex toxoid coated plates or BoNT B toxin coupled microspheres are described. Using these panning methods, we selected for binders that showed specificity for BoNT B. Phage displayed binders were screened, moved to a protein expression vector and soluble sdAb was produced. Using a Luminex flow cytometer binders were evaluated in direct binding assays. We have exploited the unique properties of sdAb and used them as biological recognition elements in immuno-based sensors that can detect BoNT B.

Analysis of the Protein Domain and Domain Architecture Content in Fungi and Its Application in the Search of New Antifungal Targets

PubMed Central

Barrera, Alejandro; Alastruey-Izquierdo, Ana; Martín, María J.; Cuesta, Isabel; Vizcaíno, Juan Antonio

2014-01-01

Over the past several years fungal infections have shown an increasing incidence in the susceptible population, and caused high mortality rates. In parallel, multi-resistant fungi are emerging in human infections. Therefore, the identification of new potential antifungal targets is a priority. The first task of this study was to analyse the protein domain and domain architecture content of the 137 fungal proteomes (corresponding to 111 species) available in UniProtKB (UniProt KnowledgeBase) by January 2013. The resulting list of core and exclusive domain and domain architectures is provided in this paper. It delineates the different levels of fungal taxonomic classification: phylum, subphylum, order, genus and species. The analysis highlighted Aspergillus as the most diverse genus in terms of exclusive domain content. In addition, we also investigated which domains could be considered promiscuous in the different organisms. As an application of this analysis, we explored three different ways to detect potential targets for antifungal drugs. First, we compared the domain and domain architecture content of the human and fungal proteomes, and identified those domains and domain architectures only present in fungi. Secondly, we looked for information regarding fungal pathways in public repositories, where proteins containing promiscuous domains could be involved. Three pathways were identified as a result: lovastatin biosynthesis, xylan degradation and biosynthesis of siroheme. Finally, we classified a subset of the studied fungi in five groups depending on their occurrence in clinical samples. We then looked for exclusive domains in the groups that were more relevant clinically and determined which of them had the potential to bind small molecules. Overall, this study provides a comprehensive analysis of the available fungal proteomes and shows three approaches that can be used as a first step in the detection of new antifungal targets. PMID:25033262

Analysis of the protein domain and domain architecture content in fungi and its application in the search of new antifungal targets.

PubMed

Barrera, Alejandro; Alastruey-Izquierdo, Ana; Martín, María J; Cuesta, Isabel; Vizcaíno, Juan Antonio

2014-07-01

Over the past several years fungal infections have shown an increasing incidence in the susceptible population, and caused high mortality rates. In parallel, multi-resistant fungi are emerging in human infections. Therefore, the identification of new potential antifungal targets is a priority. The first task of this study was to analyse the protein domain and domain architecture content of the 137 fungal proteomes (corresponding to 111 species) available in UniProtKB (UniProt KnowledgeBase) by January 2013. The resulting list of core and exclusive domain and domain architectures is provided in this paper. It delineates the different levels of fungal taxonomic classification: phylum, subphylum, order, genus and species. The analysis highlighted Aspergillus as the most diverse genus in terms of exclusive domain content. In addition, we also investigated which domains could be considered promiscuous in the different organisms. As an application of this analysis, we explored three different ways to detect potential targets for antifungal drugs. First, we compared the domain and domain architecture content of the human and fungal proteomes, and identified those domains and domain architectures only present in fungi. Secondly, we looked for information regarding fungal pathways in public repositories, where proteins containing promiscuous domains could be involved. Three pathways were identified as a result: lovastatin biosynthesis, xylan degradation and biosynthesis of siroheme. Finally, we classified a subset of the studied fungi in five groups depending on their occurrence in clinical samples. We then looked for exclusive domains in the groups that were more relevant clinically and determined which of them had the potential to bind small molecules. Overall, this study provides a comprehensive analysis of the available fungal proteomes and shows three approaches that can be used as a first step in the detection of new antifungal targets.

Developing Language Awareness for Teachers of Emergent Bilingual Learners Using Dialogic Inquiry

ERIC Educational Resources Information Center

Wallen, Matthew; Kelly-Holmes, Helen

2017-01-01

This study endeavoured to awaken mainstream teachers' awareness of language, specifically related to teaching emergent bilingual children who are learning English as an additional language (EAL) in the Republic of Ireland. Because EAL learners spend the majority of the day in the mainstream classroom, mainstream teachers' language awareness may…

The Comprehension Skills of Children Learning English as an Additional Language

ERIC Educational Resources Information Center

Burgoyne, K.; Kelly nee Hutchinson, J. M.; Whiteley, H. E.; Spooner, A.

2009-01-01

Background: Data from national test results suggests that children who are learning English as an additional language (EAL) experience relatively lower levels of educational attainment in comparison to their monolingual, English-speaking peers. Aims: The relative underachievement of children who are learning EAL demands that the literacy needs of…

English Language Proficiency and Early School Attainment among Children Learning English as an Additional Language

ERIC Educational Resources Information Center

Whiteside, Katie E.; Gooch, Debbie; Norbury, Courtenay F.

2017-01-01

Children learning English as an additional language (EAL) often experience lower academic attainment than monolingual peers. In this study, teachers provided ratings of English language proficiency and social, emotional, and behavioral functioning for 782 children with EAL and 6,485 monolingual children in reception year (ages 4-5). Academic…

The cysteine-rich domain regulates ADAM protease function in vivo.

PubMed

Smith, Katherine M; Gaultier, Alban; Cousin, Helene; Alfandari, Dominique; White, Judith M; DeSimone, Douglas W

2002-12-09

ADAMs are membrane-anchored proteases that regulate cell behavior by proteolytically modifying the cell surface and ECM. Like other membrane-anchored proteases, ADAMs contain candidate "adhesive" domains downstream of their metalloprotease domains. The mechanism by which membrane-anchored cell surface proteases utilize these putative adhesive domains to regulate protease function in vivo is not well understood. We address this important question by analyzing the relative contributions of downstream extracellular domains (disintegrin, cysteine rich, and EGF-like repeat) of the ADAM13 metalloprotease during Xenopus laevis development. When expressed in embryos, ADAM13 induces hyperplasia of the cement gland, whereas ADAM10 does not. Using chimeric constructs, we find that the metalloprotease domain of ADAM10 can substitute for that of ADAM13, but that specificity for cement gland expansion requires a downstream extracellular domain of ADAM13. Analysis of finer resolution chimeras indicates an essential role for the cysteine-rich domain and a supporting role for the disintegrin domain. These and other results reveal that the cysteine-rich domain of ADAM13 cooperates intramolecularly with the ADAM13 metalloprotease domain to regulate its function in vivo. Our findings thus provide the first evidence that a downstream extracellular adhesive domain plays an active role in regulating ADAM protease function in vivo. These findings are likely relevant to other membrane-anchored cell surface proteases.

Artificial Intelligence Techniques for the Berth Allocation and Container Stacking Problems in Container Terminals

NASA Astrophysics Data System (ADS)

Salido, Miguel A.; Rodriguez-Molins, Mario; Barber, Federico

The Container Stacking Problem and the Berth Allocation Problem are two important problems in maritime container terminal's management which are clearly related. Terminal operators normally demand all containers to be loaded into an incoming vessel should be ready and easily accessible in the terminal before vessel's arrival. Similarly, customers (i.e., vessel owners) expect prompt berthing of their vessels upon arrival. In this paper, we present an artificial intelligence based-integrated system to relate these problems. Firstly, we develop a metaheuristic algorithm for berth allocation which generates an optimized order of vessel to be served according to existing berth constraints. Secondly, we develop a domain-oriented heuristic planner for calculating the number of reshuffles needed to allocate containers in the appropriate place for a given berth ordering of vessels. By combining these optimized solutions, terminal operators can be assisted to decide the most appropriated solution in each particular case.

Chimeric Plant Calcium/Calmodulin-Dependent Protein Kinase Gene with a Neural Visinin-Like Calcium-Binding Domain

NASA Technical Reports Server (NTRS)

Patil, Shameekumar; Takezawa, D.; Poovaiah, B. W.

1995-01-01

Calcium, a universal second messenger, regulates diverse cellular processes in eukaryotes. Ca-2(+) and Ca-2(+)/calmodulin-regulated protein phosphorylation play a pivotal role in amplifying and diversifying the action of Ca-2(+)- mediated signals. A chimeric Ca-2(+)/calmodulin-dependent protein kinase (CCaMK) gene with a visinin-like Ca-2(+)- binding domain was cloned and characterized from lily. The cDNA clone contains an open reading frame coding for a protein of 520 amino acids. The predicted structure of CCaMK contains a catalytic domain followed by two regulatory domains, a calmodulin-binding domain and a visinin-like Ca-2(+)-binding domain. The amino-terminal region of CCaMK contains all 11 conserved subdomains characteristic of serine/threonine protein kinases. The calmodulin-binding region of CCaMK has high homology (79%) to alpha subunit of mammalian Ca-2(+)/calmodulin-dependent protein kinase. The calmodulin-binding region is fused to a neural visinin-like domain that contains three Ca-2(+)-binding EF-hand motifs and a biotin-binding site. The Escherichia coli-expressed protein (approx. 56 kDa) binds calmodulin in a Ca-2(+)-dependent manner. Furthermore, Ca-45-binding assays revealed that CCaMK directly binds Ca-2(+). The CCaMK gene is preferentially expressed in developing anthers. Southern blot analysis revealed that CCaMK is encoded by a single gene. The structural features of the gene suggest that it has multiple regulatory controls and could play a unique role in Ca-2(+) signaling in plants.

Structural and functional analysis of the YAP-binding domain of human TEAD2

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

Tian, Wei; Yu, Jianzhong; Tomchick, Diana R.

2010-06-15

The Hippo pathway controls organ size and suppresses tumorigenesis in metazoans by blocking cell proliferation and promoting apoptosis. The TEAD1-4 proteins (which contain a DNA-binding domain but lack an activation domain) interact with YAP (which lacks a DNA-binding domain but contains an activation domain) to form functional heterodimeric transcription factors that activate proliferative and prosurvival gene expression programs. The Hippo pathway inhibits the YAP-TEAD hybrid transcription factors by phosphorylating and promoting cytoplasmic retention of YAP. Here we report the crystal structure of the YAP-binding domain (YBD) of human TEAD2. TEAD2 YBD adopts an immunoglobulin-like {beta}-sandwich fold with two extra helix-turn-helixmore » inserts. NMR studies reveal that the TEAD-binding domain of YAP is natively unfolded and that TEAD binding causes localized conformational changes in YAP. In vitro binding and in vivo functional assays define an extensive conserved surface of TEAD2 YBD as the YAP-binding site. Therefore, our studies suggest that a short segment of YAP adopts an extended conformation and forms extensive contacts with a rigid surface of TEAD. Targeting a surface-exposed pocket of TEAD might be an effective strategy to disrupt the YAP-TEAD interaction and to reduce the oncogenic potential of YAP.« less

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.

Membrane anchoring of aminoacyl-tRNA synthetases by convergent acquisition of a novel protein domain.

PubMed

Olmedo-Verd, Elvira; Santamaría-Gómez, Javier; Ochoa de Alda, Jesús A G; Ribas de Pouplana, Lluis; Luque, Ignacio

2011-11-25

Four distinct aminoacyl-tRNA synthetases (aaRSs) found in some cyanobacterial species contain a novel protein domain that bears two putative transmembrane helices. This CAAD domain is present in glutamyl-, isoleucyl-, leucyl-, and valyl-tRNA synthetases, the latter of which has probably recruited the domain more than once during evolution. Deleting the CAAD domain from the valyl-tRNA synthetase of Anabaena sp. PCC 7120 did not significantly modify the catalytic properties of this enzyme, suggesting that it does not participate in its canonical tRNA-charging function. Multiple lines of evidence suggest that the function of the CAAD domain is structural, mediating the membrane anchorage of the enzyme, although membrane localization of aaRSs has not previously been described in any living organism. Synthetases containing the CAAD domain were localized in the intracytoplasmic thylakoid membranes of cyanobacteria and were largely absent from the plasma membrane. The CAAD domain was necessary and apparently sufficient for protein targeting to membranes. Moreover, localization of aaRSs in thylakoids was important under nitrogen limiting conditions. In Anabaena, a multicellular filamentous cyanobacterium often used as a model for prokaryotic cell differentiation, valyl-tRNA synthetase underwent subcellular relocation at the cell poles during heterocyst differentiation, a process also dependent on the CAAD domain.

Crystal structure of the motor domain of a class-I myosin

PubMed Central

Kollmar, Martin; Dürrwang, Ulrike; Kliche, Werner; Manstein, Dietmar J.; Kull, F.Jon

2002-01-01

The crystal structure of the motor domain of Dictyostelium discoideum myosin-IE, a monomeric unconventional myosin, was determined. The crystallographic asymmetric unit contains four independently resolved molecules, highlighting regions that undergo large conformational changes. Differences are particularly pronounced in the actin binding region and the converter domain. The changes in position of the converter domain reflect movements both parallel to and perpendicular to the actin axis. The orientation of the converter domain is ∼30° further up than in other myosin structures, indicating that MyoE can produce a larger power stroke by rotating its lever arm through a larger angle. The role of extended loops near the actin-binding site is discussed in the context of cellular localization. The core regions of the motor domain are similar, and the structure reveals how that core is stabilized in the absence of an N-terminal SH3-like domain. PMID:12032065

Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily

PubMed Central

Lenoir, Marc; Kufareva, Irina; Abagyan, Ruben; Overduin, Michael

2015-01-01

The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH) domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH) and Tec homology (TH) domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA) program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer. PMID:26512702

An information theory framework for dynamic functional domain connectivity.

PubMed

Vergara, Victor M; Miller, Robyn; Calhoun, Vince

2017-06-01

Dynamic functional network connectivity (dFNC) analyzes time evolution of coherent activity in the brain. In this technique dynamic changes are considered for the whole brain. This paper proposes an information theory framework to measure information flowing among subsets of functional networks call functional domains. Our method aims at estimating bits of information contained and shared among domains. The succession of dynamic functional states is estimated at the domain level. Information quantity is based on the probabilities of observing each dynamic state. Mutual information measurement is then obtained from probabilities across domains. Thus, we named this value the cross domain mutual information (CDMI). Strong CDMIs were observed in relation to the subcortical domain. Domains related to sensorial input, motor control and cerebellum form another CDMI cluster. Information flow among other domains was seldom found. Other methods of dynamic connectivity focus on whole brain dFNC matrices. In the current framework, information theory is applied to states estimated from pairs of multi-network functional domains. In this context, we apply information theory to measure information flow across functional domains. Identified CDMI clusters point to known information pathways in the basal ganglia and also among areas of sensorial input, patterns found in static functional connectivity. In contrast, CDMI across brain areas of higher level cognitive processing follow a different pattern that indicates scarce information sharing. These findings show that employing information theory to formally measured information flow through brain domains reveals additional features of functional connectivity. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification of novel protein domains required for the expression of an active dehydratase fragment from a polyunsaturated fatty acid synthase.

PubMed

Oyola-Robles, Delise; Gay, Darren C; Trujillo, Uldaeliz; Sánchez-Parés, John M; Bermúdez, Mei-Ling; Rivera-Díaz, Mónica; Carballeira, Néstor M; Baerga-Ortiz, Abel

2013-07-01

Polyunsaturated fatty acids (PUFAs) are made in some strains of deep-sea bacteria by multidomain proteins that catalyze condensation, ketoreduction, dehydration, and enoyl-reduction. In this work, we have used the Udwary-Merski Algorithm sequence analysis tool to define the boundaries that enclose the dehydratase (DH) domains in a PUFA multienzyme. Sequence analysis revealed the presence of four areas of high structure in a region that was previously thought to contain only two DH domains as defined by FabA-homology. The expression of the protein fragment containing all four protein domains resulted in an active enzyme, while shorter protein fragments were not soluble. The tetradomain fragment was capable of catalyzing the conversion of crotonyl-CoA to β-hydroxybutyryl-CoA efficiently, as shown by UV absorbance change as well as by chromatographic retention of reaction products. Sequence alignments showed that the two novel domains contain as much sequence conservation as the FabA-homology domains, suggesting that they too may play a functional role in the overall reaction. Structure predictions revealed that all domains belong to the hotdog protein family: two of them contain the active site His70 residue present in FabA-like DHs, while the remaining two do not. Replacing the active site His residues in both FabA domains for Ala abolished the activity of the tetradomain fragment, indicating that the DH activity is contained within the FabA-homology regions. Taken together, these results provide a first glimpse into a rare arrangement of DH domains which constitute a defining feature of the PUFA synthases. Copyright © 2013 The Protein Society.

Analgesic and physiological effect of electroacupuncture combined with epidural lidocaine in goats.

PubMed

Cui, Lu-Ying; Guo, Ni-Ni; Li, Yu-Lin; Li, Meng; Ding, Ming-Xing

2017-07-01

To investigate physiological and antinociceptive effects of electroacupuncture (EA) with lidocaine epidural nerve block in goats. Prospective experimental trial. Forty-eight hybrid male goats weighing 27 ± 2 kg. The goats were randomly assigned to six groups: L2.2, epidural lidocaine (2.2 mg kg -1 ); L4.4, epidural lidocaine (4.4 mg kg -1 ); EA; EA-L1.1, EA with epidural lidocaine (1.1 mg kg -1 ); EA-L2.2, EA with epidural lidocaine (2.2 mg kg -1 ); and EA-L4.4, EA with epidural lidocaine (4.4 mg kg -1 ). EA was administered for 120 minutes. Epidural lidocaine was administered 25 minutes after EA started. Nociceptive thresholds of flank and thigh regions, abdominal muscle tone, mean arterial pressure (MAP), heart rate (HR), respiratory frequency (f R ) and rectal temperature were recorded at 30, 60, 90, 120, 150 and 180 minutes. Lidocaine dose-dependently increased nociceptive thresholds. There were no differences in nociceptive thresholds between L4.4 and EA from 30 to 120 minutes. The threshold in EA-L2.2 was lower than in EA-L4.4 from 30 to 120 minutes, but higher than in EA-L1.1 from 30 to 150 minutes or in L4.4 from 30 to 180 minutes. The abdominal muscle tone in EA-L2.2 was higher at 30 minutes, but lower at 90 and 120 minutes than at 0 minutes. There were no differences in muscle tone between L4.4 and L2.2 or EA-L4.4, and between any two of the three EA-lidocaine groups from 0 to 180 minutes. The f R and HR decreased in L4.4 at 60 and 90 minutes compared with 0 minutes. No differences in f R , HR, MAP and temperature among the groups occurred from 30 to 180 minutes. EA combined with 2.2 mg kg -1 epidural lidocaine provides better antinociceptive effect than 4.4 mg kg -1 epidural lidocaine alone in goats. EA provided antinociception and allowed a decrease in epidural lidocaine dose. Copyright © 2017 Association of Veterinary Anaesthetists and American College of Veterinary Anesthesia and Analgesia. Published by Elsevier Ltd. All rights

MARS-MD: rejection based image domain material decomposition

NASA Astrophysics Data System (ADS)

Bateman, C. J.; Knight, D.; Brandwacht, B.; McMahon, J.; Healy, J.; Panta, R.; Aamir, R.; Rajendran, K.; Moghiseh, M.; Ramyar, M.; Rundle, D.; Bennett, J.; de Ruiter, N.; Smithies, D.; Bell, S. T.; Doesburg, R.; Chernoglazov, A.; Mandalika, V. B. H.; Walsh, M.; Shamshad, M.; Anjomrouz, M.; Atharifard, A.; Vanden Broeke, L.; Bheesette, S.; Kirkbride, T.; Anderson, N. G.; Gieseg, S. P.; Woodfield, T.; Renaud, P. F.; Butler, A. P. H.; Butler, P. H.

2018-05-01

This paper outlines image domain material decomposition algorithms that have been routinely used in MARS spectral CT systems. These algorithms (known collectively as MARS-MD) are based on a pragmatic heuristic for solving the under-determined problem where there are more materials than energy bins. This heuristic contains three parts: (1) splitting the problem into a number of possible sub-problems, each containing fewer materials; (2) solving each sub-problem; and (3) applying rejection criteria to eliminate all but one sub-problem's solution. An advantage of this process is that different constraints can be applied to each sub-problem if necessary. In addition, the result of this process is that solutions will be sparse in the material domain, which reduces crossover of signal between material images. Two algorithms based on this process are presented: the Segmentation variant, which uses segmented material classes to define each sub-problem; and the Angular Rejection variant, which defines the rejection criteria using the angle between reconstructed attenuation vectors.

East Asian International Student Experiences as Learners of English as an Additional Language: Implications for School Counsellors

ERIC Educational Resources Information Center

Popadiuk, Natalee E.; Marshall, Steve

2011-01-01

In the school counselling literature, little focus is placed on international students who are learners of English as an Additional Language (EAL) and on school counselling support related to their language acquisition. Using the Critical Incident Technique, we analyzed transcripts of 21 international EAL students from China, Japan, and Korea who…

The Development of Comprehension and Reading-Related Skills in Children Learning English as an Additional Language and Their Monolingual, English-Speaking Peers

ERIC Educational Resources Information Center

Burgoyne, K.; Whiteley, H. E.; Hutchinson, J. M.

2011-01-01

Background: A significant number of pupils in UK schools learn English as an additional language (EAL). Relative differences between the educational attainment of this group and monolingual, English-speaking pupils call for an exploration of the literacy needs of EAL learners. Aims: This study explores the developmental progression of reading and…

A fully automatic evolutionary classification of protein folds: Dali Domain Dictionary version 3

PubMed Central

Dietmann, Sabine; Park, Jong; Notredame, Cedric; Heger, Andreas; Lappe, Michael; Holm, Liisa

2001-01-01

The Dali Domain Dictionary (http://www.ebi.ac.uk/dali/domain) is a numerical taxonomy of all known structures in the Protein Data Bank (PDB). The taxonomy is derived fully automatically from measurements of structural, functional and sequence similarities. Here, we report the extension of the classification to match the traditional four hierarchical levels corresponding to: (i) supersecondary structural motifs (attractors in fold space), (ii) the topology of globular domains (fold types), (iii) remote homologues (functional families) and (iv) homologues with sequence identity above 25% (sequence families). The computational definitions of attractors and functional families are new. In September 2000, the Dali classification contained 10 531 PDB entries comprising 17 101 chains, which were partitioned into five attractor regions, 1375 fold types, 2582 functional families and 3724 domain sequence families. Sequence families were further associated with 99 582 unique homologous sequences in the HSSP database, which increases the number of effectively known structures several-fold. The resulting database contains the description of protein domain architecture, the definition of structural neighbours around each known structure, the definition of structurally conserved cores and a comprehensive library of explicit multiple alignments of distantly related protein families. PMID:11125048

Evaluation of rice tetraticopeptide domain-containing thioredoxin as a novel solubility-enhancing fusion tag in Escherichia coli.

PubMed

Xiao, Wenjun; Jiang, Li; Wang, Weiyu; Wang, Ruyue; Fan, Jun

2018-02-01

Fusion of solubility-enhancing tag is frequently used for improving soluble production of target protein in Escherichia coli. The Arabidopsis tetraticopeptide domain-containing thioredoxin (TDX) has been documented to exhibit functions of disulfide reductase, foldase chaperone, and holdase chaperone. Here, we identified that fusion of rice TDX with the smaller size increased soluble expression levels of three fluorescent proteins with different fluorophores in the E. coli strain BL21(DE3) or the Rosetta (DE3) strain with coexpression of six rare tRNAs, but decreased conformational quality of certain fluorescent proteins, as comparison with the His6-tagged ones. Among five maize proteins, the rice TDX fusion carrier displayed higher solubility-enhancing activity than the yeast SUMO3 tag toward three proteins in both E. coli strains. Five fusion constructs were cleaved with the co-expressed TEV protease variant, but the released target proteins were partly insolubly aggregated in vivo. Attachment of the His6-tag to the TDX tagged proteins had little impact on protein solubility. After Ni-NTA purification, five His6-TDX tagged proteins displayed different apparent purities. Taken together, our work presents that rice TDX tag is a novel solubility enhancer. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

The Kunitz-protease inhibitor domain in amyloid precursor protein reduces cellular mitochondrial enzymes expression and function.

PubMed

Chua, Li-Min; Lim, Mei-Li; Wong, Boon-Seng

2013-08-09

Mitochondrial dysfunction is a prominent feature of Alzheimer's disease (AD) and this can be contributed by aberrant metabolic enzyme function. But, the mechanism causing this enzymatic impairment is unclear. Amyloid precursor protein (APP) is known to be alternatively spliced to produce three major isoforms in the brain (APP695, APP751, APP770). Both APP770 and APP751 contain the Kunitz Protease Inhibitory (KPI) domain, but the former also contain an extra OX-2 domain. APP695 on the other hand, lacks both domains. In AD, up-regulation of the KPI-containing APP isoforms has been reported. But the functional contribution of this elevation is unclear. In the present study, we have expressed and compared the effect of the non-KPI containing APP695 and the KPI-containing APP751 on mitochondrial function. We found that the KPI-containing APP751 significantly decreased the expression of three major mitochondrial metabolic enzymes; citrate synthase, succinate dehydrogenase and cytochrome c oxidase (COX IV). This reduction lowers the NAD(+)/NADH ratio, COX IV activity and mitochondrial membrane potential. Overall, this study demonstrated that up-regulation of the KPI-containing APP isoforms is likely to contribute to the impairment of metabolic enzymes and mitochondrial function in AD. Copyright © 2013 Elsevier Inc. All rights reserved.

FOLLITROPIN RECEPTORS CONTAIN CRYPTIC LIGAND BINDING SITES1

PubMed Central

Lin, Win; Bernard, Michael P.; Cao, Donghui; Myers, Rebecca V.; Kerrigan, John E.; Moyle, William R.

2007-01-01

Human choriogonadotropin (hCG) and follitropin (hFSH) have been shown to contact different regions of the extracellular domains of G-protein coupled lutropin (LHR) and follitropin (FSHR) receptors. We report here that hCG and hFSH analogs interact with an FSHR/LHR chimera having only two unique LHR residues similar to the manners in which they dock with LHR and FSHR, respectively. This shows that although the FSHR does not normally bind hCG, it contains a cryptic lutropin binding site that has the potential to recognize hCG in a manner similar to the LHR. The presence of this cryptic site may explain why equine lutropins bind many mammalian FSHR and why mutations in the transmembrane domain distant from the extracellular domain enable the FSHR to bind hCG. The leucine-rich repeat domain (LRD) of the FSHR also appears to contain a cryptic FSH binding site that is obscured by other parts of the extracellular domain. This will explain why contacts seen in crystals of hFSH complexed with an LRD fragment of the human FSHR are hard to reconcile with the abilities of FSH analogs to interact with membrane G-protein coupled FSHR. We speculate that cryptic lutropin binding sites in the FSHR, which are also likely to be present in thyrotropin receptors (TSHR), permit the physiological regulation of ligand binding specificity. Cryptic FSH binding sites in the LRD may enable alternate spliced forms of the FSHR to interact with FSH. PMID:17059863

Frequency Domain Computer Programs for Prediction and Analysis of Rail Vehicle Dynamics : Volume 2. Appendixes

DOT National Transportation Integrated Search

1975-12-01

Frequency domain computer programs developed or acquired by TSC for the analysis of rail vehicle dynamics are described in two volumes. Volume 2 contains program listings including subroutines for the four TSC frequency domain programs described in V...

Protein domains of unknown function are essential in bacteria.

PubMed

Goodacre, Norman F; Gerloff, Dietlind L; Uetz, Peter

2013-12-31

More than 20% of all protein domains are currently annotated as "domains of unknown function" (DUFs). About 2,700 DUFs are found in bacteria compared with just over 1,500 in eukaryotes. Over 800 DUFs are shared between bacteria and eukaryotes, and about 300 of these are also present in archaea. A total of 2,786 bacterial Pfam domains even occur in animals, including 320 DUFs. Evolutionary conservation suggests that many of these DUFs are important. Here we show that 355 essential proteins in 16 model bacterial species contain 238 DUFs, most of which represent single-domain proteins, clearly establishing the biological essentiality of DUFs. We suggest that experimental research should focus on conserved and essential DUFs (eDUFs) for functional analysis given their important function and wide taxonomic distribution, including bacterial pathogens. The functional units of proteins are domains. Typically, each domain has a distinct structure and function. Genomes encode thousands of domains, and many of the domains have no known function (domains of unknown function [DUFs]). They are often ignored as of little relevance, given that many of them are found in only a few genomes. Here we show that many DUFs are essential DUFs (eDUFs) based on their presence in essential proteins. We also show that eDUFs are often essential even if they are found in relatively few genomes. However, in general, more common DUFs are more often essential than rare DUFs.

An ambiguity principle for assigning protein structural domains.

PubMed

Postic, Guillaume; Ghouzam, Yassine; Chebrek, Romain; Gelly, Jean-Christophe

2017-01-01

Ambiguity is the quality of being open to several interpretations. For an image, it arises when the contained elements can be delimited in two or more distinct ways, which may cause confusion. We postulate that it also applies to the analysis of protein three-dimensional structure, which consists in dividing the molecule into subunits called domains. Because different definitions of what constitutes a domain can be used to partition a given structure, the same protein may have different but equally valid domain annotations. However, knowledge and experience generally displace our ability to accept more than one way to decompose the structure of an object-in this case, a protein. This human bias in structure analysis is particularly harmful because it leads to ignoring potential avenues of research. We present an automated method capable of producing multiple alternative decompositions of protein structure (web server and source code available at www.dsimb.inserm.fr/sword/). Our innovative algorithm assigns structural domains through the hierarchical merging of protein units, which are evolutionarily preserved substructures that describe protein architecture at an intermediate level, between domain and secondary structure. To validate the use of these protein units for decomposing protein structures into domains, we set up an extensive benchmark made of expert annotations of structural domains and including state-of-the-art domain parsing algorithms. The relevance of our "multipartitioning" approach is shown through numerous examples of applications covering protein function, evolution, folding, and structure prediction. Finally, we introduce a measure for the structural ambiguity of protein molecules.

An ambiguity principle for assigning protein structural domains

PubMed Central

Postic, Guillaume; Ghouzam, Yassine; Chebrek, Romain; Gelly, Jean-Christophe

2017-01-01

Ambiguity is the quality of being open to several interpretations. For an image, it arises when the contained elements can be delimited in two or more distinct ways, which may cause confusion. We postulate that it also applies to the analysis of protein three-dimensional structure, which consists in dividing the molecule into subunits called domains. Because different definitions of what constitutes a domain can be used to partition a given structure, the same protein may have different but equally valid domain annotations. However, knowledge and experience generally displace our ability to accept more than one way to decompose the structure of an object—in this case, a protein. This human bias in structure analysis is particularly harmful because it leads to ignoring potential avenues of research. We present an automated method capable of producing multiple alternative decompositions of protein structure (web server and source code available at www.dsimb.inserm.fr/sword/). Our innovative algorithm assigns structural domains through the hierarchical merging of protein units, which are evolutionarily preserved substructures that describe protein architecture at an intermediate level, between domain and secondary structure. To validate the use of these protein units for decomposing protein structures into domains, we set up an extensive benchmark made of expert annotations of structural domains and including state-of-the-art domain parsing algorithms. The relevance of our “multipartitioning” approach is shown through numerous examples of applications covering protein function, evolution, folding, and structure prediction. Finally, we introduce a measure for the structural ambiguity of protein molecules. PMID:28097215

Tyrosine Phosphorylation of the Lyn Src Homology 2 (SH2) Domain Modulates Its Binding Affinity and Specificity*

PubMed Central

Jin, Lily L.; Wybenga-Groot, Leanne E.; Tong, Jiefei; Taylor, Paul; Minden, Mark D.; Trudel, Suzanne; McGlade, C. Jane; Moran, Michael F.

2015-01-01

Src homology 2 (SH2) domains are modular protein structures that bind phosphotyrosine (pY)-containing polypeptides and regulate cellular functions through protein-protein interactions. Proteomics analysis showed that the SH2 domains of Src family kinases are themselves tyrosine phosphorylated in blood system cancers, including acute myeloid leukemia, chronic lymphocytic leukemia, and multiple myeloma. Using the Src family kinase Lyn SH2 domain as a model, we found that phosphorylation at the conserved SH2 domain residue Y194 impacts the affinity and specificity of SH2 domain binding to pY-containing peptides and proteins. Analysis of the Lyn SH2 domain crystal structure supports a model wherein phosphorylation of Y194 on the EF loop modulates the binding pocket that engages amino acid side chains at the pY+2/+3 position. These data indicate another level of regulation wherein SH2-mediated protein-protein interactions are modulated by SH2 kinases and phosphatases. PMID:25587033

The number of genes encoding repeat domain-containing proteins positively correlates with genome size in amoebal giant viruses

PubMed Central

Shukla, Avi; Chatterjee, Anirvan

2018-01-01

Abstract Curiously, in viruses, the virion volume appears to be predominantly driven by genome length rather than the number of proteins it encodes or geometric constraints. With their large genome and giant particle size, amoebal viruses (AVs) are ideally suited to study the relationship between genome and virion size and explore the role of genome plasticity in their evolutionary success. Different genomic regions of AVs exhibit distinct genealogies. Although the vertically transferred core genes and their functions are universally conserved across the nucleocytoplasmic large DNA virus (NCLDV) families and are essential for their replication, the horizontally acquired genes are variable across families and are lineage-specific. When compared with other giant virus families, we observed a near–linear increase in the number of genes encoding repeat domain-containing proteins (RDCPs) with the increase in the genome size of AVs. From what is known about the functions of RDCPs in bacteria and eukaryotes and their prevalence in the AV genomes, we envisage important roles for RDCPs in the life cycle of AVs, their genome expansion, and plasticity. This observation also supports the evolution of AVs from a smaller viral ancestor by the acquisition of diverse gene families from the environment including RDCPs that might have helped in host adaption. PMID:29308275

IQGAP Proteins Reveal an Atypical Phosphoinositide (aPI) Binding Domain with a Pseudo C2 Domain Fold*

PubMed Central

Dixon, Miles J.; Gray, Alexander; Schenning, Martijn; Agacan, Mark; Tempel, Wolfram; Tong, Yufeng; Nedyalkova, Lyudmila; Park, Hee-Won; Leslie, Nicholas R.; van Aalten, Daan M. F.; Downes, C. Peter; Batty, Ian H.

2012-01-01

Class I phosphoinositide (PI) 3-kinases act through effector proteins whose 3-PI selectivity is mediated by a limited repertoire of structurally defined, lipid recognition domains. We describe here the lipid preferences and crystal structure of a new class of PI binding modules exemplified by select IQGAPs (IQ motif containing GTPase-activating proteins) known to coordinate cellular signaling events and cytoskeletal dynamics. This module is defined by a C-terminal 105–107 amino acid region of which IQGAP1 and -2, but not IQGAP3, binds preferentially to phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3). The binding affinity for PtdInsP3, together with other, secondary target-recognition characteristics, are comparable with those of the pleckstrin homology domain of cytohesin-3 (general receptor for phosphoinositides 1), an established PtdInsP3 effector protein. Importantly, the IQGAP1 C-terminal domain and the cytohesin-3 pleckstrin homology domain, each tagged with enhanced green fluorescent protein, were both re-localized from the cytosol to the cell periphery following the activation of PI 3-kinase in Swiss 3T3 fibroblasts, consistent with their common, selective recognition of endogenous 3-PI(s). The crystal structure of the C-terminal IQGAP2 PI binding module reveals unexpected topological similarity to an integral fold of C2 domains, including a putative basic binding pocket. We propose that this module integrates select IQGAP proteins with PI 3-kinase signaling and constitutes a novel, atypical phosphoinositide binding domain that may represent the first of a larger group, each perhaps structurally unique but collectively dissimilar from the known PI recognition modules. PMID:22493426

IQGAP Proteins Reveal an Atypical Phosphoinositide (aPI) Binding Domain with a Pseudo C2 Domain Fold

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

Dixon, Miles J.; Gray, Alexander; Schenning, Martijn

2012-10-16

Class I phosphoinositide (PI) 3-kinases act through effector proteins whose 3-PI selectivity is mediated by a limited repertoire of structurally defined, lipid recognition domains. We describe here the lipid preferences and crystal structure of a new class of PI binding modules exemplified by select IQGAPs (IQ motif containing GTPase-activating proteins) known to coordinate cellular signaling events and cytoskeletal dynamics. This module is defined by a C-terminal 105-107 amino acid region of which IQGAP1 and -2, but not IQGAP3, binds preferentially to phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3). The binding affinity for PtdInsP3, together with other, secondary target-recognition characteristics, are comparable with those ofmore » the pleckstrin homology domain of cytohesin-3 (general receptor for phosphoinositides 1), an established PtdInsP3 effector protein. Importantly, the IQGAP1 C-terminal domain and the cytohesin-3 pleckstrin homology domain, each tagged with enhanced green fluorescent protein, were both re-localized from the cytosol to the cell periphery following the activation of PI 3-kinase in Swiss 3T3 fibroblasts, consistent with their common, selective recognition of endogenous 3-PI(s). The crystal structure of the C-terminal IQGAP2 PI binding module reveals unexpected topological similarity to an integral fold of C2 domains, including a putative basic binding pocket. We propose that this module integrates select IQGAP proteins with PI 3-kinase signaling and constitutes a novel, atypical phosphoinositide binding domain that may represent the first of a larger group, each perhaps structurally unique but collectively dissimilar from the known PI recognition modules.« less

Yeast Ivy1p Is a Putative I-BAR-domain Protein with pH-sensitive Filament Forming Ability in vitro.

PubMed

Itoh, Yuzuru; Kida, Kazuki; Hanawa-Suetsugu, Kyoko; Suetsugu, Shiro

2016-01-01

Bin-Amphiphysin-Rvs161/167 (BAR) domains mold lipid bilayer membranes into tubules, by forming a spiral polymer on the membrane. Most BAR domains are thought to be involved in forming membrane invaginations through their concave membrane binding surfaces, whereas some members have convex membrane binding surfaces, and thereby mold membranes into protrusions. The BAR domains with a convex surface form a subtype called the inverse BAR (I-BAR) domain or IRSp53-MIM-homology domain (IMD). Although the mammalian I-BAR domains have been studied, those from other organisms remain elusive. Here, we found putative I-BAR domains in Fungi and animal-like unicellular organisms. The fungal protein containing the putative I-BAR-domain is known as Ivy1p in yeast, and is reportedly localized in the vacuole. The phylogenetic analysis of the I-BAR domains revealed that the fungal I-BAR-domain containing proteins comprise a distinct group from those containing IRSp53 or MIM. Importantly, Ivy1p formed a polymer with a diameter of approximately 20 nm in vitro, without a lipid membrane. The filaments were formed at neutral pH, but disassembled when pH was reverted to basic. Moreover, Ivy1p and the I-BAR domain expressed in mammalian HeLa cells was localized at a vacuole-like structure as filaments as revealed by super-resolved microscopy. These data indicate the pH-sensitive polymer forming ability and the functional conservation of Ivy1p in eukaryotic cells.

Structure, function and tissue forms of the C-terminal globular domain of collagen XVIII containing the angiogenesis inhibitor endostatin.

PubMed Central

Sasaki, T; Fukai, N; Mann, K; Göhring, W; Olsen, B R; Timpl, R

1998-01-01

The C-terminal domain NC1 of mouse collagen XVIII (38 kDa) and the shorter mouse and human endostatins (22 kDa) were prepared in recombinant form from transfected mammalian cells. The NC1 domain aggregated non-covalently into a globular trimer which was partially cleaved by endogenous proteolysis into several monomers (25-32 kDa) related to endostatin. Endostatins were obtained in a highly soluble, monomeric form and showed a single N-terminal sequence which, together with other data, indicated a compact folding. Endostatins and NC1 showed a comparable binding activity for the microfibrillar fibulin-1 and fibulin-2, and for heparin. Domain NC1, however, was a distinctly stronger ligand than endostatin for sulfatides and the basement membrane proteins laminin-1 and perlecan. Immunological assays demonstrated endostatin epitopes on several tissue components (22-38 kDa) and in serum (120-300 ng/ml), the latter representing the smaller variants. The data indicated that the NC1 domain consists of an N-terminal association region (approximately 50 residues), a central protease-sensitive hinge region (approximately 70 residues) and a C-terminal stable endostatin domain (approximately 180 residues). They also demonstrated that proteolytic release of endostatin can occur through several pathways, which may lead to a switch from a matrix-associated to a more soluble endocrine form. PMID:9687493

Structure and regulatory role of the C-terminal winged helix domain of the archaeal minichromosome maintenance complex

PubMed Central

Wiedemann, Christoph; Szambowska, Anna; Häfner, Sabine; Ohlenschläger, Oliver; Gührs, Karl-Heinz; Görlach, Matthias

2015-01-01

The minichromosome maintenance complex (MCM) represents the replicative DNA helicase both in eukaryotes and archaea. Here, we describe the solution structure of the C-terminal domains of the archaeal MCMs of Sulfolobus solfataricus (Sso) and Methanothermobacter thermautotrophicus (Mth). Those domains consist of a structurally conserved truncated winged helix (WH) domain lacking the two typical ‘wings’ of canonical WH domains. A less conserved N-terminal extension links this WH module to the MCM AAA+ domain forming the ATPase center. In the Sso MCM this linker contains a short α-helical element. Using Sso MCM mutants, including chimeric constructs containing Mth C-terminal domain elements, we show that the ATPase and helicase activity of the Sso MCM is significantly modulated by the short α-helical linker element and by N-terminal residues of the first α-helix of the truncated WH module. Finally, based on our structural and functional data, we present a docking-derived model of the Sso MCM, which implies an allosteric control of the ATPase center by the C-terminal domain. PMID:25712103

Epitope mapping of the domains of human angiotensin converting enzyme.

PubMed

Kugaevskaya, Elena V; Kolesanova, Ekaterina F; Kozin, Sergey A; Veselovsky, Alexander V; Dedinsky, Ilya R; Elisseeva, Yulia E

2006-06-01

Somatic angiotensin converting enzyme (sACE), contains in its single chain two homologous domains (called N- and C-domains), each bearing a functional zinc-dependent active site. The present study aims to define the differences between two sACE domains and to localize experimentally revealed antigenic determinants (B-epitopes) in the recently determined three-dimensional structure of testicular tACE. The predicted linear antigenic determinants of human sACE were determined by peptide scanning ("PEPSCAN") approach. Essential difference was demonstrated between locations of the epitopes in the N- and C-domains. Comparison of arrangement of epitopes in the human domains with the corresponding sequences of some mammalian sACEs enabled to classify the revealed antigenic determinants as variable or conserved areas. The location of antigenic determinants with respect to various structural elements and to functionally important sites of the human sACE C-domain was estimated. The majority of antigenic sites of the C-domain were located at the irregular elements and at the boundaries of secondary structure elements. The data show structural differences between the sACE domains. The experimentally revealed antigenic determinants were in agreement with the recently determined crystal tACE structure. New potential applications are open to successfully produce mono-specific and group-specific antipeptide antibodies.

CKLF-Like MARVEL Transmembrane Domain-Containing Member 3 (CMTM3) Inhibits the Proliferation and Tumorigenisis in Hepatocellular Carcinoma Cells.

PubMed

Li, Wujun; Zhang, Shaobo

2017-01-26

The CKLF-like MARVEL transmembrane domain-containing 3 (CMTM3), a member of the CMTM family, was found in several human tumors and plays an important role in the development and progression of tumors. However, the role of CMTM3 in hepatocellular carcinoma (HCC) remains largely unknown. Thus, in the present study, we explored its expression pattern in human HCC cell lines, as well as its functions in HCC cells. Our results demonstrated that the expression of CMTM3 is lowly expressed in HCC cell lines. In vitro, we found that overexpression of CMTM3 obviously inhibited the proliferation, invasion, and EMT process in HCC cells. Furthermore, overexpression of CMTM3 significantly downregulated the expression levels of phosphorylation of JAK2 and STAT3 in HepG2 cells. In vivo, overexpression of CMTM3 attenuated the tumor growth in Balb/c nude mice. In conclusion, we demonstrated that CMTM3 could play an important role in HCC metastasis by EMT induction via, at least partially, suppressing the JAK2/STAT3 signaling pathway. Therefore, CMTM3 may serve as a potential molecular target in the prevention and/or treatment of HCC invasion and metastasis.

Ciona intestinalis Noto4 contains a phosphotyrosine interaction domain and is involved in the midline intercalation of notochord cells.

PubMed

Yamada, Shigehiro; Ueno, Naoto; Satoh, Nori; Takahashi, Hiroki

2011-01-01

Brachyury plays a pivotal role in the notochord formation in ascidian embryos. Ciona intestinalis Noto4 (Ci-Noto4) was isolated as a gene downstream of Ci-Bra. This gene encodes a 307 amino-acid protein with a C-terminal phosphotyrosine interaction domain (PTB/PID). Expression of Ci-Noto4 commences at the neural plate stage and is specific to notochord cells. Suppression of Ci-Noto4 levels with specific antisense morpholino oligonucleotides resulted in the formation of two rows of notochord cells owing to a lack of midline intercalation between the bilateral populations of progenitor cells. In contrast, overexpression of Ci-Noto4 by injection of a Ci-Bra(promoter):Ci-Noto4-EGFP construct into fertilized eggs disrupted the localization of notochord cells. Ci-Noto4 overexpression did not affect cellular differentiation in the notochord, muscle, mesenchyme, or nervous system. Analysis of Ci-Noto4 regions that are responsible for its function suggested significant roles for the PTB/PID and a central region, an area with no obvious sequence similarity to other known proteins. These results suggested that PTB/PID-containing Ci-Noto4 is essential for midline intercalation of notochord cells in chordate embryos.

Functional genetic variant in the Kozak sequence of WW domain-containing oxidoreductase (WWOX) gene is associated with oral cancer risk.

PubMed

Cheng, Hsin-Lin; Liu, Yu-Fan; Su, Chun-Wen; Su, Shih-Chi; Chen, Mu-Kuan; Yang, Shun-Fa; Lin, Chiao-Wen

2016-10-25

In Taiwan, oral cancer is the fourth leading cancer in males and is associated with exposure to environmental carcinogens. WW domain-containing oxidoreductase (WWOX), a tumor suppressor gene, is associated with the development of various cancers. We hypothesized that genetic variants of WWOX influence the susceptibility to oral cancer. Five polymorphisms of WWOX gene from 761 male patients with oral cancer and 1199 male cancer-free individuals were genotyped. We observed that individuals carrying the polymorphic allele of WWOX rs11545028 are more susceptible to oral cancer. Furthermore, patients with advanced-stage oral cancer were associated with a higher frequency of WWOX rs11545028 polymorphisms with the variant genotype TT than did patients with the wild-type gene. An additional integrated in silico analysis confirmed that rs11545028 affects WWOX expression, which significantly correlates with tumor expression and subsequently with tumor development and aggressiveness. In conclusion, genetic variants of WWOX contribute to the occurrence of oral cancer, and the findings regarding these biomarkers provided a prediction model for risk assessment.

Phylogenetic Analysis and Classification of the Fungal bHLH Domain

PubMed Central

Sailsbery, Joshua K.; Atchley, William R.; Dean, Ralph A.

2012-01-01

The basic Helix-Loop-Helix (bHLH) domain is an essential highly conserved DNA-binding domain found in many transcription factors in all eukaryotic organisms. The bHLH domain has been well studied in the Animal and Plant Kingdoms but has yet to be characterized within Fungi. Herein, we obtained and evaluated the phylogenetic relationship of 490 fungal-specific bHLH containing proteins from 55 whole genome projects composed of 49 Ascomycota and 6 Basidiomycota organisms. We identified 12 major groupings within Fungi (F1–F12); identifying conserved motifs and functions specific to each group. Several classification models were built to distinguish the 12 groups and elucidate the most discerning sites in the domain. Performance testing on these models, for correct group classification, resulted in a maximum sensitivity and specificity of 98.5% and 99.8%, respectively. We identified 12 highly discerning sites and incorporated those into a set of rules (simplified model) to classify sequences into the correct group. Conservation of amino acid sites and phylogenetic analyses established that like plant bHLH proteins, fungal bHLH–containing proteins are most closely related to animal Group B. The models used in these analyses were incorporated into a software package, the source code for which is available at www.fungalgenomics.ncsu.edu. PMID:22114358

Structure of the SANT domain from the Xenopus chromatin remodeling factor ISWI

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

Horton, John R.; Elgar, Stuart J.; Khan, Seema I.

2008-09-17

The SANT (Swi3, Ada2, N-Cor, and TFIIIB) module was first described as a putative DNA-binding domain with strong similarity to the helix-turn-helix DNA binding domain of Myb-related proteins. The X-ray structure of the C-terminal one third portion of the ATPase ISWI of Drosophila melangoaster, containing both SANT and SLIDE (SANT-Like ISWI Domain), confirmed the overall helix-turn-helix structural architecture of SANT as well as SLIDE. However, the DNA-contacting residues in Myb are not conserved in SANT and the structurally corresponding residues in the ISWI SANT domain are acidic, and therefore incompatible with DNA interaction. Recent studies suggested that SANT domains mightmore » be a histone-tail-binding module, including the DNA binding SANT domain of c-Myb. Here they present the X-ray structure of Xenopus laevis ISWI SANT domain, derived from limited proteolysis of a C-terminal fragment of ISWI protein.« less

The Ubiquitin-associated Domain of Cellular Inhibitor of Apoptosis Proteins Facilitates Ubiquitylation*

PubMed Central

Budhidarmo, Rhesa; Day, Catherine L.

2014-01-01

The cellular inhibitor of apoptosis (cIAP) proteins are essential RING E3 ubiquitin ligases that regulate apoptosis and inflammatory responses. cIAPs contain a ubiquitin-associated (UBA) domain that binds ubiquitin and is implicated in the regulation of cell survival and proteasomal degradation. Here we show that mutation of the MGF and LL motifs in the UBA domain of cIAP1 caused unfolding and increased cIAP1 multimonoubiquitylation. By developing a UBA mutant that disrupted ubiquitin binding but not the structure of the UBA domain, we found that the UBA domain enhances cIAP1 and cIAP2 ubiquitylation. We demonstrate that the UBA domain binds to the UbcH5b∼Ub conjugate, and this promotes RING domain-dependent monoubiquitylation. This study establishes ubiquitin-binding modules, such as the UBA domain, as important regulatory modules that can fine tune the activity of E3 ligases. PMID:25065467

Evolutionarily conserved structural and functional roles of the FYVE domain.

PubMed

Hayakawa, Akira; Hayes, Susan; Leonard, Deborah; Lambright, David; Corvera, Silvia

2007-01-01

The FYVE domain is an approx. 80 amino acid motif that binds to the phosphoinositide PtdIns3P with high specificity and affinity. It is present in 38 predicted gene products within the human genome, but only in 12-13 in Caenorhabditis elegans and Drosophila melanogaster. Eight of these are highly conserved in all three organisms, and they include proteins that have not been characterized in any species. One of these, WDFY2, appears to play an important role in early endocytosis and was revealed in a RNAi (RNA interference) screen in C. elegans. Interestingly, some proteins contain FYVE-like domains in C. elegans and D. melanogaster, but have lost this domain during evolution. One of these is the homologue of Rabatin-5, a protein that, in mammalian cells, binds both Rab5 and Rabex-5, a guanine-nucleotide exchange factor for Rab5. Thus the Rabatin-5 homologue suggests that mechanisms to link PtdIns3P and Rab5 activation developed in evolution. In mammalian cells, these mechanisms are apparent in the existence of proteins that bind PtdIns3P and Rab GTPases, such as EEA1, Rabenosyn-5 and Rabip4'. Despite the comparable ability to bind to PtdIns3P in vitro, FYVE domains display widely variable abilities to interact with endosomes in intact cells. This variation is due to three distinct properties of FYVE domains conferred by residues that are not involved in PtdIns3P head group recognition: These properties are: (i) the propensity to oligomerize, (ii) the ability to insert into the membrane bilayer, and (iii) differing electrostatic interactions with the bilayer surface. The different binding properties are likely to regulate the extent and duration of the interaction of specific FYVE domain-containing proteins with early endosomes, and thereby their biological function.

Cross-Species Analyses Identify the BNIP-2 and Cdc42GAP Homology (BCH) Domain as a Distinct Functional Subclass of the CRAL_TRIO/Sec14 Superfamily

PubMed Central

Gupta, Anjali Bansal; Wee, Liang En; Zhou, Yi Ting; Hortsch, Michael; Low, Boon Chuan

2012-01-01

The CRAL_TRIO protein domain, which is unique to the Sec14 protein superfamily, binds to a diverse set of small lipophilic ligands. Similar domains are found in a range of different proteins including neurofibromatosis type-1, a Ras GTPase-activating Protein (RasGAP) and Rho guanine nucleotide exchange factors (RhoGEFs). Proteins containing this structural protein domain exhibit a low sequence similarity and ligand specificity while maintaining an overall characteristic three-dimensional structure. We have previously demonstrated that the BNIP-2 and Cdc42GAP Homology (BCH) protein domain, which shares a low sequence homology with the CRAL_TRIO domain, can serve as a regulatory scaffold that binds to Rho, RhoGEFs and RhoGAPs to control various cell signalling processes. In this work, we investigate 175 BCH domain-containing proteins from a wide range of different organisms. A phylogenetic analysis with ∼100 CRAL_TRIO and similar domains from eight representative species indicates a clear distinction of BCH-containing proteins as a novel subclass within the CRAL_TRIO/Sec14 superfamily. BCH-containing proteins contain a hallmark sequence motif R(R/K)h(R/K)(R/K)NL(R/K)xhhhhHPs (‘h’ is large and hydrophobic residue and ‘s’ is small and weekly polar residue) and can be further subdivided into three unique subtypes associated with BNIP-2-N, macro- and RhoGAP-type protein domains. A previously unknown group of genes encoding ‘BCH-only’ domains is also identified in plants and arthropod species. Based on an analysis of their gene-structure and their protein domain context we hypothesize that BCH domain-containing genes evolved through gene duplication, intron insertions and domain swapping events. Furthermore, we explore the point of divergence between BCH and CRAL-TRIO proteins in relation to their ability to bind small GTPases, GAPs and GEFs and lipid ligands. Our study suggests a need for a more extensive analysis of previously uncharacterized BCH, �

How much of the interannual variability of East Asian summer rainfall is forced by SST?

NASA Astrophysics Data System (ADS)

He, Chao; Wu, Bo; Li, Chunhui; Lin, Ailan; Gu, Dejun; Zheng, Bin; Zhou, Tianjun

2016-07-01

It is widely accepted that the interannual variability of East Asian summer rainfall is forced by sea surface temperature (SST), and SST anomalies are widely used as predictors of East Asian summer rainfall. But it is still not very clear what percentage of the interannual rainfall variability is contributed by SST anomalies. In this study, Atmospheric general circulation model simulations forced by observed interannual varying SST are compared with those forced by the fixed annual cycle of SST climatology, and their ratios of interannual variance (IAV) are analyzed. The output of 12 models from the 5th Phase of Coupled Model Intercomparison Project (CMIP5) are adopted, and idealized experiments are done by Community Atmosphere Model version 4 (CAM4). Both the multi-model median of CMIP5 models and CAM4 experiments show that only about 18 % of the IAV of rainfall over East Asian land (EAL) is explained by SST, which is significantly lower than the tropical western Pacific, but comparable to the mid-latitude western Pacific. There is no significant difference between the southern part and the northern part of EAL in the percentages of SST contribution. The remote SST anomalies regulates rainfall over EAL probably by modulating the horizontal water vapor transport rather than the vertical motion, since the horizontal water vapor transport into EAL is strongly modulated by SST but the vertical motion over EAL is not. Previous studies argued about the relative importance of tropical Indian Ocean and tropical Pacific Ocean to East Asian summer rainfall anomalies. Our idealized experiments performed by CAM4 suggest that the contributions from these two ocean basins are comparable to each other, both of which account for approximately 6 % of the total IAV of rainfall over EAL.

Domain atrophy creates rare cases of functional partial protein domains.

PubMed

Prakash, Ananth; Bateman, Alex

2015-04-30

Protein domains display a range of structural diversity, with numerous additions and deletions of secondary structural elements between related domains. We have observed a small number of cases of surprising large-scale deletions of core elements of structural domains. We propose a new concept called domain atrophy, where protein domains lose a significant number of core structural elements. Here, we implement a new pipeline to systematically identify new cases of domain atrophy across all known protein sequences. The output of this pipeline was carefully checked by hand, which filtered out partial domain instances that were unlikely to represent true domain atrophy due to misannotations or un-annotated sequence fragments. We identify 75 cases of domain atrophy, of which eight cases are found in a three-dimensional protein structure and 67 cases have been inferred based on mapping to a known homologous structure. Domains with structural variations include ancient folds such as the TIM-barrel and Rossmann folds. Most of these domains are observed to show structural loss that does not affect their functional sites. Our analysis has significantly increased the known cases of domain atrophy. We discuss specific instances of domain atrophy and see that there has often been a compensatory mechanism that helps to maintain the stability of the partial domain. Our study indicates that although domain atrophy is an extremely rare phenomenon, protein domains under certain circumstances can tolerate extreme mutations giving rise to partial, but functional, domains.

Pedagogy for Ethnic Minority Pupils with Special Educational Needs in England: Common yet Different?

ERIC Educational Resources Information Center

Tan, Andrea Geok Poh; Ware, Jean; Norwich, Brahm

2017-01-01

The increasing ethnic diversity in the UK has highlighted the importance of supporting primary school pupils with English as an Additional Language (EAL), some of whom also have special educational needs (SEN). However, there is relatively little research carried out in the UK on children with both EAL needs and SEN. This paper presents the…

The Multicultural Café: Enhancing Authentic Interaction for Adult English Language Learners through Service Learning

ERIC Educational Resources Information Center

Riley, Tracy; Douglas, Scott Roy

2016-01-01

While service learning platforms hold great potential for adult learners of English as an additional language (EAL), there has been little research to date related to the impact of these programs on adult newcomers' linguistic and social development. The Multicultural Café was a food service learning platform for adult EAL learners operated over a…

Changes to English as an Additional Language Writers' Research Articles: From Spoken to Written Register

ERIC Educational Resources Information Center

Koyalan, Aylin; Mumford, Simon

2011-01-01

The process of writing journal articles is increasingly being seen as a collaborative process, especially where the authors are English as an Additional Language (EAL) academics. This study examines the changes made in terms of register to EAL writers' journal articles by a native-speaker writing centre advisor at a private university in Turkey.…

Abhydrolase domain containing 2, an androgen target gene, promotes prostate cancer cell proliferation and migration.

PubMed

Obinata, Daisuke; Takada, Shogo; Takayama, Ken-ichi; Urano, Tomohiko; Ito, Akiko; Ashikari, Daisaku; Fujiwara, Kyoko; Yamada, Yuta; Murata, Taro; Kumagai, Jinpei; Fujimura, Tetsuya; Ikeda, Kazuhiro; Horie-Inoue, Kuniko; Homma, Yukio; Takahashi, Satoru; Inoue, Satoshi

2016-04-01

The androgen receptor (AR) plays a key role in the development of prostate cancer. AR signalling mediates the expression of androgen-responsive genes, which are involved in prostate cancer development and progression. Our previous chromatin immunoprecipitation study showed that the region of abhydrolase domain containing 2 (ABHD2) includes a functional androgen receptor binding site. In this study, we demonstrated that ABHD2 is a novel androgen-responsive gene that is overexpressed in human prostate cancer tissues. The expression levels of ABHD2 in androgen-sensitive cells were evaluated by quantitative reverse transcription polymerase chain reaction and western-blot analyses. LNCaP and VCaP cells with ABHD2 overexpression or short interfering RNA (siRNA) knockdown were used for functional analyses. ABHD2 expression was examined in clinical samples of prostate cancer by immunohistochemistry. We showed that ABHD2 expression is increased by androgen in LNCaP and VCaP cells. This androgen-induced ABHD2 expression was diminished by bicalutamide. While stable expression of ABHD2 affected the enhancement of LNCaP cell proliferation and migration, siRNA-mediated ABHD2 knockdown suppressed cell proliferation and migration. In addition, the siRNA treatment significantly repressed the tumour growth derived from LNCaP cells in athymic mice. Immunohistochemical analysis of ABHD2 expression in tumour specimens showed a positive correlation of ABHD2 immunoreactivity with high Gleason score and pathological N stage. Moreover, patients with high immunoreactivity of ABHD2 showed low cancer-specific survival rates and a resistance to docetaxel-based chemotherapy. ABHD2 is a novel androgen-regulated gene that can promote prostate cancer growth and resistance to chemotherapy, and is a novel target for diagnosis and treatment of prostate cancer. Copyright © 2016 Elsevier Ltd. All rights reserved.

PD-1 immunoreceptor inhibits B cell receptor-mediated signaling by recruiting src homology 2-domain-containing tyrosine phosphatase 2 to phosphotyrosine

PubMed Central

Okazaki, Taku; Maeda, Akito; Nishimura, Hiroyuki; Kurosaki, Tomohiro; Honjo, Tasuku

2001-01-01

PD-1 is an immunoreceptor that belongs to the immunoglobulin (Ig) superfamily and contains two tyrosine residues in the cytoplasmic region. Studies on PD-1-deficient mice have shown that PD-1 plays critical roles in establishment and/or maintenance of peripheral tolerance, but the mode of action is totally unknown. To study the molecular mechanism for negative regulation of lymphocytes through the PD-1 receptor, we generated chimeric molecules composed of the IgG Fc receptor type IIB (FcγRIIB) extracellular region and the PD-1 cytoplasmic region and expressed them in a B lymphoma cell line, IIA1.6. Coligation of the cytoplasmic region of PD-1 with the B cell receptor (BCR) in IIA1.6 transformants inhibited BCR-mediated growth retardation, Ca2+ mobilization, and tyrosine phosphorylation of effector molecules, including Igβ, Syk, phospholipase C-γ2 (PLCγ2), and ERK1/2, whereas phosphorylation of Lyn and Dok was not affected. Mutagenesis studies indicated that these inhibitory effects do not require the N-terminal tyrosine in the immunoreceptor tyrosine-based inhibitory motif-like sequence, but do require the other tyrosine residue in the C-terminal tail. This tyrosine was phosphorylated and recruited src homology 2-domain-containing tyrosine phosphatase 2 (SHP-2) on coligation of PD-1 with BCR. These results show that PD-1 can inhibit BCR signaling by recruiting SHP-2 to its phosphotyrosine and dephosphorylating key signal transducers of BCR signaling. PMID:11698646

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