A pomegranate (Punica granatum L.) WD40-repeat gene is a functional homologue of Arabidopsis TTG1 and is involved in the regulation of anthocyanin biosynthesis during pomegranate fruit development.

PubMed

Ben-Simhon, Zohar; Judeinstein, Sylvie; Nadler-Hassar, Talia; Trainin, Taly; Bar-Ya'akov, Irit; Borochov-Neori, Hamutal; Holland, Doron

2011-11-01

Anthocyanins are the major pigments responsible for the pomegranate (Punica granatum L.) fruit skin color. The high variability in fruit external color in pomegranate cultivars reflects variations in anthocyanin composition. To identify genes involved in the regulation of anthocyanin biosynthesis pathway in the pomegranate fruit skin we have isolated, expressed and characterized the pomegranate homologue of the Arabidopsis thaliana TRANSPARENT TESTA GLABRA1 (TTG1), encoding a WD40-repeat protein. The TTG1 protein is a regulator of anthocyanins and proanthocyanidins (PAs) biosynthesis in Arabidopsis, and acts by the formation of a transcriptional regulatory complex with two other regulatory proteins: bHLH and MYB. Our results reveal that the pomegranate gene, designated PgWD40, recovered the anthocyanin, PAs, trichome and seed coat mucilage phenotype in Arabidopsis ttg1 mutant. PgWD40 expression and anthocyanin composition in the skin were analyzed during pomegranate fruit development, in two accessions that differ in skin color intensity and timing of appearance. The results indicate high positive correlation between the total cyanidin derivatives quantity (red pigments) and the expression level of PgWD40. Furthermore, strong correlation was found between the steady state levels of PgWD40 transcripts and the transcripts of pomegranate homologues of the structural genes PgDFR and PgLDOX. PgWD40, PgDFR and PgLDOX expression also correlated with the expression of pomegranate homologues of the regulatory genes PgAn1 (bHLH) and PgAn2 (MYB). On the basis of our results we propose that PgWD40 is involved in the regulation of anthocyanin biosynthesis during pomegranate fruit development and that expression of PgWD40, PgAn1 and PgAn2 in the pomegranate fruit skin is required to regulate the expression of downstream structural genes involved in the anthocyanin biosynthesis.

WD40-repeat 47, a microtubule-associated protein, is essential for brain development and autophagy.

PubMed

Kannan, Meghna; Bayam, Efil; Wagner, Christel; Rinaldi, Bruno; Kretz, Perrine F; Tilly, Peggy; Roos, Marna; McGillewie, Lara; Bär, Séverine; Minocha, Shilpi; Chevalier, Claire; Po, Chrystelle; Chelly, Jamel; Mandel, Jean-Louis; Borgatti, Renato; Piton, Amélie; Kinnear, Craig; Loos, Ben; Adams, David J; Hérault, Yann; Collins, Stephan C; Friant, Sylvie; Godin, Juliette D; Yalcin, Binnaz

2017-10-31

The family of WD40-repeat (WDR) proteins is one of the largest in eukaryotes, but little is known about their function in brain development. Among 26 WDR genes assessed, we found 7 displaying a major impact in neuronal morphology when inactivated in mice. Remarkably, all seven genes showed corpus callosum defects, including thicker ( Atg16l1 , Coro1c , Dmxl2 , and Herc1 ), thinner ( Kif21b and Wdr89 ), or absent corpus callosum ( Wdr47 ), revealing a common role for WDR genes in brain connectivity. We focused on the poorly studied WDR47 protein sharing structural homology with LIS1, which causes lissencephaly. In a dosage-dependent manner, mice lacking Wdr47 showed lethality, extensive fiber defects, microcephaly, thinner cortices, and sensory motor gating abnormalities. We showed that WDR47 shares functional characteristics with LIS1 and participates in key microtubule-mediated processes, including neural stem cell proliferation, radial migration, and growth cone dynamics. In absence of WDR47, the exhaustion of late cortical progenitors and the consequent decrease of neurogenesis together with the impaired survival of late-born neurons are likely yielding to the worsening of the microcephaly phenotype postnatally. Interestingly, the WDR47-specific C-terminal to LisH (CTLH) domain was associated with functions in autophagy described in mammals. Silencing WDR47 in hypothalamic GT1-7 neuronal cells and yeast models independently recapitulated these findings, showing conserved mechanisms. Finally, our data identified superior cervical ganglion-10 (SCG10) as an interacting partner of WDR47. Taken together, these results provide a starting point for studying the implications of WDR proteins in neuronal regulation of microtubules and autophagy. Published under the PNAS license.

The C-terminal region of Ge-1 presents conserved structural features required for P-body localization.

PubMed

Jinek, Martin; Eulalio, Ana; Lingel, Andreas; Helms, Sigrun; Conti, Elena; Izaurralde, Elisa

2008-10-01

The removal of the 5' cap structure by the DCP1-DCP2 decapping complex irreversibly commits eukaryotic mRNAs to degradation. In human cells, the interaction between DCP1 and DCP2 is bridged by the Ge-1 protein. Ge-1 contains an N-terminal WD40-repeat domain connected by a low-complexity region to a conserved C-terminal domain. It was reported that the C-terminal domain interacts with DCP2 and mediates Ge-1 oligomerization and P-body localization. To understand the molecular basis for these functions, we determined the three-dimensional crystal structure of the most conserved region of the Drosophila melanogaster Ge-1 C-terminal domain. The region adopts an all alpha-helical fold related to ARM- and HEAT-repeat proteins. Using structure-based mutants we identified an invariant surface residue affecting P-body localization. The conservation of critical surface and structural residues suggests that the C-terminal region adopts a similar fold with conserved functions in all members of the Ge-1 protein family.

Merging of a CO WD and a He-rich WD to produce a type Ia supernovae

NASA Astrophysics Data System (ADS)

Liu, D.; Wang, B.; Wu, C.; Han, Z.

2017-10-01

Context. Although type Ia supernovae (SNe Ia) play a key role in astrophysics, the companions of the exploding carbon-oxygen white dwarfs (CO WDs) are still not completely identified. It has been suggested recently that a He-rich WD (a He WD or a hybrid HeCO WD) that merges with a CO WD may produce an SN Ia. This theory was based on the double-detonation model, in which the shock compression in the CO core caused by the surface explosion of the He-rich shell might lead to the explosion of the whole CO WD. However, so far, very few binary population synthesis (BPS) studies have been made on the merger scenario of a CO WD and a He-rich WD in the context of SNe Ia. Aims: We aim to systematically study the Galactic birthrates and delay-time distributions of SNe Ia based on the merger scenario of a CO WD and a He-rich WD. Methods: We performed a series of Monte Carlo BPS simulations to investigate the properties of SNe Ia from the merging of a CO WD and a He-rich WD based on the Hurley rapid binary evolution code. We also considered the influence of different metallicities on the final results. Results: From our simulations, we found that no more than 15% of all SNe Ia stem from the merger scenario of a CO WD and a He-rich WD, and their delay times range from 110 Myr to the Hubble time. This scenario mainly contributes to SN Ia explosions with intermediate and long delay times. The present work indicates that the merger scenario of a CO WD and a He-rich WD can roughly reproduce the birthrates of SN 1991bg-like events, and cover the range of their delay times. We also found that SN Ia birthrates from this scenario would be higher for the cases with low metallicities.

The WD40 Domain Protein MSI1 Functions in a Histone Deacetylase Complex to Fine-Tune Abscisic Acid Signaling.

PubMed

Mehdi, Saher; Derkacheva, Maria; Ramström, Margareta; Kralemann, Lejon; Bergquist, Jonas; Hennig, Lars

2016-01-01

MSI1 belongs to a family of histone binding WD40-repeat proteins. Arabidopsis thaliana contains five genes encoding MSI1-like proteins, but their functions in diverse chromatin-associated complexes are poorly understood. Here, we show that MSI1 is part of a histone deacetylase complex. We copurified HISTONE DEACETYLASE19 (HDA19) with MSI1 and transcriptional regulatory SIN3-like proteins and provide evidence that MSI1 and HDA19 associate into the same complex in vivo. These data suggest that MSI1, HDA19, and HISTONE DEACETYLATION COMPLEX1 protein form a core complex that can integrate various SIN3-like proteins. We found that reduction of MSI1 or HDA19 causes upregulation of abscisic acid (ABA) receptor genes and hypersensitivity of ABA-responsive genes. The MSI1-HDA19 complex fine-tunes ABA signaling by binding to the chromatin of ABA receptor genes and by maintaining low levels of acetylation of histone H3 at lysine 9, thereby affecting the expression levels of ABA receptor genes. Reduced MSI1 or HDA19 levels led to increased tolerance to salt stress corresponding to the increased ABA sensitivity of gene expression. Together, our results reveal the presence of an MSI1-HDA19 complex that fine-tunes ABA signaling in Arabidopsis. © 2016 American Society of Plant Biologists. All rights reserved.

WD1145+017

NASA Astrophysics Data System (ADS)

Motta, Mario

2017-06-01

WD1145 is a 17th magnitude white dwarf star 570 light years away in Virgo, that was discovered to have a disintegrating planetoid in close orbit by Andrew Vanderburg a graduate student at Harvard CFA, while data mining the Kepler 2 mission. He contacted me to obtain transit data to elucidate the nature of its rather bizarre transit light curves. I obtained multiple observations of WD1145 over the course of a year, and found a series of complex transit light curves that could only be interpreted as a ring complex or torus in close orbit around WD1145. Combined with data from other amateur astronomers, professional observations, and satellite data it became clear that WD1145 has a small planetoid in close orbit at the Roche limit and is breaking apart forming a ring of debris material that is then raining down on the white dwarf. The surface of the star is "polluted" by heavy metals by spectroscopic data. Given that in the intense gravitational field of a white dwarf any heavy metals could not for long last on the surface, this confirms that we are tracking in real time the destruction of a small planet by its host star.

WD1145+017 (Abstract)

NASA Astrophysics Data System (ADS)

Motta, M.

2017-12-01

(Abstract only) WD1145 is a 17th magnitude white dwarf star 570 light years away in Virgo that was discovered to have a disintegrating planetoid in close orbit by Andrew Vanderburg, a graduate student at Harvard CfA, while data mining the elucidate the nature of its rather bizarre transit light curves. I obtained multiple observations of WD1145 over the course of a year, and found a series of complex transit light curves that could only be interpreted as a ring complex or torus in close orbit around WD1145. Combined with data from other amateur astronomers, professional observations, and satellite data, it became clear that WD1145 has a small planetoid in close orbit at the Roche limit and is breaking apart, forming a ring of debris material that is then raining down on the white dwarf. The surface of the star is "polluted" by heavy metals, determined by spectroscopic data. Given that in the intense gravitational field of a white dwarf any heavy metals could not for long last on the surface, this confirms that we are tracking in real time the destruction of a small planet by its host star.

The Carboxy-Terminal Domain of Erb1 Is a Seven-Bladed ß-Propeller that Binds RNA

PubMed Central

Marcin, Wegrecki; Neira, Jose Luis; Bravo, Jeronimo

2015-01-01

Erb1 (Eukaryotic Ribosome Biogenesis 1) protein is essential for the maturation of the ribosomal 60S subunit. Functional studies in yeast and mammalian cells showed that altogether with Nop7 and Ytm1 it forms a stable subcomplex called PeBoW that is crucial for a correct rRNA processing. The exact function of the protein within the process remains unknown. The N-terminal region of the protein includes a well conserved region shown to be involved in PeBoW complex formation whereas the carboxy-terminal half was predicted to contain seven WD40 repeats. This first structural report on Erb1 from yeast describes the architecture of a seven-bladed β-propeller domain that revealed a characteristic extra motif formed by two α-helices and a β-strand that insert within the second WD repeat. We performed analysis of molecular surface and crystal packing, together with multiple sequence alignment and comparison of the structure with other β-propellers, in order to identify areas that are more likely to mediate protein-protein interactions. The abundance of many positively charged residues on the surface of the domain led us to investigate whether the propeller of Erb1 might be involved in RNA binding. Three independent assays confirmed that the protein interacted in vitro with polyuridilic acid (polyU), thus suggesting a possible role of the domain in rRNA rearrangement during ribosome biogenesis. PMID:25880847

Crystal structure of EML1 reveals the basis for Hsp90 dependence of oncogenic EML4-ALK by disruption of an atypical β-propeller domain

PubMed Central

Richards, Mark W.; Law, Edward W. P.; Rennalls, La’Verne P.; Busacca, Sara; O’Regan, Laura; Fry, Andrew M.; Fennell, Dean A.; Bayliss, Richard

2014-01-01

Proteins of the echinoderm microtubule-associated protein (EMAP)-like (EML) family contribute to formation of the mitotic spindle and interphase microtubule network. They contain a unique hydrophobic EML protein (HELP) motif and a variable number of WD40 repeats. Recurrent gene rearrangements in nonsmall cell lung cancer fuse EML4 to anaplastic lymphoma kinase (ALK), causing expression of several fusion oncoprotein variants. We have determined a 2.6-Å crystal structure of the representative ∼70-kDa core of EML1, revealing an intimately associated pair of β-propellers, which we term a TAPE (tandem atypical propeller in EMLs) domain. One propeller is highly atypical, having a discontinuous subdomain unrelated to a WD40 motif in place of one of its blades. This unexpected feature shows how a propeller structure can be assembled from subdomains with distinct folds. The HELP motif is not an independent domain but forms part of the hydrophobic core that joins the two β-propellers. The TAPE domain binds α/β-tubulin via its conserved, concave surface, including part of the atypical blade. Mapping the characteristic breakpoints of each EML4-ALK variant onto our structure indicates that the EML4 TAPE domain is truncated in many variants in a manner likely to make the fusion protein structurally unstable. We found that the heat shock protein 90 (Hsp90) inhibitor ganetespib induced degradation of these variants whereas others lacking a partial TAPE domain were resistant in both overexpression models and patient-derived cell lines. The Hsp90-sensitive EML4-ALK variants are exceptions to the rule that oncogenic fusion proteins involve breakpoints in disordered regions of both partners. PMID:24706829

Crystal structure of EML1 reveals the basis for Hsp90 dependence of oncogenic EML4-ALK by disruption of an atypical β-propeller domain.

PubMed

Richards, Mark W; Law, Edward W P; Rennalls, La'Verne P; Busacca, Sara; O'Regan, Laura; Fry, Andrew M; Fennell, Dean A; Bayliss, Richard

2014-04-08

Proteins of the echinoderm microtubule-associated protein (EMAP)-like (EML) family contribute to formation of the mitotic spindle and interphase microtubule network. They contain a unique hydrophobic EML protein (HELP) motif and a variable number of WD40 repeats. Recurrent gene rearrangements in nonsmall cell lung cancer fuse EML4 to anaplastic lymphoma kinase (ALK), causing expression of several fusion oncoprotein variants. We have determined a 2.6-Å crystal structure of the representative ∼70-kDa core of EML1, revealing an intimately associated pair of β-propellers, which we term a TAPE (tandem atypical propeller in EMLs) domain. One propeller is highly atypical, having a discontinuous subdomain unrelated to a WD40 motif in place of one of its blades. This unexpected feature shows how a propeller structure can be assembled from subdomains with distinct folds. The HELP motif is not an independent domain but forms part of the hydrophobic core that joins the two β-propellers. The TAPE domain binds α/β-tubulin via its conserved, concave surface, including part of the atypical blade. Mapping the characteristic breakpoints of each EML4-ALK variant onto our structure indicates that the EML4 TAPE domain is truncated in many variants in a manner likely to make the fusion protein structurally unstable. We found that the heat shock protein 90 (Hsp90) inhibitor ganetespib induced degradation of these variants whereas others lacking a partial TAPE domain were resistant in both overexpression models and patient-derived cell lines. The Hsp90-sensitive EML4-ALK variants are exceptions to the rule that oncogenic fusion proteins involve breakpoints in disordered regions of both partners.

Direct Interaction between the WD40 Repeat Protein WDR-23 and SKN-1/Nrf Inhibits Binding to Target DNA

PubMed Central

Leung, Chi K.; Hasegawa, Koichi; Wang, Ying; Deonarine, Andrew; Tang, Lanlan; Miwa, Johji

2014-01-01

SKN-1/Nrf transcription factors activate cytoprotective genes in response to reactive small molecules and strongly influence stress resistance, longevity, and development. The molecular mechanisms of SKN-1/Nrf regulation are poorly defined. We previously identified the WD40 repeat protein WDR-23 as a repressor of Caenorhabditis elegans SKN-1 that functions with a ubiquitin ligase to presumably target the factor for degradation. However, SKN-1 activity and nuclear accumulation are not always correlated, suggesting that there could be additional regulatory mechanisms. Here, we integrate forward genetics and biochemistry to gain insights into how WDR-23 interacts with and regulates SKN-1. We provide evidence that WDR-23 preferentially regulates one of three SKN-1 variants through a direct interaction that is required for normal stress resistance and development. Homology modeling predicts that WDR-23 folds into a β-propeller, and we identify the top of this structure and four motifs at the termini of SKN-1c as essential for the interaction. Two of these SKN-1 motifs are highly conserved in human Nrf1 and Nrf2 and two directly interact with target DNA. Lastly, we demonstrate that WDR-23 can block the ability of SKN-1c to interact with DNA sequences of target promoters identifying a new mechanism of regulation that is independent of the ubiquitin proteasome system, which can become occupied with damaged proteins during stress. PMID:24912676

Two novel WD40 domain–containing proteins, Ere1 and Ere2, function in the retromer-mediated endosomal recycling pathway

PubMed Central

Shi, Yufeng; Stefan, Christopher J.; Rue, Sarah M.; Teis, David; Emr, Scott D.

2011-01-01

Regulated secretion, nutrient uptake, and responses to extracellular signals depend on cell-surface proteins that are internalized and recycled back to the plasma membrane. However, the underlying mechanisms that govern membrane protein recycling to the cell surface are not fully known. Using a chemical-genetic screen in yeast, we show that the arginine transporter Can1 is recycled back to the cell surface via two independent pathways mediated by the sorting nexins Snx4/41/42 and the retromer complex, respectively. In addition, we identify two novel WD40-domain endosomal recycling proteins, Ere1 and Ere2, that function in the retromer pathway. Ere1 is required for Can1 recycling via the retromer-mediated pathway, but it is not required for the transport of other retromer cargoes, such as Vps10 and Ftr1. Biochemical studies reveal that Ere1 physically interacts with internalized Can1. Ere2 is present in a complex containing Ere1 on endosomes and functions as a regulator of Ere1. Taken together, our results suggest that Snx4/41/42 and the retromer comprise two independent pathways for the recycling of internalized cell-surface proteins. Moreover, a complex containing the two novel proteins Ere1 and Ere2 mediates cargo-specific recognition by the retromer pathway. PMID:21880895

Monitoring and modelling of white dwarfs with extremely weak magnetic fields. WD 2047+372 and WD 2359-434

NASA Astrophysics Data System (ADS)

Landstreet, J. D.; Bagnulo, S.; Valyavin, G.; Valeev, A. F.

2017-11-01

Magnetic fields are detected in a few percent of white dwarfs. The number of such magnetic white dwarfs known is now some hundreds. Fields range in strength from a few kG to several hundred MG. Almost all the known magnetic white dwarfs have a mean field modulus ≥1 MG. We are trying to fill a major gap in observational knowledge at the low field limit (≤200 kG) using circular spectro-polarimetry. In this paper we report the discovery and monitoring of strong, periodic magnetic variability in two previously discovered "super-weak field" magnetic white dwarfs, WD 2047+372 and WD 2359-434. WD 2047+372 has a mean longitudinal field that reverses between about -12 and + 15 kG, with a period of 0.243 d, while its mean field modulus appears nearly constant at 60 kG. The observations can be interpreted in terms of a dipolar field tilted with respect to the stellar rotation axis. WD 2359-434 always shows a weak positive longitudinal field with values between about 0 and + 12 kG, varying only weakly with stellar rotation, while the mean field modulus varies between about 50 and 100 kG. The rotation period is found to be 0.112 d using the variable shape of the Hα line core, consistent with available photometry. The field of this star appears to be much more complex than a dipole, and is probably not axisymmetric. Available photometry shows that WD 2359-434 is a light variable with an amplitude of only 0.005 mag; our own photometry shows that if WD 2047+372 is photometrically variable, the amplitude is below about 0.01 mag. These are the first models for magnetic white dwarfs with fields below about 100 kG based on magnetic measurements through the full stellar rotation. They reveal two very different magnetic surface configurations, and that, contrary to simple ohmic decay theory, WD 2359-434 has a much more complex surface field than the much younger WD 2047+372. Based, in part, on observations collected at the European Organisation for Astronomical Research in the

Essential role of the NH2-terminal WD/EPF motif in the phosphorylation-activated protective function of mammalian Hsp27.

PubMed

Thériault, Jimmy R; Lambert, Herman; Chávez-Zobel, Aura T; Charest, Gabriel; Lavigne, Pierre; Landry, Jacques

2004-05-28

Hsp27 is expressed at high levels after mild heat shock and contributes to making cells extremely resistant to subsequent treatments. The activity of the protein is regulated at the transcriptional level, but also by phosphorylation, which occurs rapidly during stress and is responsible for causing the dissociation of large 700-kDa Hsp27 oligomers into dimers. We investigated the mechanism by which phosphorylation and oligomerization modulate the protective activity of Chinese hamster Hsp27. In contrast to oligomer dissociation, which only required Ser90 phosphorylation, activation of Hsp27 thermoprotective activity required the phosphorylation of both Ser90 and Ser15. Replacement of Ser90 by Ala90, which prevented the dissociation of the oligomer upon stress, did cause a severe defect in the protective activity. Dissociation was, however, not a sufficient condition to activate the protein because replacement of Ser15 by Ala15, which caused little effect in the oligomeric organization of the protein, also yielded an inactive protein. Analyzes of mutants with short deletions in the NH2 terminus identified the Hsp27 WD/EPF or PF-rich domain as essential for protection, maintenance of the oligomeric structure, and in vitro chaperone activity of the protein. In light of a three-dimensional model of Hsp27 based on the crystallographic structure of wheat Hsp16.9, we propose that the conserved WD/EPF motif of mammalian Hsp27 mediates important intramolecular interactions with hydrophic surfaces of the alpha-crystallin domain of the protein. These interactions are destabilized by Ser90 phosphorylation, making the motif free to interact with heterologous molecular targets upon the additional phosphorylation of the nearby Ser15.

Discovery of a Highly Potent, Cell-Permeable Macrocyclic Peptidomimetic (MM-589) Targeting the WD Repeat Domain 5 Protein (WDR5)–Mixed Lineage Leukemia (MLL) Protein–Protein Interaction

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

Karatas, Hacer; Li, Yangbing; Liu, Liu

We report herein the design, synthesis, and evaluation of macrocyclic peptidomimetics that bind to WD repeat domain 5 (WDR5) and block the WDR5–mixed lineage leukemia (MLL) protein–protein interaction. Compound 18 (MM-589) binds to WDR5 with an IC50 value of 0.90 nM (Ki value <1 nM) and inhibits the MLL H3K4 methyltransferase (HMT) activity with an IC50 value of 12.7 nM. Compound 18 potently and selectively inhibits cell growth in human leukemia cell lines harboring MLL translocations and is >40 times better than the previously reported compound MM-401. Cocrystal structures of 16 and 18 complexed with WDR5 provide structural basis formore » their high affinity binding to WDR5. Additionally, we have developed and optimized a new AlphaLISA-based MLL HMT functional assay to facilitate the functional evaluation of these designed compounds. Compound 18 represents the most potent inhibitor of the WDR5–MLL interaction reported to date, and further optimization of 18 may yield a new therapy for acute leukemia.« less

Is WD 1437-008 a cataclysmic variable?

NASA Astrophysics Data System (ADS)

Shimansky, V. V.; Nurtdinova, D. N.; Borisov, N. V.; Spiridonova, O. I.

2011-10-01

Comprehensive observations of a close binary candidate WD 1437-008 are performed. The shape and amplitude of the observed brightness variations are shown to be inconsistent with the hypothesis of reflection effects, and the photometric period of the system, P phot = 0. d 2775, is found to differ from the period of spectral variations, P sp = 0. d 272060. As a result, WD 1437-008 has been preliminarily classified as a low-inclination cataclysmic variable.

The Molecular Chaperone TRiC/CCT Binds to the Trp-Asp 40 (WD40) Repeat Protein WDR68 and Promotes Its Folding, Protein Kinase DYRK1A Binding, and Nuclear Accumulation*

PubMed Central

Miyata, Yoshihiko; Shibata, Takeshi; Aoshima, Masato; Tsubata, Takuichi; Nishida, Eisuke

2014-01-01

Trp-Asp (WD) repeat protein 68 (WDR68) is an evolutionarily conserved WD40 repeat protein that binds to several proteins, including dual specificity tyrosine phosphorylation-regulated protein kinase (DYRK1A), MAPK/ERK kinase kinase 1 (MEKK1), and Cullin4-damage-specific DNA-binding protein 1 (CUL4-DDB1). WDR68 affects multiple and diverse physiological functions, such as controlling anthocyanin synthesis in plants, tissue growth in insects, and craniofacial development in vertebrates. However, the biochemical basis and the regulatory mechanism of WDR68 activity remain largely unknown. To better understand the cellular function of WDR68, here we have isolated and identified cellular WDR68 binding partners using a phosphoproteomic approach. More than 200 cellular proteins with wide varieties of biochemical functions were identified as WDR68-binding protein candidates. Eight T-complex protein 1 (TCP1) subunits comprising the molecular chaperone TCP1 ring complex/chaperonin-containing TCP1 (TRiC/CCT) were identified as major WDR68-binding proteins, and phosphorylation sites in both WDR68 and TRiC/CCT were identified. Co-immunoprecipitation experiments confirmed the binding between TRiC/CCT and WDR68. Computer-aided structural analysis suggested that WDR68 forms a seven-bladed β-propeller ring. Experiments with a series of deletion mutants in combination with the structural modeling showed that three of the seven β-propeller blades of WDR68 are essential and sufficient for TRiC/CCT binding. Knockdown of cellular TRiC/CCT by siRNA caused an abnormal WDR68 structure and led to reduction of its DYRK1A-binding activity. Concomitantly, nuclear accumulation of WDR68 was suppressed by the knockdown of TRiC/CCT, and WDR68 formed cellular aggregates when overexpressed in the TRiC/CCT-deficient cells. Altogether, our results demonstrate that the molecular chaperone TRiC/CCT is essential for correct protein folding, DYRK1A binding, and nuclear accumulation of WDR68. PMID

Mutation of domain III and domain VI in L gene conserved domain of Nipah virus

NASA Astrophysics Data System (ADS)

Jalani, Siti Aishah; Ibrahim, Nazlina

2016-11-01

Nipah virus (NiV) is the etiologic agent responsible for the respiratory illness and causes fatal encephalitis in human. NiV L protein subunit is thought to be responsible for the majority of enzymatic activities involved in viral transcription and replication. The L protein which is the viral RNA dependent RNA polymerase has high sequence homology among negative sense RNA viruses. In negative stranded RNA viruses, based on sequence alignment six conserved domain (domain I-IV) have been determined. Each domain is separated on variable regions that suggest the structure to consist concatenated functional domain. To directly address the roles of domains III and VI, site-directed mutations were constructed by the substitution of bases at sequences 2497, 2500, 5528 and 5532. Each mutated L gene can be used in future studies to test the ability for expression on in vitro translation.

Conserved domains and SINE diversity during animal evolution.

PubMed

Luchetti, Andrea; Mantovani, Barbara

2013-10-01

Eukaryotic genomes harbour a number of mobile genetic elements (MGEs); moving from one genomic location to another, they are known to impact on the host genome. Short interspersed elements (SINEs) are well-represented, non-autonomous retroelements and they are likely the most diversified MGEs. In some instances, sequence domains conserved across unrelated SINEs have been identified; remarkably, one of these, called Nin, has been conserved since the Radiata-Bilateria splitting. Here we report on two new domains: Inv, derived from Nin, identified in insects and in deuterostomes, and Pln, restricted to polyneopteran insects. The identification of Inv and Pln sequences allowed us to retrieve new SINEs, two in insects and one in a hemichordate. The diverse structural combination of the different domains in different SINE families, during metazoan evolution, offers a clearer view of SINE diversity and their frequent de novo emergence through module exchange, possibly underlying the high evolutionary success of SINEs. © 2013 Elsevier Inc. All rights reserved.

Evolving ONe WD+He star systems to intermediate-mass binary pulsars

NASA Astrophysics Data System (ADS)

Liu, D.; Wang, B.; Chen, W.; Zuo, Z.; Han, Z.

2018-06-01

It has been suggested that accretion-induced collapse (AIC) is a non-negligible path for the formation of the observed neutron stars (NSs). An ONe white dwarf (WD) that accretes material from a He star may experience AIC process and eventually produce intermediate-mass binary pulsars (IMBPs), named as the ONe WD+He star scenario. Note that previous studies can only account for part of the observed IMBPs with short orbital periods. In this work, we investigate the evolution of about 900 ONe WD+He star binaries to explore the distribution of IMBPs. We found that the ONe WD+He star scenario could form IMBPs including pulsars with 5-340 ms spin periods and 0.75-1.38 M_{⊙} WD companions, in which the orbital periods range from 0.04 to 900 d. Compared with the 20 observed IMBPs, this scenario can cover the parameters of 13 sources in the final orbital period-WD mass plane and the Corbet diagram, most of which have short orbital periods. We found that the ONe WD+He star scenario can explain almost all the observed IMBPs with short orbital periods. This work can well match the observed parameters of PSR J1802-2124 (one of the two precisely observed IMBPs), providing a possible evolutional path for its formation. We also speculate that the compact companion of HD 49798 (a hydrogen depleted sdO6 star) may be not a NS based on this work.

VizieR Online Data Catalog: WD+dMs from the SUPERBLINK proper motion survey (Skinner+, 2017)

NASA Astrophysics Data System (ADS)

Skinner, J. N.; Morgan, D. P.; West, A. A.; Lepine, S.; Thorstensen, J. R.

2018-06-01

To select for nearby WD+dMs, we used the SUPERBLINK proper motion survey (Lepine et al. 2002, J/AJ/124/1190; Lepine & Shara 2005, Cat. I/298), an ongoing all-sky survey that identifies and characterizes stars with proper motions μ>40 mas/yr. For this study, we used the 2011 July version of SUPERBLINK, which listed 2270481 stars, and was estimated to be >90% complete to V=19.0. We selected WD+dMs based on a combination of V magnitudes derived from the DSS plates (see Lepine & Shara 2005, Cat. I/298), near-UV magnitudes from GALEX, and Ks magnitudes from 2MASS. Using the UV-optical-IR color selection outlined in Skinner et al. (2014AJ....148..115S), we selected targets for spectroscopic follow-up (see bottom panel of Figure 1). We acquired optical spectroscopy of 178 newly identified WD+dM candidates, with the Boller and Chivens CCD spectrograph (CCDS), using both the Hiltner 2.4 m and McGraw-Hill 1.3 m telescopes located at the MDM Observatory. (3 data files).

The conservation pattern of short linear motifs is highly correlated with the function of interacting protein domains.

PubMed

Ren, Siyuan; Yang, Guang; He, Youyu; Wang, Yiguo; Li, Yixue; Chen, Zhengjun

2008-10-01

Many well-represented domains recognize primary sequences usually less than 10 amino acids in length, called Short Linear Motifs (SLiMs). Accurate prediction of SLiMs has been difficult because they are short (often < 10 amino acids) and highly degenerate. In this study, we combined scoring matrixes derived from peptide library and conservation analysis to identify protein classes enriched of functional SLiMs recognized by SH2, SH3, PDZ and S/T kinase domains. Our combined approach revealed that SLiMs are highly conserved in proteins from functional classes that are known to interact with a specific domain, but that they are not conserved in most other protein groups. We found that SLiMs recognized by SH2 domains were highly conserved in receptor kinases/phosphatases, adaptor molecules, and tyrosine kinases/phosphatases, that SLiMs recognized by SH3 domains were highly conserved in cytoskeletal and cytoskeletal-associated proteins, that SLiMs recognized by PDZ domains were highly conserved in membrane proteins such as channels and receptors, and that SLiMs recognized by S/T kinase domains were highly conserved in adaptor molecules, S/T kinases/phosphatases, and proteins involved in transcription or cell cycle control. We studied Tyr-SLiMs recognized by SH2 domains in more detail, and found that SH2-recognized Tyr-SLiMs on the cytoplasmic side of membrane proteins are more highly conserved than those on the extra-cellular side. Also, we found that SH2-recognized Tyr-SLiMs that are associated with SH3 motifs and a tyrosine kinase phosphorylation motif are more highly conserved. The interactome of protein domains is reflected by the evolutionary conservation of SLiMs recognized by these domains. Combining scoring matrixes derived from peptide libraries and conservation analysis, we would be able to find those protein groups that are more likely to interact with specific domains.

CORAL: aligning conserved core regions across domain families.

PubMed

Fong, Jessica H; Marchler-Bauer, Aron

2009-08-01

Homologous protein families share highly conserved sequence and structure regions that are frequent targets for comparative analysis of related proteins and families. Many protein families, such as the curated domain families in the Conserved Domain Database (CDD), exhibit similar structural cores. To improve accuracy in aligning such protein families, we propose a profile-profile method CORAL that aligns individual core regions as gap-free units. CORAL computes optimal local alignment of two profiles with heuristics to preserve continuity within core regions. We benchmarked its performance on curated domains in CDD, which have pre-defined core regions, against COMPASS, HHalign and PSI-BLAST, using structure superpositions and comprehensive curator-optimized alignments as standards of truth. CORAL improves alignment accuracy on core regions over general profile methods, returning a balanced score of 0.57 for over 80% of all domain families in CDD, compared with the highest balanced score of 0.45 from other methods. Further, CORAL provides E-values to aid in detecting homologous protein families and, by respecting block boundaries, produces alignments with improved 'readability' that facilitate manual refinement. CORAL will be included in future versions of the NCBI Cn3D/CDTree software, which can be downloaded at http://www.ncbi.nlm.nih.gov/Structure/cdtree/cdtree.shtml. Supplementary data are available at Bioinformatics online.

The conservation pattern of short linear motifs is highly correlated with the function of interacting protein domains

PubMed Central

Ren, Siyuan; Yang, Guang; He, Youyu; Wang, Yiguo; Li, Yixue; Chen, Zhengjun

2008-01-01

Background Many well-represented domains recognize primary sequences usually less than 10 amino acids in length, called Short Linear Motifs (SLiMs). Accurate prediction of SLiMs has been difficult because they are short (often < 10 amino acids) and highly degenerate. In this study, we combined scoring matrixes derived from peptide library and conservation analysis to identify protein classes enriched of functional SLiMs recognized by SH2, SH3, PDZ and S/T kinase domains. Results Our combined approach revealed that SLiMs are highly conserved in proteins from functional classes that are known to interact with a specific domain, but that they are not conserved in most other protein groups. We found that SLiMs recognized by SH2 domains were highly conserved in receptor kinases/phosphatases, adaptor molecules, and tyrosine kinases/phosphatases, that SLiMs recognized by SH3 domains were highly conserved in cytoskeletal and cytoskeletal-associated proteins, that SLiMs recognized by PDZ domains were highly conserved in membrane proteins such as channels and receptors, and that SLiMs recognized by S/T kinase domains were highly conserved in adaptor molecules, S/T kinases/phosphatases, and proteins involved in transcription or cell cycle control. We studied Tyr-SLiMs recognized by SH2 domains in more detail, and found that SH2-recognized Tyr-SLiMs on the cytoplasmic side of membrane proteins are more highly conserved than those on the extra-cellular side. Also, we found that SH2-recognized Tyr-SLiMs that are associated with SH3 motifs and a tyrosine kinase phosphorylation motif are more highly conserved. Conclusion The interactome of protein domains is reflected by the evolutionary conservation of SLiMs recognized by these domains. Combining scoring matrixes derived from peptide libraries and conservation analysis, we would be able to find those protein groups that are more likely to interact with specific domains. PMID:18828911

WD+RG systems as the progenitors of type Ia supernovae

NASA Astrophysics Data System (ADS)

Wang, Bo; Han, Zhan-Wen

2010-03-01

Type Ia supernovae (SNe Ia) play an important role in the study of cosmic evolution, especially in cosmology. There are several progenitor models for SNe Ia proposed in the past years. By considering the effect of accretion disk instability on the evolution of white dwarf (WD) binaries, we performed detailed binary evolution calculations for the WD + red-giant (RG) channel of SNe Ia, in which a carbon-oxygen WD accretes material from a RG star to increase its mass to the Chandrasekhar mass limit. According to these calculations, we mapped out the initial and final parameters for SNe Ia in the orbital period-secondary mass (log Pi - Mi2) plane for various WD masses for this channel. We discussed the influence of the variation of the duty cycle value on the regions for producing SNe Ia. Similar to previous studies, this work also indicates that the long-period dwarf novae offer possible ways for producing SNe Ia. Meanwhile, we find that the surviving companion stars from this channel have a low mass after the SN explosion, which may provide a means for the formation of the population of single low-mass WDs (<0.45 Modot).

50 CFR 635.40 - Restrictions to enhance conservation.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 50 Wildlife and Fisheries 12 2013-10-01 2013-10-01 false Restrictions to enhance conservation. 635.40 Section 635.40 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION, DEPARTMENT OF COMMERCE ATLANTIC HIGHLY MIGRATORY SPECIES Restrictions on Imports...

Microtubule association of EML proteins and the EML4-ALK variant 3 oncoprotein require an N-terminal trimerization domain.

PubMed

Richards, Mark W; O'Regan, Laura; Roth, Daniel; Montgomery, Jessica M; Straube, Anne; Fry, Andrew M; Bayliss, Richard

2015-05-01

Proteins of the echinoderm microtubule (MT)-associated protein (EMAP)-like (EML) family contribute to formation of the mitotic spindle and interphase MT network. EML1-4 consist of Trp-Asp 40 (WD40) repeats and an N-terminal region containing a putative coiled-coil. Recurrent gene rearrangements in non-small cell lung cancer (NSCLC) fuse EML4 to anaplastic lymphoma kinase (ALK) causing expression of several oncogenic fusion variants. The fusions have constitutive ALK activity due to self-association through the EML4 coiled-coil. We have determined crystal structures of the coiled-coils from EML2 and EML4, which describe the structural basis of both EML self-association and oncogenic EML4-ALK activation. The structures reveal a trimeric oligomerization state directed by a conserved pattern of hydrophobic residues and salt bridges. We show that the trimerization domain (TD) of EML1 is necessary and sufficient for self-association. The TD is also essential for MT binding; however, this property requires an adjacent basic region. These observations prompted us to investigate MT association of EML4-ALK and EML1-ABL1 (Abelson 1) fusions in which variable portions of the EML component are present. Uniquely, EML4-ALK variant 3, which includes the TD and basic region of EML4 but none of the WD40 repeats, was localized to MTs, both when expressed recombinantly and when expressed in a patient-derived NSCLC cell line (H2228). This raises the question of whether the mislocalization of ALK activity to MTs might influence downstream signalling and malignant properties of cells. Furthermore, the structure of EML4 TD may enable the development of protein-protein interaction inhibitors targeting the trimerization interface, providing a possible avenue towards therapeutic intervention in EML4-ALK NSCLC.

RNA polymerase II conserved protein domains as platforms for protein-protein interactions

PubMed Central

García-López, M Carmen

2011-01-01

RNA polymerase II establishes many protein-protein interactions with transcriptional regulators to coordinate gene expression, but little is known about protein domains involved in the contact with them. We use a new approach to look for conserved regions of the RNA pol II of S. cerevisiae located at the surface of the structure of the complex, hypothesizing that they might be involved in the interaction with transcriptional regulators. We defined five different conserved domains and demonstrate that all of them make contact with transcriptional regulators. PMID:21922063

HMMerThread: detecting remote, functional conserved domains in entire genomes by combining relaxed sequence-database searches with fold recognition.

PubMed

Bradshaw, Charles Richard; Surendranath, Vineeth; Henschel, Robert; Mueller, Matthias Stefan; Habermann, Bianca Hermine

2011-03-10

Conserved domains in proteins are one of the major sources of functional information for experimental design and genome-level annotation. Though search tools for conserved domain databases such as Hidden Markov Models (HMMs) are sensitive in detecting conserved domains in proteins when they share sufficient sequence similarity, they tend to miss more divergent family members, as they lack a reliable statistical framework for the detection of low sequence similarity. We have developed a greatly improved HMMerThread algorithm that can detect remotely conserved domains in highly divergent sequences. HMMerThread combines relaxed conserved domain searches with fold recognition to eliminate false positive, sequence-based identifications. With an accuracy of 90%, our software is able to automatically predict highly divergent members of conserved domain families with an associated 3-dimensional structure. We give additional confidence to our predictions by validation across species. We have run HMMerThread searches on eight proteomes including human and present a rich resource of remotely conserved domains, which adds significantly to the functional annotation of entire proteomes. We find ∼4500 cross-species validated, remotely conserved domain predictions in the human proteome alone. As an example, we find a DNA-binding domain in the C-terminal part of the A-kinase anchor protein 10 (AKAP10), a PKA adaptor that has been implicated in cardiac arrhythmias and premature cardiac death, which upon stress likely translocates from mitochondria to the nucleus/nucleolus. Based on our prediction, we propose that with this HLH-domain, AKAP10 is involved in the transcriptional control of stress response. Further remotely conserved domains we discuss are examples from areas such as sporulation, chromosome segregation and signalling during immune response. The HMMerThread algorithm is able to automatically detect the presence of remotely conserved domains in proteins based on weak

HMMerThread: Detecting Remote, Functional Conserved Domains in Entire Genomes by Combining Relaxed Sequence-Database Searches with Fold Recognition

PubMed Central

Bradshaw, Charles Richard; Surendranath, Vineeth; Henschel, Robert; Mueller, Matthias Stefan; Habermann, Bianca Hermine

2011-01-01

Conserved domains in proteins are one of the major sources of functional information for experimental design and genome-level annotation. Though search tools for conserved domain databases such as Hidden Markov Models (HMMs) are sensitive in detecting conserved domains in proteins when they share sufficient sequence similarity, they tend to miss more divergent family members, as they lack a reliable statistical framework for the detection of low sequence similarity. We have developed a greatly improved HMMerThread algorithm that can detect remotely conserved domains in highly divergent sequences. HMMerThread combines relaxed conserved domain searches with fold recognition to eliminate false positive, sequence-based identifications. With an accuracy of 90%, our software is able to automatically predict highly divergent members of conserved domain families with an associated 3-dimensional structure. We give additional confidence to our predictions by validation across species. We have run HMMerThread searches on eight proteomes including human and present a rich resource of remotely conserved domains, which adds significantly to the functional annotation of entire proteomes. We find ∼4500 cross-species validated, remotely conserved domain predictions in the human proteome alone. As an example, we find a DNA-binding domain in the C-terminal part of the A-kinase anchor protein 10 (AKAP10), a PKA adaptor that has been implicated in cardiac arrhythmias and premature cardiac death, which upon stress likely translocates from mitochondria to the nucleus/nucleolus. Based on our prediction, we propose that with this HLH-domain, AKAP10 is involved in the transcriptional control of stress response. Further remotely conserved domains we discuss are examples from areas such as sporulation, chromosome segregation and signalling during immune response. The HMMerThread algorithm is able to automatically detect the presence of remotely conserved domains in proteins based on weak

The Three Domains of Conservation Genetics: Case Histories from Hawaiian Waters

PubMed Central

2016-01-01

The scientific field of conservation biology is dominated by 3 specialties: phylogenetics, ecology, and evolution. Under this triad, phylogenetics is oriented towards the past history of biodiversity, conserving the divergent branches in the tree of life. The ecological component is rooted in the present, maintaining the contemporary life support systems for biodiversity. Evolutionary conservation (as defined here) is concerned with preserving the raw materials for generating future biodiversity. All 3 domains can be documented with genetic case histories in the waters of the Hawaiian Archipelago, an isolated chain of volcanic islands with 2 types of biodiversity: colonists, and new species that arose from colonists. This review demonstrates that 1) phylogenetic studies have identified previously unknown branches in the tree of life that are endemic to Hawaiian waters; 2) population genetic surveys define isolated marine ecosystems as management units, and 3) phylogeographic analyses illustrate the pathways of colonization that can enhance future biodiversity. Conventional molecular markers have advanced all 3 domains in conservation biology over the last 3 decades, and recent advances in genomics are especially valuable for understanding the foundations of future evolutionary diversity. PMID:27001936

THE FIRST DISTANCE CONSTRAINT ON THE RENEGADE HIGH-VELOCITY CLOUD COMPLEX WD

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

Peek, J. E. G.; Roman-Duval, Julia; Tumlinson, Jason

2016-09-10

We present medium-resolution, near-ultraviolet Very Large Telescope/FLAMES observations of the star USNO-A0600-15865535. We adapt a standard method of stellar typing to our measurement of the shape of the Balmer ϵ absorption line to demonstrate that USNO-A0600-15865535 is a blue horizontal branch star, residing in the lower stellar halo at a distance of 4.4 kpc from the Sun. We measure the H and K lines of singly ionized calcium and find two isolated velocity components, one originating in the disk, and one associated with the high-velocity cloud complex WD. This detection demonstrated that complex WD is closer than ∼4.4 kpc andmore » is the first distance constraint on the +100 km s{sup −1} Galactic complex of clouds. We find that complex WD is not in corotation with the Galactic disk, which has been assumed for decades. We examine a number of scenarios and find that the most likely scenario is that complex WD was ejected from the solar neighborhood and is only a few kiloparsecs from the Sun.« less

J domain independent functions of J proteins.

PubMed

Ajit Tamadaddi, Chetana; Sahi, Chandan

2016-07-01

Heat shock proteins of 40 kDa (Hsp40s), also called J proteins, are obligate partners of Hsp70s. Via their highly conserved and functionally critical J domain, J proteins interact and modulate the activity of their Hsp70 partners. Mutations in the critical residues in the J domain often result in the null phenotype for the J protein in question. However, as more J proteins have been characterized, it is becoming increasingly clear that a significant number of J proteins do not "completely" rely on their J domains to carry out their cellular functions, as previously thought. In some cases, regions outside the highly conserved J domain have become more important making the J domain dispensable for some, if not for all functions of a J protein. This has profound effects on the evolution of such J proteins. Here we present selected examples of J proteins that perform J domain independent functions and discuss this in the context of evolution of J proteins with dispensable J domains and J-like proteins in eukaryotes.

[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.

The Three Domains of Conservation Genetics: Case Histories from Hawaiian Waters.

PubMed

Bowen, Brian W

2016-07-01

The scientific field of conservation biology is dominated by 3 specialties: phylogenetics, ecology, and evolution. Under this triad, phylogenetics is oriented towards the past history of biodiversity, conserving the divergent branches in the tree of life. The ecological component is rooted in the present, maintaining the contemporary life support systems for biodiversity. Evolutionary conservation (as defined here) is concerned with preserving the raw materials for generating future biodiversity. All 3 domains can be documented with genetic case histories in the waters of the Hawaiian Archipelago, an isolated chain of volcanic islands with 2 types of biodiversity: colonists, and new species that arose from colonists. This review demonstrates that 1) phylogenetic studies have identified previously unknown branches in the tree of life that are endemic to Hawaiian waters; 2) population genetic surveys define isolated marine ecosystems as management units, and 3) phylogeographic analyses illustrate the pathways of colonization that can enhance future biodiversity. Conventional molecular markers have advanced all 3 domains in conservation biology over the last 3 decades, and recent advances in genomics are especially valuable for understanding the foundations of future evolutionary diversity. © The American Genetic Association. 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A conserved domain in the NH2 terminus important for assembly and functional expression of pacemaker channels.

PubMed

Tran, Neil; Proenza, Catherine; Macri, Vincenzo; Petigara, Fiona; Sloan, Erin; Samler, Shannon; Accili, Eric A

2002-11-15

Pacemaker channels are formed by co-assembly of hyperpolarization-activated cyclic nucleotide-gated (HCN) subunits. Previously, we suggested that the NH(2) termini of the mouse HCN2 isoform were important for subunit co-assembly and functional channel expression. Using an alignment strategy together with yeast two-hybrid assays, patch clamp electrophysiology, and confocal imaging, we have now identified a domain within the NH(2) terminus of the HCN2 subunit that is responsible for interactions between NH(2) termini and promoting the trafficking of functional channels to the plasma membrane. This domain is composed of 52 amino acids, is located adjacent to the putative first transmembrane segment, and is highly conserved among the mammalian HCN isoforms. This conserved domain, but not the remaining unconserved NH(2)-terminal regions of HCN2, specifically interacted with itself in yeast two-hybrid assays. Moreover, the conserved domain was important for expression of currents. Whereas relatively normal whole cell HCN2 currents were produced by channels containing only the conserved domain, further deletion of this region, leaving only a more polar and putative coiled-coil segment, eliminated HCN2 currents and resulted in proteins that localized predominantly in perinuclear compartments. Thus, we suggest that this conserved domain is the critical NH(2)-terminal determinant of subunit co-assembly and trafficking of pacemaker channels.

Conserved and variable domains of RNase MRP RNA.

PubMed

Dávila López, Marcela; Rosenblad, Magnus Alm; Samuelsson, Tore

2009-01-01

Ribonuclease MRP is a eukaryotic ribonucleoprotein complex consisting of one RNA molecule and 7-10 protein subunits. One important function of MRP is to catalyze an endonucleolytic cleavage during processing of rRNA precursors. RNase MRP is evolutionary related to RNase P which is critical for tRNA processing. A large number of MRP RNA sequences that now are available have been used to identify conserved primary and secondary structure features of the molecule. MRP RNA has structural features in common with P RNA such as a conserved catalytic core, but it also has unique features and is characterized by a domain highly variable between species. Information regarding primary and secondary structure features is of interest not only in basic studies of the function of MRP RNA, but also because mutations in the RNA give rise to human genetic diseases such as cartilage-hair hypoplasia.

WRKY domain-encoding genes of a crop legume chickpea (Cicer arietinum): comparative analysis with Medicago truncatula WRKY family and characterization of group-III gene(s).

PubMed

Kumar, Kamal; Srivastava, Vikas; Purayannur, Savithri; Kaladhar, V Chandra; Cheruvu, Purnima Jaiswal; Verma, Praveen Kumar

2016-06-01

The WRKY genes have been identified as important transcriptional modulators predominantly during the environmental stresses, but they also play critical role at various stages of plant life cycle. We report the identification of WRKY domain (WD)-encoding genes from galegoid clade legumes chickpea (Cicer arietinum L.) and barrel medic (Medicago truncatula). In total, 78 and 98 WD-encoding genes were found in chickpea and barrel medic, respectively. Comparative analysis suggests the presence of both conserved and unique WRKYs, and expansion of WRKY family in M. truncatula primarily by tandem duplication. Exclusively found in galegoid legumes, CaWRKY16 and its orthologues encode for a novel protein having a transmembrane and partial Exo70 domains flanking a group-III WD. Genomic region of galegoids, having CaWRKY16, is more dynamic when compared with millettioids. In onion cells, fused CaWRKY16-EYFP showed punctate fluorescent signals in cytoplasm. The chickpea WRKY group-III genes were further characterized for their transcript level modulation during pathogenic stress and treatments of abscisic acid, jasmonic acid, and salicylic acid (SA) by real-time PCR. Differential regulation of genes was observed during Ascochyta rabiei infection and SA treatment. Characterization of A. rabiei and SA inducible gene CaWRKY50 showed that it localizes to plant nucleus, binds to W-box, and have a C-terminal transactivation domain. Overexpression of CaWRKY50 in tobacco plants resulted in early flowering and senescence. The in-depth comparative account presented here for two legume WRKY genes will be of great utility in hastening functional characterization of crop legume WRKYs and will also help in characterization of Exo70Js. © The Author 2016. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Necroplanetology: Disrupted Planetary Material Transiting WD 1145+017

NASA Astrophysics Data System (ADS)

Manideep Duvvuri, Girish; Redfield, Seth; Veras, Dimitri

2018-06-01

The WD 1145+017 system shows irregular transit features that are consistent with the tidal disruption of differentiated asteroids with bulk densities < 4 g cm-3 and bulk masses < 1021 kg. We use the open-source N-body code REBOUND to simulate this disruption with different internal structures: varying the core volume fraction, mantle/core density ratio, and the presence/absence of a thin low-density crust. We show that these parameters have observationally distinguishable effects on the transit light curve as the asteroid is disrupted and fit the simulation-generated lightcurves to data. We find that an asteroid with a low core fraction, low mantle/density ratio, and without a crust is most consistent with the A1 feature present for multiple weeks circa April 2017. This combination of observations and simulations to study the interior structure and chemistry of exoplanetary bodies via their destruction in action is an early example of necroplanetology, a field that will hopefully grow with the discovery of other systems like WD 1145+017.

Molecular mapping of qBK1 WD , a major QTL for bakanae disease resistance in rice.

PubMed

Lee, Sais-Beul; Hur, Yeon-Jae; Cho, Jun-Hyeon; Lee, Jong-Hee; Kim, Tae-Heon; Cho, Soo-Min; Song, You-Chun; Seo, Young-Su; Lee, Jungkwan; Kim, Tae-Sung; Park, Yong-Jin; Oh, Myung-Kyu; Park, Dong-Soo

2018-01-10

Bakanae or foot rot disease is a prominent disease of rice caused by Gibberella fujikuroi. This disease may infect rice plants from the pre-emergence stage to the mature stage. In recent years, raising rice seedlings in seed boxes for mechanical transplanting has increased the incidence of many seedling diseases; only a few rice varieties have been reported to exhibit resistance to bakanae disease. In this study, we attempted to identify quantitative trait loci (QTLs) conferring bakanae disease resistance from the highly resistant japonica variety Wonseadaesoo. A primary QTL study using the genotypes/phenotypes of the recombinant inbred lines (RILs) indicated that the locus qBK1 WD conferring resistance to bakanae disease from Wonseadaesoo was located in a 1.59 Mb interval delimited on the physical map between chr01_13542347 (13.54 Mb) and chr01_15132528 (15.13 Mb). The log of odds (LOD) score of qBK1 WD was 8.29, accounting for 20.2% of the total phenotypic variation. We further identified a gene pyramiding effect of two QTLs, qBK WD and previously developed qBK1. The mean proportion of healthy plant for 31 F 4 RILs that had no resistance genes was 35.3%, which was similar to that of the susceptible check variety Ilpum. The proportion of healthy plants for the lines with only qBK WD or qBK1 was 66.1% and 55.5%, respectively, which was significantly higher than that of the lines without resistance genes and that of Ilpum. The mean proportion of the healthy plant for 15 F 4 RILs harboring both qBK WD and qBK1 was 80.2%, which was significantly higher than that of the lines with only qBK WD or qBK1. Introducing qBK WD or pyramiding the QTLs qBK WD and qBK1 could provide effective tools for breeding rice with bakanae disease resistance. To our knowledge, this is the first report on a gene pyramiding effect that provides higher resistance against bakanae disease.

Improving Assessment of Work Related Mental Health Function Using the Work Disability Functional Assessment Battery (WD-FAB).

PubMed

Marfeo, Elizabeth E; Ni, Pengsheng; McDonough, Christine; Peterik, Kara; Marino, Molly; Meterko, Mark; Rasch, Elizabeth K; Chan, Leighton; Brandt, Diane; Jette, Alan M

2018-03-01

Purpose To improve the mental health component of the Work Disability Functional Assessment Battery (WD-FAB), developed for the US Social Security Administration's (SSA) disability determination process. Specifically our goal was to expand the WD-FAB scales of mood & emotions, resilience, social interactions, and behavioral control to improve the depth and breadth of the current scales and expand the content coverage to include aspects of cognition & communication function. Methods Data were collected from a random, stratified sample of 1695 claimants applying for the SSA work disability benefits, and a general population sample of 2025 working age adults. 169 new items were developed to replenish the WD-FAB scales and analyzed using factor analysis and item response theory (IRT) analysis to construct unidimensional scales. We conducted computer adaptive test (CAT) simulations to examine the psychometric properties of the WD-FAB. Results Analyses supported the inclusion of four mental health subdomains: Cognition & Communication (68 items), Self-Regulation (34 items), Resilience & Sociability (29 items) and Mood & Emotions (34 items). All scales yielded acceptable psychometric properties. Conclusions IRT methods were effective in expanding the WD-FAB to assess mental health function. The WD-FAB has the potential to enhance work disability assessment both within the context of the SSA disability programs as well as other clinical and vocational rehabilitation settings.

Association studies of WD repeat domain 3 and chitobiosyldiphosphodolichol beta-mannosyltransferase genes with schizophrenia in a Japanese population

PubMed Central

Iwayama, Yoshimi; Yamamoto, Naoki; Toyota, Tomoko; Suzuki, Katsuaki; Kikuchi, Mitsuru; Hashimoto, Tasuku; Kanahara, Nobuhisa; Kurumaji, Akeo; Yoshikawa, Takeo

2018-01-01

Schizophrenia and schizophrenia-like symptoms induced by the dopamine agonists and N-methyl-D aspartate type glutamate receptor antagonists occur only after the adolescent period. Similarly, animal models of schizophrenia by these drugs are also induced after the critical period around postnatal week three. Based upon the development-dependent onsets of these psychotomimetic effects, by using a DNA microarray technique, we identified the WD repeat domain 3 (WDR3) and chitobiosyldiphosphodolichol beta-mannosyltransferase (ALG1) genes as novel candidates for schizophrenia-related molecules, whose mRNAs were up-regulated in the adult (postnatal week seven), but not in the infant (postnatal week one) rats by an indirect dopamine agonist, and phencyclidine, an antagonist of the NMDA receptor. WDR3 and other related proteins are the nuclear proteins presumably involved in various cellular activities, such as cell cycle progression, signal transduction, apoptosis, and gene regulation. ALG1 is presumed to be involved in the regulation of the protein N-glycosylation. To further elucidate the molecular pathophysiology of schizophrenia, we have evaluated the genetic association of WDR3 and ALG1 in schizophrenia. We examined 21 single nucleotide polymorphisms [SNPs; W1 (rs1812607)-W16 (rs6656360), A1 (rs8053916)-A10 (rs9673733)] from these genes using the Japanese case-control sample (1,808 schizophrenics and 2,170 matched controls). No significant genetic associations of these SNPs were identified. However, we detected a significant association of W4 (rs319471) in the female schizophrenics (allelic P = 0.003, genotypic P = 0.008). Based on a haplotype analysis, the observed haplotypes consisting of W4 (rs319471)–W5 (rs379058) also displayed a significant association in the female schizophrenics (P = 0.016). Even after correction for multiple testing, these associations remained significant. Our findings suggest that the WDR3 gene may likely be a sensitive factor in female

40 CFR 73.26 - Conservation and renewable energy reserve.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 17 2013-07-01 2013-07-01 false Conservation and renewable energy... renewable energy reserve. The Administrator will allocate 300,000 allowances to the Conservation and Renewable Energy Reserve subaccount of the Acid Rain Data System. Allowances from this Reserve will be...

40 CFR 73.26 - Conservation and renewable energy reserve.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 17 2012-07-01 2012-07-01 false Conservation and renewable energy... renewable energy reserve. The Administrator will allocate 300,000 allowances to the Conservation and Renewable Energy Reserve subaccount of the Acid Rain Data System. Allowances from this Reserve will be...

40 CFR 73.26 - Conservation and renewable energy reserve.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 16 2010-07-01 2010-07-01 false Conservation and renewable energy... renewable energy reserve. The Administrator will allocate 300,000 allowances to the Conservation and Renewable Energy Reserve subaccount of the Acid Rain Data System. Allowances from this Reserve will be...

40 CFR 73.26 - Conservation and renewable energy reserve.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 16 2011-07-01 2011-07-01 false Conservation and renewable energy... renewable energy reserve. The Administrator will allocate 300,000 allowances to the Conservation and Renewable Energy Reserve subaccount of the Acid Rain Data System. Allowances from this Reserve will be...

40 CFR 73.26 - Conservation and renewable energy reserve.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 17 2014-07-01 2014-07-01 false Conservation and renewable energy... renewable energy reserve. The Administrator will allocate 300,000 allowances to the Conservation and Renewable Energy Reserve subaccount of the Acid Rain Data System. Allowances from this Reserve will be...

Conservation of the Human Integrin-Type Beta-Propeller Domain in Bacteria

PubMed Central

Chouhan, Bhanupratap; Denesyuk, Alexander; Heino, Jyrki; Johnson, Mark S.; Denessiouk, Konstantin

2011-01-01

Integrins are heterodimeric cell-surface receptors with key functions in cell-cell and cell-matrix adhesion. Integrin α and β subunits are present throughout the metazoans, but it is unclear whether the subunits predate the origin of multicellular organisms. Several component domains have been detected in bacteria, one of which, a specific 7-bladed β-propeller domain, is a unique feature of the integrin α subunits. Here, we describe a structure-derived motif, which incorporates key features of each blade from the X-ray structures of human αIIbβ3 and αVβ3, includes elements of the FG-GAP/Cage and Ca2+-binding motifs, and is specific only for the metazoan integrin domains. Separately, we searched for the metazoan integrin type β-propeller domains among all available sequences from bacteria and unicellular eukaryotic organisms, which must incorporate seven repeats, corresponding to the seven blades of the β-propeller domain, and so that the newly found structure-derived motif would exist in every repeat. As the result, among 47 available genomes of unicellular eukaryotes we could not find a single instance of seven repeats with the motif. Several sequences contained three repeats, a predicted transmembrane segment, and a short cytoplasmic motif associated with some integrins, but otherwise differ from the metazoan integrin α subunits. Among the available bacterial sequences, we found five examples containing seven sequential metazoan integrin-specific motifs within the seven repeats. The motifs differ in having one Ca2+-binding site per repeat, whereas metazoan integrins have three or four sites. The bacterial sequences are more conserved in terms of motif conservation and loop length, suggesting that the structure is more regular and compact than those example structures from human integrins. Although the bacterial examples are not full-length integrins, the full-length metazoan-type 7-bladed β-propeller domains are present, and sometimes two tandem

Nonlinear (time domain) and linearized (time and frequency domain) solutions to the compressible Euler equations in conservation law form

NASA Technical Reports Server (NTRS)

Sreenivas, Kidambi; Whitfield, David L.

1995-01-01

Two linearized solvers (time and frequency domain) based on a high resolution numerical scheme are presented. The basic approach is to linearize the flux vector by expressing it as a sum of a mean and a perturbation. This allows the governing equations to be maintained in conservation law form. A key difference between the time and frequency domain computations is that the frequency domain computations require only one grid block irrespective of the interblade phase angle for which the flow is being computed. As a result of this and due to the fact that the governing equations for this case are steady, frequency domain computations are substantially faster than the corresponding time domain computations. The linearized equations are used to compute flows in turbomachinery blade rows (cascades) arising due to blade vibrations. Numerical solutions are compared to linear theory (where available) and to numerical solutions of the nonlinear Euler equations.

The HIV-1 late domain-2 S40A polymorphism in antiretroviral (or ART)-exposed individuals influences protease inhibitor susceptibility.

PubMed

Watanabe, Susan M; Simon, Viviana; Durham, Natasha D; Kemp, Brittney R; Machihara, Satoshi; Kemal, Kimdar Sherefa; Shi, Binshan; Foley, Brian; Li, Hongru; Chen, Benjamin K; Weiser, Barbara; Burger, Harold; Anastos, Kathryn; Chen, Chaoping; Carter, Carol A

2016-09-06

The p6 region of the HIV-1 structural precursor polyprotein, Gag, contains two motifs, P7TAP11 and L35YPLXSL41, designated as late (L) domain-1 and -2, respectively. These motifs bind the ESCRT-I factor Tsg101 and the ESCRT adaptor Alix, respectively, and are critical for efficient budding of virus particles from the plasma membrane. L domain-2 is thought to be functionally redundant to PTAP. To identify possible other functions of L domain-2, we examined this motif in dominant viruses that emerged in a group of 14 women who had detectable levels of HIV-1 in both plasma and genital tract despite a history of current or previous antiretroviral therapy. Remarkably, variants possessing mutations or rare polymorphisms in the highly conserved L domain-2 were identified in seven of these women. A mutation in a conserved residue (S40A) that does not reduce Gag interaction with Alix and therefore did not reduce budding efficiency was further investigated. This mutation causes a simultaneous change in the Pol reading frame but exhibits little deficiency in Gag processing and virion maturation. Whether introduced into the HIV-1 NL4-3 strain genome or a model protease (PR) precursor, S40A reduced production of mature PR. This same mutation also led to high level detection of two extended forms of PR that were fairly stable compared to the WT in the presence of IDV at various concentrations; one of the extended forms was effective in trans processing even at micromolar IDV. Our results indicate that L domain-2, considered redundant in vitro, can undergo mutations in vivo that significantly alter PR function. These may contribute fitness benefits in both the absence and presence of PR inhibitor.

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.

The structure of a conserved Piezo channel domain reveals a novel beta sandwich fold

PubMed Central

Kamajaya, Aron; Kaiser, Jens; Lee, Jonas; Reid, Michelle; Rees, Douglas C.

2014-01-01

Summary Piezo has recently been identified as a family of eukaryotic mechanosensitive channels composed of subunits containing over 2000 amino acids, without recognizable sequence similarity to other channels. Here, we present the crystal structure of a large, conserved extramembrane domain located just before the last predicted transmembrane helix of C. elegans PIEZO, which adopts a novel beta sandwich fold. The structure was also determined of a point mutation located on a conserved surface at the position equivalent to the human PIEZO1 mutation found in Dehydrated Hereditary Stomatocytosis (DHS) patients (M2225R). While the point mutation does not change the overall domain structure, it does alter the surface electrostatic potential that may perturb interactions with a yet-to-be identified ligand or protein. The lack of structural similarity between this domain and any previously characterized fold, including those of eukaryotic and bacterial channels, highlights the distinctive nature of the Piezo family of eukaryotic mechanosensitive channels. PMID:25242456

Discovery and Characterization of Cadherin Domains in Saccharophagus degradans 2-40▿ †

PubMed Central

Fraiberg, Milana; Borovok, Ilya; Weiner, Ronald M.; Lamed, Raphael

2010-01-01

Saccharophagus degradans strain 2-40 is a prominent member of newly discovered group of marine and estuarine bacteria that recycle complex polysaccharides. The S. degradans 2-40 genome codes for 15 extraordinary long polypeptides, ranging from 274 to 1,600 kDa. Five of these contain at least 52 cadherin (CA) and cadherin-like (CADG) domains, the types of which were reported to bind calcium ions and mediate protein/protein interactions in metazoan systems. In order to evaluate adhesive features of these domains, recombinant CA doublet domains (two neighboring domains) from CabC (Sde_3323) and recombinant CADG doublet domains from CabD (Sde_0798) were examined qualitatively and quantitatively for homophilic and heterophilic interactions. In addition, CA and CADG doublet domains were tested for adhesion to the surface of S. degradans 2-40. Results showed obvious homophilic and heterophilic, calcium ion-dependent interactions between CA and CADG doublet domains. Likewise, CA and CADG doublet domains adhered to the S. degradans 2-40 surface of cells that were grown on xylan from birch wood or pectin, respectively, as a sole carbon source. This research shows for the first time that bacterial cadherin homophilic and heterophilic interactions may be similar in their nature to cadherin domains from metazoan lineages. We hypothesize that S. degradans 2-40 cadherin and cadherin-like multiple domains contribute to protein-protein interactions that may mediate cell-cell contact in the marine environment. PMID:20023015

The BID Domain of Type IV Secretion Substrates Forms a Conserved Four-Helix Bundle Topped with a Hook.

PubMed

Stanger, Frédéric V; de Beer, Tjaart A P; Dranow, David M; Schirmer, Tilman; Phan, Isabelle; Dehio, Christoph

2017-01-03

The BID (Bep intracellular delivery) domain functions as secretion signal in a subfamily of protein substrates of bacterial type IV secretion (T4S) systems. It mediates transfer of (1) relaxases and the attached DNA during bacterial conjugation, and (2) numerous Bartonella effector proteins (Beps) during protein transfer into host cells infected by pathogenic Bartonella species. Furthermore, BID domains of Beps have often evolved secondary effector functions within host cells. Here, we provide crystal structures for three representative BID domains and describe a novel conserved fold characterized by a compact, antiparallel four-helix bundle topped with a hook. The conserved hydrophobic core provides a rigid scaffold to a surface that, despite a few conserved exposed residues and similarities in charge distribution, displays significant variability. We propose that the genuine function of BID domains as T4S signal may primarily depend on their rigid structure, while the plasticity of their surface may facilitate adaptation to secondary effector functions. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

PubMed

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

2018-05-26

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

Implementing the 40 Gallon Challenge to Increase Water Conservation

ERIC Educational Resources Information Center

Sheffield, Mary Carol; Bauske, Ellen; Pugliese, Paul; Kolich, Heather; Boellstorff, Diane

2016-01-01

The 40 Gallon Challenge is an easy-to-use, comprehensive indoor and outdoor water conservation educational tool. It can be used nationwide and easily incorporated into existing educational programs. Promotional materials and pledge cards are available on the 40 Gallon Challenge website and can be modified by educators. The website displays data…

The Mr 140,000 Intermediate Chain of Chlamydomonas Flagellar Inner Arm Dynein Is a WD-Repeat Protein Implicated in Dynein Arm Anchoring

PubMed Central

Yang, Pinfen; Sale, Winfield S.

1998-01-01

Previous structural and biochemical studies have revealed that the inner arm dynein I1 is targeted and anchored to a unique site located proximal to the first radial spoke in each 96-nm axoneme repeat on flagellar doublet microtubules. To determine whether intermediate chains mediate the positioning and docking of dynein complexes, we cloned and characterized the 140-kDa intermediate chain (IC140) of the I1 complex. Sequence and secondary structural analysis, with particular emphasis on β-sheet organization, predicted that IC140 contains seven WD repeats. Reexamination of other members of the dynein intermediate chain family of WD proteins indicated that these polypeptides also bear seven WD/β-sheet repeats arranged in the same pattern along each intermediate chain protein. A polyclonal antibody was raised against a 53-kDa fusion protein derived from the C-terminal third of IC140. The antibody is highly specific for IC140 and does not bind to other dynein intermediate chains or proteins in Chlamydomonas flagella. Immunofluorescent microscopy of Chlamydomonas cells confirmed that IC140 is distributed along the length of both flagellar axonemes. In vitro reconstitution experiments demonstrated that the 53-kDa C-terminal fusion protein binds specifically to axonemes lacking the I1 complex. Chemical cross-linking indicated that IC140 is closely associated with a second intermediate chain in the I1 complex. These data suggest that IC140 contains domains responsible for the assembly and docking of the I1 complex to the doublet microtubule cargo. PMID:9843573

An allosteric conduit facilitates dynamic multisite substrate recognition by the SCFCdc4 ubiquitin ligase

NASA Astrophysics Data System (ADS)

Csizmok, Veronika; Orlicky, Stephen; Cheng, Jing; Song, Jianhui; Bah, Alaji; Delgoshaie, Neda; Lin, Hong; Mittag, Tanja; Sicheri, Frank; Chan, Hue Sun; Tyers, Mike; Forman-Kay, Julie D.

2017-01-01

The ubiquitin ligase SCFCdc4 mediates phosphorylation-dependent elimination of numerous substrates by binding one or more Cdc4 phosphodegrons (CPDs). Methyl-based NMR analysis of the Cdc4 WD40 domain demonstrates that Cyclin E, Sic1 and Ash1 degrons have variable effects on the primary Cdc4WD40 binding pocket. Unexpectedly, a Sic1-derived multi-CPD substrate (pSic1) perturbs methyls around a previously documented allosteric binding site for the chemical inhibitor SCF-I2. NMR cross-saturation experiments confirm direct contact between pSic1 and the allosteric pocket. Phosphopeptide affinity measurements reveal negative allosteric communication between the primary CPD and allosteric pockets. Mathematical modelling indicates that the allosteric pocket may enhance ultrasensitivity by tethering pSic1 to Cdc4. These results suggest negative allosteric interaction between two distinct binding pockets on the Cdc4WD40 domain may facilitate dynamic exchange of multiple CPD sites to confer ultrasensitive dependence on substrate phosphorylation.

The structure of a conserved piezo channel domain reveals a topologically distinct β sandwich fold.

PubMed

Kamajaya, Aron; Kaiser, Jens T; Lee, Jonas; Reid, Michelle; Rees, Douglas C

2014-10-07

Piezo has recently been identified as a family of eukaryotic mechanosensitive channels composed of subunits containing over 2,000 amino acids, without recognizable sequence similarity to other channels. Here, we present the crystal structure of a large, conserved extramembrane domain located just before the last predicted transmembrane helix of C. elegans PIEZO, which adopts a topologically distinct β sandwich fold. The structure was also determined of a point mutation located on a conserved surface at the position equivalent to the human PIEZO1 mutation found in dehydrated hereditary stomatocytosis patients (M2225R). While the point mutation does not change the overall domain structure, it does alter the surface electrostatic potential that may perturb interactions with a yet-to-be-identified ligand or protein. The lack of structural similarity between this domain and any previously characterized fold, including those of eukaryotic and bacterial channels, highlights the distinctive nature of the Piezo family of eukaryotic mechanosensitive channels. Copyright © 2014 Elsevier Ltd. All rights reserved.

Complex structure of the fission yeast SREBP-SCAP binding domains reveals an oligomeric organization.

PubMed

Gong, Xin; Qian, Hongwu; Shao, Wei; Li, Jingxian; Wu, Jianping; Liu, Jun-Jie; Li, Wenqi; Wang, Hong-Wei; Espenshade, Peter; Yan, Nieng

2016-11-01

Sterol regulatory element-binding protein (SREBP) transcription factors are master regulators of cellular lipid homeostasis in mammals and oxygen-responsive regulators of hypoxic adaptation in fungi. SREBP C-terminus binds to the WD40 domain of SREBP cleavage-activating protein (SCAP), which confers sterol regulation by controlling the ER-to-Golgi transport of the SREBP-SCAP complex and access to the activating proteases in the Golgi. Here, we biochemically and structurally show that the carboxyl terminal domains (CTD) of Sre1 and Scp1, the fission yeast SREBP and SCAP, form a functional 4:4 oligomer and Sre1-CTD forms a dimer of dimers. The crystal structure of Sre1-CTD at 3.5 Å and cryo-EM structure of the complex at 5.4 Å together with in vitro biochemical evidence elucidate three distinct regions in Sre1-CTD required for Scp1 binding, Sre1-CTD dimerization and tetrameric formation. Finally, these structurally identified domains are validated in a cellular context, demonstrating that the proper 4:4 oligomeric complex formation is required for Sre1 activation.

Discovery of Strong EUV-induced Balmer Emission in the New WD+dM Binary EUVE J2013+40.0 (RE 2013+400)

NASA Astrophysics Data System (ADS)

Thorstensen, J. R.; Vennes, S.

1993-12-01

The binary system EUVE J2013+40.0 (= RE 2013+400) was discovered in the EUV-selected sample of white dwarfs identified in the course of the ROSAT Wide Field Camera (WFC) all-sky survey (Pounds et al. 1993, MNRAS, 260, 77). The intense extreme ultraviolet (EUV) emission from the hot white dwarf (DAO type) was also detected in the course of the Extreme Ultraviolet Explorer (EUVE) all-sky survey (Bowyer et al. 1993, ApJ, submitted), and the subsequent optical identification campaign suggested the association of EUVE J2013+40.0 with the Feige 24 class of binary systems (see Vennes & Thorstensen, these proceedings). Such systems consist of a hot H-rich white dwarf (DA/DAO) and a red dwarf companion (dM) and are characterized by strong, narrow, variable Balmer emission. We obtained spectroscopy with 4 Angstroms resolution at the Michigan-Dartmouth-MIT Hiltner 2.4 m, covering the Hα and Hβ range. The Hα emission line velocity and equivalent widths varied with a period of 0.708 +/- 0.003 d; the velocity semiamplitude is 89 +/- 3 km s(-1) . The emission equivalent width reaches maximum strength 0.251 +/- 0.007 cycle after maximum emission-line velocity, that is, when the emission source reaches superior conjunction. This is just as expected if the emission arises from reprocessing of the EUV radiation incident upon the face of the dM star facing the white dwarf, as proposed for Feige 24 by Thorstensen et al. (1978, ApJ, 223, 260). EUVE J2013+40.0 is one of a handful of WD+dM binary systems in which the illumination effect is observed with unambiguous clarity. By comparing Feige 24 and EUVE J2013+40.0, and modelling the white dwarf EUV emission and red dwarf Balmer emission, we constrain the orbital inclinations. Additional spectroscopy of EUVE J2013+40.0 is being scheduled to determine the component masses. These are important input data for the study of the close binary systems which arise from common envelope evolution. This work is supported by a forthcoming NASA

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.

Brassinosteroids control root epidermal cell fate via direct regulation of a MYB-bHLH-WD40 complex by GSK3-like kinases

PubMed Central

Cheng, Yinwei; Zhu, Wenjiao; Chen, Yuxiao; Ito, Shinsaku; Asami, Tadao; Wang, Xuelu

2014-01-01

In Arabidopsis, root hair and non-hair cell fates are determined by a MYB-bHLH-WD40 transcriptional complex and are regulated by many internal and environmental cues. Brassinosteroids play important roles in regulating root hair specification by unknown mechanisms. Here, we systematically examined root hair phenotypes in brassinosteroid-related mutants, and found that brassinosteroid signaling inhibits root hair formation through GSK3-like kinases or upstream components. We found that with enhanced brassinosteroid signaling, GL2, a cell fate marker for non-hair cells, is ectopically expressed in hair cells, while its expression in non-hair cells is suppressed when brassinosteroid signaling is reduced. Genetic analysis demonstrated that brassinosteroid-regulated root epidermal cell patterning is dependent on the WER-GL3/EGL3-TTG1 transcriptional complex. One of the GSK3-like kinases, BIN2, interacted with and phosphorylated EGL3, and EGL3s mutated at phosphorylation sites were retained in hair cell nuclei. BIN2 phosphorylated TTG1 to inhibit the activity of the WER-GL3/EGL3-TTG1 complex. Thus, our study provides insights into the mechanism of brassinosteroid regulation of root hair patterning. DOI: http://dx.doi.org/10.7554/eLife.02525.001 PMID:24771765

Influenza Virus Vaccine Based on the Conserved Hemagglutinin Stalk Domain

PubMed Central

Steel, John; Lowen, Anice C.; Wang, Taia T.; Yondola, Mark; Gao, Qinshan; Haye, Kester; García-Sastre, Adolfo; Palese, Peter

2010-01-01

ABSTRACT Although highly effective in the general population when well matched to circulating influenza virus strains, current influenza vaccines are limited in their utility due to the narrow breadth of protection they provide. The strain specificity of vaccines presently in use mirrors the exquisite specificity of the neutralizing antibodies that they induce, that is, antibodies which bind to the highly variable globular head domain of hemagglutinin (HA). Herein, we describe the construction of a novel immunogen comprising the conserved influenza HA stalk domain and lacking the globular head. Vaccination of mice with this headless HA construct elicited immune sera with broader reactivity than those obtained from mice immunized with a full-length HA. Furthermore, the headless HA vaccine provided full protection against death and partial protection against disease following lethal viral challenge. Our results suggest that the response induced by headless HA vaccines is sufficiently potent to warrant their further development toward a universal influenza virus vaccine. PMID:20689752

In vivo functional mapping of the conserved protein domains within murine Themis1.

PubMed

Zvezdova, Ekaterina; Lee, Jan; El-Khoury, Dalal; Barr, Valarie; Akpan, Itoro; Samelson, Lawrence; Love, Paul E

2014-09-01

Thymocyte development requires the coordinated input of signals that originate from numerous cell surface molecules. Although the majority of thymocyte signal-initiating receptors are lineage-specific, most trigger 'ubiquitous' downstream signaling pathways. T-lineage-specific receptors are coupled to these signaling pathways by lymphocyte-restricted adapter molecules. We and others recently identified a new putative adapter protein, Themis1, whose expression is largely restricted to the T lineage. Mice lacking Themis1 exhibit a severe block in thymocyte development and a striking paucity of mature T cells revealing a critical role for Themis1 in T-cell maturation. Themis1 orthologs contain three conserved domains: a proline-rich region (PRR) that binds to the ubiquitous cytosolic adapter Grb2, a nuclear localization sequence (NLS), and two copies of a novel cysteine-containing globular (CABIT) domain. In the present study, we evaluated the functional importance of each of these motifs by retroviral reconstitution of Themis1(-/-) progenitor cells. The results demonstrate an essential requirement for the PRR and NLS motifs but not the conserved CABIT cysteines for Themis1 function.

Domain alternation and active site remodeling are conserved structural features of ubiquitin E1.

PubMed

Lv, Zongyang; Yuan, Lingmin; Atkison, James H; Aldana-Masangkay, Grace; Chen, Yuan; Olsen, Shaun K

2017-07-21

E1 enzymes for ubiquitin (Ub) and Ub-like modifiers (Ubls) harbor two catalytic activities that are required for Ub/Ubl activation: adenylation and thioester bond formation. Structural studies of the E1 for the Ubl s mall u biquitin-like mo difier (SUMO) revealed a single active site that is transformed by a conformational switch that toggles its competency for catalysis of these two distinct chemical reactions. Although the mechanisms of adenylation and thioester bond formation revealed by SUMO E1 structures are thought to be conserved in Ub E1, there is currently a lack of structural data supporting this hypothesis. Here, we present a structure of Schizosaccharomyces pombe Uba1 in which the second catalytic cysteine half-domain (SCCH domain) harboring the catalytic cysteine has undergone a 106° rotation that results in a completely different network of intramolecular interactions between the SCCH and adenylation domains and translocation of the catalytic cysteine 12 Å closer to the Ub C terminus compared with previous Uba1 structures. SCCH domain alternation is accompanied by conformational changes within the Uba1 adenylation domains that effectively disassemble the adenylation active site. Importantly, the structural and biochemical data suggest that domain alternation and remodeling of the adenylation active site are interconnected and are intrinsic structural features of Uba1 and that the overall structural basis for adenylation and thioester bond formation exhibited by SUMO E1 is indeed conserved in Ub E1. Finally, the mechanistic insights provided by the novel conformational snapshot of Uba1 presented in this study may guide efforts to develop small molecule inhibitors of this critically important enzyme that is an active target for anticancer therapeutics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Identifying potential recommendation domains for conservation agriculture in Ethiopia, Kenya, and Malawi.

PubMed

Tesfaye, Kindie; Jaleta, Moti; Jena, Pradyot; Mutenje, Munyaradzi

2015-02-01

Conservation agriculture (CA) is being promoted as an option for reducing soil degradation, conserving water, enhancing crop productivity, and maintaining yield stability. However, CA is a knowledge- and technology-intensive practice, and may not be feasible or may not perform better than conventional agriculture under all conditions and farming systems. Using high resolution (≈1 km(2)) biophysical and socioeconomic geospatial data, this study identified potential recommendation domains (RDs) for CA in Ethiopia, Kenya, and Malawi. The biophysical variables used were soil texture, surface slope, and rainfall while the socioeconomic variables were market access and human and livestock population densities. Based on feasibility and comparative performance of CA over conventional agriculture, the biophysical and socioeconomic factors were first used to classify cultivated areas into three biophysical and three socioeconomic potential domains, respectively. Combinations of biophysical and socioeconomic domains were then used to develop potential RDs for CA based on adoption potential within the cultivated areas. About 39, 12, and 5% of the cultivated areas showed high biophysical and socioeconomic potential while 50, 39, and 21% of the cultivated areas showed high biophysical and medium socioeconomic potential for CA in Malawi, Kenya, and Ethiopia, respectively. The results indicate considerable acreages of land with high CA adoption potential in the mixed crop-livestock systems of the studied countries. However, there are large differences among countries depending on biophysical and socio-economic conditions. The information generated in this study could be used for targeting CA and prioritizing CA-related agricultural research and investment priorities in the three countries.

Identifying Potential Recommendation Domains for Conservation Agriculture in Ethiopia, Kenya, and Malawi

NASA Astrophysics Data System (ADS)

Tesfaye, Kindie; Jaleta, Moti; Jena, Pradyot; Mutenje, Munyaradzi

2015-02-01

Conservation agriculture (CA) is being promoted as an option for reducing soil degradation, conserving water, enhancing crop productivity, and maintaining yield stability. However, CA is a knowledge- and technology-intensive practice, and may not be feasible or may not perform better than conventional agriculture under all conditions and farming systems. Using high resolution (≈1 km2) biophysical and socioeconomic geospatial data, this study identified potential recommendation domains (RDs) for CA in Ethiopia, Kenya, and Malawi. The biophysical variables used were soil texture, surface slope, and rainfall while the socioeconomic variables were market access and human and livestock population densities. Based on feasibility and comparative performance of CA over conventional agriculture, the biophysical and socioeconomic factors were first used to classify cultivated areas into three biophysical and three socioeconomic potential domains, respectively. Combinations of biophysical and socioeconomic domains were then used to develop potential RDs for CA based on adoption potential within the cultivated areas. About 39, 12, and 5 % of the cultivated areas showed high biophysical and socioeconomic potential while 50, 39, and 21 % of the cultivated areas showed high biophysical and medium socioeconomic potential for CA in Malawi, Kenya, and Ethiopia, respectively. The results indicate considerable acreages of land with high CA adoption potential in the mixed crop-livestock systems of the studied countries. However, there are large differences among countries depending on biophysical and socio-economic conditions. The information generated in this study could be used for targeting CA and prioritizing CA-related agricultural research and investment priorities in the three countries.

Comparison of S. cerevisiae F-BAR domain structures reveals a conserved inositol phosphate binding site

PubMed Central

Moravcevic, Katarina; Alvarado, Diego; Schmitz, Karl R.; Kenniston, Jon A.; Mendrola, Jeannine M.; Ferguson, Kathryn M.; Lemmon, Mark A.

2015-01-01

SUMMARY F-BAR domains control membrane interactions in endocytosis, cytokinesis, and cell signaling. Although generally thought to bind curved membranes containing negatively charged phospholipids, numerous functional studies argue that differences in lipid-binding selectivities of F-BAR domains are functionally important. Here, we compare membrane-binding properties of the S. cerevisiae F-BAR domains in vitro and in vivo. Whereas some F-BAR domains (such as Bzz1p and Hof1p F-BARs) bind equally well to all phospholipids, the F-BAR domain from the RhoGAP Rgd1p preferentially binds phosphoinositides. We determined X-ray crystal structures of F-BAR domains from Hof1p and Rgd1p, the latter bound to an inositol phosphate. The structures explain phospholipid-binding selectivity differences, and reveal an F-BAR phosphoinositide binding site that is fully conserved in a mammalian RhoGAP called Gmip, and is partly retained in certain other F-BAR domains. Our findings reveal previously unappreciated determinants of F-BAR domain lipid-binding specificity, and provide a basis for its prediction from sequence. PMID:25620000

Nuclear localization and transactivation by Vitis CBF transcription factors are regulated by combinations of conserved amino acid domains.

PubMed

Carlow, Chevonne E; Faultless, J Trent; Lee, Christine; Siddiqua, Mahbuba; Edge, Alison; Nassuth, Annette

2017-09-01

The highly conserved CBF pathway is crucial in the regulation of plant responses to low temperatures. Extensive analysis of Arabidopsis CBF proteins revealed that their functions rely on several conserved amino acid domains although the exact function of each domain is disputed. The question was what functions similar domains have in CBFs from other, overwintering woody plants such as Vitis, which likely have a more involved regulation than the model plant Arabidopsis. A total of seven CBF genes were cloned and sequenced from V. riparia and the less frost tolerant V. vinifera. The deduced species-specific amino acid sequences differ in only a few amino acids, mostly in non-conserved regions. Amino acid sequence comparison and phylogenetic analysis showed two distinct groups of Vitis CBFs. One group contains CBF1, CBF2, CBF3 and CBF8 and the other group contains CBF4, CBF5 and CBF6. Transient transactivation assays showed that all Vitis CBFs except CBF5 activate via a CRT or DRE promoter element, whereby Vitis CBF3 and 4 prefer a CRT element. The hydrophobic domains in the C-terminal end of VrCBF6 were shown to be important for how well it activates. The putative nuclear localization domain of Vitis CBF1 was shown to be sufficient for nuclear localization, in contrast to previous reports for AtCBF1, and also important for transactivation. The latter highlights the value of careful analysis of domain functions instead of reliance on computer predictions and published data for other related proteins. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Structural and functional determinants of conserved lipid interaction domains of inward rectifying Kir6.2 channels.

PubMed

Cukras, Catherine A; Jeliazkova, Iana; Nichols, Colin G

2002-06-01

All members of the inward rectifiier K(+) (Kir) channel family are activated by phosphoinositides and other amphiphilic lipids. To further elucidate the mechanistic basis, we examined the membrane association of Kir6.2 fragments of K(ATP) channels, and the effects of site-directed mutations of these fragments and full-length Kir6.2 on membrane association and K(ATP) channel activity, respectively. GFP-tagged Kir6.2 COOH terminus and GFP-tagged pleckstrin homology domain from phospholipase C delta1 both associate with isolated membranes, and association of each is specifically reduced by muscarinic m1 receptor-mediated phospholipid depletion. Kir COOH termini are predicted to contain multiple beta-strands and a conserved alpha-helix (residues approximately 306-311 in Kir6.2). Systematic mutagenesis of D307-F315 reveals a critical role of E308, I309, W311 and F315, consistent with residues lying on one side of a alpha-helix. Together with systematic mutation of conserved charges, the results define critical determinants of a conserved domain that underlies phospholipid interaction in Kir channels.

WD 0158-160, a new pulsating DB white dwarf

NASA Astrophysics Data System (ADS)

Kilkenny, D.

2016-03-01

The DB star, WD 0158-160 (=EC 01585-1600) is shown to be a variable white dwarf with a rich pulsation spectrum, dominated by a strong variation with a frequency near 1637 μHz (amplitude ˜0.024 mag, period 598 s), though at least 10 frequencies are detected between about 1285 and 5747 μHz (780-173 s) in a relatively small data set. At ˜14.5 mag, the star is one of the brightest known DBV stars.

A conserved tryptophan within the WRDPLVDID domain of yeast Pah1 phosphatidate phosphatase is required for its in vivo function in lipid metabolism.

PubMed

Park, Yeonhee; Han, Gil-Soo; Carman, George M

2017-12-01

PAH1 -encoded phosphatidate phosphatase, which catalyzes the dephosphorylation of phosphatidate to produce diacylglycerol at the endoplasmic reticulum membrane, plays a major role in controlling the utilization of phosphatidate for the synthesis of triacylglycerol or membrane phospholipids. The conserved N-LIP and haloacid dehalogenase-like domains of Pah1 are required for phosphatidate phosphatase activity and the in vivo function of the enzyme. Its non-conserved regions, which are located between the conserved domains and at the C terminus, contain sites for phosphorylation by multiple protein kinases. Truncation analyses of the non-conserved regions showed that they are not essential for the catalytic activity of Pah1 and its physiological functions ( e.g. triacylglycerol synthesis). This analysis also revealed that the C-terminal region contains a previously unrecognized WRDPLVDID domain (residues 637-645) that is conserved in yeast, mice, and humans. The deletion of this domain had no effect on the catalytic activity of Pah1 but caused the loss of its in vivo function. Site-specific mutational analyses of the conserved residues within WRDPLVDID indicated that Trp-637 plays a crucial role in Pah1 function. This work also demonstrated that the catalytic activity of Pah1 is required but is not sufficient for its in vivo functions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Comparison of Saccharomyces cerevisiae F-BAR domain structures reveals a conserved inositol phosphate binding site.

PubMed

Moravcevic, Katarina; Alvarado, Diego; Schmitz, Karl R; Kenniston, Jon A; Mendrola, Jeannine M; Ferguson, Kathryn M; Lemmon, Mark A

2015-02-03

F-BAR domains control membrane interactions in endocytosis, cytokinesis, and cell signaling. Although they are generally thought to bind curved membranes containing negatively charged phospholipids, numerous functional studies argue that differences in lipid-binding selectivities of F-BAR domains are functionally important. Here, we compare membrane-binding properties of the Saccharomyces cerevisiae F-BAR domains in vitro and in vivo. Whereas some F-BAR domains (such as Bzz1p and Hof1p F-BARs) bind equally well to all phospholipids, the F-BAR domain from the RhoGAP Rgd1p preferentially binds phosphoinositides. We determined X-ray crystal structures of F-BAR domains from Hof1p and Rgd1p, the latter bound to an inositol phosphate. The structures explain phospholipid-binding selectivity differences and reveal an F-BAR phosphoinositide binding site that is fully conserved in a mammalian RhoGAP called Gmip and is partly retained in certain other F-BAR domains. Our findings reveal previously unappreciated determinants of F-BAR domain lipid-binding specificity and provide a basis for its prediction from sequence. Copyright © 2015 Elsevier Ltd. All rights reserved.

Comparison of Saccharomyces cerevisiae F-BAR Domain Structures Reveals a Conserved Inositol Phosphate Binding Site

DOE PAGES

Moravcevic, Katarina; Alvarado, Diego; Schmitz, Karl R.; ...

2015-01-22

F-BAR domains control membrane interactions in endocytosis, cytokinesis, and cell signaling. Although they are generally thought to bind curved membranes containing negatively charged phospholipids, numerous functional studies argue that differences in lipid-binding selectivities of F-BAR domains are functionally important. Here in this paper, we compare membrane-binding properties of the Saccharomyces cerevisiae F-BAR domains in vitro and in vivo. Whereas some F-BAR domains (such as Bzz1p and Hof1p F-BARs) bind equally well to all phospholipids, the F-BAR domain from the RhoGAP Rgd1p preferentially binds phosphoinositides. We determined X-ray crystal structures of F-BAR domains from Hof1p and Rgd1p, the latter bound tomore » an inositol phosphate. The structures explain phospholipid-binding selectivity differences and reveal an F-BAR phosphoinositide binding site that is fully conserved in a mammalian RhoGAP called Gmip and is partly retained in certain other F-BAR domains. In conclusion, our findings reveal previously unappreciated determinants of F-BAR domain lipid-binding specificity and provide a basis for its prediction from sequence.« less

Discovery of a Detached, Eclipsing 40 Minute Period Double White Dwarf Binary and a Friend: Implications for He+CO White Dwarf Mergers

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

Brown, Warren R.; Kilic, Mukremin; Kosakowski, Alekzander

We report the discovery of two detached double white dwarf (WD) binaries, SDSS J082239.546+304857.19 and SDSS J104336.275+055149.90, with orbital periods of 40 and 46 minutes, respectively. The 40 minute system is eclipsing; it is composed of a 0.30 M {sub ⊙} and a 0.52 M {sub ⊙} WD. The 46 minute system is a likely LISA verification binary. The short 20 ± 2 Myr and ∼34 Myr gravitational-wave merger times of the two binaries imply that many more such systems have formed and merged over the age of the Milky Way. We update the estimated Milky Way He+CO WD binarymore » merger rate and affirm our previously published result: He+CO WD binaries merge at a rate at least 40 times greater than the formation rate of stable mass-transfer AM CVn binaries, and so the majority must have unstable mass-transfer. The implication is that spin–orbit coupling in He+CO WD mergers is weak, or perhaps nova-like outbursts drive He+CO WDs into merger, as proposed by Shen.« less

WD-repeat instability and diversification of the Podospora anserina hnwd non-self recognition gene family.

PubMed

Chevanne, Damien; Saupe, Sven J; Clavé, Corinne; Paoletti, Mathieu

2010-05-06

Genes involved in non-self recognition and host defence are typically capable of rapid diversification and exploit specialized genetic mechanism to that end. Fungi display a non-self recognition phenomenon termed heterokaryon incompatibility that operates when cells of unlike genotype fuse and leads to the cell death of the fusion cell. In the fungus Podospora anserina, three genes controlling this allorecognition process het-d, het-e and het-r are paralogs belonging to the same hnwd gene family. HNWD proteins are STAND proteins (signal transduction NTPase with multiple domains) that display a WD-repeat domain controlling recognition specificity. Based on genomic sequence analysis of different P. anserina isolates, it was established that repeat regions of all members of the gene family are extremely polymorphic and undergoing concerted evolution arguing for frequent recombination within and between family members. Herein, we directly analyzed the genetic instability and diversification of this allorecognition gene family. We have constituted a collection of 143 spontaneous mutants of the het-R (HNWD2) and het-E (hnwd5) genes with altered recognition specificities. The vast majority of the mutants present rearrangements in the repeat arrays with deletions, duplications and other modifications as well as creation of novel repeat unit variants. We investigate the extreme genetic instability of these genes and provide a direct illustration of the diversification strategy of this eukaryotic allorecognition gene family.

Dual Thermosensitive Hydrogels Assembled from the Conserved C-Terminal Domain of Spider Dragline Silk.

PubMed

Qian, Zhi-Gang; Zhou, Ming-Liang; Song, Wen-Wen; Xia, Xiao-Xia

2015-11-09

Stimuli-responsive hydrogels have great potentials in biomedical and biotechnological applications. Due to the advantages of precise control over molecular weight and being biodegradable, protein-based hydrogels and their applications have been extensively studied. However, protein hydrogels with dual thermosensitive properties are rarely reported. Here we present the first report of dual thermosensitive hydrogels assembled from the conserved C-terminal domain of spider dragline silk. First, we found that recombinant C-terminal domain of major ampullate spidroin 1 (MaSp1) of the spider Nephila clavipes formed hydrogels when cooled to approximately 2 °C or heated to 65 °C. The conformational changes and self-assembly of the recombinant protein were studied to understand the mechanism of the gelation processes using multiple methods. It was proposed that the gelation in the low-temperature regime was dominated by hydrogen bonding and hydrophobic interaction between folded protein molecules, whereas the gelation in the high-temperature regime was due to cross-linking of the exposed hydrophobic patches resulting from partial unfolding of the protein upon heating. More interestingly, genetic fusion of the C-terminal domain to a short repetitive region of N. clavipes MaSp1 resulted in a chimeric protein that formed a hydrogel with significantly improved mechanical properties at low temperatures between 2 and 10 °C. Furthermore, the formation of similar hydrogels was observed for the recombinant C-terminal domains of dragline silk of different spider species, thus demonstrating the conserved ability to form dual thermosensitive hydrogels. These findings may be useful in the design and construction of novel protein hydrogels with tunable multiple thermosensitivity for applications in the future.

WD 1145+017: optical activity during 2016-2017 and limits on the X-ray flux

NASA Astrophysics Data System (ADS)

Rappaport, S.; Gary, B. L.; Vanderburg, A.; Xu, S.; Pooley, D.; Mukai, K.

2018-02-01

WD 1145+017 was observed from 2016 November through 2017 June for the purpose of further characterizing the transit behaviour of the dusty debris clouds orbiting this white dwarf. The optical observations were carried out with a small ground-based telescope run by an amateur astronomer, and covered 53 different nights over the 8-month interval. We have found that the optical activity has increased to the highest level observed since its discovery with Kepler K2, with approximately 17 per cent of the optical flux extinguished per orbit. The source exhibits some transits with depths of up to 55 per cent and durations as long as 2 h. The dominant period of the orbiting dust clouds during 2016-2017 is 4.49126 h. We present `waterfall' images for the entire 2016-2017 and 2015-2016 observing seasons. In addition, the white dwarf was observed with the Chandra X-ray Observatory for 10-ks on each of four different occasions, separated by about a month each. The upper limit on the average X-ray flux from WD 1145+017 is ≃ 5 × 10-15 erg cm-2 s-1 (unabsorbed over the range 0.1-100 keV), which translates to an upper limit on the X-ray luminosity, Lx, of ≃ 2 × 1028 erg s-1. If L_x ˜eq G M_wd \\dot{M}_acc/R_wd, where Mwd and Rwd are the mass and radius of the white dwarf, and \\dot{M}_acc is the accretion rate, then \\dot{M}_acc ≲ 2 × 10^{11} g s-1. This is just consistent with the value of \\dot{M} that is inferred from the level of dust activity.

Conservation of species, volume, and belief in patients with Alzheimer's disease: the issue of domain specificity and conceptual impairment.

PubMed

Zaitchik, Deborah; Solomon, Gregg E A

2009-09-01

Two studies investigated whether patients with Alzheimer's disease (AD) suffer high-level and category-specific impairment in the conceptual domain of living things. In Experiment 1, AD patients and healthy young and healthy elderly controls took part in three tasks: the conservation of species, volume, and belief. All 3 tasks required tracking an object's identity in the face of irrelevant but salient transformations. Healthy young and elderly controls performed at or near ceiling on all tasks. AD patients were at or near ceiling on the volume and belief tasks, but only about half succeeded on the species task. Experiment 2 demonstrated that the results were not due to simple task demands. AD patients' failure to conserve species indicates that they are impaired in their theoretical understanding of living things, and their success on the volume and belief tasks suggests that the impairment is domain-specific. Two hypotheses are put forward to explain the phenomenon: The first, a category-specific account, holds that the intuitive theory of biology undergoes pervasive degradation; the second, a hybrid domain-general/domain-specific account, holds that impairment to domain-general processes such as executive function interacts with core cognition, the primitive elements that are the foundation of domain-specific knowledge.

EBNA-2 of herpesvirus papio diverges significantly from the type A and type B EBNA-2 proteins of Epstein-Barr virus but retains an efficient transactivation domain with a conserved hydrophobic motif.

PubMed Central

Ling, P D; Ryon, J J; Hayward, S D

1993-01-01

EBNA-2 contributes to the establishment of Epstein-Barr virus (EBV) latency in B cells and to the resultant alterations in B-cell growth pattern by up-regulating expression from specific viral and cellular promoters. We have taken a comparative approach toward characterizing functional domains within EBNA-2. To this end, we have cloned and sequenced the EBNA-2 gene from the closely related baboon virus herpesvirus papio (HVP). All human EBV isolates have either a type A or type B EBNA-2 gene. However, the HVP EBNA-2 gene falls into neither the type A category nor the type B category, suggesting that the separation into these two subtypes may have been a recent evolutionary event. Comparison of the predicted amino acid sequences indicates 37% amino acid identity with EBV type A EBNA-2 and 35% amino acid identity with type B EBNA-2. To define the domains of EBNA-2 required for transcriptional activation, the DNA binding domain of the GAL4 protein was fused to overlapping segments of EBV EBNA-2. This approach identified a 40-amino-acid (40-aa) EBNA-2 activation domain located between aa 437 and 477. Transactivation ability was completely lost when the amino-terminal boundary of this domain was moved to aa 441, indicating that the motif at aa 437 to 440, Pro-Ile-Leu-Phe, contains residues critical for function. The aa 437 boundary identified in these experiments coincides precisely with a block of conserved sequences in HVP EBNA-2, and the comparable carboxy-terminal region of HVP EBNA-2 also functioned as a strong transcriptional activation domain when fused to the Gal4(1-147) protein. The EBV and HVP EBNA-2 activation domains share a mixed proline-rich, negatively charged character with a striking conservation of positionally equivalent hydrophobic residues. The importance of the individual amino acids making up the Pro-Ile-Leu-Phe motif was examined by mutagenesis. Any alteration of these residues was found to reduce transactivation efficiency, with changes at the

EBNA-2 of herpesvirus papio diverges significantly from the type A and type B EBNA-2 proteins of Epstein-Barr virus but retains an efficient transactivation domain with a conserved hydrophobic motif.

PubMed

Ling, P D; Ryon, J J; Hayward, S D

1993-06-01

EBNA-2 contributes to the establishment of Epstein-Barr virus (EBV) latency in B cells and to the resultant alterations in B-cell growth pattern by up-regulating expression from specific viral and cellular promoters. We have taken a comparative approach toward characterizing functional domains within EBNA-2. To this end, we have cloned and sequenced the EBNA-2 gene from the closely related baboon virus herpesvirus papio (HVP). All human EBV isolates have either a type A or type B EBNA-2 gene. However, the HVP EBNA-2 gene falls into neither the type A category nor the type B category, suggesting that the separation into these two subtypes may have been a recent evolutionary event. Comparison of the predicted amino acid sequences indicates 37% amino acid identity with EBV type A EBNA-2 and 35% amino acid identity with type B EBNA-2. To define the domains of EBNA-2 required for transcriptional activation, the DNA binding domain of the GAL4 protein was fused to overlapping segments of EBV EBNA-2. This approach identified a 40-amino-acid (40-aa) EBNA-2 activation domain located between aa 437 and 477. Transactivation ability was completely lost when the amino-terminal boundary of this domain was moved to aa 441, indicating that the motif at aa 437 to 440, Pro-Ile-Leu-Phe, contains residues critical for function. The aa 437 boundary identified in these experiments coincides precisely with a block of conserved sequences in HVP EBNA-2, and the comparable carboxy-terminal region of HVP EBNA-2 also functioned as a strong transcriptional activation domain when fused to the Gal4(1-147) protein. The EBV and HVP EBNA-2 activation domains share a mixed proline-rich, negatively charged character with a striking conservation of positionally equivalent hydrophobic residues. The importance of the individual amino acids making up the Pro-Ile-Leu-Phe motif was examined by mutagenesis. Any alteration of these residues was found to reduce transactivation efficiency, with changes at the

A dearth of small particles in the transiting material around the white dwarf WD 1145+017

NASA Astrophysics Data System (ADS)

Xu, S.; Rappaport, S.; van Lieshout, R.; Vanderburg, A.; Gary, B.; Hallakoun, N.; Ivanov, V. D.; Wyatt, M. C.; DeVore, J.; Bayliss, D.; Bento, J.; Bieryla, A.; Cameron, A.; Cann, J. M.; Croll, B.; Collins, K. A.; Dalba, P. A.; Debes, J.; Doyle, D.; Dufour, P.; Ely, J.; Espinoza, N.; Joner, M. D.; Jura, M.; Kaye, T.; McClain, J. L.; Muirhead, P.; Palle, E.; Panka, P. A.; Provencal, J.; Randall, S.; Rodriguez, J. E.; Scarborough, J.; Sefako, R.; Shporer, A.; Strickland, W.; Zhou, G.; Zuckerman, B.

2018-03-01

White dwarf WD 1145+017 is orbited by several clouds of dust, possibly emanating from actively disintegrating bodies. These dust clouds reveal themselves through deep, broad, and evolving transits in the star's light curve. Here, we report two epochs of multiwavelength photometric observations of WD 1145+017, including several filters in the optical, Ks and 4.5 μm bands in 2016 and 2017. The observed transit depths are different at these wavelengths. However, after correcting for excess dust emission at Ks and 4.5 μm, we find the transit depths for the white dwarf itself are the same at all wavelengths, at least to within the observational uncertainties of ˜5-10 per cent. From this surprising result, and under the assumption of low optical depth dust clouds, we conclude that there is a deficit of small particles (with radii s ≲ 1.5 μm) in the transiting material. We propose a model wherein only large particles can survive the high equilibrium temperature environment corresponding to 4.5 h orbital periods around WD 1145+017, while small particles sublimate rapidly. In addition, we evaluate dust models that are permitted by our measurements of infrared emission.

WD60/FAP163 is a dynein intermediate chain required for retrograde intraflagellar transport in cilia

PubMed Central

Patel-King, Ramila S.; Gilberti, Renée M.; Hom, Erik F. Y.; King, Stephen M.

2013-01-01

Retrograde intraflagellar transport (IFT) is required for assembly of cilia. We identify a Chlamydomonas flagellar protein (flagellar-associated protein 163 [FAP163]) as being closely related to the D1bIC(FAP133) intermediate chain (IC) of the dynein that powers this movement. Biochemical analysis revealed that FAP163 is present in the flagellar matrix and is actively trafficked by IFT. Furthermore, FAP163 copurified with D1bIC(FAP133) and the LC8 dynein light chain, indicating that it is an integral component of the retrograde IFT dynein. To assess the functional role of FAP163, we generated an RNA interference knockdown of the orthologous protein (WD60) in planaria. The Smed-wd60(RNAi) animals had a severe ciliary assembly defect that dramatically compromised whole-organism motility. Most cilia were present as short stubs that had accumulated large quantities of IFT particle–like material between the doublet microtubules and the membrane. The few remaining approximately full-length cilia had a chaotic beat with a frequency reduced from 24 to ∼10 Hz. Thus WD60/FAP163 is a dynein IC that is absolutely required for retrograde IFT and ciliary assembly. PMID:23864713

Basal body stability and ciliogenesis requires the conserved component Poc1

PubMed Central

Osborn, Daniel P.S.; Giddings, Thomas H.; Beales, Philip L.; Winey, Mark

2009-01-01

Centrioles are the foundation for centrosome and cilia formation. The biogenesis of centrioles is initiated by an assembly mechanism that first synthesizes the ninefold symmetrical cartwheel and subsequently leads to a stable cylindrical microtubule scaffold that is capable of withstanding microtubule-based forces generated by centrosomes and cilia. We report that the conserved WD40 repeat domain–containing cartwheel protein Poc1 is required for the structural maintenance of centrioles in Tetrahymena thermophila. Furthermore, human Poc1B is required for primary ciliogenesis, and in zebrafish, DrPoc1B knockdown causes ciliary defects and morphological phenotypes consistent with human ciliopathies. T. thermophila Poc1 exhibits a protein incorporation profile commonly associated with structural centriole components in which the majority of Poc1 is stably incorporated during new centriole assembly. A second dynamic population assembles throughout the cell cycle. Our experiments identify novel roles for Poc1 in centriole stability and ciliogenesis. PMID:20008567

Functional analysis of conserved aromatic amino acids in the discoidin domain of Paenibacillus β-1,3-glucanase

PubMed Central

2009-01-01

The 190-kDa Paenibacillus β-1,3-glucanase (LamA) contains a catalytic module of the glycoside hydrolase family 16 (GH16) and several auxiliary domains. Of these, a discoidin domain (DS domain), present in both eukaryotic and prokaryotic proteins with a wide variety of functions, exists at the carboxyl-terminus. To better understand the bacterial DS domain in terms of its structure and function, this domain alone was expressed in Escherichia coli and characterized. The results indicate that the DS domain binds various polysaccharides and enhances the biological activity of the GH16 module on composite substrates. We also investigated the importance of several conserved aromatic residues in the domain's stability and substrate-binding affinity. Both were affected by mutations of these residues; however, the effect on protein stability was more notable. In particular, the forces contributed by a sandwiched triad (W1688, R1756, and W1729) were critical for the presumable β-sandwich fold. PMID:19930717

The localisation of the heparin binding sites of human and murine interleukin-12 within the carboxyterminal domain of the P40 subunit.

PubMed

Garnier, Pascale; Mummery, Rosemary; Forster, Mark J; Mulloy, Barbara; Gibbs, Roslyn V; Rider, Christopher C

2018-05-09

We have previously shown that the heterodimeric cytokine interleukin-12, and the homodimer of its larger subunit p40, both bind to heparin and heparan sulfate with relatively high affinity. In the present study we characterised these interactions using a series of chemically modified heparins as competitive inhibitors. Human interleukin-12 and p40 homodimer show indistinguishable binding profiles with a panel of heparin derivatives, but that of murine interleukin-12 is distinct. Heparin markedly protects the human and murine p40 subunits, but not the p35 subunits, from cleavage by the bacterial endoprotease LysC, further implicating the larger subunit as the location of the heparin binding site. Moreover the essential role of the carboxyterminal D3 domain in heparin binding is established by the failure of a truncated construct of the p40 subunit lacking this domain to bind. Predictive docking calculations indicate that a cluster of basic residues at the tip of the exposed C'D' loop within D3 is important in heparin binding. However since the human and murine C'D' loops differ considerably in length, the mode and three dimensional orientation of heparin binding are likely to differ substantially between the human and murine p40s. Thus overall the binding of IL-12 via its p40 subunit to heparin-related polysaccharides of the extracellular matrix appears to be functionally important since it has been conserved across mammalian species despite this structural divergence. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Comparative Sequence and X-Inactivation Analyses of a Domain of Escape in Human Xp11.2 and the Conserved Segment in Mouse

PubMed Central

Tsuchiya, Karen D.; Greally, John M.; Yi, Yajun; Noel, Kevin P.; Truong, Jean-Pierre; Disteche, Christine M.

2004-01-01

We have performed X-inactivation and sequence analyses on 350 kb of sequence from human Xp11.2, a region shown previously to contain a cluster of genes that escape X inactivation, and we compared this region with the region of conserved synteny in mouse. We identified several new transcripts from this region in human and in mouse, which defined the full extent of the domain escaping X inactivation in both species. In human, escape from X inactivation involves an uninterrupted 235-kb domain of multiple genes. Despite highly conserved gene content and order between the two species, Smcx is the only mouse gene from the conserved segment that escapes inactivation. As repetitive sequences are believed to facilitate spreading of X inactivation along the chromosome, we compared the repetitive sequence composition of this region between the two species. We found that long terminal repeats (LTRs) were decreased in the human domain of escape, but not in the majority of the conserved mouse region adjacent to Smcx in which genes were subject to X inactivation, suggesting that these repeats might be excluded from escape domains to prevent spreading of silencing. Our findings indicate that genomic context, as well as gene-specific regulatory elements, interact to determine expression of a gene from the inactive X-chromosome. PMID:15197169

Annotation of Protein Domains Reveals Remarkable Conservation in the Functional Make up of Proteomes Across Superkingdoms

PubMed Central

Nasir, Arshan; Naeem, Aisha; Khan, Muhammad Jawad; Lopez-Nicora, Horacio D.; Caetano-Anollés, Gustavo

2011-01-01

The functional repertoire of a cell is largely embodied in its proteome, the collection of proteins encoded in the genome of an organism. The molecular functions of proteins are the direct consequence of their structure and structure can be inferred from sequence using hidden Markov models of structural recognition. Here we analyze the functional annotation of protein domain structures in almost a thousand sequenced genomes, exploring the functional and structural diversity of proteomes. We find there is a remarkable conservation in the distribution of domains with respect to the molecular functions they perform in the three superkingdoms of life. In general, most of the protein repertoire is spent in functions related to metabolic processes but there are significant differences in the usage of domains for regulatory and extra-cellular processes both within and between superkingdoms. Our results support the hypotheses that the proteomes of superkingdom Eukarya evolved via genome expansion mechanisms that were directed towards innovating new domain architectures for regulatory and extra/intracellular process functions needed for example to maintain the integrity of multicellular structure or to interact with environmental biotic and abiotic factors (e.g., cell signaling and adhesion, immune responses, and toxin production). Proteomes of microbial superkingdoms Archaea and Bacteria retained fewer numbers of domains and maintained simple and smaller protein repertoires. Viruses appear to play an important role in the evolution of superkingdoms. We finally identify few genomic outliers that deviate significantly from the conserved functional design. These include Nanoarchaeum equitans, proteobacterial symbionts of insects with extremely reduced genomes, Tenericutes and Guillardia theta. These organisms spend most of their domains on information functions, including translation and transcription, rather than on metabolism and harbor a domain repertoire characteristic of

Conservation of species, volume, and belief in patients with Alzheimer’s disease: the issue of domain-specificity and conceptual impairment

PubMed Central

Zaitchik, Deborah; Solomon, Gregg E. A.

2009-01-01

Two studies investigated whether patients with Alzheimer’s disease (AD) suffer high-level and category-specific impairment in the conceptual domain of living things. In Study 1, AD patients and healthy young and healthy elderly controls took part in three tasks: the Conservation of Species, Volume, and Belief. All 3 tasks required tracking an object’s identity in the face of irrelevant but salient transformations. Healthy young and elderly controls performed at or near ceiling on all tasks. AD patients were at or near ceiling on the Volume and Belief tasks, but only about half succeeded on the Species task. Study 2 demonstrated that the results were not due to simple task demands. AD patients’ failure to conserve species indicates that they are impaired in their theoretical understanding of living things, and their success on the Volume and Belief tasks suggests that the impairment is domain-specific. Two hypotheses are put forward to explain the phenomenon: the first, a category-specific account, holds that the intuitive theory of biology undergoes pervasive degradation; the second, a hybrid domain-general/domain-specific account, holds that impairment to domain-general processes such as executive function interacts with core cognition, the primitive elements that are the foundation of domain-specific knowledge. PMID:20043252

Deuterium Abundance Toward WD2211-495: Results from the Far Ultraviolet Spectroscopic Explorer (FUSE) Mission

NASA Technical Reports Server (NTRS)

Hebrard, G.; Lemoine, M.; Vidal-Madjar, A.; Desert, J. M.; LecavelierdesEtangs, A.; Ferlet, R.; Wood, B. E.; Linsky, J. L.; Kruk, J. W.; Chayer, P.;

Mapping cis-Regulatory Domains in the Human Genome UsingMulti-Species Conservation of Synteny

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

Ahituv, Nadav; Prabhakar, Shyam; Poulin, Francis

2005-06-13

Our inability to associate distant regulatory elements with the genes that they regulate has largely precluded their examination for sequence alterations contributing to human disease. One major obstacle is the large genomic space surrounding targeted genes in which such elements could potentially reside. In order to delineate gene regulatory boundaries we used whole-genome human-mouse-chicken (HMC) and human-mouse-frog (HMF) multiple alignments to compile conserved blocks of synteny (CBS), under the hypothesis that these blocks have been kept intact throughout evolution at least in part by the requirement of regulatory elements to stay linked to the genes that they regulate. A totalmore » of 2,116 and 1,942 CBS>200 kb were assembled for HMC and HMF respectively, encompassing 1.53 and 0.86 Gb of human sequence. To support the existence of complex long-range regulatory domains within these CBS we analyzed the prevalence and distribution of chromosomal aberrations leading to position effects (disruption of a genes regulatory environment), observing a clear bias not only for mapping onto CBS but also for longer CBS size. Our results provide a genome wide data set characterizing the regulatory domains of genes and the conserved regulatory elements within them.« less

Discovery of a Detached, Eclipsing 40 Minute Period Double White Dwarf Binary and a Friend: Implications for He+CO White Dwarf Mergers

NASA Astrophysics Data System (ADS)

Brown, Warren R.; Kilic, Mukremin; Kosakowski, Alekzander; Gianninas, A.

2017-09-01

We report the discovery of two detached double white dwarf (WD) binaries, SDSS J082239.546+304857.19 and SDSS J104336.275+055149.90, with orbital periods of 40 and 46 minutes, respectively. The 40 minute system is eclipsing; it is composed of a 0.30 M ⊙ and a 0.52 M ⊙ WD. The 46 minute system is a likely LISA verification binary. The short 20 ± 2 Myr and ˜34 Myr gravitational-wave merger times of the two binaries imply that many more such systems have formed and merged over the age of the Milky Way. We update the estimated Milky Way He+CO WD binary merger rate and affirm our previously published result: He+CO WD binaries merge at a rate at least 40 times greater than the formation rate of stable mass-transfer AM CVn binaries, and so the majority must have unstable mass-transfer. The implication is that spin-orbit coupling in He+CO WD mergers is weak, or perhaps nova-like outbursts drive He+CO WDs into merger, as proposed by Shen. Based on observations obtained at the MMT Observatory, a joint facility of the Smithsonian Institution and the University of Arizona, and on observations obtained with the Apache Point Observatory 3.5 m telescope, which is owned and operated by the Astrophysical Research Consortium.

Astrophysical Implications of a New Dynamical Mass for the Nearby White Dwarf 40 Eridani B

NASA Astrophysics Data System (ADS)

Bond, Howard E.; Bergeron, P.; Bédard, A.

2017-10-01

The bright, nearby DA-type white dwarf (WD) 40 Eridani B is orbited by the M dwarf 40 Eri C, allowing determination of the WD’s mass. Until recently, however, the mass depended on orbital elements determined four decades ago, and that mass was so low that it created several astrophysical puzzles. Using new astrometric measurements, the binary-star group at the U.S. Naval Observatory has revised the dynamical mass upward, to 0.573 ± 0.018 M ⊙. In this paper, we use model-atmosphere analysis to update other parameters of the WD, including effective temperature, surface gravity, radius, and luminosity. We then compare these results with WD interior models. Within the observational uncertainties, theoretical cooling tracks for CO-core WDs of its measured mass are consistent with the position of 40 Eri B in the H-R diagram; equivalently, the theoretical mass-radius relation (MRR) is consistent with the star’s location in the mass-radius plane. This consistency is, however, achieved only if we assume a “thin” outer hydrogen layer, with q H = M H/M WD ≃ 10-10. We discuss other evidence that a significant fraction of DA WDs have such thin H layers, in spite of the expectation from canonical stellar-evolution theory of “thick” H layers with q H ≃ 10-4. The cooling age of 40 Eri B is ˜122 Myr, and its total age is ˜1.8 Gyr. We present the MRRs for 40 Eri B and three other nearby WDs in visual binaries with precise mass determinations, and show that the agreement of current theory with observations is excellent in all cases.

A NEW TIMESCALE FOR PERIOD CHANGE IN THE PULSATING DA WHITE DWARF WD 0111+0018

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

Hermes, J. J.; Montgomery, M. H.; Winget, D. E.

2013-03-20

We report the most rapid rate of period change measured to date for a pulsating DA (hydrogen atmosphere) white dwarf (WD), observed in the 292.9 s mode of WD 0111+0018. The observed period change, faster than 10{sup -12} s s{sup -1}, exceeds by more than two orders of magnitude the expected rate from cooling alone for this class of slow and simply evolving pulsating WDs. This result indicates the presence of an additional timescale for period evolution in these pulsating objects. We also measure the rates of period change of nonlinear combination frequencies and show that they share the evolutionarymore » characteristics of their parent modes, confirming that these combination frequencies are not independent modes but rather artifacts of some nonlinear distortion in the outer layers of the star.« less

A conserved inter-domain communication mechanism regulates the ATPase activity of the AAA-protein Drg1.

PubMed

Prattes, Michael; Loibl, Mathias; Zisser, Gertrude; Luschnig, Daniel; Kappel, Lisa; Rössler, Ingrid; Grassegger, Manuela; Hromic, Altijana; Krieger, Elmar; Gruber, Karl; Pertschy, Brigitte; Bergler, Helmut

2017-03-17

AAA-ATPases fulfil essential roles in different cellular pathways and often act in form of hexameric complexes. Interaction with pathway-specific substrate and adaptor proteins recruits them to their targets and modulates their catalytic activity. This substrate dependent regulation of ATP hydrolysis in the AAA-domains is mediated by a non-catalytic N-terminal domain. The exact mechanisms that transmit the signal from the N-domain and coordinate the individual AAA-domains in the hexameric complex are still the topic of intensive research. Here, we present the characterization of a novel mutant variant of the eukaryotic AAA-ATPase Drg1 that shows dysregulation of ATPase activity and altered interaction with Rlp24, its substrate in ribosome biogenesis. This defective regulation is the consequence of amino acid exchanges at the interface between the regulatory N-domain and the adjacent D1 AAA-domain. The effects caused by these mutations strongly resemble those of pathological mutations of the AAA-ATPase p97 which cause the hereditary proteinopathy IBMPFD (inclusion body myopathy associated with Paget's disease of the bone and frontotemporal dementia). Our results therefore suggest well conserved mechanisms of regulation between structurally, but not functionally related members of the AAA-family.

Pwp2 mediates UTP-B assembly via two structurally independent domains.

PubMed

Boissier, Fanny; Schmidt, Christina Maria; Linnemann, Jan; Fribourg, Sébastien; Perez-Fernandez, Jorge

2017-06-09

The SSU processome constitutes a large ribonucleoprotein complex involved in the early steps of ribosome biogenesis. UTP-B is one of the first multi-subunit protein complexes that associates with the pre-ribosomal RNA to form the SSU processome. To understand the molecular basis of the hierarchical assembly of the SSU-processome, we have undergone a structural and functional analysis of the UTP-B subunit Pwp2p. We show that Pwp2p is required for the proper assembly of UTP-B and for a productive association of UTP-B with pre-rRNA. These two functions are mediated by two distinct structural domains. The N-terminal domain of Pwp2p folds into a tandem WD-repeat (tWD) that associates with Utp21p, Utp18p, and Utp6p to form a core complex. The CTDs of Pwp2p and Utp21p mediate the assembly of the heterodimer Utp12p:Utp13p that is required for the stable incorporation of the UTP-B complex in the SSU processome. Finally, we provide evidence suggesting a role of UTP-B as a platform for the binding of assembly factors during the maturation of 20S rRNA precursors.

Conserved C-Terminal Domain of Spider Tubuliform Spidroin 1 Contributes to Extensibility in Synthetic Fibers

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

Gnesa, Eric; Hsia, Yang; Yarger, Jeffery L.

2012-05-24

Spider silk is renowned for its extraordinary mechanical properties, having a balance of high tensile strength and extensibility. To date, the majority of studies have focused on the production of dragline silks from synthetic spider silk gene products. Here we report the first mechanical analysis of synthetic egg case silk fibers spun from the Latrodectus hesperus tubuliform silk proteins, TuSp1 and ECP-2. We provide evidence that recombinant ECP-2 proteins can be spun into fibers that display mechanical properties similar to other synthetic spider silks. We also demonstrate that silks spun from recombinant thioredoxin-TuSp1 fusion proteins that contain the conserved C-terminalmore » domain exhibit increased extensibility and toughness when compared to the identical fibers spun from fusion proteins lacking the C-terminus. Mechanical analyses reveal that the properties of synthetic tubuliform silks can be modulated by altering the postspin draw ratios of the fibers. Fibers subject to increased draw ratios showed elevated tensile strength and decreased extensibility but maintained constant toughness. Wide-angle X-ray diffraction studies indicate that postdrawn fibers containing the C-terminal domain of TuSp1 have more amorphous content when compared to fibers lacking the C-terminus. Taken together, these studies demonstrate that recombinant tubuliform spidroins that contain the conserved C-terminal domain with embedded protein tags can be effectively spun into fibers, resulting in similar tensile strength but increased extensibility relative to nontagged recombinant dragline silk proteins spun from equivalently sized proteins.« less

Analysis of heterogeneous gallstones using laser-induced breakdown spectroscopy (LIBS) and wavelength dispersive X-ray fluorescence (WD-XRF).

PubMed

Jaswal, Brij Bir S; Kumar, Vinay; Sharma, Jitendra; Rai, Pradeep K; Gondal, Mohammed A; Gondal, Bilal; Singh, Vivek K

2016-04-01

Laser-induced breakdown spectroscopy (LIBS) is an emerging analytical technique with numerous advantages such as rapidity, multi-elemental analysis, no specific sample preparation requirements, non-destructiveness, and versatility. It has been proven to be a robust elemental analysis tool attracting interest because of being applied to a wide range of materials including biomaterials. In this paper, we have performed spectroscopic studies on gallstones which are heterogeneous in nature using LIBS and wavelength dispersive X-ray fluorescence (WD-XRF) techniques. It has been observed that the presence and relative concentrations of trace elements in different kind of gallstones (cholesterol and pigment gallstones) can easily be determined using LIBS technique. From the experiments carried out on gallstones for trace elemental mapping and detection, it was found that LIBS is a robust tool for such biomedical applications. The stone samples studied in the present paper were classified using the Fourier transform infrared (FTIR) spectroscopy. WD-XRF spectroscopy has been applied for the qualitative and quantitative analysis of major and trace elements present in the gallstone which was compared with the LIBS data. The results obtained in the present paper show interesting prospects for LIBS and WD-XRF to study cholelithiasis better.

Astrophysical Implications of a New Dynamical Mass for the Nearby White Dwarf 40 Eridani B

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

Bond, Howard E.; Bergeron, P.; Bédard, A., E-mail: heb11@psu.edu

The bright, nearby DA-type white dwarf (WD) 40 Eridani B is orbited by the M dwarf 40 Eri C, allowing determination of the WD’s mass. Until recently, however, the mass depended on orbital elements determined four decades ago, and that mass was so low that it created several astrophysical puzzles. Using new astrometric measurements, the binary-star group at the U.S. Naval Observatory has revised the dynamical mass upward, to 0.573 ± 0.018 M {sub ☉}. In this paper, we use model-atmosphere analysis to update other parameters of the WD, including effective temperature, surface gravity, radius, and luminosity. We then comparemore » these results with WD interior models. Within the observational uncertainties, theoretical cooling tracks for CO-core WDs of its measured mass are consistent with the position of 40 Eri B in the H-R diagram; equivalently, the theoretical mass–radius relation (MRR) is consistent with the star’s location in the mass–radius plane. This consistency is, however, achieved only if we assume a “thin” outer hydrogen layer, with q {sub H} = M {sub H}/ M {sub WD} ≃ 10{sup −10}. We discuss other evidence that a significant fraction of DA WDs have such thin H layers, in spite of the expectation from canonical stellar-evolution theory of “thick” H layers with q {sub H} ≃ 10{sup −4}. The cooling age of 40 Eri B is ∼122 Myr, and its total age is ∼1.8 Gyr. We present the MRRs for 40 Eri B and three other nearby WDs in visual binaries with precise mass determinations, and show that the agreement of current theory with observations is excellent in all cases.« less

Identification of new members of the MAPK gene family in plants shows diverse conserved domains and novel activation loop variants.

PubMed

Mohanta, Tapan Kumar; Arora, Pankaj Kumar; Mohanta, Nibedita; Parida, Pratap; Bae, Hanhong

2015-02-06

Mitogen Activated Protein Kinase (MAPK) signaling is of critical importance in plants and other eukaryotic organisms. The MAPK cascade plays an indispensible role in the growth and development of plants, as well as in biotic and abiotic stress responses. The MAPKs are constitute the most downstream module of the three tier MAPK cascade and are phosphorylated by upstream MAP kinase kinases (MAPKK), which are in turn are phosphorylated by MAP kinase kinase kinase (MAPKKK). The MAPKs play pivotal roles in regulation of many cytoplasmic and nuclear substrates, thus regulating several biological processes. A total of 589 MAPKs genes were identified from the genome wide analysis of 40 species. The sequence analysis has revealed the presence of several N- and C-terminal conserved domains. The MAPKs were previously believed to be characterized by the presence of TEY/TDY activation loop motifs. The present study showed that, in addition to presence of activation loop TEY/TDY motifs, MAPKs are also contain MEY, TEM, TQM, TRM, TVY, TSY, TEC and TQY activation loop motifs. Phylogenetic analysis of all predicted MAPKs were clustered into six different groups (group A, B, C, D, E and F), and all predicted MAPKs were assigned with specific names based on their orthology based evolutionary relationships with Arabidopsis or Oryza MAPKs. We conducted global analysis of the MAPK gene family of plants from lower eukaryotes to higher eukaryotes and analyzed their genomic and evolutionary aspects. Our study showed the presence of several new activation loop motifs and diverse conserved domains in MAPKs. Advance study of newly identified activation loop motifs can provide further information regarding the downstream signaling cascade activated in response to a wide array of stress conditions, as well as plant growth and development.

Structural mapping of the coiled-coil domain of a bacterial condensin and comparative analyses across all domains of life suggest conserved features of SMC proteins.

PubMed

Waldman, Vincent M; Stanage, Tyler H; Mims, Alexandra; Norden, Ian S; Oakley, Martha G

2015-06-01

The structural maintenance of chromosomes (SMC) proteins form the cores of multisubunit complexes that are required for the segregation and global organization of chromosomes in all domains of life. These proteins share a common domain structure in which N- and C- terminal regions pack against one another to form a globular ATPase domain. This "head" domain is connected to a central, globular, "hinge" or dimerization domain by a long, antiparallel coiled coil. To date, most efforts for structural characterization of SMC proteins have focused on the globular domains. Recently, however, we developed a method to map interstrand interactions in the 50-nm coiled-coil domain of MukB, the divergent SMC protein found in γ-proteobacteria. Here, we apply that technique to map the structure of the Bacillus subtilis SMC (BsSMC) coiled-coil domain. We find that, in contrast to the relatively complicated coiled-coil domain of MukB, the BsSMC domain is nearly continuous, with only two detectable coiled-coil interruptions. Near the middle of the domain is a break in coiled-coil structure in which there are three more residues on the C-terminal strand than on the N-terminal strand. Close to the head domain, there is a second break with a significantly longer insertion on the same strand. These results provide an experience base that allows an informed interpretation of the output of coiled-coil prediction algorithms for this family of proteins. A comparison of such predictions suggests that these coiled-coil deviations are highly conserved across SMC types in a wide variety of organisms, including humans. © 2015 Wiley Periodicals, Inc.

The Escherichia coli Lpt transenvelope protein complex for lipopolysaccharide export is assembled via conserved structurally homologous domains.

PubMed

Villa, Riccardo; Martorana, Alessandra M; Okuda, Suguru; Gourlay, Louise J; Nardini, Marco; Sperandeo, Paola; Dehò, Gianni; Bolognesi, Martino; Kahne, Daniel; Polissi, Alessandra

2013-03-01

Lipopolysaccharide is a major glycolipid component in the outer leaflet of the outer membrane (OM), a peculiar permeability barrier of Gram-negative bacteria that prevents many toxic compounds from entering the cell. Lipopolysaccharide transport (Lpt) across the periplasmic space and its assembly at the Escherichia coli cell surface are carried out by a transenvelope complex of seven essential Lpt proteins spanning the inner membrane (LptBCFG), the periplasm (LptA), and the OM (LptDE), which appears to operate as a unique machinery. LptC is an essential inner membrane-anchored protein with a large periplasm-protruding domain. LptC binds the inner membrane LptBFG ABC transporter and interacts with the periplasmic protein LptA. However, its role in lipopolysaccharide transport is unclear. Here we show that LptC lacking the transmembrane region is viable and can bind the LptBFG inner membrane complex; thus, the essential LptC functions are located in the periplasmic domain. In addition, we characterize two previously described inactive single mutations at two conserved glycines (G56V and G153R, respectively) of the LptC periplasmic domain, showing that neither mutant is able to assemble the transenvelope machinery. However, while LptCG56V failed to copurify any Lpt component, LptCG153R was able to interact with the inner membrane protein complex LptBFG. Overall, our data further support the model whereby the bridge connecting the inner and outer membranes would be based on the conserved structurally homologous jellyroll domain shared by five out of the seven Lpt components.

The Escherichia coli Lpt Transenvelope Protein Complex for Lipopolysaccharide Export Is Assembled via Conserved Structurally Homologous Domains

PubMed Central

Villa, Riccardo; Martorana, Alessandra M.; Okuda, Suguru; Gourlay, Louise J.; Nardini, Marco; Sperandeo, Paola; Dehò, Gianni; Bolognesi, Martino; Kahne, Daniel

2013-01-01

Lipopolysaccharide is a major glycolipid component in the outer leaflet of the outer membrane (OM), a peculiar permeability barrier of Gram-negative bacteria that prevents many toxic compounds from entering the cell. Lipopolysaccharide transport (Lpt) across the periplasmic space and its assembly at the Escherichia coli cell surface are carried out by a transenvelope complex of seven essential Lpt proteins spanning the inner membrane (LptBCFG), the periplasm (LptA), and the OM (LptDE), which appears to operate as a unique machinery. LptC is an essential inner membrane-anchored protein with a large periplasm-protruding domain. LptC binds the inner membrane LptBFG ABC transporter and interacts with the periplasmic protein LptA. However, its role in lipopolysaccharide transport is unclear. Here we show that LptC lacking the transmembrane region is viable and can bind the LptBFG inner membrane complex; thus, the essential LptC functions are located in the periplasmic domain. In addition, we characterize two previously described inactive single mutations at two conserved glycines (G56V and G153R, respectively) of the LptC periplasmic domain, showing that neither mutant is able to assemble the transenvelope machinery. However, while LptCG56V failed to copurify any Lpt component, LptCG153R was able to interact with the inner membrane protein complex LptBFG. Overall, our data further support the model whereby the bridge connecting the inner and outer membranes would be based on the conserved structurally homologous jellyroll domain shared by five out of the seven Lpt components. PMID:23292770

Bleeding score in type 1 von Willebrand disease patients using the condensed MCMDM-1 vWD validated questionnaire.

PubMed

Pathare, A; Al Hajri, F; Al Omrani, S; Al Obaidani, N; Al Balushi, B; Al Falahi, K

2018-05-13

Assessment of the severity of bleeding symptom has led to the evolution of bleeding assessment tools which are now validated. To administer the condensed molecular and clinical markers for the diagnosis and management of type 1 von Willebrand disease VWD (MCMDM-1 vWD) questionnaire to the Omani type 1 vWD patients and correlate it with the laboratory parameters. Patients and controls were personally interviewed and the condensed MCMDM-1 vWD questionnaire administered by a single investigator. Bleeding score (BS) was calculated, based on the presence or absence of the bleeding symptoms according to a standard validated questionnaire in both the patients and the controls. The median age of the patient cohort was 27 (range, 7-49) years with 60.87% of females. The median time to administer condensed MCMDM-1 BS questionnaire was 11 minutes (interquartile range-IQR;7,16). Overall, bleeding from the oral cavity was the most predominant symptom (63%). The median BS was 5 (IQR;1,8) although individual scores ranged between 0 and 29. However, there was no statistically significant difference in BS between genders (males: median 4; IQR 1,6 and females: median 5, IQR 1,10) (P > .05, Kruskal-Wallis test) The Spearman's correlation value of BS was weak with FVIII:C levels and von Willebrand Ristocetin co-factor activity; very weak with von Willebrand Antigen level, and moderate with vonWillebrand Collagen Binding activity being -0.29, -0.28, -0.14 and -0.43, respectively. The BS reflects the severity of bleeding among the vWD patients. Although the BS was abnormal, it did not correlate significantly with the surrogate laboratory parameters [P > .05]. © 2018 John Wiley & Sons Ltd.

Cysteine-rich Domain 1 of CD40 Mediates Receptor Self-assembly*

PubMed Central

Smulski, Cristian R.; Beyrath, Julien; Decossas, Marion; Chekkat, Neila; Wolff, Philippe; Estieu-Gionnet, Karine; Guichard, Gilles; Speiser, Daniel; Schneider, Pascal; Fournel, Sylvie

2013-01-01

The activation of CD40 on B cells, macrophages, and dendritic cells by its ligand CD154 (CD40L) is essential for the development of humoral and cellular immune responses. CD40L and other TNF superfamily ligands are noncovalent homotrimers, but the form under which CD40 exists in the absence of ligand remains to be elucidated. Here, we show that both cell surface-expressed and soluble CD40 self-assemble, most probably as noncovalent dimers. The cysteine-rich domain 1 (CRD1) of CD40 participated to dimerization and was also required for efficient receptor expression. Modelization of a CD40 dimer allowed the identification of lysine 29 in CRD1, whose mutation decreased CD40 self-interaction without affecting expression or response to ligand. When expressed alone, recombinant CD40-CRD1 bound CD40 with a KD of 0.6 μm. This molecule triggered expression of maturation markers on human dendritic cells and potentiated CD40L activity. These results suggest that CD40 self-assembly modulates signaling, possibly by maintaining the receptor in a quiescent state. PMID:23463508

HCV IRES domain IIb affects the configuration of coding RNA in the 40S subunit's decoding groove

PubMed Central

Filbin, Megan E.; Kieft, Jeffrey S.

2011-01-01

Hepatitis C virus (HCV) uses a structured internal ribosome entry site (IRES) RNA to recruit the translation machinery to the viral RNA and begin protein synthesis without the ribosomal scanning process required for canonical translation initiation. Different IRES structural domains are used in this process, which begins with direct binding of the 40S ribosomal subunit to the IRES RNA and involves specific manipulation of the translational machinery. We have found that upon initial 40S subunit binding, the stem–loop domain of the IRES that contains the start codon unwinds and adopts a stable configuration within the subunit's decoding groove. This configuration depends on the sequence and structure of a different stem–loop domain (domain IIb) located far from the start codon in sequence, but spatially proximal in the IRES•40S complex. Mutation of domain IIb results in misconfiguration of the HCV RNA in the decoding groove that includes changes in the placement of the AUG start codon, and a substantial decrease in the ability of the IRES to initiate translation. Our results show that two distal regions of the IRES are structurally communicating at the initial step of 40S subunit binding and suggest that this is an important step in driving protein synthesis. PMID:21606179

HCV IRES domain IIb affects the configuration of coding RNA in the 40S subunit's decoding groove.

PubMed

Filbin, Megan E; Kieft, Jeffrey S

2011-07-01

Hepatitis C virus (HCV) uses a structured internal ribosome entry site (IRES) RNA to recruit the translation machinery to the viral RNA and begin protein synthesis without the ribosomal scanning process required for canonical translation initiation. Different IRES structural domains are used in this process, which begins with direct binding of the 40S ribosomal subunit to the IRES RNA and involves specific manipulation of the translational machinery. We have found that upon initial 40S subunit binding, the stem-loop domain of the IRES that contains the start codon unwinds and adopts a stable configuration within the subunit's decoding groove. This configuration depends on the sequence and structure of a different stem-loop domain (domain IIb) located far from the start codon in sequence, but spatially proximal in the IRES•40S complex. Mutation of domain IIb results in misconfiguration of the HCV RNA in the decoding groove that includes changes in the placement of the AUG start codon, and a substantial decrease in the ability of the IRES to initiate translation. Our results show that two distal regions of the IRES are structurally communicating at the initial step of 40S subunit binding and suggest that this is an important step in driving protein synthesis.

A conserved gene family encodes transmembrane proteins with fibronectin, immunoglobulin and leucine-rich repeat domains (FIGLER)

PubMed Central

Munfus, Delicia L; Haga, Christopher L; Burrows, Peter D; Cooper, Max D

2007-01-01

Background In mouse the cytokine interleukin-7 (IL-7) is required for generation of B lymphocytes, but human IL-7 does not appear to have this function. A bioinformatics approach was therefore used to identify IL-7 receptor related genes in the hope of identifying the elusive human cytokine. Results Our database search identified a family of nine gene candidates, which we have provisionally named fibronectin immunoglobulin leucine-rich repeat (FIGLER). The FIGLER 1–9 genes are predicted to encode type I transmembrane glycoproteins with 6–12 leucine-rich repeats (LRR), a C2 type Ig domain, a fibronectin type III domain, a hydrophobic transmembrane domain, and a cytoplasmic domain containing one to four tyrosine residues. Members of this multichromosomal gene family possess 20–47% overall amino acid identity and are differentially expressed in cell lines and primary hematopoietic lineage cells. Genes for FIGLER homologs were identified in macaque, orangutan, chimpanzee, mouse, rat, dog, chicken, toad, and puffer fish databases. The non-human FIGLER homologs share 38–99% overall amino acid identity with their human counterpart. Conclusion The extracellular domain structure and absence of recognizable cytoplasmic signaling motifs in members of the highly conserved FIGLER gene family suggest a trophic or cell adhesion function for these molecules. PMID:17854505

The E2 Domains of APP and APLP1 Share a Conserved Mode of Dimerization

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

S Lee; Y Xue; J Hulbert

2011-12-31

Amyloid precursor protein (APP) is genetically linked to Alzheimer's disease. APP is a type I membrane protein, and its oligomeric structure is potentially important because this property may play a role in its function or affect the processing of the precursor by the secretases to generate amyloid {beta}-peptide. Several independent studies have shown that APP can form dimers in the cell, but how it dimerizes remains controversial. At least three regions of the precursor, including a centrally located and conserved domain called E2, have been proposed to contribute to dimerization. Here we report two new crystal structures of E2, onemore » from APP and the other from APLP1, a mammalian APP homologue. Comparison with an earlier APP structure, which was determined in a different space group, shows that the E2 domains share a conserved and antiparallel mode of dimerization. Biophysical measurements in solution show that heparin binding induces E2 dimerization. The 2.1 {angstrom} resolution electron density map also reveals phosphate ions that are bound to the protein surface. Mutational analysis shows that protein residues interacting with the phosphate ions are also involved in heparin binding. The locations of two of these residues, Arg-369 and His-433, at the dimeric interface suggest a mechanism for heparin-induced protein dimerization.« less

Functional evidence for the critical amino-terminal conserved domain and key amino acids of Arabidopsis 4-HYDROXY-3-METHYLBUT-2-ENYL DIPHOSPHATE REDUCTASE.

PubMed

Hsieh, Wei-Yu; Sung, Tzu-Ying; Wang, Hsin-Tzu; Hsieh, Ming-Hsiun

2014-09-01

The plant 4-HYDROXY-3-METHYLBUT-2-ENYL DIPHOSPHATE REDUCTASE (HDR) catalyzes the last step of the methylerythritol phosphate pathway to synthesize isopentenyl diphosphate and its allyl isomer dimethylallyl diphosphate, which are common precursors for the synthesis of plastid isoprenoids. The Arabidopsis (Arabidopsis thaliana) genomic HDR transgene-induced gene-silencing lines are albino, variegated, or pale green, confirming that HDR is essential for plants. We used Escherichia coli isoprenoid synthesis H (Protein Data Bank code 3F7T) as a template for homology modeling to identify key amino acids of Arabidopsis HDR. The predicted model reveals that cysteine (Cys)-122, Cys-213, and Cys-350 are involved in iron-sulfur cluster formation and that histidine (His)-152, His-241, glutamate (Glu)-242, Glu-243, threonine (Thr)-244, Thr-312, serine-379, and asparagine-381 are related to substrate binding or catalysis. Glu-242 and Thr-244 are conserved only in cyanobacteria, green algae, and land plants, whereas the other key amino acids are absolutely conserved from bacteria to plants. We used site-directed mutagenesis and complementation assay to confirm that these amino acids, except His-152 and His-241, were critical for Arabidopsis HDR function. Furthermore, the Arabidopsis HDR contains an extra amino-terminal domain following the transit peptide that is highly conserved from cyanobacteria, and green algae to land plants but not existing in the other bacteria. We demonstrated that the amino-terminal conserved domain was essential for Arabidopsis and cyanobacterial HDR function. Further analysis of conserved amino acids in the amino-terminal conserved domain revealed that the tyrosine-72 residue was critical for Arabidopsis HDR. These results suggest that the structure and reaction mechanism of HDR evolution have become specific for oxygen-evolving photosynthesis organisms and that HDR probably evolved independently in cyanobacteria versus other prokaryotes. © 2014

Far-ultraviolet spectroscopy of the hot DA white dwarf WD 2218+706 (DeHt5) with STIS

NASA Astrophysics Data System (ADS)

Barstow, M. A.; Bannister, N. P.; Holberg, J. B.; Hubeny, I.; Bruhweiler, F. C.; Napiwotzki, R.

2001-08-01

We report a study of the photospheric composition of the hot DAQ3 white dwarf WD 2218+706, which is also the central star of the old planetary nebula DeHt5. Helium is detected in the far-UV spectrumQ4. In addition, the star clearly contains significant quantities of elements heavier than He at abundances generally a factor of 2 to 10 higher than those found in the archetypal heavy element-rich DA G191-B2B. This is the first detection of trace He using the Heii λ1640 line in an isolated DA white dwarf, but the low surface gravity is more indicative of a binary evolution route from the red giant branch rather than a path along the asymptotic giant branch (AGB) as a single star. However, the absence of any evidence for a companion star and the uncertainty in the measured mass for WD 2218+706 still allow the possibility of an origin along an AGB evolutionary track. We reanalyse the existing optical spectra of WD 2218+706 using our latest pure H and heavy element-rich model atmospheres, obtaining a good match between the observed and synthetic spectra with either set of models. We find little evidence of any inconsistency in the temperature required to fit individual Balmer lines, as reported elsewhere for this star. Any discrepancies we see are confined to the Hα line and the core of Hβ but they do not compromise our analysis.

The Conserved Foot Domain of RNA Pol II Associates with Proteins Involved in Transcriptional Initiation and/or Early Elongation

PubMed Central

García-López, M. Carmen; Pelechano, Vicent; Mirón-García, M. Carmen; Garrido-Godino, Ana I.; García, Alicia; Calvo, Olga; Werner, Michel; Pérez-Ortín, José E.; Navarro, Francisco

2011-01-01

RNA polymerase (pol) II establishes many protein–protein interactions with transcriptional regulators to coordinate different steps of transcription. Although some of these interactions have been well described, little is known about the existence of RNA pol II regions involved in contact with transcriptional regulators. We hypothesize that conserved regions on the surface of RNA pol II contact transcriptional regulators. We identified such an RNA pol II conserved region that includes the majority of the “foot” domain and identified interactions of this region with Mvp1, a protein required for sorting proteins to the vacuole, and Spo14, a phospholipase D. Deletion of MVP1 and SPO14 affects the transcription of their target genes and increases phosphorylation of Ser5 in the carboxy-terminal domain (CTD). Genetic, phenotypic, and functional analyses point to a role for these proteins in transcriptional initiation and/or early elongation, consistent with their genetic interactions with CEG1, a guanylyltransferase subunit of the Saccharomyces cerevisiae capping enzyme. PMID:21954159

A conserved glutamine plays a central role in LOV domain signal transmission and duration

PubMed Central

Nash, Abigail I.; Ko, Wen-Huang; Harper, Shannon M.; Gardner, Kevin H.

2009-01-01

Light is a key stimulus for plant biological functions, several of which are controlled by light-activated kinases known as phototropins, a group of kinases that contain two light-sensing domains (LOV, Light-Oxygen-Voltage domains) and a C-terminal serine/threonine kinase domain. The second sensory domain, LOV2, plays a key role in regulating kinase enzymatic activity via the photochemical formation of a covalent adduct between a LOV2 cysteine residue and an internally-bound flavin mononucleotide (FMN) chromophore. Subsequent conformational changes in LOV2 lead to the unfolding of a peripheral Jα helix, and ultimately, phototropin kinase activation. To date, the mechanism coupling bond formation and helix dissociation has remained unclear. Previous studies found that a conserved glutamine residue (Q513 in the Avena sativa phototropin 1 LOV2 (AsLOV2) domain) switches its hydrogen-bonding pattern with FMN upon light stimulation. Located in the immediate vicinity of the FMN binding site, this Gln residue is provided by the Iβ strand that interacts with the Jα helix, suggesting a route for signal propagation from the core of the LOV domain to its peripheral Jα helix. To test whether Q513 plays a key role in tuning the photochemical and transduction properties of AsLOV2, we designed two point mutations, Q513L and Q513N, and monitored the effects on the chromophore and protein using a combination of UV-visible absorbance and circular dichroism spectroscopy, limited proteolysis, and solution NMR. The results show that these mutations significantly dampen the changes between the dark and lit state AsLOV2 structures, leaving the protein in a pseudo-dark state (Q513L) or a pseudo-lit state (Q513N) conformation. Further, both mutations changed the photochemical properties of this receptor, particularly the lifetime of the photoexcited signaling states. Together, these data establish that this residue plays a central role in both spectral tuning and signal propagation from

The White-Dwarf Mass-Radius Relation from 40 Eridani B and Other Nearby Visual Binaries

NASA Astrophysics Data System (ADS)

Bond, Howard E.; Bergeron, P.; Bedard, A.

2018-01-01

The bright, nearby DA-type white dwarf (WD) 40 Eridani B is orbited by the M dwarf 40 Eri C, allowing determination of the WD's mass. Until recently, however, the mass depended on orbital elements determined four decades ago, and that mass was so low that it created several astrophysical puzzles. Using new astrometric measurements, the binary-star group at the U.S. Naval Observatory has revised the dynamical mass upward, to 0.573 ± 0.018 M⊙. We have used model-atmosphere analysis to update other parameters of the WD, including effective temperature, surface gravity, radius, and luminosity. We then comparethese results with WD interior models.Within the observational uncertainties, theoretical cooling tracks for CO-core WDs of its measured mass are consistent with the position of 40 Eri B in the H-R diagram; equivalently, the theoretical mass-radius relation (MRR) is consistent with the star's location in the mass-radius plane. This consistency is, however, achieved only if we assume a "thin'' outer hydrogen layer, with qH = MH/MWD ∼ 10–10.We discuss other evidence that a significant fraction of DA WDs have such thin H layers, in spite of expectation from canonical stellar-evolution theory of "thick'' H layers with qH ∼ 10–4 . The cooling age of 40 Eri B is ~122 Myr, and its total age is ~1.8 Gyr. We present the MRRs for 40 Eri B and three other nearby WDs in visual binaries with precise mass determinations, and show that the agreement of current theory with observation is excellent in all cases.However, astrophysical puzzles remain. The eccentricity of the BC orbit has remained high (0.43), even though the progenitor of B ought to have interacted tidally with C when it was an AGB star. This puzzle exists also for the Sirius and Procyon systems. If thin hydrogen layers are common among WDs, the mass scale will need to be shifted downwards by a few hundredths of a solar mass.

The conserved RNA recognition motif and C3H1 domain of the Not4 ubiquitin ligase regulate in vivo ligase function.

PubMed

Chen, Hongfeng; Sirupangi, Tirupataiah; Wu, Zhao-Hui; Johnson, Daniel L; Laribee, R Nicholas

2018-05-25

The Ccr4-Not complex controls RNA polymerase II (Pol II) dependent gene expression and proteasome function. The Not4 ubiquitin ligase is a Ccr4-Not subunit that has both a RING domain and a conserved RNA recognition motif and C3H1 domain (referred to as the RRM-C domain) with unknown function. We demonstrate that while individual Not4 RING or RRM-C mutants fail to replicate the proteasomal defects found in Not4 deficient cells, mutation of both exhibits a Not4 loss of function phenotype. Transcriptome analysis revealed that the Not4 RRM-C affects a specific subset of Pol II-regulated genes, including those involved in transcription elongation, cyclin-dependent kinase regulated nutrient responses, and ribosomal biogenesis. The Not4 RING, RRM-C, or RING/RRM-C mutations cause a generalized increase in Pol II binding at a subset of these genes, yet their impact on gene expression does not always correlate with Pol II recruitment which suggests Not4 regulates their expression through additional mechanisms. Intriguingly, we find that while the Not4 RRM-C is dispensable for Ccr4-Not association with RNA Pol II, the Not4 RING domain is required for these interactions. Collectively, these data elucidate previously unknown roles for the conserved Not4 RRM-C and RING domains in regulating Ccr4-Not dependent functions in vivo.

Function of the conserved FHIPEP domain of the flagellar type III export apparatus, protein FlhA.

PubMed

Barker, Clive S; Inoue, Tomoharu; Meshcheryakova, Irina V; Kitanobo, Seiya; Samatey, Fadel A

2016-04-01

The Type III flagellar protein export apparatus of bacteria consists of five or six membrane proteins, notably FlhA, which controls the export of other proteins and is homologous to the large family of FHIPEP export proteins. FHIPEP proteins contain a highly-conserved cytoplasmic domain. We mutagenized the cloned Salmonella flhA gene for the 692 amino acid FlhA, changing a single, conserved amino acid in the 68-amino acid FHIPEP region. Fifty-two mutations at 30 positions mostly led to loss of motility and total disappearance of microscopically visible flagella, also Western blot protein/protein hybridization showed no detectable export of hook protein and flagellin. There were two exceptions: a D199A mutant strain, which produced short-stubby flagella; and a V151L mutant strain, which did not produce flagella and excreted mainly un-polymerized hook protein. The V151L mutant strain also exported a reduced amount of hook-cap protein FlgD, but when grown with exogenous FlgD it produced polyhooks and polyhook-filaments. A suppressor mutant in the cytoplasmic domain of the export apparatus membrane protein FlhB rescued export of hook-length control protein FliK and facilitated growth of full-length flagella. These results suggested that the FHIPEP region is part of the gate regulating substrate entry into the export apparatus pore. © 2015 John Wiley & Sons Ltd.

Phosphorylation of a conserved serine in the deoxyribonucleic acid binding domain of nuclear receptors alters intracellular localization.

PubMed

Sun, Kai; Montana, Vedrana; Chellappa, Karthikeyani; Brelivet, Yann; Moras, Dino; Maeda, Yutaka; Parpura, Vladimir; Paschal, Bryce M; Sladek, Frances M

2007-06-01

Nuclear receptors (NRs) are a superfamily of transcription factors whose genomic functions are known to be activated by lipophilic ligands, but little is known about how to deactivate them or how to turn on their nongenomic functions. One obvious mechanism is to alter the nuclear localization of the receptors. Here, we show that protein kinase C (PKC) phosphorylates a highly conserved serine (Ser) between the two zinc fingers of the DNA binding domain of orphan receptor hepatocyte nuclear factor 4alpha (HNF4alpha). This Ser (S78) is adjacent to several positively charged residues (Arg or Lys), which we show here are involved in nuclear localization of HNF4alpha and are conserved in nearly all other NRs, along with the Ser/threonine (Thr). A phosphomimetic mutant of HNF4alpha (S78D) reduced DNA binding, transactivation ability, and protein stability. It also impaired nuclear localization, an effect that was greatly enhanced in the MODY1 mutant Q268X. Treatment of the hepatocellular carcinoma cell line HepG2 with PKC activator phorbol 12-myristate 13-acetate also resulted in increased cytoplasmic localization of HNF4alpha as well as decreased endogenous HNF4alpha protein levels in a proteasome-dependent fashion. We also show that PKC phosphorylates the DNA binding domain of other NRs (retinoic acid receptor alpha, retinoid X receptor alpha, and thyroid hormone receptor beta) and that phosphomimetic mutants of the same Ser/Thr result in cytoplasmic localization of retinoid X receptor alpha and peroxisome proliferator-activated receptor alpha. Thus, phosphorylation of this conserved Ser between the two zinc fingers may be a common mechanism for regulating the function of NRs.

Species-Specific Elements in the Large T-Antigen J Domain Are Required for Cellular Transformation and DNA Replication by Simian Virus 40

PubMed Central

Sullivan, Christopher S.; Tremblay, James D.; Fewell, Sheara W.; Lewis, John A.; Brodsky, Jeffrey L.; Pipas, James M.

2000-01-01

The J domain of simian virus 40 (SV40) large T antigen is required for efficient DNA replication and transformation. Despite previous reports demonstrating the promiscuity of J domains in heterologous systems, results presented here show the requirement for specific J-domain sequences in SV40 large-T-antigen-mediated activities. In particular, chimeric-T-antigen constructs in which the SV40 T-antigen J domain was replaced with that from the yeast Ydj1p or Escherichia coli DnaJ proteins failed to replicate in BSC40 cells and did not transform REF52 cells. However, T antigen containing the JC virus J domain was functional in these assays, although it was less efficient than the wild type. The inability of some large-T-antigen chimeras to promote DNA replication and elicit cellular transformation was not due to a failure to interact with hsc70, since a nonfunctional chimera, containing the DnaJ J domain, bound hsc70. However, this nonfunctional chimeric T antigen was reduced in its ability to stimulate hsc70 ATPase activity and unable to liberate E2F from p130, indicating that transcriptional activation of factors required for cell growth and DNA replication may be compromised. Our data suggest that the T-antigen J domain harbors species-specific elements required for viral activities in vivo. PMID:10891510

Effects of disrupting the polyketide synthase gene WdPKS1 in Wangiella [Exophiala] dermatitidis on melanin production and resistance to killing by antifungal compounds, enzymatic degradation, and extremes in temperature

PubMed Central

Paolo, William F; Dadachova, Ekaterina; Mandal, Piyali; Casadevall, Arturo; Szaniszlo, Paul J; Nosanchuk, Joshua D

2006-01-01

Background Wangiella dermatitidis is a human pathogenic fungus that is an etiologic agent of phaeohyphomycosis. W. dermatitidis produces a black pigment that has been identified as a dihydroxynaphthalene melanin and the production of this pigment is associated with its virulence. Cell wall pigmentation in W. dermatitidis depends on the WdPKS1 gene, which encodes a polyketide synthase required for generating the key precursor for dihydroxynaphthalene melanin biosynthesis. Results We analyzed the effects of disrupting WdPKS1 on dihydroxynaphthalene melanin production and resistance to antifungal compounds. Transmission electron microscopy revealed that wdpks1Δ-1 yeast had thinner cell walls that lacked an electron-opaque layer compared to wild-type cells. However, digestion of the wdpks1Δ-1 yeast revealed small black particles that were consistent with a melanin-like compound, because they were acid-resistant, reacted with melanin-binding antibody, and demonstrated a free radical signature by electron spin resonance analysis. Despite lacking the WdPKS1 gene, the mutant yeast were capable of catalyzing the formation of melanin from L-3,4-dihyroxyphenylalanine. The wdpks1Δ-1 cells were significantly more susceptible to killing by voriconazole, amphotericin B, NP-1 [a microbicidal peptide], heat and cold, and lysing enzymes than the heavily melanized parental or complemented strains. Conclusion In summary, W. dermatitidis makes WdPKS-dependent and -independent melanins, and the WdPKS1-dependent deposition of melanin in the cell wall confers protection against antifungal agents and environmental stresses. The biological role of the WdPKS-independent melanin remains unclear. PMID:16784529

40 CFR 256.31 - Recommendations for developing and implementing resource conservation and recovery programs.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 26 2013-07-01 2013-07-01 false Recommendations for developing and implementing resource conservation and recovery programs. 256.31 Section 256.31 Protection of Environment... are identified. These studies should review various technological approaches, environmental...

40 CFR 256.31 - Recommendations for developing and implementing resource conservation and recovery programs.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 26 2012-07-01 2011-07-01 true Recommendations for developing and implementing resource conservation and recovery programs. 256.31 Section 256.31 Protection of Environment... are identified. These studies should review various technological approaches, environmental...

40 CFR 256.31 - Recommendations for developing and implementing resource conservation and recovery programs.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 25 2014-07-01 2014-07-01 false Recommendations for developing and implementing resource conservation and recovery programs. 256.31 Section 256.31 Protection of Environment... are identified. These studies should review various technological approaches, environmental...

40 CFR 256.31 - Recommendations for developing and implementing resource conservation and recovery programs.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 25 2011-07-01 2011-07-01 false Recommendations for developing and implementing resource conservation and recovery programs. 256.31 Section 256.31 Protection of Environment... are identified. These studies should review various technological approaches, environmental...

Cadherin Domains in the Polysaccharide-Degrading Marine Bacterium Saccharophagus degradans 2-40 Are Carbohydrate-Binding Modules▿

PubMed Central

Fraiberg, Milana; Borovok, Ilya; Bayer, Edward A.; Weiner, Ronald M.; Lamed, Raphael

2011-01-01

The complex polysaccharide-degrading marine bacterium Saccharophagus degradans strain 2-40 produces putative proteins that contain numerous cadherin and cadherin-like domains involved in intercellular contact interactions. The current study reveals that both domain types exhibit reversible calcium-dependent binding to different complex polysaccharides which serve as growth substrates for the bacterium. PMID:21036994

Conservation and divergence of C-terminal domain structure in the retinoblastoma protein family

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

Liban, Tyler J.; Medina, Edgar M.; Tripathi, Sarvind

The retinoblastoma protein (Rb) and the homologous pocket proteins p107 and p130 negatively regulate cell proliferation by binding and inhibiting members of the E2F transcription factor family. The structural features that distinguish Rb from other pocket proteins have been unclear but are critical for understanding their functional diversity and determining why Rb has unique tumor suppressor activities. We describe here important differences in how the Rb and p107 C-terminal domains (CTDs) associate with the coiled-coil and marked-box domains (CMs) of E2Fs. We find that although CTD–CM binding is conserved across protein families, Rb and p107 CTDs show clear preferences formore » different E2Fs. A crystal structure of the p107 CTD bound to E2F5 and its dimer partner DP1 reveals the molecular basis for pocket protein–E2F binding specificity and how cyclin-dependent kinases differentially regulate pocket proteins through CTD phosphorylation. Our structural and biochemical data together with phylogenetic analyses of Rb and E2F proteins support the conclusion that Rb evolved specific structural motifs that confer its unique capacity to bind with high affinity those E2Fs that are the most potent activators of the cell cycle.« less

Roles of conserved arginines in ATP-binding domains of AAA+ chaperone ClpB from Thermus thermophilus.

PubMed

Yamasaki, Takashi; Nakazaki, Yosuke; Yoshida, Masasuke; Watanabe, Yo-hei

2011-07-01

ClpB, a member of the expanded superfamily of ATPases associated with diverse cellular activities (AAA+), forms a ring-shaped hexamer and cooperates with the DnaK chaperone system to reactivate aggregated proteins in an ATP-dependent manner. The ClpB protomer consists of an N-terminal domain, an AAA+ module (AAA-1), a middle domain, and a second AAA+ module (AAA-2). Each AAA+ module contains highly conserved WalkerA and WalkerB motifs, and two arginines (AAA-1) or one arginine (AAA-2). Here, we investigated the roles of these arginines (Arg322, Arg323, and Arg747) of ClpB from Thermus thermophilus in the ATPase cycle and chaperone function by alanine substitution. These mutations did not affect nucleotide binding, but did inhibit the hydrolysis of the bound ATP and slow the threading of the denatured protein through the central pore of the T. thermophilus ClpB ring, which severely impaired the chaperone functions. Previously, it was demonstrated that ATP binding to the AAA-1 module induced motion of the middle domain and stabilized the ClpB hexamer. However, the arginine mutations of the AAA-1 module destabilized the ClpB hexamer, even though ATP-induced motion of the middle domain was not affected. These results indicated that the three arginines are crucial for ATP hydrolysis and chaperone activity, but not for ATP binding. In addition, the two arginines in AAA-1 and the ATP-induced motion of the middle domain independently contribute to the stabilization of the hexamer. © 2011 The Authors Journal compilation © 2011 FEBS.

Co-Conserved MAPK Features Couple D-Domain Docking Groove to Distal Allosteric Sites via the C-Terminal Flanking Tail

PubMed Central

Nguyen, Tuan; Ruan, Zheng; Oruganty, Krishnadev; Kannan, Natarajan

2015-01-01

Mitogen activated protein kinases (MAPKs) form a closely related family of kinases that control critical pathways associated with cell growth and survival. Although MAPKs have been extensively characterized at the biochemical, cellular, and structural level, an integrated evolutionary understanding of how MAPKs differ from other closely related protein kinases is currently lacking. Here, we perform statistical sequence comparisons of MAPKs and related protein kinases to identify sequence and structural features associated with MAPK functional divergence. We show, for the first time, that virtually all MAPK-distinguishing sequence features, including an unappreciated short insert segment in the β4-β5 loop, physically couple distal functional sites in the kinase domain to the D-domain peptide docking groove via the C-terminal flanking tail (C-tail). The coupling mediated by MAPK-specific residues confers an allosteric regulatory mechanism unique to MAPKs. In particular, the regulatory αC-helix conformation is controlled by a MAPK-conserved salt bridge interaction between an arginine in the αC-helix and an acidic residue in the C-tail. The salt-bridge interaction is modulated in unique ways in individual sub-families to achieve regulatory specificity. Our study is consistent with a model in which the C-tail co-evolved with the D-domain docking site to allosterically control MAPK activity. Our study provides testable mechanistic hypotheses for biochemical characterization of MAPK-conserved residues and new avenues for the design of allosteric MAPK inhibitors. PMID:25799139

Conserved intron positions in FGFR genes reflect the modular structure of FGFR and reveal stepwise addition of domains to an already complex ancestral FGFR.

PubMed

Rebscher, Nicole; Deichmann, Christina; Sudhop, Stefanie; Fritzenwanker, Jens Holger; Green, Stephen; Hassel, Monika

2009-10-01

We have analyzed the evolution of fibroblast growth factor receptor (FGFR) tyrosine kinase genes throughout a wide range of animal phyla. No evidence for an FGFR gene was found in Porifera, but we tentatively identified an FGFR gene in the placozoan Trichoplax adhaerens. The gene encodes a protein with three immunoglobulin-like domains, a single-pass transmembrane, and a split tyrosine kinase domain. By superimposing intron positions of 20 FGFR genes from Placozoa, Cnidaria, Protostomia, and Deuterostomia over the respective protein domain structure, we identified ten ancestral introns and three conserved intron groups. Our analysis shows (1) that the position of ancestral introns correlates to the modular structure of FGFRs, (2) that the acidic domain very likely evolved in the last common ancestor of triploblasts, (3) that splicing of IgIII was enabled by a triploblast-specific insertion, and (4) that IgI is subject to substantial loss or duplication particularly in quickly evolving genomes. Moreover, intron positions in the catalytic domain of FGFRs map to the borders of protein subdomains highly conserved in other serine/threonine kinases. Nevertheless, these introns were introduced in metazoan receptor tyrosine kinases exclusively. Our data support the view that protein evolution dating back to the Cambrian explosion took place in such a short time window that only subtle changes in the domain structure are detectable in extant representatives of animal phyla. We propose that the first multidomain FGFR originated in the last common ancestor of Placozoa, Cnidaria, and Bilateria. Additional domains were introduced mainly in the ancestor of triploblasts and in the Ecdysozoa.

Interfacial Partitioning of a Loop Hinge Residue Contributes to Diacylglycerol Affinity of Conserved Region 1 Domains*

PubMed Central

Stewart, Mikaela D.; Cole, Taylor R.; Igumenova, Tatyana I.

2014-01-01

Conventional and novel isoenzymes of PKC are activated by the membrane-embedded second messenger diacylglycerol (DAG) through its interactions with the C1 regulatory domain. The affinity of C1 domains to DAG varies considerably among PKCs. To gain insight into the origin of differential DAG affinities, we conducted high-resolution NMR studies of C1B domain from PKCδ (C1Bδ) and its W252Y variant. The W252Y mutation was previously shown to render C1Bδ less responsive to DAG (Dries, D. R., Gallegos, L. L., and Newton, A. C. (2007) A single residue in the C1 domain sensitizes novel protein kinase C isoforms to cellular diacylglycerol production. J. Biol. Chem. 282, 826–830) and thereby emulate the behavior of C1B domains from conventional PKCs that have a conserved Tyr at the equivalent position. Our data revealed that W252Y mutation did not perturb the conformation of C1Bδ in solution but significantly reduced its propensity to partition into a membrane-mimicking environment in the absence of DAG. Using detergent micelles doped with a paramagnetic lipid, we determined that both the residue identity at position 252 and complexation with diacylglycerol influence the geometry of C1Bδ-micelle interactions. In addition, we identified the C-terminal helix α1 of C1Bδ as an interaction site with the head groups of phosphatidylserine, a known activator of PKCδ. Taken together, our studies (i) reveal the identities of C1Bδ residues involved in interactions with membrane-mimicking environment, DAG, and phosphatidylserine, as well as the affinities associated with each event and (ii) suggest that the initial ligand-independent membrane recruitment of C1B domains, which is greatly facilitated by the interfacial partitioning of Trp-252, is responsible, at least in part, for the differential DAG affinities. PMID:25124034

The conserved N-terminal domain of herpes simplex virus 1 UL24 protein is sufficient to induce the spatial redistribution of nucleolin.

PubMed

Bertrand, Luc; Pearson, Angela

2008-05-01

UL24 is widely conserved among herpesviruses but its function during infection is poorly understood. Previously, we discovered a genetic link between UL24 and the herpes simplex virus 1-induced dispersal of the nucleolar protein nucleolin. Here, we report that in the absence of viral infection, transiently expressed UL24 accumulated in both the nucleus and the Golgi apparatus. In the majority of transfected cells, nuclear staining for UL24 was diffuse, but a minor staining pattern, whereby UL24 was present in nuclear foci corresponding to nucleoli, was also observed. Expression of UL24 correlated with the dispersal of nucleolin. This dispersal did not appear to be a consequence of a general disaggregation of nucleoli, as foci of fibrillarin staining persisted in cells expressing UL24. The conserved N-terminal region of UL24 was sufficient to cause this change in subcellular distribution of nucleolin. Interestingly, a bipartite nuclear localization signal predicted within the C terminus of UL24 was dispensable for nuclear localization. None of the five individual UL24 homology domains was required for nuclear or Golgi localization, but deletion of these domains resulted in the loss of nucleolin-dispersal activity. We determined that a nucleolar-targeting signal was contained within the first 60 aa of UL24. Our results show that the conserved N-terminal domain of UL24 is sufficient to specifically induce dispersal of nucleolin in the absence of other viral proteins or virus-induced cellular modifications. These results suggest that UL24 directly targets cellular factors that affect the composition of nucleoli.

The LOTUS domain is a conserved DEAD-box RNA helicase regulator essential for the recruitment of Vasa to the germ plasm and nuage

PubMed Central

Jeske, Mandy; Müller, Christoph W.; Ephrussi, Anne

2017-01-01

DEAD-box RNA helicases play important roles in a wide range of metabolic processes. Regulatory proteins can stimulate or block the activity of DEAD-box helicases. Here, we show that LOTUS (Limkain, Oskar, and Tudor containing proteins 5 and 7) domains present in the germline proteins Oskar, TDRD5 (Tudor domain-containing 5), and TDRD7 bind and stimulate the germline-specific DEAD-box RNA helicase Vasa. Our crystal structure of the LOTUS domain of Oskar in complex with the C-terminal RecA-like domain of Vasa reveals that the LOTUS domain occupies a surface on a DEAD-box helicase not implicated previously in the regulation of the enzyme's activity. We show that, in vivo, the localization of Drosophila Vasa to the nuage and germ plasm depends on its interaction with LOTUS domain proteins. The binding and stimulation of Vasa DEAD-box helicases by LOTUS domains are widely conserved. PMID:28536148

FBW7 targets KLF10 for ubiquitin-dependent degradation.

PubMed

Yu, Su; Wang, Feng; Tan, Xiao; Gao, Guo-Li; Pan, Wei-Juan; Luan, Yi; Ge, Xin

2018-01-08

FBW7, a key component of SCF FBW7 E3 ubiquitin ligase, targets various proteins for degradation via the conserved Cdc4 phosphodegron (CPD) in substrates. In this study, we report that KLF10 is degraded by FBW7 via a conserved CPD. Through systematic analysis of the degradation of KLF transcription factors by FBW7, we identified KLF10 as a novel degradation target of FBW7. Ectopic expression of FBW7 markedly promoted the degradation of KLF10 while knockdown of endogenous FBW7 increased the protein levels of KLF10. In addition, simultaneous mutations of both threonine 82 (T82) and serine 86 (S86) significantly reduced the FBW7-mediated KLF10 degradation. Moreover, KLF10 containing a conserved putative CPD (TPPXSP) from amino acids 82 to 87, directly interacted with WD40 domain of FBW7 in a phosphorylation-dependent manner. Importantly, FBW7 could reverse the KLF10-mediated inhibition of Smad7 activity. Thus, our study uncovers a novel regulatory mechanism underlying which KLF10 stability and its biological function are mediated by FBW7. Copyright © 2017 Elsevier Inc. All rights reserved.

Diversified Structural Basis of a Conserved Molecular Mechanism for pH-Dependent Dimerization in Spider Silk N-Terminal Domains.

PubMed

Otikovs, Martins; Chen, Gefei; Nordling, Kerstin; Landreh, Michael; Meng, Qing; Jörnvall, Hans; Kronqvist, Nina; Rising, Anna; Johansson, Jan; Jaudzems, Kristaps

2015-08-17

Conversion of spider silk proteins from soluble dope to insoluble fibers involves pH-dependent dimerization of the N-terminal domain (NT). This conversion is tightly regulated to prevent premature precipitation and enable rapid silk formation at the end of the duct. Three glutamic acid residues that mediate this process in the NT from Euprosthenops australis major ampullate spidroin 1 are well conserved among spidroins. However, NTs of minor ampullate spidroins from several species, including Araneus ventricosus ((Av)MiSp NT), lack one of the glutamic acids. Here we investigate the pH-dependent structural changes of (Av)MiSp NT, revealing that it uses the same mechanism but involves a non-conserved glutamic acid residue instead. Homology modeling of the structures of other MiSp NTs suggests that these harbor different compensatory residues. This indicates that, despite sequence variations, the molecular mechanism underlying pH-dependent dimerization of NT is conserved among different silk types. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

High-resolution Crystal Structure of Dimeric VP40 From Sudan ebolavirus.

PubMed

Clifton, Matthew C; Bruhn, Jessica F; Atkins, Kateri; Webb, Terry L; Baydo, Ruth O; Raymond, Amy; Lorimer, Donald D; Edwards, Thomas E; Myler, Peter J; Saphire, Erica Ollmann

2015-10-01

Ebolaviruses cause severe hemorrhagic fever. Central to the Ebola life cycle is the matrix protein VP40, which oligomerizes and drives viral budding. Here we present the crystal structure of the Sudan virus (SUDV) matrix protein. This structure is higher resolution (1.6 Å) than previously achievable. Despite differences in the protein purification, we find that it still forms a stable dimer in solution, as was noted for other Ebola VP40s. Although the N-terminal domain interface by which VP40 dimerizes is conserved between Ebola virus and SUDV, the C-terminal domain interface by which VP40 dimers may further assemble is significantly smaller in this SUDV assembly. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

A conserved truncated isoform of the ATR-X syndrome protein lacking the SWI/SNF-homology domain.

PubMed

Garrick, David; Samara, Vassiliki; McDowell, Tarra L; Smith, Andrew J H; Dobbie, Lorraine; Higgs, Douglas R; Gibbons, Richard J

2004-02-04

Mutations in the ATRX gene cause a severe X-linked mental retardation syndrome that is frequently associated with alpha thalassemia (ATR-X syndrome). The previously characterized ATRX protein (approximately 280 kDa) contains both a Plant homeodomain (PHD)-like zinc finger motif as well as an ATPase domain of the SNF2 family. These motifs suggest that ATRX may function as a regulator of gene expression, probably by exerting an effect on chromatin structure, although the exact cellular role of ATRX has not yet been fully elucidated. Here we characterize a truncated (approximately 200 kDa) isoform of ATRX (called here ATRXt) that has been highly conserved between mouse and human. In both species, ATRXt arises due to the failure to splice intron 11 from the primary transcript, and the use of a proximal intronic poly(A) signal. We show that the relative expression of the full length and ATRXt isoforms is subject to tissue-specific regulation. The ATRXt isoform contains the PHD-like domain but not the SWI/SNF-like motifs and is therefore unlikely to be functionally equivalent to the full length protein. We used indirect immunofluorescence to demonstrate that the full length and ATRXt isoforms are colocalized at blocks of pericentromeric heterochromatin but unlike full length ATRX, the truncated isoform does not associate with promyelocytic leukemia (PML) nuclear bodies. The high degree of conservation of ATRXt and the tight regulation of its expression relative to the full length protein suggest that this truncated isoform fulfills an important biological function.

Mechanism of Mediator recruitment by tandem Gcn4 activation domains and three Gal11 activator-binding domains.

PubMed

Herbig, Eric; Warfield, Linda; Fish, Lisa; Fishburn, James; Knutson, Bruce A; Moorefield, Beth; Pacheco, Derek; Hahn, Steven

2010-05-01

Targets of the tandem Gcn4 acidic activation domains in transcription preinitiation complexes were identified by site-specific cross-linking. The individual Gcn4 activation domains cross-link to three common targets, Gal11/Med15, Taf12, and Tra1, which are subunits of four conserved coactivator complexes, Mediator, SAGA, TFIID, and NuA4. The Gcn4 N-terminal activation domain also cross-links to the Mediator subunit Sin4/Med16. The contribution of the two Gcn4 activation domains to transcription was gene specific and varied from synergistic to less than additive. Gcn4-dependent genes had a requirement for Gal11 ranging from 10-fold dependence to complete Gal11 independence, while the Gcn4-Taf12 interaction did not significantly contribute to the expression of any gene studied. Complementary methods identified three conserved Gal11 activator-binding domains that bind each Gcn4 activation domain with micromolar affinity. These Gal11 activator-binding domains contribute additively to transcription activation and Mediator recruitment at Gcn4- and Gal11-dependent genes. Although we found that the conserved Gal11 KIX domain contributes to Gal11 function, we found no evidence of specific Gcn4-KIX interaction and conclude that the Gal11 KIX domain does not function by specific interaction with Gcn4. Our combined results show gene-specific coactivator requirements, a surprising redundancy in activator-target interactions, and an activator-coactivator interaction mediated by multiple low-affinity protein-protein interactions.

Three-dimensional reconstructions of the bacteriophage CUS-3 virion reveal a conserved coat protein I-domain but a distinct tailspike receptor-binding domain

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

Parent, Kristin N., E-mail: kparent@msu.edu; Tang, Jinghua; Cardone, Giovanni

CUS-3 is a short-tailed, dsDNA bacteriophage that infects serotype K1 Escherichia coli. We report icosahedrally averaged and asymmetric, three-dimensional, cryo-electron microscopic reconstructions of the CUS-3 virion. Its coat protein structure adopts the “HK97-fold” shared by other tailed phages and is quite similar to that in phages P22 and Sf6 despite only weak amino acid sequence similarity. In addition, these coat proteins share a unique extra external domain (“I-domain”), suggesting that the group of P22-like phages has evolved over a very long time period without acquiring a new coat protein gene from another phage group. On the other hand, the morphologymore » of the CUS-3 tailspike differs significantly from that of P22 or Sf6, but is similar to the tailspike of phage K1F, a member of the extremely distantly related T7 group of phages. We conclude that CUS-3 obtained its tailspike gene from a distantly related phage quite recently. - Highlights: • Asymmetric and symmetric three-dimensional reconstructions of phage CUS-3 are presented. • CUS-3 major capsid protein has a conserved I-domain, which is found in all three categories of “P22-like phage”. • CUS-3 has very different tailspike receptor binding domain from those of P22 and Sf6. • The CUS-3 tailspike likely was acquired by horizontal gene transfer.« less

Recombinant Influenza Virus Carrying the Conserved Domain of Respiratory Syncytial Virus (RSV) G Protein Confers Protection against RSV without inflammatory disease

PubMed Central

Lee, Yu-Na; Hwang, Hye Suk; Kim, Min-Chul; Lee, Young-Tae; Cho, Min-Kyoung; Kwon, Young-Man; Lee, Jong Seok; Plemper, Richard K.; Kang, Sang-Moo

2014-01-01

Respiratory syncytial virus (RSV) is one of the most important causes for viral lower respiratory tract disease in humans. There is no licensed RSV vaccine. Here, we generated recombinant influenza viruses (PR8/RSV.HA-G) carrying the chimeric constructs of hemagglutinin (HA) and central conserved-domains of the RSV G protein. PR8/RSV.HA-G virus showed lower pathogenicity without compromising immunogenicity in mice. Single intranasal inoculation of mice with PR8/RSV.HA-G induced IgG2a isotype dominant antibodies and RSV neutralizing activity. Mice with single intranasal inoculation of PR8/RSV.HA-G were protected against RSV infection as evidenced by significant reduction of lung viral loads to a detection limit upon RSV challenge. PR8/RSV.HA-G inoculation of mice did not induce pulmonary eosinophilia and inflammation upon RSV infection. These findings support a concept that recombinant influenza viruses carrying the RSV G conserved-domain can be developed as a promising RSV vaccine candidate without pulmonary disease. PMID:25553517

Evaluating the Effects of Restraint Systems on 4WD Testing Methodologies: A Collaborative Effort between the NVFEL and ANL

EPA Science Inventory

Testing vehicles for emissions and fuel economy has traditionally been conducted with a single-axle chassis dynamometer. The 2006 SAE All Wheel Drive Symposium cited four wheel drive (4WD) and all wheel drive (AWD) sales as climbing from 20% toward 30% of a motor vehicle mar...

The box C/D sRNP dimeric architecture is conserved across domain Archaea

PubMed Central

Bower-Phipps, Kathleen R.; Taylor, David W.; Wang, Hong-Wei; Baserga, Susan J.

2012-01-01

Box C/D small (nucleolar) ribonucleoproteins [s(no)RNPs] catalyze RNA-guided 2′-O-ribose methylation in two of the three domains of life. Recent structural studies have led to a controversy over whether box C/D sRNPs functionally assemble as monomeric or dimeric macromolecules. The archaeal box C/D sRNP from Methanococcus jannaschii (Mj) has been shown by glycerol gradient sedimentation, gel filtration chromatography, native gel analysis, and single-particle electron microscopy (EM) to adopt a di-sRNP architecture, containing four copies of each box C/D core protein and two copies of the Mj sR8 sRNA. Subsequently, investigators used a two-stranded artificial guide sRNA, CD45, to assemble a box C/D sRNP from Sulfolobus solfataricus with a short RNA methylation substrate, yielding a crystal structure of a mono-sRNP. To more closely examine box C/D sRNP architecture, we investigate the role of the omnipresent sRNA loop as a structural determinant of sRNP assembly. We show through sRNA mutagenesis, native gel electrophoresis, and single-particle EM that a di-sRNP is the near exclusive architecture obtained when reconstituting box C/D sRNPs with natural or artificial sRNAs containing an internal loop. Our results span three distantly related archaeal species—Sulfolobus solfataricus, Pyrococcus abyssi, and Archaeoglobus fulgidus—indicating that the di-sRNP architecture is broadly conserved across the entire archaeal domain. PMID:22753779

The box C/D sRNP dimeric architecture is conserved across domain Archaea.

PubMed

Bower-Phipps, Kathleen R; Taylor, David W; Wang, Hong-Wei; Baserga, Susan J

2012-08-01

Box C/D small (nucleolar) ribonucleoproteins [s(no)RNPs] catalyze RNA-guided 2'-O-ribose methylation in two of the three domains of life. Recent structural studies have led to a controversy over whether box C/D sRNPs functionally assemble as monomeric or dimeric macromolecules. The archaeal box C/D sRNP from Methanococcus jannaschii (Mj) has been shown by glycerol gradient sedimentation, gel filtration chromatography, native gel analysis, and single-particle electron microscopy (EM) to adopt a di-sRNP architecture, containing four copies of each box C/D core protein and two copies of the Mj sR8 sRNA. Subsequently, investigators used a two-stranded artificial guide sRNA, CD45, to assemble a box C/D sRNP from Sulfolobus solfataricus with a short RNA methylation substrate, yielding a crystal structure of a mono-sRNP. To more closely examine box C/D sRNP architecture, we investigate the role of the omnipresent sRNA loop as a structural determinant of sRNP assembly. We show through sRNA mutagenesis, native gel electrophoresis, and single-particle EM that a di-sRNP is the near exclusive architecture obtained when reconstituting box C/D sRNPs with natural or artificial sRNAs containing an internal loop. Our results span three distantly related archaeal species--Sulfolobus solfataricus, Pyrococcus abyssi, and Archaeoglobus fulgidus--indicating that the di-sRNP architecture is broadly conserved across the entire archaeal domain.

Feature-based classification of amino acid substitutions outside conserved functional protein domains.

PubMed

Gemovic, Branislava; Perovic, Vladimir; Glisic, Sanja; Veljkovic, Nevena

2013-01-01

There are more than 500 amino acid substitutions in each human genome, and bioinformatics tools irreplaceably contribute to determination of their functional effects. We have developed feature-based algorithm for the detection of mutations outside conserved functional domains (CFDs) and compared its classification efficacy with the most commonly used phylogeny-based tools, PolyPhen-2 and SIFT. The new algorithm is based on the informational spectrum method (ISM), a feature-based technique, and statistical analysis. Our dataset contained neutral polymorphisms and mutations associated with myeloid malignancies from epigenetic regulators ASXL1, DNMT3A, EZH2, and TET2. PolyPhen-2 and SIFT had significantly lower accuracies in predicting the effects of amino acid substitutions outside CFDs than expected, with especially low sensitivity. On the other hand, only ISM algorithm showed statistically significant classification of these sequences. It outperformed PolyPhen-2 and SIFT by 15% and 13%, respectively. These results suggest that feature-based methods, like ISM, are more suitable for the classification of amino acid substitutions outside CFDs than phylogeny-based tools.

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

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

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

Liao Maofu; Kielian, Margaret

2005-02-05

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

Cooperative control between AtRGS1 and AtHXK1 in a WD40-repeat protein pathway in Arabidopsis thaliana

DOE PAGES

Huang, Jian -Ping; Tunc-Ozdemir, Meral; Chang, Ying; ...

2015-10-13

HEXOKINASE 1 (AtHXK1) and Regulator of G-protein Signaling 1 (AtRGS1) pathways, mediate D-glucose signaling in Arabidopsis. However, it is not known the degree, if any, that these pathways overlap and how. We show modest signaling crosstalk between these pathways, albeit complex with both epistatic interactions and additive effects that may be indirect. The action of HXK1 on AtRGS1 signaling lies downstream of the primary step in G protein-mediated sugar signaling in which the WD-repeat protein, AGB1, is the propelling signaling element. RHIP1, a previously unknown protein predicted here to have a 3-stranded helical structure, interacts with both AtRGS1 and AtHXK1more » in planta and is required for some glucose-regulated gene expression, providing a physical connection between these two proteins in sugar signaling. The rhip1 null mutant displays similar seedling growth phenotypes as rgs1-2 in response to glucose, further suggesting a role for RHIP1 in glucose signaling. Lastly, glucose signaling is a complex hierarchical relationship which is specific to the target gene and sugar phenotype and suggests that there are two glycolysis-independent glucose signaling sensors: AtRGS1 and AtHXK1 that weakly communicate with each other via feed-back and feed-forward loops to fine tune the response to glucose.« less

The Runt domain of AML1 (RUNX1) binds a sequence-conserved RNA motif that mimics a DNA element.

PubMed

Fukunaga, Junichi; Nomura, Yusuke; Tanaka, Yoichiro; Amano, Ryo; Tanaka, Taku; Nakamura, Yoshikazu; Kawai, Gota; Sakamoto, Taiichi; Kozu, Tomoko

2013-07-01

AML1 (RUNX1) is a key transcription factor for hematopoiesis that binds to the Runt-binding double-stranded DNA element (RDE) of target genes through its N-terminal Runt domain. Aberrations in the AML1 gene are frequently found in human leukemia. To better understand AML1 and its potential utility for diagnosis and therapy, we obtained RNA aptamers that bind specifically to the AML1 Runt domain. Enzymatic probing and NMR analyses revealed that Apt1-S, which is a truncated variant of one of the aptamers, has a CACG tetraloop and two stem regions separated by an internal loop. All the isolated aptamers were found to contain the conserved sequence motif 5'-NNCCAC-3' and 5'-GCGMGN'N'-3' (M:A or C; N and N' form Watson-Crick base pairs). The motif contains one AC mismatch and one base bulged out. Mutational analysis of Apt1-S showed that three guanines of the motif are important for Runt binding as are the three guanines of RDE, which are directly recognized by three arginine residues of the Runt domain. Mutational analyses of the Runt domain revealed that the amino acid residues used for Apt1-S binding were similar to those used for RDE binding. Furthermore, the aptamer competed with RDE for binding to the Runt domain in vitro. These results demonstrated that the Runt domain of the AML1 protein binds to the motif of the aptamer that mimics DNA. Our findings should provide new insights into RNA function and utility in both basic and applied sciences.

Near-infrared Detection of WD 0806-661 B with the Hubble Space Telescope

NASA Astrophysics Data System (ADS)

Luhman, K. L.; Morley, C. V.; Burgasser, A. J.; Esplin, T. L.; Bochanski, J. J.

2014-10-01

WD 0806-661 B is one of the coldest known brown dwarfs (T eff = 300-345 K) based on previous mid-infrared photometry from the Spitzer Space Telescope. In addition, it is a benchmark for testing theoretical models of brown dwarfs because its age and distance are well constrained via its primary star (2 ± 0.5 Gyr, 19.2 ± 0.6 pc). We present the first near-infrared detection of this object, which has been achieved through F110W imaging (~Y + J) with the Wide Field Camera 3 on board the Hubble Space Telescope. We measure a Vega magnitude of m 110 = 25.70 ± 0.08, which implies J ~ 25.0. When combined with the Spitzer photometry, our estimate of J helps to better define the empirical sequence of the coldest brown dwarfs in M 4.5 versus J - [4.5]. The positions of WD 0806-661 B and other Y dwarfs in that diagram are best matched by the cloudy models of Burrows et al. and the cloudless models of Saumon et al., both of which employ chemical equilibrium. The calculations by Morley et al. for 50% cloud coverage differ only modestly from the data. Spectroscopy would enable a more stringent test of the models, but based on our F110W measurement, such observations are currently possible only with Hubble, and would require at least ~10 orbits to reach a signal-to-noise ratio of ~5. Based on observations made with the NASA/ESA Hubble Space Telescope through program 12815, obtained at the Space Telescope Science Institute, which is operated by the Association of Universities for Research in Astronomy, Inc., under NASA contract NAS 5-26555, and observations with the ESO Telescopes at Paranal Observatory under programs ID 089.C-0428 and ID 089.C-0597.

Presence of tannins in sorghum grains is conditioned by different natural alleles of Tannin1

PubMed Central

Wu, Yuye; Li, Xianran; Xiang, Wenwen; Zhu, Chengsong; Lin, Zhongwei; Wu, Yun; Li, Jiarui; Pandravada, Satchidanand; Ridder, Dustan D.; Bai, Guihua; Wang, Ming L.; Trick, Harold N.; Bean, Scott R.; Tuinstra, Mitchell R.; Tesso, Tesfaye T.; Yu, Jianming

2012-01-01

Sorghum, an ancient old-world cereal grass, is the dietary staple of over 500 million people in more than 30 countries in the tropics and semitropics. Its C4 photosynthesis, drought resistance, wide adaptation, and high nutritional value hold the promise to alleviate hunger in Africa. Not present in other major cereals, such as rice, wheat, and maize, condensed tannins (proanthocyanidins) in the pigmented testa of some sorghum cultivars have been implicated in reducing protein digestibility but recently have been shown to promote human health because of their high antioxidant capacity and ability to fight obesity through reduced digestion. Combining quantitative trait locus mapping, meta-quantitative trait locus fine-mapping, and association mapping, we showed that the nucleotide polymorphisms in the Tan1 gene, coding a WD40 protein, control the tannin biosynthesis in sorghum. A 1-bp G deletion in the coding region, causing a frame shift and a premature stop codon, led to a nonfunctional allele, tan1-a. Likewise, a different 10-bp insertion resulted in a second nonfunctional allele, tan1-b. Transforming the sorghum Tan1 ORF into a nontannin Arabidopsis mutant restored the tannin phenotype. In addition, reduction in nucleotide diversity from wild sorghum accessions to landraces and cultivars was found at the region that codes the highly conserved WD40 repeat domains and the C-terminal region of the protein. Genetic research in crops, coupled with nutritional and medical research, could open the possibility of producing different levels and combinations of phenolic compounds to promote human health. PMID:22699509

Investigations by the U.S. Geological Survey of soil and moisture conservation on public domain lands, 1941-1964

USGS Publications Warehouse

Peterson, H.V.; Melin, K.R.

1979-01-01

The passage of the Taylor Grazing Act in 1934 marked the end of an era in the land policies in the United States in that disposal of the public lands by homesteading was terminated except under rigidly prescribed procedures, and the remaining public lands covering about 175 million acres in the western conterminous states were brought under regulatory authority for grazing use. In 1934 the lands were mostly in a severe state of deterioration as a result of overgrazing and drought. In addition to reducing numbers of livestock using the lands, successive programs of conservation practices were established of which the Soil and Moisture Conservation Program of the Department of the Interior is of particular interest here. The services of the Geological Survey, in an investigational and advisory capacity were enlisted in this program. The work of the Geological Survey has consisted of the collection of hydrologic data, investigations of range-water supplies to facilitate management and provide information for design of structures and land-treatment measures. Appraisal of the effects of treatment practices has also been an important activity. Conservation on the public domain involves mainly growing vegetation for forage and reducing erosion. The two elements are intimately related--accomplishment in one is usually reflected by an improvement in the other. Erosion is a serious problem on most of the public domain, but particularly in the Colorado River and Rio Grande basins where, despite low annual water yields, the public domain and similar lands on the Indian reservations contribute the major part of the sediment measured at the downstream gaging stations. In parts of the Missouri River basin also, erosion is obviously very active but the sediment yield contributed by the public domain cannot be as readily isolated. The reasons for the erosion are generally evident--the erodibility of the rock and soils and the sparsity of vegetation as a result of low precipitation

Evolutionary dynamics of protein domain architecture in plants

PubMed Central

2012-01-01

Background Protein domains are the structural, functional and evolutionary units of the protein. Protein domain architectures are the linear arrangements of domain(s) in individual proteins. Although the evolutionary history of protein domain architecture has been extensively studied in microorganisms, the evolutionary dynamics of domain architecture in the plant kingdom remains largely undefined. To address this question, we analyzed the lineage-based protein domain architecture content in 14 completed green plant genomes. Results Our analyses show that all 14 plant genomes maintain similar distributions of species-specific, single-domain, and multi-domain architectures. Approximately 65% of plant domain architectures are universally present in all plant lineages, while the remaining architectures are lineage-specific. Clear examples are seen of both the loss and gain of specific protein architectures in higher plants. There has been a dynamic, lineage-wise expansion of domain architectures during plant evolution. The data suggest that this expansion can be largely explained by changes in nuclear ploidy resulting from rounds of whole genome duplications. Indeed, there has been a decrease in the number of unique domain architectures when the genomes were normalized into a presumed ancestral genome that has not undergone whole genome duplications. Conclusions Our data show the conservation of universal domain architectures in all available plant genomes, indicating the presence of an evolutionarily conserved, core set of protein components. However, the occurrence of lineage-specific domain architectures indicates that domain architecture diversity has been maintained beyond these core components in plant genomes. Although several features of genome-wide domain architecture content are conserved in plants, the data clearly demonstrate lineage-wise, progressive changes and expansions of individual protein domain architectures, reinforcing the notion that plant

Phosphoinositide-binding proteins in autophagy.

PubMed

Lystad, Alf Håkon; Simonsen, Anne

2016-08-01

Phosphoinositides represent a very small fraction of membrane phospholipids, having fast turnover rates and unique subcellular distributions, which make them perfect for initiating local temporal effects. Seven different phosphoinositide species are generated through reversible phosphorylation of the inositol ring of phosphatidylinositol (PtdIns). The negative charge generated by the phosphates provides specificity for interaction with various protein domains that commonly contain a cluster of basic residues. Examples of domains that bind phosphoinositides include PH domains, WD40 repeats, PX domains, and FYVE domains. Such domains often display specificity toward a certain species or subset of phosphoinositides. Here we will review the current literature of different phosphoinositide-binding proteins involved in autophagy. © 2016 Federation of European Biochemical Societies.

Protein domain organisation: adding order.

PubMed

Kummerfeld, Sarah K; Teichmann, Sarah A

2009-01-29

Domains are the building blocks of proteins. During evolution, they have been duplicated, fused and recombined, to produce proteins with novel structures and functions. Structural and genome-scale studies have shown that pairs or groups of domains observed together in a protein are almost always found in only one N to C terminal order and are the result of a single recombination event that has been propagated by duplication of the multi-domain unit. Previous studies of domain organisation have used graph theory to represent the co-occurrence of domains within proteins. We build on this approach by adding directionality to the graphs and connecting nodes based on their relative order in the protein. Most of the time, the linear order of domains is conserved. However, using the directed graph representation we have identified non-linear features of domain organization that are over-represented in genomes. Recognising these patterns and unravelling how they have arisen may allow us to understand the functional relationships between domains and understand how the protein repertoire has evolved. We identify groups of domains that are not linearly conserved, but instead have been shuffled during evolution so that they occur in multiple different orders. We consider 192 genomes across all three kingdoms of life and use domain and protein annotation to understand their functional significance. To identify these features and assess their statistical significance, we represent the linear order of domains in proteins as a directed graph and apply graph theoretical methods. We describe two higher-order patterns of domain organisation: clusters and bi-directionally associated domain pairs and explore their functional importance and phylogenetic conservation. Taking into account the order of domains, we have derived a novel picture of global protein organization. We found that all genomes have a higher than expected degree of clustering and more domain pairs in forward and

Top surface blade residues and the central channel water molecules are conserved in every repeat of the integrin-like β-propeller structures.

PubMed

Denesyuk, Alexander; Denessiouk, Konstantin; Johnson, Mark S

2018-02-01

An integrin-like β-propeller domain contains seven repeats of a four-stranded antiparallel β-sheet motif (blades). Previously we described a 3D structural motif within each blade of the integrin-type β-propeller. Here, we show unique structural links that join different blades of the β-propeller structure, which together with the structural motif for a single blade are repeated in a β-propeller to provide the functional top face of the barrel, found to be involved in protein-protein interactions and substrate recognition. We compare functional top face diagrams of the integrin-type β-propeller domain and two non-integrin type β-propeller domains of virginiamycin B lyase and WD Repeat-Containing Protein 5. Copyright © 2017 Elsevier Inc. All rights reserved.

CDD/SPARCLE: functional classification of proteins via subfamily domain architectures.

PubMed

Marchler-Bauer, Aron; Bo, Yu; Han, Lianyi; He, Jane; Lanczycki, Christopher J; Lu, Shennan; Chitsaz, Farideh; Derbyshire, Myra K; Geer, Renata C; Gonzales, Noreen R; Gwadz, Marc; Hurwitz, David I; Lu, Fu; Marchler, Gabriele H; Song, James S; Thanki, Narmada; Wang, Zhouxi; Yamashita, Roxanne A; Zhang, Dachuan; Zheng, Chanjuan; Geer, Lewis Y; Bryant, Stephen H

2017-01-04

NCBI's Conserved Domain Database (CDD) aims at annotating biomolecular sequences with the location of evolutionarily conserved protein domain footprints, and functional sites inferred from such footprints. An archive of pre-computed domain annotation is maintained for proteins tracked by NCBI's Entrez database, and live search services are offered as well. CDD curation staff supplements a comprehensive collection of protein domain and protein family models, which have been imported from external providers, with representations of selected domain families that are curated in-house and organized into hierarchical classifications of functionally distinct families and sub-families. CDD also supports comparative analyses of protein families via conserved domain architectures, and a recent curation effort focuses on providing functional characterizations of distinct subfamily architectures using SPARCLE: Subfamily Protein Architecture Labeling Engine. CDD can be accessed at https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml. Published by Oxford University Press on behalf of Nucleic Acids Research 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Conservation and diversification of Msx protein in metazoan evolution.

PubMed

Takahashi, Hirokazu; Kamiya, Akiko; Ishiguro, Akira; Suzuki, Atsushi C; Saitou, Naruya; Toyoda, Atsushi; Aruga, Jun

2008-01-01

Msx (/msh) family genes encode homeodomain (HD) proteins that control ontogeny in many animal species. We compared the structures of Msx genes from a wide range of Metazoa (Porifera, Cnidaria, Nematoda, Arthropoda, Tardigrada, Platyhelminthes, Mollusca, Brachiopoda, Annelida, Echiura, Echinodermata, Hemichordata, and Chordata) to gain an understanding of the role of these genes in phylogeny. Exon-intron boundary analysis suggested that the position of the intron located N-terminally to the HDs was widely conserved in all the genes examined, including those of cnidarians. Amino acid (aa) sequence comparison revealed 3 new evolutionarily conserved domains, as well as very strong conservation of the HDs. Two of the three domains were associated with Groucho-like protein binding in both a vertebrate and a cnidarian Msx homolog, suggesting that the interaction between Groucho-like proteins and Msx proteins was established in eumetazoan ancestors. Pairwise comparison among the collected HDs and their C-flanking aa sequences revealed that the degree of sequence conservation varied depending on the animal taxa from which the sequences were derived. Highly conserved Msx genes were identified in the Vertebrata, Cephalochordata, Hemichordata, Echinodermata, Mollusca, Brachiopoda, and Anthozoa. The wide distribution of the conserved sequences in the animal phylogenetic tree suggested that metazoan ancestors had already acquired a set of conserved domains of the current Msx family genes. Interestingly, although strongly conserved sequences were recovered from the Vertebrata, Cephalochordata, and Anthozoa, the sequences from the Urochordata and Hydrozoa showed weak conservation. Because the Vertebrata-Cephalochordata-Urochordata and Anthozoa-Hydrozoa represent sister groups in the Chordata and Cnidaria, respectively, Msx sequence diversification may have occurred differentially in the course of evolution. We speculate that selective loss of the conserved domains in Msx family

The Runt domain of AML1 (RUNX1) binds a sequence-conserved RNA motif that mimics a DNA element

PubMed Central

Fukunaga, Junichi; Nomura, Yusuke; Tanaka, Yoichiro; Amano, Ryo; Tanaka, Taku; Nakamura, Yoshikazu; Kawai, Gota; Sakamoto, Taiichi; Kozu, Tomoko

2013-01-01

AML1 (RUNX1) is a key transcription factor for hematopoiesis that binds to the Runt-binding double-stranded DNA element (RDE) of target genes through its N-terminal Runt domain. Aberrations in the AML1 gene are frequently found in human leukemia. To better understand AML1 and its potential utility for diagnosis and therapy, we obtained RNA aptamers that bind specifically to the AML1 Runt domain. Enzymatic probing and NMR analyses revealed that Apt1-S, which is a truncated variant of one of the aptamers, has a CACG tetraloop and two stem regions separated by an internal loop. All the isolated aptamers were found to contain the conserved sequence motif 5′-NNCCAC-3′ and 5′-GCGMGN′N′-3′ (M:A or C; N and N′ form Watson–Crick base pairs). The motif contains one AC mismatch and one base bulged out. Mutational analysis of Apt1-S showed that three guanines of the motif are important for Runt binding as are the three guanines of RDE, which are directly recognized by three arginine residues of the Runt domain. Mutational analyses of the Runt domain revealed that the amino acid residues used for Apt1-S binding were similar to those used for RDE binding. Furthermore, the aptamer competed with RDE for binding to the Runt domain in vitro. These results demonstrated that the Runt domain of the AML1 protein binds to the motif of the aptamer that mimics DNA. Our findings should provide new insights into RNA function and utility in both basic and applied sciences. PMID:23709277

Functional Information Stored in the Conserved Structural RNA Domains of Flavivirus Genomes

PubMed Central

Fernández-Sanlés, Alba; Ríos-Marco, Pablo; Romero-López, Cristina; Berzal-Herranz, Alfredo

2017-01-01

The genus Flavivirus comprises a large number of small, positive-sense single-stranded, RNA viruses able to replicate in the cytoplasm of certain arthropod and/or vertebrate host cells. The genus, which has some 70 member species, includes a number of emerging and re-emerging pathogens responsible for outbreaks of human disease around the world, such as the West Nile, dengue, Zika, yellow fever, Japanese encephalitis, St. Louis encephalitis, and tick-borne encephalitis viruses. Like other RNA viruses, flaviviruses have a compact RNA genome that efficiently stores all the information required for the completion of the infectious cycle. The efficiency of this storage system is attributable to supracoding elements, i.e., discrete, structural units with essential functions. This information storage system overlaps and complements the protein coding sequence and is highly conserved across the genus. It therefore offers interesting potential targets for novel therapeutic strategies. This review summarizes our knowledge of the features of flavivirus genome functional RNA domains. It also provides a brief overview of the main achievements reported in the design of antiviral nucleic acid-based drugs targeting functional genomic RNA elements. PMID:28421048

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.

A prefoldin-associated WD-repeat protein (WDR92) is required for the correct architectural assembly of motile cilia

PubMed Central

Patel-King, Ramila S.; King, Stephen M.

2016-01-01

WDR92 is a highly conserved WD-repeat protein that has been proposed to be involved in apoptosis and also to be part of a prefoldin-like cochaperone complex. We found that WDR92 has a phylogenetic signature that is generally compatible with it playing a role in the assembly or function of specifically motile cilia. To test this hypothesis, we performed an RNAi-based knockdown of WDR92 gene expression in the planarian Schmidtea mediterranea and were able to achieve a robust reduction in mRNA expression to levels undetectable under our standard RT-PCR conditions. We found that this treatment resulted in a dramatic reduction in the rate of organismal movement that was caused by a switch in the mode of locomotion from smooth, cilia-driven gliding to muscle-based, peristaltic contractions. Although the knockdown animals still assembled cilia of normal length and in similar numbers to controls, these structures had reduced beat frequency and did not maintain hydrodynamic coupling. By transmission electron microscopy we observed that many cilia had pleiomorphic defects in their architecture, including partial loss of dynein arms, incomplete closure of the B-tubule, and occlusion or replacement of the central pair complex by accumulated electron-dense material. These observations suggest that WDR92 is part of a previously unrecognized cytoplasmic chaperone system that is specifically required to fold key components necessary to build motile ciliary axonemes. PMID:26912790

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.

An adaptive actuator failure compensation scheme for two linked 2WD mobile robots

NASA Astrophysics Data System (ADS)

Ma, Yajie; Al-Dujaili, Ayad; Cocquempot, Vincent; El Badaoui El Najjar, Maan

2017-01-01

This paper develops a new adaptive compensation control scheme for two linked mobile robots with actuator failurs. A configuration with two linked two-wheel drive (2WD) mobile robots is proposed, and the modelling of its kinematics and dynamics are given. An adaptive failure compensation scheme is developed to compensate actuator failures, consisting of a kinematic controller and a multi-design integration based dynamic controller. The kinematic controller is a virtual one, and based on which, multiple adaptive dynamic control signals are designed which covers all possible failure cases. By combing these dynamic control signals, the dynamic controller is designed, which ensures system stability and asymptotic tracking properties. Simulation results verify the effectiveness of the proposed adaptive failure compensation scheme.

ALIX Rescues Budding of a Double PTAP/PPEY L-Domain Deletion Mutant of Ebola VP40: A Role for ALIX in Ebola Virus Egress.

PubMed

Han, Ziying; Madara, Jonathan J; Liu, Yuliang; Liu, Wenbo; Ruthel, Gordon; Freedman, Bruce D; Harty, Ronald N

2015-10-01

Ebola (EBOV) is an enveloped, negative-sense RNA virus belonging to the family Filoviridae that causes hemorrhagic fever syndromes with high-mortality rates. To date, there are no licensed vaccines or therapeutics to control EBOV infection and prevent transmission. Consequently, the need to better understand the mechanisms that regulate virus transmission is critical to developing countermeasures. The EBOV VP40 matrix protein plays a central role in late stages of virion assembly and egress, and independent expression of VP40 leads to the production of virus-like particles (VLPs) by a mechanism that accurately mimics budding of live virus. VP40 late (L) budding domains mediate efficient virus-cell separation by recruiting host ESCRT and ESCRT-associated proteins to complete the membrane fission process. L-domains consist of core consensus amino acid motifs including PPxY, P(T/S)AP, and YPx(n)L/I, and EBOV VP40 contains overlapping PPxY and PTAP motifs whose interactions with Nedd4 and Tsg101, respectively, have been characterized extensively. Here, we present data demonstrating for the first time that EBOV VP40 possesses a third L-domain YPx(n)L/I consensus motif that interacts with the ESCRT-III protein Alix. We show that the YPx(n)L/I motif mapping to amino acids 18-26 of EBOV VP40 interacts with the Alix Bro1-V fragment, and that siRNA knockdown of endogenous Alix expression inhibits EBOV VP40 VLP egress. Furthermore, overexpression of Alix Bro1-V rescues VLP production of the budding deficient EBOV VP40 double PTAP/PPEY L-domain deletion mutant to wild-type levels. Together, these findings demonstrate that EBOV VP40 recruits host Alix via a YPx(n)L/I motif that can function as an alternative L-domain to promote virus egress. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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

Crystal Structure of Marburg Virus VP40 Reveals a Broad, Basic Patch for Matrix Assembly and a Requirement of the N-Terminal Domain for Immunosuppression.

PubMed

Oda, Shun-Ichiro; Noda, Takeshi; Wijesinghe, Kaveesha J; Halfmann, Peter; Bornholdt, Zachary A; Abelson, Dafna M; Armbrust, Tammy; Stahelin, Robert V; Kawaoka, Yoshihiro; Saphire, Erica Ollmann

2016-02-15

Marburg virus (MARV), a member of the filovirus family, causes severe hemorrhagic fever with up to 90% lethality. MARV matrix protein VP40 is essential for assembly and release of newly copied viruses and also suppresses immune signaling in the infected cell. Here we report the crystal structure of MARV VP40. We found that MARV VP40 forms a dimer in solution, mediated by N-terminal domains, and that formation of this dimer is essential for budding of virus-like particles. We also found the N-terminal domain to be necessary and sufficient for immune antagonism. The C-terminal domains of MARV VP40 are dispensable for immunosuppression but are required for virus assembly. The C-terminal domains are only 16% identical to those of Ebola virus, differ in structure from those of Ebola virus, and form a distinct broad and flat cationic surface that likely interacts with the cell membrane during virus assembly. Marburg virus, a cousin of Ebola virus, causes severe hemorrhagic fever, with up to 90% lethality seen in recent outbreaks. Molecular structures and visual images of the proteins of Marburg virus are essential for the development of antiviral drugs. One key protein in the Marburg virus life cycle is VP40, which both assembles the virus and suppresses the immune system. Here we provide the molecular structure of Marburg virus VP40, illustrate differences from VP40 of Ebola virus, and reveal surfaces by which Marburg VP40 assembles progeny and suppresses immune function. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Conserved Tryptophan Motifs in the Large Tegument Protein pUL36 Are Required for Efficient Secondary Envelopment of Herpes Simplex Virus Capsids

PubMed Central

Ivanova, Lyudmila; Buch, Anna; Döhner, Katinka; Pohlmann, Anja; Binz, Anne; Prank, Ute; Sandbaumhüter, Malte

2016-01-01

ABSTRACT Herpes simplex virus (HSV) replicates in the skin and mucous membranes, and initiates lytic or latent infections in sensory neurons. Assembly of progeny virions depends on the essential large tegument protein pUL36 of 3,164 amino acid residues that links the capsids to the tegument proteins pUL37 and VP16. Of the 32 tryptophans of HSV-1-pUL36, the tryptophan-acidic motifs 1766WD1767 and 1862WE1863 are conserved in all HSV-1 and HSV-2 isolates. Here, we characterized the role of these motifs in the HSV life cycle since the rare tryptophans often have unique roles in protein function due to their large hydrophobic surface. The infectivity of the mutants HSV-1(17+)Lox-pUL36-WD/AA-WE/AA and HSV-1(17+)Lox-CheVP26-pUL36-WD/AA-WE/AA, in which the capsid has been tagged with the fluorescent protein Cherry, was significantly reduced. Quantitative electron microscopy shows that there were a larger number of cytosolic capsids and fewer enveloped virions compared to their respective parental strains, indicating a severe impairment in secondary capsid envelopment. The capsids of the mutant viruses accumulated in the perinuclear region around the microtubule-organizing center and were not dispersed to the cell periphery but still acquired the inner tegument proteins pUL36 and pUL37. Furthermore, cytoplasmic capsids colocalized with tegument protein VP16 and, to some extent, with tegument protein VP22 but not with the envelope glycoprotein gD. These results indicate that the unique conserved tryptophan-acidic motifs in the central region of pUL36 are required for efficient targeting of progeny capsids to the membranes of secondary capsid envelopment and for efficient virion assembly. IMPORTANCE Herpesvirus infections give rise to severe animal and human diseases, especially in young, immunocompromised, and elderly individuals. The structural hallmark of herpesvirus virions is the tegument, which contains evolutionarily conserved proteins that are essential for several

Pub1p C-Terminal RRM Domain Interacts with Tif4631p through a Conserved Region Neighbouring the Pab1p Binding Site

PubMed Central

Rico-Lastres, Palma; Pérez-Cañadillas, José Manuel

2011-01-01

Pub1p, a highly abundant poly(A)+ mRNA binding protein in Saccharomyces cerevisiae, influences the stability and translational control of many cellular transcripts, particularly under some types of environmental stresses. We have studied the structure, RNA and protein recognition modes of different Pub1p constructs by NMR spectroscopy. The structure of the C-terminal RRM domain (RRM3) shows a non-canonical N-terminal helix that packs against the canonical RRM fold in an original fashion. This structural trait is conserved in Pub1p metazoan homologues, the TIA-1 family, defining a new class of RRM-type domains that we propose to name TRRM (TIA-1 C-terminal domain-like RRM). Pub1p TRRM and the N-terminal RRM1-RRM2 tandem bind RNA with high selectivity for U-rich sequences, with TRRM showing additional preference for UA-rich ones. RNA-mediated chemical shift changes map to β-sheet and protein loops in the three RRMs. Additionally, NMR titration and biochemical in vitro cross-linking experiments determined that Pub1p TRRM interacts specifically with the N-terminal region (1–402) of yeast eIF4G1 (Tif4631p), very likely through the conserved Box1, a short sequence motif neighbouring the Pab1p binding site in Tif4631p. The interaction involves conserved residues of Pub1p TRRM, which define a protein interface that mirrors the Pab1p-Tif4631p binding mode. Neither protein nor RNA recognition involves the novel N-terminal helix, whose functional role remains unclear. By integrating these new results with the current knowledge about Pub1p, we proposed different mechanisms of Pub1p recruitment to the mRNPs and Pub1p-mediated mRNA stabilization in which the Pub1p/Tif4631p interaction would play an important role. PMID:21931728

Prediction of conserved sites and domains in glycoproteins B, C and D of herpes viruses.

PubMed

Rasheed, Muhammad Asif; Ansari, Abdur Rahman; Ihsan, Awais; Navid, Muhammad Tariq; Ur-Rehman, Shahid; Raza, Sohail

2018-03-01

Glycoprotein B (gB), C (gC) and D (gD) of herpes simplex virus are implicated in virus adsorption and penetration. The gB, gC and gD are glycoproteins for different processes of virus binding and attachment to the host cells. Moreover, their expression is necessary and sufficient to induce cell fusion in the absence of other glycoproteins. Egress of herpes simplex virus (HSV) and other herpes viruses from cells involves extensive modification of cellular membranes and sequential envelopment, de-envelopment and re-envelopment steps. Viral glycoproteins are important in these processes, and frequently two or more glycoproteins can largely suffice in any step. Hence, we target the 3 important glycoproteins (B, C and D) of eight different herpes viruses of different species. These species include human (HSV1 and 2), bovine (BHV1), equine (EHV1 and 4), chicken (ILT1 and MDV2) and pig (PRV1). By applying different bioinformatics tools, we highlighted the conserved sites in these glycoproteins which might be most significant regarding attachment and infection of the viruses. Moreover the conserved domains in these glycoproteins are also highlighted. From this study, we will able to analyze the role of different viral glycoproteins of different species during herpes virus adsorption and penetration. Moreover, this study will help to construct the antivirals that target the glycoproteins of different herpes viruses. Copyright © 2018 Elsevier Ltd. All rights reserved.

Antiviral drug acyclovir exhibits antitumor activity via targeting βTrCP1: Molecular docking and dynamics simulation study.

PubMed

Shafique, Shagufta; Rashid, Sajid

2017-03-01

The critical role of βTrCP1 in cancer development makes it a discerning target for the development of small drug like molecules. Currently, no inhibitor exists that is able to target its substrate binding site. Through molecular docking and dynamics simulation assays, we explored the comparative binding pattern of βTrCP1-WD40 domain with ACV and its phospho-derivatives (ACVMP, ACVDP and ACVTP). Consequently, through principal component analysis, βTrCP1-ACVTP was found to be more stable complex by obscuring a reduced conformational space than other systems. Thus based on the residual contribution and hydrogen bonding pattern, ACVTP was considered as a noteworthy inhibitor which demarcated binding in the cleft formed by βTrCP1-WD40 specific β-propeller. The outcomes of this study may provide a platform for rational design of specific and potent inhibitor against βTrCP1, with special emphasis on anticancer activity. Copyright © 2016 Elsevier Inc. All rights reserved.

Molecular dynamics simulations of site point mutations in the TPR domain of cyclophilin 40 identify conformational states with distinct dynamic and enzymatic properties

NASA Astrophysics Data System (ADS)

Gur, Mert; Blackburn, Elizabeth A.; Ning, Jia; Narayan, Vikram; Ball, Kathryn L.; Walkinshaw, Malcolm D.; Erman, Burak

2018-04-01

Cyclophilin 40 (Cyp40) is a member of the immunophilin family that acts as a peptidyl-prolyl-isomerase enzyme and binds to the heat shock protein 90 (Hsp90). Its structure comprises an N-terminal cyclophilin domain and a C-terminal tetratricopeptide (TPR) domain. Cyp40 is overexpressed in prostate cancer and certain T-cell lymphomas. The groove for Hsp90 binding on the TPR domain includes residues Lys227 and Lys308, referred to as the carboxylate clamp, and is essential for Cyp40-Hsp90 binding. In this study, the effect of two mutations, K227A and K308A, and their combinative mutant was investigated by performing a total of 5.76 μs of all-atom molecular dynamics (MD) simulations in explicit solvent. All simulations, except the K308A mutant, were found to adopt two distinct (extended or compact) conformers defined by different cyclophilin-TPR interdomain distances. The K308A mutant was only observed in the extended form which is observed in the Cyp40 X-ray structure. The wild-type, K227A, and combined mutant also showed bimodal distributions. The experimental melting temperature, Tm, values of the mutants correlate with the degree of compactness with the K308A extended mutant having a marginally lower melting temperature. Another novel measure of compactness determined from the MD data, the "coordination shell volume," also shows a direct correlation with Tm. In addition, the MD simulations show an allosteric effect with the mutations in the remote TPR domain having a pronounced effect on the molecular motions of the enzymatic cyclophilin domain which helps rationalise the experimentally observed increase in enzyme activity measured for all three mutations.

Comprehensively Surveying Structure and Function of RING Domains from Drosophila melanogaster

PubMed Central

Wu, Yuehao; Wan, Fusheng; Huang, Chunhong; Jie, Kemin

2011-01-01

Using a complete set of RING domains from Drosophila melanogaster, all the solved RING domains and cocrystal structures of RING-containing ubiquitin-ligases (RING-E3) and ubiquitin-conjugating enzyme (E2) pairs, we analyzed RING domains structures from their primary to quarternary structures. The results showed that: i) putative orthologs of RING domains between Drosophila melanogaster and the human largely occur (118/139, 84.9%); ii) of the 118 orthologous pairs from Drosophila melanogaster and the human, 117 pairs (117/118, 99.2%) were found to retain entirely uniform domain architectures, only Iap2/Diap2 experienced evolutionary expansion of domain architecture; iii) 4 evolutionary structurally conserved regions (SCRs) are responsible for homologous folding of RING domains at the superfamily level; iv) besides the conserved Cys/His chelating zinc ions, 6 equivalent residues (4 hydrophobic and 2 polar residues) in the SCRs possess good-consensus and conservation- these 4 SCRs function in the structural positioning of 6 equivalent residues as determinants for RING-E3 catalysis; v) members of these RING proteins located nucleus, multiple subcellular compartments, membrane protein and mitochondrion are respectively 42 (42/139, 30.2%), 71 (71/139, 51.1%), 22 (22/139, 15.8%) and 4 (4/139, 2.9%); vi) CG15104 (Topors) and CG1134 (Mul1) in C3HC4, and CG3929 (Deltex) in C3H2C3 seem to display broader E2s binding profiles than other RING-E3s; vii) analyzing intermolecular interfaces of E2/RING-E3 complexes indicate that residues directly interacting with E2s are all from the SCRs in RING domains. Of the 6 residues, 2 hydrophobic ones contribute to constructing the conserved hydrophobic core, while the 2 hydrophobic and 2 polar residues directly participate in E2/RING-E3 interactions. Based on sequence and structural data, SCRs, conserved equivalent residues and features of intermolecular interfaces were extracted, highlighting the presence of a nucleus for RING domain fold

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

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

ELMO Domains, Evolutionary and Functional Characterization of a Novel GTPase-activating Protein (GAP) Domain for Arf Protein Family GTPases*

PubMed Central

East, Michael P.; Bowzard, J. Bradford; Dacks, Joel B.; Kahn, Richard A.

2012-01-01

The human family of ELMO domain-containing proteins (ELMODs) consists of six members and is defined by the presence of the ELMO domain. Within this family are two subclassifications of proteins, based on primary sequence conservation, protein size, and domain architecture, deemed ELMOD and ELMO. In this study, we used homology searching and phylogenetics to identify ELMOD family homologs in genomes from across eukaryotic diversity. This demonstrated not only that the protein family is ancient but also that ELMOs are potentially restricted to the supergroup Opisthokonta (Metazoa and Fungi), whereas proteins with the ELMOD organization are found in diverse eukaryotes and thus were likely the form present in the last eukaryotic common ancestor. The segregation of the ELMO clade from the larger ELMOD group is consistent with their contrasting functions as unconventional Rac1 guanine nucleotide exchange factors and the Arf family GTPase-activating proteins, respectively. We used unbiased, phylogenetic sorting and sequence alignments to identify the most highly conserved residues within the ELMO domain to identify a putative GAP domain within the ELMODs. Three independent but complementary assays were used to provide an initial characterization of this domain. We identified a highly conserved arginine residue critical for both the biochemical and cellular GAP activity of ELMODs. We also provide initial evidence of the function of human ELMOD1 as an Arf family GAP at the Golgi. These findings provide the basis for the future study of the ELMOD family of proteins and a new avenue for the study of Arf family GTPases. PMID:23014990

ST proteins, a new family of plant tandem repeat proteins with a DUF2775 domain mainly found in Fabaceae and Asteraceae.

PubMed

Albornos, Lucía; Martín, Ignacio; Iglesias, Rebeca; Jiménez, Teresa; Labrador, Emilia; Dopico, Berta

2012-11-07

Many proteins with tandem repeats in their sequence have been described and classified according to the length of the repeats: I) Repeats of short oligopeptides (from 2 to 20 amino acids), including structural cell wall proteins and arabinogalactan proteins. II) Repeats that range in length from 20 to 40 residues, including proteins with a well-established three-dimensional structure often involved in mediating protein-protein interactions. (III) Longer repeats in the order of 100 amino acids that constitute structurally and functionally independent units. Here we analyse ShooT specific (ST) proteins, a family of proteins with tandem repeats of unknown function that were first found in Leguminosae, and their possible similarities to other proteins with tandem repeats. ST protein sequences were only found in dicotyledonous plants, limited to several plant families, mainly the Fabaceae and the Asteraceae. ST mRNAs accumulate mainly in the roots and under biotic interactions. Most ST proteins have one or several Domain(s) of Unknown Function 2775 (DUF2775). All deduced ST proteins have a signal peptide, indicating that these proteins enter the secretory pathway, and the mature proteins have tandem repeat oligopeptides that share a hexapeptide (E/D)FEPRP followed by 4 partially conserved amino acids, which could determine a putative N-glycosylation signal, and a fully conserved tyrosine. In a phylogenetic tree, the sequences clade according to taxonomic group. A possible involvement in symbiosis and abiotic stress as well as in plant cell elongation is suggested, although different STs could play different roles in plant development. We describe a new family of proteins called ST whose presence is limited to the plant kingdom, specifically to a few families of dicotyledonous plants. They present 20 to 40 amino acid tandem repeat sequences with different characteristics (signal peptide, DUF2775 domain, conservative repeat regions) from the described group of 20 to 40

ST proteins, a new family of plant tandem repeat proteins with a DUF2775 domain mainly found in Fabaceae and Asteraceae

PubMed Central

2012-01-01

Background Many proteins with tandem repeats in their sequence have been described and classified according to the length of the repeats: I) Repeats of short oligopeptides (from 2 to 20 amino acids), including structural cell wall proteins and arabinogalactan proteins. II) Repeats that range in length from 20 to 40 residues, including proteins with a well-established three-dimensional structure often involved in mediating protein-protein interactions. (III) Longer repeats in the order of 100 amino acids that constitute structurally and functionally independent units. Here we analyse ShooT specific (ST) proteins, a family of proteins with tandem repeats of unknown function that were first found in Leguminosae, and their possible similarities to other proteins with tandem repeats. Results ST protein sequences were only found in dicotyledonous plants, limited to several plant families, mainly the Fabaceae and the Asteraceae. ST mRNAs accumulate mainly in the roots and under biotic interactions. Most ST proteins have one or several Domain(s) of Unknown Function 2775 (DUF2775). All deduced ST proteins have a signal peptide, indicating that these proteins enter the secretory pathway, and the mature proteins have tandem repeat oligopeptides that share a hexapeptide (E/D)FEPRP followed by 4 partially conserved amino acids, which could determine a putative N-glycosylation signal, and a fully conserved tyrosine. In a phylogenetic tree, the sequences clade according to taxonomic group. A possible involvement in symbiosis and abiotic stress as well as in plant cell elongation is suggested, although different STs could play different roles in plant development. Conclusions We describe a new family of proteins called ST whose presence is limited to the plant kingdom, specifically to a few families of dicotyledonous plants. They present 20 to 40 amino acid tandem repeat sequences with different characteristics (signal peptide, DUF2775 domain, conservative repeat regions) from the

A conserved serine residue regulates the stability of Drosophila Salvador and human WW domain-containing adaptor 45 through proteasomal degradation

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

Wu, Di, E-mail: DiWu@mail.nankai.edu.cn; Wu, Shian

2013-04-19

Highlights: •Ser-17 is key for the stability of Drosophila Sav. •Ala mutation of Ser-17 promotes the proteasomal degradation of Sav. •Ser-17 residue is not the main target of Hpo-induced Sav stabilization. •Hpo-dependent and -independent mechanisms regulate Sav stability. •This mechanism is conserved in the homologue of Sav, human WW45. -- Abstract: The Hippo (Hpo) pathway is a conserved tumor suppressor pathway that controls organ size through the coordinated regulation of apoptosis and proliferation. Drosophila Salvador (Sav), which limits organ size, is a core component of the Hpo pathway. In this study, Ser-17 was shown to be important for the stabilitymore » of Sav. Alanine mutation of Ser-17 promoted the proteasomal degradation of Sav. Destabilization and stabilization of the Sav protein mediated by alanine mutation of Ser-17 and by Hpo, respectively, were independent of each other. This implies that the stability of Sav is controlled by two mechanisms, one that is Ser-17-dependent and Hpo-independent, and another that is Ser-17-independent and Hpo-dependent. These dual mechanisms also regulated the human counterpart of Drosophila Sav, WW domain-containing adaptor 45 (WW45). The conservation of this regulation adds to its significance in normal physiology and tumorigenesis.« less

Phytoplasma-conserved phyllogen proteins induce phyllody across the Plantae by degrading floral MADS domain proteins.

PubMed

Kitazawa, Yugo; Iwabuchi, Nozomu; Himeno, Misako; Sasano, Momoka; Koinuma, Hiroaki; Nijo, Takamichi; Tomomitsu, Tatsuya; Yoshida, Tetsuya; Okano, Yukari; Yoshikawa, Nobuyuki; Maejima, Kensaku; Oshima, Kenro; Namba, Shigetou

2017-05-17

ABCE-class MADS domain transcription factors (MTFs) are key regulators of floral organ development in angiosperms. Aberrant expression of these genes can result in abnormal floral traits such as phyllody. Phyllogen is a virulence factor conserved in phytoplasmas, plant pathogenic bacteria of the class Mollicutes. It triggers phyllody in Arabidopsis thaliana by inducing degradation of A- and E-class MTFs. However, it is still unknown whether phyllogen can induce phyllody in plants other than A. thaliana, although phytoplasma-associated phyllody symptoms are observed in a broad range of angiosperms. In this study, phyllogen was shown to cause phyllody phenotypes in several eudicot species belonging to three different families. Moreover, phyllogen can interact with MTFs of not only angiosperm species including eudicots and monocots but also gymnosperms and a fern, and induce their degradation. These results suggest that phyllogen induces phyllody in angiosperms and inhibits MTF function in diverse plant species. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

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

PubMed

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

2008-08-01

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

Functional characterisation of Arabidopsis SPL7 conserved protein domains suggests novel regulatory mechanisms in the Cu deficiency response.

PubMed

Garcia-Molina, Antoni; Xing, Shuping; Huijser, Peter

2014-08-30

The Arabidopsis SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) transcription factor SPL7 reprograms cellular gene expression to adapt plant growth and cellular metabolism to copper (Cu) limited culture conditions. Plant cells require Cu to maintain essential processes, such as photosynthesis, scavenging reactive oxygen species, cell wall lignification and hormone sensing. More specifically, SPL7 activity promotes a high-affinity Cu-uptake system and optimizes Cu (re-)distribution to essential Cu-proteins by means of specific miRNAs targeting mRNA transcripts for those dispensable. However, the functional mechanism underlying SPL7 activation is still to be elucidated. As SPL7 transcript levels are largely non-responsive to Cu availability, post-translational modification seems an obvious possibility. Previously, it was reported that the SPL7 SBP domain does not bind to DNA in vitro in the presence of Cu ions and that SPL7 interacts with a kin17 domain protein to raise SPL7-target gene expression upon Cu deprivation. Here we report how additional conserved SPL7 protein domains may contribute to the Cu deficiency response in Arabidopsis. Cytological and biochemical approaches confirmed an operative transmembrane domain (TMD) and uncovered a dual localisation of SPL7 between the nucleus and an endomembrane system, most likely the endoplasmic reticulum (ER). This new perspective unveiled a possible link between Cu deficit and ER stress, a metabolic dysfunction found capable of inducing SPL7 targets in an SPL7-dependent manner. Moreover, in vivo protein-protein interaction assays revealed that SPL7 is able to homodimerize, probably mediated by the IRPGC domain. These observations, in combination with the constitutive activation of SPL7 targets, when ectopically expressing the N-terminal part of SPL7 including the SBP domain, shed some light on the mechanisms governing SPL7 function. Here, we propose a revised model of SPL7 activation and regulation. According to our

Selecting soluble/foldable protein domains through single-gene or genomic ORF filtering: structure of the head domain of Burkholderia pseudomallei antigen BPSL2063.

PubMed

Gourlay, Louise J; Peano, Clelia; Deantonio, Cecilia; Perletti, Lucia; Pietrelli, Alessandro; Villa, Riccardo; Matterazzo, Elena; Lassaux, Patricia; Santoro, Claudio; Puccio, Simone; Sblattero, Daniele; Bolognesi, Martino

2015-11-01

The 1.8 Å resolution crystal structure of a conserved domain of the potential Burkholderia pseudomallei antigen and trimeric autotransporter BPSL2063 is presented as a structural vaccinology target for melioidosis vaccine development. Since BPSL2063 (1090 amino acids) hosts only one conserved domain, and the expression/purification of the full-length protein proved to be problematic, a domain-filtering library was generated using β-lactamase as a reporter gene to select further BPSL2063 domains. As a result, two domains (D1 and D2) were identified and produced in soluble form in Escherichia coli. Furthermore, as a general tool, a genomic open reading frame-filtering library from the B. pseudomallei genome was also constructed to facilitate the selection of domain boundaries from the entire ORFeome. Such an approach allowed the selection of three potential protein antigens that were also produced in soluble form. The results imply the further development of ORF-filtering methods as a tool in protein-based research to improve the selection and production of soluble proteins or domains for downstream applications such as X-ray crystallography.

The impact of young age on locoregional recurrence after doxorubicin-based breast conservation therapy in patients 40 years old or younger: How young is 'young'?

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

Oh, Julia L.; Bonnen, Mark; Outlaw, Elesyia D.

2006-08-01

Purpose: The aim of this study was to investigate whether patients <35 years old have similar risk of locoregional recurrence after breast conservation therapy compared with patients 35 to 40 years old. Methods and materials: We retrospectively reviewed records of 196 consecutive patients {<=}40 years old who received breast conservation therapy (BCT) from 1987 to 2000 for breast cancer and compared outcomes between patients <35 years old with patients 35 to 40 years old. The majority of patients received neoadjuvant chemotherapy as part of their treatment. Multivariate analysis was performed to assess risk factors for locoregional recurrence. Results: After amore » median follow-up of 64 months, 22 locoregional recurrences (LRR) were observed. Twenty patients developed locoregional recurrence as their first site of relapse. Two patients had bone-only metastases before their locoregional recurrence. On multivariate analysis, age <35 years was associated with a statistically significant increased risk of locoregional recurrence. The 5-year rate of locoregional control was 87.9% in patients <35 years old compared with 91.7% in patients 35 to 40 years old (p = 0.042). Conclusions: Our finding supports an increased risk of locoregional recurrence as a function of younger age after breast conservation therapy, even among young patients 40 years old and younger.« less

The Caenorhabditis elegans Iodotyrosine Deiodinase Ortholog SUP-18 Functions through a Conserved Channel SC-Box to Regulate the Muscle Two-Pore Domain Potassium Channel SUP-9

PubMed Central

de la Cruz, Ignacio Perez; Ma, Long; Horvitz, H. Robert

2014-01-01

Loss-of-function mutations in the Caenorhabditis elegans gene sup-18 suppress the defects in muscle contraction conferred by a gain-of-function mutation in SUP-10, a presumptive regulatory subunit of the SUP-9 two-pore domain K+ channel associated with muscle membranes. We cloned sup-18 and found that it encodes the C. elegans ortholog of mammalian iodotyrosine deiodinase (IYD), an NADH oxidase/flavin reductase that functions in iodine recycling and is important for the biosynthesis of thyroid hormones that regulate metabolism. The FMN-binding site of mammalian IYD is conserved in SUP-18, which appears to require catalytic activity to function. Genetic analyses suggest that SUP-10 can function with SUP-18 to activate SUP-9 through a pathway that is independent of the presumptive SUP-9 regulatory subunit UNC-93. We identified a novel evolutionarily conserved serine-cysteine-rich region in the C-terminal cytoplasmic domain of SUP-9 required for its specific activation by SUP-10 and SUP-18 but not by UNC-93. Since two-pore domain K+ channels regulate the resting membrane potentials of numerous cell types, we suggest that the SUP-18 IYD regulates the activity of the SUP-9 channel using NADH as a coenzyme and thus couples the metabolic state of muscle cells to muscle membrane excitability. PMID:24586202

Anthrax Toxin Receptor 1 / Tumor Endothelial Marker 8: Mutation of Conserved Inserted Domain Residues Overrides Cytosolic Control of Protective Antigen Binding†

PubMed Central

Ramey, Jordan D.; Villareal, Valerie A.; Ng, Charles; Ward, Sabrina; Xiong, Jian-Ping; Clubb, Robert T.; Bradley, Kenneth A.

2010-01-01

Anthrax toxin receptor 1 (ANTXR1) / tumor endothelial marker 8 (TEM8) is one of two known proteinaceous cell surface anthrax toxin receptors. A metal ion dependent adhesion site (MIDAS) present in the integrin-like inserted (I) domain of ANTXR1 mediates the binding of the anthrax toxin subunit, protective antigen (PA). Here we provide evidence that single point mutations in the I domain can override regulation of ANTXR1 ligand-binding activity mediated by intracellular signals. A previously reported MIDAS-mutant of ANTXR1 (T118A) was found to retain normal metal ion binding and secondary structure but failed to bind PA, consistent with a locked inactive state. Conversely, mutation of a conserved I domain phenylalanine residue to a tryptophan (F205W) increased the proportion of cell-surface ANTXR1 that bound PA, consistent with a locked active state. Interestingly, the KD and total amount of PA bound by the isolated ANTXR1 I domain was not affected by the F205W mutation, indicating that ANTXR1 is preferentially found in the active state in the absence of inside-out signaling. Circular dichroism (CD) spectroscopy and 1H-15N heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) revealed that structural changes between T118A, F205W and WT I domains were minor despite a greater than 103-fold difference in their abilities to bind toxin. Regulation of toxin binding has important implications for the design of toxin inhibitors and for the targeting of ANTXR1 for anti-tumor therapies. PMID:20690680

Domain duplication, divergence, and loss events in vertebrate Msx paralogs reveal phylogenomically informed disease markers

PubMed Central

Finnerty, John R; Mazza, Maureen E; Jezewski, Peter A

2009-01-01

Background Msx originated early in animal evolution and is implicated in human genetic disorders. To reconstruct the functional evolution of Msx and inform the study of human mutations, we analyzed the phylogeny and synteny of 46 metazoan Msx proteins and tracked the duplication, diversification and loss of conserved motifs. Results Vertebrate Msx sequences sort into distinct Msx1, Msx2 and Msx3 clades. The sister-group relationship between MSX1 and MSX2 reflects their derivation from the 4p/5q chromosomal paralogon, a derivative of the original "MetaHox" cluster. We demonstrate physical linkage between Msx and other MetaHox genes (Hmx, NK1, Emx) in a cnidarian. Seven conserved domains, including two Groucho repression domains (N- and C-terminal), were present in the ancestral Msx. In cnidarians, the Groucho domains are highly similar. In vertebrate Msx1, the N-terminal Groucho domain is conserved, while the C-terminal domain diverged substantially, implying a novel function. In vertebrate Msx2 and Msx3, the C-terminal domain was lost. MSX1 mutations associated with ectodermal dysplasia or orofacial clefting disorders map to conserved domains in a non-random fashion. Conclusion Msx originated from a MetaHox ancestor that also gave rise to Tlx, Demox, NK, and possibly EHGbox, Hox and ParaHox genes. Duplication, divergence or loss of domains played a central role in the functional evolution of Msx. Duplicated domains allow pleiotropically expressed proteins to evolve new functions without disrupting existing interaction networks. Human missense sequence variants reside within evolutionarily conserved domains, likely disrupting protein function. This phylogenomic evaluation of candidate disease markers will inform clinical and functional studies. PMID:19154605

Domain duplication, divergence, and loss events in vertebrate Msx paralogs reveal phylogenomically informed disease markers.

PubMed

Finnerty, John R; Mazza, Maureen E; Jezewski, Peter A

2009-01-20

Msx originated early in animal evolution and is implicated in human genetic disorders. To reconstruct the functional evolution of Msx and inform the study of human mutations, we analyzed the phylogeny and synteny of 46 metazoan Msx proteins and tracked the duplication, diversification and loss of conserved motifs. Vertebrate Msx sequences sort into distinct Msx1, Msx2 and Msx3 clades. The sister-group relationship between MSX1 and MSX2 reflects their derivation from the 4p/5q chromosomal paralogon, a derivative of the original "MetaHox" cluster. We demonstrate physical linkage between Msx and other MetaHox genes (Hmx, NK1, Emx) in a cnidarian. Seven conserved domains, including two Groucho repression domains (N- and C-terminal), were present in the ancestral Msx. In cnidarians, the Groucho domains are highly similar. In vertebrate Msx1, the N-terminal Groucho domain is conserved, while the C-terminal domain diverged substantially, implying a novel function. In vertebrate Msx2 and Msx3, the C-terminal domain was lost. MSX1 mutations associated with ectodermal dysplasia or orofacial clefting disorders map to conserved domains in a non-random fashion. Msx originated from a MetaHox ancestor that also gave rise to Tlx, Demox, NK, and possibly EHGbox, Hox and ParaHox genes. Duplication, divergence or loss of domains played a central role in the functional evolution of Msx. Duplicated domains allow pleiotropically expressed proteins to evolve new functions without disrupting existing interaction networks. Human missense sequence variants reside within evolutionarily conserved domains, likely disrupting protein function. This phylogenomic evaluation of candidate disease markers will inform clinical and functional studies.

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

Automated hierarchical classification of protein domain subfamilies based on functionally-divergent residue signatures

PubMed Central

2012-01-01

Background The NCBI Conserved Domain Database (CDD) consists of a collection of multiple sequence alignments of protein domains that are at various stages of being manually curated into evolutionary hierarchies based on conserved and divergent sequence and structural features. These domain models are annotated to provide insights into the relationships between sequence, structure and function via web-based BLAST searches. Results Here we automate the generation of conserved domain (CD) hierarchies using a combination of heuristic and Markov chain Monte Carlo (MCMC) sampling procedures and starting from a (typically very large) multiple sequence alignment. This procedure relies on statistical criteria to define each hierarchy based on the conserved and divergent sequence patterns associated with protein functional-specialization. At the same time this facilitates the sequence and structural annotation of residues that are functionally important. These statistical criteria also provide a means to objectively assess the quality of CD hierarchies, a non-trivial task considering that the protein subgroups are often very distantly related—a situation in which standard phylogenetic methods can be unreliable. Our aim here is to automatically generate (typically sub-optimal) hierarchies that, based on statistical criteria and visual comparisons, are comparable to manually curated hierarchies; this serves as the first step toward the ultimate goal of obtaining optimal hierarchical classifications. A plot of runtimes for the most time-intensive (non-parallelizable) part of the algorithm indicates a nearly linear time complexity so that, even for the extremely large Rossmann fold protein class, results were obtained in about a day. Conclusions This approach automates the rapid creation of protein domain hierarchies and thus will eliminate one of the most time consuming aspects of conserved domain database curation. At the same time, it also facilitates protein domain

A Proteome-wide Domain-centric Perspective on Protein Phosphorylation *

PubMed Central

Palmeri, Antonio; Ausiello, Gabriele; Ferrè, Fabrizio; Helmer-Citterich, Manuela; Gherardini, Pier Federico

2014-01-01

Phosphorylation is a widespread post-translational modification that modulates the function of a large number of proteins. Here we show that a significant proportion of all the domains in the human proteome is significantly enriched or depleted in phosphorylation events. A substantial improvement in phosphosites prediction is achieved by leveraging this observation, which has not been tapped by existing methods. Phosphorylation sites are often not shared between multiple occurrences of the same domain in the proteome, even when the phosphoacceptor residue is conserved. This is partly because of different functional constraints acting on the same domain in different protein contexts. Moreover, by augmenting domain alignments with structural information, we were able to provide direct evidence that phosphosites in protein-protein interfaces need not be positionally conserved, likely because they can modulate interactions simply by sitting in the same general surface area. PMID:24830415

Contrasting tectonothermal domains and faulting in the Potomac terrane, Virginia-Maryland - Discrimination by 40Ar/39Ar and fission-track thermochronology

USGS Publications Warehouse

Kunk, Michael J.; Wintsch, R.P.; Naeser, C.W.; Naeser, N.D.; Southworth, C.S.; Drake, Avery A.; Becker, J.L.

2005-01-01

New 40Ar/39Ar data reveal ages and thermal discontinuities that identify mapped and unmapped fault boundaries in the Potomac terrane in northern Virginia, thus confirming previous interpretations that it is a composite terrane. The rocks of the Potomac terrane were examined along the Potomac River, where it has been previously subdivided into three units: the Mather Gorge, Sykesville, and Laurel Formations. In the Mather Gorge Formation, at least two metamorphic thermal domains were identified, the Blockhouse Point and Bear Island domains, separated by a fault active in the late Devonian. Early Ordovician (ca. 475 Ma) cooling ages of amphibole in the Bear Island domain reflect cooling from Taconic metamorphism, whereas the Blockhouse Point domain was first metamorphosed in the Devonian. The 40Ar/39Ar data from muscovites in a third (eastern) domain within the Mather Gorge Formation, the Stubblefield Falls domain, record thrusting of the Sykesville Formation over the Mather Gorge Formation on the Plummers Island fault in the Devonian. The existence of two distinctly different thermal domains separated by a tectonic boundary within the Mather Gorge argues against its status as a formation. Hornblende cooling ages in the Sykesville Formation are Early Devonian (ca. 400 Ma), reflecting cooling from Taconic and Acadian metamorphism. The ages of retrograde and overprinting muscovite in phyllonites from domain-bounding faults are late Devonian (Acadian) and late Pennsylvanian (Alleghanian), marking the time of assembly of these domains and subsequent movement on the Plummers Island fault. Our data indicate that net vertical motion between the Bear Island domain of the Mather Gorge complex and the Sykesville Formation across the Plummers Island fault is east-side-up. Zircon fission-track cooling ages demonstrate thermal equillbrium across the Potomac terrane in the early Permian, and apatite fission-track cooling ages record tilting of the Potomac terrane in the Cretaceous

Formin homology 2 domains occur in multiple contexts in angiosperms

PubMed Central

Cvrčková, Fatima; Novotný, Marian; Pícková, Denisa; Žárský, Viktor

2004-01-01

Background Involvement of conservative molecular modules and cellular mechanisms in the widely diversified processes of eukaryotic cell morphogenesis leads to the intriguing question: how do similar proteins contribute to dissimilar morphogenetic outputs. Formins (FH2 proteins) play a central part in the control of actin organization and dynamics, providing a good example of evolutionarily versatile use of a conserved protein domain in the context of a variety of lineage-specific structural and signalling interactions. Results In order to identify possible plant-specific sequence features within the FH2 protein family, we performed a detailed analysis of angiosperm formin-related sequences available in public databases, with particular focus on the complete Arabidopsis genome and the nearly finished rice genome sequence. This has led to revision of the current annotation of half of the 22 Arabidopsis formin-related genes. Comparative analysis of the two plant genomes revealed a good conservation of the previously described two subfamilies of plant formins (Class I and Class II), as well as several subfamilies within them that appear to predate the separation of monocot and dicot plants. Moreover, a number of plant Class II formins share an additional conserved domain, related to the protein phosphatase/tensin/auxilin fold. However, considerable inter-species variability sets limits to generalization of any functional conclusions reached on a single species such as Arabidopsis. Conclusions The plant-specific domain context of the conserved FH2 domain, as well as plant-specific features of the domain itself, may reflect distinct functional requirements in plant cells. The variability of formin structures found in plants far exceeds that known from both fungi and metazoans, suggesting a possible contribution of FH2 proteins in the evolution of the plant type of multicellularity. PMID:15256004

OST-HTH: a novel predicted RNA-binding domain

PubMed Central

2010-01-01

Background The mechanism by which the arthropod Oskar and vertebrate TDRD5/TDRD7 proteins nucleate or organize structurally related ribonucleoprotein (RNP) complexes, the polar granule and nuage, is poorly understood. Using sequence profile searches we identify a novel domain in these proteins that is widely conserved across eukaryotes and bacteria. Results Using contextual information from domain architectures, sequence-structure superpositions and available functional information we predict that this domain is likely to adopt the winged helix-turn-helix fold and bind RNA with a potential specificity for dsRNA. We show that in eukaryotes this domain is often combined in the same polypeptide with protein-protein- or lipid- interaction domains that might play a role in anchoring these proteins to specific cytoskeletal structures. Conclusions Thus, proteins with this domain might have a key role in the recognition and localization of dsRNA, including miRNAs, rasiRNAs and piRNAs hybridized to their targets. In other cases, this domain is fused to ubiquitin-binding, E3 ligase and ubiquitin-like domains indicating a previously under-appreciated role for ubiquitination in regulating the assembly and stability of nuage-like RNP complexes. Both bacteria and eukaryotes encode a conserved family of proteins that combines this predicted RNA-binding domain with a previously uncharacterized domain (DUF88). We present evidence that it is an RNAse belonging to the superfamily that includes the 5'->3' nucleases, PIN and NYN domains and might be recruited to degrade certain RNAs. Reviewers This article was reviewed by Sandor Pongor and Arcady Mushegian. PMID:20302647

VizieR Online Data Catalog: The ELM survey. VI. 11 new ELM WD binaries (Gianninas+, 2015)

NASA Astrophysics Data System (ADS)

Gianninas, A.; Kilic, M.; Brown, W. R.; Canton, P.; Kenyon, S. J.

2016-02-01

We used the 6.5m MMT telescope equipped with the Blue Channel spectrograph, the 200 inch Hale telescope equipped with the Double spectrograph, the Kitt Peak National Observatory 4m telescope equipped with the R-C spectrograph, and more recently with Kitt Peak Ohio State Multi-Object Spectrograph (KOSMOS), to obtain spectroscopy of our 11 targets in several observing runs. We have also been obtaining radial-velocity measurements for candidates from other sources including the Large Sky Area Multi-Object Spectroscopy Telescope (LAMOST). Those 11 new Extremely low-mass white dwarf (ELM WD) binaries bring the total of ELM WDs identified by the ELM Survey up to 73. (4 data files).

Evidence of Distinct Channel Conformations and Substrate Binding Affinities for the Mitochondrial Outer Membrane Protein Translocase Pore Tom40*

PubMed Central

Kuszak, Adam J.; Jacobs, Daniel; Gurnev, Philip A.; Shiota, Takuya; Louis, John M.; Lithgow, Trevor; Bezrukov, Sergey M.; Rostovtseva, Tatiana K.; Buchanan, Susan K.

2015-01-01

Nearly all mitochondrial proteins are coded by the nuclear genome and must be transported into mitochondria by the translocase of the outer membrane complex. Tom40 is the central subunit of the translocase complex and forms a pore in the mitochondrial outer membrane. To date, the mechanism it utilizes for protein transport remains unclear. Tom40 is predicted to comprise a membrane-spanning β-barrel domain with conserved α-helical domains at both the N and C termini. To investigate Tom40 function, including the role of the N- and C-terminal domains, recombinant forms of the Tom40 protein from the yeast Candida glabrata, and truncated constructs lacking the N- and/or C-terminal domains, were functionally characterized in planar lipid membranes. Our results demonstrate that each of these Tom40 constructs exhibits at least four distinct conductive levels and that full-length and truncated Tom40 constructs specifically interact with a presequence peptide in a concentration- and voltage-dependent manner. Therefore, neither the first 51 amino acids of the N terminus nor the last 13 amino acids of the C terminus are required for Tom40 channel formation or for the interaction with a presequence peptide. Unexpectedly, substrate binding affinity was dependent upon the Tom40 state corresponding to a particular conductive level. A model where two Tom40 pores act in concert as a dimeric protein complex best accounts for the observed biochemical and electrophysiological data. These results provide the first evidence for structurally distinct Tom40 conformations playing a role in substrate recognition and therefore in transport function. PMID:26336107

The structure of the nucleoprotein binding domain of lyssavirus phosphoprotein reveals a structural relationship between the N-RNA binding domains of Rhabdoviridae and Paramyxoviridae.

PubMed

Delmas, Olivier; Assenberg, Rene; Grimes, Jonathan M; Bourhy, Hervé

2010-01-01

The phosphoprotein P of non-segmented negative-sense RNA viruses is an essential component of the replication and transcription complex and acts as a co-factor for the viral RNA-dependent RNA polymerase. P recruits the viral polymerase to the nucleoprotein-bound viral RNA (N-RNA) via an interaction between its C-terminal domain and the N-RNA complex. We have obtained the structure of the C-terminal domain of P of Mokola virus (MOKV), a lyssavirus that belongs to the Rhabdoviridae family and mapped at the amino acid level the crucial positions involved in interaction with N and in the formation of the viral replication complex. Comparison of the N-RNA binding domains of P solved to date suggests that the N-RNA binding domains are structurally conserved among paramyxoviruses and rhabdoviruses in spite of low sequence conservation. We also review the numerous other functions of this domain and more generally of the phosphoprotein.

Transcriptional activation is a conserved feature of the early embryonic factor Zelda that requires a cluster of four zinc fingers for DNA binding and a low-complexity activation domain.

PubMed

Hamm, Danielle C; Bondra, Eliana R; Harrison, Melissa M

2015-02-06

Delayed transcriptional activation of the zygotic genome is a nearly universal phenomenon in metazoans. Immediately following fertilization, development is controlled by maternally deposited products, and it is not until later stages that widespread activation of the zygotic genome occurs. Although the mechanisms driving this genome activation are currently unknown, the transcriptional activator Zelda (ZLD) has been shown to be instrumental in driving this process in Drosophila melanogaster. Here we define functional domains of ZLD required for both DNA binding and transcriptional activation. We show that the C-terminal cluster of four zinc fingers mediates binding to TAGteam DNA elements in the promoters of early expressed genes. All four zinc fingers are required for this activity, and splice isoforms lacking three of the four zinc fingers fail to activate transcription. These truncated splice isoforms dominantly suppress activation by the full-length, embryonically expressed isoform. We map the transcriptional activation domain of ZLD to a central region characterized by low complexity. Despite relatively little sequence conservation within this domain, ZLD orthologs from Drosophila virilis, Anopheles gambiae, and Nasonia vitripennis activate transcription in D. melanogaster cells. Transcriptional activation by these ZLD orthologs suggests that ZLD functions through conserved interactions with a protein cofactor(s). We have identified distinct DNA-binding and activation domains within the critical transcription factor ZLD that controls the initial activation of the zygotic genome. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

TOPDOM: database of conservatively located domains and motifs in proteins.

PubMed

Varga, Julia; Dobson, László; Tusnády, Gábor E

2016-09-01

The TOPDOM database-originally created as a collection of domains and motifs located consistently on the same side of the membranes in α-helical transmembrane proteins-has been updated and extended by taking into consideration consistently localized domains and motifs in globular proteins, too. By taking advantage of the recently developed CCTOP algorithm to determine the type of a protein and predict topology in case of transmembrane proteins, and by applying a thorough search for domains and motifs as well as utilizing the most up-to-date version of all source databases, we managed to reach a 6-fold increase in the size of the whole database and a 2-fold increase in the number of transmembrane proteins. TOPDOM database is available at http://topdom.enzim.hu The webpage utilizes the common Apache, PHP5 and MySQL software to provide the user interface for accessing and searching the database. The database itself is generated on a high performance computer. tusnady.gabor@ttk.mta.hu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

When a domain isn’t a domain, and why it’s important to properly filter proteins in databases

PubMed Central

Towse, Clare-Louise; Daggett, Valerie

2013-01-01

Summary Membership in a protein domain database does not a domain make; a feature we realized when generating a consensus view of protein fold space with our Consensus Domain Dictionary (CDD). This dictionary was used to select representative structures for characterization of the protein dynameome: the Dynameomics initiative. Through this endeavor we rejected a surprising 40% of the 1695 folds in the CDD as being non-autonomous folding units. Although some of this was due to the challenges of grouping similar fold topologies, the dissonance between the cataloguing and structural qualification of protein domains remains surprising. Another potential factor is previously overlooked intrinsic disorder; predicted estimates suggest 40% of proteins to have either local or global disorder. One thing is clear, filtering a structural database and ensuring a consistent definition for protein domains is crucial, and caution is prescribed when generalizations of globular domains are drawn from unfiltered protein domain datasets. PMID:23108912

The U-box family genes in Medicago truncatula: Key elements in response to salt, cold, and drought stresses.

PubMed

Song, Jianbo; Mo, Xiaowei; Yang, Haiqi; Yue, Luming; Song, Jun; Mo, Beixin

2017-01-01

The ubiquitination pathway regulates growth, development, and stress responses in plants, and the U-box protein family of ubiquitin ligases has important roles in this pathway. Here, 64 putative U-box proteins were identified in the Medicago truncatula genome. In addition to the conserved U-box motif, other functional domains, such as the ARM, kinase, KAP, and WD40 domains, were also detected. Phylogenetic analysis of the M. truncatula U-box proteins grouped them into six subfamilies, and chromosomal mapping and synteny analyses indicated that tandem and segmental duplications may have contributed to the expansion and evolution of the U-box gene family in this species. Using RNA-seq data from M. truncatula seedlings subjected to three different abiotic stresses, we identified 33 stress-inducible plant U-box genes (MtPUBs). Specifically, 25 salinity-, 15 drought-, and 16 cold-regulated MtPUBs were detected. Among them, MtPUB10, MtPUB17, MtPUB18, MtPUB35, MtPUB42, and MtPUB44 responded to all three stress conditions. Expression profiling by qRT-PCR was consistent with the RNA-seq data, and stress-related elements were identified in the promoter regions. The present findings strongly indicate that U-box proteins play critical roles in abiotic stress response in M. truncatula.

The U-box family genes in Medicago truncatula: Key elements in response to salt, cold, and drought stresses

PubMed Central

Yang, Haiqi; Yue, Luming; Song, Jun

2017-01-01

The ubiquitination pathway regulates growth, development, and stress responses in plants, and the U-box protein family of ubiquitin ligases has important roles in this pathway. Here, 64 putative U-box proteins were identified in the Medicago truncatula genome. In addition to the conserved U-box motif, other functional domains, such as the ARM, kinase, KAP, and WD40 domains, were also detected. Phylogenetic analysis of the M. truncatula U-box proteins grouped them into six subfamilies, and chromosomal mapping and synteny analyses indicated that tandem and segmental duplications may have contributed to the expansion and evolution of the U-box gene family in this species. Using RNA-seq data from M. truncatula seedlings subjected to three different abiotic stresses, we identified 33 stress-inducible plant U-box genes (MtPUBs). Specifically, 25 salinity-, 15 drought-, and 16 cold-regulated MtPUBs were detected. Among them, MtPUB10, MtPUB17, MtPUB18, MtPUB35, MtPUB42, and MtPUB44 responded to all three stress conditions. Expression profiling by qRT-PCR was consistent with the RNA-seq data, and stress-related elements were identified in the promoter regions. The present findings strongly indicate that U-box proteins play critical roles in abiotic stress response in M. truncatula. PMID:28771553

40 CFR 40.115-6 - State.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 1 2010-07-01 2010-07-01 false State. 40.115-6 Section 40.115-6... DEMONSTRATION GRANTS § 40.115-6 State. (a) Under the Federal Water Pollution Control Act, a State, the District... Territory of the Pacific Islands. (b) Under the Resource Conservation and Recovery Act, a State, the...

Core Histones and HIRIP3, a Novel Histone-Binding Protein, Directly Interact with WD Repeat Protein HIRA

PubMed Central

Lorain, Stéphanie; Quivy, Jean-Pierre; Monier-Gavelle, Frédérique; Scamps, Christine; Lécluse, Yann; Almouzni, Geneviève; Lipinski, Marc

1998-01-01

The human HIRA gene has been named after Hir1p and Hir2p, two corepressors which together appear to act on chromatin structure to control gene transcription in Saccharomyces cerevisiae. HIRA homologs are expressed in a regulated fashion during mouse and chicken embryogenesis, and the human gene is a major candidate for the DiGeorge syndrome and related developmental disorders caused by a reduction to single dose of a fragment of chromosome 22q. Western blot analysis and double-immunofluorescence experiments using a specific antiserum revealed a primary nuclear localization of HIRA. Similar to Hir1p, HIRA contains seven amino-terminal WD repeats and probably functions as part of a multiprotein complex. HIRA and core histone H2B were found to physically interact in a yeast double-hybrid protein interaction trap, in GST pull-down assays, and in coimmunoprecipitation experiments performed from cellular extracts. In vitro, HIRA also interacted with core histone H4. H2B- and H4-binding domains were overlapping but distinguishable in the carboxy-terminal region of HIRA, and the region for HIRA interaction was mapped to the amino-terminal tail of H2B and the second α helix of H4. HIRIP3 (HIRA-interacting protein 3) is a novel gene product that was identified from its HIRA-binding properties in the yeast protein interaction trap. In vitro, HIRIP3 directly interacted with HIRA but also with core histones H2B and H3, suggesting that a HIRA-HIRIP3-containing complex could function in some aspects of chromatin and histone metabolism. Insufficient production of HIRA, which we report elsewhere interacts with homeodomain-containing DNA-binding factors during mammalian embryogenesis, could perturb the stoichiometric assembly of multimolecular complexes required for normal embryonic development. PMID:9710638

A Fast Alignment-Free Approach for De Novo Detection of Protein Conserved Regions

PubMed Central

Abnousi, Armen; Broschat, Shira L.; Kalyanaraman, Ananth

2016-01-01

Background Identifying conserved regions in protein sequences is a fundamental operation, occurring in numerous sequence-driven analysis pipelines. It is used as a way to decode domain-rich regions within proteins, to compute protein clusters, to annotate sequence function, and to compute evolutionary relationships among protein sequences. A number of approaches exist for identifying and characterizing protein families based on their domains, and because domains represent conserved portions of a protein sequence, the primary computation involved in protein family characterization is identification of such conserved regions. However, identifying conserved regions from large collections (millions) of protein sequences presents significant challenges. Methods In this paper we present a new, alignment-free method for detecting conserved regions in protein sequences called NADDA (No-Alignment Domain Detection Algorithm). Our method exploits the abundance of exact matching short subsequences (k-mers) to quickly detect conserved regions, and the power of machine learning is used to improve the prediction accuracy of detection. We present a parallel implementation of NADDA using the MapReduce framework and show that our method is highly scalable. Results We have compared NADDA with Pfam and InterPro databases. For known domains annotated by Pfam, accuracy is 83%, sensitivity 96%, and specificity 44%. For sequences with new domains not present in the training set an average accuracy of 63% is achieved when compared to Pfam. A boost in results in comparison with InterPro demonstrates the ability of NADDA to capture conserved regions beyond those present in Pfam. We have also compared NADDA with ADDA and MKDOM2, assuming Pfam as ground-truth. On average NADDA shows comparable accuracy, more balanced sensitivity and specificity, and being alignment-free, is significantly faster. Excluding the one-time cost of training, runtimes on a single processor were 49s, 10,566s, and 456s

The GP(Y/F) domain of TF1 integrase multimerizes when present in a fragment, and substitutions in this domain reduce enzymatic activity of the full-length protein.

PubMed

Ebina, Hirotaka; Chatterjee, Atreyi Ghatak; Judson, Robert L; Levin, Henry L

2008-06-06

Integrases (INs) of retroviruses and long terminal repeat retrotransposons possess a C-terminal domain with DNA binding activity. Other than this binding activity, little is known about how the C-terminal domain contributes to integration. A stretch of conserved amino acids called the GP(Y/F) domain has been identified within the C-terminal IN domains of two distantly related families, the gamma-retroviruses and the metavirus retrotransposons. To enhance understanding of the C-terminal domain, we examined the function of the GP(Y/F) domain in the IN of Tf1, a long terminal repeat retrotransposon of Schizosaccharomyces pombe. The activities of recombinant IN were measured with an assay that modeled the reverse of integration called disintegration. Although deletion of the entire C-terminal domain disrupted disintegration activity, an alanine substitution (P365A) in a conserved amino acid of the GP(Y/F) domain did not significantly reduce disintegration. When assayed for the ability to join two molecules of DNA in a reaction that modeled forward integration, the P365A substitution disrupted activity. UV cross-linking experiments detected DNA binding activity in the C-terminal domain and found that this activity was not reduced by substitutions in two conserved amino acids of the GP(Y/F) domain, G364A and P365A. Gel filtration and cross-linking of a 71-amino acid fragment containing the GP(Y/F) domain revealed a surprising ability to form dimers, trimers, and tetramers that was disrupted by the G364A and P365A substitutions. These results suggest that the GP(Y/F) residues may play roles in promoting multimerization and intermolecular strand joining.

Analysis of the linker region joining the adenylation and carrier protein domains of the modular nonribosomal peptide synthetases.

PubMed

Miller, Bradley R; Sundlov, Jesse A; Drake, Eric J; Makin, Thomas A; Gulick, Andrew M

2014-10-01

Nonribosomal peptide synthetases (NRPSs) are multimodular proteins capable of producing important peptide natural products. Using an assembly line process, the amino acid substrate and peptide intermediates are passed between the active sites of different catalytic domains of the NRPS while bound covalently to a peptidyl carrier protein (PCP) domain. Examination of the linker sequences that join the NRPS adenylation and PCP domains identified several conserved proline residues that are not found in standalone adenylation domains. We examined the roles of these proline residues and neighboring conserved sequences through mutagenesis and biochemical analysis of the reaction catalyzed by the adenylation domain and the fully reconstituted NRPS pathway. In particular, we identified a conserved LPxP motif at the start of the adenylation-PCP linker. The LPxP motif interacts with a region on the adenylation domain to stabilize a critical catalytic lysine residue belonging to the A10 motif that immediately precedes the linker. Further, this interaction with the C-terminal subdomain of the adenylation domain may coordinate movement of the PCP with the conformational change of the adenylation domain. Through this work, we extend the conserved A10 motif of the adenylation domain and identify residues that enable proper adenylation domain function. © 2014 Wiley Periodicals, Inc.

Structure and Function of Vps15 in the Endosomal G Protein Signaling Pathway

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

Heenan, Erin J.; Vanhooke, Janeen L.; Temple, Brenda R.

2009-09-11

G protein-coupled receptors mediate cellular responses to a wide variety of stimuli, including taste, light, and neurotransmitters. In the yeast Saccharomyces cerevisiae, activation of the pheromone pathway triggers events leading to mating. The view had long been held that the G protein-mediated signal occurs principally at the plasma membrane. Recently, it has been shown that the G protein {alpha} subunit Gpa1 can promote signaling at endosomes and requires two components of the sole phosphatidylinositol-3-kinase in yeast, Vps15 and Vps34. Vps15 contains multiple WD repeats and also binds to Gpa1 preferentially in the GDP-bound state; these observations led us to hypothesizemore » that Vps15 may function as a G protein {beta} subunit at the endosome. Here we show an X-ray crystal structure of the Vps15 WD domain that reveals a seven-bladed propeller resembling that of typical G{beta} subunits. We show further that the WD domain is sufficient to bind Gpa1 as well as to Atg14, a potential G{gamma} protein that exists in a complex with Vps15. The Vps15 kinase domain together with the intermediate domain (linking the kinase and WD domains) also contributes to Gpa1 binding and is necessary for Vps15 to sustain G protein signaling. These findings reveal that the Vps15 G{beta}-like domain serves as a scaffold to assemble Gpa1 and Atg14, whereas the kinase and intermediate domains are required for proper signaling at the endosome.« less

Conservation of an ATP-binding domain among recA proteins from Proteus vulgaris, erwinia carotovora, Shigella flexneri, and Escherichia coli K-12 and B/r

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

Knight, K.L.; Hess, R.M.; McEntee, K.

1988-06-01

The purified RecA proteins encoded by the cloned genes from Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli B/r were compared with the RecA protein from E. coli K-12. Each of the proteins hydrolyzed ATP in the presence of single-stranded DNA, and each was covalently modified with the photoaffinity ATP analog 8-azidoadenosine 5'-triphosphate (8N/sub 3/ATP). Two-dimensional tryptic maps of the four heterologous RecA proteins demonstrated considerable structural conservation among these bacterial genera. Moreover, when the (..cap alpha..-/sup 32/P)8N/sub 3/ATP-modified proteins were digested with trypsin and analyzed by high-performance liquid chromatography, a single peak of radioactivity was detected in eachmore » of the digests and these peptides eluted identically with the tryptic peptide T/sub 31/ of the E. coli K-12 RecA protein, which was the unique site of 8N/sub 3/ATP photolabeling. Each of the heterologous recA genes hybridized to oligonucleotide probes derived from the ATP-binding domain sequence of the E. coli K-12 gene. These last results demonstrate that the ATP-binding domain of the RecA protein has been strongly conserved for greater than 10/sup 7/ years.« less

Parallel CE/SE Computations via Domain Decomposition

NASA Technical Reports Server (NTRS)

Himansu, Ananda; Jorgenson, Philip C. E.; Wang, Xiao-Yen; Chang, Sin-Chung

2000-01-01

This paper describes the parallelization strategy and achieved parallel efficiency of an explicit time-marching algorithm for solving conservation laws. The Space-Time Conservation Element and Solution Element (CE/SE) algorithm for solving the 2D and 3D Euler equations is parallelized with the aid of domain decomposition. The parallel efficiency of the resultant algorithm on a Silicon Graphics Origin 2000 parallel computer is checked.

Functional diversity of potassium channel voltage-sensing domains.

PubMed

Islas, León D

2016-01-01

Voltage-gated potassium channels or Kv's are membrane proteins with fundamental physiological roles. They are composed of 2 main functional protein domains, the pore domain, which regulates ion permeation, and the voltage-sensing domain, which is in charge of sensing voltage and undergoing a conformational change that is later transduced into pore opening. The voltage-sensing domain or VSD is a highly conserved structural motif found in all voltage-gated ion channels and can also exist as an independent feature, giving rise to voltage sensitive enzymes and also sustaining proton fluxes in proton-permeable channels. In spite of the structural conservation of VSDs in potassium channels, there are several differences in the details of VSD function found across variants of Kvs. These differences are mainly reflected in variations in the electrostatic energy needed to open different potassium channels. In turn, the differences in detailed VSD functioning among voltage-gated potassium channels might have physiological consequences that have not been explored and which might reflect evolutionary adaptations to the different roles played by Kv channels in cell physiology.

Functional diversity of potassium channel voltage-sensing domains

PubMed Central

Islas, León D.

2016-01-01

Abstract Voltage-gated potassium channels or Kv's are membrane proteins with fundamental physiological roles. They are composed of 2 main functional protein domains, the pore domain, which regulates ion permeation, and the voltage-sensing domain, which is in charge of sensing voltage and undergoing a conformational change that is later transduced into pore opening. The voltage-sensing domain or VSD is a highly conserved structural motif found in all voltage-gated ion channels and can also exist as an independent feature, giving rise to voltage sensitive enzymes and also sustaining proton fluxes in proton-permeable channels. In spite of the structural conservation of VSDs in potassium channels, there are several differences in the details of VSD function found across variants of Kvs. These differences are mainly reflected in variations in the electrostatic energy needed to open different potassium channels. In turn, the differences in detailed VSD functioning among voltage-gated potassium channels might have physiological consequences that have not been explored and which might reflect evolutionary adaptations to the different roles played by Kv channels in cell physiology. PMID:26794852

A novel application of cultural consensus models to evaluate conservation education programs.

PubMed

Nekaris, K A I; McCabe, Sharon; Spaan, Denise; Ali, Muhammad Imron; Nijman, Vincent

2018-04-01

Conservation professionals recognize the need to evaluate education initiatives with a flexible approach that is culturally appropriate. Cultural-consensus theory (CCT) provides a framework for measuring the extent to which beliefs are communally held and has long been applied by social scientists. In a conservation-education context, we applied CCT and used free lists (i.e., a list of items on a topic stated in order of cultural importance) and domain analysis (analysis of how free lists go together within a cultural group) to evaluate a conservation education program in which we used a children's picture book to increase knowledge about and empathy for a critically endangered mammal, the Javan slow loris (Nycticebus javanicus). We extracted free lists of keywords generated by students (n = 580 in 18 schools) from essays they wrote before and after the education program. In 2 classroom sessions conducted approximately 18 weeks apart, we asked students to write an essay about their knowledge of the target species and then presented a book and several activities about slow loris ecology. Prior to the second session, we asked students to write a second essay. We generated free lists from both essays, quantified salience of terms used, and conducted minimal residuals factor analysis to determine presence of cultural domains surrounding slow lorises in each session. Students increased their use of words accurately associated with slow loris ecology and conservation from 43% in initial essays to 76% in final essays. Domain coherence increased from 22% to 47% across schools. Fifteen factors contributed to the domain slow loris. Between the first and second essays, factors that showed the greatest change were feeding ecology and slow loris as a forest protector, which increased 7-fold, and the humancentric factor, which decreased 5-fold. As demonstrated by knowledge retention and creation of unique stories and conservation opinions, children achieved all six levels of

The GP(Y/F) Domain of TF1 Integrase Multimerizes when Present in a Fragment, and Substitutions in This Domain Reduce Enzymatic Activity of the Full-length Protein*S⃞

PubMed Central

Ebina, Hirotaka; Chatterjee, Atreyi Ghatak; Judson, Robert L.; Levin, Henry L.

2008-01-01

Integrases (INs) of retroviruses and long terminal repeat retrotransposons possess a C-terminal domain with DNA binding activity. Other than this binding activity, little is known about how the C-terminal domain contributes to integration. A stretch of conserved amino acids called the GP(Y/F) domain has been identified within the C-terminal IN domains of two distantly related families, the γ-retroviruses and the metavirus retrotransposons. To enhance understanding of the C-terminal domain, we examined the function of the GP(Y/F) domain in the IN of Tf1, a long terminal repeat retrotransposon of Schizosaccharomyces pombe. The activities of recombinant IN were measured with an assay that modeled the reverse of integration called disintegration. Although deletion of the entire C-terminal domain disrupted disintegration activity, an alanine substitution (P365A) in a conserved amino acid of the GP(Y/F) domain did not significantly reduce disintegration. When assayed for the ability to join two molecules of DNA in a reaction that modeled forward integration, the P365A substitution disrupted activity. UV cross-linking experiments detected DNA binding activity in the C-terminal domain and found that this activity was not reduced by substitutions in two conserved amino acids of the GP(Y/F) domain, G364A and P365A. Gel filtration and cross-linking of a 71-amino acid fragment containing the GP(Y/F) domain revealed a surprising ability to form dimers, trimers, and tetramers that was disrupted by the G364A and P365A substitutions. These results suggest that the GP(Y/F) residues may play roles in promoting multimerization and intermolecular strand joining. PMID:18397885

The outer-membrane export signal of Porphyromonas gingivalis type IX secretion system (T9SS) is a conserved C-terminal β-sandwich domain

PubMed Central

de Diego, Iñaki; Ksiazek, Miroslaw; Mizgalska, Danuta; Koneru, Lahari; Golik, Przemyslaw; Szmigielski, Borys; Nowak, Magdalena; Nowakowska, Zuzanna; Potempa, Barbara; Houston, John A.; Enghild, Jan J.; Thøgersen, Ida B.; Gao, Jinlong; Kwan, Ann H.; Trewhella, Jill; Dubin, Grzegorz; Gomis-Rüth, F. Xavier; Nguyen, Ky-Anh; Potempa, Jan

2016-01-01

In the recently characterized Type IX Secretion System (T9SS), the conserved C-terminal domain (CTD) in secreted proteins functions as an outer membrane translocation signal for export of virulence factors to the cell surface in the Gram-negative Bacteroidetes phylum. In the periodontal pathogen Porphyromonas gingivalis, the CTD is cleaved off by PorU sortase in a sequence-independent manner, and anionic lipopolysaccharide (A-LPS) is attached to many translocated proteins, thus anchoring them to the bacterial surface. Here, we solved the atomic structure of the CTD of gingipain B (RgpB) from P. gingivalis, alone and together with a preceding immunoglobulin-superfamily domain (IgSF). The CTD was found to possess a typical Ig-like fold encompassing seven antiparallel β-strands organized in two β-sheets, packed into a β-sandwich structure that can spontaneously dimerise through C-terminal strand swapping. Small angle X-ray scattering (SAXS) revealed no fixed orientation of the CTD with respect to the IgSF. By introducing insertion or substitution of residues within the inter-domain linker in the native protein, we were able to show that despite the region being unstructured, it nevertheless is resistant to general proteolysis. These data suggest structural motifs located in the two adjacent Ig-like domains dictate the processing of CTDs by the T9SS secretion pathway. PMID:27005013

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.

Molecular dynamics simulations of Hsp40 J-domain mutants identifies disruption of the critical HPD-motif as the key factor for impaired curing in vivo of the yeast prion [URE3].

PubMed

Xue, You-Lin; Wang, Hao; Riedy, Michael; Roberts, Brittany-Lee; Sun, Yuna; Song, Yong-Bo; Jones, Gary W; Masison, Daniel C; Song, Youtao

2018-05-01

Genetic screens using Saccharomyces cerevisiae have identified an array of Hsp40 (Ydj1p) J-domain mutants that are impaired in the ability to cure the yeast [URE3] prion through disrupting functional interactions with Hsp70. However, biochemical analysis of some of these Hsp40 J-domain mutants has so far failed to provide major insight into the specific functional changes in Hsp40-Hsp70 interactions. To explore the detailed structural and dynamic properties of the Hsp40 J-domain, 20 ns molecular dynamic simulations of 4 mutants (D9A, D36A, A30T, and F45S) and wild-type J-domain were performed, followed by Hsp70 docking simulations. Results demonstrated that although the Hsp70 interaction mechanism of the mutants may vary, the major structural change was targeted to the critical HPD motif of the J-domain. Our computational analysis fits well with previous yeast genetics studies regarding highlighting the importance of J-domain function in prion propagation. During the molecular dynamics simulations several important residues were identified and predicted to play an essential role in J-domain structure. Among these residues, Y26 and F45 were confirmed, using both in silico and in vivo methods, as being critical for Ydj1p function.

Spectroscopic Evolution of Disintegrating Planetesimals: Minute to Month Variability in the Circumstellar Gas Associated with WD 1145+017

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

Redfield, Seth; Cauley, P. Wilson; Duvvuri, Girish M.

With the recent discovery of transiting planetary material around WD 1145+017, a critical target has been identified that links the evolution of planetary systems with debris disks and their accretion onto the star. We present a series of observations, five epochs over a year, taken with Keck and the VLT, which for the first time show variability of circumstellar absorption in the gas disk surrounding WD 1145+017 on timescales of minutes to months. Circumstellar absorption is measured in more than 250 lines of 14 ions among 10 different elements associated with planetary composition, e.g., O, Mg, Ca, Ti, Cr, Mn,more » Fe, and Ni. Broad circumstellar gas absorption with a velocity spread of 225 km s{sup −1} is detected, but over the course of a year blueshifted absorption disappears, while redshifted absorption systematically increases. A correlation of equivalent width and oscillator strength indicates that the gas is not highly optically thick (median τ ≈ 2). We discuss simple models of an eccentric disk coupled with magnetospheric accretion to explain the basic observed characteristics of these high-resolution and high signal-to-noise observations. Variability is detected on timescales of minutes in the two most recent observations, showing a loss of redshifted absorption for tens of minutes, coincident with major transit events and consistent with gas hidden behind opaque transiting material. This system currently presents a unique opportunity to learn how the gas causing the spectroscopic, circumstellar absorption is associated with the ongoing accretion evidenced by photospheric contamination, as well as the transiting planetary material detected in photometric observations.« less

Once in a blue moon: detection of `bluing' during debris transits in the white dwarf WD 1145+017

NASA Astrophysics Data System (ADS)

Hallakoun, N.; Xu, S.; Maoz, D.; Marsh, T. R.; Ivanov, V. D.; Dhillon, V. S.; Bours, M. C. P.; Parsons, S. G.; Kerry, P.; Sharma, S.; Su, K.; Rengaswamy, S.; Pravec, P.; Kušnirák, P.; Kučáková, H.; Armstrong, J. D.; Arnold, C.; Gerard, N.; Vanzi, L.

2017-08-01

The first transiting planetesimal orbiting a white dwarf was recently detected in K2 data of WD 1145+017 and has been followed up intensively. The multiple, long and variable transits suggest the transiting objects are dust clouds, probably produced by a disintegrating asteroid. In addition, the system contains circumstellar gas, evident by broad absorption lines, mostly in the u΄ band, and a dust disc, indicated by an infrared excess. Here we present the first detection of a change in colour of WD 1145+017 during transits, using simultaneous multiband fast-photometry ULTRACAM measurements over the u΄g΄r΄I΄ bands. The observations reveal what appears to be 'bluing' during transits; transits are deeper in the redder bands, with a u΄ - r΄ colour difference of up to ˜-0.05 mag. We explore various possible explanations for the bluing, including limb darkening or peculiar dust properties. 'Spectral' photometry obtained by integrating over bandpasses in the spectroscopic data in and out of transit, compared to the photometric data, shows that the observed colour difference is most likely the result of reduced circumstellar absorption in the spectrum during transits. This indicates that the transiting objects and the gas share the same line of sight and that the gas covers the white dwarf only partially, as would be expected if the gas, the transiting debris and the dust emitting the infrared excess are part of the same general disc structure (although possibly at different radii). In addition, we present the results of a week-long monitoring campaign of the system using a global network of telescopes.

Saving the spandrels? Adaptive genomic variation in conservation and fisheries management.

PubMed

Pearse, D E

2016-12-01

As highlighted by many of the papers in this issue, research on the genomic basis of adaptive phenotypic variation in natural populations has made spectacular progress in the past few years, largely due to the advances in sequencing technology and analysis. Without question, the resulting genomic data will improve the understanding of regions of the genome under selection and extend knowledge of the genetic basis of adaptive evolution. What is far less clear, but has been the focus of active discussion, is how such information can or should transfer into conservation practice to complement more typical conservation applications of genetic data. Before such applications can be realized, the evolutionary importance of specific targets of selection relative to the genome-wide diversity of the species as a whole must be evaluated. The key issues for the incorporation of adaptive genomic variation in conservation and management are discussed here, using published examples of adaptive genomic variation associated with specific phenotypes in salmonids and other taxa to highlight practical considerations for incorporating such information into conservation programmes. Scenarios are described in which adaptive genomic data could be used in conservation or restoration, constraints on its utility and the importance of validating inferences drawn from new genomic data before applying them in conservation practice. Finally, it is argued that an excessive focus on preserving the adaptive variation that can be measured, while ignoring the vast unknown majority that cannot, is a modern twist on the adaptationist programme that Gould and Lewontin critiqued almost 40 years ago. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

The Structure of a Conserved Domain of TamB Reveals a Hydrophobic β Taco Fold.

PubMed

Josts, Inokentijs; Stubenrauch, Christopher James; Vadlamani, Grishma; Mosbahi, Khedidja; Walker, Daniel; Lithgow, Trevor; Grinter, Rhys

2017-12-05

The translocation and assembly module (TAM) plays a role in the transport and insertion of proteins into the bacterial outer membrane. TamB, a component of this system spans the periplasmic space to engage with its partner protein TamA. Despite efforts to characterize the TAM, the structure and mechanism of action of TamB remained enigmatic. Here we present the crystal structure of TamB amino acids 963-1,138. This region represents half of the conserved DUF490 domain, the defining feature of TamB. TamB 963-1138 consists of a concave, taco-shaped β sheet with a hydrophobic interior. This β taco structure is of dimensions capable of accommodating and shielding the hydrophobic side of an amphipathic β strand, potentially allowing TamB to chaperone nascent membrane proteins from the aqueous environment. In addition, sequence analysis suggests that the structure of TamB 963-1138 is shared by a large portion of TamB. This architecture could allow TamB to act as a conduit for membrane proteins. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

An allelic series reveals essential roles for FY in plant development in addition to flowering-time control.

PubMed

Henderson, Ian R; Liu, Fuquan; Drea, Sinead; Simpson, Gordon G; Dean, Caroline

2005-08-01

The autonomous pathway functions to promote flowering in Arabidopsis by limiting the accumulation of the floral repressor FLOWERING LOCUS C (FLC). Within this pathway FCA is a plant-specific, nuclear RNA-binding protein, which interacts with FY, a highly conserved eukaryotic polyadenylation factor. FCA and FY function to control polyadenylation site choice during processing of the FCA transcript. Null mutations in the yeast FY homologue Pfs2p are lethal. This raises the question as to whether these essential RNA processing functions are conserved in plants. Characterisation of an allelic series of fy mutations reveals that null alleles are embryo lethal. Furthermore, silencing of FY, but not FCA, is deleterious to growth in Nicotiana. The late-flowering fy alleles are hypomorphic and indicate a requirement for both intact FY WD repeats and the C-terminal domain in repression of FLC. The FY C-terminal domain binds FCA and in vitro assays demonstrate a requirement for both C-terminal FY-PPLPP repeats during this interaction. The expression domain of FY supports its roles in essential and flowering-time functions. Hence, FY may mediate both regulated and constitutive RNA 3'-end processing.

The structure and dynamics of tandem WW domains in a negative regulator of notch signaling, Suppressor of deltex.

PubMed

Fedoroff, Oleg Y; Townson, Sharon A; Golovanov, Alexander P; Baron, Martin; Avis, Johanna M

2004-08-13

WW domains mediate protein recognition, usually though binding to proline-rich sequences. In many proteins, WW domains occur in tandem arrays. Whether or how individual domains within such arrays cooperate to recognize biological partners is, as yet, poorly characterized. An important question is whether functional diversity of different WW domain proteins is reflected in the structural organization and ligand interaction mechanisms of their multiple domains. We have determined the solution structure and dynamics of a pair of WW domains (WW3-4) from a Drosophila Nedd4 family protein called Suppressor of deltex (Su(dx)), a regulator of Notch receptor signaling. We find that the binding of a type 1 PPPY ligand to WW3 stabilizes the structure with effects propagating to the WW4 domain, a domain that is not active for ligand binding. Both WW domains adopt the characteristic triple-stranded beta-sheet structure, and significantly, this is the first example of a WW domain structure to include a domain (WW4) lacking the second conserved Trp (replaced by Phe). The domains are connected by a flexible linker, which allows a hinge-like motion of domains that may be important for the recognition of functionally relevant targets. Our results contrast markedly with those of the only previously determined three-dimensional structure of tandem WW domains, that of the rigidly oriented WW domain pair from the RNA-splicing factor Prp40. Our data illustrate that arrays of WW domains can exhibit a variety of higher order structures and ligand interaction mechanisms.

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.

Interface conditions for domain decomposition with radical grid refinement

NASA Technical Reports Server (NTRS)

Scroggs, Jeffrey S.

1991-01-01

Interface conditions for coupling the domains in a physically motivated domain decomposition method are discussed. The domain decomposition is based on an asymptotic-induced method for the numerical solution of hyperbolic conservation laws with small viscosity. The method consists of multiple stages. The first stage is to obtain a first approximation using a first-order method, such as the Godunov scheme. Subsequent stages of the method involve solving internal-layer problem via a domain decomposition. The method is derived and justified via singular perturbation techniques.

RapA2 Is a Calcium-binding Lectin Composed of Two Highly Conserved Cadherin-like Domains That Specifically Recognize Rhizobium leguminosarum Acidic Exopolysaccharides*

PubMed Central

Abdian, Patricia L.; Caramelo, Julio J.; Ausmees, Nora; Zorreguieta, Angeles

2013-01-01

In silico analyses have revealed a conserved protein domain (CHDL) widely present in bacteria that has significant structural similarity to eukaryotic cadherins. A CHDL domain was shown to be present in RapA, a protein that is involved in autoaggregation of Rhizobium cells, biofilm formation, and adhesion to plant roots as shown by us and others. Structural similarity to cadherins suggested calcium-dependent oligomerization of CHDL domains as a mechanistic basis for RapA action. Here we show by circular dichroism spectroscopy, light scattering, isothermal titration calorimetry, and other methods that RapA2 from Rhizobium leguminosarum indeed exhibits a cadherin-like β-sheet conformation and that its proper folding and stability are dependent on the binding of one calcium ion per protein molecule. By further in silico analysis we also reveal that RapA2 consists of two CHDL domains and expand the range of CHDL-containing proteins in bacteria and archaea. However, light scattering assays at various concentrations of added calcium revealed that RapA2 formed neither homo-oligomers nor hetero-oligomers with RapB (a distinct CHDL protein), indicating that RapA2 does not mediate cellular interactions through a cadherin-like mechanism. Instead, we demonstrate that RapA2 interacts specifically with the acidic exopolysaccharides (EPSs) produced by R. leguminosarum in a calcium-dependent manner, sustaining a role of these proteins in the development of the biofilm matrix made of EPS. Because EPS binding by RapA2 can only be attributed to its two CHDL domains, we propose that RapA2 is a calcium-dependent lectin and that CHDL domains in various bacterial and archaeal proteins confer carbohydrate binding activity to these proteins. PMID:23235153

Genomic positional conservation identifies topological anchor point RNAs linked to developmental loci.

PubMed

Amaral, Paulo P; Leonardi, Tommaso; Han, Namshik; Viré, Emmanuelle; Gascoigne, Dennis K; Arias-Carrasco, Raúl; Büscher, Magdalena; Pandolfini, Luca; Zhang, Anda; Pluchino, Stefano; Maracaja-Coutinho, Vinicius; Nakaya, Helder I; Hemberg, Martin; Shiekhattar, Ramin; Enright, Anton J; Kouzarides, Tony

2018-03-15

The mammalian genome is transcribed into large numbers of long noncoding RNAs (lncRNAs), but the definition of functional lncRNA groups has proven difficult, partly due to their low sequence conservation and lack of identified shared properties. Here we consider promoter conservation and positional conservation as indicators of functional commonality. We identify 665 conserved lncRNA promoters in mouse and human that are preserved in genomic position relative to orthologous coding genes. These positionally conserved lncRNA genes are primarily associated with developmental transcription factor loci with which they are coexpressed in a tissue-specific manner. Over half of positionally conserved RNAs in this set are linked to chromatin organization structures, overlapping binding sites for the CTCF chromatin organiser and located at chromatin loop anchor points and borders of topologically associating domains (TADs). We define these RNAs as topological anchor point RNAs (tapRNAs). Characterization of these noncoding RNAs and their associated coding genes shows that they are functionally connected: they regulate each other's expression and influence the metastatic phenotype of cancer cells in vitro in a similar fashion. Furthermore, we find that tapRNAs contain conserved sequence domains that are enriched in motifs for zinc finger domain-containing RNA-binding proteins and transcription factors, whose binding sites are found mutated in cancers. This work leverages positional conservation to identify lncRNAs with potential importance in genome organization, development and disease. The evidence that many developmental transcription factors are physically and functionally connected to lncRNAs represents an exciting stepping-stone to further our understanding of genome regulation.

Evolution of SH2 domains and phosphotyrosine signalling networks

PubMed Central

Liu, Bernard A.; Nash, Piers D.

2012-01-01

Src homology 2 (SH2) domains mediate selective protein–protein interactions with tyrosine phosphorylated proteins, and in doing so define specificity of phosphotyrosine (pTyr) signalling networks. SH2 domains and protein-tyrosine phosphatases expand alongside protein-tyrosine kinases (PTKs) to coordinate cellular and organismal complexity in the evolution of the unikont branch of the eukaryotes. Examination of conserved families of PTKs and SH2 domain proteins provides fiduciary marks that trace the evolutionary landscape for the development of complex cellular systems in the proto-metazoan and metazoan lineages. The evolutionary provenance of conserved SH2 and PTK families reveals the mechanisms by which diversity is achieved through adaptations in tissue-specific gene transcription, altered ligand binding, insertions of linear motifs and the gain or loss of domains following gene duplication. We discuss mechanisms by which pTyr-mediated signalling networks evolve through the development of novel and expanded families of SH2 domain proteins and the elaboration of connections between pTyr-signalling proteins. These changes underlie the variety of general and specific signalling networks that give rise to tissue-specific functions and increasingly complex developmental programmes. Examination of SH2 domains from an evolutionary perspective provides insight into the process by which evolutionary expansion and modification of molecular protein interaction domain proteins permits the development of novel protein-interaction networks and accommodates adaptation of signalling networks. PMID:22889907

Structural Insight into the Mechanism of c-di-GMP hydrolysis by EAL domain phosphodiesterases.

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

Tchigvintsev, A.; Xu, X.; Singer, A.

2010-08-01

Cyclic diguanylate (or bis-(3'-5') cyclic dimeric guanosine monophosphate; c-di-GMP) is a ubiquitous second messenger that regulates diverse cellular functions, including motility, biofilm formation, cell cycle progression, and virulence in bacteria. In the cell, degradation of c-di-GMP is catalyzed by highly specific EAL domain phosphodiesterases whose catalytic mechanism is still unclear. Here, we purified 13 EAL domain proteins from various organisms and demonstrated that their catalytic activity is associated with the presence of 10 conserved EAL domain residues. The crystal structure of the TBD1265 EAL domain was determined in free state (1.8 {angstrom}) and in complex with c-di-GMP (2.35 {angstrom}), andmore » unveiled the role of conserved residues in substrate binding and catalysis. The structure revealed the presence of two metal ions directly coordinated by six conserved residues, two oxygens of c-di-GMP phosphate, and potential catalytic water molecule. Our results support a two-metal-ion catalytic mechanism of c-di-GMP hydrolysis by EAL domain phosphodiesterases.« less

A Conserved Acidic Motif in the N-Terminal Domain of Nitrate Reductase Is Necessary for the Inactivation of the Enzyme in the Dark by Phosphorylation and 14-3-3 Binding1

PubMed Central

Pigaglio, Emmanuelle; Durand, Nathalie; Meyer, Christian

1999-01-01

It has previously been shown that the N-terminal domain of tobacco (Nicotiana tabacum) nitrate reductase (NR) is involved in the inactivation of the enzyme by phosphorylation, which occurs in the dark (L. Nussaume, M. Vincentz, C. Meyer, J.P. Boutin, and M. Caboche [1995] Plant Cell 7: 611–621). The activity of a mutant NR protein lacking this N-terminal domain was no longer regulated by light-dark transitions. In this study smaller deletions were performed in the N-terminal domain of tobacco NR that removed protein motifs conserved among higher plant NRs. The resulting truncated NR-coding sequences were then fused to the cauliflower mosaic virus 35S RNA promoter and introduced in NR-deficient mutants of the closely related species Nicotiana plumbaginifolia. We found that the deletion of a conserved stretch of acidic residues led to an active NR protein that was more thermosensitive than the wild-type enzyme, but it was relatively insensitive to the inactivation by phosphorylation in the dark. Therefore, the removal of this acidic stretch seems to have the same effects on NR activation state as the deletion of the N-terminal domain. A hypothetical explanation for these observations is that a specific factor that impedes inactivation remains bound to the truncated enzyme. A synthetic peptide derived from this acidic protein motif was also found to be a good substrate for casein kinase II. PMID:9880364

Interactions between the S-Domain Receptor Kinases and AtPUB-ARM E3 Ubiquitin Ligases Suggest a Conserved Signaling Pathway in Arabidopsis1[W][OA

PubMed Central

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

2008-01-01

The Arabidopsis (Arabidopsis thaliana) genome encompasses multiple receptor kinase families with highly variable extracellular domains. Despite their large numbers, the various ligands and the downstream interacting partners for these kinases have been deciphered only for a few members. One such member, the S-receptor kinase, is known to mediate the self-incompatibility (SI) response in Brassica. S-receptor kinase has been shown to interact and phosphorylate a U-box/ARM-repeat-containing E3 ligase, ARC1, which, in turn, acts as a positive regulator of the SI response. In an effort to identify conserved signaling pathways in Arabidopsis, we performed yeast two-hybrid analyses of various S-domain receptor kinase family members with representative Arabidopsis plant U-box/ARM-repeat (AtPUB-ARM) E3 ligases. The kinase domains from S-domain receptor kinases were found to interact with ARM-repeat domains from AtPUB-ARM proteins. These kinase domains, along with M-locus protein kinase, a positive regulator of SI response, were also able to phosphorylate the ARM-repeat domains in in vitro phosphorylation assays. Subcellular localization patterns were investigated using transient expression assays in tobacco (Nicotiana tabacum) BY-2 cells and changes were detected in the presence of interacting kinases. Finally, potential links to the involvement of these interacting modules to the hormone abscisic acid (ABA) were investigated. Interestingly, AtPUB9 displayed redistribution to the plasma membrane of BY-2 cells when either treated with ABA or coexpressed with the active kinase domain of ARK1. As well, T-DNA insertion mutants for ARK1 and AtPUB9 lines were altered in their ABA sensitivity during germination and acted at or upstream of ABI3, indicating potential involvement of these proteins in ABA responses. PMID:18552232

Evaluating, Comparing, and Interpreting Protein Domain Hierarchies

PubMed Central

2014-01-01

Abstract Arranging protein domain sequences hierarchically into evolutionarily divergent subgroups is important for investigating evolutionary history, for speeding up web-based similarity searches, for identifying sequence determinants of protein function, and for genome annotation. However, whether or not a particular hierarchy is optimal is often unclear, and independently constructed hierarchies for the same domain can often differ significantly. This article describes methods for statistically evaluating specific aspects of a hierarchy, for probing the criteria underlying its construction and for direct comparisons between hierarchies. Information theoretical notions are used to quantify the contributions of specific hierarchical features to the underlying statistical model. Such features include subhierarchies, sequence subgroups, individual sequences, and subgroup-associated signature patterns. Underlying properties are graphically displayed in plots of each specific feature's contributions, in heat maps of pattern residue conservation, in “contrast alignments,” and through cross-mapping of subgroups between hierarchies. Together, these approaches provide a deeper understanding of protein domain functional divergence, reveal uncertainties caused by inconsistent patterns of sequence conservation, and help resolve conflicts between competing hierarchies. PMID:24559108

Proteins with an Euonymus lectin-like domain are ubiquitous in Embryophyta

PubMed Central

2009-01-01

Background Cloning of the Euonymus lectin led to the discovery of a novel domain that also occurs in some stress-induced plant proteins. The distribution and the diversity of proteins with an Euonymus lectin (EUL) domain were investigated using detailed analysis of sequences in publicly accessible genome and transcriptome databases. Results Comprehensive in silico analyses indicate that the recently identified Euonymus europaeus lectin domain represents a conserved structural unit of a novel family of putative carbohydrate-binding proteins, which will further be referred to as the Euonymus lectin (EUL) family. The EUL domain is widespread among plants. Analysis of retrieved sequences revealed that some sequences consist of a single EUL domain linked to an unrelated N-terminal domain whereas others comprise two in tandem arrayed EUL domains. A new classification system for these lectins is proposed based on the overall domain architecture. Evolutionary relationships among the sequences with EUL domains are discussed. Conclusion The identification of the EUL family provides the first evidence for the occurrence in terrestrial plants of a highly conserved plant specific domain. The widespread distribution of the EUL domain strikingly contrasts the more limited or even narrow distribution of most other lectin domains found in plants. The apparent omnipresence of the EUL domain is indicative for a universal role of this lectin domain in plants. Although there is unambiguous evidence that several EUL domains possess carbohydrate-binding activity further research is required to corroborate the carbohydrate-binding properties of different members of the EUL family. PMID:19930663

Conserved enzymes mediate the early reactions of carotenoid biosynthesis in nonphotosynthetic and photosynthetic prokaryotes

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

Armstrong, G.A.; Hearst, J.E.; Alberti, M.

1990-12-01

Carotenoids comprise one of the most widespread classes of pigments found in nature. The first reactions of C{sub 40} carotenoid biosynthesis proceed through common intermediates in all organisms, suggesting the evolutionary conservation of early enzymes from this pathway. The authors report here the nucleotide sequence of three genes from the carotenoid biosynthesis gene cluster of Erwinia herbicola, a nonphotosynthetic epiphytic bacterium, which encode homologs of the CrtB, CrtE, and CrtI proteins of Rhodobacter capsulatus, a purple nonsulfur photosynthetic bacterium. CrtB (prephytoene pyrophosphate synthase), CrtE (phytoene synthase), and CrtI (phytoene dehydrogenase) are required for the first three reactions specific to themore » carotenoid branch of general isoprenoid metabolism. All three dehydrogenases possess a hydrophobic N-terminal domain containing a putative ADP-binding {beta}{alpha}{beta} fold characteristic of enzymes known to bind FAD or NAD(P) cofactors. These data indicate the structural conservation of early carotenoid biosynthesis enzymes in evolutionary diverse organisms.« less

Cognitive profile in Wilson's disease: a case series of 31 patients.

PubMed

Wenisch, E; De Tassigny, A; Trocello, J-M; Beretti, J; Girardot-Tinant, N; Woimant, F

2013-12-01

Wilson's disease (WD) is a rare autosomal recessive disorder of copper metabolism. If untreated, WD, which is initially a liver disease, can turn into a multi-systemic disease with neurological involvement. Very few studies have described cognitive impairment in WD. The aim of this study is to report the cognitive profile of 31 treated WD patients. Patients were classed into two groups using the Unified Wilson Disease Rating Scale (UWDRS): WD patients without neurological signs (WD-N(-)) (n=13), and WD patients with neurological signs (WD-N(+)) (n=18). The patients participated in a neuropsychological assessment evaluating memory, executive function and visuo-spatial abilities. Both groups performed well for verbal intelligence and episodic memory skills. However, the majority of these patients exhibited altered performance for at least one cognitive test, particularly in the executive domain. The WD-N(+) group performed less well than the WD-N(-) group on cognitive tests involving rapid motor function, abstract thinking, working memory and top-down inhibitory control. Cognitive impairment in treated WD patients essentially affects executive function involving fronto-striatal circuits. Verbal intelligence and episodic memory abilities seem to be remarkably preserved. Neuropsychological assessment is a valuable tool to evaluate the presence and the consequences of these cognitive impairments in WD patients with or without neurological signs in the course of this chronic disease. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Climate-induced change of environmentally defined floristic domains: A conservation based vulnerability framework

Treesearch

Debbie Jewitt; Barend F.N. Erasmus; Peter S. Goodman; Timothy G. O' Connor; William W. Hargrove; Damian M. Maddalena; Ed. T.F. Witkowski

2015-01-01

Global climate change is having marked influences on species distributions, phenology and ecosystem composition and raises questions as to the effectiveness of current conservation strategies. Conservation planning has only recently begun to adequately account for dynamic threats such as climate change. We propose a method to incorporate climate-dynamic environmental...

Characterization of STIP, a multi-domain nuclear protein, highly conserved in metazoans, and essential for embryogenesis in Caenorhabditis elegans.

PubMed

Ji, Qiongmei; Huang, Cheng-Han; Peng, Jianbin; Hashmi, Sarwar; Ye, Tianzhang; Chen, Ying

2007-04-15

We report here the identification and characterization of STIP, a multi-domain nuclear protein that contains a G-patch, a coiled-coil, and several short tryptophan-tryptophan repeats highly conserved in metazoan species. To analyze their functional role in vivo, we cloned nematode stip-1 genes and determined the spatiotemporal pattern of Caenorhabditis elegans STIP-1 protein. RNA analyses and Western blots revealed that stip-1 mRNA was produced via trans-splicing and translated as a 95-kDa protein. Using reporter constructs, we found STIP-1 to be expressed at all developmental stages and in many tissue/cell types including worm oocyte nuclei. We found that STIP-1 is targeted to the nucleus and forms large polymers with a rod-like shape when expressed in mammalian cells. Using deletion mutants, we mapped the regions of STIP-1 involved in nuclear import and polymer assembly. We further showed that knockdown of C. elegans stip-1 by RNA interference arrested development and resulted in morphologic abnormalities around the 16-cell stage followed by 100% lethality, suggesting its essential role in worm embryogenesis. Importantly, the embryonic lethal phenotype could be faithfully rescued with Drosophila and human genes via transgenic expression. Our data provide the first direct evidence that STIP have a conserved essential nuclear function across metazoans from worms to humans.

18 CFR 35.40 - Ancillary services.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Ancillary services. 35.40 Section 35.40 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION... Sales of Electric Energy, Capacity and Ancillary Services at Market-Based Rates § 35.40 Ancillary...

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

Regulation of the expression of plant resistance gene SNC1 by a protein with a conserved BAT2 domain.

PubMed

Li, Yingzhong; Tessaro, Mark J; Li, Xin; Zhang, Yuelin

2010-07-01

Plant Resistance (R) genes encode immune receptors that recognize pathogens and activate defense responses. Because of fitness costs associated with maintaining R protein-mediated resistance, expression levels of R genes have to be tightly regulated. However, mechanisms on how R-gene expression is regulated are poorly understood. Here we show that MODIFIER OF snc1, 1 (MOS1) regulates the expression of SUPPRESSOR OF npr1-1, CONSTITUTIVE1 (SNC1), which encodes a Toll/interleukin receptor-nucleotide binding site-leucine-rich repeat type of R protein in Arabidopsis (Arabidopsis thaliana). In the mos1 loss-of-function mutant plants, snc1 expression is repressed and constitutive resistance responses mediated by snc1 are lost. The repression of snc1 expression in mos1 is released by knocking out DECREASE IN DNA METHYLATION1. In mos1 mutants, DNA methylation in a region upstream of SNC1 is altered. Furthermore, expression of snc1 transgenes using the native promoter does not require MOS1, indicating that regulation of SNC1 expression by MOS1 is at the chromatin level. Map-based cloning of MOS1 revealed that it encodes a novel protein with a HLA-B ASSOCIATED TRANSCRIPT2 (BAT2) domain that is conserved in plants and animals. Our study on MOS1 suggests that BAT2 domain-containing proteins may function in regulation of gene expression at chromatin level.

Conserved TCP domain of Sas-4/CPAP is essential for pericentriolar material tethering during centrosome biogenesis

PubMed Central

Zheng, Xiangdong; Gooi, Li Ming; Wason, Arpit; Gabriel, Elke; Mehrjardi, Narges Zare; Yang, Qian; Zhang, Xingrun; Debec, Alain; Basiri, Marcus L.; Avidor-Reiss, Tomer; Pozniakovsky, Andrei; Poser, Ina; Šarić, Tomo; Hyman, Anthony A.; Li, Haitao; Gopalakrishnan, Jay

2014-01-01

Pericentriolar material (PCM) recruitment to centrioles forms a key step in centrosome biogenesis. Deregulation of this process leads to centrosome aberrations causing disorders, one of which is autosomal recessive primary microcephaly (MCPH), a neurodevelopmental disorder where brain size is reduced. During PCM recruitment, the conserved centrosomal protein Sas-4/CPAP/MCPH6, known to play a role in centriole formation, acts as a scaffold for cytoplasmic PCM complexes to bind and then tethers them to centrioles to form functional centrosomes. To understand Sas-4’s tethering role, we determined the crystal structure of its T complex protein 10 (TCP) domain displaying a solvent-exposed single-layer of β-sheets fold. This unique feature of the TCP domain suggests that it could provide an “extended surface-like” platform to tether the Sas-4–PCM scaffold to a centriole. Functional studies in Drosophila, human cells, and human induced pluripotent stem cell-derived neural progenitor cells were used to test this hypothesis, where point mutations within the 9–10th β-strands (β9–10 mutants including a MCPH-associated mutation) perturbed PCM tethering while allowing Sas-4/CPAP to scaffold cytoplasmic PCM complexes. Specifically, the Sas-4 β9–10 mutants displayed perturbed interactions with Ana2, a centrosome duplication factor, and Bld-10, a centriole microtubule-binding protein, suggesting a role for the β9–10 surface in mediating protein–protein interactions for efficient Sas-4–PCM scaffold centriole tethering. Hence, we provide possible insights into how centrosomal protein defects result in human MCPH and how Sas-4 proteins act as a vehicle to tether PCM complexes to centrioles independent of its well-known role in centriole duplication. PMID:24385583

Role of conserved cysteine residues in Herbaspirillum seropedicae NifA activity.

PubMed

Oliveira, Marco A S; Baura, Valter A; Aquino, Bruno; Huergo, Luciano F; Kadowaki, Marco A S; Chubatsu, Leda S; Souza, Emanuel M; Dixon, Ray; Pedrosa, Fábio O; Wassem, Roseli; Monteiro, Rose A

2009-01-01

Herbaspirillum seropedicae is an endophytic diazotrophic bacterium that associates with economically important crops. NifA protein, the transcriptional activator of nif genes in H. seropedicae, binds to nif promoters and, together with RNA polymerase-sigma(54) holoenzyme, catalyzes the formation of open complexes to allow transcription initiation. The activity of H. seropedicae NifA is controlled by ammonium and oxygen levels, but the mechanisms of such control are unknown. Oxygen sensitivity is attributed to a conserved motif of cysteine residues in NifA that spans the central AAA+ domain and the interdomain linker that connects the AAA+ domain to the C-terminal DNA binding domain. Here we mutagenized this conserved motif of cysteines and assayed the activity of mutant proteins in vivo. We also purified the mutant variants of NifA and tested their capacity to bind to the nifB promoter region. Chimeric proteins between H. seropedicae NifA, an oxygen-sensitive protein, and Azotobacter vinelandii NifA, an oxygen-tolerant protein, were constructed and showed that the oxygen response is conferred by the central AAA+ and C-terminal DNA binding domains of H. seropedicae NifA. We conclude that the conserved cysteine motif is essential for NifA activity, although single cysteine-to-serine mutants are still competent at binding DNA.

Alternative dimerization interfaces in the glucocorticoid receptor-α ligand binding domain.

PubMed

Bianchetti, Laurent; Wassmer, Bianca; Defosset, Audrey; Smertina, Anna; Tiberti, Marion L; Stote, Roland H; Dejaegere, Annick

2018-04-30

Nuclear hormone receptors (NRs) constitute a large family of multi-domain ligand-activated transcription factors. Dimerization is essential for their regulation, and both DNA binding domain (DBD) and ligand binding domain (LBD) are implicated in dimerization. Intriguingly, the glucocorticoid receptor-α (GRα) presents a DBD dimeric architecture similar to that of the homologous estrogen receptor-α (ERα), but an atypical dimeric architecture for the LBD. The physiological relevance of the proposed GRα LBD dimer is a subject of debate. We analyzed all GRα LBD homodimers observed in crystals using an energetic analysis based on the PISA and on the MM/PBSA methods and a sequence conservation analysis, using the ERα LBD dimer as a reference point. Several dimeric assemblies were observed for GRα LBD. The assembly generally taken to be physiologically relevant showed weak binding free energy and no significant residue conservation at the contact interface, while an alternative homodimer mediated by both helix 9 and C-terminal residues showed significant binding free energy and residue conservation. However, none of the GRα LBD assemblies found in crystals are as stable or conserved as the canonical ERα LBD dimer. GRα C-terminal sequence (F-domain) forms a steric obstacle to the canonical dimer assembly in all available structures. Our analysis calls for a re-examination of the currently accepted GRα homodimer structure and experimental investigations of the alternative architectures. This work questions the validity of the currently accepted architecture. This has implications for interpreting physiological data and for therapeutic design pertaining to glucocorticoid research. Copyright © 2018. Published by Elsevier B.V.

Effects of mutations within the SV40 large T antigen ATPase/p53 binding domain on viral replication and transformation.

PubMed

Peden, K W; Srinivasan, A; Vartikar, J V; Pipas, J M

1998-01-01

The simian virus 40 (SV40) large T antigen is a 708 amino-acid protein possessing multiple biochemical activities that play distinct roles in productive infection or virus-induced cell transformation. The carboxy-terminal portion of T antigen includes a domain that carries the nucleotide binding and ATPase activities of the protein, as well as sequences required for T antigen to associate with the cellular tumor suppressor p53. Consequently this domain functions both in viral DNA replication and cellular transformation. We have generated a collection of SV40 mutants with amino-acid deletions, insertions or substitutions in specific domains of the protein. Here we report the properties of nine mutants with single or multiple substitutions between amino acids 402 and 430, a region thought to be important for both the p53 binding and ATPase functions. The mutants were examined for the ability to produce infectious progeny virions, replicate viral DNA in vivo, perform in trans complementation tests, and transform established cell lines. Two of the mutants exhibited a wild-type phenotype in all these tests. The remaining seven mutants were defective for plaque formation and viral DNA replication, but in each case these defects could be complemented by a wild-type T antigen supplied in trans. One of these replication-defective mutants efficiently transformed the REF52 and C3H10T1/2 cell lines as assessed by the dense-focus assay. The remaining six mutants were defective for transforming REF52 cells and transformed the C3H10T1/2 line with a reduced efficiency. The ability of mutant T antigen to transform REF52 cells correlated with their ability to induce increased levels of p53.

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

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

PubMed Central

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

2008-01-01

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

Replication domains are self-interacting structural chromatin units of human chromosomes

NASA Astrophysics Data System (ADS)

Arneodo, Alain

2011-03-01

In higher eukaryotes, the absence of specific sequence motifs marking the origins of replication has been a serious hindrance to the understanding of the mechanisms that regulate the initiation and the maintenance of the replication program in different cell types. In silico analysis of nucleotide compositional skew has predicted the existence, in the germline, of replication N-domains bordered by putative replication origins and where the skew decreases rather linearly as the signature of a progressive inversion of the average fork polarity. Here, from the demonstration that the average fork polarity can be directly extracted from the derivative of replication timing profiles, we develop a wavelet-based pattern recognition methodology to delineate replication U-domains where the replication timing profile is shaped as a U and its derivative as a N. Replication U-domains are robustly found in seven cell lines as covering a significant portion (40-50%) of the human genome where the replication timing data actually displays some plasticity between cell lines. The early replication initiation zones at U-domains borders are found to be hypersensitive to DNase I cleavage, to be associated with transcriptional activity and to present a significant enrichment in insular-binding proteins CTCF, the hallmark of an open chromatin structure. A comparative analysis of genome-wide chromatin interaction (HiC) data shows that replication-U domains correspond to self-interacting structural high order chromatin units of megabase characteristic size. Taken together, these findings provide evidence that the epigenetic compartmentalization of the human genome into autonomous replication U-domains comes along with an extensive remodelling of the threedimensional chromosome architecture during development or in specific diseases. The observed cell specific conservation of the replication timing between the human and mouse genomes strongly suggests that this chromosome organization into

Simultaneous infrared and optical observations of the transiting debris cloud around WD 1145+017

NASA Astrophysics Data System (ADS)

Zhou, G.; Kedziora-Chudczer, L.; Bailey, J.; Marshall, J. P.; Bayliss, D. D. R.; Stockdale, C.; Nelson, P.; Tan, T. G.; Rodriguez, J. E.; Tinney, C. G.; Dragomir, D.; Colon, K.; Shporer, A.; Bento, J.; Sefako, R.; Horne, K.; Cochran, W.

2016-12-01

We present multiwavelength photometric monitoring of WD 1145+017, a white dwarf exhibiting periodic dimming events interpreted to be the transits of orbiting, disintegrating planetesimals. Our observations include the first set of near-infrared light curves for the object, obtained on multiple nights over the span of 1 month, and recorded multiple transit events with depths varying between ˜20 and 50 per cent. Simultaneous near-infrared and optical observations of the deepest and longest duration transit event were obtained on two epochs with the Anglo-Australian Telescope and three optical facilities, over the wavelength range of 0.5-1.2 μm. These observations revealed no measurable difference in transit depths for multiple photometric pass bands, allowing us to place a 2σ lower limit of 0.8 μm on the grain size in the putative transiting debris cloud. This conclusion is consistent with the spectral energy distribution of the system, which can be fit with an optically thin debris disc with minimum particle sizes of 10^{+5}_{-3} μm.

Plant homologs of mammalian MBT-domain protein-regulated KDM1 histone lysine demethylases do not interact with plant Tudor/PWWP/MBT-domain proteins

PubMed Central

Sadiq, Irfan; Keren, Ido; Citovsky, Vitaly

2016-01-01

Histone lysine demethylases of the LSD1/KDM1 family play important roles in epigenetic regulation of eukaryotic chromatin, and they are conserved between plants and animals. Mammalian LSD1 is thought to be targeted to its substrates, i.e., methylated histones, by an MBT-domain protein SFMBT1 that represents a component of the LSD1-based repressor complex and binds methylated histones. Because MBT-domain proteins are conserved between different organisms, from animals to plants, we examined whether the KDM1-type histone lysine demethylases KDM1C and FLD of Arabidopsis interact with the Arabidopsis Tudor/PWWP/MBT-domain SFMBT1-like proteins SL1, SL2, SL3, and SL4. No such interaction was detected using the bimolecular fluorescence complementation assay in living plant cells. Thus, plants most likely direct their KDM1 chromatin-modifying enzymes to methylated histones of the target chromatin by a mechanism different from that employed by the mammalian cells. PMID:26826387

Plant homologs of mammalian MBT-domain protein-regulated KDM1 histone lysine demethylases do not interact with plant Tudor/PWWP/MBT-domain proteins.

PubMed

Sadiq, Irfan; Keren, Ido; Citovsky, Vitaly

2016-02-19

Histone lysine demethylases of the LSD1/KDM1 family play important roles in epigenetic regulation of eukaryotic chromatin, and they are conserved between plants and animals. Mammalian LSD1 is thought to be targeted to its substrates, i.e., methylated histones, by an MBT-domain protein SFMBT1 that represents a component of the LSD1-based repressor complex and binds methylated histones. Because MBT-domain proteins are conserved between different organisms, from animals to plants, we examined whether the KDM1-type histone lysine demethylases KDM1C and FLD of Arabidopsis interact with the Arabidopsis Tudor/PWWP/MBT-domain SFMBT1-like proteins SL1, SL2, SL3, and SL4. No such interaction was detected using the bimolecular fluorescence complementation assay in living plant cells. Thus, plants most likely direct their KDM1 chromatin-modifying enzymes to methylated histones of the target chromatin by a mechanism different from that employed by the mammalian cells. Copyright © 2016 Elsevier Inc. All rights reserved.

50 CFR Table 3 (south) to Part 660... - Non-Trawl Rockfish Conservation Areas and Trip Limits for Open Access Gears South of 40°10′ N. Lat.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 50 Wildlife and Fisheries 13 2014-10-01 2014-10-01 false Non-Trawl Rockfish Conservation Areas and Trip Limits for Open Access Gears South of 40°10ⲠN. Lat. 3 Table 3 (South) to Part 660, Subpart F Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION...

50 CFR Table 3 (north) to Part 660... - Non-Trawl Rockfish Conservation Areas and Trip Limits for Open Access Gears North of 40°10′ N. Lat.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 50 Wildlife and Fisheries 13 2014-10-01 2014-10-01 false Non-Trawl Rockfish Conservation Areas and Trip Limits for Open Access Gears North of 40°10ⲠN. Lat. 3 Table 3 (North) to Part 660, Subpart F Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION...

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].

Homozygosity mapping and targeted genomic sequencing reveal the gene responsible for cerebellar hypoplasia and quadrupedal locomotion in a consanguineous kindred

PubMed Central

Gulsuner, Suleyman; Tekinay, Ayse Begum; Doerschner, Katja; Boyaci, Huseyin; Bilguvar, Kaya; Unal, Hilal; Ors, Aslihan; Onat, O. Emre; Atalar, Ergin; Basak, A. Nazli; Topaloglu, Haluk; Kansu, Tulay; Tan, Meliha; Tan, Uner; Gunel, Murat; Ozcelik, Tayfun

2011-01-01

The biological basis for the development of the cerebro-cerebellar structures required for posture and gait in humans is poorly understood. We investigated a large consanguineous family from Turkey exhibiting an extremely rare phenotype associated with quadrupedal locomotion, mental retardation, and cerebro-cerebellar hypoplasia, linked to a 7.1-Mb region of homozygosity on chromosome 17p13.1–13.3. Diffusion weighted imaging and fiber tractography of the patients' brains revealed morphological abnormalities in the cerebellum and corpus callosum, in particular atrophy of superior, middle, and inferior peduncles of the cerebellum. Structural magnetic resonance imaging showed additional morphometric abnormalities in several cortical areas, including the corpus callosum, precentral gyrus, and Brodmann areas BA6, BA44, and BA45. Targeted sequencing of the entire homozygous region in three affected individuals and two obligate carriers uncovered a private missense mutation, WDR81 p.P856L, which cosegregated with the condition in the extended family. The mutation lies in a highly conserved region of WDR81, flanked by an N-terminal BEACH domain and C-terminal WD40 beta-propeller domains. WDR81 is predicted to be a transmembrane protein. It is highly expressed in the cerebellum and corpus callosum, in particular in the Purkinje cell layer of the cerebellum. WDR81 represents the third gene, after VLDLR and CA8, implicated in quadrupedal locomotion in humans. PMID:21885617

Improvement in Protein Domain Identification Is Reached by Breaking Consensus, with the Agreement of Many Profiles and Domain Co-occurrence

PubMed Central

Bernardes, Juliana; Zaverucha, Gerson; Vaquero, Catherine; Carbone, Alessandra

2016-01-01

Traditional protein annotation methods describe known domains with probabilistic models representing consensus among homologous domain sequences. However, when relevant signals become too weak to be identified by a global consensus, attempts for annotation fail. Here we address the fundamental question of domain identification for highly divergent proteins. By using high performance computing, we demonstrate that the limits of state-of-the-art annotation methods can be bypassed. We design a new strategy based on the observation that many structural and functional protein constraints are not globally conserved through all species but might be locally conserved in separate clades. We propose a novel exploitation of the large amount of data available: 1. for each known protein domain, several probabilistic clade-centered models are constructed from a large and differentiated panel of homologous sequences, 2. a decision-making protocol combines outcomes obtained from multiple models, 3. a multi-criteria optimization algorithm finds the most likely protein architecture. The method is evaluated for domain and architecture prediction over several datasets and statistical testing hypotheses. Its performance is compared against HMMScan and HHblits, two widely used search methods based on sequence-profile and profile-profile comparison. Due to their closeness to actual protein sequences, clade-centered models are shown to be more specific and functionally predictive than the broadly used consensus models. Based on them, we improved annotation of Plasmodium falciparum protein sequences on a scale not previously possible. We successfully predict at least one domain for 72% of P. falciparum proteins against 63% achieved previously, corresponding to 30% of improvement over the total number of Pfam domain predictions on the whole genome. The method is applicable to any genome and opens new avenues to tackle evolutionary questions such as the reconstruction of ancient domain

The crystal structure of the regulatory domain of the human sodium-driven chloride/bicarbonate exchanger.

PubMed

Alvadia, Carolina M; Sommer, Theis; Bjerregaard-Andersen, Kaare; Damkier, Helle Hasager; Montrasio, Michele; Aalkjaer, Christian; Morth, J Preben

2017-09-21

The sodium-driven chloride/bicarbonate exchanger (NDCBE) is essential for maintaining homeostatic pH in neurons. The crystal structure at 2.8 Å resolution of the regulatory N-terminal domain of human NDCBE represents the first crystal structure of an electroneutral sodium-bicarbonate cotransporter. The crystal structure forms an equivalent dimeric interface as observed for the cytoplasmic domain of Band 3, and thus establishes that the consensus motif VTVLP is the key minimal dimerization motif. The VTVLP motif is highly conserved and likely to be the physiologically relevant interface for all other members of the SLC4 family. A novel conserved Zn 2+ -binding motif present in the N-terminal domain of NDCBE is identified and characterized in vitro. Cellular studies confirm the Zn 2+ dependent transport of two electroneutral bicarbonate transporters, NCBE and NBCn1. The Zn 2+ site is mapped to a cluster of histidines close to the conserved ETARWLKFEE motif and likely plays a role in the regulation of this important motif. The combined structural and bioinformatics analysis provides a model that predicts with additional confidence the physiologically relevant interface between the cytoplasmic domain and the transmembrane domain.

On the role of second number-conserving functional derivatives

NASA Astrophysics Data System (ADS)

Gál, Tamás

2006-06-01

It is found that number-conserving second derivatives, of functional differentiation constrained to the domain of functional variables ρ(x) of a given norm ∫ρ(x)dx, are not obtained via two successive number-conserving differentiations, contrary to the case of unrestricted second derivatives. Investigating the role of second number-conserving derivatives, with the density-functional formulation of time-dependent quantum mechanics in focus, it is shown how number-conserving differentiation handles the dual nature of the Kohn Sham potential arising in the practical use of the theory. On the other hand, it is pointed out that number-conserving derivatives cannot resolve the causality paradox connected with the second derivative of the exchange-correlation part of the action density functional.

The Aspartate-Less Receiver (ALR) Domains: Distribution, Structure and Function

PubMed Central

Weiner, Joshua J.; Han, Lanlan; Peterson, Francis C.; Volkman, Brian F.; Silvaggi, Nicholas R.; Ulijasz, Andrew T.

2015-01-01

Two-component signaling systems are ubiquitous in bacteria, Archaea and plants and play important roles in sensing and responding to environmental stimuli. To propagate a signaling response the typical system employs a sensory histidine kinase that phosphorylates a Receiver (REC) domain on a conserved aspartate (Asp) residue. Although it is known that some REC domains are missing this Asp residue, it remains unclear as to how many of these divergent REC domains exist, what their functional roles are and how they are regulated in the absence of the conserved Asp. Here we have compiled all deposited REC domains missing their phosphorylatable Asp residue, renamed here as the Aspartate-Less Receiver (ALR) domains. Our data show that ALRs are surprisingly common and are enriched for when attached to more rare effector outputs. Analysis of our informatics and the available ALR atomic structures, combined with structural, biochemical and genetic data of the ALR archetype RitR from Streptococcus pneumoniae presented here suggest that ALRs have reorganized their active pockets to instead take on a constitutive regulatory role or accommodate input signals other than Asp phosphorylation, while largely retaining the canonical post-phosphorylation mechanisms and dimeric interface. This work defines ALRs as an atypical REC subclass and provides insights into shared mechanisms of activation between ALR and REC domains. PMID:25875291

Identification of an evolutionarily conserved extracellular threonine residue critical for surface expression and its potential coupling of adjacent voltage-sensing and gating domains in voltage-gated potassium channels.

PubMed

Mckeown, Lynn; Burnham, Matthew P; Hodson, Charlotte; Jones, Owen T

2008-10-31

The dynamic expression of voltage-gated potassium channels (Kvs) at the cell surface is a fundamental factor controlling membrane excitability. In exploring possible mechanisms controlling Kv surface expression, we identified a region in the extracellular linker between the first and second of the six (S1-S6) transmembrane-spanning domains of the Kv1.4 channel, which we hypothesized to be critical for its biogenesis. Using immunofluorescence microscopy, flow cytometry, patch clamp electrophysiology, and mutagenesis, we identified a single threonine residue at position 330 within the Kv1.4 S1-S2 linker that is absolutely required for cell surface expression. Mutation of Thr-330 to an alanine, aspartate, or lysine prevented surface expression. However, surface expression occurred upon co-expression of mutant and wild type Kv1.4 subunits or mutation of Thr-330 to a serine. Mutation of the corresponding residue (Thr-211) in Kv3.1 to alanine also caused intracellular retention, suggesting that the conserved threonine plays a generalized role in surface expression. In support of this idea, sequence comparisons showed conservation of the critical threonine in all Kv families and in organisms across the evolutionary spectrum. Based upon the Kv1.2 crystal structure, further mutagenesis, and the partial restoration of surface expression in an electrostatic T330K bridging mutant, we suggest that Thr-330 hydrogen bonds to equally conserved outer pore residues, which may include a glutamate at position 502 that is also critical for surface expression. We propose that Thr-330 serves to interlock the voltage-sensing and gating domains of adjacent monomers, thereby yielding a structure competent for the surface expression of functional tetramers.

The DIMA web resource--exploring the protein domain network.

PubMed

Pagel, Philipp; Oesterheld, Matthias; Stümpflen, Volker; Frishman, Dmitrij

2006-04-15

Conserved domains represent essential building blocks of most known proteins. Owing to their role as modular components carrying out specific functions they form a network based both on functional relations and direct physical interactions. We have previously shown that domain interaction networks provide substantially novel information with respect to networks built on full-length protein chains. In this work we present a comprehensive web resource for exploring the Domain Interaction MAp (DIMA), interactively. The tool aims at integration of multiple data sources and prediction techniques, two of which have been implemented so far: domain phylogenetic profiling and experimentally demonstrated domain contacts from known three-dimensional structures. A powerful yet simple user interface enables the user to compute, visualize, navigate and download domain networks based on specific search criteria. http://mips.gsf.de/genre/proj/dima

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

PubMed Central

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

2012-01-01

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

Origin and evolution of group XI secretory phospholipase A2 from flax (Linum usitatissimum) based on phylogenetic analysis of conserved domains.

PubMed

Gupta, Payal; Saini, Raman; Dash, Prasanta K

2017-07-01

Phospholipase A 2 (PLA 2 ) belongs to class of lipolytic enzymes (EC 3.1.1.4). Lysophosphatidic acid (LPA) and free fatty acids (FFAs) are the products of PLA 2 catalyzed hydrolysis of phosphoglycerides at sn-2 position. LPA and FFA that act as second mediators involved in the development and maturation of plants and animals. Mining of flax genome identified two phospholipase A 2 encoding genes, viz., LusPLA 2 I and LusPLA 2 II (Linum usitatissimum secretory phospholipase A 2 ). Molecular simulation of LusPLA 2 s with already characterized plant sPLA 2 s revealed the presence of conserved motifs and signature domains necessary to classify them as secretory phospholipase A 2 . Phylogenetic analysis of flax sPLA 2 with representative sPLA 2 s from other organisms revealed that they evolved rapidly via gene duplication/deletion events and shares a common ancestor. Our study is the first report of detailed phylogenetic analysis for secretory phospholipase A 2 in flax. Comparative genomic analysis of two LusPLA 2 s with earlier reported plant sPLA 2 s, based on their gene architectures, sequence similarities, and domain structures are presented elucidating the uniqueness of flax sPLA 2 .

Application of MRIL-WD (Magnetic Resonance Imaging Logging While Drilling) for irreducible water saturation, total reservoir, free-fluid, bound-fluid porosity measurements and its value for the petrophysical analysis of RT/RM data from the Shah Deniz well

NASA Astrophysics Data System (ADS)

Amirov, Elnur

2016-04-01

Sperry-Sun (Sperry Drilling Services) is the leader in MWD/LWD reliability, has developed the industry's first LWD NMR/MRIL-WD (nuclear magnetic resonance) tool. The MRIL-WD (magnetic resonance imaging logging-while-drilling) service directly measures the T1 component of hydrogen in subsurface rock units while drilling to obtain total reservoir porosity and to dissect the observed total porosity into its respective components of free fluid and bound fluid porosity. These T1 data are used to secure accurate total, free-fluid, capillary-bound water, and clay-bound water porosity of the reservoir sections which can be drilled in the several Runs. Over the last decade, results from Magnetic Resonance Imaging logs (NMR) have added significant value to petrophysical analysis and understanding by providing total, free-fluid and bound-fluid porosities, combined with fluid typing capabilities. With MRIL-WD very valuable Real-Time or Recorded Memory data/information is now available during or shortly after the drilling operation (formation properties measurement can be taken right after a drill bit penetration), while trip in and trip out as well. A key point in utilizing MRIL in an LWD environment is motion-tolerant measurements. Recent MRIL-WD logging runs from the Shah Deniz wells located in the Khazarian-Caspian Sea of the Azerbaijan Republic helped to delineate and assess hydrocarbon bearing zones. Acquired results demonstrate how MRIL data can be acquired while-drilling and provide reliable/high quality measurements. Magnetic Resonance Imaging logs at some developments wells have become a cornerstone in formation evaluation and petrophysical understanding. By providing total, free-fluid, and bound-fluid porosities together with fluid typing, MRIL results have significantly added to the assessment of reservoirs. In order to reduce NPT (Non-Productive Time) and save the rig operations time, there is always the desire to obtain logging results as soon as possible

Detection of lipid-induced structural changes of the Marburg virus matrix protein VP40 using hydrogen/deuterium exchange-mass spectrometry

PubMed Central

Wijesinghe, Kaveesha J.; Urata, Sarah; Bhattarai, Nisha; Kooijman, Edgar E.; Gerstman, Bernard S.; Chapagain, Prem P.; Li, Sheng; Stahelin, Robert V.

2017-01-01

Marburg virus (MARV) is a lipid-enveloped virus from the Filoviridae family containing a negative sense RNA genome. One of the seven MARV genes encodes the matrix protein VP40, which forms a matrix layer beneath the plasma membrane inner leaflet to facilitate budding from the host cell. MARV VP40 (mVP40) has been shown to be a dimeric peripheral protein with a broad and flat basic surface that can associate with anionic phospholipids such as phosphatidylserine. Although a number of mVP40 cationic residues have been shown to facilitate binding to membranes containing anionic lipids, much less is known on how mVP40 assembles to form the matrix layer following membrane binding. Here we have used hydrogen/deuterium exchange (HDX) mass spectrometry to determine the solvent accessibility of mVP40 residues in the absence and presence of phosphatidylserine and phosphatidylinositol 4,5-bisphosphate. HDX analysis demonstrates that two basic loops in the mVP40 C-terminal domain make important contributions to anionic membrane binding and also reveals a potential oligomerization interface in the C-terminal domain as well as a conserved oligomerization interface in the mVP40 N-terminal domain. Lipid binding assays confirm the role of the two basic patches elucidated with HD/X measurements, whereas molecular dynamics simulations and membrane insertion measurements complement these studies to demonstrate that mVP40 does not appreciably insert into the hydrocarbon region of anionic membranes in contrast to the matrix protein from Ebola virus. Taken together, we propose a model by which association of the mVP40 dimer with the anionic plasma membrane facilitates assembly of mVP40 oligomers. PMID:28167534

Detection of lipid-induced structural changes of the Marburg virus matrix protein VP40 using hydrogen/deuterium exchange-mass spectrometry.

PubMed

Wijesinghe, Kaveesha J; Urata, Sarah; Bhattarai, Nisha; Kooijman, Edgar E; Gerstman, Bernard S; Chapagain, Prem P; Li, Sheng; Stahelin, Robert V

2017-04-14

Marburg virus (MARV) is a lipid-enveloped virus from the Filoviridae family containing a negative sense RNA genome. One of the seven MARV genes encodes the matrix protein VP40, which forms a matrix layer beneath the plasma membrane inner leaflet to facilitate budding from the host cell. MARV VP40 (mVP40) has been shown to be a dimeric peripheral protein with a broad and flat basic surface that can associate with anionic phospholipids such as phosphatidylserine. Although a number of mVP40 cationic residues have been shown to facilitate binding to membranes containing anionic lipids, much less is known on how mVP40 assembles to form the matrix layer following membrane binding. Here we have used hydrogen/deuterium exchange (HDX) mass spectrometry to determine the solvent accessibility of mVP40 residues in the absence and presence of phosphatidylserine and phosphatidylinositol 4,5-bisphosphate. HDX analysis demonstrates that two basic loops in the mVP40 C-terminal domain make important contributions to anionic membrane binding and also reveals a potential oligomerization interface in the C-terminal domain as well as a conserved oligomerization interface in the mVP40 N-terminal domain. Lipid binding assays confirm the role of the two basic patches elucidated with HD/X measurements, whereas molecular dynamics simulations and membrane insertion measurements complement these studies to demonstrate that mVP40 does not appreciably insert into the hydrocarbon region of anionic membranes in contrast to the matrix protein from Ebola virus. Taken together, we propose a model by which association of the mVP40 dimer with the anionic plasma membrane facilitates assembly of mVP40 oligomers. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

50 CFR Table 3 (north) to Part 660... - Non-Trawl Rockfish Conservation Areas and Trip Limits for Open Access Gears North of 40°10′ N. Lat.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 50 Wildlife and Fisheries 11 2011-10-01 2011-10-01 false Non-Trawl Rockfish Conservation Areas and Trip Limits for Open Access Gears North of 40°10â² N. Lat. 3 Table 3 (North) to Part 660, Subpart F...) to Part 660, Subpart F—Non-Trawl Rockfish Conservation Areas and Trip Limits for Open Access Gears...

50 CFR Table 3 (south) to Part 660... - Non-Trawl Rockfish Conservation Areas and Trip Limits for Open Access Gears South of 40°10′ N. Lat.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 50 Wildlife and Fisheries 11 2011-10-01 2011-10-01 false Non-Trawl Rockfish Conservation Areas and Trip Limits for Open Access Gears South of 40°10â² N. Lat. 3 Table 3 (South) to Part 660, Subpart F...) to Part 660, Subpart F—Non-Trawl Rockfish Conservation Areas and Trip Limits for Open Access Gears...

Identification and Characterization of Functionally Critical, Conserved Motifs in the Internal Repeats and N-terminal Domain of Yeast Translation Initiation Factor 4B (yeIF4B)*

PubMed Central

Zhou, Fujun; Walker, Sarah E.; Mitchell, Sarah F.; Lorsch, Jon R.; Hinnebusch, Alan G.

2014-01-01

eIF4B has been implicated in attachment of the 43 S preinitiation complex (PIC) to mRNAs and scanning to the start codon. We recently determined that the internal seven repeats (of ∼26 amino acids each) of Saccharomyces cerevisiae eIF4B (yeIF4B) compose the region most critically required to enhance mRNA recruitment by 43 S PICs in vitro and stimulate general translation initiation in yeast. Moreover, although the N-terminal domain (NTD) of yeIF4B contributes to these activities, the RNA recognition motif is dispensable. We have now determined that only two of the seven internal repeats are sufficient for wild-type (WT) yeIF4B function in vivo when all other domains are intact. However, three or more repeats are needed in the absence of the NTD or when the functions of eIF4F components are compromised. We corroborated these observations in the reconstituted system by demonstrating that yeIF4B variants with only one or two repeats display substantial activity in promoting mRNA recruitment by the PIC, whereas additional repeats are required at lower levels of eIF4A or when the NTD is missing. These findings indicate functional overlap among the 7-repeats and NTD domains of yeIF4B and eIF4A in mRNA recruitment. Interestingly, only three highly conserved positions in the 26-amino acid repeat are essential for function in vitro and in vivo. Finally, we identified conserved motifs in the NTD and demonstrate functional overlap of two such motifs. These results provide a comprehensive description of the critical sequence elements in yeIF4B that support eIF4F function in mRNA recruitment by the PIC. PMID:24285537

Membrane-Sculpting BAR Domains Generate Stable Lipid Microdomains

PubMed Central

Zhao, Hongxia; Michelot, Alphée; Koskela, Essi V.; Tkach, Vadym; Stamou, Dimitrios; Drubin, David G.; Lappalainen, Pekka

2014-01-01

SUMMARY Bin-Amphiphysin-Rvs (BAR) domain proteins are central regulators of many cellular processes involving membrane dynamics. BAR domains sculpt phosphoinositide-rich membranes to generate membrane protrusions or invaginations. Here, we report that, in addition to regulating membrane geometry, BAR domains can generate extremely stable lipid microdomains by “freezing” phosphoinositide dynamics. This is a general feature of BAR domains, because the yeast endocytic BAR and Fes/CIP4 homology BAR (F-BAR) domains, the inverse BAR domain of Pinkbar, and the eisosomal BAR protein Lsp1 induced phosphoinositide clustering and halted lipid diffusion, despite differences in mechanisms of membrane interactions. Lsp1 displays comparable low diffusion rates in vitro and in vivo, suggesting that BAR domain proteins also generate stable phosphoinositide microdomains in cells. These results uncover a conserved role for BAR superfamily proteins in regulating lipid dynamics within membranes. Stable microdomains induced by BAR domain scaffolds and specific lipids can generate phase boundaries and diffusion barriers, which may have profound impacts on diverse cellular processes. PMID:24055060

Membrane-sculpting BAR domains generate stable lipid microdomains.

PubMed

Zhao, Hongxia; Michelot, Alphée; Koskela, Essi V; Tkach, Vadym; Stamou, Dimitrios; Drubin, David G; Lappalainen, Pekka

2013-09-26

Bin-Amphiphysin-Rvs (BAR) domain proteins are central regulators of many cellular processes involving membrane dynamics. BAR domains sculpt phosphoinositide-rich membranes to generate membrane protrusions or invaginations. Here, we report that, in addition to regulating membrane geometry, BAR domains can generate extremely stable lipid microdomains by "freezing" phosphoinositide dynamics. This is a general feature of BAR domains, because the yeast endocytic BAR and Fes/CIP4 homology BAR (F-BAR) domains, the inverse BAR domain of Pinkbar, and the eisosomal BAR protein Lsp1 induced phosphoinositide clustering and halted lipid diffusion, despite differences in mechanisms of membrane interactions. Lsp1 displays comparable low diffusion rates in vitro and in vivo, suggesting that BAR domain proteins also generate stable phosphoinositide microdomains in cells. These results uncover a conserved role for BAR superfamily proteins in regulating lipid dynamics within membranes. Stable microdomains induced by BAR domain scaffolds and specific lipids can generate phase boundaries and diffusion barriers, which may have profound impacts on diverse cellular processes. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

40 CFR 73.81 - Qualified conservation measures and renewable energy generation.

Code of Federal Regulations, 2012 CFR

2012-07-01

... renewable energy generation. 73.81 Section 73.81 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) SULFUR DIOXIDE ALLOWANCE SYSTEM Energy Conservation and Renewable Energy Reserve § 73.81 Qualified conservation measures and renewable energy generation. (a) Qualified energy...

40 CFR 73.81 - Qualified conservation measures and renewable energy generation.

Code of Federal Regulations, 2014 CFR

2014-07-01

... renewable energy generation. 73.81 Section 73.81 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) SULFUR DIOXIDE ALLOWANCE SYSTEM Energy Conservation and Renewable Energy Reserve § 73.81 Qualified conservation measures and renewable energy generation. (a) Qualified energy...

40 CFR 73.81 - Qualified conservation measures and renewable energy generation.

Code of Federal Regulations, 2013 CFR

2013-07-01

... renewable energy generation. 73.81 Section 73.81 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) SULFUR DIOXIDE ALLOWANCE SYSTEM Energy Conservation and Renewable Energy Reserve § 73.81 Qualified conservation measures and renewable energy generation. (a) Qualified energy...

Between-domain relations of students' academic emotions and their judgments of school domain similarity

PubMed Central

Goetz, Thomas; Haag, Ludwig; Lipnevich, Anastasiya A.; Keller, Melanie M.; Frenzel, Anne C.; Collier, Antonie P. M.

2014-01-01

With the aim to deepen our understanding of the between-domain relations of academic emotions, a series of three studies was conducted. We theorized that between-domain relations of trait (i.e., habitual) emotions reflected students' judgments of domain similarities, whereas between-domain relations of state (i.e., momentary) emotions did not. This supposition was based on the accessibility model of emotional self-report, according to which individuals' beliefs tend to strongly impact trait, but not state emotions. The aim of Study 1 (interviews; N = 40; 8th and 11th graders) was to gather salient characteristics of academic domains from students' perspective. In Study 2 (N = 1709; 8th and 11th graders) the 13 characteristics identified in Study 1 were assessed along with academic emotions in four different domains (mathematics, physics, German, and English) using a questionnaire-based trait assessment. With respect to the same domains, state emotions were assessed in Study 3 (N = 121; 8th and 11th graders) by employing an experience sampling approach. In line with our initial assumptions, between-domain relations of trait but not state academic emotions reflected between-domain relations of domain characteristics. Implications for research and practice are discussed. PMID:25374547

18 CFR 40.3 - Availability of Reliability Standards.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Availability of Reliability Standards. 40.3 Section 40.3 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY... THE BULK-POWER SYSTEM § 40.3 Availability of Reliability Standards. The Electric Reliability...

Mutations in the FMN domain modulate MCD spectra of the heme site in the oxygenase domain of inducible nitric oxide synthase.

PubMed

Sempombe, Joseph; Elmore, Bradley O; Sun, Xi; Dupont, Andrea; Ghosh, Dipak K; Guillemette, J Guy; Kirk, Martin L; Feng, Changjian

2009-05-27

The nitric oxide synthase (NOS) output state for NO production is a complex of the flavin mononucleotide (FMN)-binding domain and the heme domain, and thereby it facilitates the interdomain electron transfer from the FMN to the catalytic heme site. Emerging evidence suggests that interdomain FMN-heme interactions are important in the formation of the output state because they guide the docking of the FMN domain to the heme domain. In this study, notable effects of mutations in the adjacent FMN domain on the heme structure in a human iNOS bidomain oxygenase/FMN construct have been observed by using low-temperature magnetic circular dichroism (MCD) spectroscopy. The comparative MCD study of wild-type and mutant proteins clearly indicates that a properly docked FMN domain contributes to the observed L-Arg perturbation of the heme MCD spectrum in the wild-type protein and that the conserved surface residues in the FMN domain (E546 and E603) play key roles in facilitating a productive alignment of the FMN and heme domains in iNOS.

Type-specific and cross-reactive antibodies and T cell responses in norovirus VLP immunized mice are targeted both to conserved and variable domains of capsid VP1 protein.

PubMed

Malm, Maria; Tamminen, Kirsi; Vesikari, Timo; Blazevic, Vesna

2016-10-01

Norovirus (NoV)-specific antibodies, which block binding of the virus-like particles (VLPs) to the cell receptors are conformation dependent and directed towards the most exposed domain of the NoV capsid VP1 protein, the P2 domain. Limited data are available on the antibodies directed to other domains of the VP1, and even less on the NoV VP1-specific T cell epitopes. In here, BALB/c mice were immunized with six VLPs derived from NoV GII.4-1999, GII.4-2009 (New Orleans), GII.4-2012 (Sydney), GII.12, GI.1, and G1.3. Serum immunoglobulin G binding antibodies, histo-blood group antigen blocking antibodies and T cell responses using type-specific and heterologous NoV VLPs, P-dimers and 76 overlapping synthetic peptides, spanning the entire 539 amino acid sequence of GII.4 VP1, were determined. The results showed that at least half of the total antibody content is directed towards conserved S domain of the VP1. Only a small fraction (<1%) of the VP1 binding antibodies were blocking/neutralizing. With the use of matrix peptide pools and individual peptides, seven CD4 + and CD8 + T cell restricted epitopes were mapped, two located in S domain, four in P2 domain and one in P1 domain of NoV VP1. The epitopes were GII.4 strain-specific but also common GII.4 genotype-specific T cell epitopes were identified. More importantly, the results suggest a 9-amino acids long sequence ( 318 PAPLGTPDF 326 ) in P2 domain of VP1 as a universal NoV genogroup II-specific CD8 + T cell epitope. Distribution of the T cell epitopes alongside the capsid VP1 indicates the need of the complete protein for high immunogenicity. Copyright © 2016 Elsevier Ltd. All rights reserved.

High-Order Entropy Stable Finite Difference Schemes for Nonlinear Conservation Laws: Finite Domains

NASA Technical Reports Server (NTRS)

Fisher, Travis C.; Carpenter, Mark H.

2013-01-01

Developing stable and robust high-order finite difference schemes requires mathematical formalism and appropriate methods of analysis. In this work, nonlinear entropy stability is used to derive provably stable high-order finite difference methods with formal boundary closures for conservation laws. Particular emphasis is placed on the entropy stability of the compressible Navier-Stokes equations. A newly derived entropy stable weighted essentially non-oscillatory finite difference method is used to simulate problems with shocks and a conservative, entropy stable, narrow-stencil finite difference approach is used to approximate viscous terms.

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

PubMed

Sharma, Monika; Anirudh, C R

2017-10-03

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

Conservation of tubulin-binding sequences in TRPV1 throughout evolution.

PubMed

Sardar, Puspendu; Kumar, Abhishek; Bhandari, Anita; Goswami, Chandan

2012-01-01

Transient Receptor Potential Vanilloid sub type 1 (TRPV1), commonly known as capsaicin receptor can detect multiple stimuli ranging from noxious compounds, low pH, temperature as well as electromagnetic wave at different ranges. In addition, this receptor is involved in multiple physiological and sensory processes. Therefore, functions of TRPV1 have direct influences on adaptation and further evolution also. Availability of various eukaryotic genomic sequences in public domain facilitates us in studying the molecular evolution of TRPV1 protein and the respective conservation of certain domains, motifs and interacting regions that are functionally important. Using statistical and bioinformatics tools, our analysis reveals that TRPV1 has evolved about ∼420 million years ago (MYA). Our analysis reveals that specific regions, domains and motifs of TRPV1 has gone through different selection pressure and thus have different levels of conservation. We found that among all, TRP box is the most conserved and thus have functional significance. Our results also indicate that the tubulin binding sequences (TBS) have evolutionary significance as these stretch sequences are more conserved than many other essential regions of TRPV1. The overall distribution of positively charged residues within the TBS motifs is conserved throughout evolution. In silico analysis reveals that the TBS-1 and TBS-2 of TRPV1 can form helical structures and may play important role in TRPV1 function. Our analysis identifies the regions of TRPV1, which are important for structure-function relationship. This analysis indicates that tubulin binding sequence-1 (TBS-1) near the TRP-box forms a potential helix and the tubulin interactions with TRPV1 via TBS-1 have evolutionary significance. This interaction may be required for the proper channel function and regulation and may also have significance in the context of Taxol®-induced neuropathy.

Functional analysis of the global repressor Tup1 for maltose metabolism in Saccharomyces cerevisiae: different roles of the functional domains.

PubMed

Lin, Xue; Yu, Ai-Qun; Zhang, Cui-Ying; Pi, Li; Bai, Xiao-Wen; Xiao, Dong-Guang

2017-11-09

Tup1 is a general transcriptional repressor of diverse gene families coordinately controlled by glucose repression, mating type, and other mechanisms in Saccharomyces cerevisiae. Several functional domains of Tup1 have been identified, each of which has differing effects on transcriptional repression. In this study, we aim to investigate the role of Tup1 and its domains in maltose metabolism of industrial baker's yeast. To this end, a battery of in-frame truncations in the TUP1 gene coding region were performed in the industrial baker's yeasts with different genetic background, and the maltose metabolism, leavening ability, MAL gene expression levels, and growth characteristics were investigated. The results suggest that the TUP1 gene is essential to maltose metabolism in industrial baker's yeast. Importantly, different domains of Tup1 play different roles in glucose repression and maltose metabolism of industrial baker's yeast cells. The Ssn6 interaction, N-terminal repression and C-terminal repression domains might play roles in the regulation of MAL transcription by Tup1 for maltose metabolism of baker's yeast. The WD region lacking the first repeat could influence the regulation of maltose metabolism directly, rather than indirectly through glucose repression. These findings lay a foundation for the optimization of industrial baker's yeast strains for accelerated maltose metabolism and facilitate future research on glucose repression in other sugar metabolism.

Isolation and characterization of a J domain protein that interacts with ARC1 from ornamental kale (Brassica oleracea var. acephala).

PubMed

Lan, Xingguo; Yang, Jia; Cao, Mingming; Wang, Yanhong; Kawabata, Saneyuki; Li, Yuhua

2015-05-01

A novel J domain protein, JDP1, was isolated from ornamental kale. The C-terminus of JDP1 specifically interacted with ARC1, which has a conserved role in self-incompatibility signaling. Armadillo (ARM)-repeat containing 1 (ARC1) plays a conserved role in self-incompatibility signaling across the Brassicaceae and functions downstream of the S-locus receptor kinase. Here, we identified a J domain protein 1 (JDP1) that interacts with ARC1 using a yeast two-hybrid screen against a stigma cDNA library from ornamental kale (Brassica oleracea var. acephala). JDP1, a 38.4-kDa protein with 344 amino acids, is a member of the Hsp40 family. Fragment JDP1(57-344), originally isolated from a yeast two-hybrid cDNA library, interacted specifically with ARC1 in yeast two-hybrid assays. The N-terminus of JDP1 (JDP1(1-68)) contains a J domain, and the C-terminus of JDP1 (JDP1(69-344)) contains an X domain of unknown function. However, JDP1(69-344) was required and sufficient for interaction with ARC1 in yeast two-hybrid assays and in vitro binding assays. Moreover, JDP1(69-344) regulated the trafficking of ARC1 from the cytoplasm to the plasma membrane by interacting with ARC1 in Arabidopsis mesophyll protoplasts. Finally, Tyr(8) in the JDP1 N-terminal region was identified to be the specific site for regulating the interaction between JDP1 and BoARC1 in yeast two-hybrid assays. Possible roles of JDP1 as an interactor with ARC1 in Brassica are discussed.

Enhanced Expression of WD Repeat-Containing Protein 35 via Nuclear Factor-Kappa B Activation in Bupivacaine-Treated Neuro2a Cells

PubMed Central

Huang, Lei; Kondo, Fumio; Harato, Misako; Feng, Guo-Gang; Ishikawa, Naoshisa; Fujiwara, Yoshihiro; Okada, Shoshiro

2014-01-01

The family of WD repeat proteins comprises a large number of proteins and is involved in a wide variety of cellular processes such as signal transduction, cell growth, proliferation, and apoptosis. Bupivacaine is a sodium channel blocker administered for local infiltration, nerve block, epidural, and intrathecal anesthesia. Recently, we reported that bupivacaine induces reactive oxygen species (ROS) generation and p38 mitogen-activated protein kinase (MAPK) activation, resulting in an increase in the expression of WD repeat-containing protein 35 (WDR35) in mouse neuroblastoma Neuro2a cells. It has been shown that ROS activate MAPK through phosphorylation, followed by activation of nuclear factor-kappa B (NF-κB) and activator protein 1 (AP-1). The present study was undertaken to test whether NF-κB and c-Jun/AP-1 are involved in bupivacaine-induced WDR35 expression in Neuro2a cells. Bupivacaine activated both NF-κB and c-Jun in Neuro2a cells. APDC, an NF-κB inhibitor, attenuated the increase in NF-κB activity and WDR35 protein expression in bupivacaine-treated Neuro2a cells. GW9662, a selective peroxisome proliferator-activated receptor-γ antagonist, enhanced the increase in NF-κB activity and WDR35 protein expression in bupivacaine-treated Neuro2a cells. In contrast, c-Jun siRNA did not inhibit the bupivacaine-induced increase in WDR35 mRNA expression. These results indicate that bupivacaine induces the activation of transcription factors NF-κB and c-Jun/AP-1 in Neuro2a cells, while activation of NF-κB is involved in bupivacaine-induced increases in WDR35 expression. PMID:24466034

Modelling conservation in the Amazon basin.

PubMed

Soares-Filho, Britaldo Silveira; Nepstad, Daniel Curtis; Curran, Lisa M; Cerqueira, Gustavo Coutinho; Garcia, Ricardo Alexandrino; Ramos, Claudia Azevedo; Voll, Eliane; McDonald, Alice; Lefebvre, Paul; Schlesinger, Peter

2006-03-23

Expansion of the cattle and soy industries in the Amazon basin has increased deforestation rates and will soon push all-weather highways into the region's core. In the face of this growing pressure, a comprehensive conservation strategy for the Amazon basin should protect its watersheds, the full range of species and ecosystem diversity, and the stability of regional climates. Here we report that protected areas in the Amazon basin--the central feature of prevailing conservation approaches--are an important but insufficient component of this strategy, based on policy-sensitive simulations of future deforestation. By 2050, current trends in agricultural expansion will eliminate a total of 40% of Amazon forests, including at least two-thirds of the forest cover of six major watersheds and 12 ecoregions, releasing 32 +/- 8 Pg of carbon to the atmosphere. One-quarter of the 382 mammalian species examined will lose more than 40% of the forest within their Amazon ranges. Although an expanded and enforced network of protected areas could avoid as much as one-third of this projected forest loss, conservation on private lands is also essential. Expanding market pressures for sound land management and prevention of forest clearing on lands unsuitable for agriculture are critical ingredients of a strategy for comprehensive conservation.

TRANSPARENT TESTA GLABRA1 and GLABRA1 Compete for Binding to GLABRA3 in Arabidopsis

PubMed Central

Pesch, Martina; Schultheiß, Ilka; Klopffleisch, Karsten; Clemen, Christoph S.; Hülskamp, Martin

2015-01-01

The MBW (for R2R3MYB, basic helix-loop-helix [bHLH], and WD40) genes comprise an evolutionarily conserved gene cassette that regulates several traits such as (pro)anthocyanin and anthocyanin biosynthesis and epidermal cell differentiation in plants. Trichome differentiation in Arabidopsis (Arabidopsis thaliana) is governed by GLABRA1 (GL1; R2R3MYB), GL3 (bHLH), and TRANSPARENT TESTA GLABRA1 (TTG1; WD40). They are thought to form a trimeric complex that acts as a transcriptional activation complex. We provide evidence that these three MBW proteins form either GL1 GL3 or GL3 TTG1 dimers. The formation of each dimer is counteracted by the respective third protein in yeast three-hybrid assays, pulldown experiments (luminescence-based mammalian interactome), and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer studies. We further show that two target promoters, TRIPTYCHON (TRY) and CAPRICE (CPC), are differentially regulated: GL1 represses the activation of the TRY promoter by GL3 and TTG1, and TTG1 suppresses the activation of the CPC promoter by GL1 and GL3. Our data suggest that the transcriptional activation by the MBW complex involves alternative complex formation and that the two dimers can differentially regulate downstream genes. PMID:25926482

Putting people on the map through an approach that integrates social data in conservation planning.

PubMed

Stephanson, Sheri L; Mascia, Michael B

2014-10-01

Conservation planning is integral to strategic and effective operations of conservation organizations. Drawing upon biological sciences, conservation planning has historically made limited use of social data. We offer an approach for integrating data on social well-being into conservation planning that captures and places into context the spatial patterns and trends in human needs and capacities. This hierarchical approach provides a nested framework for characterizing and mapping data on social well-being in 5 domains: economic well-being, health, political empowerment, education, and culture. These 5 domains each have multiple attributes; each attribute may be characterized by one or more indicators. Through existing or novel data that display spatial and temporal heterogeneity in social well-being, conservation scientists, planners, and decision makers may measure, benchmark, map, and integrate these data within conservation planning processes. Selecting indicators and integrating these data into conservation planning is an iterative, participatory process tailored to the local context and planning goals. Social well-being data complement biophysical and threat-oriented social data within conservation planning processes to inform decisions regarding where and how to conserve biodiversity, provide a structure for exploring socioecological relationships, and to foster adaptive management. Building upon existing conservation planning methods and insights from multiple disciplines, this approach to putting people on the map can readily merge with current planning practices to facilitate more rigorous decision making. © 2014 Society for Conservation Biology.

Importance of Conserved Cysteine Residues in the Coronavirus Envelope Protein▿

PubMed Central

Lopez, Lisa A.; Riffle, Ambere J.; Pike, Steven L.; Gardner, Douglas; Hogue, Brenda G.

2008-01-01

Coronavirus envelope (E) proteins play an important, not fully understood role(s) in the virus life cycle. All E proteins have conserved cysteine residues located on the carboxy side of the long hydrophobic domain, suggesting functional significance. In this study, we confirmed that mouse hepatitis coronavirus A59 E protein is palmitoylated. To understand the role of the conserved residues and the necessity of palmitoylation, three cysteines at positions 40, 44, and 47 were changed singly and in various combinations to alanine. Double- and triple-mutant E proteins resulted in decreased virus-like particle output when coexpressed with the membrane (M) protein. Mutant E proteins were also studied in the context of a full-length infectious clone. Single-substitution viruses exhibited growth characteristics virtually identical to those of the wild-type virus, while the double-substitution mutations gave rise to viruses with less robust growth phenotypes indicated by smaller plaques and decreased virus yields. In contrast, replacement of all three cysteines resulted in crippled virus with significantly reduced yields. Triple-mutant viruses did not exhibit impairment in entry. Mutant E proteins localized properly in infected cells. A comparison of intracellular and extracellular virus yields suggested that release is only slightly impaired. E protein lacking all three cysteines exhibited an increased rate of degradation compared to that of the wild-type protein, suggesting that palmitoylation is important for the stability of the protein. Altogether, the results indicate that the conserved cysteines and presumably palmitoylation are functionally important for virus production. PMID:18184703

Improving measures of work-related physical functioning.

PubMed

McDonough, Christine M; Ni, Pengsheng; Peterik, Kara; Marfeo, Elizabeth E; Marino, Molly E; Meterko, Mark; Rasch, Elizabeth K; Brandt, Diane E; Jette, Alan M; Chan, Leighton

2017-03-01

To expand content of the physical function domain of the Work Disability Functional Assessment Battery (WD-FAB), developed for the US Social Security Administration's (SSA) disability determination process. Newly developed questions were administered to 3532 recent SSA applicants for work disability benefits and 2025 US adults. Factor analyses and item response theory (IRT) methods were used to calibrate and link the new items to the existing WD-FAB, and computer-adaptive test simulations were conducted. Factor and IRT analyses supported integration of 44 new items into three existing WD-FAB scales and the addition of a new 11-item scale (Community Mobility). The final physical function domain consisting of: Basic Mobility (56 items), Upper Body Function (34 items), Fine Motor Function (45 items), and Community Mobility (11 items) demonstrated acceptable psychometric properties. The WD-FAB offers an important tool for enhancement of work disability determination. The FAB could provide relevant information about work-related functioning for initial assessment of claimants; identifying denied applicants who may benefit from interventions to improve work and health outcomes; enhancing periodic review of work disability beneficiaries; and assessing outcomes for policies, programs and services targeting people with work disability.

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

PubMed Central

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

2014-01-01

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

Using XAS and SXRF to Study Copper in Wilson Disease at the Molecular and Tissue Level

NASA Astrophysics Data System (ADS)

Ralle, Martina; Blackburn, Ninian J.; Lutsenko, Svetlana

2007-02-01

Wilson disease (WD) is a genetic disorder of copper metabolism associated with severe hepatic, neurological, and psychiatric abnormalities. In WD, the billiary copper excretion is impaired and copper accumulates in tissues, particularly in the liver and the brain. The affected gene, ATP7B, encodes the copper transporting ATPase, Wilson disease protein (WNDP). WNDP has six copper binding sites in the N-terminal portion of the molecule. Each site includes the conserved amino acid sequence MXCXXC, and binds 1 Cu(I) through its 2 cysteine residues. We performed X-ray absorption studies at the Cu Kα-edge on the recombinant N-terminal domain of WNDP (N-WNDP). Copper was bound to N-WNDP either in vivo or in vitro in the presence of different reducing agents. We found that in N-WNDP copper is predominantly coordinated in a linear fashion by two cysteines, with the appearance of a Cu-Cu interaction when all metal binding sites are filled. Increasing amounts of reducing agents containing sulfide or phosphine groups led to binding of the exogenous ligands to copper thereby increasing the coordination number of copper from two to three. To better understand the role of copper in WD, we utilized livers of the 6-weeks-old Atp7b-/- mice (an animal model for WD) in which the copper concentration was 10-20-fold higher compared to that of the control mice. The distribution of copper in hepatocytes was evaluated by synchrotron based X-ray fluorescence microprobe (SXRF). We demonstrate that we can prepare liver slices that retain copper and can detect copper with subcellular resolution. On the same sections μ-XANES (spot size: 5 micron) was used to determine the oxidation state of copper.

Discretely Conservative Finite-Difference Formulations for Nonlinear Conservation Laws in Split Form: Theory and Boundary Conditions

NASA Technical Reports Server (NTRS)

Fisher, Travis C.; Carpenter, Mark H.; Nordstroem, Jan; Yamaleev, Nail K.; Swanson, R. Charles

2011-01-01

Simulations of nonlinear conservation laws that admit discontinuous solutions are typically restricted to discretizations of equations that are explicitly written in divergence form. This restriction is, however, unnecessary. Herein, linear combinations of divergence and product rule forms that have been discretized using diagonal-norm skew-symmetric summation-by-parts (SBP) operators, are shown to satisfy the sufficient conditions of the Lax-Wendroff theorem and thus are appropriate for simulations of discontinuous physical phenomena. Furthermore, special treatments are not required at the points that are near physical boundaries (i.e., discrete conservation is achieved throughout the entire computational domain, including the boundaries). Examples are presented of a fourth-order, SBP finite-difference operator with second-order boundary closures. Sixth- and eighth-order constructions are derived, and included in E. Narrow-stencil difference operators for linear viscous terms are also derived; these guarantee the conservative form of the combined operator.

Ubiquitin--conserved protein or selfish gene?

PubMed

Catic, André; Ploegh, Hidde L

2005-11-01

The posttranslational modifier ubiquitin is encoded by a multigene family containing three primary members, which yield the precursor protein polyubiquitin and two ubiquitin moieties, Ub(L40) and Ub(S27), that are fused to the ribosomal proteins L40 and S27, respectively. The gene encoding polyubiquitin is highly conserved and, until now, those encoding Ub(L40) and Ub(S27) have been generally considered to be equally invariant. The evolution of the ribosomal ubiquitin moieties is, however, proving to be more dynamic. It seems that the genes encoding Ub(L40) and Ub(S27) are actively maintained by homologous recombination with the invariant polyubiquitin locus. Failure to recombine leads to deterioration of the sequence of the ribosomal ubiquitin moieties in several phyla, although this deterioration is evidently constrained by the structural requirements of the ubiquitin fold. Only a few amino acids in ubiquitin are vital for its function, and we propose that conservation of all three ubiquitin genes is driven not only by functional properties of the ubiquitin protein, but also by the propensity of the polyubiquitin locus to act as a 'selfish gene'.

Work disability in Argentinian patients with systemic lupus erythematosus is prevalent and it is due to ethnic, socioeconomic and disease-related factors.

PubMed

Pisoni, Cecilia N; Muñoz, Sebastián A; Tamborenea, María N; García, Mercedes; Curti, Ana; Cappuccio, Ana; Rillo, Oscar; Imamura, Patricia M; Schneeberger, Emilce; Ballent, Marcela; Cousseau, Mario L; Velasco Zamora, Jorge; Saurit, Verónica; Toloza, Sergio; Danielsen, María C; Bellomio, Verónica I; Graf, Cesar; Paira, Sergio; Cavallasca, Javier; Pons Estel, Bernardo; Moreno, José L C; Díaz, Mónica; Alba, Paula; Verando, Marcela; Tate, Guillermo; Mysler, Eduardo; Sarano, Judith; Civit, Emma E; Risueño, Fabián; Álvarez Sepúlveda, Pablo; Larroude, María S; Méndez, Marcos F; Conforti, Andrea; Sohn, Débora

2018-04-02

To study the prevalence and the associated factors of work disability (WD) in systemic lupus erythematosus (SLE) patients. A sample of 419 SLE patients from an observational cross-sectional multicenter study was included. Sociodemographic features, disease characteristics, comorbidities, quality of life, unhealthy behaviors, and work-related factors were measured in a standardized interview. Work disability was defined by patient self-report of not being able to work because of SLE. To identify variables associated with work disability, two different multivariate regression models using a stepwise backward method were performed. Prevalence of WD due to SLE was 24.3%. Eighty-nine percent were female and 51% were Caucasians. Mean disease duration was 8.9 ± 7.2 years, and median System Lupus International Collaborating Clinics/American College of Rheumatology damage index SLICC-SDI was 1.5 (range 0-17). In stepwise multivariate logistic regression, living below the poverty line (odds ratio [OR] = 4.65), less than 12 years of education (OR = 2.84), Mestizo ethnicity (OR = 1.94) and SLICC-SDI (OR = 1.25) were predictors of WD. A second model was performed including patient-derived measures; in this model sedentary lifestyle (OR = 2.69) and lower emotional health domain score of the Lupus Quality of Life (LupusQoL) questionnaire (OR = 1.03) were found to be associated to WD and a higher score in LupusQoL physical health domain (OR = 0.93) was protective. The prevalence of WD in Argentinian SLE patients was 24.3%. WD was associated with ethnic (Mestizo), socioeconomic (poverty) and disease-related factors. Patient-related outcomes such us sedentary lifestyle and poor emotional quality of life were also associated with WD. © 2018 Asia Pacific League of Associations for Rheumatology and John Wiley & Sons Australia, Ltd.

New insight into the architecture of oxy-anion pocket in unliganded conformation of GAT domains: A MD-simulation study.

PubMed

Bairagya, Hridoy R; Bansal, Manju

2016-03-01

Human Guanine Monophosphate Synthetase (hGMPS) converts XMP to GMP, and acts as a bifunctional enzyme with N-terminal "glutaminase" (GAT) and C-terminal "synthetase" domain. The enzyme is identified as a potential target for anti-cancer and immunosuppressive therapies. GAT domain of enzyme plays central role in metabolism, and contains conserved catalytic residues Cys104, His190, and Glu192. MD simulation studies on GAT domain suggest that position of oxyanion in unliganded conformation is occupied by one conserved water molecule (W1), which also stabilizes that pocket. This position is occupied by a negatively charged atom of the substrate or ligand in ligand bound crystal structures. In fact, MD simulation study of Ser75 to Val indicates that W1 conserved water molecule is stabilized by Ser75, while Thr152, and His190 also act as anchor residues to maintain appropriate architecture of oxyanion pocket through water mediated H-bond interactions. Possibly, four conserved water molecules stabilize oxyanion hole in unliganded state, but they vacate these positions when the enzyme (hGMPS)-substrate complex is formed. Thus this study not only reveals functionally important role of conserved water molecules in GAT domain, but also highlights essential role of other non-catalytic residues such as Ser75 and Thr152 in this enzymatic domain. The results from this computational study could be of interest to experimental community and provide a testable hypothesis for experimental validation. Conserved sites of water molecules near and at oxyanion hole highlight structural importance of water molecules and suggest a rethink of the conventional definition of chemical geometry of inhibitor binding site. © 2016 Wiley Periodicals, Inc.

Structure of the SPRY domain of the human RNA helicase DDX1, a putative interaction platform within a DEAD-box protein

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

Kellner, Julian N.; Meinhart, Anton, E-mail: anton.meinhart@mpimf-heidelberg.mpg.de

The structure of the SPRY domain of the human RNA helicase DDX1 was determined at 2.0 Å resolution. The SPRY domain provides a putative protein–protein interaction platform within DDX1 that differs from other SPRY domains in its structure and conserved regions. The human RNA helicase DDX1 in the DEAD-box family plays an important role in RNA processing and has been associated with HIV-1 replication and tumour progression. Whereas previously described DEAD-box proteins have a structurally conserved core, DDX1 shows a unique structural feature: a large SPRY-domain insertion in its RecA-like consensus fold. SPRY domains are known to function as protein–proteinmore » interaction platforms. Here, the crystal structure of the SPRY domain of human DDX1 (hDSPRY) is reported at 2.0 Å resolution. The structure reveals two layers of concave, antiparallel β-sheets that stack onto each other and a third β-sheet beneath the β-sandwich. A comparison with SPRY-domain structures from other eukaryotic proteins showed that the general β-sandwich fold is conserved; however, differences were detected in the loop regions, which were identified in other SPRY domains to be essential for interaction with cognate partners. In contrast, in hDSPRY these loop regions are not strictly conserved across species. Interestingly, though, a conserved patch of positive surface charge is found that may replace the connecting loops as a protein–protein interaction surface. The data presented here comprise the first structural information on DDX1 and provide insights into the unique domain architecture of this DEAD-box protein. By providing the structure of a putative interaction domain of DDX1, this work will serve as a basis for further studies of the interaction network within the hetero-oligomeric complexes of DDX1 and of its recruitment to the HIV-1 Rev protein as a viral replication factor.« less

Expression of simian virus 40 T antigen in Escherichia coli: localization of T-antigen origin DNA-binding domain to within 129 amino acids.

PubMed Central

Arthur, A K; Höss, A; Fanning, E

1988-01-01

The genomic coding sequence of the large T antigen of simian virus 40 (SV40) was cloned into an Escherichia coli expression vector by joining new restriction sites, BglII and BamHI, introduced at the intron boundaries of the gene. Full-length large T antigen, as well as deletion and amino acid substitution mutants, were inducibly expressed from the lac promoter of pUC9, albeit with different efficiencies and protein stabilities. Specific interaction with SV40 origin DNA was detected for full-length T antigen and certain mutants. Deletion mutants lacking T-antigen residues 1 to 130 and 260 to 708 retained specific origin-binding activity, demonstrating that the region between residues 131 and 259 must carry the essential binding domain for DNA-binding sites I and II. A sequence between residues 302 and 320 homologous to a metal-binding "finger" motif is therefore not required for origin-specific binding. However, substitution of serine for either of two cysteine residues in this motif caused a dramatic decrease in origin DNA-binding activity. This region, as well as other regions of the full-length protein, may thus be involved in stabilizing the DNA-binding domain and altering its preference for binding to site I or site II DNA. Images PMID:2835505

DIPA-family coiled-coils bind conserved isoform-specific head domain of p120-catenin family: potential roles in hydrocephalus and heterotopia.

PubMed

Markham, Nicholas O; Doll, Caleb A; Dohn, Michael R; Miller, Rachel K; Yu, Huapeng; Coffey, Robert J; McCrea, Pierre D; Gamse, Joshua T; Reynolds, Albert B

2014-09-01

p120-catenin (p120) modulates adherens junction (AJ) dynamics by controlling the stability of classical cadherins. Among all p120 isoforms, p120-3A and p120-1A are the most prevalent. Both stabilize cadherins, but p120-3A is preferred in epithelia, whereas p120-1A takes precedence in neurons, fibroblasts, and macrophages. During epithelial-to-mesenchymal transition, E- to N-cadherin switching coincides with p120-3A to -1A alternative splicing. These isoforms differ by a 101-amino acid "head domain" comprising the p120-1A N-terminus. Although its exact role is unknown, the head domain likely mediates developmental and cancer-associated events linked to p120-1A expression (e.g., motility, invasion, metastasis). Here we identified delta-interacting protein A (DIPA) as the first head domain-specific binding partner and candidate mediator of isoform 1A activity. DIPA colocalizes with AJs in a p120-1A- but not 3A-dependent manner. Moreover, all DIPA family members (Ccdc85a, Ccdc85b/DIPA, and Ccdc85c) interact reciprocally with p120 family members (p120, δ-catenin, p0071, and ARVCF), suggesting significant functional overlap. During zebrafish neural tube development, both knockdown and overexpression of DIPA phenocopy N-cadherin mutations, an effect bearing functional ties to a reported mouse hydrocephalus phenotype associated with Ccdc85c. These studies identify a novel, highly conserved interaction between two protein families that may participate either individually or collectively in N-cadherin-mediated development. © 2014 Markham et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Evolution of the PWWP-domain encoding genes in the plant and animal lineages

PubMed Central

2012-01-01

Background Conserved domains are recognized as the building blocks of eukaryotic proteins. Domains showing a tendency to occur in diverse combinations (‘promiscuous’ domains) are involved in versatile architectures in proteins with different functions. Current models, based on global-level analyses of domain combinations in multiple genomes, have suggested that the propensity of some domains to associate with other domains in high-level architectures increases with organismal complexity. Alternative models using domain-based phylogenetic trees propose that domains have become promiscuous independently in different lineages through convergent evolution and are, thus, random with no functional or structural preferences. Here we test whether complex protein architectures have occurred by accretion from simpler systems and whether the appearance of multidomain combinations parallels organismal complexity. As a model, we analyze the modular evolution of the PWWP domain and ask whether its appearance in combinations with other domains into multidomain architectures is linked with the occurrence of more complex life-forms. Whether high-level combinations of domains are conserved and transmitted as stable units (cassettes) through evolution is examined in the genomes of plant or metazoan species selected for their established position in the evolution of the respective lineages. Results Using the domain-tree approach, we analyze the evolutionary origins and distribution patterns of the promiscuous PWWP domain to understand the principles of its modular evolution and its existence in combination with other domains in higher-level protein architectures. We found that as a single module the PWWP domain occurs only in proteins with a limited, mainly, species-specific distribution. Earlier, it was suggested that domain promiscuity is a fast-changing (volatile) feature shaped by natural selection and that only a few domains retain their promiscuity status throughout

Biochemical and Structural Characterization of a Five-domain GH115 α-Glucuronidase from the Marine Bacterium Saccharophagus degradans 2-40T*

PubMed Central

Wang, Weijun; Yan, Ruoyu; Nocek, Boguslaw P.; Vuong, Thu V.; Di Leo, Rosa; Xu, Xiaohui; Cui, Hong; Gatenholm, Paul; Toriz, Guillermo; Tenkanen, Maija; Savchenko, Alexei; Master, Emma R.

2016-01-01

Glucuronic acid (GlcAp) and/or methylglucuronic acid (MeGlcAp) decorate the major forms of xylan in hardwood and coniferous softwoods as well as many cereal grains. Accordingly, the complete utilization of glucuronoxylans or conversion to sugar precursors requires the action of main chain xylanases as well as α-glucuronidases that release the α- (1→2)-linked (Me)GlcAp side groups. Herein, a family GH115 enzymefrom the marine bacterium Saccharophagus degradans 2-40T, SdeAgu115A, demonstrated activity toward glucuronoxylan and oligomers thereof with preference toward MeGlcAp linked to internal xylopyranosyl residues. Unique biochemical characteristics of NaCl activation were also observed. The crystal structure of SdeAgu115A revealed a five-domain architecture, with an additional insertion C+ domain that had significant impact on the domain arrangement of SdeAgu115A monomer and its dimerization. The participation of domain C+ in substrate binding was supported by reduced substrate inhibition upon introducing W773A, W689A, and F696A substitutions within this domain. In addition to Asp-335, the catalytic essentiality of Glu-216 was revealed by site-specific mutagenesis. A primary sequence analysis suggested that the SdeAgu115A architecture is shared by more than half of GH115 members, thus defining a distinct archetype for GH115 enzymes. PMID:27129264

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.

A low-complexity region in the YTH domain protein Mmi1 enhances RNA binding.

PubMed

Stowell, James A W; Wagstaff, Jane L; Hill, Chris H; Yu, Minmin; McLaughlin, Stephen H; Freund, Stefan M V; Passmore, Lori A

2018-06-15

Mmi1 is an essential RNA-binding protein in the fission yeast Schizosaccharomyces pombe that eliminates meiotic transcripts during normal vegetative growth. Mmi1 contains a YTH domain that binds specific RNA sequences, targeting mRNAs for degradation. The YTH domain of Mmi1 uses a noncanonical RNA-binding surface that includes contacts outside the conserved fold. Here, we report that an N-terminal extension that is proximal to the YTH domain enhances RNA binding. Using X-ray crystallography, NMR, and biophysical methods, we show that this low-complexity region becomes more ordered upon RNA binding. This enhances the affinity of the interaction of the Mmi1 YTH domain with specific RNAs by reducing the dissociation rate of the Mmi1-RNA complex. We propose that the low-complexity region influences RNA binding indirectly by reducing dynamic motions of the RNA-binding groove and stabilizing a conformation of the YTH domain that binds to RNA with high affinity. Taken together, our work reveals how a low-complexity region proximal to a conserved folded domain can adopt an ordered structure to aid nucleic acid binding. © 2018 Stowell et al.

A structural role for the PHP domain in E. coli DNA polymerase III

PubMed Central

2013-01-01

Background In addition to the core catalytic machinery, bacterial replicative DNA polymerases contain a Polymerase and Histidinol Phosphatase (PHP) domain whose function is not entirely understood. The PHP domains of some bacterial replicases are active metal-dependent nucleases that may play a role in proofreading. In E. coli DNA polymerase III, however, the PHP domain has lost several metal-coordinating residues and is likely to be catalytically inactive. Results Genomic searches show that the loss of metal-coordinating residues in polymerase PHP domains is likely to have coevolved with the presence of a separate proofreading exonuclease that works with the polymerase. Although the E. coli Pol III PHP domain has lost metal-coordinating residues, the structure of the domain has been conserved to a remarkable degree when compared to that of metal-binding PHP domains. This is demonstrated by our ability to restore metal binding with only three point mutations, as confirmed by the metal-bound crystal structure of this mutant determined at 2.9 Å resolution. We also show that Pol III, a large multi-domain protein, unfolds cooperatively and that mutations in the degenerate metal-binding site of the PHP domain decrease the overall stability of Pol III and reduce its activity. Conclusions While the presence of a PHP domain in replicative bacterial polymerases is strictly conserved, its ability to coordinate metals and to perform proofreading exonuclease activity is not, suggesting additional non-enzymatic roles for the domain. Our results show that the PHP domain is a major structural element in Pol III and its integrity modulates both the stability and activity of the polymerase. PMID:23672456

A structural role for the PHP domain in E. coli DNA polymerase III.

PubMed

Barros, Tiago; Guenther, Joel; Kelch, Brian; Anaya, Jordan; Prabhakar, Arjun; O'Donnell, Mike; Kuriyan, John; Lamers, Meindert H

2013-05-14

In addition to the core catalytic machinery, bacterial replicative DNA polymerases contain a Polymerase and Histidinol Phosphatase (PHP) domain whose function is not entirely understood. The PHP domains of some bacterial replicases are active metal-dependent nucleases that may play a role in proofreading. In E. coli DNA polymerase III, however, the PHP domain has lost several metal-coordinating residues and is likely to be catalytically inactive. Genomic searches show that the loss of metal-coordinating residues in polymerase PHP domains is likely to have coevolved with the presence of a separate proofreading exonuclease that works with the polymerase. Although the E. coli Pol III PHP domain has lost metal-coordinating residues, the structure of the domain has been conserved to a remarkable degree when compared to that of metal-binding PHP domains. This is demonstrated by our ability to restore metal binding with only three point mutations, as confirmed by the metal-bound crystal structure of this mutant determined at 2.9 Å resolution. We also show that Pol III, a large multi-domain protein, unfolds cooperatively and that mutations in the degenerate metal-binding site of the PHP domain decrease the overall stability of Pol III and reduce its activity. While the presence of a PHP domain in replicative bacterial polymerases is strictly conserved, its ability to coordinate metals and to perform proofreading exonuclease activity is not, suggesting additional non-enzymatic roles for the domain. Our results show that the PHP domain is a major structural element in Pol III and its integrity modulates both the stability and activity of the polymerase.

Distinct Contributions of Conserved Modules to Runt Transcription Factor Activity

PubMed Central

Walrad, Pegine B.; Hang, Saiyu; Joseph, Genevieve S.; Salas, Julia

2010-01-01

Runx proteins play vital roles in regulating transcription in numerous developmental pathways throughout the animal kingdom. Two Runx protein hallmarks are the DNA-binding Runt domain and a C-terminal VWRPY motif that mediates interaction with TLE/Gro corepressor proteins. A phylogenetic analysis of Runt, the founding Runx family member, identifies four distinct regions C-terminal to the Runt domain that are conserved in Drosophila and other insects. We used a series of previously described ectopic expression assays to investigate the functions of these different conserved regions in regulating gene expression during embryogenesis and in controlling axonal projections in the developing eye. The results indicate each conserved region is required for a different subset of activities and identify distinct regions that participate in the transcriptional activation and repression of the segmentation gene sloppy-paired-1 (slp1). Interestingly, the C-terminal VWRPY-containing region is not required for repression but instead plays a role in slp1 activation. Genetic experiments indicating that Groucho (Gro) does not participate in slp1 regulation further suggest that Runt's conserved C-terminus interacts with other factors to promote transcriptional activation. These results provide a foundation for further studies on the molecular interactions that contribute to the context-dependent properties of Runx proteins as developmental regulators. PMID:20462957

Mechanisms of pseudosubstrate inhibition of the anaphase promoting complex by Acm1

PubMed Central

Burton, Janet L; Xiong, Yong; Solomon, Mark J

2011-01-01

The anaphase promoting complex (APC) is a ubiquitin ligase that promotes the degradation of cell-cycle regulators by the 26S proteasome. Cdc20 and Cdh1 are WD40-containing APC co-activators that bind destruction boxes (DB) and KEN boxes within substrates to recruit them to the APC for ubiquitination. Acm1 is an APCCdh1 inhibitor that utilizes a DB and a KEN box to bind Cdh1 and prevent substrate binding, although Acm1 itself is not a substrate. We investigated what differentiates an APC substrate from an inhibitor. We identified the Acm1 A-motif that interacts with Cdh1 and together with the DB and KEN box is required for APCCdh1 inhibition. A genetic screen identified Cdh1 WD40 domain residues important for Acm1 A-motif interaction and inhibition that appears to reside near Cdh1 residues important for DB recognition. Specific lysine insertion mutations within Acm1 promoted its ubiquitination by APCCdh1 whereas lysine removal from the APC substrate Hsl1 converted it into a potent APCCdh1 inhibitor. These findings suggest that tight Cdh1 binding combined with the inaccessibility of ubiquitinatable lysines contributes to pseudosubstrate inhibition of APCCdh1. PMID:21460798

Topologically associating domains are stable units of replication-timing regulation.

PubMed

Pope, Benjamin D; Ryba, Tyrone; Dileep, Vishnu; Yue, Feng; Wu, Weisheng; Denas, Olgert; Vera, Daniel L; Wang, Yanli; Hansen, R Scott; Canfield, Theresa K; Thurman, Robert E; Cheng, Yong; Gülsoy, Günhan; Dennis, Jonathan H; Snyder, Michael P; Stamatoyannopoulos, John A; Taylor, James; Hardison, Ross C; Kahveci, Tamer; Ren, Bing; Gilbert, David M

2014-11-20

Eukaryotic chromosomes replicate in a temporal order known as the replication-timing program. In mammals, replication timing is cell-type-specific with at least half the genome switching replication timing during development, primarily in units of 400-800 kilobases ('replication domains'), whose positions are preserved in different cell types, conserved between species, and appear to confine long-range effects of chromosome rearrangements. Early and late replication correlate, respectively, with open and closed three-dimensional chromatin compartments identified by high-resolution chromosome conformation capture (Hi-C), and, to a lesser extent, late replication correlates with lamina-associated domains (LADs). Recent Hi-C mapping has unveiled substructure within chromatin compartments called topologically associating domains (TADs) that are largely conserved in their positions between cell types and are similar in size to replication domains. However, TADs can be further sub-stratified into smaller domains, challenging the significance of structures at any particular scale. Moreover, attempts to reconcile TADs and LADs to replication-timing data have not revealed a common, underlying domain structure. Here we localize boundaries of replication domains to the early-replicating border of replication-timing transitions and map their positions in 18 human and 13 mouse cell types. We demonstrate that, collectively, replication domain boundaries share a near one-to-one correlation with TAD boundaries, whereas within a cell type, adjacent TADs that replicate at similar times obscure replication domain boundaries, largely accounting for the previously reported lack of alignment. Moreover, cell-type-specific replication timing of TADs partitions the genome into two large-scale sub-nuclear compartments revealing that replication-timing transitions are indistinguishable from late-replicating regions in chromatin composition and lamina association and accounting for the

Anomaly inflow on QCD axial domain-walls and vortices

NASA Astrophysics Data System (ADS)

Fukushima, Kenji; Imaki, Shota

2018-06-01

We study the chiral effective theory in the presence of quantum chromodynamics (QCD) vortices. Gauge invariance requires novel terms from vortex singularities in the gauged Wess-Zumino-Witten action, which incorporate anomaly-induced currents along the vortices. We examine these terms for systems with QCD axial domain-walls bounded by vortices (vortons) under magnetic fields. We discuss how the baryon and electric charge conservations are satisfied in these systems through interplay between domain-walls and vortices, manifesting Callan-Harvey's mechanism of anomaly inflow.

Estimating and Modelling Bias of the Hierarchical Partitioning Public-Domain Software: Implications in Environmental Management and Conservation

PubMed Central

Olea, Pedro P.; Mateo-Tomás, Patricia; de Frutos, Ángel

2010-01-01

Background Hierarchical partitioning (HP) is an analytical method of multiple regression that identifies the most likely causal factors while alleviating multicollinearity problems. Its use is increasing in ecology and conservation by its usefulness for complementing multiple regression analysis. A public-domain software “hier.part package” has been developed for running HP in R software. Its authors highlight a “minor rounding error” for hierarchies constructed from >9 variables, however potential bias by using this module has not yet been examined. Knowing this bias is pivotal because, for example, the ranking obtained in HP is being used as a criterion for establishing priorities of conservation. Methodology/Principal Findings Using numerical simulations and two real examples, we assessed the robustness of this HP module in relation to the order the variables have in the analysis. Results indicated a considerable effect of the variable order on the amount of independent variance explained by predictors for models with >9 explanatory variables. For these models the nominal ranking of importance of the predictors changed with variable order, i.e. predictors declared important by its contribution in explaining the response variable frequently changed to be either most or less important with other variable orders. The probability of changing position of a variable was best explained by the difference in independent explanatory power between that variable and the previous one in the nominal ranking of importance. The lesser is this difference, the more likely is the change of position. Conclusions/Significance HP should be applied with caution when more than 9 explanatory variables are used to know ranking of covariate importance. The explained variance is not a useful parameter to use in models with more than 9 independent variables. The inconsistency in the results obtained by HP should be considered in future studies as well as in those already published

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.

The EARP Complex and Its Interactor EIPR-1 Are Required for Cargo Sorting to Dense-Core Vesicles

PubMed Central

Topalidou, Irini; Cattin-Ortolá, Jérôme; MacCoss, Michael J.

2016-01-01

The dense-core vesicle is a secretory organelle that mediates the regulated release of peptide hormones, growth factors, and biogenic amines. Dense-core vesicles originate from the trans-Golgi of neurons and neuroendocrine cells, but it is unclear how this specialized organelle is formed and acquires its specific cargos. To identify proteins that act in dense-core vesicle biogenesis, we performed a forward genetic screen in Caenorhabditis elegans for mutants defective in dense-core vesicle function. We previously reported the identification of two conserved proteins that interact with the small GTPase RAB-2 to control normal dense-core vesicle cargo-sorting. Here we identify several additional conserved factors important for dense-core vesicle cargo sorting: the WD40 domain protein EIPR-1 and the endosome-associated recycling protein (EARP) complex. By assaying behavior and the trafficking of dense-core vesicle cargos, we show that mutants that lack EIPR-1 or EARP have defects in dense-core vesicle cargo-sorting similar to those of mutants in the RAB-2 pathway. Genetic epistasis data indicate that RAB-2, EIPR-1 and EARP function in a common pathway. In addition, using a proteomic approach in rat insulinoma cells, we show that EIPR-1 physically interacts with the EARP complex. Our data suggest that EIPR-1 is a new interactor of the EARP complex and that dense-core vesicle cargo sorting depends on the EARP-dependent trafficking of cargo through an endosomal sorting compartment. PMID:27191843

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

PubMed

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

2014-07-18

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

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

Structure of a two-CAP-domain protein from the human hookworm parasite Necator americanus

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

Asojo, Oluwatoyin A., E-mail: oasojo@unmc.edu

2011-05-01

The first structure of a two-CAP-domain protein, Na-ASP-1, from the major human hookworm parasite N. americanus refined to a resolution limit of 2.2 Å is presented. Major proteins secreted by the infective larval stage hookworms upon host entry include Ancylostoma secreted proteins (ASPs), which are characterized by one or two CAP (cysteine-rich secretory protein/antigen 5/pathogenesis related-1) domains. The CAP domain has been reported in diverse phylogenetically unrelated proteins, but has no confirmed function. The first structure of a two-CAP-domain protein, Na-ASP-1, from the major human hookworm parasite Necator americanus was refined to a resolution limit of 2.2 Å. The structuremore » was solved by molecular replacement (MR) using Na-ASP-2, a one-CAP-domain ASP, as the search model. The correct MR solution could only be obtained by truncating the polyalanine model of Na-ASP-2 and removing several loops. The structure reveals two CAP domains linked by an extended loop. Overall, the carboxyl-terminal CAP domain is more similar to Na-ASP-2 than to the amino-terminal CAP domain. A large central cavity extends from the amino-terminal CAP domain to the carboxyl-terminal CAP domain, encompassing the putative CAP-binding cavity. The putative CAP-binding cavity is a characteristic cavity in the carboxyl-terminal CAP domain that contains a His and Glu pair. These residues are conserved in all single-CAP-domain proteins, but are absent in the amino-terminal CAP domain. The conserved His residues are oriented such that they appear to be capable of directly coordinating a zinc ion as observed for CAP proteins from reptile venoms. This first structure of a two-CAP-domain ASP can serve as a template for homology modeling of other two-CAP-domain proteins.« less

Evolution and Conservation of Plant NLR Functions

PubMed Central

Jacob, Florence; Vernaldi, Saskia; Maekawa, Takaki

2013-01-01

In plants and animals, nucleotide-binding domain and leucine-rich repeats (NLR)-containing proteins play pivotal roles in innate immunity. Despite their similar biological functions and protein architecture, comparative genome-wide analyses of NLRs and genes encoding NLR-like proteins suggest that plant and animal NLRs have independently arisen in evolution. Furthermore, the demonstration of interfamily transfer of plant NLR functions from their original species to phylogenetically distant species implies evolutionary conservation of the underlying immune principle across plant taxonomy. In this review we discuss plant NLR evolution and summarize recent insights into plant NLR-signaling mechanisms, which might constitute evolutionarily conserved NLR-mediated immune mechanisms. PMID:24093022

Animal-specific C-terminal domain links myeloblastosis oncoprotein (Myb) to an ancient repressor complex

PubMed Central

Andrejka, Laura; Wen, Hong; Ashton, Jonathan; Grant, Megan; Iori, Kevin; Wang, Amy; Manak, J. Robert; Lipsick, Joseph S.

2011-01-01

Members of the Myb oncoprotein and E2F-Rb tumor suppressor protein families are present within the same highly conserved multiprotein transcriptional repressor complex, named either as Myb and synthetic multivuval class B (Myb-MuvB) or as Drosophila Rb E2F and Myb-interacting proteins (dREAM). We now report that the animal-specific C terminus of Drosophila Myb but not the more highly conserved N-terminal DNA-binding domain is necessary and sufficient for (i) adult viability, (ii) proper localization to chromosomes in vivo, (iii) regulation of gene expression in vivo, and (iv) interaction with the highly conserved core of the MuvB/dREAM transcriptional repressor complex. In addition, we have identified a conserved peptide motif that is required for this interaction. Our results imply that an ancient function of Myb in regulating G2/M genes in both plants and animals appears to have been transferred from the DNA-binding domain to the animal-specific C-terminal domain. Increased expression of B-MYB/MYBL2, the human ortholog of Drosophila Myb, correlates with poor prognosis in human patients with breast cancer. Therefore, our results imply that the specific interaction of the C terminus of Myb with the MuvB/dREAM core complex may provide an attractive target for the development of cancer therapeutics. PMID:21969598

MyTH4-FERM myosins have an ancient and conserved role in filopod formation

PubMed Central

Goodson, Holly V.; Arthur, Ashley L.; Luxton, G. W. Gant; Houdusse, Anne; Titus, Margaret A.

2016-01-01

The formation of filopodia in Metazoa and Amoebozoa requires the activity of myosin 10 (Myo10) in mammalian cells and of Dictyostelium unconventional myosin 7 (DdMyo7) in the social amoeba Dictyostelium. However, the exact roles of these MyTH4-FERM myosins (myosin tail homology 4-band 4.1, ezrin, radixin, moesin; MF) in the initiation and elongation of filopodia are not well defined and may reflect conserved functions among phylogenetically diverse MF myosins. Phylogenetic analysis of MF myosin domains suggests that a single ancestral MF myosin existed with a structure similar to DdMyo7, which has two MF domains, and that subsequent duplications in the metazoan lineage produced its functional homolog Myo10. The essential functional features of the DdMyo7 myosin were identified using quantitative live-cell imaging to characterize the ability of various mutants to rescue filopod formation in myo7-null cells. The two MF domains were found to function redundantly in filopod formation with the C-terminal FERM domain regulating both the number of filopodia and their elongation velocity. DdMyo7 mutants consisting solely of the motor plus a single MyTH4 domain were found to be capable of rescuing the formation of filopodia, establishing the minimal elements necessary for the function of this myosin. Interestingly, a chimeric myosin with the Myo10 MF domain fused to the DdMyo7 motor also was capable of rescuing filopod formation in the myo7-null mutant, supporting fundamental functional conservation between these two distant myosins. Together, these findings reveal that MF myosins have an ancient and conserved role in filopod formation. PMID:27911821

WW domain-binding protein 2: an adaptor protein closely linked to the development of breast cancer.

PubMed

Chen, Shuai; Wang, Han; Huang, Yu-Fan; Li, Ming-Li; Cheng, Jiang-Hong; Hu, Peng; Lu, Chuan-Hui; Zhang, Ya; Liu, Na; Tzeng, Chi-Meng; Zhang, Zhi-Ming

2017-07-19

The WW domain is composed of 38 to 40 semi-conserved amino acids shared with structural, regulatory, and signaling proteins. WW domain-binding protein 2 (WBP2), as a binding partner of WW domain protein, interacts with several WW-domain-containing proteins, such as Yes kinase-associated protein (Yap), paired box gene 8 (Pax8), WW-domain-containing transcription regulator protein 1 (TAZ), and WW-domain-containing oxidoreductase (WWOX) through its PPxY motifs within C-terminal region, and further triggers the downstream signaling pathway in vitro and in vivo. Studies have confirmed that phosphorylated form of WBP2 can move into nuclei and activate the transcription of estrogen receptor (ER) and progesterone receptor (PR), whose expression were the indicators of breast cancer development, indicating that WBP2 may participate in the progression of breast cancer. Both overexpression of WBP2 and activation of tyrosine phosphorylation upregulate the signal cascades in the cross-regulation of the Wnt and ER signaling pathways in breast cancer. Following the binding of WBP2 to the WW domain region of TAZ which can accelerate migration, invasion and is required for the transformed phenotypes of breast cancer cells, the transformation of epithelial to mesenchymal of MCF10A is activated, suggesting that WBP2 is a key player in regulating cell migration. When WBP2 binds with WWOX, a tumor suppressor, ER transactivation and tumor growth can be suppressed. Thus, WBP2 may serve as a molecular on/off switch that controls the crosstalk between E2, WWOX, Wnt, TAZ, and other oncogenic signaling pathways. This review interprets the relationship between WBP2 and breast cancer, and provides comprehensive views about the function of WBP2 in the regulation of the pathogenesis of breast cancer and endocrine therapy in breast cancer treatment.

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.

Aperiodic topological order in the domain configurations of functional materials

NASA Astrophysics Data System (ADS)

Huang, Fei-Ting; Cheong, Sang-Wook

2017-03-01

In numerous functional materials, such as steels, ferroelectrics and magnets, new functionalities can be achieved through the engineering of the domain structures, which are associated with the ordering of certain parameters within the material. The recent progress in technologies that enable imaging at atomic-scale spatial resolution has transformed our understanding of domain topology, revealing that, along with simple stripe-like or irregularly shaped domains, intriguing vortex-type topological domain configurations also exist. In this Review, we present a new classification scheme of 'Zm Zn domains with Zl vortices' for 2D macroscopic domain structures with m directional variants and n translational antiphases. This classification, together with the concepts of topological protection and topological charge conservation, can be applied to a wide range of materials, such as multiferroics, improper ferroelectrics, layered transition metal dichalcogenides and magnetic superconductors, as we discuss using selected examples. The resulting topological considerations provide a new basis for the understanding of the formation, kinetics, manipulation and property optimization of domains and domain boundaries in functional materials.

Flux-vector splitting algorithm for chain-rule conservation-law form

NASA Technical Reports Server (NTRS)

Shih, T. I.-P.; Nguyen, H. L.; Willis, E. A.; Steinthorsson, E.; Li, Z.

1991-01-01

A flux-vector splitting algorithm with Newton-Raphson iteration was developed for the 'full compressible' Navier-Stokes equations cast in chain-rule conservation-law form. The algorithm is intended for problems with deforming spatial domains and for problems whose governing equations cannot be cast in strong conservation-law form. The usefulness of the algorithm for such problems was demonstrated by applying it to analyze the unsteady, two- and three-dimensional flows inside one combustion chamber of a Wankel engine under nonfiring conditions. Solutions were obtained to examine the algorithm in terms of conservation error, robustness, and ability to handle complex flows on time-dependent grid systems.

A Bayesian Sampler for Optimization of Protein Domain Hierarchies

PubMed Central

2014-01-01

Abstract The process of identifying and modeling functionally divergent subgroups for a specific protein domain class and arranging these subgroups hierarchically has, thus far, largely been done via manual curation. How to accomplish this automatically and optimally is an unsolved statistical and algorithmic problem that is addressed here via Markov chain Monte Carlo sampling. Taking as input a (typically very large) multiple-sequence alignment, the sampler creates and optimizes a hierarchy by adding and deleting leaf nodes, by moving nodes and subtrees up and down the hierarchy, by inserting or deleting internal nodes, and by redefining the sequences and conserved patterns associated with each node. All such operations are based on a probability distribution that models the conserved and divergent patterns defining each subgroup. When we view these patterns as sequence determinants of protein function, each node or subtree in such a hierarchy corresponds to a subgroup of sequences with similar biological properties. The sampler can be applied either de novo or to an existing hierarchy. When applied to 60 protein domains from multiple starting points in this way, it converged on similar solutions with nearly identical log-likelihood ratio scores, suggesting that it typically finds the optimal peak in the posterior probability distribution. Similarities and differences between independently generated, nearly optimal hierarchies for a given domain help distinguish robust from statistically uncertain features. Thus, a future application of the sampler is to provide confidence measures for various features of a domain hierarchy. PMID:24494927

The haloarchaeal MCM proteins: bioinformatic analysis and targeted mutagenesis of the β7-β8 and β9-β10 hairpin loops and conserved zinc binding domain cysteines.

PubMed

Kristensen, Tatjana P; Maria Cherian, Reeja; Gray, Fiona C; MacNeill, Stuart A

2014-01-01

The hexameric MCM complex is the catalytic core of the replicative helicase in eukaryotic and archaeal cells. Here we describe the first in vivo analysis of archaeal MCM protein structure and function relationships using the genetically tractable haloarchaeon Haloferax volcanii as a model system. Hfx. volcanii encodes a single MCM protein that is part of the previously identified core group of haloarchaeal MCM proteins. Three structural features of the N-terminal domain of the Hfx. volcanii MCM protein were targeted for mutagenesis: the β7-β8 and β9-β10 β-hairpin loops and putative zinc binding domain. Five strains carrying single point mutations in the β7-β8 β-hairpin loop were constructed, none of which displayed impaired cell growth under normal conditions or when treated with the DNA damaging agent mitomycin C. However, short sequence deletions within the β7-β8 β-hairpin were not tolerated and neither was replacement of the highly conserved residue glutamate 187 with alanine. Six strains carrying paired alanine substitutions within the β9-β10 β-hairpin loop were constructed, leading to the conclusion that no individual amino acid within that hairpin loop is absolutely required for MCM function, although one of the mutant strains displays greatly enhanced sensitivity to mitomycin C. Deletions of two or four amino acids from the β9-β10 β-hairpin were tolerated but mutants carrying larger deletions were inviable. Similarly, it was not possible to construct mutants in which any of the conserved zinc binding cysteines was replaced with alanine, underlining the likely importance of zinc binding for MCM function. The results of these studies demonstrate the feasibility of using Hfx. volcanii as a model system for reverse genetic analysis of archaeal MCM protein function and provide important confirmation of the in vivo importance of conserved structural features identified by previous bioinformatic, biochemical and structural studies.

The haloarchaeal MCM proteins: bioinformatic analysis and targeted mutagenesis of the β7-β8 and β9-β10 hairpin loops and conserved zinc binding domain cysteines

PubMed Central

Kristensen, Tatjana P.; Maria Cherian, Reeja; Gray, Fiona C.; MacNeill, Stuart A.

2014-01-01

The hexameric MCM complex is the catalytic core of the replicative helicase in eukaryotic and archaeal cells. Here we describe the first in vivo analysis of archaeal MCM protein structure and function relationships using the genetically tractable haloarchaeon Haloferax volcanii as a model system. Hfx. volcanii encodes a single MCM protein that is part of the previously identified core group of haloarchaeal MCM proteins. Three structural features of the N-terminal domain of the Hfx. volcanii MCM protein were targeted for mutagenesis: the β7-β8 and β9-β10 β-hairpin loops and putative zinc binding domain. Five strains carrying single point mutations in the β7-β8 β-hairpin loop were constructed, none of which displayed impaired cell growth under normal conditions or when treated with the DNA damaging agent mitomycin C. However, short sequence deletions within the β7-β8 β-hairpin were not tolerated and neither was replacement of the highly conserved residue glutamate 187 with alanine. Six strains carrying paired alanine substitutions within the β9-β10 β-hairpin loop were constructed, leading to the conclusion that no individual amino acid within that hairpin loop is absolutely required for MCM function, although one of the mutant strains displays greatly enhanced sensitivity to mitomycin C. Deletions of two or four amino acids from the β9-β10 β-hairpin were tolerated but mutants carrying larger deletions were inviable. Similarly, it was not possible to construct mutants in which any of the conserved zinc binding cysteines was replaced with alanine, underlining the likely importance of zinc binding for MCM function. The results of these studies demonstrate the feasibility of using Hfx. volcanii as a model system for reverse genetic analysis of archaeal MCM protein function and provide important confirmation of the in vivo importance of conserved structural features identified by previous bioinformatic, biochemical and structural studies. PMID:24723920

Identification of influenza A nucleoprotein body domain residues essential for viral RNA expression expose antiviral target.

PubMed

Davis, Alicia M; Ramirez, Jose; Newcomb, Laura L

2017-02-07

Influenza A virus is controlled with yearly vaccination while emerging global pandemics are kept at bay with antiviral medications. Unfortunately, influenza A viruses have emerged resistance to approved influenza antivirals. Accordingly, there is an urgent need for novel antivirals to combat emerging influenza A viruses resistant to current treatments. Conserved viral proteins are ideal targets because conserved protein domains are present in most, if not all, influenza subtypes, and are presumed less prone to evolve viable resistant versions. The threat of an antiviral resistant influenza pandemic justifies our study to identify and characterize antiviral targets within influenza proteins that are highly conserved. Influenza A nucleoprotein (NP) is highly conserved and plays essential roles throughout the viral lifecycle, including viral RNA synthesis. Using NP crystal structure, we targeted accessible amino acids for substitution. To characterize the NP proteins, reconstituted viral ribonucleoproteins (vRNPs) were expressed in 293 T cells, RNA was isolated, and reverse transcription - quantitative PCR (RT-qPCR) was employed to assess viral RNA expressed from reconstituted vRNPs. Location was confirmed using cellular fractionation and western blot, along with observation of NP-GFP fusion proteins. Nucleic acid binding, oligomerization, and vRNP formation, were each assessed with native gel electrophoresis. Here we report characterization of an accessible and conserved five amino acid region within the NP body domain that plays a redundant but essential role in viral RNA synthesis. Our data demonstrate substitutions in this domain did not alter NP localization, oligomerization, or ability to bind nucleic acids, yet resulted in a defect in viral RNA expression. To define this region further, single and double amino acid substitutions were constructed and investigated. All NP single substitutions were functional, suggesting redundancy, yet different combinations of

Improving Measures of Work-Related Physical Functioning

PubMed Central

McDonough, Christine M.; Ni, Pengsheng; Peterik, Kara; Marfeo, Elizabeth E.; Marino, Molly E.; Meterko, Mark; Rasch, Elizabeth K; Brandt, Diane E.; Jette, Alan M; Chan, Leighton

2016-01-01

Purpose To expand content of the physical function domain of the Work Disability Functional Assessment Battery (WD-FAB), developed for the US Social Security Administration’s (SSA) disability determination process. Methods Newly developed questions were administered to 3,532 recent SSA applicants for work disability benefits and 2,025 US adults. Factor analyses and item response theory (IRT) methods were used to calibrate and link the new items to existing WD-FAB, and computer-adaptive test simulations were conducted. Results Factor and IRT analyses supported integration of 44 new items into 3 existing WD-FAB scales and the addition of a new 11-item scale (Community Mobility). The final physical function domain consisting of: Basic Mobility (56 items), Upper Body Function (34 items), Fine Motor Function (45 items), and Community Mobility (11 items) demonstrated acceptable psychometric properties. Conclusions The WD-FAB offers an important tool for enhancement of work disability determination. The FAB could provide relevant information about work-related functioning for initial assessment of claimants, identifying denied applicants who may benefit from interventions to improve work and health outcomes; enhancing periodic review of work disability beneficiaries; and assessing outcomes for policies, programs and services targeting people with work disability. PMID:28005243

18 CFR 401.40 - Informal conferences and emergencies.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 18 Conservation of Power and Water Resources 2 2011-04-01 2011-04-01 false Informal conferences and emergencies. 401.40 Section 401.40 Conservation of Power and Water Resources DELAWARE RIVER BASIN COMMISSION ADMINISTRATIVE MANUAL RULES OF PRACTICE AND PROCEDURE Project Review Under Section 3.8 of the...

Plumbing the depths: extracellular water storage in specialized leaf structures and its functional expression in a three-domain pressure -volume relationship.

PubMed

Nguyen, Hoa T; Meir, Patrick; Wolfe, Joe; Mencuccini, Maurizio; Ball, Marilyn C

2017-07-01

A three-domain pressure-volume relationship (PV curve) was studied in relation to leaf anatomical structure during dehydration in the grey mangrove, Avicennia marina. In domain 1, relative water content (RWC) declined 13% with 0.85 MPa decrease in leaf water potential, reflecting a decrease in extracellular water stored primarily in trichomes and petiolar cisternae. In domain 2, RWC decreased by another 12% with a further reduction in leaf water potential to -5.1 MPa, the turgor loss point. Given the osmotic potential at full turgor (-4.2 MPa) and the effective modulus of elasticity (~40 MPa), domain 2 emphasized the role of cell wall elasticity in conserving cellular hydration during leaf water loss. Domain 3 was dominated by osmotic effects and characterized by plasmolysis in most tissues and cell types without cell wall collapse. Extracellular and cellular water storage could support an evaporation rate of 1 mmol m -2 s -1 for up to 54 and 50 min, respectively, before turgor loss was reached. This study emphasized the importance of leaf anatomy for the interpretation of PV curves, and identified extracellular water storage sites that enable transient water use without substantive turgor loss when other factors, such as high soil salinity, constrain rates of water transport. © 2016 John Wiley & Sons Ltd.

40 CFR 256.31 - Recommendations for developing and implementing resource conservation and recovery programs.

Code of Federal Regulations, 2010 CFR

2010-07-01

... implementing resource conservation and recovery programs. 256.31 Section 256.31 Protection of Environment... SOLID WASTE MANAGEMENT PLANS Resource Conservation and Resource Recovery Programs § 256.31 Recommendations for developing and implementing resource conservation and recovery programs. (a) In order to...

The conserved phenylalanine in the K+ channel voltage-sensor domain creates a barrier with unidirectional effects.

PubMed

Schwaiger, Christine S; Liin, Sara I; Elinder, Fredrik; Lindahl, Erik

2013-01-08

Voltage-gated ion channels are crucial for regulation of electric activity of excitable tissues such as nerve cells, and play important roles in many diseases. During activation, the charged S4 segment in the voltage sensor domain translates across a hydrophobic core forming a barrier for the gating charges. This barrier is critical for channel function, and a conserved phenylalanine in segment S2 has previously been identified to be highly sensitive to substitutions. Here, we have studied the kinetics of K(v)1-type potassium channels (Shaker and K(v)1.2/2.1 chimera) through site-directed mutagenesis, electrophysiology, and molecular simulations. The F290L mutation in Shaker (F233L in K(v)1.2/2.1) accelerates channel closure by at least a factor 50, although opening is unaffected. Free energy profiles with the hydrophobic neighbors of F233 mutated to alanine indicate that the open state with the fourth arginine in S4 above the hydrophobic core is destabilized by ∼17 kJ/mol compared to the first closed intermediate. This significantly lowers the barrier of the first deactivation step, although the last step of activation is unaffected. Simulations of wild-type F233 show that the phenyl ring always rotates toward the extracellular side both for activation and deactivation, which appears to help stabilize a well-defined open state. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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

40 CFR 73.80 - Operation of allowance reserve program for conservation and renewable energy.

Code of Federal Regulations, 2011 CFR

2011-07-01

... for conservation and renewable energy. 73.80 Section 73.80 Protection of Environment ENVIRONMENTAL... and Renewable Energy Reserve § 73.80 Operation of allowance reserve program for conservation and renewable energy. (a) General. The Administrator will allocate allowances from the Conservation and...

40 CFR 73.80 - Operation of allowance reserve program for conservation and renewable energy.

Code of Federal Regulations, 2013 CFR

2013-07-01

... for conservation and renewable energy. 73.80 Section 73.80 Protection of Environment ENVIRONMENTAL... and Renewable Energy Reserve § 73.80 Operation of allowance reserve program for conservation and renewable energy. (a) General. The Administrator will allocate allowances from the Conservation and...

40 CFR 73.80 - Operation of allowance reserve program for conservation and renewable energy.

Code of Federal Regulations, 2014 CFR

2014-07-01

... for conservation and renewable energy. 73.80 Section 73.80 Protection of Environment ENVIRONMENTAL... and Renewable Energy Reserve § 73.80 Operation of allowance reserve program for conservation and renewable energy. (a) General. The Administrator will allocate allowances from the Conservation and...

40 CFR 73.80 - Operation of allowance reserve program for conservation and renewable energy.

Code of Federal Regulations, 2012 CFR

2012-07-01

... for conservation and renewable energy. 73.80 Section 73.80 Protection of Environment ENVIRONMENTAL... and Renewable Energy Reserve § 73.80 Operation of allowance reserve program for conservation and renewable energy. (a) General. The Administrator will allocate allowances from the Conservation and...

40 CFR 73.80 - Operation of allowance reserve program for conservation and renewable energy.

Code of Federal Regulations, 2010 CFR

2010-07-01

... for conservation and renewable energy. 73.80 Section 73.80 Protection of Environment ENVIRONMENTAL... and Renewable Energy Reserve § 73.80 Operation of allowance reserve program for conservation and renewable energy. (a) General. The Administrator will allocate allowances from the Conservation and...

The HARP domain dictates the annealing helicase activity of HARP/SMARCAL1.

PubMed

Ghosal, Gargi; Yuan, Jingsong; Chen, Junjie

2011-06-01

Mutations in HepA-related protein (HARP, or SMARCAL1) cause Schimke immunoosseous dysplasia (SIOD). HARP has ATP-dependent annealing helicase activity, which helps to stabilize stalled replication forks and facilitate DNA repair during replication. Here, we show that the conserved tandem HARP (2HP) domain dictates this annealing helicase activity. Furthermore, chimeric proteins generated by fusing the 2HP domain of HARP with the SNF2 domain of BRG1 or HELLS show annealing helicase activity in vitro and, when targeted to replication forks, mimic the functions of HARP in vivo. We propose that the HARP domain endows HARP with this ATP-driven annealing helicase activity.

Biochemical and Structural Characterization of a Five-domain GH115 α-Glucuronidase from the Marine Bacterium Saccharophagus degradans 2-40 T

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

Wang, Weijun; Yan, Ruoyu; Nocek, Boguslaw P.

Glucuronic acid (GlcAp) and/or methylglucuronic acid (MeGlcAp) decorate the major forms of xylan in hardwood and coniferous softwoods as well as many cereal grains. Accordingly, the complete utilization of glucuronoxylans or conversion to sugar precursors requires the action of main chain xylanases as well as -glucuronidases that release the - (132)-linked (Me)GlcAp side groups. Herein, a family GH115 enzyme from the marine bacterium Saccharophagus degradans 2-40T, SdeAgu115A, demonstrated activity toward glucuronoxylan and oligomers thereof with preference toward MeGlcAp linked to internal xylopyranosyl residues. Unique biochemical characteristics of NaCl activation were also observed. The crystal structure of SdeAgu115A revealed a five-domainmore » architecture, with an additional insertion C domain that had significant impact on the domain arrangement of SdeAgu115A monomer and its dimerization. The participation of domain C in substrate binding was supported by reduced substrate inhibition upon introducing W773A, W689A, and F696A substitutions within this domain. In addition to Asp-335, the catalytic essentiality of Glu-216 was revealed by site-specific mutagenesis. A primary sequence analysis suggested that the SdeAgu115A architecture is shared by more than half of GH115 members, thus defining a distinct archetype for GH115 enzymes.« less

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

Phylogenetic Relationships within the Opisthokonta Based on Phylogenomic Analyses of Conserved Single-Copy Protein Domains

PubMed Central

Torruella, Guifré; Derelle, Romain; Paps, Jordi; Lang, B. Franz; Roger, Andrew J.; Shalchian-Tabrizi, Kamran; Ruiz-Trillo, Iñaki

2012-01-01

Many of the eukaryotic phylogenomic analyses published to date were based on alignments of hundreds to thousands of genes. Frequently, in such analyses, the most realistic evolutionary models currently available are often used to minimize the impact of systematic error. However, controversy remains over whether or not idiosyncratic gene family dynamics (i.e., gene duplications and losses) and incorrect orthology assignments are always appropriately taken into account. In this paper, we present an innovative strategy for overcoming orthology assignment problems. Rather than identifying and eliminating genes with paralogy problems, we have constructed a data set comprised exclusively of conserved single-copy protein domains that, unlike most of the commonly used phylogenomic data sets, should be less confounded by orthology miss-assignments. To evaluate the power of this approach, we performed maximum likelihood and Bayesian analyses to infer the evolutionary relationships within the opisthokonts (which includes Metazoa, Fungi, and related unicellular lineages). We used this approach to test 1) whether Filasterea and Ichthyosporea form a clade, 2) the interrelationships of early-branching metazoans, and 3) the relationships among early-branching fungi. We also assessed the impact of some methods that are known to minimize systematic error, including reducing the distance between the outgroup and ingroup taxa or using the CAT evolutionary model. Overall, our analyses support the Filozoa hypothesis in which Ichthyosporea are the first holozoan lineage to emerge followed by Filasterea, Choanoflagellata, and Metazoa. Blastocladiomycota appears as a lineage separate from Chytridiomycota, although this result is not strongly supported. These results represent independent tests of previous phylogenetic hypotheses, highlighting the importance of sophisticated approaches for orthology assignment in phylogenomic analyses. PMID:21771718

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.

Crowdfunding biodiversity conservation.

PubMed

Gallo-Cajiao, E; Archibald, C; Friedman, R; Steven, R; Fuller, R A; Game, E T; Morrison, T H; Ritchie, E G

2018-05-26

Raising funds is critical for conserving biodiversity and hence so too is scrutinizing emerging financial mechanisms that might help achieve this goal. In this context, anecdotal evidence indicates crowdfunding is being used to support a variety of activities needed for biodiversity conservation, yet its magnitude and allocation remain largely unknown. We conducted a global analysis to help address this knowledge gap, based on empirical data from conservation-focused projects extracted from crowdfunding platforms. For each project, we determined the funds raised, date, country of implementation, proponent characteristics, activity type, biodiversity realm, and target taxa. We identified 72 relevant platforms and 577 conservation-focused projects that have raised US$4 790 634 since 2009. Whilst proponents were based in 38 countries, projects were delivered across 80 countries, indicating a potential mechanism of resource mobilization. Proponents were from non-governmental organizations (35%), universities (30%), or were freelancers (26%). Most projects were for research (40%), persuasion (31%), and on-ground actions (21%). Projects have focused primarily on species (57.7%) and terrestrial ecosystems (20.3%), and less on marine (8.8%) and freshwater ecosystems (3.6%). Projects have focused on 208 species, including a disproportionate number of threatened bird and mammal species. Crowdfunding for biodiversity conservation has now become a global phenomenon and presents signals for potential expansion, despite possible pitfalls. Opportunities arise from its spatial amplifying effect, steady increase over time, inclusion of Cinderella species, adoption by multiple actors, and funding of a range of activities beyond research. Our study paves the way for further research on key questions, such as campaign success rates, effectiveness, and drivers of adoption. Even though the capital input of crowdfunding so far has been modest compared to other conservation finance

Evaluation of genetic and metabolic role of SKIP11 in Arabidopsis thaliana

NASA Astrophysics Data System (ADS)

Hassan, Muhammad Naeem ul; Ismail, Ismanizan

2015-09-01

Most of the regulatory proteins are degraded by 26S proteasome complex, only when they are tagged by Ubiquitin. A complex of four proteins, SKP1-Cullin-Ring box-F box (SCF) catalyses the final step to link the Ubiquitin tag with the target proteins. SCF complex interacts with the target proteins through F-box proteins, which confer the overall substrate specificity to the complex. F-box proteins, one of the largest family of proteins in plants have an N-terminal F-box domain and variable C-terminal domains, like leucine-rich repeat, WD-40 repeat and the kelch-repeat domains. In this study, we analysed the role of SKIP11, a kelch containing F-box protein (KFB) from Arabidopsis thaliana, by using reverse genetics strategy. The results show that SKIP11 is involved in the down-regulation of oxylipin pathway, possibly through the degradation of enzymes or/ and the regulatory factors of the pathway.

Draft whole genome sequence of groundnut stem rot fungus Athelia rolfsii revealing genetic architect of its pathogenicity and virulence.

PubMed

Iquebal, M A; Tomar, Rukam S; Parakhia, M V; Singla, Deepak; Jaiswal, Sarika; Rathod, V M; Padhiyar, S M; Kumar, Neeraj; Rai, Anil; Kumar, Dinesh

2017-07-13

Groundnut (Arachis hypogaea L.) is an important oil seed crop having major biotic constraint in production due to stem rot disease caused by fungus, Athelia rolfsii causing 25-80% loss in productivity. As chemical and biological combating strategies of this fungus are not very effective, thus genome sequencing can reveal virulence and pathogenicity related genes for better understanding of the host-parasite interaction. We report draft assembly of Athelia rolfsii genome of ~73 Mb having 8919 contigs. Annotation analysis revealed 16830 genes which are involved in fungicide resistance, virulence and pathogenicity along with putative effector and lethal genes. Secretome analysis revealed CAZY genes representing 1085 enzymatic genes, glycoside hydrolases, carbohydrate esterases, carbohydrate-binding modules, auxillary activities, glycosyl transferases and polysaccharide lyases. Repeat analysis revealed 11171 SSRs, LTR, GYPSY and COPIA elements. Comparative analysis with other existing ascomycotina genome predicted conserved domain family of WD40, CYP450, Pkinase and ABC transporter revealing insight of evolution of pathogenicity and virulence. This study would help in understanding pathogenicity and virulence at molecular level and development of new combating strategies. Such approach is imperative in endeavour of genome based solution in stem rot disease management leading to better productivity of groundnut crop in tropical region of world.

Platelet glycoprotein Ibalpha forms catch bonds with human WT vWF but not with type 2B von Willebrand disease vWF.

PubMed

Yago, Tadayuki; Lou, Jizhong; Wu, Tao; Yang, Jun; Miner, Jonathan J; Coburn, Leslie; López, José A; Cruz, Miguel A; Dong, Jing-Fei; McIntire, Larry V; McEver, Rodger P; Zhu, Cheng

2008-09-01

Arterial blood flow enhances glycoprotein Ibalpha (GPIbalpha) binding to vWF, which initiates platelet adhesion to injured vessels. Mutations in the vWF A1 domain that cause type 2B von Willebrand disease (vWD) reduce the flow requirement for adhesion. Here we show that increasing force on GPIbalpha/vWF bonds first prolonged ("catch") and then shortened ("slip") bond lifetimes. Two type 2B vWD A1 domain mutants, R1306Q and R1450E, converted catch bonds to slip bonds by prolonging bond lifetimes at low forces. Steered molecular dynamics simulations of GPIbalpha dissociating from the A1 domain suggested mechanisms for catch bonds and their conversion by the A1 domain mutations. Catch bonds caused platelets and GPIbalpha-coated microspheres to roll more slowly on WT vWF and WT A1 domains as flow increased from suboptimal levels, explaining flow-enhanced rolling. Longer bond lifetimes at low forces eliminated the flow requirement for rolling on R1306Q and R1450E mutant A1 domains. Flowing platelets agglutinated with microspheres bearing R1306Q or R1450E mutant A1 domains, but not WT A1 domains. Therefore, catch bonds may prevent vWF multimers from agglutinating platelets. A disintegrin and metalloproteinase with a thrombospondin type 1 motif-13 (ADAMTS-13) reduced platelet agglutination with microspheres bearing a tridomain A1A2A3 vWF fragment with the R1450E mutation in a shear-dependent manner. We conclude that in type 2B vWD, prolonged lifetimes of vWF bonds with GPIbalpha on circulating platelets may allow ADAMTS-13 to deplete large vWF multimers, causing bleeding.

Platelet glycoprotein Ibα forms catch bonds with human WT vWF but not with type 2B von Willebrand disease vWF

PubMed Central

Yago, Tadayuki; Lou, Jizhong; Wu, Tao; Yang, Jun; Miner, Jonathan J.; Coburn, Leslie; López, José A.; Cruz, Miguel A.; Dong, Jing-Fei; McIntire, Larry V.; McEver, Rodger P.; Zhu, Cheng

2008-01-01

Arterial blood flow enhances glycoprotein Ibα (GPIbα) binding to vWF, which initiates platelet adhesion to injured vessels. Mutations in the vWF A1 domain that cause type 2B von Willebrand disease (vWD) reduce the flow requirement for adhesion. Here we show that increasing force on GPIbα/vWF bonds first prolonged (“catch”) and then shortened (“slip”) bond lifetimes. Two type 2B vWD A1 domain mutants, R1306Q and R1450E, converted catch bonds to slip bonds by prolonging bond lifetimes at low forces. Steered molecular dynamics simulations of GPIbα dissociating from the A1 domain suggested mechanisms for catch bonds and their conversion by the A1 domain mutations. Catch bonds caused platelets and GPIbα-coated microspheres to roll more slowly on WT vWF and WT A1 domains as flow increased from suboptimal levels, explaining flow-enhanced rolling. Longer bond lifetimes at low forces eliminated the flow requirement for rolling on R1306Q and R1450E mutant A1 domains. Flowing platelets agglutinated with microspheres bearing R1306Q or R1450E mutant A1 domains, but not WT A1 domains. Therefore, catch bonds may prevent vWF multimers from agglutinating platelets. A disintegrin and metalloproteinase with a thrombospondin type 1 motif–13 (ADAMTS-13) reduced platelet agglutination with microspheres bearing a tridomain A1A2A3 vWF fragment with the R1450E mutation in a shear-dependent manner. We conclude that in type 2B vWD, prolonged lifetimes of vWF bonds with GPIbα on circulating platelets may allow ADAMTS-13 to deplete large vWF multimers, causing bleeding. PMID:18725999

Structure of the GH1 domain of guanylate kinase-associated protein from Rattus norvegicus

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

Tong, Junsen; Yang, Huiseon; Eom, Soo Hyun

2014-09-12

Graphical abstract: - Highlights: • The crystal structure of GKAP homology domain 1 (GH1) was determined. • GKAP GH1 is a three-helix bundle connected by short flexible loops. • The predicted helix α4 associates weakly with the helix α3, suggesting dynamic nature of the GH1 domain. - Abstract: Guanylate-kinase-associated protein (GKAP) is a scaffolding protein that links NMDA receptor-PSD-95 to Shank–Homer complexes by protein–protein interactions at the synaptic junction. GKAP family proteins are characterized by the presence of a C-terminal conserved GKAP homology domain 1 (GH1) of unknown structure and function. In this study, crystal structure of the GH1 domainmore » of GKAP from Rattus norvegicus was determined in fusion with an N-terminal maltose-binding protein at 2.0 Å resolution. The structure of GKAP GH1 displays a three-helix bundle connected by short flexible loops. The predicted helix α4 which was not visible in the crystal structure associates weakly with the helix α3 suggesting dynamic nature of the GH1 domain. The strict conservation of GH1 domain across GKAP family members and the lack of a catalytic active site required for enzyme activity imply that the GH1 domain might serve as a protein–protein interaction module for the synaptic protein clustering.« less

Biochemical and bioinformatic analysis of the MYO19 motor domain

PubMed Central

Adikes, Rebecca C.; Unrath, William C.; Yengo, Christopher M.; Quintero, Omar A.

2014-01-01

Mitochondrial dynamics are dependent on both the microtubule and actin cytoskeletal systems. Evidence for the involvement of myosin motors has been described in many systems, and until recently a candidate mitochondrial transport motor had not been described in vertebrates. Myosin-XIX (MYO19) was predicted to represent a novel class of myosin and had previously been shown to bind to mitochondria and increase mitochondrial network dynamics when ectopically expressed. Our analyses comparing ∼40 MYO19 orthologs to ∼2000 other myosin motor domain sequences identified instances of homology well-conserved within class XIX myosins that were not found in other myosin classes, suggesting MYO19-specific mechanochemistry. Steady-state biochemical analyses of the MYO19 motor domain indicate that Homo sapiens MYO19 is a functional motor. Insect cell-expressed constructs bound calmodulin as a light chain at the predicted stoichiometry and displayed actin-activated ATPase activity. MYO19 constructs demonstrated high actin affinity in the presence of ATP in actin-cosedimentation assays, and translocated actin filaments in gliding assays. Expression of GFP-MYO19 containing a mutation impairing ATPase activity did not enhance mitochondrial network dynamics, as occurs with wild-type MYO19, indicating that myosin motor activity is required for mitochondrial motility. The measured biochemical properties of MYO19 suggest it is a high-duty ratio motor that could serve to transport mitochondria or anchor mitochondria, depending upon the cellular microenvironment. PMID:23568824

Top 40 priorities for science to inform conservation and management policy in the United States

USGS Publications Warehouse

Fleishman, Erica; Blockstein, David E.; Hall, John A.; Mascia, Michael B.; Rudd, Murray A.; Scott, J. Michael; Sutherland, William J.; Bartuska, Ann M.; Brown, A. Gordon; Christen, Catherine A.; Clement, Joel P.; DellaSala, Dominick; Duke, Clifford D.; Fiske, Shirley J.; Gosnell, Hannah; Haney, J. Christopher; Hutchins, Michael; Klein, Mary L.; Marqusee, Jeffrey; Noon, Barry R.; Nordgren, John R.; Orbuch, Paul M.; Powell, Jimmie; Quarles, Steven P.; Saterson, Kathryn A.; Stein, Bruce A.; Webster, Michael S.; Vedder, Amy

2011-01-01

To maximize the utility of research to decisionmaking, especially given limited financial resources, scientists must set priorities for their efforts. We present a list of the top 40 high-priority, multidisciplinary research questions directed toward informing some of the most important current and future decisions about management of species, communities, and ecological processes in the United States. The questions were generated by an open, inclusive process that included personal interviews with decisionmakers, broad solicitation of research needs from scientists and policymakers, and an intensive workshop that included scientifically oriented individuals responsible for managing and developing policy related to natural resources. The process differed from previous efforts to set priorities for conservation research in its focus on the engagement of decisionmakers in addition to researchers. The research priorities emphasized the importance of addressing societal context and exploration of trade-offs among alternative policies and actions, as well as more traditional questions related to ecological processes and functions.

UXDs-Driven Transferring Method from TRIZ Solution to Domain Solution

NASA Astrophysics Data System (ADS)

Ma, Lihui; Cao, Guozhong; Chang, Yunxia; Wei, Zihui; Ma, Kai

The translation process from TRIZ solutions to domain solutions is an analogy-based process. TRIZ solutions, such as 40 inventive principles and the related cases, are medium-solutions for domain problems. Unexpected discoveries (UXDs) are the key factors to trigger designers to generate new ideas for domain solutions. The Algorithm of UXD resolving based on Means-Ends Analysis(MEA) is studied and an UXDs-driven transferring method from TRIZ solution to domain solution is formed. A case study shows the application of the process.

Combining protein sequence, structure, and dynamics: A novel approach for functional evolution analysis of PAS domain superfamily.

PubMed

Dong, Zheng; Zhou, Hongyu; Tao, Peng

2018-02-01

PAS domains are widespread in archaea, bacteria, and eukaryota, and play important roles in various functions. In this study, we aim to explore functional evolutionary relationship among proteins in the PAS domain superfamily in view of the sequence-structure-dynamics-function relationship. We collected protein sequences and crystal structure data from RCSB Protein Data Bank of the PAS domain superfamily belonging to three biological functions (nucleotide binding, photoreceptor activity, and transferase activity). Protein sequences were aligned and then used to select sequence-conserved residues and build phylogenetic tree. Three-dimensional structure alignment was also applied to obtain structure-conserved residues. The protein dynamics were analyzed using elastic network model (ENM) and validated by molecular dynamics (MD) simulation. The result showed that the proteins with same function could be grouped by sequence similarity, and proteins in different functional groups displayed statistically significant difference in their vibrational patterns. Interestingly, in all three functional groups, conserved amino acid residues identified by sequence and structure conservation analysis generally have a lower fluctuation than other residues. In addition, the fluctuation of conserved residues in each biological function group was strongly correlated with the corresponding biological function. This research suggested a direct connection in which the protein sequences were related to various functions through structural dynamics. This is a new attempt to delineate functional evolution of proteins using the integrated information of sequence, structure, and dynamics. © 2017 The Protein Society.

3D model for Cancerous Inhibitor of Protein Phosphatase 2A armadillo domain unveils highly conserved protein-protein interaction characteristics.

PubMed

Dahlström, Käthe M; Salminen, Tiina A

2015-12-07

Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) is a human oncoprotein, which exerts its cancer-promoting function through interaction with other proteins, for example Protein Phosphatase 2A (PP2A) and MYC. The lack of structural information for CIP2A significantly prevents the design of anti-cancer therapeutics targeting this protein. In an attempt to counteract this fact, we modeled the three-dimensional structure of the N-terminal domain (CIP2A-ArmRP), analyzed key areas and amino acids, and coupled the results to the existing literature. The model reliably shows a stable armadillo repeat fold with a positively charged groove. The fact that this conserved groove highly likely binds peptides is corroborated by the presence of a conserved polar ladder, which is essential for the proper peptide-binding mode of armadillo repeat proteins and, according to our results, several known CIP2A interaction partners appropriately possess an ArmRP-binding consensus motif. Moreover, we show that Arg229Gln, which has been linked to the development of cancer, causes a significant change in charge and surface properties of CIP2A-ArmRP. In conclusion, our results reveal that CIP2A-ArmRP shares the typical fold, protein-protein interaction site and interaction patterns with other natural armadillo proteins and that, presumably, several interaction partners bind into the central groove of the modeled CIP2A-ArmRP. By providing essential structural characteristics of CIP2A, the present study significantly increases our knowledge on how CIP2A interacts with other proteins in cancer progression and how to develop new therapeutics targeting CIP2A. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Rock bream (Oplegnathus fasciatus) IL-12p40: identification, expression, and effect on bacterial infection.

PubMed

Zhang, Lu; Zhang, Bao-Cun; Hu, Yong-Hua

2014-08-01

IL-12p40, also called IL-12β, is a subunit of the proinflammatory cytokines interleukin (IL)-12 and IL-23. In teleost, IL-12p40 homologues have been identified in several species, however, the biological function of fish IL-12p40 is essentially unknown. In this work, we reported the identification and analysis of an IL-12p40, OfIL-12p40, from rock bream (Oplegnathus fasciatus). OfIL-12p40 is composed of 361 amino acids and possesses a conserved IL-12p40 domain and a WSxWS signature motif characteristic of known IL-12p40. Constitutive expression of OfIL-12p40 occurred in multiple tissues and was highest in kidney. Experimental infection with bacterial pathogen upregulated the expression of OfIL-12p40 in kidney and spleen in a time-dependent manner. Purified recombinant OfIL-12p40 (rOfIL-12p40) stimulated the respiratory burst activity of peripheral blood leukocytes in a dose-dependent manner. rOfIL-12p40 also enhanced the resistance of rock bream against bacterial infection and upregulated the expression of innate immune genes in kidney. Taken together, these results indicate that OfIL-12p40 possesses cytokine-like property and plays a role in immune defense against bacterial infection. Copyright © 2014 Elsevier Ltd. All rights reserved.

Structure/Function Analysis of Recurrent Mutations in SETD2 Protein Reveals a Critical and Conserved Role for a SET Domain Residue in Maintaining Protein Stability and Histone H3 Lys-36 Trimethylation*

PubMed Central

Hacker, Kathryn E.; Fahey, Catherine C.; Shinsky, Stephen A.; Chiang, Yun-Chen J.; DiFiore, Julia V.; Jha, Deepak Kumar; Vo, Andy H.; Shavit, Jordan A.; Davis, Ian J.; Strahl, Brian D.; Rathmell, W. Kimryn

2016-01-01

The yeast Set2 histone methyltransferase is a critical enzyme that plays a number of key roles in gene transcription and DNA repair. Recently, the human homologue, SETD2, was found to be recurrently mutated in a significant percentage of renal cell carcinomas, raising the possibility that the activity of SETD2 is tumor-suppressive. Using budding yeast and human cell line model systems, we examined the functional significance of two evolutionarily conserved residues in SETD2 that are recurrently mutated in human cancers. Whereas one of these mutations (R2510H), located in the Set2 Rpb1 interaction domain, did not result in an observable defect in SETD2 enzymatic function, a second mutation in the catalytic domain of this enzyme (R1625C) resulted in a complete loss of histone H3 Lys-36 trimethylation (H3K36me3). This mutant showed unchanged thermal stability as compared with the wild type protein but diminished binding to the histone H3 tail. Surprisingly, mutation of the conserved residue in Set2 (R195C) similarly resulted in a complete loss of H3K36me3 but did not affect dimethylated histone H3 Lys-36 (H3K36me2) or functions associated with H3K36me2 in yeast. Collectively, these data imply a critical role for Arg-1625 in maintaining the protein interaction with H3 and specific H3K36me3 function of this enzyme, which is conserved from yeast to humans. They also may provide a refined biochemical explanation for how H3K36me3 loss leads to genomic instability and cancer. PMID:27528607

Structure of the kinase domain of Gilgamesh from Drosophila melanogaster

PubMed Central

Han, Ni; Chen, CuiCui; Shi, Zhubing; Cheng, Dianlin

2014-01-01

The CK1 family kinases regulate multiple cellular aspects and play important roles in Wnt/Wingless and Hedgehog signalling. The kinase domain of Drosophila Gilgamesh isoform I (Gilgamesh-I), a homologue of human CK1-γ, was purified and crystallized. Crystals of methylated Gilgamesh-I kinase domain with a D210A mutation diffracted to 2.85 Å resolution and belonged to space group P43212, with unit-cell parameters a = b = 52.025, c = 291.727 Å. The structure of Gilgamesh-I kinase domain, which was determined by molecular replacement, has conserved catalytic elements and an active conformation. Structural comparison indicates that an extended loop between the α1 helix and the β4 strand exists in the Gilgamesh-I kinase domain. This extended loop may regulate the activity and function of Gilgamesh-I. PMID:24699734

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

PubMed Central

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

2016-01-01

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

COOLAIR Antisense RNAs Form Evolutionarily Conserved Elaborate Secondary Structures

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

Hawkes, Emily J.; Hennelly, Scott P.; Novikova, Irina V.

There is considerable debate about the functionality of long non-coding RNAs (lncRNAs). Lack of sequence conservation has been used to argue against functional relevance. Here, we investigated antisense lncRNAs, called COOLAIR, at the A. thaliana FLC locus and experimentally determined their secondary structure. The major COOLAIR variants are highly structured, organized by exon. The distally polyadenylated transcript has a complex multi-domain structure, altered by a single non-coding SNP defining a functionally distinct A. thaliana FLC haplotype. The A. thaliana COOLAIR secondary structure was used to predict COOLAIR exons in evolutionarily divergent Brassicaceae species. These predictions were validated through chemical probingmore » and cloning. Despite the relatively low nucleotide sequence identity, the structures, including multi-helix junctions, show remarkable evolutionary conservation. In a number of places, the structure is conserved through covariation of a non-contiguous DNA sequence. This structural conservation supports a functional role for COOLAIR transcripts rather than, or in addition to, antisense transcription.« less

COOLAIR Antisense RNAs Form Evolutionarily Conserved Elaborate Secondary Structures

DOE PAGES

Hawkes, Emily J.; Hennelly, Scott P.; Novikova, Irina V.; ...

2016-09-20

There is considerable debate about the functionality of long non-coding RNAs (lncRNAs). Lack of sequence conservation has been used to argue against functional relevance. Here, we investigated antisense lncRNAs, called COOLAIR, at the A. thaliana FLC locus and experimentally determined their secondary structure. The major COOLAIR variants are highly structured, organized by exon. The distally polyadenylated transcript has a complex multi-domain structure, altered by a single non-coding SNP defining a functionally distinct A. thaliana FLC haplotype. The A. thaliana COOLAIR secondary structure was used to predict COOLAIR exons in evolutionarily divergent Brassicaceae species. These predictions were validated through chemical probingmore » and cloning. Despite the relatively low nucleotide sequence identity, the structures, including multi-helix junctions, show remarkable evolutionary conservation. In a number of places, the structure is conserved through covariation of a non-contiguous DNA sequence. This structural conservation supports a functional role for COOLAIR transcripts rather than, or in addition to, antisense transcription.« less

Nanobody Binding to a Conserved Epitope Promotes Norovirus Particle Disassembly

PubMed Central

Koromyslova, Anna D.

2014-01-01

ABSTRACT Human noroviruses are icosahedral single-stranded RNA viruses. The capsid protein is divided into shell (S) and protruding (P) domains, which are connected by a flexible hinge region. There are numerous genetically and antigenically distinct noroviruses, and the dominant strains evolve every other year. Vaccine and antiviral development is hampered by the difficulties in growing human norovirus in cell culture and the continually evolving strains. Here, we show the X-ray crystal structures of human norovirus P domains in complex with two different nanobodies. One nanobody, Nano-85, was broadly reactive, while the other, Nano-25, was strain specific. We showed that both nanobodies bound to the lower region on the P domain and had nanomolar affinities. The Nano-85 binding site mainly comprised highly conserved amino acids among the genetically distinct genogroup II noroviruses. Several of the conserved residues also were recognized by a broadly reactive monoclonal antibody, which suggested this region contained a dominant epitope. Superposition of the P domain nanobody complex structures into a cryoelectron microscopy particle structure revealed that both nanobodies bound at occluded sites on the particles. The flexible hinge region, which contained ∼10 to 12 amino acids, likely permitted a certain degree of P domain movement on the particles in order to accommodate the nanobodies. Interestingly, the Nano-85 binding interaction with intact particles caused the particles to disassemble in vitro. Altogether, these results suggested that the highly conserved Nano-85 binding epitope contained a trigger mechanism for particle disassembly. Principally, this epitope represents a potential site of norovirus vulnerability. IMPORTANCE We characterized two different nanobodies (Nano-85 and Nano-25) that bind to human noroviruses. Both nanobodies bound with high affinities to the lower region of the P domain, which was occluded on intact particles. Nano-25 was

Source-Receptor Relationship Analysis of the Atmospheric Deposition of PAHs Subject to Long-Range Transport in Northeast Asia.

PubMed

Inomata, Yayoi; Kajino, Mizuo; Sato, Keiichi; Kurokawa, Junichi; Tang, Ning; Ohara, Toshimasa; Hayakawa, Kazuichi; Ueda, Hiromasa

2017-07-18

The source-receptor relationship analysis of PAH deposition in Northeast Asia was investigated using an Eulerian regional-scale aerosol chemical transport model. Dry deposition (DD) of PAH was controlled by wind flow patterns, whereas wet deposition (WD) depended on precipitation in addition to wind flow patterns. The contribution of WD was approximately 50-90% of the total deposition, except during winter in Northern China (NCHN) and Eastern Russia (ERUS) because of the low amount of precipitation. The amount of PAH deposition showed clear seasonal variation and was high in winter and low in summer in downwind (South Korea, Japan) and oceanic-receptor regions. In the downwind region, the contributions from NCHN (WD 28-52%; DD 54-55%) and Central China (CCHN) (WD 43-65%; DD 33-38%) were large in winter, whereas self-contributions (WD 20-51%; DD 79-81%) were relatively high in summer. In the oceanic-receptor region, the deposition amount decreased with distance from the Asian continent. The amount of DD was strongly influenced by emissions from neighboring domains. The contributions of WD from NCHN (16-20%) and CCHN (28-35%) were large. The large contributions from China in summer to the downwind region were linked to vertical transport of PAHs over the Asian continent associated with convection.

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

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

Distribution and cluster analysis of predicted intrinsically disordered protein Pfam domains

PubMed Central

Williams, Robert W; Xue, Bin; Uversky, Vladimir N; Dunker, A Keith

2013-01-01

The Pfam database groups regions of proteins by how well hidden Markov models (HMMs) can be trained to recognize similarities among them. Conservation pressure is probably in play here. The Pfam seed training set includes sequence and structure information, being drawn largely from the PDB. A long standing hypothesis among intrinsically disordered protein (IDP) investigators has held that conservation pressures are also at play in the evolution of different kinds of intrinsic disorder, but we find that predicted intrinsic disorder (PID) is not always conserved across Pfam domains. Here we analyze distributions and clusters of PID regions in 193024 members of the version 23.0 Pfam seed database. To include the maximum information available for proteins that remain unfolded in solution, we employ the 10 linearly independent Kidera factors1–3 for the amino acids, combined with PONDR4 predictions of disorder tendency, to transform the sequences of these Pfam members into an 11 column matrix where the number of rows is the length of each Pfam region. Cluster analyses of the set of all regions, including those that are folded, show 6 groupings of domains. Cluster analyses of domains with mean VSL2b scores greater than 0.5 (half predicted disorder or more) show at least 3 separated groups. It is hypothesized that grouping sets into shorter sequences with more uniform length will reveal more information about intrinsic disorder and lead to more finely structured and perhaps more accurate predictions. HMMs could be trained to include this information. PMID:28516017

18 CFR 35.40 - Ancillary services.

Code of Federal Regulations, 2011 CFR

2011-04-01

....40 Section 35.40 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY REGULATIONS UNDER THE FEDERAL POWER ACT FILING OF RATE SCHEDULES AND TARIFFS Wholesale Sales of Electric Energy, Capacity and Ancillary Services at Market-Based Rates § 35.40 Ancillary...

Multiple conserved domains of the nucleoporin Nup124p and its orthologs Nup1p and Nup153 are critical for nuclear import and activity of the fission yeast Tf1 retrotransposon.

PubMed

Sistla, Srivani; Pang, Junxiong Vincent; Wang, Cui Xia; Balasundaram, David

2007-09-01

The nucleoporin Nup124p is a host protein required for the nuclear import of both, retrotransposon Tf1-Gag as well as the retroviral HIV-1 Vpr in fission yeast. The human nucleoporin Nup153 and the Saccharomyces cerevisiae Nup1p were identified as orthologs of Nup124p. In this study, we show that all three nucleoporins share a large FG/FXFG-repeat domain and a C-terminal peptide sequence, GRKIxxxxxRRKx, that are absolutely essential for Tf1 retrotransposition. Though the FXFG domain was essential, the FXFG repeats themselves could be eliminated without loss of retrotransposon activity, suggesting the existence of a common element unrelated to FG/FXFG motifs. The Nup124p C-terminal peptide, GRKIAVPRSRRKR, was extremely sensitive to certain single amino acid changes within stretches of the basic residues. On the basis of our comparative study of Nup124p, Nup1p, and Nup153 domains, we have developed peptides that specifically knockdown retrotransposon activity by disengaging the Tf1-Gag from its host nuclear transport machinery without any harmful consequence to the host itself. Our results imply that those domains challenged a specific pathway affecting Tf1 transposition. Although full-length Nup1p or Nup153 does not complement Nup124p, the functionality of their conserved domains with reference to Tf1 activity suggests that these three proteins evolved from a common ancestor.

Multiple Conserved Domains of the Nucleoporin Nup124p and Its Orthologs Nup1p and Nup153 Are Critical for Nuclear Import and Activity of the Fission Yeast Tf1 Retrotransposon

PubMed Central

Sistla, Srivani; Pang, Junxiong Vincent; Wang, Cui Xia

2007-01-01

The nucleoporin Nup124p is a host protein required for the nuclear import of both, retrotransposon Tf1-Gag as well as the retroviral HIV-1 Vpr in fission yeast. The human nucleoporin Nup153 and the Saccharomyces cerevisiae Nup1p were identified as orthologs of Nup124p. In this study, we show that all three nucleoporins share a large FG/FXFG-repeat domain and a C-terminal peptide sequence, GRKIxxxxxRRKx, that are absolutely essential for Tf1 retrotransposition. Though the FXFG domain was essential, the FXFG repeats themselves could be eliminated without loss of retrotransposon activity, suggesting the existence of a common element unrelated to FG/FXFG motifs. The Nup124p C-terminal peptide, GRKIAVPRSRRKR, was extremely sensitive to certain single amino acid changes within stretches of the basic residues. On the basis of our comparative study of Nup124p, Nup1p, and Nup153 domains, we have developed peptides that specifically knockdown retrotransposon activity by disengaging the Tf1-Gag from its host nuclear transport machinery without any harmful consequence to the host itself. Our results imply that those domains challenged a specific pathway affecting Tf1 transposition. Although full-length Nup1p or Nup153 does not complement Nup124p, the functionality of their conserved domains with reference to Tf1 activity suggests that these three proteins evolved from a common ancestor. PMID:17615301

Stress-Related Gene Expression Reflects Morphophysiological Responses to Water Deficit1[OPEN

PubMed Central

Vile, Denis; Bediee, Alexis; Dauzat, Myriam; Luchaire, Nathalie; Kamrowska, Dominika; Granier, Christine

2017-01-01

Acclimation to water deficit (WD) enables plants to maintain growth under unfavorable environmental conditions, although the mechanisms are not completely understood. In this study, the natural variation of long-term acclimation to moderate and severe soil WD was investigated in 18 Arabidopsis (Arabidopsis thaliana) accessions using PHENOPSIS, an automated phenotyping platform. Soil water content was adjusted at an early stage of plant development and maintained at a constant level until reproductive age was achieved. The accessions were selected based on the expression levels of ANNEXIN1, a drought-related marker. Severe WD conditions had a greater effect on most of the measured morphophysiological traits than moderate WD conditions. Multivariate analyses indicated that trait responses associated with plant size and water management drove most of the variation. Accessions with similar responses at these two levels were grouped in clusters that displayed different response strategies to WD. The expression levels of selected stress-response genes revealed large natural variation under WD conditions. Responses of morphophysiological traits, such as projected rosette area, transpiration rate, and rosette water content, were correlated with changes in the expression of stress-related genes, such as NINE-CIS-EPOXYCAROTENOID DIOXYGENASE3 and N-MYC DOWNREGULATED-LIKE1 (NDL1), in response to WD. Interestingly, the morphophysiological acclimation response to WD also was reflected in the gene expression levels (most notably those of NDL1, CHALCONE SYNTHASE, and MYB DOMAIN PROTEIN44) in plants cultivated under well-watered conditions. Our results may lead to the development of biomarkers and predictors of plant morphophysiological responses based on gene expression patterns. PMID:28522456

Domain Evolution and Functional Diversification of Sulfite Reductases

NASA Astrophysics Data System (ADS)

Dhillon, Ashita; Goswami, Sulip; Riley, Monica; Teske, Andreas; Sogin, Mitchell

2005-02-01

Sulfite reductases are key enzymes of assimilatory and dissimilatory sulfur metabolism, which occur in diverse bacterial and archaeal lineages. They share a highly conserved domain "C-X5-C-n-C-X3-C" for binding siroheme and iron-sulfur clusters that facilitate electron transfer to the substrate. For each sulfite reductase cluster, the siroheme-binding domain is positioned slightly differently at the N-terminus of dsrA and dsrB, while in the assimilatory proteins the siroheme domain is located at the C-terminus. Our sequence and phylogenetic analysis of the siroheme-binding domain shows that sulfite reductase sequences diverged from a common ancestor into four separate clusters (aSir, alSir, dsr, and asrC) that are biochemically distinct; each serves a different assimilatory or dissimilatory role in sulfur metabolism. The phylogenetic distribution and functional grouping in sulfite reductase clusters (dsrA and dsrB vs. aSiR, asrC, and alSir) suggest that their functional diversification during evolution may have preceded the bacterial/archaeal divergence.

Dissociation of Paramyxovirus Interferon Evasion Activities: Universal and Virus-Specific Requirements for Conserved V Protein Amino Acids in MDA5 Interference ▿

PubMed Central

Ramachandran, Aparna; Horvath, Curt M.

2010-01-01

The V protein of the paramyxovirus subfamily Paramyxovirinae is an important virulence factor that can interfere with host innate immunity by inactivating the cytosolic pathogen recognition receptor MDA5. This interference is a result of a protein-protein interaction between the highly conserved carboxyl-terminal domain of the V protein and the helicase domain of MDA5. The V protein C-terminal domain (CTD) is an evolutionarily conserved 49- to 68-amino-acid region that coordinates two zinc atoms per protein chain. Site-directed mutagenesis of conserved residues in the V protein CTD has revealed both universal and virus-specific requirements for zinc coordination in MDA5 engagement and has also identified other conserved residues as critical for MDA5 interaction and interference. Mutation of these residues produces V proteins that are specifically defective for MDA5 interference and not impaired in targeting STAT1 for proteasomal degradation via the VDC ubiquitin ligase complex. Results demonstrate that mutation of conserved charged residues in the V proteins of Nipah virus, measles virus, and mumps virus also abolishes MDA5 interaction. These findings clearly define molecular determinants for MDA5 inhibition by the paramyxovirus V proteins. PMID:20719949

SH3 interactome conserves general function over specific form

PubMed Central

Xin, Xiaofeng; Gfeller, David; Cheng, Jackie; Tonikian, Raffi; Sun, Lin; Guo, Ailan; Lopez, Lianet; Pavlenco, Alevtina; Akintobi, Adenrele; Zhang, Yingnan; Rual, Jean-François; Currell, Bridget; Seshagiri, Somasekar; Hao, Tong; Yang, Xinping; Shen, Yun A; Salehi-Ashtiani, Kourosh; Li, Jingjing; Cheng, Aaron T; Bouamalay, Dryden; Lugari, Adrien; Hill, David E; Grimes, Mark L; Drubin, David G; Grant, Barth D; Vidal, Marc; Boone, Charles; Sidhu, Sachdev S; Bader, Gary D

2013-01-01

Src homology 3 (SH3) domains bind peptides to mediate protein–protein interactions that assemble and regulate dynamic biological processes. We surveyed the repertoire of SH3 binding specificity using peptide phage display in a metazoan, the worm Caenorhabditis elegans, and discovered that it structurally mirrors that of the budding yeast Saccharomyces cerevisiae. We then mapped the worm SH3 interactome using stringent yeast two-hybrid and compared it with the equivalent map for yeast. We found that the worm SH3 interactome resembles the analogous yeast network because it is significantly enriched for proteins with roles in endocytosis. Nevertheless, orthologous SH3 domain-mediated interactions are highly rewired. Our results suggest a model of network evolution where general function of the SH3 domain network is conserved over its specific form. PMID:23549480

Conserved Kv4 N-terminal domain critical for effects of Kv channel-interacting protein 2.2 on channel expression and gating.

PubMed

Bähring, R; Dannenberg, J; Peters, H C; Leicher, T; Pongs, O; Isbrandt, D

2001-06-29

Association of Kv channel-interacting proteins (KChIPs) with Kv4 channels leads to modulation of these A-type potassium channels (An, W. F., Bowlby, M. R., Betty, M., Cao, J., Ling, H. P., Mendoza, G., Hinson, J. W., Mattsson, K. I., Strassle, B. W., Trimmer, J. S., and Rhodes, K. J. (2000) Nature 403, 553-556). We cloned a KChIP2 splice variant (KChIP2.2) from human ventricle. In comparison with KChIP2.1, coexpression of KChIP2.2 with human Kv4 channels in mammalian cells slowed the onset of Kv4 current inactivation (2-3-fold), accelerated the recovery from inactivation (5-7-fold), and shifted Kv4 steady-state inactivation curves by 8-29 mV to more positive potentials. The features of Kv4.2/KChIP2.2 currents closely resemble those of cardiac rapidly inactivating transient outward currents. KChIP2.2 stimulated the Kv4 current density in Chinese hamster ovary cells by approximately 55-fold. This correlated with a redistribution of immunoreactivity from perinuclear areas to the plasma membrane. Increased Kv4 cell-surface expression and current density were also obtained in the absence of KChIP2.2 when the highly conserved proximal Kv4 N terminus was deleted. The same domain is required for association of KChIP2.2 with Kv4 alpha-subunits. We propose that an efficient transport of Kv4 channels to the cell surface depends on KChIP binding to the Kv4 N-terminal domain. Our data suggest that the binding is necessary, but not sufficient, for the functional activity of KChIPs.

Structure of the USP15 N-terminal domains: a β-hairpin mediates close association between the DUSP and UBL domains.

PubMed

Harper, Stephen; Besong, Tabot M D; Emsley, Jonas; Scott, David J; Dreveny, Ingrid

2011-09-20

Ubiquitin specific protease 15 (USP15) functions in COP9 signalosome mediated regulation of protein degradation and cellular signaling through catalyzing the ubiquitin deconjugation reaction of a discrete number of substrates. It influences the stability of adenomatous polyposis coli, IκBα, caspase-3, and the human papillomavirus type 16 E6. USP15 forms a subfamily with USP4 and USP11 related through a shared presence of N-terminal "domain present in ubiquitin specific proteases" (DUSP) and "ubiquitin-like" (UBL) domains (DU subfamily). Here we report the 1.5 Å resolution crystal structure of the human USP15 N-terminal domains revealing a 80 Å elongated arrangement with the DU domains aligned in tandem. This architecture is generated through formation of a defined interface that is dominated by an intervening β-hairpin structure (DU finger) that engages in an intricate hydrogen-bonding network between the domains. The UBL domain is closely related to ubiquitin among β-grasp folds but is characterized by the presence of longer loop regions and different surface characteristics, indicating that this domain is unlikely to act as ubiquitin mimic. Comparison with the related murine USP4 DUSP-UBL crystal structure reveals that the main DU interdomain contacts are conserved. Analytical ultracentrifugation, small-angle X-ray scattering, and gel filtration experiments revealed that USP15 DU is monomeric in solution. Our data provide a framework to advance study of the structure and function of the DU subfamily. © 2011 American Chemical Society

The primary structure of stinging nettle (Urtica dioica) agglutinin. A two-domain member of the hevein family.

PubMed

Beintema, J J; Peumans, W J

1992-03-09

The primary structure of stinging nettle (Urtica dioica) agglutinin has been determined by sequence analysis of peptides obtained from three overlapping proteolytic digests. The sequence of 80 residues consists of two hevein-like domains with the same spacing of half-cystine residues and several other conserved residues as observed earlier in other proteins with hevein-like domains. The hinge region between the two domains is four residues longer than those between the four domains in cereal lectins like wheat germ agglutinin.

White Dwarf WD-1145+17 "Zombie Star" Consumes Asteroid

NASA Astrophysics Data System (ADS)

Kaye, Thomas G.; Gary, Bruce L.; Rappaport, Saul A.; Foote, Jerry, Benni, Paul

2016-05-01

It has long been suspected that white dwarfs accrete asteroid debris as evidenced by heavy metals in many white dwarf spectra. WD1145 was initially detected in Kepler data as an exoplanet candidate with a repeating 1.3% dip over the course of the Jul-Sep 2014 observing season. Follow-up ground based observations were conducted with professional telescopes during March through May of 2015, and these showed that the Kepler dip must likely consist of deeper and shorter dips which come and go with an uncertain pattern. It was hypothesized that the observations were due to an asteroid in a 4.5 hour orbit. In anticipation of its return to nighttime visibility, major observatories scheduled time starting in 2016 Feb. A pro/am collaboration was formed in late 2015 for amateur observations prior to the 2016 Feb professional observations in order to determine an ephemeris for fade activity for the purpose of scheduling relatively short observations with professional telescopes. The amateur observations began in 2015 Nov, sooner than requested, and they showed that the fade activity level had exploded, becoming 20 times the level measured by Kepler. As many as 13 different fades per 4.5-hour orbit were measured, and these varied in depth from night to night. The amateur project turned into a full assault on the star with as many as 4 amateur telescopes observing on the same night. Continuous monitoring mysteriously showed that the clouds drifted in phase with respect to the dominant period i.e., they have a shorter period than measured by Kepler; this would imply that the orbiting dust clouds were located inside the orbit of the parent planetesimal. The best model indicated that the parent planetesimal was releasing fragments from inside its Hill sphere at the L1 Lagrange point, causing them to fall into an inner orbit. New astrophysics was described for the first time when the team used the diameter of the planetesimal orbit, and the diameter of the drift fragment orbit, to

Analysis of cellulose synthase genes from domesticated apple identifies collinear genes WDR53 and CesA8A: partial co-expression, bicistronic mRNA, and alternative splicing of CESA8A

PubMed Central

Guerriero, Gea; Spadiut, Oliver; Kerschbamer, Christine; Giorno, Filomena; Baric, Sanja; Ezcurra, Inés

2016-01-01

Cellulose synthase (CesA) genes constitute a complex multigene family with six major phylogenetic clades in angiosperms. The recently sequenced genome of domestic apple, Malus×domestica, was mined for CesA genes, by blasting full-length cellulose synthase protein (CESA) sequences annotated in the apple genome against protein databases from the plant models Arabidopsis thaliana and Populus trichocarpa. Thirteen genes belonging to the six angiosperm CesA clades and coding for proteins with conserved residues typical of processive glycosyltransferases from family 2 were detected. Based on their phylogenetic relationship to Arabidopsis CESAs, as well as expression patterns, a nomenclature is proposed to facilitate further studies. Examination of their genomic organization revealed that MdCesA8-A is closely linked and co-oriented with WDR53, a gene coding for a WD40 repeat protein. The WDR53 and CesA8 genes display conserved collinearity in dicots and are partially co-expressed in the apple xylem. Interestingly, the presence of a bicistronic WDR53–CesA8A transcript was detected in phytoplasma-infected phloem tissues of apple. The bicistronic transcript contains a spliced intergenic sequence that is predicted to fold into hairpin structures typical of internal ribosome entry sites, suggesting its potential cap-independent translation. Surprisingly, the CesA8A cistron is alternatively spliced and lacks the zinc-binding domain. The possible roles of WDR53 and the alternatively spliced CESA8 variant during cellulose biosynthesis in M.×domestica are discussed. PMID:23048131

Chimeric Saccharomyces cerevisiae Msh6 protein with an Msh3 mispair-binding domain combines properties of both proteins.

PubMed

Shell, Scarlet S; Putnam, Christopher D; Kolodner, Richard D

2007-06-26

Msh2-Msh3 and Msh2-Msh6 are two partially redundant mispair-recognition complexes that initiate mismatch repair in eukaryotes. Crystal structures of the prokaryotic homolog MutS suggest the mechanism by which Msh6 interacts with mispairs because key mispair-contacting residues are conserved in these two proteins. Because Msh3 lacks these conserved residues, we constructed a series of mutants to investigate the requirements for mispair interaction by Msh3. We found that a chimeric protein in which the mispair-binding domain (MBD) of Msh6 was replaced by the equivalent domain of Msh3 was functional for mismatch repair. This chimera possessed the mispair-binding specificity of Msh3 and revealed that communication between the MBD and the ATPase domain is conserved between Msh2-Msh3 and Msh2-Msh6. Further, the chimeric protein retained Msh6-like properties with respect to genetic interactions with the MutL homologs and an Msh2 MBD deletion mutant, indicating that Msh3-like behaviors beyond mispair specificity are not features controlled by the MBD.

A novel essential domain perspective for exploring gene essentiality.

PubMed

Lu, Yao; Lu, Yulan; Deng, Jingyuan; Peng, Hai; Lu, Hui; Lu, Long Jason

2015-09-15

Genes with indispensable functions are identified as essential; however, the traditional gene-level studies of essentiality have several limitations. In this study, we characterized gene essentiality from a new perspective of protein domains, the independent structural or functional units of a polypeptide chain. To identify such essential domains, we have developed an Expectation-Maximization (EM) algorithm-based Essential Domain Prediction (EDP) Model. With simulated datasets, the model provided convergent results given different initial values and offered accurate predictions even with noise. We then applied the EDP model to six microbial species and predicted 1879 domains to be essential in at least one species, ranging 10-23% in each species. The predicted essential domains were more conserved than either non-essential domains or essential genes. Comparing essential domains in prokaryotes and eukaryotes revealed an evolutionary distance consistent with that inferred from ribosomal RNA. When utilizing these essential domains to reproduce the annotation of essential genes, we received accurate results that suggest protein domains are more basic units for the essentiality of genes. Furthermore, we presented several examples to illustrate how the combination of essential and non-essential domains can lead to genes with divergent essentiality. In summary, we have described the first systematic analysis on gene essentiality on the level of domains. huilu.bioinfo@gmail.com or Long.Lu@cchmc.org Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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.

Conservation for Children [Levels 1-6 and an All-Levels Supplement].

ERIC Educational Resources Information Center

Cupertino Union School District, CA.

Developed to promote conservation awareness in elementary students, each of the six grade-level-sequenced activity guides provides: (1) a list of conservation concepts; (2) a criterion-referenced test; (3) a class record sheet; (4) a content guide; and (5) 90 student worksheets (40 for language arts, 20 for mathematics, 20 for social studies and…

ERp57 interacts with conserved cysteine residues in the MHC class I peptide-binding groove.

PubMed

Antoniou, Antony N; Santos, Susana G; Campbell, Elaine C; Lynch, Sarah; Arosa, Fernando A; Powis, Simon J

2007-05-15

The oxidoreductase ERp57 is a component of the major histocompatibility complex (MHC) class I peptide-loading complex. ERp57 can interact directly with MHC class I molecules, however, little is known about which of the cysteine residues within the MHC class I molecule are relevant to this interaction. MHC class I molecules possess conserved disulfide bonds between cysteines 101-164, and 203-259 in the peptide-binding and alpha3 domain, respectively. By studying a series of mutants of these conserved residues, we demonstrate that ERp57 predominantly associates with cysteine residues in the peptide-binding domain, thus indicating ERp57 has direct access to the peptide-binding groove of MHC class I molecules during assembly.

Fertility and obstetric outcome after conservative management of placenta accreta.

PubMed

Provansal, Magali; Courbiere, Blandine; Agostini, Aubert; D'Ercole, Claude; Boubli, Léon; Bretelle, Florence

2010-05-01

To determine the fertility and obstetric outcomes after conservative management of placenta accreta. A retrospective observational cohort study of all identified cases of placenta accreta from 1993 to 2007 in 2 tertiary university hospitals in France. For patients treated conservatively, maternal and fetal morbidity, reproductive function, fertility, and subsequent pregnancies were recorded. During the study period, 46 patients were treated by conservative management; 6 patients underwent a secondary hysterectomy. Of the remaining 40 patients, 35 were followed up for a median of 65 months (range 18-156 months). Patients resumed their menstrual cycles after a median of 130 days (range 48-176 days). Menses were irregular in 11 patients (31%), but none had amenorrhea. Twelve of the 14 patients desiring another pregnancy achieved a total of 15 pregnancies; 2 patients had recurrent placenta accreta. Five spontaneous abortions and 1 termination of pregnancy occurred during the first trimester. The median term at delivery was 37 weeks (range, 35-40 weeks). Four patients delivered prematurely. Conservative management of placenta accreta can preserve fertility, although the risk of recurrent placenta accreta appears to be high. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

Regulation of Telomere Length Requires a Conserved N-Terminal Domain of Rif2 in Saccharomyces cerevisiae

PubMed Central

Kaizer, Hannah; Connelly, Carla J.; Bettridge, Kelsey; Viggiani, Christopher; Greider, Carol W.

2015-01-01

The regulation of telomere length equilibrium is essential for cell growth and survival since critically short telomeres signal DNA damage and cell cycle arrest. While the broad principles of length regulation are well established, the molecular mechanism of how these steps occur is not fully understood. We mutagenized the RIF2 gene in Saccharomyces cerevisiae to understand how this protein blocks excess telomere elongation. We identified an N-terminal domain in Rif2 that is essential for length regulation, which we have termed BAT domain for Blocks Addition of Telomeres. Tethering this BAT domain to Rap1 blocked telomere elongation not only in rif2Δ mutants but also in rif1Δ and rap1C-terminal deletion mutants. Mutation of a single amino acid in the BAT domain, phenylalanine at position 8 to alanine, recapitulated the rif2Δ mutant phenotype. Substitution of F8 with tryptophan mimicked the wild-type phenylalanine, suggesting the aromatic amino acid represents a protein interaction site that is essential for telomere length regulation. PMID:26294668

Structural Characterization of the Boca/Mesd Maturation Factors for LDL-Receptor-Type beta Propeller Domains

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

M Collins; W Hendrickson

2011-12-31

Folding and trafficking of low-density lipoprotein receptor (LDLR) family members, which play essential roles in development and homeostasis, are mediated by specific chaperones. The Boca/Mesd chaperone family specifically promotes folding and trafficking of the YWTD {beta} propeller-EGF domain pair found in the ectodomain of all LDLR members. Limited proteolysis, NMR spectroscopy, analytical ultracentrifugation, and X-ray crystallography were used to define a conserved core composed of a structured domain that is preceded by a disordered N-terminal region. High-resolution structures of the ordered domain were determined for homologous proteins from three metazoans. Seven independent protomers reveal a novel ferrodoxin-like superfamily fold withmore » two distinct {beta} sheet topologies. A conserved hydrophobic surface forms a dimer interface in each crystal, but these differ substantially at the atomic level, indicative of nonspecific hydrophobic interactions that may play a role in the chaperone activity of the Boca/Mesd family.« less

40 CFR 40.115-3 - Interstate agency.

Code of Federal Regulations, 2010 CFR

2010-07-01

... RESEARCH AND DEMONSTRATION GRANTS § 40.115-3 Interstate agency. (a) Under the Clean Air Act, an agency... substantial powers or duties pertaining to the prevention and control of air pollution. (b) Under the Federal... powers or duties pertaining to the control of pollution of waters. (c) Under the Resource Conservation...

Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair

PubMed Central

Redpath, G. M. I.; Woolger, N.; Piper, A. K.; Lemckert, F. A.; Lek, A.; Greer, P. A.; North, K. N.; Cooper, S. T.

2014-01-01

Dysferlin and calpain are important mediators of the emergency response to repair plasma membrane injury. Our previous research revealed that membrane injury induces cleavage of dysferlin to release a synaptotagmin-like C-terminal module we termed mini-dysferlinC72. Here we show that injury-activated cleavage of dysferlin is mediated by the ubiquitous calpains via a cleavage motif encoded by alternately spliced exon 40a. An exon 40a–specific antibody recognizing cleaved mini-dysferlinC72 intensely labels the circumference of injury sites, supporting a key role for dysferlinExon40a isoforms in membrane repair and consistent with our evidence suggesting that the calpain-cleaved C-terminal module is the form specifically recruited to injury sites. Calpain cleavage of dysferlin is a ubiquitous response to membrane injury in multiple cell lineages and occurs independently of the membrane repair protein MG53. Our study links calpain and dysferlin in the calcium-activated vesicle fusion of membrane repair, placing calpains as upstream mediators of a membrane repair cascade that elicits cleaved dysferlin as an effector. Of importance, we reveal that myoferlin and otoferlin are also cleaved enzymatically to release similar C-terminal modules, bearing two C2 domains and a transmembrane domain. Evolutionary preservation of this feature highlights its functional importance and suggests that this highly conserved C-terminal region of ferlins represents a functionally specialized vesicle fusion module. PMID:25143396

A small cellulose binding domain protein in Phytophtora is cell wall localized

USDA-ARS?s Scientific Manuscript database

Cellulose binding domains (CBD) are structurally conserved regions linked to catalytic regions of cellulolytic enzymes. While widespread amongst saprophytic fungi that subsist on plant cell wall polysaccharides, they are not generally present in plant pathogenic fungi. A genome wide survey of CBDs w...

The prokaryotic antecedents of the ubiquitin-signaling system and the early evolution of ubiquitin-like β-grasp domains

PubMed Central

Iyer, Lakshminarayan M; Burroughs, A Maxwell; Aravind, L

2006-01-01

Background Ubiquitin (Ub)-mediated signaling is one of the hallmarks of all eukaryotes. Prokaryotic homologs of Ub (ThiS and MoaD) and E1 ligases have been studied in relation to sulfur incorporation reactions in thiamine and molybdenum/tungsten cofactor biosynthesis. However, there is no evidence for entire protein modification systems with Ub-like proteins and deconjugation by deubiquitinating enzymes in prokaryotes. Hence, the evolutionary assembly of the eukaryotic Ub-signaling apparatus remains unclear. Results We systematically analyzed prokaryotic Ub-related β-grasp fold proteins using sensitive sequence profile searches and structural analysis. Consequently, we identified novel Ub-related proteins beyond the characterized ThiS, MoaD, TGS, and YukD domains. To understand their functional associations, we sought and recovered several conserved gene neighborhoods and domain architectures. These included novel associations involving diverse sulfur metabolism proteins, siderophore biosynthesis and the gene encoding the transfer mRNA binding protein SmpB, as well as domain fusions between Ub-like domains and PIN-domain related RNAses. Most strikingly, we found conserved gene neighborhoods in phylogenetically diverse bacteria combining genes for JAB domains (the primary de-ubiquitinating isopeptidases of the proteasomal complex), along with E1-like adenylating enzymes and different Ub-related proteins. Further sequence analysis of other conserved genes in these neighborhoods revealed several Ub-conjugating enzyme/E2-ligase related proteins. Genes for an Ub-like protein and a JAB domain peptidase were also found in the tail assembly gene cluster of certain caudate bacteriophages. Conclusion These observations imply that members of the Ub family had already formed strong functional associations with E1-like proteins, UBC/E2-related proteins, and JAB peptidases in the bacteria. Several of these Ub-like proteins and the associated protein families are likely to

[Identification of new conserved and variable regions in the 16S rRNA gene of acetic acid bacteria and acetobacteraceae family].

PubMed

Chakravorty, S; Sarkar, S; Gachhui, R

2015-01-01

The Acetobacteraceae family of the class Alpha Proteobacteria is comprised of high sugar and acid tolerant bacteria. The Acetic Acid Bacteria are the economically most significant group of this family because of its association with food products like vinegar, wine etc. Acetobacteraceae are often hard to culture in laboratory conditions and they also maintain very low abundances in their natural habitats. Thus identification of the organisms in such environments is greatly dependent on modern tools of molecular biology which require a thorough knowledge of specific conserved gene sequences that may act as primers and or probes. Moreover unconserved domains in genes also become markers for differentiating closely related genera. In bacteria, the 16S rRNA gene is an ideal candidate for such conserved and variable domains. In order to study the conserved and variable domains of the 16S rRNA gene of Acetic Acid Bacteria and the Acetobacteraceae family, sequences from publicly available databases were aligned and compared. Near complete sequences of the gene were also obtained from Kombucha tea biofilm, a known Acetobacteraceae family habitat, in order to corroborate the domains obtained from the alignment studies. The study indicated that the degree of conservation in the gene is significantly higher among the Acetic Acid Bacteria than the whole Acetobacteraceae family. Moreover it was also observed that the previously described hypervariable regions V1, V3, V5, V6 and V7 were more or less conserved in the family and the spans of the variable regions are quite distinct as well.