Roles of F-box proteins in human digestive system tumors (Review).

Science.gov (United States)

Gong, Jian; Lv, Liang; Huo, Jirong

2014-12-01

F-box proteins (FBPs), the substrate-recognition subunit of E3 ubiquitin (Ub) ligase, are the important components of Ub proteasome system (UPS). FBPs are involved in multiple cellular processes through ubiquitylation and subsequent degradation of their target proteins. Many studies have described the roles of FBPs in human cancers. Digestive system tumors account for a large proportion of all the tumors, and their mortality is very high. This review summarizes for the first time the roles of FBPs in digestive system tumorige-nesis and tumor progression, aiming at finding new routes for the rational design of targeted anticancer therapies in digestive system tumors.

Wheat F-Box Protein Gene TaFBA1 Is Involved in Plant Tolerance to Heat Stress

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Qinxue Li

2018-04-01

Full Text Available Adverse environmental conditions, including high temperature, often affect the growth and production of crops worldwide. F-box protein, a core component of the Skp1-Cullin-F-box (SCF E3 ligase complex, plays an important role in abiotic stress responses. A previously cloned gene from wheat, TaFBA1, encodes a homologous F-box protein. A Yeast two-Hybrid (Y2H assay showed that TaFBA1 interacted with other SCF proteins. We found that the expression of TaFBA1 could be induced by heat stress (45°C. Overexpression of TaFBA1 enhanced heat stress tolerance in transgenic tobacco, because growth inhibition was reduced and photosynthesis increased as compared with those in the wild type (WT plants. Furthermore, the accumulation of H2O2, O2-, and carbonyl protein decreased and cell damage was alleviated in transgenic plants under heat stress, which resulted in less oxidative damage. However, the transgenic plants contained more enzymatic antioxidants after heat stress, which might be related to the regulation of some antioxidant gene expressions. The qRT-PCR analysis showed that the overexpression of TaFBA1 upregulated the expression of genes involved in reactive oxygen species (ROS scavenging, proline biosynthesis, and abiotic stress responses. We identified the interaction of TaFBA1 with Triticum aestivum stress responsive protein 1 (TaASRP1 by Y2H assay and bimolecular fluorescence complementation (BiFC assay. The results suggested that TaFBA1 may improve enzymatic antioxidant levels and regulate gene expression by interacting with other proteins, such as TaASRP1, which leads to the enhanced heat stress tolerance seen in the transgenic plants.

SCF Ubiquitin Ligase F-box Protein Fbx15 Controls Nuclear Co-repressor Localization, Stress Response and Virulence of the Human Pathogen Aspergillus fumigatus.

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Bastian Jöhnk

2016-09-01

Full Text Available F-box proteins share the F-box domain to connect substrates of E3 SCF ubiquitin RING ligases through the adaptor Skp1/A to Cul1/A scaffolds. F-box protein Fbx15 is part of the general stress response of the human pathogenic mold Aspergillus fumigatus. Oxidative stress induces a transient peak of fbx15 expression, resulting in 3x elevated Fbx15 protein levels. During non-stress conditions Fbx15 is phosphorylated and F-box mediated interaction with SkpA preferentially happens in smaller subpopulations in the cytoplasm. The F-box of Fbx15 is required for an appropriate oxidative stress response, which results in rapid dephosphorylation of Fbx15 and a shift of the cellular interaction with SkpA to the nucleus. Fbx15 binds SsnF/Ssn6 as part of the RcoA/Tup1-SsnF/Ssn6 co-repressor and is required for its correct nuclear localization. Dephosphorylated Fbx15 prevents SsnF/Ssn6 nuclear localization and results in the derepression of gliotoxin gene expression. fbx15 deletion mutants are unable to infect immunocompromised mice in a model for invasive aspergillosis. Fbx15 has a novel dual molecular function by controlling transcriptional repression and being part of SCF E3 ubiquitin ligases, which is essential for stress response, gliotoxin production and virulence in the opportunistic human pathogen A. fumigatus.

A Large Complement of the Predicted Arabidopsis ARM Repeat Proteins Are Members of the U-Box E3 Ubiquitin Ligase Family1[w

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Mudgil, Yashwanti; Shiu, Shin-Han; Stone, Sophia L.; Salt, Jennifer N.; Goring, Daphne R.

2004-01-01

The Arabidopsis genome was searched to identify predicted proteins containing armadillo (ARM) repeats, a motif known to mediate protein-protein interactions in a number of different animal proteins. Using domain database predictions and models generated in this study, 108 Arabidopsis proteins were identified that contained a minimum of two ARM repeats with the majority of proteins containing four to eight ARM repeats. Clustering analysis showed that the 108 predicted Arabidopsis ARM repeat proteins could be divided into multiple groups with wide differences in their domain compositions and organizations. Interestingly, 41 of the 108 Arabidopsis ARM repeat proteins contained a U-box, a motif present in a family of E3 ligases, and these proteins represented the largest class of Arabidopsis ARM repeat proteins. In 14 of these U-box/ARM repeat proteins, there was also a novel conserved domain identified in the N-terminal region. Based on the phylogenetic tree, representative U-box/ARM repeat proteins were selected for further study. RNA-blot analyses revealed that these U-box/ARM proteins are expressed in a variety of tissues in Arabidopsis. In addition, the selected U-box/ARM proteins were found to be functional E3 ubiquitin ligases. Thus, these U-box/ARM proteins represent a new family of E3 ligases in Arabidopsis. PMID:14657406

E2F-6: a novel member of the E2F family is an inhibitor of E2F-dependent transcription

DEFF Research Database (Denmark)

Cartwright, P; Müller, H; Wagener, C

1998-01-01

with E2Fs 1-5, especially within the DNA binding, heterodimerization and marked box domains. Unlike E2Fs 1-5, E2F-6 lacks a transactivation and a pocket protein binding domain, hence, forms a unique third group within the E2F family. E2F-6 is a nuclear protein that can form heterodimers with the DP......The E2F family of transcription factors are essential for the regulation of genes required for appropriate progression through the cell cycle. Five members of the E2F family have been previously reported, namely E2F1-5. All five are key elements in transcriptional regulation of essential genes......, and they can be divided into two functional groups, those that induce S-phase progression when overexpressed in quiescent cells (E2Fs 1-3), and those that do not (E2Fs 4-5). Here, we describe the identification of a novel member of this family, which we refer to as E2F-6. E2F-6 shares significant homology...

A photo-responsive F-box protein FOF2 regulates floral initiation by promoting FLC expression in Arabidopsis.

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He, Reqing; Li, Xinmei; Zhong, Ming; Yan, Jindong; Ji, Ronghuan; Li, Xu; Wang, Qin; Wu, Dan; Sun, Mengsi; Tang, Dongying; Lin, Jianzhong; Li, Hongyu; Liu, Bin; Liu, Hongtao; Liu, Xuanming; Zhao, Xiaoying; Lin, Chentao

2017-09-01

Floral initiation is regulated by various genetic pathways in response to light, temperature, hormones and developmental status; however, the molecular mechanisms underlying the interactions between different genetic pathways are not fully understood. Here, we show that the photoresponsive gene FOF2 (F-box of flowering 2) negatively regulates flowering. FOF2 encodes a putative F-box protein that interacts specifically with ASK14, and its overexpression results in later flowering under both long-day and short-day photoperiods. Conversely, transgenic plants expressing the F-box domain deletion mutant of FOF2 (FOF2ΔF), or double loss of function mutant of FOF2 and FOL1 (FOF2-LIKE 1) present early flowering phenotypes. The late flowering phenotype of the FOF2 overexpression lines is suppressed by the flc-3 loss-of-function mutation. Furthermore, FOF2 mRNA expression is regulated by autonomous pathway gene FCA, and the repressive effect of FOF2 in flowering can be overcome by vernalization. Interestingly, FOF2 expression is regulated by light. The protein level of FOF2 accumulates in response to light, whereas it is degraded under dark conditions via the 26S proteasome pathway. Our findings suggest a possible mechanistic link between light conditions and the autonomous floral promotion pathway in Arabidopsis. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

The F-box Protein KIB1 Mediates Brassinosteroid-Induced Inactivation and Degradation of GSK3-like Kinases in Arabidopsis.

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Zhu, Jia-Ying; Li, Yuyao; Cao, Dong-Mei; Yang, Hongjuan; Oh, Eunkyoo; Bi, Yang; Zhu, Shengwei; Wang, Zhi-Yong

2017-06-01

The glycogen synthase kinase-3 (GSK3) family kinases are central cellular regulators highly conserved in all eukaryotes. In Arabidopsis, the GSK3-like kinase BIN2 phosphorylates a range of proteins to control broad developmental processes, and BIN2 is degraded through unknown mechanism upon receptor kinase-mediated brassinosteroid (BR) signaling. Here we identify KIB1 as an F-box E3 ubiquitin ligase that promotes the degradation of BIN2 while blocking its substrate access. Loss-of-function mutations of KIB1 and its homologs abolished BR-induced BIN2 degradation and caused severe BR-insensitive phenotypes. KIB1 directly interacted with BIN2 in a BR-dependent manner and promoted BIN2 ubiquitination in vitro. Expression of an F-box-truncated KIB1 caused BIN2 accumulation but dephosphorylation of its substrate BZR1 and activation of BR responses because KIB1 blocked BIN2 binding to BZR1. Our study demonstrates that KIB1 plays an essential role in BR signaling by inhibiting BIN2 through dual mechanisms of blocking substrate access and promoting degradation. Copyright © 2017 Elsevier Inc. All rights reserved.

F-box protein interactions with the hallmark pathways in cancer.

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Randle, Suzanne J; Laman, Heike

2016-02-01

F-box proteins (FBP) are the substrate specifying subunit of Skp1-Cul1-FBP (SCF)-type E3 ubiquitin ligases and are responsible for directing the ubiquitination of numerous proteins essential for cellular function. Due to their ability to regulate the expression and activity of oncogenes and tumour suppressor genes, FBPs themselves play important roles in cancer development and progression. In this review, we provide a comprehensive overview of FBPs and their targets in relation to their interaction with the hallmarks of cancer cell biology, including the regulation of proliferation, epigenetics, migration and invasion, metabolism, angiogenesis, cell death and DNA damage responses. Each cancer hallmark is revealed to have multiple FBPs which converge on common signalling hubs or response pathways. We also highlight the complex regulatory interplay between SCF-type ligases and other ubiquitin ligases. We suggest six highly interconnected FBPs affecting multiple cancer hallmarks, which may prove sensible candidates for therapeutic intervention. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Molecular dissection of the APC/C inhibitor Rca1 shows a novel F-box-dependent function.

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Zielke, Norman; Querings, Silvia; Grosskortenhaus, Ruth; Reis, Tânia; Sprenger, Frank

2006-12-01

Rca1 (regulator of Cyclin A)/Emi (early mitotic inhibitor) proteins are essential inhibitors of the anaphase-promoting complex/cyclosome (APC/C). In Drosophila, Rca1 is required during G2 to prevent premature cyclin degradation by the Fizzy-related (Fzr)-dependent APC/C activity. Here, we present a structure and function analysis of Rca1 showing that a carboxy-terminal fragment is sufficient for APC/C inhibition. Rca1/Emi proteins contain a conserved F-box and interact with components of the Skp-Cullin-F-box (SCF) complex. So far, no function has been ascribed to this domain. We find that the F-box of Rca1 is dispensable for APC/C-Fzr inhibition during G2. Nevertheless, we show that Rca1 has an additional function at the G1-S transition, which requires the F-box. Overexpression of Rca1 accelerates the G1-S transition in an F-box-dependent manner. Conversely, S-phase entry is delayed in cells in which endogenous Rca1 is replaced by a transgene lacking the F-box. We propose that Rca1 acts as an F-box protein in an as yet uncharacterized SCF complex, which promotes S-phase entry.

The F-box protein Fbp1 functions in the invasive growth and cell wall integrity mitogen-activated protein kinase (MAPK) pathways in Fusarium oxysporum.

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Miguel-Rojas, Cristina; Hera, Concepcion

2016-01-01

F-box proteins determine substrate specificity of the ubiquitin-proteasome system. Previous work has demonstrated that the F-box protein Fbp1, a component of the SCF(Fbp1) E3 ligase complex, is essential for invasive growth and virulence of the fungal plant pathogen Fusarium oxysporum. Here, we show that, in addition to invasive growth, Fbp1 also contributes to vegetative hyphal fusion and fungal adhesion to tomato roots. All of these functions have been shown previously to require the mitogen-activated protein kinase (MAPK) Fmk1. We found that Fbp1 is required for full phosphorylation of Fmk1, indicating that Fbp1 regulates virulence and invasive growth via the Fmk1 pathway. Moreover, the Δfbp1 mutant is hypersensitive to sodium dodecylsulfate (SDS) and calcofluor white (CFW) and shows reduced phosphorylation levels of the cell wall integrity MAPK Mpk1 after SDS treatment. Collectively, these results suggest that Fbp1 contributes to both the invasive growth and cell wall integrity MAPK pathways of F. oxysporum. © 2015 BSPP AND JOHN WILEY & SONS LTD.

F-BAR family proteins, emerging regulators for cell membrane dynamic changes-from structure to human diseases.

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Liu, Suxuan; Xiong, Xinyu; Zhao, Xianxian; Yang, Xiaofeng; Wang, Hong

2015-05-09

Eukaryotic cell membrane dynamics change in curvature during physiological and pathological processes. In the past ten years, a novel protein family, Fes/CIP4 homology-Bin/Amphiphysin/Rvs (F-BAR) domain proteins, has been identified to be the most important coordinators in membrane curvature regulation. The F-BAR domain family is a member of the Bin/Amphiphysin/Rvs (BAR) domain superfamily that is associated with dynamic changes in cell membrane. However, the molecular basis in membrane structure regulation and the biological functions of F-BAR protein are unclear. The pathophysiological role of F-BAR protein is unknown. This review summarizes the current understanding of structure and function in the BAR domain superfamily, classifies F-BAR family proteins into nine subfamilies based on domain structure, and characterizes F-BAR protein structure, domain interaction, and functional relevance. In general, F-BAR protein binds to cell membrane via F-BAR domain association with membrane phospholipids and initiates membrane curvature and scission via Src homology-3 (SH3) domain interaction with its partner proteins. This process causes membrane dynamic changes and leads to seven important cellular biological functions, which include endocytosis, phagocytosis, filopodium, lamellipodium, cytokinesis, adhesion, and podosome formation, via distinct signaling pathways determined by specific domain-binding partners. These cellular functions play important roles in many physiological and pathophysiological processes. We further summarize F-BAR protein expression and mutation changes observed in various diseases and developmental disorders. Considering the structure feature and functional implication of F-BAR proteins, we anticipate that F-BAR proteins modulate physiological and pathophysiological processes via transferring extracellular materials, regulating cell trafficking and mobility, presenting antigens, mediating extracellular matrix degradation, and transmitting

Assaying Auxin Receptor Activity Using SPR Assays with F-Box Proteins and Aux/IAA Degrons.

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Quareshy, Mussa; Uzunova, Veselina; Prusinska, Justyna M; Napier, Richard M

2017-01-01

The identification of TIR1 as an auxin receptor combined with advanced biophysical instrumentation has led to the development of real-time activity assays for auxins. Traditionally, molecules have been assessed for auxinic activity using bioassays, and agrochemical compound discovery continues to be based on "spray and pray" technologies. Here, we describe the methodology behind an SPR-based assay that uses TIR1 and related F-box proteins with surface plasmon resonance spectrometry for rapid compound screening. In addition, methods for collecting kinetic binding data and data processing are given so that they may support programs for rational design of novel auxin ligands.

Biochemical function of typical and variant Arabidopsis thaliana U-box E3 ubiquitin-protein ligases

DEFF Research Database (Denmark)

Wiborg, Jakob; O'Shea, Charlotte; Skriver, Karen

2008-01-01

of the distant U-box protein, AtPUB49, representing a large family of eukaryotic proteins containing a U-box linked to a cyclophilin-like peptidyl-prolyl cis-trans isomerase domain, was characterized biochemically. AtPUB49 functioned both as a prolyl isomerase and a chaperone by catalysing cis......The variance of the U-box domain in 64 Arabidopsis thaliana (thale cress) E3s (ubiquitin-protein ligases) was used to examine the interactions between E3s and E2s (ubiquitin-conjugating enzymes). E2s and E3s are components of the ubiquitin protein degradation pathway. Seven U-box proteins were...... analysed for their ability to ubiquitinate proteins in vitro in co-operation with different E2s. All U-box domains exhibited ubiquitination activity and interacted productively with UBC4/5-type E2s. Three and four of the U-box domains mediated ubiquitin addition in the presence of UBC13 and UBC7 E2s...

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

Energy Technology Data Exchange (ETDEWEB)

Liban, Tyler J.; Medina, Edgar M.; Tripathi, Sarvind; Sengupta, Satyaki; Henry, R. William; Buchler, Nicolas E.; Rubin, Seth M. (UCSC); (Duke); (MSU)

2017-04-24

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 for 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.

F-box only protein 2 (Fbxo2) regulates amyloid precursor protein levels and processing.

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Atkin, Graham; Hunt, Jack; Minakawa, Eiko; Sharkey, Lisa; Tipper, Nathan; Tennant, William; Paulson, Henry L

2014-03-07

The amyloid precursor protein (APP) is an integral membrane glycoprotein whose cleavage products, particularly amyloid-β, accumulate in Alzheimer disease (AD). APP is present at synapses and is thought to play a role in both the formation and plasticity of these critical neuronal structures. Despite the central role suggested for APP in AD pathogenesis, the mechanisms regulating APP in neurons and its processing into cleavage products remain incompletely understood. F-box only protein 2 (Fbxo2), a neuron-enriched ubiquitin ligase substrate adaptor that preferentially binds high-mannose glycans on glycoproteins, was previously implicated in APP processing by facilitating the degradation of the APP-cleaving β-secretase, β-site APP-cleaving enzyme. Here, we sought to determine whether Fbxo2 plays a similar role for other glycoproteins in the amyloid processing pathway. We present in vitro and in vivo evidence that APP is itself a substrate for Fbxo2. APP levels were decreased in the presence of Fbxo2 in non-neuronal cells, and increased in both cultured hippocampal neurons and brain tissue from Fbxo2 knock-out mice. The processing of APP into its cleavage products was also increased in hippocampi and cultured hippocampal neurons lacking Fbxo2. In hippocampal slices, this increase in cleavage products was accompanied by a significant reduction in APP at the cell surface. Taken together, these results suggest that Fbxo2 regulates APP levels and processing in the brain and may play a role in modulating AD pathogenesis.

Virulence factor NSs of rift valley fever virus recruits the F-box protein FBXO3 to degrade subunit p62 of general transcription factor TFIIH.

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Kainulainen, Markus; Habjan, Matthias; Hubel, Philipp; Busch, Laura; Lau, Simone; Colinge, Jacques; Superti-Furga, Giulio; Pichlmair, Andreas; Weber, Friedemann

2014-03-01

The nonstructural protein NSs is the main virulence factor of Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus), a serious pathogen of livestock and humans in Africa. RVFV NSs blocks transcriptional upregulation of antiviral type I interferons (IFN) and destroys the general transcription factor TFIIH subunit p62 via the ubiquitin/proteasome pathway. Here, we identified a subunit of E3 ubiquitin ligases, F-box protein FBXO3, as a host cell interactor of NSs. Small interfering RNA (siRNA)-mediated depletion of FBXO3 rescued p62 protein levels in RVFV-infected cells and elevated IFN transcription by 1 order of magnitude. NSs interacts with the full-length FBXO3 protein as well as with a truncated isoform that lacks the C-terminal acidic and poly(R)-rich domains. These isoforms are present in both the nucleus and the cytoplasm. NSs exclusively removes the nuclear pool of full-length FBXO3, likely due to consumption during the degradation process. F-box proteins form the variable substrate recognition subunit of the so-called SCF ubiquitin ligases, which also contain the constant components Skp1, cullin 1 (or cullin 7), and Rbx1. siRNA knockdown of Skp1 also protected p62 from degradation, suggesting involvement in NSs action. However, knockdown of cullin 1, cullin 7, or Rbx1 could not rescue p62 degradation by NSs. Our data show that the enzymatic removal of p62 via the host cell factor FBXO3 is a major mechanism of IFN suppression by RVFV. Rift Valley fever virus is a serious emerging pathogen of animals and humans. Its main virulence factor, NSs, enables unhindered virus replication by suppressing the antiviral innate immune system. We identified the E3 ubiquitin ligase FBXO3 as a novel host cell interactor of NSs. NSs recruits FBXO3 to destroy the general host cell transcription factor TFIIH-p62, resulting in suppression of the transcriptional upregulation of innate immunity.

Apple F-box Protein MdMAX2 Regulates Plant Photomorphogenesis and Stress Response

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Jian-Ping An

2016-11-01

Full Text Available MAX2 (MORE AXILLARY GROWTH2 is involved in diverse physiological processes, including photomorphogenesis, the abiotic stress response, as well as karrikin and strigolactone signaling-mediated shoot branching. In this study, MdMAX2, an F-box protein that is a homolog of Arabidopsis MAX2, was identified and characterized. Overexpression of MdMAX2 in apple calli enhanced the accumulation of anthocyanin. Ectopic expression of MdMAX2 in Arabidopsis exhibited photomorphogenesis phenotypes, including increased anthocyanin content and decreased hypocotyl length. Further study indicated that MdMAX2 might promote plant photomorphogenesis by affecting the auxin signaling as well as other plant hormones. Transcripts of MdMAX2 were noticeably up-regulated in response to NaCl and Mannitol treatments. Moreover, compared with the wild type, the MdMAX2-overexpressing apple calli and Arabidopsis exhibited increased tolerance to salt and drought stresses. Taken together, these results suggest that MdMAX2 plays a positive regulatory role in plant photomorphogenesis and stress response.

Classification of EA1-box proteins and new insights into their role during reproduction in grasses.

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Uebler, Susanne; Márton, Mihaela L; Dresselhaus, Thomas

2015-12-01

EA1-box protein classification. Success in reproduction and vegetative development in flowering plants strongly depends on precise cell-to-cell signaling events mediated by secreted peptides.A small peptide family named as EA1-like (EAL) has been first described 10 years ago and includes EA1 involved in pollen tubes attraction by the female gametophyte and EAL1-regulating germ cell identity in maize. EALs consist of an N-terminal endoplasmic reticulum-targeting motif, the highly conserved EA1-box and a short C-terminal alanine-rich domain. Whereas EAL peptides are exclusively found in the Gramineae, the EA1-box is widely distributed throughout the plant kingdom. Based on in silico analysis and subcellular localization studies, we report here a new classification of EA1-box proteins in flowering plants. They can be distinguished into three protein classes: the already defined EAL proteins, the EAG (EA1-box glycine-rich) proteins and the EAC (EA1-box containing)proteins. While fusion proteins of EAL and EAC classes locate to the secretory pathway, EAGs are cytoplasmic and locate also to the nucleus. Moreover, we further show that the third EAL protein of Zea mays, EAL2, appears to be also involved in processes related to late embryogenic development as its peptide level increases after formation of leaf primordia. Immunohistochemical studies indicate its presence in the scutellar parenchyma and around the vasculature, where it is secreted to the extracellular space. In conclusion, the members of the maize EAL family possess very diverse functions during reproduction and it will now be exciting to elucidate the functions of EAGs and EACs in plants.

Genomic Organization, Phylogenetic Comparison and Differential Expression of the SBP-Box Family Genes in Grape

Science.gov (United States)

Hou, Hongmin; Li, Jun; Gao, Min; Singer, Stacy D.; Wang, Hao; Mao, Linyong; Fei, Zhangjun; Wang, Xiping

2013-01-01

Background The SBP-box gene family is specific to plants and encodes a class of zinc finger-containing transcription factors with a broad range of functions. Although SBP-box genes have been identified in numerous plants including green algae, moss, silver birch, snapdragon, Arabidopsis, rice and maize, there is little information concerning SBP-box genes, or the corresponding miR156/157, function in grapevine. Methodology/Principal Findings Eighteen SBP-box gene family members were identified in Vitis vinifera, twelve of which bore sequences that were complementary to miRNA156/157. Phylogenetic reconstruction demonstrated that plant SBP-domain proteins could be classified into seven subgroups, with the V. vinifera SBP-domain proteins being more closely related to SBP-domain proteins from dicotyledonous angiosperms than those from monocotyledonous angiosperms. In addition, synteny analysis between grape and Arabidopsis demonstrated that homologs of several grape SBP genes were found in corresponding syntenic blocks of Arabidopsis. Expression analysis of the grape SBP-box genes in various organs and at different stages of fruit development in V. quinquangularis ‘Shang-24’ revealed distinct spatiotemporal patterns. While the majority of the grape SBP-box genes lacking a miR156/157 target site were expressed ubiquitously and constitutively, most genes bearing a miR156/157 target site exhibited distinct expression patterns, possibly due to the inhibitory role of the microRNA. Furthermore, microarray data mining and quantitative real-time RT-PCR analysis identified several grape SBP-box genes that are potentially involved in the defense against biotic and abiotic stresses. Conclusion The results presented here provide a further understanding of SBP-box gene function in plants, and yields additional insights into the mechanism of stress management in grape, which may have important implications for the future success of this crop. PMID:23527172

Genome-wide analysis of the SBP-box gene family in Chinese cabbage (Brassica rapa subsp. pekinensis).

Science.gov (United States)

Tan, Hua-Wei; Song, Xiao-Ming; Duan, Wei-Ke; Wang, Yan; Hou, Xi-Lin

2015-11-01

The SQUAMOSA PROMOTER BINDING PROTEIN (SBP)-box gene family contains highly conserved plant-specific transcription factors that play an important role in plant development, especially in flowering. Chinese cabbage (Brassica rapa subsp. pekinensis) is a leafy vegetable grown worldwide and is used as a model crop for research in genome duplication. The present study aimed to characterize the SBP-box transcription factor genes in Chinese cabbage. Twenty-nine SBP-box genes were identified in the Chinese cabbage genome and classified into six groups. We identified 23 orthologous and 5 co-orthologous SBP-box gene pairs between Chinese cabbage and Arabidopsis. An interaction network among these genes was constructed. Sixteen SBP-box genes were expressed more abundantly in flowers than in other tissues, suggesting their involvement in flowering. We show that the MiR156/157 family members may regulate the coding regions or 3'-UTR regions of Chinese cabbage SBP-box genes. As SBP-box genes were found to potentially participate in some plant development pathways, quantitative real-time PCR analysis was performed and showed that Chinese cabbage SBP-box genes were also sensitive to the exogenous hormones methyl jasmonic acid and salicylic acid. The SBP-box genes have undergone gene duplication and loss, evolving a more refined regulation for diverse stimulation in plant tissues. Our comprehensive genome-wide analysis provides insights into the SBP-box gene family of Chinese cabbage.

The Polerovirus F box protein P0 targets ARGONAUTE1 to suppress RNA silencing.

Science.gov (United States)

Bortolamiol, Diane; Pazhouhandeh, Maghsoud; Marrocco, Katia; Genschik, Pascal; Ziegler-Graff, Véronique

2007-09-18

Plants employ post-transcriptional gene silencing (PTGS) as an antiviral defense response. In this mechanism, viral-derived small RNAs are incorporated into the RNA-induced silencing complex (RISC) to guide degradation of the corresponding viral RNAs. ARGONAUTE1 (AGO1) is a key component of RISC: it carries the RNA slicer activity. As a counter-defense, viruses have evolved various proteins that suppress PTGS. Recently, we showed that the Polerovirus P0 protein carries an F box motif required to form an SCF-like complex, which is also essential for P0's silencing suppressor function. Here, we investigate the molecular mechanism by which P0 impairs PTGS. First we show that P0's expression does not affect the biogenesis of primary siRNAs in an inverted repeat-PTGS assay, but it does affect their activity. Moreover, P0's expression in transformed Arabidopsis plants leads to various developmental abnormalities reminiscent of mutants affected in miRNA pathways, which is accompanied by enhanced levels of several miRNA-target transcripts, suggesting that P0 acts at the level of RISC. Interestingly, ectopic expression of P0 triggered AGO1 protein decay in planta. Finally, we provide evidence that P0 physically interacts with AGO1. Based on these results, we propose that P0 hijacks the host SCF machinery to modulate gene silencing by destabilizing AGO1.

Biochemical function of typical and variant Arabidopsis thaliana U-box E3 ubiquitin-protein ligases.

Science.gov (United States)

Wiborg, Jakob; O'Shea, Charlotte; Skriver, Karen

2008-08-01

The variance of the U-box domain in 64 Arabidopsis thaliana (thale cress) E3s (ubiquitin-protein ligases) was used to examine the interactions between E3s and E2s (ubiquitin-conjugating enzymes). E2s and E3s are components of the ubiquitin protein degradation pathway. Seven U-box proteins were analysed for their ability to ubiquitinate proteins in vitro in co-operation with different E2s. All U-box domains exhibited ubiquitination activity and interacted productively with UBC4/5-type E2s. Three and four of the U-box domains mediated ubiquitin addition in the presence of UBC13 and UBC7 E2s respectively, but no productive interaction was observed with the UBC15 E2 tested. The activity of AtPUB54 [Arabidopsis thaliana (thale cress) plant U-box 54 protein] was dependent on Trp(266) in the E2-binding cleft, and the E2 selectivity was changed by substitution of this position. The function of the distant U-box protein, AtPUB49, representing a large family of eukaryotic proteins containing a U-box linked to a cyclophilin-like peptidyl-prolyl cis-trans isomerase domain, was characterized biochemically. AtPUB49 functioned both as a prolyl isomerase and a chaperone by catalysing cis-trans isomerization of peptidyl-prolyl bonds and dissolving protein aggregates. In conclusion, both typical and atypical Arabidopsis U-box proteins were active E3s. The overlap in the E3/E2 selectivity suggests that in vivo specificity is not determined only by the E3-E2 interactions, but also by other parameters, e.g. co-existence or interactions with additional domains. The biochemical functions of AtPUB49 suggest that the protein can be involved in folding or degradation of protein substrates. Similar functions can also be retained within a protein complex with separate chaperone and U-box proteins.

Beyond ubiquitination: the atypical functions of Fbxo7 and other F-box proteins.

Science.gov (United States)

Nelson, David E; Randle, Suzanne J; Laman, Heike

2013-10-09

F-box proteins (FBPs) are substrate-recruiting subunits of Skp1-cullin1-FBP (SCF)-type E3 ubiquitin ligases. To date, 69 FBPs have been identified in humans, but ubiquitinated substrates have only been identified for a few, with the majority of FBPs remaining 'orphans'. In recent years, a growing body of work has identified non-canonical, SCF-independent roles for about 12% of the human FBPs. These atypical FBPs affect processes as diverse as transcription, cell cycle regulation, mitochondrial dynamics and intracellular trafficking. Here, we provide a general review of FBPs, with a particular emphasis on these expanded functions. We review Fbxo7 as an exemplar of this special group as it has well-defined roles in both SCF and non-SCF complexes. We review its function as a cell cycle regulator, via its ability to stabilize p27 protein and Cdk6 complexes, and as a proteasome regulator, owing to its high affinity binding to PI31. We also highlight recent advances in our understanding of Fbxo7 function in Parkinson's disease, where it functions in the regulation of mitophagy with PINK1 and Parkin. We postulate that a few extraordinary FBPs act as platforms that seamlessly segue their canonical and non-canonical functions to integrate different cellular pathways and link their regulation.

Pollen S-locus F-box proteins of Petunia involved in S-RNase-based self-incompatibility are themselves subject to ubiquitin-mediated degradation.

Science.gov (United States)

Sun, Penglin; Li, Shu; Lu, Dihong; Williams, Justin S; Kao, Teh-Hui

2015-07-01

Many flowering plants show self-incompatibility, an intra-specific reproductive barrier by which pistils reject self-pollen to prevent inbreeding and accept non-self pollen to promote out-crossing. In Petunia, the polymorphic S-locus determines self/non-self recognition. The locus contains a gene encoding an S-RNase, which controls pistil specificity, and multiple S-locus F-box (SLF) genes that collectively control pollen specificity. Each SLF is a component of an SCF (Skp1/Cullin/F-box) complex that is responsible for mediating degradation of non-self S-RNase(s), with which the SLF interacts, via the ubiquitin-26S proteasome pathway. A complete set of SLFs is required to detoxify all non-self S-RNases to allow cross-compatible pollination. Here, we show that SLF1 of Petunia inflata is itself subject to degradation via the ubiquitin-26S proteasome pathway, and identify an 18 amino acid sequence in the C-terminal region of S2 -SLF1 (SLF1 of S2 haplotype) that contains a degradation motif. Seven of the 18 amino acids are conserved among all 17 SLF proteins of S2 haplotype and S3 haplotype involved in pollen specificity, suggesting that all SLF proteins are probably subject to similar degradation. Deleting the 18 amino acid sequence from S2 -SLF1 stabilized the protein but abolished its function in self-incompatibility, suggesting that dynamic cycling of SLF proteins is an integral part of their function in self-incompatibility. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

Members of the YjgF/YER057c/UK114 family of proteins inhibit phosphoribosylamine synthesis in vitro.

Science.gov (United States)

Lambrecht, Jennifer A; Browne, Beth Ann; Downs, Diana M

2010-11-05

The YjgF/YER057c/UK114 family of proteins is highly conserved across all three domains of life and currently lacks a consensus biochemical function. Analysis of Salmonella enterica strains lacking yjgF has led to a working model in which YjgF functions to remove potentially toxic secondary products of cellular enzymes. Strains lacking yjgF synthesize the thiamine precursor phosphoribosylamine (PRA) by a TrpD-dependent mechanism that is not present in wild-type strains. Here, PRA synthesis was reconstituted in vitro with anthranilate phosphoribosyltransferase (TrpD), threonine dehydratase (IlvA), threonine, and phosphoribosyl pyrophosphate. TrpD-dependent PRA formation in vitro was inhibited by S. enterica YjgF and the human homolog UK114. Thus, the work herein describes the first biochemical assay for diverse members of the highly conserved YjgF/YER057c/UK114 family of proteins and provides a means to dissect the cellular functions of these proteins.

F-box protein FBXO31 is a dedicated checkpoint protein to facilitate cell cycle arrest through activation of regulators in radiation induced DNA damage

International Nuclear Information System (INIS)

Santra, Manas Kumar

2017-01-01

In response to radiation-induced DNA damage, eukaryotic cells initiate a complex signalling pathway, termed the DNA damage response (DDR), which coordinates cell cycle arrest with DNA repair. Previous study showed that induction of G1 arrest in response to radiation induced DNA damage is minimally a two-step process: a fast p53-independent initiation of G1 arrest mediated by cyclin D1 proteolysis and a slower maintenance of arrest resulting from increased p53 stability. We elucidated the molecular mechanism of slow and fast response of radiation induced DDR. We showed that FBXO31, a member of F-box family proteins, plays important role in DDR induced by ionizing radiation. We show that FBXO31 is responsible for promoting MDM2 degradation following radiation. FBXO31 interacts with and directs the degradation of MDM2 in ATM dependent phosphorylation of MDM2. FBXO31-mediated loss of MDM2 leads to elevated levels of p53, resulting in growth arrest. In cells depleted of FBXO31, MDM2 is not degraded and p53 levels do not increase following genotoxic stress. Thus, FBXO31 is essential for the classic robust increase in p53 levels following DNA damage

The F box protein Fbx6 regulates Chk1 stability and cellular sensitivity to replication stress.

Science.gov (United States)

Zhang, You-Wei; Brognard, John; Coughlin, Chris; You, Zhongsheng; Dolled-Filhart, Marisa; Aslanian, Aaron; Manning, Gerard; Abraham, Robert T; Hunter, Tony

2009-08-28

ATR and Chk1 are two key protein kinases in the replication checkpoint. Activation of ATR-Chk1 has been extensively investigated, but checkpoint termination and replication fork restart are less well understood. Here, we report that DNA damage not only activates Chk1, but also exposes a degron-like region at the carboxyl terminus of Chk1 to an Fbx6-containing SCF (Skp1-Cul1-F box) E3 ligase, which mediates the ubiquitination and degradation of Chk1 and, in turn, terminates the checkpoint. The protein levels of Chk1 and Fbx6 showed an inverse correlation in both cultured cancer cells and in human breast tumor tissues. Further, we show that low levels of Fbx6 and consequent impairment of replication stress-induced Chk1 degradation are associated with cancer cell resistance to the chemotherapeutic agent, camptothecin. We propose that Fbx6-dependent Chk1 degradation contributes to S phase checkpoint termination and that a defect in this mechanism might increase tumor cell resistance to certain anticancer drugs.

Transcriptome analysis uncovers Arabidopsis F-BOX STRESS INDUCED 1 as a regulator of jasmonic acid and abscisic acid stress gene expression.

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Gonzalez, Lauren E; Keller, Kristen; Chan, Karen X; Gessel, Megan M; Thines, Bryan C

2017-07-17

The ubiquitin 26S proteasome system (UPS) selectively degrades cellular proteins, which results in physiological changes to eukaryotic cells. F-box proteins are substrate adaptors within the UPS and are responsible for the diversity of potential protein targets. Plant genomes are enriched in F-box genes, but the vast majority of these have unknown roles. This work investigated the Arabidopsis F-box gene F-BOX STRESS INDUCED 1 (FBS1) for its effects on gene expression in order elucidate its previously unknown biological function. Using publically available Affymetrix ATH1 microarray data, we show that FBS1 is significantly co-expressed in abiotic stresses with other well-characterized stress response genes, including important stress-related transcriptional regulators. This gene suite is most highly expressed in roots under cold and salt stresses. Transcriptome analysis of fbs1-1 knock-out plants grown at a chilling temperature shows that hundreds of genes require FBS1 for appropriate expression, and that these genes are enriched in those having roles in both abiotic and biotic stress responses. Based on both this genome-wide expression data set and quantitative real-time PCR (qPCR) analysis, it is apparent that FBS1 is required for elevated expression of many jasmonic acid (JA) genes that have established roles in combatting environmental stresses, and that it also controls a subset of JA biosynthesis genes. FBS1 also significantly impacts abscisic acid (ABA) regulated genes, but this interaction is more complex, as FBS1 has both positive and negative effects on ABA-inducible and ABA-repressible gene modules. One noteworthy effect of FBS1 on ABA-related stress processes, however, is the restraint it imposes on the expression of multiple class I LIPID TRANSFER PROTEIN (LTP) gene family members that have demonstrated protective effects in water deficit-related stresses. FBS1 impacts plant stress responses by regulating hundreds of genes that respond to the plant

Poxviral Ankyrin Proteins

Directory of Open Access Journals (Sweden)

Michael H. Herbert

2015-02-01

Full Text Available Multiple repeats of the ankyrin motif (ANK are ubiquitous throughout the kingdoms of life but are absent from most viruses. The main exception to this is the poxvirus family, and specifically the chordopoxviruses, with ANK repeat proteins present in all but three species from separate genera. The poxviral ANK repeat proteins belong to distinct orthologue groups spread over different species, and align well with the phylogeny of their genera. This distribution throughout the chordopoxviruses indicates these proteins were present in an ancestral vertebrate poxvirus, and have since undergone numerous duplication events. Most poxviral ANK repeat proteins contain an unusual topology of multiple ANK motifs starting at the N-terminus with a C-terminal poxviral homologue of the cellular F-box enabling interaction with the cellular SCF ubiquitin ligase complex. The subtle variations between ANK repeat proteins of individual poxviruses suggest an array of different substrates may be bound by these protein-protein interaction domains and, via the F-box, potentially directed to cellular ubiquitination pathways and possible degradation. Known interaction partners of several of these proteins indicate that the NF-κB coordinated anti-viral response is a key target, whilst some poxviral ANK repeat domains also have an F-box independent affect on viral host-range.

Protein Interaction Screening for the Ankyrin Repeats and Suppressor of Cytokine Signaling (SOCS) Box (ASB) Family Identify Asb11 as a Novel Endoplasmic Reticulum Resident Ubiquitin Ligase

DEFF Research Database (Denmark)

Andresen, Christina Aaen; Smedegaard, Stine; Sylvestersen, Kathrine Beck

2014-01-01

The Ankyrin and SOCS (Suppressor of Cytokine Signaling) box (ASB) family of proteins function as the substrate recognition subunit in a subset of Elongin-Cullin-SOCS (ECS) E3 ubiquitin ligases. Despite counting with 18 members in humans, the identity of the physiological targets of the Asb protei...

Characterization of an AGAMOUS-like MADS Box Protein, a Probable Constituent of Flowering and Fruit Ripening Regulatory System in Banana

Science.gov (United States)

Roy Choudhury, Swarup; Roy, Sujit; Nag, Anish; Singh, Sanjay Kumar; Sengupta, Dibyendu N.

2012-01-01

The MADS-box family of genes has been shown to play a significant role in the development of reproductive organs, including dry and fleshy fruits. In this study, the molecular properties of an AGAMOUS like MADS box transcription factor in banana cultivar Giant governor (Musa sp, AAA group, subgroup Cavendish) has been elucidated. We have detected a CArG-box sequence binding AGAMOUS MADS-box protein in banana flower and fruit nuclear extracts in DNA-protein interaction assays. The protein fraction in the DNA-protein complex was analyzed by mass spectrometry and using this information we have obtained the full length cDNA of the corresponding protein. The deduced protein sequence showed ∼95% amino acid sequence homology with MA-MADS5, a MADS-box protein described previously from banana. We have characterized the domains of the identified AGAMOUS MADS-box protein involved in DNA binding and homodimer formation in vitro using full-length and truncated versions of affinity purified recombinant proteins. Furthermore, in order to gain insight about how DNA bending is achieved by this MADS-box factor, we performed circular permutation and phasing analysis using the wild type recombinant protein. The AGAMOUS MADS-box protein identified in this study has been found to predominantly accumulate in the climacteric fruit pulp and also in female flower ovary. In vivo and in vitro assays have revealed specific binding of the identified AGAMOUS MADS-box protein to CArG-box sequence in the promoters of major ripening genes in banana fruit. Overall, the expression patterns of this MADS-box protein in banana female flower ovary and during various phases of fruit ripening along with the interaction of the protein to the CArG-box sequence in the promoters of major ripening genes lead to interesting assumption about the possible involvement of this AGAMOUS MADS-box factor in banana fruit ripening and floral reproductive organ development. PMID:22984496

Analyse der F-box Funktion von Rca1 in Drosophila melanogaster

OpenAIRE

Frank, Martina

2015-01-01

Rca1 ist ein wichtiger Inhibitor für die Ubiqutin-E3-Ligase APC/C mit dem Aktivatorproteine Fzr (Fizzy-related, ortholog zu Cdh1), vor allem in der G2-Phase des Zellzyklus, und ermöglicht die Akkumulati-on von Cyclinen für die darauffolgende Mitose. Embryonen, die die rca1-Allele rca1[2] und rca1[C1474] tragen, arretieren deshalb im Zellzyklus 16 und können nicht in die Mitose 16 eintreten. Durch Überexpression von Rca1 und Rca1 mit deletierter F-box (Rca1ΔF-box) konnte dieser Phänotyp komple...

Functions of DEAD-box proteins in bacteria: current knowledge and pending questions.

Science.gov (United States)

Iost, Isabelle; Bizebard, Thierry; Dreyfus, Marc

2013-08-01

DEAD-box proteins are RNA-dependent ATPases that are widespread in all three kingdoms of life. They are thought to rearrange the structures of RNA or ribonucleoprotein complexes but their exact mechanism of action is rarely known. Whereas in yeast most DEAD-box proteins are essential, no example of an essential bacterial DEAD-box protein has been reported so far; at most, their absence results in cold-sensitive growth. Moreover, whereas yeast DEAD-box proteins are implicated in virtually all reactions involving RNA, in E. coli (the bacterium where DEAD-box proteins have been mostly studied) their role is limited to ribosome biogenesis, mRNA degradation, and possibly translation initiation. Plausible reasons for these differences are discussed here. In spite of their dispensability, E. coli DEAD-box proteins are valuable models for the mechanism of action of DEAD-box proteins in general because the reactions in which they participate can be reproduced in vitro. Here we review our present understanding of this mechanism of action. Using selected examples for which information is available: (i) we describe how, by interacting directly with a particular RNA motif or by binding to proteins that themselves recognize such a motif, DEAD-box proteins are brought to their specific RNA substrate(s); (ii) we discuss the nature of the structural transitions that DEAD-box proteins induce on their substrates; and (iii) we analyze the reasons why these proteins are mostly important at low temperatures. This article is part of a Special Issue entitled: The Biology of RNA helicases-Modulation for life. Copyright © 2013 Elsevier B.V. All rights reserved.

The F-box protein Cdc4/Fbxw7 is a novel regulator of neural crest development in Xenopus laevis

Directory of Open Access Journals (Sweden)

Hartley Rebecca S

2010-01-01

Full Text Available Abstract Background The neural crest is a unique population of cells that arise in the vertebrate ectoderm at the neural plate border after which they migrate extensively throughout the embryo, giving rise to a wide range of derivatives. A number of proteins involved in neural crest development have dynamic expression patterns, and it is becoming clear that ubiquitin-mediated protein degradation is partly responsible for this. Results Here we demonstrate a novel role for the F-box protein Cdc4/Fbxw7 in neural crest development. Two isoforms of Xenopus laevis Cdc4 were identified, and designated xCdc4α and xCdc4β. These are highly conserved with vertebrate Cdc4 orthologs, and the Xenopus proteins are functionally equivalent in terms of their ability to degrade Cyclin E, an established vertebrate Cdc4 target. Blocking xCdc4 function specifically inhibited neural crest development at an early stage, prior to expression of c-Myc, Snail2 and Snail. Conclusions We demonstrate that Cdc4, an ubiquitin E3 ligase subunit previously identified as targeting primarily cell cycle regulators for proteolysis, has additional roles in control of formation of the neural crest. Hence, we identify Cdc4 as a protein with separable but complementary functions in control of cell proliferation and differentiation.

Molecular dissection of the APC/C inhibitor Rca1 shows a novel F-box-dependent function

OpenAIRE

Zielke, Norman; Querings, Silvia; Grosskortenhaus, Ruth; Reis, Tânia; Sprenger, Frank

2006-01-01

Rca1 (regulator of Cyclin A)/Emi (early mitotic inhibitor) proteins are essential inhibitors of the anaphase-promoting complex/cyclosome (APC/C). In Drosophila, Rca1 is required during G2 to prevent premature cyclin degradation by the Fizzy-related (Fzr)-dependent APC/C activity. Here, we present a structure and function analysis of Rca1 showing that a carboxy-terminal fragment is sufficient for APC/C inhibition. Rca1/Emi proteins contain a conserved F-box and interact with components of the ...

Pollen-expressed F-box gene family and mechanism of S-RNase-based gametophytic self-incompatibility (GSI) in Rosaceae.

Science.gov (United States)

Sassa, Hidenori; Kakui, Hiroyuki; Minamikawa, Mai

2010-03-01

Many species of Rosaceae, Solanaceae, and Plantaginaceae exhibit S-RNase-based self-incompatibility (SI) in which pistil-part specificity is controlled by S locus-encoded ribonuclease (S-RNase). Although recent findings revealed that S locus-encoded F-box protein, SLF/SFB, determines pollen-part specificity, how these pistil- and pollen-part S locus products interact in vivo and elicit the SI reaction is largely unclear. Furthermore, genetic studies suggested that pollen S function can differ among species. In Solanaceae and the rosaceous subfamily Maloideae (e.g., apple and pear), the coexistence of two different pollen S alleles in a pollen breaks down SI of the pollen, a phenomenon known as competitive interaction. However, competitive interaction seems not to occur in the subfamily Prunoideae (e.g., cherry and almond) of Rosaceae. Furthermore, the effect of the deletion of pollen S seems to vary among taxa. This review focuses on the potential differences in pollen-part function between subfamilies of Rosaceae, Maloideae, and Prunoideae, and discusses implications for the mechanistic divergence of the S-RNase-based SI.

Genome-wide identification and analysis of the SBP-box family genes in apple (Malus × domestica Borkh.).

Science.gov (United States)

Li, Jun; Hou, Hongmin; Li, Xiaoqin; Xiang, Jiang; Yin, Xiangjing; Gao, Hua; Zheng, Yi; Bassett, Carole L; Wang, Xiping

2013-09-01

SQUAMOSA promoter binding protein (SBP)-box genes encode a family of plant-specific transcription factors and play many crucial roles in plant development. In this study, 27 SBP-box gene family members were identified in the apple (Malus × domestica Borkh.) genome, 15 of which were suggested to be putative targets of MdmiR156. Plant SBPs were classified into eight groups according to the phylogenetic analysis of SBP-domain proteins. Gene structure, gene chromosomal location and synteny analyses of MdSBP genes within the apple genome demonstrated that tandem and segmental duplications, as well as whole genome duplications, have likely contributed to the expansion and evolution of the SBP-box gene family in apple. Additionally, synteny analysis between apple and Arabidopsis indicated that several paired homologs of MdSBP and AtSPL genes were located in syntenic genomic regions. Tissue-specific expression analysis of MdSBP genes in apple demonstrated their diversified spatiotemporal expression patterns. Most MdmiR156-targeted MdSBP genes, which had relatively high transcript levels in stems, leaves, apical buds and some floral organs, exhibited a more differential expression pattern than most MdmiR156-nontargeted MdSBP genes. Finally, expression analysis of MdSBP genes in leaves upon various plant hormone treatments showed that many MdSBP genes were responsive to different plant hormones, indicating that MdSBP genes may be involved in responses to hormone signaling during stress or in apple development. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Comparative structural analysis of human DEAD-box RNA helicases.

Science.gov (United States)

Schütz, Patrick; Karlberg, Tobias; van den Berg, Susanne; Collins, Ruairi; Lehtiö, Lari; Högbom, Martin; Holmberg-Schiavone, Lovisa; Tempel, Wolfram; Park, Hee-Won; Hammarström, Martin; Moche, Martin; Thorsell, Ann-Gerd; Schüler, Herwig

2010-09-30

DEAD-box RNA helicases play various, often critical, roles in all processes where RNAs are involved. Members of this family of proteins are linked to human disease, including cancer and viral infections. DEAD-box proteins contain two conserved domains that both contribute to RNA and ATP binding. Despite recent advances the molecular details of how these enzymes convert chemical energy into RNA remodeling is unknown. We present crystal structures of the isolated DEAD-domains of human DDX2A/eIF4A1, DDX2B/eIF4A2, DDX5, DDX10/DBP4, DDX18/myc-regulated DEAD-box protein, DDX20, DDX47, DDX52/ROK1, and DDX53/CAGE, and of the helicase domains of DDX25 and DDX41. Together with prior knowledge this enables a family-wide comparative structural analysis. We propose a general mechanism for opening of the RNA binding site. This analysis also provides insights into the diversity of DExD/H- proteins, with implications for understanding the functions of individual family members.

Comparative structural analysis of human DEAD-box RNA helicases.

Directory of Open Access Journals (Sweden)

Patrick Schütz

2010-09-01

Full Text Available DEAD-box RNA helicases play various, often critical, roles in all processes where RNAs are involved. Members of this family of proteins are linked to human disease, including cancer and viral infections. DEAD-box proteins contain two conserved domains that both contribute to RNA and ATP binding. Despite recent advances the molecular details of how these enzymes convert chemical energy into RNA remodeling is unknown. We present crystal structures of the isolated DEAD-domains of human DDX2A/eIF4A1, DDX2B/eIF4A2, DDX5, DDX10/DBP4, DDX18/myc-regulated DEAD-box protein, DDX20, DDX47, DDX52/ROK1, and DDX53/CAGE, and of the helicase domains of DDX25 and DDX41. Together with prior knowledge this enables a family-wide comparative structural analysis. We propose a general mechanism for opening of the RNA binding site. This analysis also provides insights into the diversity of DExD/H- proteins, with implications for understanding the functions of individual family members.

The retinoblastoma protein binds to a family of E2F transcription factors

DEFF Research Database (Denmark)

Lees, J A; Saito, M; Vidal, M

1993-01-01

E2F is a transcription factor that helps regulate the expression of a number of genes that are important in cell proliferation. Recently, several laboratories have isolated a cDNA clone that encodes an E2F-like protein, known as E2F-1. Subsequent characterization of this protein showed that it had...... the properties of E2F, but it was difficult to account for all of the suggested E2F activities through the function of this one protein. Using low-stringency hybridization, we have isolated cDNA clones that encode two additional E2F-like proteins, called E2F-2 and E2F-3. The chromosomal locations of the genes...... protein in vivo. Finally, E2F-2 and E2F-3 were able to activate transcription of E2F-responsive genes in a manner that was dependent upon the presence of at least one functional E2F binding site. These observations suggest that the E2F activities described previously result from the combined action...

The F-box-containing protein UFO and AGAMOUS participate in antagonistic pathways governing early petal development in Arabidopsis.

Science.gov (United States)

Durfee, Tim; Roe, Judith L; Sessions, R Allen; Inouye, Carla; Serikawa, Kyle; Feldmann, Kenneth A; Weigel, Detlef; Zambryski, Patricia C

2003-07-08

The UNUSUAL FLORAL ORGANS (UFO) gene is required for multiple processes in the developing Arabidopsis flower, including the proper patterning and identity of both petals and stamens. The gene encodes an F-box-containing protein, UFO, which interacts physically and genetically with the Skp1 homolog, ASK1. In this report, we describe four ufo alleles characterized by the absence of petals, which uncover another role for UFO in promoting second whorl development. This UFO-dependent pathway is required regardless of the second whorl organ to be formed, arguing that it affects a basic process acting in parallel with those establishing organ identity. However, the pathway is dispensable in the absence of AGAMOUS (AG), a known inhibitor of petal development. In situ hybridization results argue that AG is not transcribed in the petal region, suggesting that it acts non-cell-autonomously to inhibit second whorl development in ufo mutants. These results are combined into a genetic model explaining early second whorl initiation/proliferation, in which UFO functions to inhibit an AG-dependent activity.

ABC-F Proteins Mediate Antibiotic Resistance through Ribosomal Protection.

Science.gov (United States)

Sharkey, Liam K R; Edwards, Thomas A; O'Neill, Alex J

2016-03-22

Members of the ABC-F subfamily of ATP-binding cassette proteins mediate resistance to a broad array of clinically important antibiotic classes that target the ribosome of Gram-positive pathogens. The mechanism by which these proteins act has been a subject of long-standing controversy, with two competing hypotheses each having gained considerable support: antibiotic efflux versus ribosomal protection. Here, we report on studies employing a combination of bacteriological and biochemical techniques to unravel the mechanism of resistance of these proteins, and provide several lines of evidence that together offer clear support to the ribosomal protection hypothesis. Of particular note, we show that addition of purified ABC-F proteins to anin vitrotranslation assay prompts dose-dependent rescue of translation, and demonstrate that such proteins are capable of displacing antibiotic from the ribosomein vitro To our knowledge, these experiments constitute the first direct evidence that ABC-F proteins mediate antibiotic resistance through ribosomal protection.IMPORTANCEAntimicrobial resistance ranks among the greatest threats currently facing human health. Elucidation of the mechanisms by which microorganisms resist the effect of antibiotics is central to understanding the biology of this phenomenon and has the potential to inform the development of new drugs capable of blocking or circumventing resistance. Members of the ABC-F family, which includelsa(A),msr(A),optr(A), andvga(A), collectively yield resistance to a broader range of clinically significant antibiotic classes than any other family of resistance determinants, although their mechanism of action has been controversial since their discovery 25 years ago. Here we present the first direct evidence that proteins of the ABC-F family act to protect the bacterial ribosome from antibiotic-mediated inhibition. Copyright © 2016 Sharkey et al.

Protein Kinase R Degradation Is Essential for Rift Valley Fever Virus Infection and Is Regulated by SKP1-CUL1-F-box (SCF)FBXW11-NSs E3 Ligase.

Science.gov (United States)

Mudhasani, Rajini; Tran, Julie P; Retterer, Cary; Kota, Krishna P; Whitehouse, Chris A; Bavari, Sina

2016-02-01

Activated protein kinase R (PKR) plays a vital role in antiviral defense primarily by inhibiting protein synthesis and augmenting interferon responses. Many viral proteins have adopted unique strategies to counteract the deleterious effects of PKR. The NSs (Non-structural s) protein which is encoded by Rift Valley fever virus (RVFV) promotes early PKR proteasomal degradation through a previously undefined mechanism. In this study, we demonstrate that NSs carries out this activity by assembling the SCF (SKP1-CUL1-F-box)(FBXW11) E3 ligase. NSs binds to the F-box protein, FBXW11, via the six amino acid sequence DDGFVE called the degron sequence and recruits PKR through an alternate binding site to the SCF(FBXW11) E3 ligase. We further show that disrupting the assembly of the SCF(FBXW11-NSs) E3 ligase with MLN4924 (a small molecule inhibitor of SCF E3 ligase activity) or NSs degron viral mutants or siRNA knockdown of FBXW11 can block PKR degradation. Surprisingly, under these conditions when PKR degradation was blocked, NSs was essential and sufficient to activate PKR causing potent inhibition of RVFV infection by suppressing viral protein synthesis. These antiviral effects were antagonized by the loss of PKR expression or with a NSs deleted mutant virus. Therefore, early PKR activation by disassembly of SCF(FBXW11-NSs) E3 ligase is sufficient to inhibit RVFV infection. Furthermore, FBXW11 and BTRC are the two homologues of the βTrCP (Beta-transducin repeat containing protein) gene that were previously described to be functionally redundant. However, in RVFV infection, among the two homologues of βTrCP, FBXW11 plays a dominant role in PKR degradation and is the limiting factor in the assembly of the SCF(FBXW11) complex. Thus, FBXW11 serves as a master regulator of RVFV infection by promoting PKR degradation. Overall these findings provide new insights into NSs regulation of PKR activity and offer potential opportunities for therapeutic intervention of RVFV infection.

Genome-wide identification and expression analysis of the B-box gene family in the Apple (Malus domestica Borkh.) genome.

Science.gov (United States)

Liu, Xin; Li, Rong; Dai, Yaqing; Chen, Xuesen; Wang, Xiaoyun

2018-04-01

The B-box proteins (BBXs) are a family of zinc finger proteins containing one/two B-box domain(s). Compared with intensive studies of animal BBXs, investigations of the plant BBX family are limited, though some specific plant BBXs have been demonstrated to act as transcription factors in the regulation of flowering and photomorphogenesis. In this study, using a global search of the apple (Malus domestica Borkh.) genome, a total of 64 members of BBX (MdBBX) were identified. All the MdBBXs were divided into five groups based on the phylogenetic relationship, numbers of B-boxes contained and whether there was with an additional CCT domain. According to the characteristics of organ-specific expression, MdBBXs were divided into three groups based on the microarray information. An analysis of cis-acting elements showed that elements related to the stress response were prevalent in the promoter sequences of most MdBBXs. Twelve MdBBX members from different groups were randomly selected and exposed to abiotic stresses. Their expressions were up-regulated to some extent in the roots and leaves. Six among 12 MdBBXs were sensitive to osmotic pressure, salt, cold stress and exogenous abscisic acid treatment, with their expressions enhanced more than 20-fold. Our results suggested that MdBBXs may take part in response to abiotic stress.

Electrostatic potentials of the S-locus F-box proteins contribute to the pollen S specificity in self-incompatibility in Petunia hybrida.

Science.gov (United States)

Li, Junhui; Zhang, Yue; Song, Yanzhai; Zhang, Hui; Fan, Jiangbo; Li, Qun; Zhang, Dongfen; Xue, Yongbiao

2017-01-01

Self-incompatibility (SI) is a self/non-self discrimination system found widely in angiosperms and, in many species, is controlled by a single polymorphic S-locus. In the Solanaceae, Rosaceae and Plantaginaceae, the S-locus encodes a single S-RNase and a cluster of S-locus F-box (SLF) proteins to control the pistil and pollen expression of SI, respectively. Previous studies have shown that their cytosolic interactions determine their recognition specificity, but the physical force between their interactions remains unclear. In this study, we show that the electrostatic potentials of SLF contribute to the pollen S specificity through a physical mechanism of 'like charges repel and unlike charges attract' between SLFs and S-RNases in Petunia hybrida. Strikingly, the alteration of a single C-terminal amino acid of SLF reversed its surface electrostatic potentials and subsequently the pollen S specificity. Collectively, our results reveal that the electrostatic potentials act as a major physical force between cytosolic SLFs and S-RNases, providing a mechanistic insight into the self/non-self discrimination between cytosolic proteins in angiosperms. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Genome-wide identification and characterization of the SBP-box gene family in Petunia.

Science.gov (United States)

Zhou, Qin; Zhang, Sisi; Chen, Feng; Liu, Baojun; Wu, Lan; Li, Fei; Zhang, Jiaqi; Bao, Manzhu; Liu, Guofeng

2018-03-12

SQUAMOSA PROMOTER BINDING PROTEIN (SBP)-box genes encode a family of plant-specific transcription factors (TFs) that play important roles in many growth and development processes including phase transition, leaf initiation, shoot and inflorescence branching, fruit development and ripening etc. The SBP-box gene family has been identified and characterized in many species, but has not been well studied in Petunia, an important ornamental genus. We identified 21 putative SPL genes of Petunia axillaris and P. inflata from the reference genome of P. axillaris N and P. inflata S6, respectively, which were supported by the transcriptome data. For further confirmation, all the 21 genes were also cloned from P. hybrida line W115 (Mitchel diploid). Phylogenetic analysis based on the highly conserved SBP domains arranged PhSPLs in eight groups, analogous to those from Arabidopsis and tomato. Furthermore, the Petunia SPL genes had similar exon-intron structure and the deduced proteins contained very similar conserved motifs within the same subgroup. Out of 21 PhSPL genes, fourteen were predicted to be potential targets of PhmiR156/157, and the putative miR156/157 response elements (MREs) were located in the coding region of group IV, V, VII and VIII genes, but in the 3'-UTR regions of group VI genes. SPL genes were also identified from another two wild Petunia species, P. integrifolia and P. exserta, based on their transcriptome databases to investigate the origin of PhSPLs. Phylogenetic analysis and multiple alignments of the coding sequences of PhSPLs and their orthologs from wild species indicated that PhSPLs were originated mainly from P. axillaris. qRT-PCR analysis demonstrated differential spatiotemperal expression patterns of PhSPL genes in petunia and many were expressed predominantly in the axillary buds and/or inflorescences. In addition, overexpression of PhSPL9a and PhSPL9b in Arabidopsis suggested that these genes play a conserved role in promoting the vegetative

Isolation and characterization of multiple F-box genes linked to the S9- and S10-RNase in apple (Malus × domestica Borkh.).

Science.gov (United States)

Okada, Kazuma; Moriya, Shigeki; Haji, Takashi; Abe, Kazuyuki

2013-06-01

Using 11 consensus primer pairs designed from S-linked F-box genes of apple and Japanese pear, 10 new F-box genes (MdFBX21 to 30) were isolated from the apple cultivar 'Spartan' (S(9)S(10)). MdFBX21 to 23 and MdFBX24 to 30 were completely linked to the S(9) -RNase and S(10-)RNase, respectively, and showed pollen-specific expression and S-haplotype-specific polymorphisms. Therefore, these 10 F-box genes are good candidates for the pollen determinant of self-incompatibility in apple. Phylogenetic analysis and comparison of deduced amino acid sequences of MdFBX21 to 30 with those of 25 S-linked F-box genes previously isolated from apple showed that a deduced amino acid identity of greater than 88.0 % can be used as the tentative criterion to classify F-box genes into one type. Using this criterion, 31 of 35 F-box genes of apple were classified into 11 types (SFBB1-11). All types included F-box genes derived from S(3-) and S(9-)haplotypes, and seven types included F-box genes derived from S(3-), S(9-), and S(10-)haplotypes. Moreover, comparison of nucleotide sequences of S-RNases and multiple F-box genes among S(3-), S(9-), and S(10-)haplotypes suggested that F-box genes within each type showed high nucleotide identity regardless of the identity of the S-RNase. The large number of F-box genes as candidates for the pollen determinant and the high degree of conservation within each type are consistent with the collaborative non-self-recognition model reported for Petunia. These findings support that the collaborative non-self-recognition system also exists in apple.

A STE12 homologue of the homothallic ascomycete Sordaria macrospora interacts with the MADS box protein MCM1 and is required for ascosporogenesis.

Science.gov (United States)

Nolting, Nicole; Pöggeler, Stefanie

2006-11-01

The MADS box protein MCM1 controls diverse developmental processes and is essential for fruiting body formation in the homothallic ascomycete Sordaria macrospora. MADS box proteins derive their regulatory specificity from a wide range of different protein interactions. We have recently shown that the S. macrospora MCM1 is able to interact with the alpha-domain mating-type protein SMTA-1. To further evaluate the functional roles of MCM1, we used the yeast two-hybrid approach to identify MCM1-interacting proteins. From this screen, we isolated a protein with a putative N-terminal homeodomain and C-terminal C2/H2-Zn2+ finger domains. The protein is a member of the highly conserved fungal STE12 transcription factor family of proteins and was therefore termed STE12. Furthermore, we demonstrate by means of two-hybrid and far western analysis that in addition to MCM1, the S. macrospora STE12 protein is able to interact with the mating-type protein SMTA-1. Unlike the situation in the closely related heterothallic ascomycete Neurospora crassa, deletion (Delta) of the ste12 gene in S. macrospora neither affects vegetative growth nor fruiting body formation. However, ascus and ascospore development are highly impaired by the Deltaste12 mutation. Our data provide another example of the functional divergence within the fungal STE12 transcription factor family.

E2F family members are differentially regulated by reversible acetylation

DEFF Research Database (Denmark)

Marzio, G; Wagener, C; Gutierrez, M I

2000-01-01

of the other E2F family members. Here we report that E2F-1, -2, and -3, but not E2F-4, -5, and -6, associate with and are acetylated by p300 and cAMP-response element-binding protein acetyltransferases. Acetylation occurs at three conserved lysine residues located at the N-terminal boundary of their DNA......The six members of the E2F family of transcription factors play a key role in the control of cell cycle progression by regulating the expression of genes involved in DNA replication and cell proliferation. E2F-1, -2, and -3 belong to a structural and functional subfamily distinct from those...

Insights into the Prunus-Specific S-RNase-Based Self-Incompatibility System from a Genome-Wide Analysis of the Evolutionary Radiation of S Locus-Related F-box Genes.

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Akagi, Takashi; Henry, Isabelle M; Morimoto, Takuya; Tao, Ryutaro

2016-06-01

Self-incompatibility (SI) is an important plant reproduction mechanism that facilitates the maintenance of genetic diversity within species. Three plant families, the Solanaceae, Rosaceae and Plantaginaceae, share an S-RNase-based gametophytic SI (GSI) system that involves a single S-RNase as the pistil S determinant and several F-box genes as pollen S determinants that act via non-self-recognition. Previous evidence has suggested a specific self-recognition mechanism in Prunus (Rosaceae), raising questions about the generality of the S-RNase-based GSI system. We investigated the evolution of the pollen S determinant by comparing the sequences of the Prunus S haplotype-specific F-box gene (SFB) with those of its orthologs in other angiosperm genomes. Our results indicate that the Prunus SFB does not cluster with the pollen S of other plants and diverged early after the establishment of the Eudicots. Our results further indicate multiple F-box gene duplication events, specifically in the Rosaceae family, and suggest that the Prunus SFB gene originated in a recent Prunus-specific gene duplication event. Transcriptomic and evolutionary analyses of the Prunus S paralogs are consistent with the establishment of a Prunus-specific SI system, and the possibility of subfunctionalization differentiating the newly generated SFB from the original pollen S determinant. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Genome-wide identification, characterisation and expression analysis of the MADS-box gene family in Prunus mume.

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Xu, Zongda; Zhang, Qixiang; Sun, Lidan; Du, Dongliang; Cheng, Tangren; Pan, Huitang; Yang, Weiru; Wang, Jia

2014-10-01

MADS-box genes encode transcription factors that play crucial roles in plant development, especially in flower and fruit development. To gain insight into this gene family in Prunus mume, an important ornamental and fruit plant in East Asia, and to elucidate their roles in flower organ determination and fruit development, we performed a genome-wide identification, characterisation and expression analysis of MADS-box genes in this Rosaceae tree. In this study, 80 MADS-box genes were identified in P. mume and categorised into MIKC, Mα, Mβ, Mγ and Mδ groups based on gene structures and phylogenetic relationships. The MIKC group could be further classified into 12 subfamilies. The FLC subfamily was absent in P. mume and the six tandemly arranged DAM genes might experience a species-specific evolution process in P. mume. The MADS-box gene family might experience an evolution process from MIKC genes to Mδ genes to Mα, Mβ and Mγ genes. The expression analysis suggests that P. mume MADS-box genes have diverse functions in P. mume development and the functions of duplicated genes diverged after the duplication events. In addition to its involvement in the development of female gametophytes, type I genes also play roles in male gametophytes development. In conclusion, this study adds to our understanding of the roles that the MADS-box genes played in flower and fruit development and lays a foundation for selecting candidate genes for functional studies in P. mume and other species. Furthermore, this study also provides a basis to study the evolution of the MADS-box family.

Direct modulation of T-box riboswitch-controlled transcription by protein synthesis inhibitors.

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Stamatopoulou, Vassiliki; Apostolidi, Maria; Li, Shuang; Lamprinou, Katerina; Papakyriakou, Athanasios; Zhang, Jinwei; Stathopoulos, Constantinos

2017-09-29

Recently, it was discovered that exposure to mainstream antibiotics activate numerous bacterial riboregulators that control antibiotic resistance genes including metabolite-binding riboswitches and other transcription attenuators. However, the effects of commonly used antibiotics, many of which exhibit RNA-binding properties, on the widespread T-box riboswitches, remain unknown. In Staphylococcus aureus, a species-specific glyS T-box controls the supply of glycine for both ribosomal translation and cell wall synthesis, making it a promising target for next-generation antimicrobials. Here, we report that specific protein synthesis inhibitors could either significantly increase T-box-mediated transcription antitermination, while other compounds could suppress it, both in vitro and in vivo. In-line probing of the full-length T-box combined with molecular modelling and docking analyses suggest that the antibiotics that promote transcription antitermination stabilize the T-box:tRNA complex through binding specific positions on stem I and the Staphylococcal-specific stem Sa. By contrast, the antibiotics that attenuate T-box transcription bind to other positions on stem I and do not interact with stem Sa. Taken together, our results reveal that the transcription of essential genes controlled by T-box riboswitches can be directly modulated by commonly used protein synthesis inhibitors. These findings accentuate the regulatory complexities of bacterial response to antimicrobials that involve multiple riboregulators. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

F-Type Lectins: A Highly Diversified Family of Fucose-Binding Proteins with a Unique Sequence Motif and Structural Fold, Involved in Self/Non-Self-Recognition

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Gerardo R. Vasta

2017-11-01

Full Text Available The F-type lectin (FTL family is one of the most recent to be identified and structurally characterized. Members of the FTL family are characterized by a fucose recognition domain [F-type lectin domain (FTLD] that displays a novel jellyroll fold (“F-type” fold and unique carbohydrate- and calcium-binding sequence motifs. This novel lectin family comprises widely distributed proteins exhibiting single, double, or greater multiples of the FTLD, either tandemly arrayed or combined with other structurally and functionally distinct domains, yielding lectin subunits of pleiotropic properties even within a single species. Furthermore, the extraordinary variability of FTL sequences (isoforms that are expressed in a single individual has revealed genetic mechanisms of diversification in ligand recognition that are unique to FTLs. Functions of FTLs in self/non-self-recognition include innate immunity, fertilization, microbial adhesion, and pathogenesis, among others. In addition, although the F-type fold is distinctive for FTLs, a structure-based search revealed apparently unrelated proteins with minor sequence similarity to FTLs that displayed the FTLD fold. In general, the phylogenetic analysis of FTLD sequences from viruses to mammals reveals clades that are consistent with the currently accepted taxonomy of extant species. However, the surprisingly discontinuous distribution of FTLDs within each taxonomic category suggests not only an extensive structural/functional diversification of the FTLs along evolutionary lineages but also that this intriguing lectin family has been subject to frequent gene duplication, secondary loss, lateral transfer, and functional co-option.

The DEAD-Box Protein CYT-19 Uses Arginine Residues in Its C-Tail To Tether RNA Substrates.

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Busa, Veronica F; Rector, Maxwell J; Russell, Rick

2017-07-18

DEAD-box proteins are nonprocessive RNA helicases that play diverse roles in cellular processes. The Neurospora crassa DEAD-box protein CYT-19 promotes mitochondrial group I intron splicing and functions as a general RNA chaperone. CYT-19 includes a disordered, arginine-rich "C-tail" that binds RNA, positioning the helicase core to capture and unwind nearby RNA helices. Here we probed the C-tail further by varying the number and positions of arginines within it. We found that removing sets of as few as four of the 11 arginines reduced RNA unwinding activity (k cat /K M ) to a degree equivalent to that seen upon removal of the C-tail, suggesting that a minimum or "threshold" number of arginines is required. In addition, a mutant with 16 arginines displayed RNA unwinding activity greater than that of wild-type CYT-19. The C-tail modifications impacted unwinding only of RNA helices within constructs that included an adjacent helix or structured RNA element that would allow C-tail binding, indicating that the helicase core remained active in the mutants. In addition, changes in RNA unwinding efficiency of the mutants were mirrored by changes in functional RNA affinity, as determined from the RNA concentration dependence of ATPase activity, suggesting that the C-tail functions primarily to increase RNA affinity. Interestingly, the salt concentration dependence of RNA unwinding activity is unaffected by C-tail composition, suggesting that the C-tail uses primarily hydrogen bonding, not electrostatic interactions, to bind double-stranded RNA. Our results provide insights into how an unstructured C-tail contributes to DEAD-box protein activity and suggest parallels with other families of RNA- and DNA-binding proteins.

IGSF9 Family Proteins

DEFF Research Database (Denmark)

Hansen, Maria; Walmod, Peter Schledermann

2013-01-01

The Drosophila protein Turtle and the vertebrate proteins immunoglobulin superfamily (IgSF), member 9 (IGSF9/Dasm1) and IGSF9B are members of an evolutionarily ancient protein family. A bioinformatics analysis of the protein family revealed that invertebrates contain only a single IGSF9 family gene......, the longest isoforms of the proteins have the same general organization as the neural cell adhesion molecule family of cell adhesion molecule proteins, and like this family of proteins, IGSF9 family members are expressed in the nervous system. A review of the literature revealed that Drosophila Turtle...... facilitates homophilic cell adhesion. Moreover, IGSF9 family proteins have been implicated in the outgrowth and branching of neurites, axon guidance, synapse maturation, self-avoidance, and tiling. However, despite the few published studies on IGSF9 family proteins, reports on the functions of both Turtle...

All 17 S-locus F-box proteins of the S2 - and S3 -haplotypes of Petunia inflata are assembled into similar SCF complexes with a specific function in self-incompatibility.

Science.gov (United States)

Li, Shu; Williams, Justin S; Sun, Penglin; Kao, Teh-Hui

2016-09-01

The collaborative non-self-recognition model for S-RNase-based self-incompatibility predicts that multiple S-locus F-box proteins (SLFs) produced by pollen of a given S-haplotype collectively mediate ubiquitination and degradation of all non-self S-RNases, but not self S-RNases, in the pollen tube, thereby resulting in cross-compatible pollination but self-incompatible pollination. We had previously used pollen extracts containing GFP-fused S2 -SLF1 (SLF1 with an S2 -haplotype) of Petunia inflata for co-immunoprecipitation (Co-IP) and mass spectrometry (MS), and identified PiCUL1-P (a pollen-specific Cullin1), PiSSK1 (a pollen-specific Skp1-like protein) and PiRBX1 (a conventional Rbx1) as components of the SCF(S) (2-) (SLF) (1) complex. Using pollen extracts containing PiSSK1:FLAG:GFP for Co-IP/MS, we identified two additional SLFs (SLF4 and SLF13) that were assembled into SCF(SLF) complexes. As 17 SLF genes (SLF1 to SLF17) have been identified in S2 and S3 pollen, here we examined whether all 17 SLFs are assembled into similar complexes and, if so, whether these complexes are unique to SLFs. We modified the previous Co-IP/MS procedure, including the addition of style extracts from four different S-genotypes to pollen extracts containing PiSSK1:FLAG:GFP, to perform four separate experiments. The results taken together show that all 17 SLFs and an SLF-like protein, SLFLike1 (encoded by an S-locus-linked gene), co-immunoprecipitated with PiSSK1:FLAG:GFP. Moreover, of the 179 other F-box proteins predicted by S2 and S3 pollen transcriptomes, only a pair with 94.9% identity and another pair with 99.7% identity co-immunoprecipitated with PiSSK1:FLAG:GFP. These results suggest that SCF(SLF) complexes have evolved specifically to function in self-incompatibility. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Differential expression of members of the E2F family of transcription factors in rodent testes

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Toppari Jorma

2006-12-01

Full Text Available Abstract Background The E2F family of transcription factors is required for the activation or repression of differentially expressed gene programs during the cell cycle in normal and abnormal development of tissues. We previously determined that members of the retinoblastoma protein family that interacts with the E2F family are differentially expressed and localized in almost all the different cell types and tissues of the testis and in response to known endocrine disruptors. In this study, the cell-specific and stage-specific expression of members of the E2F proteins has been elucidated. Methods We used immunohistochemical (IHC analysis of tissue sections and Western blot analysis of proteins, from whole testis and microdissected stages of seminiferous tubules to study the differential expression of the E2F proteins. Results For most of the five E2F family members studied, the localizations appear conserved in the two most commonly studied rodent models, mice and rats, with some notable differences. Comparisons between wild type and E2F-1 knockout mice revealed that the level of E2F-1 protein is stage-specific and most abundant in leptotene to early pachytene spermatocytes of stages IX to XI of mouse while strong staining of E2F-1 in some cells close to the basal lamina of rat tubules suggest that it may also be expressed in undifferentiated spermatogonia. The age-dependent development of a Sertoli-cell-only phenotype in seminiferous tubules of E2F-1 knockout males corroborates this, and indicates that E2F-1 is required for spermatogonial stem cell renewal. Interestingly, E2F-3 appears in both terminally differentiated Sertoli cells, as well as spermatogonial cells in the differentiative pathway, while the remaining member of the activating E2Fs, E2F-2 is most concentrated in spermatocytes of mid to late prophase of meiosis. Comparisons between wildtype and E2F-4 knockout mice demonstrated that the level of E2F-4 protein displays a distinct

Essential and distinct roles of the F-box and helicase domains of Fbh1 in DNA damage repair

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Shinagawa Hideo

2008-03-01

Full Text Available Abstract Background DNA double-strand breaks (DSBs are induced by exogenous insults such as ionizing radiation and chemical exposure, and they can also arise as a consequence of stalled or collapsed DNA replication forks. Failure to repair DSBs can lead to genomic instability or cell death and cancer in higher eukaryotes. The Schizosaccharomyces pombe fbh1 gene encodes an F-box DNA helicase previously described to play a role in the Rhp51 (an orthologue of S. cerevisiae RAD51-dependent recombinational repair of DSBs. Fbh1 fused to GFP localizes to discrete nuclear foci following DNA damage. Results To determine the functional roles of the highly conserved F-box and helicase domains, we have characterized fbh1 mutants carrying specific mutations in these domains. We show that the F-box mutation fbh1-fb disturbs the nuclear localization of Fbh1, conferring an fbh1 null-like phenotype. Moreover, nuclear foci do not form in fbh1-fb cells with DNA damage even if Fbh1-fb is targeted to the nucleus by fusion to a nuclear localization signal sequence. In contrast, the helicase mutation fbh1-hl causes the accumulation of Fbh1 foci irrespective of the presence of DNA damage and confers damage sensitivity greater than that conferred by the null allele. Additional mutation of the F-box alleviates the hypermorphic phenotype of the fbh1-hl mutant. Conclusion These results suggest that the F-box and DNA helicase domains play indispensable but distinct roles in Fbh1 function. Assembly of the SCFFbh1 complex is required for both the nuclear localization and DNA damage-induced focus formation of Fbh1 and is therefore prerequisite for the Fbh1 recombination function.

Molecular Cloning of a cDNA Encoding for Taenia solium TATA-Box Binding Protein 1 (TsTBP1) and Study of Its Interactions with the TATA-Box of Actin 5 and Typical 2-Cys Peroxiredoxin Genes.

Science.gov (United States)

Rodríguez-Lima, Oscar; García-Gutierrez, Ponciano; Jiménez, Lucía; Zarain-Herzberg, Ángel; Lazzarini, Roberto; Landa, Abraham

2015-01-01

TATA-box binding protein (TBP) is an essential regulatory transcription factor for the TATA-box and TATA-box-less gene promoters. We report the cloning and characterization of a full-length cDNA that encodes a Taenia solium TATA-box binding protein 1 (TsTBP1). Deduced amino acid composition from its nucleotide sequence revealed that encodes a protein of 238 residues with a predicted molecular weight of 26.7 kDa, and a theoretical pI of 10.6. The NH2-terminal domain shows no conservation when compared with to pig and human TBP1s. However, it shows high conservation in size and amino acid identity with taeniids TBP1s. In contrast, the TsTBP1 COOH-terminal domain is highly conserved among organisms, and contains the amino acids involved in interactions with the TATA-box, as well as with TFIIA and TFIIB. In silico TsTBP1 modeling reveals that the COOH-terminal domain forms the classical saddle structure of the TBP family, with one α-helix at the end, not present in pig and human. Native TsTBP1 was detected in T. solium cysticerci´s nuclear extract by western blot using rabbit antibodies generated against two synthetic peptides located in the NH2 and COOH-terminal domains of TsTBP1. These antibodies, through immunofluorescence technique, identified the TBP1 in the nucleus of cells that form the bladder wall of cysticerci of Taenia crassiceps, an organism close related to T. solium. Electrophoretic mobility shift assays using nuclear extracts from T. solium cysticerci and antibodies against the NH2-terminal domain of TsTBP1 showed the interaction of native TsTBP1 with the TATA-box present in T. solium actin 5 (pAT5) and 2-Cys peroxiredoxin (Ts2-CysPrx) gene promoters; in contrast, when antibodies against the anti-COOH-terminal domain of TsTBP1 were used, they inhibited the binding of TsTBP1 to the TATA-box of the pAT5 promoter gene.

ErpC, a member of the complement regulator-acquiring family of surface proteins from Borrelia burgdorferi, possesses an architecture previously unseen in this protein family

International Nuclear Information System (INIS)

Caesar, Joseph J. E.; Johnson, Steven; Kraiczy, Peter; Lea, Susan M.

2013-01-01

The structure of ErpC, a member of the complement regulator-acquiring surface protein family from B. burgdorferi, has been solved, providing insights into the strategies of complement evasion by this zoonotic bacterium and suggesting a common architecture for other members of this protein family. Borrelia burgdorferi is a spirochete responsible for Lyme disease, the most commonly occurring vector-borne disease in Europe and North America. The bacterium utilizes a set of proteins, termed complement regulator-acquiring surface proteins (CRASPs), to aid evasion of the human complement system by recruiting and presenting complement regulator factor H on its surface in a manner that mimics host cells. Presented here is the atomic resolution structure of a member of this protein family, ErpC. The structure provides new insights into the mechanism of recruitment of factor H and other factor H-related proteins by acting as a molecular mimic of host glycosaminoglycans. It also describes the architecture of other CRASP proteins belonging to the OspE/F-related paralogous protein family and suggests that they have evolved to bind specific complement proteins, aiding survival of the bacterium in different hosts

Cyclin B1 Destruction Box-Mediated Protein Instability: The Enhanced Sensitivity of Fluorescent-Protein-Based Reporter Gene System

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Chao-Hsun Yang

2013-01-01

Full Text Available The periodic expression and destruction of several cyclins are the most important steps for the exact regulation of cell cycle. Cyclins are degraded by the ubiquitin-proteasome system during cell cycle. Besides, a short sequence near the N-terminal of cyclin B called the destruction box (D-box; CDB is also required. Fluorescent-protein-based reporter gene system is insensitive to analysis because of the overly stable fluorescent proteins. Therefore, in this study, we use human CDB fused with both enhanced green fluorescent protein (EGFP at C-terminus and red fluorescent protein (RFP, DsRed at N-terminus in the transfected human melanoma cells to examine the effects of CDB on different fluorescent proteins. Our results indicated that CDB-fused fluorescent protein can be used to examine the slight gene regulations in the reporter gene system and have the potential to be the system for screening of functional compounds in the future.

The role of the F-box gene TaFBA1 from wheat (Triticum aestivum L.) in drought tolerance.

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Zhou, Shumei; Sun, Xiudong; Yin, Suhong; Kong, Xiangzhu; Zhou, Shan; Xu, Ying; Luo, Yin; Wang, Wei

2014-11-01

Drought is one of the most important factors limiting plant growth and development. We identified a gene in wheat (Triticum aestivum L.) under drought stress named TaFBA1. TaFBA1 encodes a putative 325-amino-acid F-box protein with a conserved N-terminal F-box domain and a C-terminal AMN1 domain. Real-time RT-PCR analysis revealed that TaFBA1 transcript accumulation was upregulated by high-salinity, water stress, and abscisic acid (ABA) treatment. To evaluate the functions of TaFBA1 in the regulation of drought stress responses, we produced transgenic tobacco lines overexpressing TaFBA1. Under water stress conditions, the transgenic tobacco plants had a higher germination rate, higher relative water content, net photosynthesis rate (Pn), less chlorophyll loss, and less growth inhibition than WT. These results demonstrate the high tolerance of the transgenic plants to drought stress compared to the WT. The enhanced oxidative stress tolerance of these plants, which may be involved in their drought tolerance, was indicated by their lower levels of reactive oxygen species (ROS) accumulation, MDA content, and cell membrane damage under drought stress compared to WT. The antioxidant enzyme activities were higher in the transgenic plants than in WT, which may be related to the upregulated expression of some antioxidant genes via overexpression of TaFBA1. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Cyclin F suppresses B-Myb activity to promote cell cycle checkpoint control

DEFF Research Database (Denmark)

Klein, Ditte Kjærsgaard; Hoffmann, Saskia; Ahlskog, Johanna K

2015-01-01

an important role in checkpoint control following ionizing radiation. Cyclin F-depleted cells initiate checkpoint signalling after ionizing radiation, but fail to maintain G2 phase arrest and progress into mitosis prematurely. Importantly, cyclin F suppresses the B-Myb-driven transcriptional programme...... that promotes accumulation of crucial mitosis-promoting proteins. Cyclin F interacts with B-Myb via the cyclin box domain. This interaction is important to suppress cyclin A-mediated phosphorylation of B-Myb, a key step in B-Myb activation. In summary, we uncover a regulatory mechanism linking the F-box protein...

Phylogenetic comparison of F-Box (FBX gene superfamily within the plant kingdom reveals divergent evolutionary histories indicative of genomic drift.

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Zhihua Hua

Full Text Available The emergence of multigene families has been hypothesized as a major contributor to the evolution of complex traits and speciation. To help understand how such multigene families arose and diverged during plant evolution, we examined the phylogenetic relationships of F-Box (FBX genes, one of the largest and most polymorphic superfamilies known in the plant kingdom. FBX proteins comprise the target recognition subunit of SCF-type ubiquitin-protein ligases, where they individually recruit specific substrates for ubiquitylation. Through the extensive analysis of 10,811 FBX loci from 18 plant species, ranging from the alga Chlamydomonas reinhardtii to numerous monocots and eudicots, we discovered strikingly diverse evolutionary histories. The number of FBX loci varies widely and appears independent of the growth habit and life cycle of land plants, with a little as 198 predicted for Carica papaya to as many as 1350 predicted for Arabidopsis lyrata. This number differs substantially even among closely related species, with evidence for extensive gains/losses. Despite this extraordinary inter-species variation, one subset of FBX genes was conserved among most species examined. Together with evidence of strong purifying selection and expression, the ligases synthesized from these conserved loci likely direct essential ubiquitylation events. Another subset was much more lineage specific, showed more relaxed purifying selection, and was enriched in loci with little or no evidence of expression, suggesting that they either control more limited, species-specific processes or arose from genomic drift and thus may provide reservoirs for evolutionary innovation. Numerous FBX loci were also predicted to be pseudogenes with their numbers tightly correlated with the total number of FBX genes in each species. Taken together, it appears that the FBX superfamily has independently undergone substantial birth/death in many plant lineages, with its size and rapid

A human Polycomb isoform lacking the Pc box does not participate to PRC1 complexes but forms protein assemblies and represses transcription

DEFF Research Database (Denmark)

Völkel, Pamela; Le Faou, Perrine; Vandamme, Julien

2012-01-01

site for the PRC1 protein complex. Drosophila core PRC1 is composed of four subunits: Polycomb (Pc), Posterior sex combs (Psc), Polyhomeotic (Ph) and Sex combs extra (Sce). Each of these proteins has multiple orthologs in vertebrates, thus generating an enormous scope for potential combinatorial...... diversity. In particular, mammalian genomes encode five Pc family members: CBX2, CBX4, CBX6, CBX7 and CBX8. To complicate matters further, distinct isoforms might arise from single genes. Here, we address the functional role of the two human CBX2 isoforms. Owing to different polyadenylation sites...... and alternative splicing events, the human CBX2 locus produces two transcripts: a 5-exon transcript that encodes the 532-amino acid CBX2-1 isoform that contains the conserved chromodomain and Pc box and a 4-exon transcript encoding a shorter isoform, CBX2-2, lacking the Pc box but still possessing a chromodomain...

Bioactive L acidissima protein hydrolysates using Box-Behnken design.

Science.gov (United States)

Sonawane, Sachin K; Arya, Shalini S

2017-07-01

This study examines the extraction and hydrolysis of proteins using single factor and Box-Behnken Design (BBD). From single factor tests, optimised extraction parameters were 1% alkali concentration, 40 °C temperature, 60 min time, and 1:20 solid to alkali ratio. Under these conditions; 924.31 mg/g of total protein was obtained from Limonia acidissima (L acidissima). The maximum degree of hydrolysis was 39.82% at pH 2, enzyme to substrate ratio 2.5% (w/w), and hydrolysis time was 42.41 min using BBD design. L acidissima seed protein hydrolysate showed 32.94% DPPH and 88.18% of ABTS activity at concentration of 100 µg/ml and 1 mg/ml, respectively. Reducing power of 0.16 and metal chelating activity of 87.39% was obtained from 5 mg/ml protein hydrolysates. This implied that L acidissima seed protein hydrolysate could be utilised in protein rich product or as protein supplements.

Self-incompatibility in Petunia inflata: the relationship between a self-incompatibility locus F-box protein and its non-self S-RNases.

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Sun, Penglin; Kao, Teh-hui

2013-02-01

The highly polymorphic S (for self-incompatibility) locus regulates self-incompatibility in Petunia inflata; the S-RNase regulates pistil specificity, and multiple S-locus F-box (SLF) genes regulate pollen specificity. The collaborative non-self recognition model predicts that, for any S-haplotype, an unknown number of SLFs collectively recognize all non-self S-RNases to mediate their ubiquitination and degradation. Using a gain-of-function assay, we examined the relationships between S2-SLF1 (for S2-allelic product of Type-1 SLF) and four S-RNases. The results suggest that S2-SLF1 interacts with S7- and S13-RNases, and the previously identified S1- and S3-RNases, but not with S5- or S11-RNase. An artificial microRNA expressed by the S2-SLF1 promoter, but not by the vegetative cell-specific promoter, Late Anther Tomato 52, suppressed expression of S2-SLF1 in S2 pollen, suggesting that SLF1 is specific to the generative cell. The S2 pollen with S2-SLF1 suppressed was compatible with S3-, S5-, S7-, S11-, and S13-carrying pistils, confirming that other SLF proteins are responsible for detoxifying S5- and S11-RNases and suggesting that S2-SLF1 is not the only SLF in S2 pollen that interacts with S3-, S7-, and S13-RNases. Petunia may have evolved at least two types of SLF proteins to detoxify any non-self S-RNase to minimize the deleterious effects of mutation in any SLF.

A Point Mutation in an F-Box Domain-Containing Protein Is Responsible for Brown Hull Phenotype in Rice

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Xu Xia

2016-01-01

Full Text Available The accumulation of pigments affects the color of rice hulls while only limited information is known about its underlying mechanisms. In the present study, a rice brown hull 6 (bh6 mutant was isolated from an ethane methyl sulfonate (EMS-induced IR64 mutant bank. Brown pigments started to accumulate in bh6 rice hulls after heading and reached a higher level in mature seeds. Some major agronomic traits including panicle length and 1000-grain weight in bh6 were significantly lower than those in its corresponding wild type IR64, while other agronomic traits such as plant height, growth duration and seed-setting rate were largely similar between the two genotypes. The analysis of pigment content showed that the contents of total flavonoids and anthocyanin in bh6 hulls were significantly higher than those in IR64 hulls. Our results showed that the brown hull phenotype in bh6 was controlled by a single recessive gene which locates on the long arm of chromosome 9. Sequencing analysis detected a single base substitution (G/A at position 1013 of the candidate gene (LOC_Os09g12150 encoding an F-box domain-containing protein (FBX310. Functional complementation experiment using the wild type allele can rescue the phenotype in bh6. Thus, we named this mutated gene as OsFBX310bh6, an allele of OsFBX310 functioning as an inhibitor of brown hull. The isolation of OsFBX310bh6 and its wild type allele can provide useful experimental materials and will facilitate the studies on revealing the mechanisms of flavonoid metabolism in monocot plants.

Crystal structure of the UBR-box from UBR6/FBXO11 reveals domain swapping mediated by zinc binding.

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Muñoz-Escobar, Juliana; Kozlov, Guennadi; Gehring, Kalle

2017-10-01

The UBR-box is a 70-residue zinc finger domain present in the UBR family of E3 ubiquitin ligases that directly binds N-terminal degradation signals in substrate proteins. UBR6, also called FBXO11, is an UBR-box containing E3 ubiquitin ligase that does not bind N-terminal signals. Here, we present the crystal structure of the UBR-box domain from human UBR6. The dimeric crystal structure reveals a unique form of domain swapping mediated by zinc coordination, where three independent protein chains come together to regenerate the topology of the monomeric UBR-box fold. Analysis of the structure suggests that the absence of N-terminal residue binding arises from the lack of an amino acid binding pocket. © 2017 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.

PspF-binding domain PspA1-144 and the PspA·F complex: New insights into the coiled-coil-dependent regulation of AAA+ proteins.

Science.gov (United States)

Osadnik, Hendrik; Schöpfel, Michael; Heidrich, Eyleen; Mehner, Denise; Lilie, Hauke; Parthier, Christoph; Risselada, H Jelger; Grubmüller, Helmut; Stubbs, Milton T; Brüser, Thomas

2015-11-01

Phage shock protein A (PspA) belongs to the highy conserved PspA/IM30 family and is a key component of the stress inducible Psp system in Escherichia coli. One of its central roles is the regulatory interaction with the transcriptional activator of this system, the σ(54) enhancer-binding protein PspF, a member of the AAA+ protein family. The PspA/F regulatory system has been intensively studied and serves as a paradigm for AAA+ enzyme regulation by trans-acting factors. However, the molecular mechanism of how exactly PspA controls the activity of PspF and hence σ(54) -dependent expression of the psp genes is still unclear. To approach this question, we identified the minimal PspF-interacting domain of PspA, solved its structure, determined its affinity to PspF and the dissociation kinetics, identified residues that are potentially important for PspF regulation and analyzed effects of their mutation on PspF in vivo and in vitro. Our data indicate that several characteristics of AAA+ regulation in the PspA·F complex resemble those of the AAA+ unfoldase ClpB, with both proteins being regulated by a structurally highly conserved coiled-coil domain. The convergent evolution of both regulatory domains points to a general mechanism to control AAA+ activity for divergent physiologic tasks via coiled-coil domains. © 2015 John Wiley & Sons Ltd.

A MADS box protein interacts with a mating-type protein and is required for fruiting body development in the homothallic ascomycete Sordaria macrospora.

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Nolting, Nicole; Pöggeler, Stefanie

2006-07-01

MADS box transcription factors control diverse developmental processes in plants, metazoans, and fungi. To analyze the involvement of MADS box proteins in fruiting body development of filamentous ascomycetes, we isolated the mcm1 gene from the homothallic ascomycete Sordaria macrospora, which encodes a putative homologue of the Saccharomyces cerevisiae MADS box protein Mcm1p. Deletion of the S. macrospora mcm1 gene resulted in reduced biomass, increased hyphal branching, and reduced hyphal compartment length during vegetative growth. Furthermore, the S. macrospora Deltamcm1 strain was unable to produce fruiting bodies or ascospores during sexual development. A yeast two-hybrid analysis in conjugation with in vitro analyses demonstrated that the S. macrospora MCM1 protein can interact with the putative transcription factor SMTA-1, encoded by the S. macrospora mating-type locus. These results suggest that the S. macrospora MCM1 protein is involved in the transcriptional regulation of mating-type-specific genes as well as in fruiting body development.

Differentiation and injury-repair signals modulate the interaction of E2F and pRB proteins with novel target genes in keratinocytes.

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Chang, Wing Y; Andrews, Joseph; Carter, David E; Dagnino, Lina

2006-08-01

E2F transcription factors are central to epidermal morphogenesis and regeneration after injury. The precise nature of E2F target genes involved in epidermal formation and repair has yet to be determined. Identification of these genes is essential to understand how E2F proteins regulate fundamental aspects of epidermal homeostasis and transformation. We have conducted a genome-wide screen using CpG island microarray analysis to identify novel promoters bound by E2F3 and E2F5 in human keratinocytes. We further characterized several of these genes, and determined that multiple E2F and retinoblastoma (pRb) family proteins associate with them in exponentially proliferating cells. We also assessed the effect on E2F and pRb binding to those genes in response to differentiation induced by bone morphogenetic protein-6 (BMP-6), or to activation of repair mechanisms induced by transforming growth factor-beta (TGF-beta). These studies demonstrate promoter- and cytokine-specific changes in binding profiles of E2F and/or pRb family proteins. For example, E2F1, 3, 4 and p107 were recruited to the N-myc promoter in cells treated with BMP-6, whereas E2F1, 3, 4, 5, p107 and p130 were bound to this promoter in the presence of TGF-beta. Functionally, these different interactions resulted in transcriptional repression by BMP-6 and TGF-beta of the N-myc gene, via mechanisms that involved E2F binding to the promoter and association with pRb-family proteins. Thus, multiple combinations of E2F and pRb family proteins may associate with and transcriptionally regulate a given target promoter in response to differentiation and injury-repair stimuli in epidermal keratinocytes.

Multiple cis-regulatory elements are involved in the complex regulation of the sieve element-specific MtSEO-F1 promoter from Medicago truncatula.

Science.gov (United States)

Bucsenez, M; Rüping, B; Behrens, S; Twyman, R M; Noll, G A; Prüfer, D

2012-09-01

The sieve element occlusion (SEO) gene family includes several members that are expressed specifically in immature sieve elements (SEs) in the developing phloem of dicotyledonous plants. To determine how this restricted expression profile is achieved, we analysed the SE-specific Medicago truncatula SEO-F1 promoter (PMtSEO-F1) by constructing deletion, substitution and hybrid constructs and testing them in transgenic tobacco plants using green fluorescent protein as a reporter. This revealed four promoter regions, each containing cis-regulatory elements that activate transcription in SEs. One of these segments also contained sufficient information to suppress PMtSEO-F1 transcription in the phloem companion cells (CCs). Subsequent in silico analysis revealed several candidate cis-regulatory elements that PMtSEO-F1 shares with other SEO promoters. These putative sieve element boxes (PSE boxes) are promising candidates for cis-regulatory elements controlling the SE-specific expression of PMtSEO-F1. © 2012 German Botanical Society and The Royal Botanical Society of the Netherlands.

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

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Kellner, Julian N.; Meinhart, Anton, E-mail: anton.meinhart@mpimf-heidelberg.mpg.de [Max Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg (Germany)

2015-08-25

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–protein 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.

The effect of box shape on the dynamic properties of proteins simulated under periodic boundary conditions

NARCIS (Netherlands)

Wassenaar, T.A.; Mark, A.E.

The effect of the box shape on the dynamic behavior of proteins simulated under periodic boundary conditions is evaluated. In particular, the influence of simulation boxes defined by the near-densest lattice packing (NDLP) in conjunction with rotational constraints is compared to that of standard

Forkhead-box transcription factors and their role in the immune system

NARCIS (Netherlands)

Coffer, PJ; Burgering, BMT

2004-01-01

It is more than a decade since the discovery of the first forkhead-box (FOX) transcription factor in the fruit fly Drosophila melanogaster. In the intervening time, there has been an explosion in the identification and characterization of members of this family of proteins. Importantly, in the past

A human Polycomb isoform lacking the Pc box does not participate to PRC1 complexes but forms protein assemblies and represses transcription.

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Völkel, Pamela; Le Faou, Perrine; Vandamme, Julien; Pira, Dorcas; Angrand, Pierre-Olivier

2012-05-01

Polycomb repression controls the expression of hundreds of genes involved in development and is mediated by essentially two classes of chromatin-associated protein complexes. The Polycomb repressive complex 2 (PRC2) trimethylates histone H3 at lysine 27, an epigenetic mark that serves as a docking site for the PRC1 protein complex. Drosophila core PRC1 is composed of four subunits: Polycomb (Pc), Posterior sex combs (Psc), Polyhomeotic (Ph) and Sex combs extra (Sce). Each of these proteins has multiple orthologs in vertebrates, thus generating an enormous scope for potential combinatorial diversity. In particular, mammalian genomes encode five Pc family members: CBX2, CBX4, CBX6, CBX7 and CBX8. To complicate matters further, distinct isoforms might arise from single genes. Here, we address the functional role of the two human CBX2 isoforms. Owing to different polyadenylation sites and alternative splicing events, the human CBX2 locus produces two transcripts: a 5-exon transcript that encodes the 532-amino acid CBX2-1 isoform that contains the conserved chromodomain and Pc box and a 4-exon transcript encoding a shorter isoform, CBX2-2, lacking the Pc box but still possessing a chromodomain. Using biochemical approaches and a novel in vivo imaging assay, we show that the short CBX2-2 isoform lacking the Pc box, does not participate in PRC1 protein complexes, but self-associates in vivo and forms complexes of high molecular weight. Furthermore, the CBX2 short isoform is still able to repress transcription, suggesting that Polycomb repression might occur in the absence of PRC1 formation.

Thermal performance study of a box type solar cooker: evaluation of second figure of merit, F2

International Nuclear Information System (INIS)

Subudh Kumar

2006-01-01

The thermal performance of a box type solar cooker can be evaluated through the determination of two figures of merit-F 1 and F 2 . The F 1 is defined as the ratio of optical efficiency factor (η o ) the overall heat loss coefficient (U L ) and F 2 relates to the effectiveness of heat exchange between cooker interiors and contents of the pots. The values of F 1 and F 2 can be found experimentally from the stagnation (no-load) and load (water) tests respectively. The sensible heating curve (a plot between pot water temperature and time) from the load test is used for determination of F 2 . An accurate determination of F 2 is necessary for making the correct and reliable assessment of solar cooker performance. In the present work, the thermal analysis has been carried out to simulate the sensible heating curves for different loads of water in the commercially available, fibre body double-glazed box type solar cooker (size 0.245 m 2 ) by using the heat balance equation. The comparison between the simulated and experimental sensible heating curves is presented. The close agreement in the results shows that the simulated heating curve can be employed for predicting the thermal performance (or F 2 ) of the solar cooker. The F 2 for different loads of water in the solar cooker have been obtained using the computer simulation, while considering the variable overall heat loss coefficient U L (a function of pot water temperature). The results indicate that F 2 increases linearly up to a load of 1.5 kg. Thereafter, the increase in the value of F 2 decreases gradually with the load

Nuclear detection of Y-box protein-1 (YB-1) closely associates with progesterone receptor negativity and is a strong adverse survival factor in human breast cancer

International Nuclear Information System (INIS)

Dahl, Edgar; Dunn, Sandra E; Mertens, Peter R; En-Nia, Abdelaziz; Wiesmann, Frank; Krings, Renate; Djudjaj, Sonja; Breuer, Elisabeth; Fuchs, Thomas; Wild, Peter J; Hartmann, Arndt

2009-01-01

Y-box binding protein-1 (YB-1) is the prototypic member of the cold shock protein family that fulfills numerous cellular functions. In the nucleus YB-1 protein orchestrates transcription of proliferation-related genes, whereas in the cytoplasm it associates with mRNA and directs translation. In human tumor entities, such as breast, lung and prostate cancer, cellular YB-1 expression indicates poor clinical outcome, suggesting that YB-1 is an attractive marker to predict patients' prognosis and, potentially, is suitable to individualize treatment protocols. Given these predictive qualities of YB-1 detection we sought to establish a highly specific monoclonal antibody (Mab) for diagnostic testing and its characterization towards outcome prediction (relapse-free and overall survival). Hybridoma cell generation was carried out with recombinant YB-1 protein as immunogen and Mab characterization was performed using immunoblotting and ELISA with recombinant and tagged YB-1 proteins, as well as immunohistochemistry of healthy and breast cancer specimens. Breast tumor tissue array staining results were analyzed for correlations with receptor expression and outcome parameters. YB-1-specific Mab F-E2G5 associates with conformational binding epitopes mapping to two domains within the N-terminal half of the protein and detects nuclear YB-1 protein by immunohistochemistry in paraffin-embedded breast cancer tissues. Prognostic evaluation of Mab F-E2G5 was performed by immunohistochemistry of a human breast cancer tissue microarray comprising 179 invasive breast cancers, 8 ductal carcinoma in situ and 37 normal breast tissue samples. Nuclear YB-1 detection in human breast cancer cells was associated with poor overall survival (p = 0.0046). We observed a close correlation between nuclear YB-1 detection and absence of progesterone receptor expression (p = 0.002), indicating that nuclear YB-1 detection marks a specific subgroup of breast cancer. Likely due to limitation of sample

Differential stability of TATA box binding proteins from archaea with different optimal growth temperatures

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Kopitz, Annette; Soppa, Jörg; Krejtschi, Carsten; Hauser, Karin

2009-09-01

The TATA box binding protein (TBP) is involved in promoter recognition, the first step of transcription initiation. TBP is universally conserved and essential in archaea and eukaryotes. In archaea, TBPs have to be stable and to function in species that cover an extremely wide range of optimal growth temperatures (OGTs), from below 0 °C to more than 100 °C. Thus, the archaeal TBP family is ideally suited to study the evolutionary adaptation of proteins to an extremely wide range of temperatures. We characterized the thermostability of one mesophilic and one thermophilic TBP by infrared spectroscopy. Transition temperatures ( Tms) of thermal unfolding have been determined using TBPs from Methanosarcina mazei (OGT 37 °C) and from Methanothermobacter thermautotrophicus (OGT 65 °C). Furthermore, the influence of protein and salt concentration on thermostability has been characterized. Together with previous studies, our results reveal that the Tms of archaeal TBPs are closely correlated with the OGTs of the respective species. Noteworthy, this is also true for the TBP from M. mazei representing the first characterized TBP from a mesophilic archaeon. In contrast, the only characterized eukaryotic TBP of the mesophilic plant Arabidopsis thaliana has a Tm more than 40 °C above the OGT.

The Prenylated Rab GTPase Receptor PRA1.F4 Contributes to Protein Exit from the Golgi Apparatus.

Science.gov (United States)

Lee, Myoung Hui; Yoo, Yun-Joo; Kim, Dae Heon; Hanh, Nguyen Hong; Kwon, Yun; Hwang, Inhwan

2017-07-01

Prenylated Rab acceptor1 (PRA1) functions in the recruitment of prenylated Rab proteins to their cognate organelles. Arabidopsis ( Arabidopsis thaliana ) contains a large number of proteins belonging to the AtPRA1 family. However, their physiological roles remain largely unknown. Here, we investigated the physiological role of AtPRA1.F4, a member of the AtPRA1 family. A T-DNA insertion knockdown mutant of AtPRA1.F4 , atpra1.f4 , was smaller in stature than parent plants and possessed shorter roots, whereas transgenic plants overexpressing HA:AtPRA1.F4 showed enhanced development of secondary roots and root hairs. However, both overexpression and knockdown plants exhibited increased sensitivity to high-salt stress, lower vacuolar Na + /K + -ATPase and plasma membrane ATPase activities, lower and higher pH in the vacuole and apoplast, respectively, and highly vesiculated Golgi apparatus. HA:AtPRA1.F4 localized to the Golgi apparatus and assembled into high-molecular-weight complexes. atpra1.f4 plants displayed a defect in vacuolar trafficking, which was complemented by low but not high levels of HA : AtPRA1.F4 Overexpression of HA:AtPRA1.F4 also inhibited protein trafficking at the Golgi apparatus, albeit differentially depending on the final destination or type of protein: trafficking of vacuolar proteins, plasma membrane proteins, and trans-Golgi network (TGN)-localized SYP61 was strongly inhibited; trafficking of TGN-localized SYP51 was slightly inhibited; and trafficking of secretory proteins and TGN-localized SYP41 was negligibly or not significantly inhibited. Based on these results, we propose that Golgi-localized AtPRA1.F4 is involved in the exit of many but not all types of post-Golgi proteins from the Golgi apparatus. Additionally, an appropriate level of AtPRA1.F4 is crucial for its function at the Golgi apparatus. © 2017 American Society of Plant Biologists. All Rights Reserved.

Genome-wide characterization of the MADS-box gene family in radish (Raphanus sativus L. and assessment of its roles in flowering and floral organogenesis

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Chao Li

2016-09-01

Full Text Available The MADS-box gene family is an important transcription factor (TF family that is involved in various aspects of plant growth and development, especially flowering time and floral organogenesis. Although it has been reported in many plant species, the systematic identification and characterization of MADS-box TF family is still limited in radish (Raphanus sativus L.. In the present study, a comprehensive analysis of MADS-box genes was performed, and a total of 144 MADS-box family members were identified from the whole radish genome. Meanwhile, a detailed list of MADS-box genes from other 28 plant species was also investigated. Through the phylogenetic analysis between radish and Arabidopsis thaliana, all the RsMADS genes were classified into two groups including 68 type I (31 Mα, 12 Mβ and 25Mγ and 76 type II (70 MIKCC and 6 MIKC*. Among them, 41 (28.47% RsMADS genes were located in nine linkage groups of radish from R1 to R9. Moreover, the homologous MADS-box gene pairs were identified among radish, A. thaliana, Chinese cabbage and rice. Additionally, the expression profiles of RsMADS genes were systematically investigated in different tissues and growth stages. Furthermore, quantitative real-time PCR analysis was employed to validate expression patterns of some crucial RsMADS genes. These results could provide a valuable resource to explore the potential functions of RsMADS genes in radish, and facilitate dissecting MADS-box gene-mediated molecular mechanisms underlying flowering and floral organogenesis in root vegetable crops.

A specific type of cyclin-like F-box domain gene is involved in the cryogenic autolysis of Volvariella volvacea.

Science.gov (United States)

Gong, Ming; Chen, Mingjie; Wang, Hong; Zhu, Qiuming; Tan, Qi

2015-01-01

Cryogenic autolysis is a typical phenomenon of abnormal metabolism in Volvariella volvacea. Recent studies have identified 20 significantly up-regulated genes via high-throughput sequencing of the mRNAs expressed in the mycelia of V. volvacea after cold exposure. Among these significantly up-regulated genes, 15 annotated genes were used for functional annotation cluster analysis. Our results showed that the cyclin-like F-box domain (FBDC) formed the functional cluster with the lowest P-value. We also observed a significant expansion of FBDC families in V. volvacea. Among these, the FBDC3 family displayed the maximal gene expansion in V. volvacea. Gene expression profiling analysis revealed only one FBDC gene in V. volvacea (FBDV1) that was significantly up-regulated, which is located in the FBDC3 family. Comparative genomics analysis revealed the homologous sequences of FBDV1 with high similarity were clustered on the same scaffold. However, FBDV1 was located far from these clusters, indicating the divergence of duplicated genes. Relative time estimation and rate test provided evidence for the divergence of FBDV1 after recent duplications. Real-time RT-PCR analysis confirmed that the expression of the FBDV1 was significantly up-regulated (P autolysis of V. volvacea. © 2015 by The Mycological Society of America.

Protein arginine methyltransferase 1 regulates herpes simplex virus replication through ICP27 RGG-box methylation

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Yu, Jungeun; Shin, Bongjin; Park, Eui-Soon; Yang, Sujeong; Choi, Seunga [Department of Microbiology, Chungnam National University, 220 Gung-dong, Yuseong-gu, Daejon 305-764 (Korea, Republic of); BK21 Bio Brain Center, Chungnam National University, 220 Gung-dong, Yuseong-gu, Daejon 305-764 (Korea, Republic of); Kang, Misun [Department of Microbiology, Chungnam National University, 220 Gung-dong, Yuseong-gu, Daejon 305-764 (Korea, Republic of); Rho, Jaerang, E-mail: jrrho@cnu.ac.kr [Department of Microbiology, Chungnam National University, 220 Gung-dong, Yuseong-gu, Daejon 305-764 (Korea, Republic of); BK21 Bio Brain Center, Chungnam National University, 220 Gung-dong, Yuseong-gu, Daejon 305-764 (Korea, Republic of); GRAST, Chungnam National University, 220 Gung-dong, Yuseong-gu, Daejon 305-764 (Korea, Republic of)

2010-01-01

Protein arginine methylation is involved in viral infection and replication through the modulation of diverse cellular processes including RNA metabolism, cytokine signaling, and subcellular localization. It has been suggested previously that the protein arginine methylation of the RGG-box of ICP27 is required for herpes simplex virus type-1 (HSV-1) viral replication and gene expression in vivo. However, a cellular mediator for this process has not yet been identified. In our current study, we show that the protein arginine methyltransferase 1 (PRMT1) is a cellular mediator of the arginine methylation of ICP27 RGG-box. We generated arginine substitution mutants in this domain and examined which arginine residues are required for methylation by PRMT1. R138, R148 and R150 were found to be the major sites of this methylation but additional arginine residues serving as minor methylation sites are still required to sustain the fully methylated form of ICP27 RGG. We also demonstrate that the nuclear foci-like structure formation, SRPK interactions, and RNA-binding activity of ICP27 are modulated by the arginine methylation of the ICP27 RGG-box. Furthermore, HSV-1 replication is inhibited by hypomethylation of this domain resulting from the use of general PRMT inhibitors or arginine mutations. Our data thus suggest that the PRMT1 plays a key role as a cellular regulator of HSV-1 replication through ICP27 RGG-box methylation.

In silico studies on structure-function of DNA GCC- box binding domain of brassica napus DREB1 protein

International Nuclear Information System (INIS)

Qamarunnisa, S.; Hussain, M.

2012-01-01

DREB1 is a transcriptional factor, which selectively binds with the promoters of the genes involved in stress response in the plants. Homology of DREB protein and its binding element have been detected in the genome of many plants. However, only a few reports exist that discusses the binding properties of this protein with the gene (s) promoter. In the present study, we have undertaken studies exploring the structure-function relationship of Brassica napus DREB1. Multiple sequence alignment, protein homology modeling and intermolecular docking of GCC-box binding domain (GBD) of the said protein was carried out using atomic coordinates of GBD from Arabdiopsis thaliana and GCC-box containing DNA respectively. Similarities and/or identities in multiple, sequence alignment, particularly at the functionally important amino acids, strongly suggested the binding specificity of B. napus DREB1 to GCC-box. Similarly, despite 56% sequence homology, tertiary structures of both template and modeled protein were found to be extremely similar as indicated by root mean square deviation of 0.34 A. More similarities were established between GBD of both A. thaliana and B. napus DREB1 by conducting protein docking with the DNA containing GCC-box. It appears that both proteins interact through their beta-sheet with the major DNA groove including both nitrogen bases and phosphate and sugar moieties. Additionally, in most cases the interacting residues were also found to be identical. Briefly, this study attempts to elucidate the molecular basis of DREB1 interaction with its target sequence in the promoter. (author)

A novel missense mutation of the paired box 3 gene in a Turkish family with Waardenburg syndrome type 1.

Science.gov (United States)

Hazan, Filiz; Ozturk, A Taylan; Adibelli, Hamit; Unal, Nurettin; Tukun, Ajlan

2013-01-01

Screening of mutations in the paired box 3 (PAX3) gene in three generations of a Turkish family with Waardenburg syndrome type 1 (WS1). WS1 was diagnosed in a 13-month-old girl according to the WS Consortium criteria. Detailed family history of the proband revealed eight affected members in three generations. Routine clinical and audiological examination and ophthalmologic evaluation were performed on eight affected and five healthy members of the study family. Dystopia canthorum was detected in all affected patients; however, a brilliant blue iris was present in five patients who also had mild retinal hypopigmentation. Genomic DNA was extracted from the peripheral blood of affected and unaffected individuals in the family as well as 50 unrelated healthy volunteers. All coding exons and adjacent intronic regions of PAX3 were sequenced directly. A novel missense heterozygous c.788T>G mutation was identified in eight patients. This nucleotide alteration was not found in unaffected members of the study family or in the 50 unrelated control subjects. The mutation causes V263G amino-acid substitution in the homeodomain of the PAX3 protein, which represents the 45(th) residue of helix 3. We identified a novel missense c.788T>G mutation in PAX3 in a family with Waardenburg syndrome with intrafamilial phenotypic heterogeneity.

Heritability and genetic advance studies for biochemical traits in F2-3 introgressed families of Brassica

International Nuclear Information System (INIS)

Farhatullah, N.K.; Khalil, I.H.; Nahed, H.

2015-01-01

Higher heritability estimates along with high genetic advance values are effective in envisaging gain under selection in developing genotypes. The objective of the present study was to evaluate variability, heritability and genetic advance in 10 interspecific F2-3 families of Brassica species (B. napus * B. juncea, B. napus * B. rapa). These families were studied for heterospecific introgression of biochemical traits. Low to high heritability estimates were recorded for seed quality traits. Considerable variations within F2-3 families were observed for biochemical traits. Most of the F2-3 families for oil content and erucic showed moderate to high heritability indicating the slightest influence of environment thus modification of trait by selection would be more effective. Among F2-3 introgressed families Bn-510 x Bj-109 produced high oil i.e., 49.5% while Bn-532 x Br-118 (24.4%), Bn-533 x Bj-109 (24.1%) and high protein percentage in terms of mean performance. In the present research, individual segregating progenies of interspecific cross populations i.e., which possessed combination of desirable traits, were identified which could be incorporated in the future Breeding programs and it may facilitate varietal development. (author)

Calmodulin-dependent nuclear import of HMG-box family nuclear factors: importance of the role of SRY in sex reversal.

Science.gov (United States)

Kaur, Gurpreet; Delluc-Clavieres, Aurelie; Poon, Ivan K H; Forwood, Jade K; Glover, Dominic J; Jans, David A

2010-08-15

The HMG (high-mobility group)-box-containing chromatin-remodelling factor SRY (sex-determining region on the Y chromosome) plays a key role in sex determination. Its role in the nucleus is critically dependent on two NLSs (nuclear localization signals) that flank its HMG domain: the C-terminally located 'beta-NLS' that mediates nuclear transport through Impbeta1 (importin beta1) and the N-terminally located 'CaM-NLS' which is known to recognize the calcium-binding protein CaM (calmodulin). In the present study, we examined a number of missense mutations in the SRY CaM-NLS from human XY sex-reversed females for the first time, showing that they result in significantly reduced nuclear localization of GFP (green fluorescent protein)-SRY fusion proteins in transfected cells compared with wild-type. The CaM antagonist CDZ (calmidazolium chloride) was found to significantly reduce wild-type SRY nuclear accumulation, indicating dependence of SRY nuclear import on CaM. Intriguingly, the CaM-NLS mutants were all resistant to CDZ's effects, implying a loss of interaction with CaM, which was confirmed by direct binding experiments. CaM-binding/resultant nuclear accumulation was the only property of SRY found to be impaired by two of the CaM-NLS mutations, implying that inhibition of CaM-dependent nuclear import is the basis of sex reversal in these cases. Importantly, the CaM-NLS is conserved in other HMG-box-domain-containing proteins such as SOX-2, -9, -10 and HMGN1, all of which were found for the first time to rely on CaM for optimal nuclear localization. CaM-dependent nuclear translocation is thus a common mechanism for this family of important transcription factors.

Shaping 3-D boxes

DEFF Research Database (Denmark)

Stenholt, Rasmus; Madsen, Claus B.

2011-01-01

Enabling users to shape 3-D boxes in immersive virtual environments is a non-trivial problem. In this paper, a new family of techniques for creating rectangular boxes of arbitrary position, orientation, and size is presented and evaluated. These new techniques are based solely on position data...

Combining native MS approaches to decipher archaeal box H/ACA ribonucleoprotein particle structure and activity.

Science.gov (United States)

Saliou, Jean-Michel; Manival, Xavier; Tillault, Anne-Sophie; Atmanene, Cédric; Bobo, Claude; Branlant, Christiane; Van Dorsselaer, Alain; Charpentier, Bruno; Cianférani, Sarah

2015-08-01

Site-specific isomerization of uridines into pseudouridines in RNAs is catalyzed either by stand-alone enzymes or by box H/ACA ribonucleoprotein particles (sno/sRNPs). The archaeal box H/ACA sRNPs are five-component complexes that consist of a guide RNA and the aCBF5, aNOP10, L7Ae, and aGAR1 proteins. In this study, we performed pairwise incubations of individual constituents of archaeal box H/ACA sRNPs and analyzed their interactions by native MS to build a 2D-connectivity map of direct binders. We describe the use of native MS in combination with ion mobility-MS to monitor the in vitro assembly of the active H/ACA sRNP particle. Real-time native MS was used to monitor how box H/ACA particle functions in multiple-turnover conditions. Native MS also unambiguously revealed that a substrate RNA containing 5-fluorouridine (f(5) U) was hydrolyzed into 5-fluoro-6-hydroxy-pseudouridine (f(5) ho(6) Ψ). In terms of enzymatic mechanism, box H/ACA sRNP was shown to catalyze the pseudouridylation of a first RNA substrate, then to release the RNA product (S22 f(5) ho(6) ψ) from the RNP enzyme and reload a new substrate RNA molecule. Altogether, our native MS-based approaches provide relevant new information about the potential assembly process and catalytic mechanism of box H/ACA RNPs. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Perspectives on community gardens, community kitchens and the Good Food Box program in a community-based sample of low-income families.

Science.gov (United States)

Loopstra, Rachel; Tarasuk, Valerie

2013-01-08

Growing recognition of the problem of household food insecurity in Canada has meant public health practitioners are looking for effective ways to ameliorate this problem in their communities. Community gardens, community kitchens, and food box programs can offer nutritious foods for comparably lower costs, however, the uptake and perceptions of these programs in populations at risk of food insecurity have not been evaluated. Building on a previous finding of low program participation among 485 families living in high-poverty neighbourhoods in Toronto, the objective of this study was to understand reasons for non-participation. One year after the baseline study, 371 families were interviewed a second time and were asked to provide their reasons for not participating in community gardens, community kitchens, or the Good Food Box program. Responses were analyzed by inductive content analysis. At follow-up, only 12 families had participated in a community garden, 16 in a community kitchen, and 4 in the Good Food Box program. Reasons for non-participation grouped under two themes. First, families expressed that programs were not accessible because they lacked the knowledge of how or where to participate or because programs were not in their neighbourhoods. Second, programs lacked fit for families, as they were not suited to busy schedules, interests, or needs. This study provides unique perspective on participation in community food programs among food-insecure families and suggests that these programs may not be effective options for these families to improve their food access.

Neurochemical aftermath of amateur boxing.

Science.gov (United States)

Zetterberg, Henrik; Hietala, M Albert; Jonsson, Michael; Andreasen, Niels; Styrud, Ewa; Karlsson, Ingvar; Edman, Ake; Popa, Cornel; Rasulzada, Abdullah; Wahlund, Lars-Olof; Mehta, Pankaj D; Rosengren, Lars; Blennow, Kaj; Wallin, Anders

2006-09-01

Little solid information is available on the possible risks for neuronal injury in amateur boxing. To determine whether amateur boxing and severity of hits are associated with elevated levels of biochemical markers for neuronal injury in cerebrospinal fluid. Longitudinal study. Referral center specializing in evaluation of neurodegenerative disorders. Fourteen amateur boxers (11 men and 3 women) and 10 healthy male nonathletic control subjects. The boxers underwent lumbar puncture 7 to 10 days and 3 months after a bout. The control subjects underwent LP once. Neurofilament light protein, total tau, glial fibrillary acidic protein, phosphorylated tau, and beta-amyloid protein 1-40 (Abeta([1-40])) and 1-42 (Abeta([1-42])) concentrations in cerebrospinal fluid were measured. Increased levels after a bout compared with after 3 months of rest from boxing were found for 2 markers for neuronal and axonal injury, neurofilament light protein (mean +/- SD, 845 +/- 1140 ng/L vs 208 +/- 108 ng/L; P = .008) and total tau (mean +/- SD, 449 +/- 176 ng/L vs 306 +/- 78 ng/L; P = .006), and for the astroglial injury marker glial fibrillary acidic protein (mean +/- SD, 541 +/- 199 ng/L vs 405 +/- 138 ng/L; P = .003). The increase was significantly higher among boxers who had received many hits (>15) or high-impact hits to the head compared with boxers who reported few hits. In the boxers, concentrations of neurofilament light protein and glial fibrillary acidic protein, but not total tau, were significantly elevated after a bout compared with the nonathletic control subjects. With the exception of neurofilament light protein, there were no significant differences between boxers after 3 months of rest from boxing and the nonathletic control subjects. Amateur boxing is associated with acute neuronal and astroglial injury. If verified in longitudinal studies with extensive follow-up regarding the clinical outcome, analyses of cerebrospinal fluid may provide a scientific basis for

POPDC1S201F causes muscular dystrophy and arrhythmia by affecting protein trafficking

Science.gov (United States)

Schindler, Roland F.R.; Scotton, Chiara; Zhang, Jianguo; Passarelli, Chiara; Ortiz-Bonnin, Beatriz; Simrick, Subreena; Schwerte, Thorsten; Poon, Kar-Lai; Fang, Mingyan; Rinné, Susanne; Froese, Alexander; Nikolaev, Viacheslav O.; Grunert, Christiane; Müller, Thomas; Tasca, Giorgio; Sarathchandra, Padmini; Drago, Fabrizio; Dallapiccola, Bruno; Rapezzi, Claudio; Arbustini, Eloisa; Di Raimo, Francesca Romana; Neri, Marcella; Selvatici, Rita; Gualandi, Francesca; Fattori, Fabiana; Pietrangelo, Antonello; Li, Wenyan; Jiang, Hui; Xu, Xun; Bertini, Enrico; Decher, Niels; Wang, Jun; Brand, Thomas; Ferlini, Alessandra

2015-01-01

The Popeye domain–containing 1 (POPDC1) gene encodes a plasma membrane–localized cAMP-binding protein that is abundantly expressed in striated muscle. In animal models, POPDC1 is an essential regulator of structure and function of cardiac and skeletal muscle; however, POPDC1 mutations have not been associated with human cardiac and muscular diseases. Here, we have described a homozygous missense variant (c.602C>T, p.S201F) in POPDC1, identified by whole-exome sequencing, in a family of 4 with cardiac arrhythmia and limb-girdle muscular dystrophy (LGMD). This allele was absent in known databases and segregated with the pathological phenotype in this family. We did not find the allele in a further screen of 104 patients with a similar phenotype, suggesting this mutation to be family specific. Compared with WT protein, POPDC1S201F displayed a 50% reduction in cAMP affinity, and in skeletal muscle from patients, both POPDC1S201F and WT POPDC2 displayed impaired membrane trafficking. Forced expression of POPDC1S201F in a murine cardiac muscle cell line (HL-1) increased hyperpolarization and upstroke velocity of the action potential. In zebrafish, expression of the homologous mutation (popdc1S191F) caused heart and skeletal muscle phenotypes that resembled those observed in patients. Our study therefore identifies POPDC1 as a disease gene causing a very rare autosomal recessive cardiac arrhythmia and LGMD, expanding the genetic causes of this heterogeneous group of inherited rare diseases. PMID:26642364

A truncated F-box protein confers the dwarfism in cucumber

Science.gov (United States)

Dwarfism is an important plant architecture trait for cucumber breeding. In the present study, we identified a dwarf mutant 406M in cucumber which showed a shorter internode length as compared with its wild type. In a BC1F2 population from the cross of 406M with its wild type parental line 406, the ...

Heterodimerization of the transcription factors E2F-1 and DP-1 is required for binding to the adenovirus E4 (ORF6/7) protein

DEFF Research Database (Denmark)

Helin, K; Harlow, E

1994-01-01

Adenovirus infection leads to E1A-dependent activation of the transcription factor E2F. E2F has recently been identified in complexes with cellular proteins such as the retinoblastoma protein (pRB) and the two pRB family members p107 and p130. E1A dissociates E2F from these cellular proteins...

49 CFR 178.515 - Standards for reconstituted wood boxes.

Science.gov (United States)

2010-10-01

... wood boxes. (a) The identification code for a reconstituted wood box is 4F. (b) Construction requirements for reconstituted wood boxes are as follows: (1) The walls of boxes must be made of water... 49 Transportation 2 2010-10-01 2010-10-01 false Standards for reconstituted wood boxes. 178.515...

Crystal structure of CbpF, a bifunctional choline-binding protein and autolysis regulator from Streptococcus pneumoniae.

Science.gov (United States)

Molina, Rafael; González, Ana; Stelter, Meike; Pérez-Dorado, Inmaculada; Kahn, Richard; Morales, María; Moscoso, Miriam; Campuzano, Susana; Campillo, Nuria E; Mobashery, Shahriar; García, José L; García, Pedro; Hermoso, Juan A

2009-03-01

Phosphorylcholine, a crucial component of the pneumococcal cell wall, is essential in bacterial physiology and in human pathogenesis because it binds to serum components of the immune system and acts as a docking station for the family of surface choline-binding proteins. The three-dimensional structure of choline-binding protein F (CbpF), one of the most abundant proteins in the pneumococcal cell wall, has been solved in complex with choline. CbpF shows a new modular structure composed both of consensus and non-consensus choline-binding repeats, distributed along its length, which markedly alter its shape, charge distribution and binding ability, and organizing the protein into two well-defined modules. The carboxy-terminal module is involved in cell wall binding and the amino-terminal module is crucial for inhibition of the autolytic LytC muramidase, providing a regulatory function for pneumococcal autolysis.

A family of splice variants of CstF-64 expressed in vertebrate nervous systems

Science.gov (United States)

Shankarling, Ganesh S; Coates, Penelope W; Dass, Brinda; MacDonald, Clinton C

2009-01-01

Background Alternative splicing and polyadenylation are important mechanisms for creating the proteomic diversity necessary for the nervous system to fulfill its specialized functions. The contribution of alternative splicing to proteomic diversity in the nervous system has been well documented, whereas the role of alternative polyadenylation in this process is less well understood. Since the CstF-64 polyadenylation protein is known to be an important regulator of tissue-specific polyadenylation, we examined its expression in brain and other organs. Results We discovered several closely related splice variants of CstF-64 – collectively called βCstF-64 – that could potentially contribute to proteomic diversity in the nervous system. The βCstF-64 splice variants are found predominantly in the brains of several vertebrate species including mice and humans. The major βCstF-64 variant mRNA is generated by inclusion of two alternate exons (that we call exons 8.1 and 8.2) found between exons 8 and 9 of the CstF-64 gene, and contains an additional 147 nucleotides, encoding 49 additional amino acids. Some variants of βCstF-64 contain only the first alternate exon (exon 8.1) while other variants contain both alternate exons (8.1 and 8.2). In mice, the predominant form of βCstF-64 also contains a deletion of 78 nucleotides from exon 9, although that variant is not seen in any other species examined, including rats. Immunoblot and 2D-PAGE analyses of mouse nuclear extracts indicate that a protein corresponding to βCstF-64 is expressed in brain at approximately equal levels to CstF-64. Since βCstF-64 splice variant family members were found in the brains of all vertebrate species examined (including turtles and fish), this suggests that βCstF-64 has an evolutionarily conserved function in these animals. βCstF-64 was present in both pre- and post-natal mice and in different regions of the nervous system, suggesting an important role for βCstF-64 in neural gene

Paramyxovirus F1 protein has two fusion peptides: implications for the mechanism of membrane fusion.

Science.gov (United States)

Peisajovich, S G; Samuel, O; Shai, Y

2000-03-10

Viral fusion proteins contain a highly hydrophobic segment, named the fusion peptide, which is thought to be responsible for the merging of the cellular and viral membranes. Paramyxoviruses are believed to contain a single fusion peptide at the N terminus of the F1 protein. However, here we identified an additional internal segment in the Sendai virus F1 protein (amino acids 214-226) highly homologous to the fusion peptides of HIV-1 and RSV. A synthetic peptide, which includes this region, was found to induce membrane fusion of large unilamellar vesicles, at concentrations where the known N-terminal fusion peptide is not effective. A scrambled peptide as well as several peptides from other regions of the F1 protein, which strongly bind to membranes, are not fusogenic. The functional and structural characterization of this active segment suggest that the F1 protein has an additional internal fusion peptide that could participate in the actual fusion event. The presence of homologous regions in other members of the same family suggests that the concerted action of two fusion peptides, one N-terminal and the other internal, is a general feature of paramyxoviruses. Copyright 2000 Academic Press.

Interaction of Arabidopsis Trihelix-Domain Transcription Factors VFP3 and VFP5 with Agrobacterium Virulence Protein VirF.

Directory of Open Access Journals (Sweden)

Elena García-Cano

Full Text Available Agrobacterium is a natural genetic engineer of plants that exports several virulence proteins into host cells in order to take advantage of the cell machinery to facilitate transformation and support bacterial growth. One of these effectors is the F-box protein VirF, which presumably uses the host ubiquitin/proteasome system (UPS to uncoat the packaging proteins from the invading bacterial T-DNA. By analogy to several other bacterial effectors, VirF most likely has several functions in the host cell and, therefore, several interacting partners among host proteins. Here we identify one such interactor, an Arabidopsis trihelix-domain transcription factor VFP3, and further show that its very close homolog VFP5 also interacted with VirF. Interestingly, interactions of VirF with either VFP3 or VFP5 did not activate the host UPS, suggesting that VirF might play other UPS-independent roles in bacterial infection. To better understand the potential scope of VFP3 function, we used RNAi to reduce expression of the VFP3 gene. Transcriptome profiling of these VFP3-silenced plants using high-throughput cDNA sequencing (RNA-seq revealed that VFP3 substantially affected plant gene expression; specifically, 1,118 genes representing approximately 5% of all expressed genes were significantly either up- or down-regulated in the VFP3 RNAi line compared to wild-type Col-0 plants. Among the 507 up-regulated genes were genes implicated in the regulation of transcription, protein degradation, calcium signaling, and hormone metabolism, whereas the 611 down-regulated genes included those involved in redox regulation, light reactions of photosynthesis, and metabolism of lipids, amino acids, and cell wall. Overall, this pattern of changes in gene expression is characteristic of plants under stress. Thus, VFP3 likely plays an important role in controlling plant homeostasis.

Interaction of Arabidopsis Trihelix-Domain Transcription Factors VFP3 and VFP5 with Agrobacterium Virulence Protein VirF

Science.gov (United States)

García-Cano, Elena; Magori, Shimpei; Sun, Qi; Ding, Zehong; Lazarowitz, Sondra G.; Citovsky, Vitaly

2015-01-01

Agrobacterium is a natural genetic engineer of plants that exports several virulence proteins into host cells in order to take advantage of the cell machinery to facilitate transformation and support bacterial growth. One of these effectors is the F-box protein VirF, which presumably uses the host ubiquitin/proteasome system (UPS) to uncoat the packaging proteins from the invading bacterial T-DNA. By analogy to several other bacterial effectors, VirF most likely has several functions in the host cell and, therefore, several interacting partners among host proteins. Here we identify one such interactor, an Arabidopsis trihelix-domain transcription factor VFP3, and further show that its very close homolog VFP5 also interacted with VirF. Interestingly, interactions of VirF with either VFP3 or VFP5 did not activate the host UPS, suggesting that VirF might play other UPS-independent roles in bacterial infection. To better understand the potential scope of VFP3 function, we used RNAi to reduce expression of the VFP3 gene. Transcriptome profiling of these VFP3-silenced plants using high-throughput cDNA sequencing (RNA-seq) revealed that VFP3 substantially affected plant gene expression; specifically, 1,118 genes representing approximately 5% of all expressed genes were significantly either up- or down-regulated in the VFP3 RNAi line compared to wild-type Col-0 plants. Among the 507 up-regulated genes were genes implicated in the regulation of transcription, protein degradation, calcium signaling, and hormone metabolism, whereas the 611 down-regulated genes included those involved in redox regulation, light reactions of photosynthesis, and metabolism of lipids, amino acids, and cell wall. Overall, this pattern of changes in gene expression is characteristic of plants under stress. Thus, VFP3 likely plays an important role in controlling plant homeostasis. PMID:26571494

The tubby family proteins

OpenAIRE

Mukhopadhyay, Saikat; Jackson, Peter K

2011-01-01

The tubby mouse shows a tripartite syndrome characterized by maturity-onset obesity, blindness and deafness. The causative gene Tub is the founding member of a family of related proteins present throughout the animal and plant kingdoms, each characterized by a signature carboxy-terminal tubby domain. This domain consists of a β barrel enclosing a central α helix and binds selectively to specific membrane phosphoinositides. The vertebrate family of tubby-like proteins (TULPs) includes the foun...

Functional conservation and divergence of four ginger AP1/AGL9 MADS-box genes revealed by analysis of their expression and protein-protein interaction, and ectopic expression of AhFUL gene in Arabidopsis.

Directory of Open Access Journals (Sweden)

Xiumei Li

Full Text Available Alpinia genus are known generally as ginger-lilies for showy flowers in the ginger family, Zingiberaceae, and their floral morphology diverges from typical monocotyledon flowers. However, little is known about the functions of ginger MADS-box genes in floral identity. In this study, four AP1/AGL9 MADS-box genes were cloned from Alpinia hainanensis, and protein-protein interactions (PPIs and roles of the four genes in floral homeotic conversion and in floral evolution are surveyed for the first time. AhFUL is clustered to the AP1 lineage, AhSEP4 and AhSEP3b to the SEP lineage, and AhAGL6-like to the AGL6 lineage. The four genes showed conserved and divergent expression patterns, and their encoded proteins were localized in the nucleus. Seven combinations of PPI (AhFUL-AhSEP4, AhFUL-AhAGL6-like, AhFUL-AhSEP3b, AhSEP4-AhAGL6-like, AhSEP4-AhSEP3b, AhAGL6-like-AhSEP3b, and AhSEP3b-AhSEP3b were detected, and the PPI patterns in the AP1/AGL9 lineage revealed that five of the 10 possible combinations are conserved and three are variable, while conclusions cannot yet be made regarding the other two. Ectopic expression of AhFUL in Arabidopsis thaliana led to early flowering and floral organ homeotic conversion to sepal-like or leaf-like. Therefore, we conclude that the four A. hainanensis AP1/AGL9 genes show functional conservation and divergence in the floral identity from other MADS-box genes.

Effects of Quercetin Supplementation on Lipid and Protein Metabolism after Classic Boxing Training

Science.gov (United States)

Demirci, Nevzat

2017-01-01

The metabolic fitness (MF) is a component of athletes' physical conditioning. This study aims to investigate the effects of quercetin supplementation on Turkish Junior athletes' lipid and protein metabolism relating to MF after one month classic boxing training. Totally 20 voluntary junior male athletes were separated into two equal groups as the…

SCF(KMD) controls cytokinin signaling by regulating the degradation of type-B response regulators.

Science.gov (United States)

Kim, Hyo Jung; Chiang, Yi-Hsuan; Kieber, Joseph J; Schaller, G Eric

2013-06-11

Cytokinins are plant hormones that play critical roles in growth and development. In Arabidopsis, the transcriptional response to cytokinin is regulated by action of type-B Arabidopsis response regulators (ARRs). Although central elements in the cytokinin signal transduction pathway have been identified, mechanisms controlling output remain to be elucidated. Here we demonstrate that a family of F-box proteins, called the kiss me deadly (KMD) family, targets type-B ARR proteins for degradation. KMD proteins form an S-phase kinase-associated PROTEIN1 (SKP1)/Cullin/F-box protein (SCF) E3 ubiquitin ligase complex and directly interact with type-B ARR proteins. Loss-of-function KMD mutants stabilize type-B ARRs and exhibit an enhanced cytokinin response. In contrast, plants with elevated KMD expression destabilize type-B ARR proteins leading to cytokinin insensitivity. Our results support a model in which an SCF(KMD) complex negatively regulates cytokinin responses by controlling levels of a key family of transcription factors.

Characterization of E2F8, a novel E2F-like cell-cycle regulated repressor of E2F-activated transcription

DEFF Research Database (Denmark)

Christensen, Jesper; Cloos, Paul; Toftegaard, Ulla

2005-01-01

The E2F family of transcription factors are downstream effectors of the retinoblastoma protein, pRB, pathway and are essential for the timely regulation of genes necessary for cell-cycle progression. Here we describe the characterization of human and murine E2F8, a new member of the E2F family...

A multi-layer box model of carbon dynamics in soil

International Nuclear Information System (INIS)

Kuc, T.

2005-01-01

A multi-layer box model (MLB) for quantification of carbon fluxes between soil and atmosphere has been developed. In the model, soil carbon reservoir is represented by two boxes: fast decomposition box (FDB) and slow decomposition box (SDB), characterised by substantially different turnover time (TT) of carbon compounds. Each box has an internal structure (sub-compartments) accounting for carbon deposited in consecutive time intervals. The rate of decomposition of carbon compounds in each sub-compartment is proportional to the carbon content. With the aid of the MLB model and the 14 C signature of carbon dioxide, the fluxes entering and leaving the boxes, turnover time of carbon in each box, and the ratio of mass of carbon in the slow and fast box (M s /M f ) were calculated. The MBL model yields the turnover time of carbon in the FDB (TT f ) ca. 14 for typical investigated soils of temperate climate ecosystems. The calculated contribution of the CO 2 flux originating from the slow box (F s ) to the total CO 2 flux into the atmosphere ranges from 12% to 22%. These values are in agreement with experimental observations at different locations. Assuming that the input flux of carbon (F i n) to the soil system is doubled within the period of 100 years, the soil buffering capacity for excess carbon predicted by the MLB model for typical soil parameters may vary in the range between 26% and 52%. The highest values are obtained for soils characterised by long TTf, and well developed old carbon pool. (author)

Deregulated expression of E2F family members induces S-phase entry and overcomes p16INK4A-mediated growth suppression

DEFF Research Database (Denmark)

Lukas, J; Petersen, B O; Holm, K

1996-01-01

The E2F family of transcription factors regulate genes, whose products are essential for progression through the mammalian cell cycle. The transcriptional activity of the E2Fs is inhibited through the specific binding of the retinoblastoma protein, pRB, and the pRB homologs p107 and p130 to their......The E2F family of transcription factors regulate genes, whose products are essential for progression through the mammalian cell cycle. The transcriptional activity of the E2Fs is inhibited through the specific binding of the retinoblastoma protein, pRB, and the pRB homologs p107 and p130...

Insights into the quality of DnaA boxes and their cooperativity

DEFF Research Database (Denmark)

Hansen, Flemming G.; Christensen, Bjarke Bak; Nielsen, Christina Bang

2006-01-01

Plasmids carrying the mioC promoter region with its two DnaA boxes are as efficient in titration of DnaA protein as plasmids carrying a replicationinactivated oriC region with its five DnaA boxes. The two DnaA boxes upstream of the mioC promoter were mutated in various ways to study the cooperati......Plasmids carrying the mioC promoter region with its two DnaA boxes are as efficient in titration of DnaA protein as plasmids carrying a replicationinactivated oriC region with its five DnaA boxes. The two DnaA boxes upstream of the mioC promoter were mutated in various ways to study...... the cooperativity between the DnaA boxes, and to study in vivo the in vitrodefined 9mer DnaA box consensus sequence TTA/TTNCACA). The quality and cooperativity of the DnaA oxes were determined in two complementary ways: as titration of DnaA protein leading to derepression of the dnaA promoter, and as repression...... of the mioC promoter caused by the DnaA protein binding to the DnaA boxes. Titration of DnaA protein correlated with repression of the mioC promoter. The level of titration and repression with the normal promoter-proximal box (TTTTCCACA) depends strongly on the presence and the quality of a DnaA box...

The human protein disulfide isomerase gene family

Directory of Open Access Journals (Sweden)

Galligan James J

2012-07-01

Full Text Available Abstract Enzyme-mediated disulfide bond formation is a highly conserved process affecting over one-third of all eukaryotic proteins. The enzymes primarily responsible for facilitating thiol-disulfide exchange are members of an expanding family of proteins known as protein disulfide isomerases (PDIs. These proteins are part of a larger superfamily of proteins known as the thioredoxin protein family (TRX. As members of the PDI family of proteins, all proteins contain a TRX-like structural domain and are predominantly expressed in the endoplasmic reticulum. Subcellular localization and the presence of a TRX domain, however, comprise the short list of distinguishing features required for gene family classification. To date, the PDI gene family contains 21 members, varying in domain composition, molecular weight, tissue expression, and cellular processing. Given their vital role in protein-folding, loss of PDI activity has been associated with the pathogenesis of numerous disease states, most commonly related to the unfolded protein response (UPR. Over the past decade, UPR has become a very attractive therapeutic target for multiple pathologies including Alzheimer disease, Parkinson disease, alcoholic and non-alcoholic liver disease, and type-2 diabetes. Understanding the mechanisms of protein-folding, specifically thiol-disulfide exchange, may lead to development of a novel class of therapeutics that would help alleviate a wide range of diseases by targeting the UPR.

Isolation and characterization of an Aux/IAA gene (LaIAA2) from Larix

African Journals Online (AJOL)

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... an F-box protein, TIR1, which is a component of the SCFTIR1 ubiquitin ligase ... protein-protein interactions (Ulmasov et al., 1997b; Tiwari et al., 2003). In plant .... Statistical analysis .... main classes in the family (Abel and Theologis, 1995;.

An apple B-box protein, MdCOL11, is involved in UV-B- and temperature-induced anthocyanin biosynthesis.

Science.gov (United States)

Bai, Songling; Saito, Takanori; Honda, Chikako; Hatsuyama, Yoshimichi; Ito, Akiko; Moriguchi, Takaya

2014-11-01

Our studies showed that an apple B-box protein, MdCOL11, the homolog of AtBBX22, is involved in UV-B- and temperature-induced anthocyanin biosynthesis in apple peel. Anthocyanin is responsible for the red pigmentation in apple peel and a R2R3 MYB gene, MdMYBA/1/10, a homolog of MdMYBA, controls its accumulation. Arabidopsis PAP1 is under the control of a series of upstream factors involved in light signal transduction and photomorphogenesis, such as ELONGATED HYPOCOTYL 5 (HY5) and B-box family (BBX) proteins. In this study, we identified and characterized the homolog of Arabidopsis BBX22 in apple, designated as MdCOL11. Overexpression of MdCOL11 in Arabidopsis enhanced the accumulation of anthocyanin. In apples, MdCOL11 was differentially expressed in all tissues, with the highest expression in petals and the lowest expression in the xylem. Transcripts of MdCOL11 noticeably accumulated at the ripening stage, concomitant with increases in the expressions of anthocyanin biosynthesis-related genes. In an in vitro treatment experiment, MdCOL11 was upregulated in an ultra-violet (UV)-B- and temperature-dependent manner, together with the inductions of anthocyanin biosynthesis-related genes and anthocyanin accumulation in apple peel. Furthermore, a dual-luciferase assay indicated that (1) MdCOL11 regulated the expression of MdMYBA and (2) MdCOL11 was a target of MdHY5. Taken together, our results suggest that MdCOL11 is involved in MdHY5-mediated signal transduction and regulates anthocyanin accumulation in apple peel, which sheds new light on anthocyanin accumulation in apples.

Resistance of Sendai virus (HVJ) F-protein against gamma irradiation

International Nuclear Information System (INIS)

Hosokawa, Yasushi

1980-01-01

Sendai virus envelope proteins (hemagglutinin and F-protein), containing disulfide bonds in their molecules, were almost caused neither cleavage of protein molecules nor disappearance of antigenisitics by 10 5 R of γ-irradiation, but the liberated inner proteins (P, NP and M-protein) were severed into low molecules. By irradiation, hemagglutinin and F-protein of the virions were polymerized or aggregated together in such a way that the aggregate was not dissociated by SDS-2ME treatment on Slab gel electrophoresis, but in the liberated states hemagglutinin alone was aggregated and F-protein was separated with little loss of its anigenisity. From above results, the interaction between hemagglutinin and F-protein, and the utility of irradiation for isolation of F-protein were discussed. (author)

Spliced X-box binding protein 1 couples the unfolded protein response to hexosamine biosynthetic pathway.

Science.gov (United States)

Wang, Zhao V; Deng, Yingfeng; Gao, Ningguo; Pedrozo, Zully; Li, Dan L; Morales, Cyndi R; Criollo, Alfredo; Luo, Xiang; Tan, Wei; Jiang, Nan; Lehrman, Mark A; Rothermel, Beverly A; Lee, Ann-Hwee; Lavandero, Sergio; Mammen, Pradeep P A; Ferdous, Anwarul; Gillette, Thomas G; Scherer, Philipp E; Hill, Joseph A

2014-03-13

The hexosamine biosynthetic pathway (HBP) generates uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) for glycan synthesis and O-linked GlcNAc (O-GlcNAc) protein modifications. Despite the established role of the HBP in metabolism and multiple diseases, regulation of the HBP remains largely undefined. Here, we show that spliced X-box binding protein 1 (Xbp1s), the most conserved signal transducer of the unfolded protein response (UPR), is a direct transcriptional activator of the HBP. We demonstrate that the UPR triggers HBP activation via Xbp1s-dependent transcription of genes coding for key, rate-limiting enzymes. We further establish that this previously unrecognized UPR-HBP axis is triggered in a variety of stress conditions. Finally, we demonstrate a physiologic role for the UPR-HBP axis by showing that acute stimulation of Xbp1s in heart by ischemia/reperfusion confers robust cardioprotection in part through induction of the HBP. Collectively, these studies reveal that Xbp1s couples the UPR to the HBP to protect cells under stress. Copyright © 2014 Elsevier Inc. All rights reserved.

Forkhead box transcription factors in embryonic heart development and congenital heart disease.

Science.gov (United States)

Zhu, Hong

2016-01-01

Embryonic heart development is a very complicated process regulated precisely by a network composed of many genes and signaling pathways in time and space. Forkhead box (Fox, FOX) proteins are a family of transcription factors characterized by the presence of an evolutionary conserved "forkhead"or "winged-helix" DNA-binding domain and able to organize temporal and spatial gene expression during development. They are involved in a wide variety of cellular processes, such as cell cycle progression, proliferation, differentiation, migration, metabolism and DNA damage response. An abundance of studies in model organisms and systems has established that Foxa2, Foxc1/c2, Foxh1 and Foxm1, Foxos and Foxps are important components of the signaling pathways that instruct cardiogenesis and embryonic heart development, playing paramount roles in heart development. The previous studies also have demonstrated that mutations in some of the forkhead box genes and the aberrant expression of forkhead box gene are heavily implicated in the congenital heart disease (CHD) of humans. This review primarily focuses on the current understanding of heart development regulated by forkhead box transcription factors and molecular genetic mechanisms by which forkhead box factors modulate heart development during embryogenesis and organogenesis. This review also summarizes human CHD related mutations in forkhead box genes as well as the abnormal expression of forkhead box gene, and discusses additional possible regulatory mechanisms of the forkhead box genes during embryonic heart development that warrant further investigation. Copyright © 2015 Elsevier Inc. All rights reserved.

The Y-Box Binding Protein 1 Suppresses Alzheimer's Disease Progression in Two Animal Models.

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N V Bobkova

Full Text Available The Y-box binding protein 1 (YB-1 is a member of the family of DNA- and RNA binding proteins. It is involved in a wide variety of DNA/RNA-dependent events including cell proliferation and differentiation, stress response, and malignant cell transformation. Previously, YB-1 was detected in neurons of the neocortex and hippocampus, but its precise role in the brain remains undefined. Here we show that subchronic intranasal injections of recombinant YB-1, as well as its fragment YB-11-219, suppress impairment of spatial memory in olfactory bulbectomized (OBX mice with Alzheimer's type degeneration and improve learning in transgenic 5XFAD mice used as a model of cerebral amyloidosis. YB-1-treated OBX and 5XFAD mice showed a decreased level of brain β-amyloid. In OBX animals, an improved morphological state of neurons was revealed in the neocortex and hippocampus; in 5XFAD mice, a delay in amyloid plaque progression was observed. Intranasally administered YB-1 penetrated into the brain and could enter neurons. In vitro co-incubation of YB-1 with monomeric β-amyloid (1-42 inhibited formation of β-amyloid fibrils, as confirmed by electron microscopy. This suggests that YB-1 interaction with β-amyloid prevents formation of filaments that are responsible for neurotoxicity and neuronal death. Our data are the first evidence for a potential therapeutic benefit of YB-1 for treatment of Alzheimer's disease.

Shot-gun proteome and transcriptome mapping of the jujube floral organ and identification of a pollen-specific S-locus F-box gene

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Ruihong Chen

2017-07-01

Full Text Available The flower is a plant reproductive organ that forms part of the fruit produced as the flowering season ends. While the number and identity of proteins expressed in a jujube (Ziziphus jujuba Mill. flower is currently unknown, integrative proteomic and transcriptomic analyses provide a systematic strategy of characterizing the floral biology of plants. We conducted a shotgun proteomic analysis on jujube flowers by using a filter-aided sample preparation tryptic digestion, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS. In addition, transcriptomics analyses were performed on HiSeq2000 sequencers. In total, 7,853 proteins were identified accounting for nearly 30% of the ‘Junzao’ gene models (27,443. Genes identified in proteome generally showed higher RPKM (reads per kilobase per million mapped reads values than undetected genes. Gene ontology categories showed that ribosomes and intracellular organelles were the most dominant classes and accounted for 17.0% and 14.0% of the proteome mass, respectively. The top-ranking proteins with iBAQ >1010 included non-specific lipid transfer proteins, histones, actin-related proteins, fructose-bisphosphate aldolase, Bet v I type allergens, etc. In addition, we identified one pollen-specificity S-locus F-box-like gene located on the same chromosome as the S-RNase gene. Both of these may activate the behaviour of gametophyte self-incompatibility in jujube. These results reflected the protein profile features of jujube flowers and contributes new information important to the jujube breeding system.

The multifaceted activity of the VirF regulatory protein in the Shigella lifestyle

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Maria Letizia Di Martino

2016-09-01

Full Text Available Shigella is a highly adapted human pathogen, mainly found in the developing world and causing a severe enteric syndrome. The highly sophisticated infectious strategy of Shigella banks on the capacity to invade the intestinal epithelial barrier and cause its inflammatory destruction. The cellular pathogenesis and clinical presentation of shigellosis are the sum of the complex action of a large number of bacterial virulence factors mainly located on a large virulence plasmid (pINV. The expression of pINV genes is controlled by multiple environmental stimuli through a regulatory cascade involving proteins and sRNAs encoded by both the pINV and the chromosome. The primary regulator of the virulence phenotype is VirF, a DNA-binding protein belonging to the AraC family of transcriptional regulators. The virF gene, located on the pINV, is expressed only within the host, mainly in response to the temperature transition occurring when the bacterium transits from the outer environment to the intestinal milieu. VirF then acts as anti-H-NS protein and directly activates the icsA and virB genes, triggering the full expression of the invasion program of Shigella. In this review we will focus on the structure of VirF, on its sophisticated regulation, and on its role as major player in the path leading from the non invasive to the invasive phenotype of Shigella. We will address also the involvement of VirF in mechanisms aimed at withstanding adverse conditions inside the host, indicating that this protein is emerging as a global regulator whose action is not limited to virulence systems. Finally, we will discuss recent observations conferring VirF the potential of a novel antibacterial target for shigellosis.

Characterization of paralogous protein families in rice

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Zhu Wei

2008-02-01

Full Text Available Abstract Background High gene numbers in plant genomes reflect polyploidy and major gene duplication events. Oryza sativa, cultivated rice, is a diploid monocotyledonous species with a ~390 Mb genome that has undergone segmental duplication of a substantial portion of its genome. This, coupled with other genetic events such as tandem duplications, has resulted in a substantial number of its genes, and resulting proteins, occurring in paralogous families. Results Using a computational pipeline that utilizes Pfam and novel protein domains, we characterized paralogous families in rice and compared these with paralogous families in the model dicotyledonous diploid species, Arabidopsis thaliana. Arabidopsis, which has undergone genome duplication as well, has a substantially smaller genome (~120 Mb and gene complement compared to rice. Overall, 53% and 68% of the non-transposable element-related rice and Arabidopsis proteins could be classified into paralogous protein families, respectively. Singleton and paralogous family genes differed substantially in their likelihood of encoding a protein of known or putative function; 26% and 66% of singleton genes compared to 73% and 96% of the paralogous family genes encode a known or putative protein in rice and Arabidopsis, respectively. Furthermore, a major skew in the distribution of specific gene function was observed; a total of 17 Gene Ontology categories in both rice and Arabidopsis were statistically significant in their differential distribution between paralogous family and singleton proteins. In contrast to mammalian organisms, we found that duplicated genes in rice and Arabidopsis tend to have more alternative splice forms. Using data from Massively Parallel Signature Sequencing, we show that a significant portion of the duplicated genes in rice show divergent expression although a correlation between sequence divergence and correlation of expression could be seen in very young genes. Conclusion

A bifunctional archaeal protein that is a component of 30S ribosomal subunits and interacts with C/D box small RNAs

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Andrea Ciammaruconi

2008-01-01

Full Text Available We have identified a novel archaeal protein that apparently plays two distinct roles in ribosome metabolism. It is a polypeptide of about 18 kDa (termed Rbp18 that binds free cytosolic C/D box sRNAs in vivo and in vitro and behaves as a structural ribosomal protein, specifically a component of the 30S ribosomal subunit. As Rbp18 is selectively present in Crenarcheota and highly thermophilic Euryarchaeota, we propose that it serves to protect C/D box sRNAs from degradation and perhaps to stabilize thermophilic 30S subunits.

The Arabidopsis thaliana F-box gene HAWAIIAN SKIRT is a new player in the microRNA pathway.

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Xuebin Zhang

Full Text Available In Arabidopsis, the F-box HAWAIIAN SKIRT (HWS protein is important for organ growth. Loss of function of HWS exhibits pleiotropic phenotypes including sepal fusion. To dissect the HWS role, we EMS-mutagenized hws-1 seeds and screened for mutations that suppress hws-1 associated phenotypes. We identified shs-2 and shs-3 (suppressor of hws-2 and 3 mutants in which the sepal fusion phenotype of hws-1 was suppressed. shs-2 and shs-3 (renamed hst-23/hws-1 and hst-24/hws-1 carry transition mutations that result in premature terminations in the plant homolog of Exportin-5 HASTY (HST, known to be important in miRNA biogenesis, function and transport. Genetic crosses between hws-1 and mutant lines for genes in the miRNA pathway also suppress the phenotypes associated with HWS loss of function, corroborating epistatic relations between the miRNA pathway genes and HWS. In agreement with these data, accumulation of miRNA is modified in HWS loss or gain of function mutants. Our data propose HWS as a new player in the miRNA pathway, important for plant growth.

Production of recombinant proteins in Escherichia coli tagged with the fusion protein CusF3H.

Science.gov (United States)

Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Zarate, Xristo

2017-04-01

Recombinant protein expression in the bacterium Escherichia coli still is the number one choice for large-scale protein production. Nevertheless, many complications can arise using this microorganism, such as low yields, the formation of inclusion bodies, and the requirement for difficult purification steps. Most of these problems can be solved with the use of fusion proteins. Here, the use of the metal-binding protein CusF3H+ is described as a new fusion protein for recombinant protein expression and purification in E. coli. We have previously shown that CusF produces large amounts of soluble protein, with low levels of formation of inclusion bodies, and that proteins can be purified using IMAC resins charged with Cu(II) ions. CusF3H+ is an enhanced variant of CusF, formed by the addition of three histidine residues at the N-terminus. These residues then can bind Ni(II) ions allowing improved purity after affinity chromatography. Expression and purification of Green Fluorescent Protein tagged with CusF3H+ showed that the mutation did not alter the capacity of the fusion protein to increase protein expression, and purity improved considerably after affinity chromatography with immobilized nickel ions; high yields are obtained after tag-removal since CusF3H+ is a small protein of just 10 kDa. Furthermore, the results of experiments involving expression of tagged proteins having medium to large molecular weights indicate that the presence of the CusF3H+ tag improves protein solubility, as compared to a His-tag. We therefore endorse CusF3H+ as a useful alternative fusion protein/affinity tag for production of recombinant proteins in E. coli. Copyright © 2017 Elsevier Inc. All rights reserved.

Structure and biochemical function of a prototypical Arabidopsis U-box domain

DEFF Research Database (Denmark)

Andersen, Pernille; Kragelund, Birthe B; Olsen, Addie N

2004-01-01

U-box proteins, as well as other proteins involved in regulated protein degradation, are apparently over-represented in Arabidopsis compared with other model eukaryotes. The Arabidopsis protein AtPUB14 contains a typical U-box domain followed by an Armadillo repeat region, a domain organization t...

Crystallization and preliminary X-ray diffraction analysis of central structure domains from mumps virus F protein

International Nuclear Information System (INIS)

Liu, Yueyong; Xu, Yanhui; Zhu, Jieqing; Qiu, Bingsheng; Rao, Zihe; Gao, George F.; Tien, Po

2005-01-01

Single crystals of the central structure domains from mumps virus F protein have been obtained by the hanging-drop vapour-diffusion method. A diffraction data set has been collected to 2.2 Å resolution. Fusion of members of the Paramyxoviridae family involves two glycoproteins: the attachment protein and the fusion protein. Changes in the fusion-protein conformation were caused by binding of the attachment protein to the cellular receptor. In the membrane-fusion process, two highly conserved heptad-repeat (HR) regions, HR1 and HR2, are believed to form a stable six-helix coiled-coil bundle. However, no crystal structure has yet been determined for this state in the mumps virus (MuV, a member of the Paramyxoviridae family). In this study, a single-chain protein consisting of two HR regions connected by a flexible amino-acid linker (named 2-Helix) was expressed, purified and crystallized by the hanging-drop vapour-diffusion method. A complete X-ray data set was obtained in-house to 2.2 Å resolution from a single crystal. The crystal belongs to space group C2, with unit-cell parameters a = 161.2, b = 60.8, c = 40.1 Å, β = 98.4°. The crystal structure will help in understanding the molecular mechanism of Paramyxoviridae family membrane fusion

Two particle states in an asymmetric box

OpenAIRE

Li, Xin; Liu, Chuan

2004-01-01

The exact two-particle energy eigenstates in an asymmetric rectangular box with periodic boundary conditions in all three directions are studied. Their relation with the elastic scattering phases of the two particles in the continuum are obtained. These results can be viewed as a generalization of the corresponding formulae in a cubic box obtained by L\\"uscher before. In particular, the s-wave scattering length is related to the energy shift in the finite box. Possible applications of these f...

Integral UBL domain proteins: a family of proteasome interacting proteins

DEFF Research Database (Denmark)

Hartmann-Petersen, Rasmus; Gordon, Colin

2004-01-01

The family of ubiquitin-like (UBL) domain proteins (UDPs) comprises a conserved group of proteins involved in a multitude of different cellular activities. However, recent studies on UBL-domain proteins indicate that these proteins appear to share a common property in their ability to interact...

A hybrid clustering approach to recognition of protein families in 114 microbial genomes

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Gogarten J Peter

2004-04-01

Full Text Available Abstract Background Grouping proteins into sequence-based clusters is a fundamental step in many bioinformatic analyses (e.g., homology-based prediction of structure or function. Standard clustering methods such as single-linkage clustering capture a history of cluster topologies as a function of threshold, but in practice their usefulness is limited because unrelated sequences join clusters before biologically meaningful families are fully constituted, e.g. as the result of matches to so-called promiscuous domains. Use of the Markov Cluster algorithm avoids this non-specificity, but does not preserve topological or threshold information about protein families. Results We describe a hybrid approach to sequence-based clustering of proteins that combines the advantages of standard and Markov clustering. We have implemented this hybrid approach over a relational database environment, and describe its application to clustering a large subset of PDB, and to 328577 proteins from 114 fully sequenced microbial genomes. To demonstrate utility with difficult problems, we show that hybrid clustering allows us to constitute the paralogous family of ATP synthase F1 rotary motor subunits into a single, biologically interpretable hierarchical grouping that was not accessible using either single-linkage or Markov clustering alone. We describe validation of this method by hybrid clustering of PDB and mapping SCOP families and domains onto the resulting clusters. Conclusion Hybrid (Markov followed by single-linkage clustering combines the advantages of the Markov Cluster algorithm (avoidance of non-specific clusters resulting from matches to promiscuous domains and single-linkage clustering (preservation of topological information as a function of threshold. Within the individual Markov clusters, single-linkage clustering is a more-precise instrument, discerning sub-clusters of biological relevance. Our hybrid approach thus provides a computationally efficient

Metagenome and Metatranscriptome Analyses Using Protein Family Profiles.

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Cuncong Zhong

2016-07-01

Full Text Available Analyses of metagenome data (MG and metatranscriptome data (MT are often challenged by a paucity of complete reference genome sequences and the uneven/low sequencing depth of the constituent organisms in the microbial community, which respectively limit the power of reference-based alignment and de novo sequence assembly. These limitations make accurate protein family classification and abundance estimation challenging, which in turn hamper downstream analyses such as abundance profiling of metabolic pathways, identification of differentially encoded/expressed genes, and de novo reconstruction of complete gene and protein sequences from the protein family of interest. The profile hidden Markov model (HMM framework enables the construction of very useful probabilistic models for protein families that allow for accurate modeling of position specific matches, insertions, and deletions. We present a novel homology detection algorithm that integrates banded Viterbi algorithm for profile HMM parsing with an iterative simultaneous alignment and assembly computational framework. The algorithm searches a given profile HMM of a protein family against a database of fragmentary MG/MT sequencing data and simultaneously assembles complete or near-complete gene and protein sequences of the protein family. The resulting program, HMM-GRASPx, demonstrates superior performance in aligning and assembling homologs when benchmarked on both simulated marine MG and real human saliva MG datasets. On real supragingival plaque and stool MG datasets that were generated from healthy individuals, HMM-GRASPx accurately estimates the abundances of the antimicrobial resistance (AMR gene families and enables accurate characterization of the resistome profiles of these microbial communities. For real human oral microbiome MT datasets, using the HMM-GRASPx estimated transcript abundances significantly improves detection of differentially expressed (DE genes. Finally, HMM

Structure of Escherichia coli RutC, a member of the YjgF family and putative aminoacrylate peracid reductase of the rut operon

International Nuclear Information System (INIS)

Knapik, Aleksandra Alicja; Petkowski, Janusz Jurand; Otwinowski, Zbyszek; Cymborowski, Marcin Tadeusz; Cooper, David Robert; Chruszcz, Maksymilian; Krajewska, Wanda Małgorzata; Minor, Wladek

2012-01-01

The structure of the putative aminoacrylate peracid reductase RutC of the rut operon, a member of the YjgF family, is reported. RutC is the third enzyme in the Escherichia coli rut pathway of uracil degradation. RutC belongs to the highly conserved YjgF family of proteins. The structure of the RutC protein was determined and refined to 1.95 Å resolution. The crystal belonged to space group P2 1 2 1 2 and contained six molecules in the asymmetric unit. The structure was solved by SAD phasing and was refined to an R work of 19.3% (R free = 21.7%). The final model revealed that this protein has a Bacillus chorismate mutase-like fold and forms a homotrimer with a hydrophobic cavity in the center of the structure and ligand-binding clefts between two subunits. A likely function for RutC is the reduction of peroxy-aminoacrylate to aminoacrylate as a part of a detoxification process

PIP degron proteins, substrates of CRL4Cdt2, and not PIP boxes, interfere with DNA polymerase η and κ focus formation on UV damage

OpenAIRE

Tsanov, Nikolay; Kermi, Chames; Coulombe, Philippe; Van der Laan, Siem; Hodroj, Dana; Maiorano, Domenico

2014-01-01

Proliferating cell nuclear antigen (PCNA) is a well-known scaffold for many DNA replication and repair proteins, but how the switch between partners is regulated is currently unclear. Interaction with PCNA occurs via a domain known as a PCNA-Interacting Protein motif (PIP box). More recently, an additional specialized PIP box has been described, the « PIP degron », that targets PCNA-interacting proteins for proteasomal degradation via the E3 ubiquitin ligase CRL4Cdt2. Here we provide evidence...

Family symmetries in F-theory GUTs

CERN Document Server

King, S F; Ross, G G

2010-01-01

We discuss F-theory SU(5) GUTs in which some or all of the quark and lepton families are assigned to different curves and family symmetry enforces a leading order rank one structure of the Yukawa matrices. We consider two possibilities for the suppression of baryon and lepton number violation. The first is based on Flipped SU(5) with gauge group SU(5)\\times U(1)_\\chi \\times SU(4)_{\\perp} in which U(1)_{\\chi} plays the role of a generalised matter parity. We present an example which, after imposing a Z_2 monodromy, has a U(1)_{\\perp}^2 family symmetry. Even in the absence of flux, spontaneous breaking of the family symmetry leads to viable quark, charged lepton and neutrino masses and mixing. The second possibility has an R-parity associated with the symmetry of the underlying compactification manifold and the flux. We construct an example of a model with viable masses and mixing angles based on the gauge group SU(5)\\times SU(5)_{\\perp} with a U(1)_{\\perp}^3 family symmetry after imposing a Z_2 monodromy.

Comparative evaluation of low-molecular-mass proteins from Mycobacterium tuberculosis identifies members of the ESAT-6 family as immunodominant T-cell antigens

DEFF Research Database (Denmark)

Skjøt, Rikke L. V.; Oettinger, Thomas; Rosenkrands, Ida

2000-01-01

. The molecules were characterized, mapped in a two-dimensional electrophoresis reference map of short-term culture filtrate, and compared with another recently identified low-mass protein, CFP10 (F. X. Berthet, P, B. Rasmussen, I. Rosenkrands, P. Andersen, and B. Gicquel. Microbiology 144:3195-3203, 1998......), and the well-described ESAT-6 antigen. Genetic analyses demonstrated that TB10.4 as well as CFP10 belongs to the ESAT-6 family of low-mass proteins, whereas TB7.3 is a low-molecular-mass protein outside this family. The proteins were expressed in Escherichia coli, and their immunogenicity was tested...

To control and to be controlled – understanding the Arabidopsis SLIM1 function in sulfur deficiency through comprehensive investigation of the EIL protein family.

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Anna eWawrzyńska

2014-10-01

Full Text Available SSLIM1, a member of the EIN3-like (EIL family of transcription factors in Arabidopsis, is the regulator of many sulfur-deficiency responsive genes. Among the five other proteins of the family, three regulate ethylene responses and two have unassigned functions. Contrary to the well-defined ethylene signaling, the pathway leading from sensing sulfate status to the activation of its acquisition via SLIM1 is completely unknown. SLIM1 binds to the 20 nt-long specific UPE-box sequence; however, it also recognizes the shorter TEIL sequence, unique for the whole EIL family. SLIM1 takes part in the upregulation and downregulation of various sulfur metabolism genes, but also it controls the degradation of glucosinolates under sulfur deficient conditions. Besides facilitating the increased flux through the sulfate assimilation pathway, SLIM1 induces microRNA395, specifically targeting ATP sulfurylases and a low-affinity sulfate transporter, SULTR2;1, thus affecting sulfate translocation to the shoot. Here, we briefly review the identification, structural characteristics and molecular function of SLIM1 from the perspective of the whole EIL protein family.

Evidence for multiple major histocompatibility class II X-box binding proteins.

OpenAIRE

Celada, A; Maki, R

1989-01-01

The X box is a loosely conserved DNA sequence that is located upstream of all major histocompatibility class II genes and is one of the cis-acting regulatory elements. Despite the similarity between all X-box sequences, each promoter-proximal X box in the mouse appears to bind a separate nuclear factor.

Annotation and analysis of a large cuticular protein family with the R&R Consensus in Anopheles gambiae

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He Ningjia

2008-01-01

Full Text Available Abstract Background The most abundant family of insect cuticular proteins, the CPR family, is recognized by the R&R Consensus, a domain of about 64 amino acids that binds to chitin and is present throughout arthropods. Several species have now been shown to have more than 100 CPR genes, inviting speculation as to the functional importance of this large number and diversity. Results We have identified 156 genes in Anopheles gambiae that code for putative cuticular proteins in this CPR family, over 1% of the total number of predicted genes in this species. Annotation was verified using several criteria including identification of TATA boxes, INRs, and DPEs plus support from proteomic and gene expression analyses. Two previously recognized CPR classes, RR-1 and RR-2, form separate, well-supported clades with the exception of a small set of genes with long branches whose relationships are poorly resolved. Several of these outliers have clear orthologs in other species. Although both clades are under purifying selection, the RR-1 variant of the R&R Consensus is evolving at twice the rate of the RR-2 variant and is structurally more labile. In contrast, the regions flanking the R&R Consensus have diversified in amino-acid composition to a much greater extent in RR-2 genes compared with RR-1 genes. Many genes are found in compact tandem arrays that may include similar or dissimilar genes but always include just one of the two classes. Tandem arrays of RR-2 genes frequently contain subsets of genes coding for highly similar proteins (sequence clusters. Properties of the proteins indicated that each cluster may serve a distinct function in the cuticle. Conclusion The complete annotation of this large gene family provides insight on the mechanisms of gene family evolution and clues about the need for so many CPR genes. These data also should assist annotation of other Anopheles genes.

Differential binding of calmodulin-related proteins to their targets revealed through high-density Arabidopsis protein microarrays

Science.gov (United States)

Popescu, Sorina C.; Popescu, George V.; Bachan, Shawn; Zhang, Zimei; Seay, Montrell; Gerstein, Mark; Snyder, Michael; Dinesh-Kumar, S. P.

2007-01-01

Calmodulins (CaMs) are the most ubiquitous calcium sensors in eukaryotes. A number of CaM-binding proteins have been identified through classical methods, and many proteins have been predicted to bind CaMs based on their structural homology with known targets. However, multicellular organisms typically contain many CaM-like (CML) proteins, and a global identification of their targets and specificity of interaction is lacking. In an effort to develop a platform for large-scale analysis of proteins in plants we have developed a protein microarray and used it to study the global analysis of CaM/CML interactions. An Arabidopsis thaliana expression collection containing 1,133 ORFs was generated and used to produce proteins with an optimized medium-throughput plant-based expression system. Protein microarrays were prepared and screened with several CaMs/CMLs. A large number of previously known and novel CaM/CML targets were identified, including transcription factors, receptor and intracellular protein kinases, F-box proteins, RNA-binding proteins, and proteins of unknown function. Multiple CaM/CML proteins bound many binding partners, but the majority of targets were specific to one or a few CaMs/CMLs indicating that different CaM family members function through different targets. Based on our analyses, the emergent CaM/CML interactome is more extensive than previously predicted. Our results suggest that calcium functions through distinct CaM/CML proteins to regulate a wide range of targets and cellular activities. PMID:17360592

Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF.

Science.gov (United States)

Cantu-Bustos, J Enrique; Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Galbraith, David W; McEvoy, Megan M; Zarate, Xristo

2016-05-01

Production of recombinant proteins in Escherichia coli has been improved considerably through the use of fusion proteins, because they increase protein solubility and facilitate purification via affinity chromatography. In this article, we propose the use of CusF as a new fusion partner for expression and purification of recombinant proteins in E. coli. Using a cell-free protein expression system, based on the E. coli S30 extract, Green Fluorescent Protein (GFP) was expressed with a series of different N-terminal tags, immobilized on self-assembled protein microarrays, and its fluorescence quantified. GFP tagged with CusF showed the highest fluorescence intensity, and this was greater than the intensities from corresponding GFP constructs that contained MBP or GST tags. Analysis of protein production in vivo showed that CusF produces large amounts of soluble protein with low levels of inclusion bodies. Furthermore, fusion proteins can be exported to the cellular periplasm, if CusF contains the signal sequence. Taking advantage of its ability to bind copper ions, recombinant proteins can be purified with readily available IMAC resins charged with this metal ion, producing pure proteins after purification and tag removal. We therefore recommend the use of CusF as a viable alternative to MBP or GST as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli. Copyright © 2016 Elsevier Inc. All rights reserved.

Olympic boxing is associated with elevated levels of the neuronal protein tau in plasma.

Science.gov (United States)

Neselius, Sanna; Zetterberg, Henrik; Blennow, Kaj; Randall, Jeffrey; Wilson, David; Marcusson, Jan; Brisby, Helena

2013-01-01

The aim of this study was to investigate if olympic (amateur) boxing is associated with elevation of brain injury biomarkers in peripheral blood compared to controls. Thirty olympic boxers competing in at least 47 bouts were compared to 25 controls. Blood was collected from the controls at one occasion and from the boxers within 1-6 days after a bout and after a rest period of at least 14 days. Tau concentration in plasma was determined using a novel single molecule ELISA assay and S-100B, glial fibrillary acidic protein, brain-derived neurotrophic factor and amyloid β 1-42 were determined using standard immunoassays. None of the boxers had been knocked-out during the bout. Plasma-tau was significantly increased in the boxers after a bout compared to controls (mean ± SD, 2.46 ± 5.10 vs. 0.79 ± 0.961 ng L(-1), p = 0.038). The other brain injury markers did not differ between the groups. Plasma-tau decreased significantly in the boxers after a resting period compared to after a bout (p = 0.030). Olympic boxing is associated with elevation of tau in plasma. The repetitive minimal head injury in boxing may lead to axonal injuries that can be diagnosed with a blood test.

The SKP1-like gene family of Arabidopsis exhibits a high degree of differential gene expression and gene product interaction during development.

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Mohammad H Dezfulian

Full Text Available The Arabidopsis thaliana genome encodes several families of polypeptides that are known or predicted to participate in the formation of the SCF-class of E3-ubiquitin ligase complexes. One such gene family encodes the Skp1-like class of polypeptide subunits, where 21 genes have been identified and are known to be expressed in Arabidopsis. Phylogenetic analysis based on deduced polypeptide sequence organizes the family of ASK proteins into 7 clades. The complexity of the ASK gene family, together with the close structural similarity among its members raises the prospect of significant functional redundancy among select paralogs. We have assessed the potential for functional redundancy within the ASK gene family by analyzing an expanded set of criteria that define redundancy with higher resolution. The criteria used include quantitative expression of locus-specific transcripts using qRT-PCR, assessment of the sub-cellular localization of individual ASK:YFP auto-fluorescent fusion proteins expressed in vivo as well as the in planta assessment of individual ASK-F-Box protein interactions using bimolecular fluorescent complementation techniques in combination with confocal imagery in live cells. The results indicate significant functional divergence of steady state transcript abundance and protein-protein interaction specificity involving ASK proteins in a pattern that is poorly predicted by sequence-based phylogeny. The information emerging from this and related studies will prove important for defining the functional intersection of expression, localization and gene product interaction that better predicts the formation of discrete SCF complexes, as a prelude to investigating their molecular mode of action.

Protein-protein interactions in the regulation of WRKY transcription factors.

Science.gov (United States)

Chi, Yingjun; Yang, Yan; Zhou, Yuan; Zhou, Jie; Fan, Baofang; Yu, Jing-Quan; Chen, Zhixiang

2013-03-01

It has been almost 20 years since the first report of a WRKY transcription factor, SPF1, from sweet potato. Great progress has been made since then in establishing the diverse biological roles of WRKY transcription factors in plant growth, development, and responses to biotic and abiotic stress. Despite the functional diversity, almost all analyzed WRKY proteins recognize the TTGACC/T W-box sequences and, therefore, mechanisms other than mere recognition of the core W-box promoter elements are necessary to achieve the regulatory specificity of WRKY transcription factors. Research over the past several years has revealed that WRKY transcription factors physically interact with a wide range of proteins with roles in signaling, transcription, and chromatin remodeling. Studies of WRKY-interacting proteins have provided important insights into the regulation and mode of action of members of the important family of transcription factors. It has also emerged that the slightly varied WRKY domains and other protein motifs conserved within each of the seven WRKY subfamilies participate in protein-protein interactions and mediate complex functional interactions between WRKY proteins and between WRKY and other regulatory proteins in the modulation of important biological processes. In this review, we summarize studies of protein-protein interactions for WRKY transcription factors and discuss how the interacting partners contribute, at different levels, to the establishment of the complex regulatory and functional network of WRKY transcription factors.

E2F6: a member of the E2F family that does not modulate squamous differentiation

International Nuclear Information System (INIS)

Wong, C.F.; Barnes, Liam M.; Smith, Louise; Popa, Claudia; Serewko-Auret, Magdalena M.; Saunders, Nicholas A.

2004-01-01

The inhibition of E2F has been demonstrated to be important in the initiation of squamous differentiation by two independent manners: promotion of growth arrest and the relief of the differentiation-suppressive properties of E2Fs. E2F6 is reported to behave as a transcriptional repressor of the E2F family. In this study, we examined the ability of E2F6 to act as the molecular switch required for E2F inhibition in order for keratinocytes to enter a terminal differentiation programme. Results demonstrated that whilst E2F6 was able to suppress E2F activity in proliferating keratinocytes, it did not modulate squamous differentiation in a differentiated keratinocyte. Furthermore, inhibition of E2F, by overexpressing E2F6, was not sufficient to sensitise either proliferating keratinocytes or the squamous cell carcinoma cell line, KJD-1/SV40, to differentiation-inducing agents. Significantly, although E2F6 could suppress E2F activity in proliferating cells, it could not inhibit proliferation of KJD-1/SV40 cells. These results demonstrate that E2F6 does not contain the domains required for modulation of squamous differentiation and imply isoform-specific functions for individual E2F family members

The DEAD-box protein MEL-46 is required in the germ line of the nematode Caenorhabditis elegans.

Science.gov (United States)

Minasaki, Ryuji; Puoti, Alessandro; Streit, Adrian

2009-06-17

In the hermaphrodite of the nematode Caenorhabditis elegans, the first germ cells differentiate as sperm. Later the germ line switches to the production of oocytes. This process requires the activity of a genetic regulatory network that includes among others the fem, fog and mog genes. The function of some of these genes is germline specific while others also act in somatic tissues. DEAD box proteins have been shown to be involved in the control of gene expression at different steps such as transcription and pre-mRNA processing. We show that the Caenorhabditis elegans gene mel-46 (maternal effect lethal) encodes a DEAD box protein that is related to the mammalian DDX20/Gemin3/DP103 genes. mel-46 is expressed throughout development and mutations in mel-46 display defects at multiple developmental stages. Here we focus on the role of mel-46 in the hermaphrodite germ line. mel-46(yt5) mutant hermaphrodites are partially penetrant sterile and fully penetrant maternal effect lethal. The germ line of mutants shows variable defects in oogenesis. Further, mel-46(yt5) suppresses the complete feminization caused by mutations in fog-2 and fem-3, two genes that are at the top and the center, respectively, of the genetic germline sex determining cascade, but not fog-1 that is at the bottom of this cascade. The C. elegans gene mel-46 encodes a DEAD box protein that is required maternally for early embryogenesis and zygotically for postembryonic development. In the germ line, it is required for proper oogenesis. Although it interacts genetically with genes of the germline sex determination machinery its primary function appears to be in oocyte differentiation rather than sex determination.

E2F1 is crucial for E2F-dependent apoptosis

DEFF Research Database (Denmark)

Lazzerini Denchi, Eros; Helin, Kristian

2005-01-01

Loss of the retinoblastoma protein, pRB, leads to apoptosis, and several results have suggested that this is dependent on the E2F transcription factors. However, so far, the ability of the different E2F family members to contribute to apoptosis is controversial. Here, we show that ectopic...

Revisiting the description of Protein-Protein interfaces. Part II: Experimental study

OpenAIRE

Cazals , Frédéric; Proust , Flavien

2006-01-01

This paper provides a detailed experimental study of an interface model developed in the companion article F. Cazals and F. Proust, Revisiting the description of Protein-Protein interfaces. Part I: algorithms. Our experimental study is concerned with the usual database of protein-protein complexes, split into five families (Proteases, Immune system, Enzyme Complexes, Signal transduction, Misc.) Our findings, which bear some contradictions with usual statements are the following: (i)Connectivi...

TIF1alpha: a possible link between KRAB zinc finger proteins and nuclear receptors

DEFF Research Database (Denmark)

Le Douarin, B; You, J; Nielsen, Anders Lade

1998-01-01

Ligand-induced gene activation by nuclear receptors (NRs) is thought to be mediated by transcriptional intermediary factors (TIFs), that interact with their ligand-dependent AF-2 activating domain. Included in the group of the putative AF-2 TIFs identified so far is TIF1alpha, a member of a new...... family of proteins which contains an N-terminal RBCC (RING finger-B boxes-coiled coil) motif and a C-terminal bromodomain preceded by a PHD finger. In addition to these conserved domains present in a number of transcriptional regulatory proteins, TIF1alpha was found to contain several protein......-protein interaction sites. Of these, one specifically interacts with NRs bound to their agonistic ligand and not with NR mutants that are defective in the AF-2 activity. Immediately adjacent to this 'NR box', TIF1alpha contains an interaction site for members of the chromatin organization modifier (chromo) family, HP...

Nanobody-Directed Specific Degradation of Proteins by the 26S-Proteasome in Plants

OpenAIRE

Baudisch, Bianca; Pfort, Ingrid; Sorge, Eberhard; Conrad, Udo

2018-01-01

Here, we present data showing the directed degradation of target proteins recognized by a specific nanobody in transgenic plants. Green fluorescent protein was depleted by a chimeric nanobody fused to a distinct F-box domain, which enables protein degradation via the ubiquitin proteasome pathway. This technique could thus be used to knock out other proteins of interest in planta using specific, high-affinity binding proteins.

Nanobody-Directed Specific Degradation of Proteins by the 26S-Proteasome in Plants.

Science.gov (United States)

Baudisch, Bianca; Pfort, Ingrid; Sorge, Eberhard; Conrad, Udo

2018-01-01

Here, we present data showing the directed degradation of target proteins recognized by a specific nanobody in transgenic plants. Green fluorescent protein was depleted by a chimeric nanobody fused to a distinct F-box domain, which enables protein degradation via the ubiquitin proteasome pathway. This technique could thus be used to knock out other proteins of interest in planta using specific, high-affinity binding proteins.

Monoubiquitination of Tob/BTG family proteins competes with degradation-targeting polyubiquitination

Energy Technology Data Exchange (ETDEWEB)

Suzuki, Toru, E-mail: toru@ims.u-tokyo.ac.jp [Division of Oncology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Kim, Minsoo [Division of Bacterial Infection, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Kozuka-Hata, Hiroko [Medical Proteomics Laboratory, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Watanabe, Masato [Department of Medical Genome Science, School of Frontier Sciences, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa 277-8562 (Japan); Oyama, Masaaki [Medical Proteomics Laboratory, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Tsumoto, Kouhei [Medical Proteomics Laboratory, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Department of Medical Genome Science, School of Frontier Sciences, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa 277-8562 (Japan); Yamamoto, Tadashi, E-mail: tyamamot@ims.u-tokyo.ac.jp [Division of Oncology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Cell Signal Unit, Okinawa Institute of Science and Technology, 1919-1 Onna-son, Kunigami, Okinawa 904-0412 (Japan)

2011-05-27

Highlights: {yields} Tob/BTG family proteins are monoubiquitinated in the absence of E3s in vitro. {yields} Monoubiquitination sites of Tob are identified by mass spectrometry. {yields} The monoubiquitination event correlates with lower levels of polyubiquitination. -- Abstract: Tob belongs to the anti-proliferative Tob/BTG protein family. The expression level of Tob family proteins is strictly regulated both transcriptionally and through post-translational modification. Ubiquitin (Ub)/proteosome-dependent degradation of Tob family proteins is critical in controlling cell cycle progression and DNA damage responses. Various Ub ligases (E3s) are responsible for degradation of Tob protein. Here, we show that Tob family proteins undergo monoubiquitination even in the absence of E3s in vitro. Determination of the ubiquitination site(s) in Tob by mass spectrometric analysis revealed that two lysine residues (Lys48 and Lys63) located in Tob/BTG homology domain are ubiquitinated. A mutant Tob, in which both Lys48 and Lys63 are substituted with alanine, is more strongly polyubiquitinated than wild-type Tob in vivo. These data suggest that monoubiquitination of Tob family proteins confers resistance against polyubiquitination, which targets proteins for degradation. The strategy for regulating the stability of Tob family proteins suggests a novel role for monoubiquitination.

Monoubiquitination of Tob/BTG family proteins competes with degradation-targeting polyubiquitination

International Nuclear Information System (INIS)

Suzuki, Toru; Kim, Minsoo; Kozuka-Hata, Hiroko; Watanabe, Masato; Oyama, Masaaki; Tsumoto, Kouhei; Yamamoto, Tadashi

2011-01-01

Highlights: → Tob/BTG family proteins are monoubiquitinated in the absence of E3s in vitro. → Monoubiquitination sites of Tob are identified by mass spectrometry. → The monoubiquitination event correlates with lower levels of polyubiquitination. -- Abstract: Tob belongs to the anti-proliferative Tob/BTG protein family. The expression level of Tob family proteins is strictly regulated both transcriptionally and through post-translational modification. Ubiquitin (Ub)/proteosome-dependent degradation of Tob family proteins is critical in controlling cell cycle progression and DNA damage responses. Various Ub ligases (E3s) are responsible for degradation of Tob protein. Here, we show that Tob family proteins undergo monoubiquitination even in the absence of E3s in vitro. Determination of the ubiquitination site(s) in Tob by mass spectrometric analysis revealed that two lysine residues (Lys48 and Lys63) located in Tob/BTG homology domain are ubiquitinated. A mutant Tob, in which both Lys48 and Lys63 are substituted with alanine, is more strongly polyubiquitinated than wild-type Tob in vivo. These data suggest that monoubiquitination of Tob family proteins confers resistance against polyubiquitination, which targets proteins for degradation. The strategy for regulating the stability of Tob family proteins suggests a novel role for monoubiquitination.

Phylogenetic distribution of plant snoRNA families.

Science.gov (United States)

Patra Bhattacharya, Deblina; Canzler, Sebastian; Kehr, Stephanie; Hertel, Jana; Grosse, Ivo; Stadler, Peter F

2016-11-24

Small nucleolar RNAs (snoRNAs) are one of the most ancient families amongst non-protein-coding RNAs. They are ubiquitous in Archaea and Eukarya but absent in bacteria. Their main function is to target chemical modifications of ribosomal RNAs. They fall into two classes, box C/D snoRNAs and box H/ACA snoRNAs, which are clearly distinguished by conserved sequence motifs and the type of chemical modification that they govern. Similarly to microRNAs, snoRNAs appear in distinct families of homologs that affect homologous targets. In animals, snoRNAs and their evolution have been studied in much detail. In plants, however, their evolution has attracted comparably little attention. In order to chart the phylogenetic distribution of individual snoRNA families in plants, we applied a sophisticated approach for identifying homologs of known plant snoRNAs across the plant kingdom. In response to the relatively fast evolution of snoRNAs, information on conserved sequence boxes, target sequences, and secondary structure is combined to identify additional snoRNAs. We identified 296 families of snoRNAs in 24 species and traced their evolution throughout the plant kingdom. Many of the plant snoRNA families comprise paralogs. We also found that targets are well-conserved for most snoRNA families. The sequence conservation of snoRNAs is sufficient to establish homologies between phyla. The degree of this conservation tapers off, however, between land plants and algae. Plant snoRNAs are frequently organized in highly conserved spatial clusters. As a resource for further investigations we provide carefully curated and annotated alignments for each snoRNA family under investigation.

Chips in black boxes? Convenience life span, parafood, brandwidth, families, and co-creation.

Science.gov (United States)

Jacobs, Marc

2015-11-01

Any consumer who opens a bag of potato or corn chips (or crisps in the UK) knows there is no time to waste to enjoy or share them. The convenience life span of chips is limited: it is the shelf or storage life and a very limited time once outside the bag. Many technologies converge to generate the desired effect as a black box, not only of the packaging but also of the chips themselves. The concept of paratext can be applied to printed messages on the package, including the brand name and other texts like advertising (epitexts), which can be expanded into the concept of parafood. These concepts help to discuss technological developments and interpret why this has recently become a negotiation zone for co-creation (see the Do us a flavor campaigns). They are symptoms of changing relations between production, research and development, marketing, and consumption. This paper pays special attention to back stories, underdog brand biographies and narratives about origin. The concept of brandwidth is introduced to sensitize about the limits of combining different stories about chips. A recent brand biography, a family history and a cookery book are used to discuss the phenomenon of cooking with Fritos. Together with the concepts of parafood, brandwidth and black boxes, more reflection and dialogue about the role of history and heritage in marketing put new challenging perspectives on the agenda. Copyright © 2015 Elsevier Ltd. All rights reserved.

Isolation of three B-box zinc finger proteins that interact with STF1 and COP1 defines a HY5/COP1 interaction network involved in light control of development in soybean

International Nuclear Information System (INIS)

Shin, Su Young; Kim, Seong Hee; Kim, Hye Jin; Jeon, Su Jeong; Sim, Soon Ae; Ryu, Gyeong Ryul; Yoo, Cheol Min; Cheong, Yong Hwa; Hong, Jong Chan

2016-01-01

LONG HYPOCOTYL5 (HY5) and STF1 (Soybean TGACG-motif binding Factor 1) are two related bZIP transcription factors that play a positive role in photomorphogenesis and hormonal signaling. In this study, we compared full length STF1 and truncated STF1 overexpression lines and found that the C-terminal 133 amino acids (194–306) possess all the HY5-like function in Arabidopsis. The STF1-DC1 mutant (1–306), with a 20 amino acid deletion at the carboxy terminus, failed to complement the hy5 mutant phenotype, which suggests an intact C-terminus is required for STF1 function. To understand the role of the C-terminal domain in photomorphogenesis we used a yeast two-hybrid screen to isolate proteins that bind to the STF1 C-terminus. We isolated three soybean cDNAs encoding the zinc-finger proteins GmSTO, GmSTH, and GmSTH2, which interact with STF1. These proteins belong to a family of B-box zinc finger proteins that include Arabidopsis SALT TOLERANCE (STO) and STO HOMOLOG (STH) and STH2, which play a role in light-dependent development and gene expression. The C-terminal 63 amino acids of STF1, containing a leucine zipper and the two N-terminal B-boxes, contains the domain involved in interactions between STF1 and GmSTO. In addition, we identified an interaction between soybean COP1 (GmCOP1) and GmSTO and GmSTH, as well as STF1, which strongly suggests the presence of a similar regulatory circuit for light signaling in soybean as in Arabidopsis. This study shows that photomorphogenic control requires complex molecular interactions among several different classes of transcription factors such as bZIP, B-box factors, and COP1, a ubiquitin ligase. - Highlights: • STF1 interact with GmSTO, GmSTH and GmSTH2. • The bZIP transcription factor STF1 requires an intact C-terminal domain for STF1 function. • STF1 and GmSTO are nuclear proteins.

Functional analysis of the cell cycle regulator Rca1 in Drosophila melanogaster

OpenAIRE

Zielke, Norman

2007-01-01

Tight regulation of APC/C activity is essential for cell cycle progression. An important class of negative APC/C regulators are the Rca1/Emi1 family proteins. All members of the Rca1/Emi1 family share a conserved zinc binding region (ZBR) which is essential for their inhibitory activity. The Rca1/Emi1 proteins belong to the class of F-box proteins that are known to act as substrate recognition subunits in SCF-E3-ligase complexes. Emi1 and Rca1 interact in vitro with members of the Skp family ...

C-terminal motif prediction in eukaryotic proteomes using comparative genomics and statistical over-representation across protein families

Directory of Open Access Journals (Sweden)

Cutler Sean R

2007-06-01

Full Text Available Abstract Background The carboxy termini of proteins are a frequent site of activity for a variety of biologically important functions, ranging from post-translational modification to protein targeting. Several short peptide motifs involved in protein sorting roles and dependent upon their proximity to the C-terminus for proper function have already been characterized. As a limited number of such motifs have been identified, the potential exists for genome-wide statistical analysis and comparative genomics to reveal novel peptide signatures functioning in a C-terminal dependent manner. We have applied a novel methodology to the prediction of C-terminal-anchored peptide motifs involving a simple z-statistic and several techniques for improving the signal-to-noise ratio. Results We examined the statistical over-representation of position-specific C-terminal tripeptides in 7 eukaryotic proteomes. Sequence randomization models and simple-sequence masking were applied to the successful reduction of background noise. Similarly, as C-terminal homology among members of large protein families may artificially inflate tripeptide counts in an irrelevant and obfuscating manner, gene-family clustering was performed prior to the analysis in order to assess tripeptide over-representation across protein families as opposed to across all proteins. Finally, comparative genomics was used to identify tripeptides significantly occurring in multiple species. This approach has been able to predict, to our knowledge, all C-terminally anchored targeting motifs present in the literature. These include the PTS1 peroxisomal targeting signal (SKL*, the ER-retention signal (K/HDEL*, the ER-retrieval signal for membrane bound proteins (KKxx*, the prenylation signal (CC* and the CaaX box prenylation motif. In addition to a high statistical over-representation of these known motifs, a collection of significant tripeptides with a high propensity for biological function exists

Identification of a Cullin5-ElonginB-ElonginC E3 complex in degradation of feline immunodeficiency virus Vif-mediated feline APOBEC3 proteins.

Science.gov (United States)

Wang, Jiawen; Zhang, Wenyan; Lv, Mingyu; Zuo, Tao; Kong, Wei; Yu, Xianghui

2011-12-01

Various feline APOBEC3 (fA3) proteins exhibit broad antiviral activities against a wide range of viruses, such as feline immunodeficiency virus (FIV), feline foamy virus (FFV), and feline leukemia virus (FeLV), as well as those of other species. This activity can be counteracted by the FIV Vif protein, but the mechanism by which FIV Vif suppresses fA3s is unknown. In the present study, we demonstrated that FIV Vif could act via a proteasome-dependent pathway to overcome fA3s. FIV Vif interacted with feline cellular proteins Cullin5 (Cul5), ElonginB, and ElonginC to form an E3 complex to induce degradation of fA3s. Both the dominant-negative Cul5 mutant and a C-terminal hydrophilic replacement ElonginC mutant potently disrupted the FIV Vif activity against fA3s. Furthermore, we identified a BC-box motif in FIV Vif that was essential for the recruitment of E3 ubiquitin ligase and also required for FIV Vif-mediated degradation of fA3s. Moreover, despite the lack of either a Cul5-box or a HCCH zinc-binding motif, FIV Vif specifically selected Cul5. Therefore, FIV Vif may interact with Cul5 via a novel mechanism. These finding imply that SOCS proteins may possess distinct mechanisms to bind Cul5 during formation of the Elongin-Cullin-SOCS box complex.

The PANTHER database of protein families, subfamilies, functions and pathways

OpenAIRE

Mi, Huaiyu; Lazareva-Ulitsky, Betty; Loo, Rozina; Kejariwal, Anish; Vandergriff, Jody; Rabkin, Steven; Guo, Nan; Muruganujan, Anushya; Doremieux, Olivier; Campbell, Michael J.; Kitano, Hiroaki; Thomas, Paul D.

2004-01-01

PANTHER is a large collection of protein families that have been subdivided into functionally related subfamilies, using human expertise. These subfamilies model the divergence of specific functions within protein families, allowing more accurate association with function (ontology terms and pathways), as well as inference of amino acids important for functional specificity. Hidden Markov models (HMMs) are built for each family and subfamily for classifying additional protein sequences. The l...

In silico modeling of the yeast protein and protein family interaction network

Science.gov (United States)

Goh, K.-I.; Kahng, B.; Kim, D.

2004-03-01

Understanding of how protein interaction networks of living organisms have evolved or are organized can be the first stepping stone in unveiling how life works on a fundamental ground. Here we introduce an in silico ``coevolutionary'' model for the protein interaction network and the protein family network. The essential ingredient of the model includes the protein family identity and its robustness under evolution, as well as the three previously proposed: gene duplication, divergence, and mutation. This model produces a prototypical feature of complex networks in a wide range of parameter space, following the generalized Pareto distribution in connectivity. Moreover, we investigate other structural properties of our model in detail with some specific values of parameters relevant to the yeast Saccharomyces cerevisiae, showing excellent agreement with the empirical data. Our model indicates that the physical constraints encoded via the domain structure of proteins play a crucial role in protein interactions.

Nanobody-Directed Specific Degradation of Proteins by the 26S-Proteasome in Plants

Directory of Open Access Journals (Sweden)

Bianca Baudisch

2018-02-01

Full Text Available Here, we present data showing the directed degradation of target proteins recognized by a specific nanobody in transgenic plants. Green fluorescent protein was depleted by a chimeric nanobody fused to a distinct F-box domain, which enables protein degradation via the ubiquitin proteasome pathway. This technique could thus be used to knock out other proteins of interest in planta using specific, high-affinity binding proteins.

Genome-wide analysis of the MADS-box gene family in polyploid cotton (Gossypium hirsutum) and in its diploid parental species (Gossypium arboreum and Gossypium raimondii).

Science.gov (United States)

Nardeli, Sarah Muniz; Artico, Sinara; Aoyagi, Gustavo Mitsunori; de Moura, Stéfanie Menezes; da Franca Silva, Tatiane; Grossi-de-Sa, Maria Fatima; Romanel, Elisson; Alves-Ferreira, Marcio

2018-06-01

The MADS-box gene family encodes transcription factors that share a highly conserved domain known to bind to DNA. Members of this family control various processes of development in plants, from root formation to fruit ripening. In this work, a survey of diploid (Gossypium raimondii and Gossypium arboreum) and tetraploid (Gossypium hirsutum) cotton genomes found a total of 147, 133 and 207 MADS-box genes, respectively, distributed in the MIKC, Mα, Mβ, Mγ, and Mδ subclades. A comparative phylogenetic analysis among cotton species, Arabidopsis, poplar and grapevine MADS-box homologous genes allowed us to evaluate the evolution of each MADS-box lineage in cotton plants and identify sequences within well-established subfamilies. Chromosomal localization and phylogenetic analysis revealed that G. raimondii and G. arboreum showed a conserved evolution of the MIKC subclade and a distinct pattern of duplication events in the Mα, Mγ and Mδ subclades. Additionally, G. hirsutum showed a combination of its parental subgenomes followed by a distinct evolutionary history including gene gain and loss in each subclade. qPCR analysis revealed the expression patterns of putative homologs in the AP1, AP3, AGL6, SEP4, AGL15, AG, AGL17, TM8, SVP, SOC and TT16 subfamilies of G. hirsutum. The identification of putative cotton orthologs is discussed in the light of evolution and gene expression data from other plants. This analysis of the MADS-box genes in Gossypium species opens an avenue to understanding the origin and evolution of each gene subfamily within diploid and polyploid species and paves the way for functional studies in cotton species. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Investigation of the milling capabilities of the F10 Fine Grind mill using Box-Behnken designs.

Science.gov (United States)

Tan, Bernice Mei Jin; Tay, Justin Yong Soon; Wong, Poh Mun; Chan, Lai Wah; Heng, Paul Wan Sia

2015-01-01

Size reduction or milling of the active is often the first processing step in the design of a dosage form. The ability of a mill to convert coarse crystals into the target size and size distribution efficiently is highly desirable as the quality of the final pharmaceutical product after processing is often still dependent on the dimensional attributes of its component constituents. The F10 Fine Grind mill is a mechanical impact mill designed to produce unimodal mid-size particles by utilizing a single-pass two-stage size reduction process for fine grinding of raw materials needed in secondary processing. Box-Behnken designs were used to investigate the effects of various mill variables (impeller, blower and feeder speeds and screen aperture size) on the milling of coarse crystals. Response variables included the particle size parameters (D10, D50 and D90), span and milling rate. Milled particles in the size range of 5-200 μm, with D50 ranging from 15 to 60 μm, were produced. The impeller and feeder speeds were the most critical factors influencing the particle size and milling rate, respectively. Size distributions of milled particles were better described by their goodness-of-fit to a log-normal distribution (i.e. unimodality) rather than span. Milled particles with symmetrical unimodal distributions were obtained when the screen aperture size was close to the median diameter of coarse particles employed. The capacity for high throughput milling of particles to a mid-size range, which is intermediate between conventional mechanical impact mills and air jet mills, was demonstrated in the F10 mill. Prediction models from the Box-Behnken designs will aid in providing a better guide to the milling process and milled product characteristics. Copyright © 2014 Elsevier B.V. All rights reserved.

Elevated temperature triggers human respiratory syncytial virus F protein six-helix bundle formation

International Nuclear Information System (INIS)

Yunus, Abdul S.; Jackson, Trent P.; Crisafi, Katherine; Burimski, Irina; Kilgore, Nicole R.; Zoumplis, Dorian; Allaway, Graham P.; Wild, Carl T.; Salzwedel, Karl

2010-01-01

Human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infection in infants, immunocompromised patients, and the elderly. The RSV fusion (F) protein mediates fusion of the viral envelope with the target cell membrane during virus entry and is a primary target for antiviral drug and vaccine development. The F protein contains two heptad repeat regions, HR1 and HR2. Peptides corresponding to these regions form a six-helix bundle structure that is thought to play a critical role in membrane fusion. However, characterization of six-helix bundle formation in native RSV F protein has been hindered by the fact that a trigger for F protein conformational change has yet to be identified. Here we demonstrate that RSV F protein on the surface of infected cells undergoes a conformational change following exposure to elevated temperature, resulting in the formation of the six-helix bundle structure. We first generated and characterized six-helix bundle-specific antibodies raised against recombinant peptides modeling the RSV F protein six-helix bundle structure. We then used these antibodies as probes to monitor RSV F protein six-helix bundle formation in response to a diverse array of potential triggers of conformational changes. We found that exposure of 'membrane-anchored' RSV F protein to elevated temperature (45-55 deg. C) was sufficient to trigger six-helix bundle formation. Antibody binding to the six-helix bundle conformation was detected by both flow cytometry and cell-surface immunoprecipitation of the RSV F protein. None of the other treatments, including interaction with a number of potential receptors, resulted in significant binding by six-helix bundle-specific antibodies. We conclude that native, untriggered RSV F protein exists in a metastable state that can be converted in vitro to the more stable, fusogenic six-helix bundle conformation by an increase in thermal energy. These findings help to better define the mechanism of

Two high-mobility group box domains act together to underwind and kink DNA

Energy Technology Data Exchange (ETDEWEB)

Sánchez-Giraldo, R.; Acosta-Reyes, F. J. [Universitat Politecnica de Catalunya, 08028 Barcelona (Spain); Malarkey, C. S. [University of Colorado School of Medicine, Aurora, CO 80045 (United States); Saperas, N. [Universitat Politecnica de Catalunya, 08028 Barcelona (Spain); Churchill, M. E. A., E-mail: mair.churchill@ucdenver.edu [University of Colorado School of Medicine, Aurora, CO 80045 (United States); Campos, J. L., E-mail: mair.churchill@ucdenver.edu [Universitat Politecnica de Catalunya, 08028 Barcelona (Spain)

2015-06-30

The crystal structure of HMGB1 box A bound to an unmodified AT-rich DNA fragment is reported at a resolution of 2 Å. A new mode of DNA recognition for HMG box proteins is found in which two box A domains bind in an unusual configuration generating a highly kinked DNA structure. High-mobility group protein 1 (HMGB1) is an essential and ubiquitous DNA architectural factor that influences a myriad of cellular processes. HMGB1 contains two DNA-binding domains, box A and box B, which have little sequence specificity but have remarkable abilities to underwind and bend DNA. Although HMGB1 box A is thought to be responsible for the majority of HMGB1–DNA interactions with pre-bent or kinked DNA, little is known about how it recognizes unmodified DNA. Here, the crystal structure of HMGB1 box A bound to an AT-rich DNA fragment is reported at a resolution of 2 Å. Two box A domains of HMGB1 collaborate in an unusual configuration in which the Phe37 residues of both domains stack together and intercalate the same CG base pair, generating highly kinked DNA. This represents a novel mode of DNA recognition for HMGB proteins and reveals a mechanism by which structure-specific HMG boxes kink linear DNA.

Two high-mobility group box domains act together to underwind and kink DNA

International Nuclear Information System (INIS)

Sánchez-Giraldo, R.; Acosta-Reyes, F. J.; Malarkey, C. S.; Saperas, N.; Churchill, M. E. A.; Campos, J. L.

2015-01-01

The crystal structure of HMGB1 box A bound to an unmodified AT-rich DNA fragment is reported at a resolution of 2 Å. A new mode of DNA recognition for HMG box proteins is found in which two box A domains bind in an unusual configuration generating a highly kinked DNA structure. High-mobility group protein 1 (HMGB1) is an essential and ubiquitous DNA architectural factor that influences a myriad of cellular processes. HMGB1 contains two DNA-binding domains, box A and box B, which have little sequence specificity but have remarkable abilities to underwind and bend DNA. Although HMGB1 box A is thought to be responsible for the majority of HMGB1–DNA interactions with pre-bent or kinked DNA, little is known about how it recognizes unmodified DNA. Here, the crystal structure of HMGB1 box A bound to an AT-rich DNA fragment is reported at a resolution of 2 Å. Two box A domains of HMGB1 collaborate in an unusual configuration in which the Phe37 residues of both domains stack together and intercalate the same CG base pair, generating highly kinked DNA. This represents a novel mode of DNA recognition for HMGB proteins and reveals a mechanism by which structure-specific HMG boxes kink linear DNA

INK4 proteins, a family of mammalian CDK inhibitors with novel biological functions.

Science.gov (United States)

Cánepa, Eduardo T; Scassa, María E; Ceruti, Julieta M; Marazita, Mariela C; Carcagno, Abel L; Sirkin, Pablo F; Ogara, María F

2007-07-01

The cyclin D-Cdk4-6/INK4/Rb/E2F pathway plays a key role in controlling cell growth by integrating multiple mitogenic and antimitogenic stimuli. The members of INK4 family, comprising p16(INK4a), p15(INK4b), p18(INK4c), and p19(INK4d), block the progression of the cell cycle by binding to either Cdk4 or Cdk6 and inhibiting the action of cyclin D. These INK4 proteins share a similar structure dominated by several ankyrin repeats. Although they appear to be structurally redundant and equally potent as inhibitors, the INK4 family members are differentially expressed during mouse development. The striking diversity in the pattern of expression of INK4 genes suggested that this family of cell cycle inhibitors might have cell lineage-specific or tissue-specific functions. The INK4 proteins are commonly lost or inactivated by mutations in diverse types of cancer, and they represent established or candidate tumor suppressors. Apart from their capacity to arrest cells in the G1-phase of the cell cycle they have been shown to participate in an increasing number of cellular processes. Given their emerging roles in fundamental physiological as well as pathological processes, it is interesting to explore the diverse roles for the individual INK4 family members in different functions other than cell cycle regulation. Extensive studies, over the past few years, uncover the involvement of INK4 proteins in senescence, apoptosis, DNA repair, and multistep oncogenesis. We will focus the discussion here on these unexpected issues.

Identification and analysis of YELLOW protein family genes in the silkworm, Bombyx mori

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Yi Yong-Zhu

2006-08-01

Full Text Available Abstract Background The major royal jelly proteins/yellow (MRJP/YELLOW family possesses several physiological and chemical functions in the development of Apis mellifera and Drosophila melanogaster. Each protein of the family has a conserved domain named MRJP. However, there is no report of MRJP/YELLOW family proteins in the Lepidoptera. Results Using the YELLOW protein sequence in Drosophila melanogaster to BLAST silkworm EST database, we found a gene family composed of seven members with a conserved MRJP domain each and named it YELLOW protein family of Bombyx mori. We completed the cDNA sequences with RACE method. The protein of each member possesses a MRJP domain and a putative cleavable signal peptide consisting of a hydrophobic sequence. In view of genetic evolution, the whole Bm YELLOW protein family composes a monophyletic group, which is distinctly separate from Drosophila melanogaster and Apis mellifera. We then showed the tissue expression profiles of Bm YELLOW protein family genes by RT-PCR. Conclusion A Bombyx mori YELLOW protein family is found to be composed of at least seven members. The low homogeneity and unique pattern of gene expression by each member among the family ensure us to prophesy that the members of Bm YELLOW protein family would play some important physiological functions in silkworm development.

Structural and functional studies of a family of Dictyostelium discoideum developmentally regulated, prestalk genes coding for small proteins

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Escalante Ricardo

2008-01-01

Full Text Available Abstract Background The social amoeba Dictyostelium discoideum executes a multicellular development program upon starvation. This morphogenetic process requires the differential regulation of a large number of genes and is coordinated by extracellular signals. The MADS-box transcription factor SrfA is required for several stages of development, including slug migration and spore terminal differentiation. Results Subtractive hybridization allowed the isolation of a gene, sigN (SrfA-induced gene N, that was dependent on the transcription factor SrfA for expression at the slug stage of development. Homology searches detected the existence of a large family of sigN-related genes in the Dictyostelium discoideum genome. The 13 most similar genes are grouped in two regions of chromosome 2 and have been named Group1 and Group2 sigN genes. The putative encoded proteins are 87–89 amino acids long. All these genes have a similar structure, composed of a first exon containing a 13 nucleotides long open reading frame and a second exon comprising the remaining of the putative coding region. The expression of these genes is induced at10 hours of development. Analyses of their promoter regions indicate that these genes are expressed in the prestalk region of developing structures. The addition of antibodies raised against SigN Group 2 proteins induced disintegration of multi-cellular structures at the mound stage of development. Conclusion A large family of genes coding for small proteins has been identified in D. discoideum. Two groups of very similar genes from this family have been shown to be specifically expressed in prestalk cells during development. Functional studies using antibodies raised against Group 2 SigN proteins indicate that these genes could play a role during multicellular development.

The SGS3 protein involved in PTGS finds a family

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Bateman Alex

2002-08-01

Full Text Available Abstract Background Post transcriptional gene silencing (PTGS is a recently discovered phenomenon that is an area of intense research interest. Components of the PTGS machinery are being discovered by genetic and bioinformatics approaches, but the picture is not yet complete. Results The gene for the PTGS impaired Arabidopsis mutant sgs3 was recently cloned and was not found to have similarity to any other known protein. By a detailed analysis of the sequence of SGS3 we have defined three new protein domains: the XH domain, the XS domain and the zf-XS domain, that are shared with a large family of uncharacterised plant proteins. This work implicates these plant proteins in PTGS. Conclusion The enigmatic SGS3 protein has been found to contain two predicted domains in common with a family of plant proteins. The other members of this family have been predicted to be transcription factors, however this function seems unlikely based on this analysis. A bioinformatics approach has implicated a new family of plant proteins related to SGS3 as potential candidates for PTGS related functions.

Interactions between globular proteins and F-actin in isotonic saline solution.

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Lakatos, S; Minton, A P

1991-10-05

Solutions of each of three different globular proteins (cytochrome c, chromophorically labeled serum albumin, and chromophorically labeled aldolase), mixed with another unlabeled globular protein or with fibrous actin, were prepared in pH 8.0 Tris-HCl buffer containing 0.15 M NaCl. Each solution was centrifuged at low speed, at 5 degrees C, until unassociated globular protein in solution achieved sedimentation equilibrium. Individual absorbance gradients of both macrosolutes in the mixtures subsequent to centrifugation were obtained via optical scans of the centrifuge tubes at two wavelengths. The gradients of each macrosolute in mixtures of two globular proteins revealed no association of globular proteins under the conditions of these experiments, but perturbation of the gradients of serum albumin, aldolase, and cytochrome c in the presence of F-actin indicated association of all three globular proteins with F-actin. Perturbation of actin gradients in the presence of serum albumin and aldolase suggested partial depolymerization of the F-actin by the globular protein. Analysis of the data with a simple phenomenological model relating free globular protein, bound globular protein, and total actin concentration provided estimates of the respective equilibrium constants for association of serum albumin and aldolase with F-actin, under the conditions of these experiments, of the order of 0.1 microM-1.

Cell differentiation by interaction of two HMG-box proteins: Mat1-Mc activates M cell-specific genes in S.pombe by recruiting the ubiquitous transcription factor Ste11 to weak binding sites

DEFF Research Database (Denmark)

Kjaerulff, S; Dooijes, D; Clevers, H

1997-01-01

The Schizosaccharomyces pombe mfm1 gene is expressed in an M cell-specific fashion. This regulation requires two HMG-box proteins: the ubiquitous Ste11 transcription factor and the M cell-controlling protein Mat1-Mc. Here we report that the mfm1 promoter contains a single, weak Stell-binding site...... where we could not detect Mat1-Mc in the resulting protein-DNA complex. When we changed a single base in the mfm1 TR-box, such that it resembled those boxes found in ubiquitously expressed genes, Ste11 binding was enhanced, and in vivo the mfm1 gene also became expressed in P cells where Mat1-Mc...

PIP degron proteins, substrates of CRL4Cdt2, and not PIP boxes, interfere with DNA polymerase η and κ focus formation on UV damage.

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Tsanov, Nikolay; Kermi, Chames; Coulombe, Philippe; Van der Laan, Siem; Hodroj, Dana; Maiorano, Domenico

2014-04-01

Proliferating cell nuclear antigen (PCNA) is a well-known scaffold for many DNA replication and repair proteins, but how the switch between partners is regulated is currently unclear. Interaction with PCNA occurs via a domain known as a PCNA-Interacting Protein motif (PIP box). More recently, an additional specialized PIP box has been described, the « PIP degron », that targets PCNA-interacting proteins for proteasomal degradation via the E3 ubiquitin ligase CRL4(Cdt2). Here we provide evidence that CRL4(Cdt2)-dependent degradation of PIP degron proteins plays a role in the switch of PCNA partners during the DNA damage response by facilitating accumulation of translesion synthesis DNA polymerases into nuclear foci. We show that expression of a nondegradable PIP degron (Cdt1) impairs both Pol η and Pol κ focus formation on ultraviolet irradiation and reduces cell viability, while canonical PIP box-containing proteins have no effect. Furthermore, we identify PIP degron-containing peptides from several substrates of CRL4(Cdt2) as efficient inhibitors of Pol η foci formation. By site-directed mutagenesis we show that inhibition depends on a conserved threonine residue that confers high affinity for PCNA-binding. Altogether these findings reveal an important regulative role for the CRL4(Cdt2) pathway in the switch of PCNA partners on DNA damage.

Receptor-interacting protein (RIP) kinase family

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Zhang, Duanwu; Lin, Juan; Han, Jiahuai

2010-01-01

Receptor-interacting protein (RIP) kinases are a group of threonine/serine protein kinases with a relatively conserved kinase domain but distinct non-kinase regions. A number of different domain structures, such as death and caspase activation and recruitment domain (CARD) domains, were found in different RIP family members, and these domains should be keys in determining the specific function of each RIP kinase. It is known that RIP kinases participate in different biological processes, including those in innate immunity, but their downstream substrates are largely unknown. This review will give an overview of the structures and functions of RIP family members, and an update of recent progress in RIP kinase research. PMID:20383176

E2F/Rb Family Proteins Mediate Interferon Induced Repression of Adenovirus Immediate Early Transcription to Promote Persistent Viral Infection.

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Yueting Zheng

2016-01-01

Full Text Available Interferons (IFNs are cytokines that have pleiotropic effects and play important roles in innate and adaptive immunity. IFNs have broad antiviral properties and function by different mechanisms. IFNs fail to inhibit wild-type Adenovirus (Ad replication in established cancer cell lines. In this study, we analyzed the effects of IFNs on Ad replication in normal human cells. Our data demonstrate that both IFNα and IFNγ blocked wild-type Ad5 replication in primary human bronchial epithelial cells (NHBEC and TERT-immortalized normal human diploid fibroblasts (HDF-TERT. IFNs inhibited the replication of divergent adenoviruses. The inhibition of Ad5 replication by IFNα and IFNγ is the consequence of repression of transcription of the E1A immediate early gene product. Both IFNα and IFNγ impede the association of the transactivator GABP with the E1A enhancer region during the early phase of infection. The repression of E1A expression by IFNs requires a conserved E2F binding site in the E1A enhancer, and IFNs increased the enrichment of the E2F-associated pocket proteins, Rb and p107, at the E1A enhancer in vivo. PD0332991 (Pabociclib, a specific CDK4/6 inhibitor, dephosphoryles pocket proteins to promote their interaction with E2Fs and inhibited wild-type Ad5 replication dependent on the conserved E2F binding site. Consistent with this result, expression of the small E1A oncoprotein, which abrogates E2F/pocket protein interactions, rescued Ad replication in the presence of IFNα or IFNγ. Finally, we established a persistent Ad infection model in vitro and demonstrated that IFNγ suppresses productive Ad replication in a manner dependent on the E2F binding site in the E1A enhancer. This is the first study that probes the molecular basis of persistent adenovirus infection and reveals a novel mechanism by which adenoviruses utilize IFN signaling to suppress lytic virus replication and to promote persistent infection.

Y-box protein-1/p18 fragment identifies malignancies in patients with chronic liver disease

International Nuclear Information System (INIS)

Tacke, Frank; Kanig, Nicolas; En-Nia, Abdelaziz; Kaehne, Thilo; Eberhardt, Christiane S; Shpacovitch, Victoria; Trautwein, Christian; Mertens, Peter R

2011-01-01

Immunohistochemical detection of cold shock proteins is predictive for deleterious outcome in various malignant diseases. We recently described active secretion of a family member, denoted Y-box (YB) protein-1. We tested the clinical and diagnostic value of YB-1 protein fragment p18 (YB-1/p18) detection in blood for malignant diseases. We used a novel monoclonal anti-YB-1 antibody to detect YB-1/p18 by immunoblotting in plasma samples of healthy volunteers (n = 33), patients with non-cancerous, mostly inflammatory diseases (n = 60), hepatocellular carcinoma (HCC; n = 25) and advanced solid tumors (n = 20). YB-1/p18 was then tested in 111 patients with chronic liver diseases, alongside established tumor markers and various diagnostic measures, during evaluation for potential liver transplantation. We developed a novel immunoblot to detect the 18 kD fragment of secreted YB-1 in human plasma (YB-1/p18) that contains the cold-shock domains (CSD) 1-3 of the full-length protein. YB-1/p18 was detected in 11/25 HCC and 16/20 advanced carcinomas compared to 0/33 healthy volunteers and 10/60 patients with non-cancerous diseases. In 111 patients with chronic liver disease, YB-1/p18 was detected in 20 samples. Its occurrence was not associated with advanced Child stages of liver cirrhosis or liver function. In this cohort, YB-1/p18 was not a good marker for HCC, but proved most powerful in detecting malignancies other than HCC (60% positive) with a lower rate of false-positive results compared to established tumor markers. Alpha-fetoprotein (AFP) was most sensitive in detecting HCC, but simultaneous assessment of AFP, CA19-9 and YB-1/p18 improved overall identification of HCC patients. Plasma YB-1/p18 can identify patients with malignancies, independent of acute inflammation, renal impairment or liver dysfunction. The detection of YB-1/p18 in human plasma may have potential as a tumor marker for screening of high-risk populations, e.g. before organ transplantation, and should

Sub-grouping and sub-functionalization of the RIFIN multi-copy protein family

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Sonnhammer Erik L

2008-01-01

Full Text Available Abstract Background Parasitic protozoans possess many multicopy gene families which have central roles in parasite survival and virulence. The number and variability of members of these gene families often make it difficult to predict possible functions of the encoded proteins. The families of extra-cellular proteins that are exposed to a host immune response have been driven via immune selection to become antigenically variant, and thereby avoid immune recognition while maintaining protein function to establish a chronic infection. Results We have combined phylogenetic and function shift analyses to study the evolution of the RIFIN proteins, which are antigenically variant and are encoded by the largest multicopy gene family in Plasmodium falciparum. We show that this family can be subdivided into two major groups that we named A- and B-RIFIN proteins. This suggested sub-grouping is supported by a recently published study that showed that, despite the presence of the Plasmodium export (PEXEL motif in all RIFIN variants, proteins from each group have different cellular localizations during the intraerythrocytic life cycle of the parasite. In the present study we show that function shift analysis, a novel technique to predict functional divergence between sub-groups of a protein family, indicates that RIFINs have undergone neo- or sub-functionalization. Conclusion These results question the general trend of clustering large antigenically variant protein groups into homogenous families. Assigning functions to protein families requires their subdivision into meaningful groups such as we have shown for the RIFIN protein family. Using phylogenetic and function shift analysis methods, we identify new directions for the investigation of this broad and complex group of proteins.

The Polerovirus silencing suppressor P0 targets ARGONAUTE proteins for degradation.

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Baumberger, Nicolas; Tsai, Ching-Hsui; Lie, Miranda; Havecker, Ericka; Baulcombe, David C

2007-09-18

Plant and animal viruses encode suppressor proteins of an adaptive immunity mechanism in which viral double-stranded RNA is processed into 21-25 nt short interfering (si)RNAs. The siRNAs guide ARGONAUTE (AGO) proteins so that they target viral RNA. Most viral suppressors bind long dsRNA or siRNAs and thereby prevent production of siRNA or binding of siRNA to AGO. The one exception is the 2b suppressor of Cucumoviruses that binds to and inhibits AGO1. Here we describe a novel suppressor mechanism in which a Polerovirus-encoded F box protein (P0) targets the PAZ motif and its adjacent upstream sequence in AGO1 and mediates its degradation. F box proteins are components of E3 ubiquitin ligase complexes that add polyubiquitin tracts on selected lysine residues and thereby mark a protein for proteasome-mediated degradation. With P0, however, the targeted degradation of AGO is insensitive to inhibition of the proteasome, indicating that the proteasome is not involved. We also show that P0 does not block a mobile signal of silencing, indicating that the signal molecule does not have AGO protein components. The ability of P0 to block silencing without affecting signal movement may contribute to the phloem restriction of viruses in the Polerovirus group.

Cloning and analysis of two Ceratopteris thalictroides MADS-box genes

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XU Daolan

2014-06-01

Full Text Available MADS-box transcription factors,as a large gene family,play an important role in plant growth and development,especially act as key regulators in controlling the identities of floral organs in flowering plants.They are also significant in the evolutionary revelation.In order to understand MADS-box genes,we need more information of MADS-box genes in non flowering plant.MADS-box genes of Ceratopteris thalictroides were selected to clone and analysis by using RACE method.Two MADS-box genes,designated CtMADS1 and CtMADS2 in C. thalictroides,were cloned.Analysis indicates that CtMADS1 is belonged to MIKC*-clade,while CtMADS2 is belonged to MIKCc-clade.Phylogeny suggests that these two MADS-box genes of C. thalictroides have a close relationship with flowering plants,the data indicates that at least two different MADS-box genes are homologous to floral homeotic genes existed in the last common ancestor of contemporary vascular plants.

The Pentapeptide Repeat Proteins

OpenAIRE

Vetting, Matthew W.; Hegde, Subray S.; Fajardo, J. Eduardo; Fiser, Andras; Roderick, Steven L.; Takiff, Howard E.; Blanchard, John S.

2006-01-01

The Pentapeptide Repeat Protein (PRP) family has over 500 members in the prokaryotic and eukaryotic kingdoms. These proteins are composed of, or contain domains composed of, tandemly repeated amino acid sequences with a consensus sequence of [S,T,A,V][D,N][L,F]-[S,T,R][G]. The biochemical function of the vast majority of PRP family members is unknown. The three-dimensional structure of the first member of the PRP family was determined for the fluoroquinolone resistance protein (MfpA) from Myc...

Extensive Mutagenesis of the Conserved Box E Motif in Duck Hepatitis B Virus P Protein Reveals Multiple Functions in Replication and a Common Structure with the Primer Grip in HIV-1 Reverse Transcriptase

OpenAIRE

Wang, Yong-Xiang; Luo, Cheng; Zhao, Dan; Beck, Jürgen; Nassal, Michael

2012-01-01

Hepadnaviruses, including the pathogenic hepatitis B virus (HBV), replicate their small DNA genomes through protein-primed reverse transcription, mediated by the terminal protein (TP) domain in their P proteins and an RNA stem-loop, ϵ, on the pregenomic RNA (pgRNA). No direct structural data are available for P proteins, but their reverse transcriptase (RT) domains contain motifs that are conserved in all RTs (box A to box G), implying a similar architecture; however, experimental support for...

The Puf-family RNA-binding protein Puf2 controls sporozoite conversion to liver stages in the malaria parasite.

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Katja Müller

Full Text Available Malaria is a vector-borne infectious disease caused by unicellular, obligate intracellular parasites of the genus Plasmodium. During host switch the malaria parasite employs specialized latent stages that colonize the new host environment. Previous work has established that gametocytes, sexually differentiated stages that are taken up by the mosquito vector, control expression of genes required for mosquito colonization by translational repression. Sexual parasite development is controlled by a DEAD-box RNA helicase of the DDX6 family, termed DOZI. Latency of sporozoites, the transmission stage injected during an infectious blood meal, is controlled by the eIF2alpha kinase IK2, a general inhibitor of protein synthesis. Whether RNA-binding proteins participate in translational regulation in sporozoites remains to be studied. Here, we investigated the roles of two RNA-binding proteins of the Puf-family, Plasmodium Puf1 and Puf2, during sporozoite stage conversion. Our data reveal that, in the rodent malaria parasite P. berghei, Puf2 participates in the regulation of IK2 and inhibits premature sporozoite transformation. Inside mosquito salivary glands puf2⁻ sporozoites transform over time to round forms resembling early intra-hepatic stages. As a result, mutant parasites display strong defects in initiating a malaria infection. In contrast, Puf1 is dispensable in vivo throughout the entire Plasmodium life cycle. Our findings support the notion of a central role for Puf2 in parasite latency during switch between the insect and mammalian hosts.

PATtyFams: Protein families for the microbial genomes in the PATRIC database

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James J Davis

2016-02-01

Full Text Available The ability to build accurate protein families is a fundamental operation in bioinformatics that influences comparative analyses, genome annotation and metabolic modeling. For several years we have been maintaining protein families for all microbial genomes in the PATRIC database (Pathosystems Resource Integration Center, patricbrc.org in order to drive many of the comparative analysis tools that are available through the PATRIC website. However, due to the burgeoning number of genomes, traditional approaches for generating protein families are becoming prohibitive. In this report, we describe a new approach for generating protein families, which we call PATtyFams. This method uses the k-mer-based function assignments available through RAST (Rapid Annotation using Subsystem Technology to rapidly guide family formation, and then differentiates the function-based groups into families using a Markov Cluster algorithm (MCL. This new approach for generating protein families is rapid, scalable and has properties that are consistent with alignment-based methods.

The netrin protein family.

Science.gov (United States)

Rajasekharan, Sathyanath; Kennedy, Timothy E

2009-01-01

The name netrin is derived from the Sanskrit Netr, meaning 'guide'. Netrins are a family of extracellular proteins that direct cell and axon migration during embryogenesis. Three secreted netrins (netrins 1, 3 and 4), and two glycosylphosphatidylinositol (GPI)-anchored membrane proteins, netrins G1 and G2, have been identified in mammals. The secreted netrins are bifunctional, acting as attractants for some cell types and repellents for others. Receptors for the secreted netrins include the Deleted in Colorectal Cancer (DCC) family, the Down's syndrome cell adhesion molecule (DSCAM), and the UNC-5 homolog family: Unc5A, B, C and D in mammals. Netrin Gs do not appear to interact with these receptors, but regulate synaptic interactions between neurons by binding to the transmembrane netrin G ligands NGL1 and 2. The chemotropic function of secreted netrins has been best characterized with regard to axon guidance during the development of the nervous system. Extending axons are tipped by a flattened, membranous structure called the growth cone. Multiple extracellular guidance cues direct axonal growth cones to their ultimate targets where synapses form. Such cues can be locally derived (short-range), or can be secreted diffusible cues that allow target cells to signal axons from a distance (long-range). The secreted netrins function as short-range and long-range guidance cues in different circumstances. In addition to directing cell migration, functional roles for netrins have been identified in the regulation of cell adhesion, the maturation of cell morphology, cell survival and tumorigenesis.

Analysis of substructural variation in families of enzymatic proteins with applications to protein function prediction

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Fofanov Viacheslav Y

2010-05-01

Full Text Available Abstract Background Structural variations caused by a wide range of physico-chemical and biological sources directly influence the function of a protein. For enzymatic proteins, the structure and chemistry of the catalytic binding site residues can be loosely defined as a substructure of the protein. Comparative analysis of drug-receptor substructures across and within species has been used for lead evaluation. Substructure-level similarity between the binding sites of functionally similar proteins has also been used to identify instances of convergent evolution among proteins. In functionally homologous protein families, shared chemistry and geometry at catalytic sites provide a common, local point of comparison among proteins that may differ significantly at the sequence, fold, or domain topology levels. Results This paper describes two key results that can be used separately or in combination for protein function analysis. The Family-wise Analysis of SubStructural Templates (FASST method uses all-against-all substructure comparison to determine Substructural Clusters (SCs. SCs characterize the binding site substructural variation within a protein family. In this paper we focus on examples of automatically determined SCs that can be linked to phylogenetic distance between family members, segregation by conformation, and organization by homology among convergent protein lineages. The Motif Ensemble Statistical Hypothesis (MESH framework constructs a representative motif for each protein cluster among the SCs determined by FASST to build motif ensembles that are shown through a series of function prediction experiments to improve the function prediction power of existing motifs. Conclusions FASST contributes a critical feedback and assessment step to existing binding site substructure identification methods and can be used for the thorough investigation of structure-function relationships. The application of MESH allows for an automated

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of universal stress protein F (YnaF) from Salmonella typhimurium

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Sagurthi, Someswar Rao; Panigrahi, Rashmi Rekha; Gowda, Giri [Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012 (India); Savithri, H. S. [Department of Biochemistry, Indian Institute of Science, Bangalore 560012 (India); Murthy, M. R. N., E-mail: mrn@mbu.iisc.ernet.in [Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012 (India)

2007-11-01

The cloning, purification and crystallization of YnaF from S. typhimurium are reported along with preliminary X-ray crystallographic studies. The universal stress protein UspF (YnaF) is a small cytoplasmic bacterial protein. The expression of stress proteins is enhanced when cells are exposed to heat shock, nutrition starvation and certain other stress-inducing agents. YnaF promotes cell survival during prolonged exposure to stress and may activate a general mechanism for stress endurance. This manuscript reports preliminary crystallographic studies on YnaF from Salmonella typhimurium. The gene coding for YnaF was cloned and overexpressed and the protein was purified by Ni–NTA affinity chromatography. Purified YnaF was crystallized using vapour-diffusion and microbatch methods. The crystals belong to space group P2{sub 1}, with unit-cell parameters a = 37.51, b = 77.18, c = 56.34 Å, β = 101.8°. A data set was collected to 2.5 Å resolution with 94.6% completeness using an image-plate detector system mounted on a rotating-anode X-ray generator. Attempts to determine the structure are in progress.

The Role of Y-Box Binding Protein 1 in Kidney Injury: Friend or Foe?

Science.gov (United States)

Ke, Ben; Fan, Chuqiao; Tu, Weiping; Fang, Xiangdong

2018-01-01

Y-box-binding protein 1 (YB-1) is a multifunctional protein involved in various cellular processes via the transcriptional and translational regulation of target gene expression. YB-1 promotes acute or chronic kidney injury through multiple molecular pathways; however, accumulating evidence suggests that significantly increased YB-1 levels are of great importance in renoprotection. In addition, YB-1 may contribute to obesity-related kidney disease by promoting adipogenesis. Thus, the role of YB-1 in kidney injury is complicated, and no comprehensive review is currently available. In this review, we summarise recent progress in our understanding of the function of YB-1 in kidney injury and provide an overview of the dual role of YB-1 in kidney disease. Moreover, we propose that YB-1 is a potential therapeutic target to restrict kidney disease. © 2018 The Author(s). Published by S. Karger AG, Basel.

DAZ Family Proteins, Key Players for Germ Cell Development

Science.gov (United States)

Fu, Xia-Fei; Cheng, Shun-Feng; Wang, Lin-Qing; Yin, Shen; De Felici, Massimo; Shen, Wei

2015-01-01

DAZ family proteins are found almost exclusively in germ cells in distant animal species. Deletion or mutations of their encoding genes usually severely impair either oogenesis or spermatogenesis or both. The family includes Boule (or Boll), Dazl (or Dazla) and DAZ genes. Boule and Dazl are situated on autosomes while DAZ, exclusive of higher primates, is located on the Y chromosome. Deletion of DAZ gene is the most common causes of infertility in humans. These genes, encoding for RNA binding proteins, contain a highly conserved RNA recognition motif and at least one DAZ repeat encoding for a 24 amino acids sequence able to bind other mRNA binding proteins. Basically, Daz family proteins function as adaptors for target mRNA transport and activators of their translation. In some invertebrate species, BOULE protein play a pivotal role in germline specification and a conserved regulatory role in meiosis. Depending on the species, DAZL is expressed in primordial germ cells (PGCs) and/or pre-meiotic and meiotic germ cells of both sexes. Daz is found in fetal gonocytes, spermatogonia and spermatocytes of adult testes. Here we discuss DAZ family genes in a phylogenic perspective, focusing on the common and distinct features of these genes, and their pivotal roles during gametogenesis evolved during evolution. PMID:26327816

Both core and F proteins of hepatitis C virus could enhance cell proliferation in transgenic mice

Energy Technology Data Exchange (ETDEWEB)

Hu, Wen-Ta [Graduate Institute of Medical Biotechnology, Tzu Chi University, Hualien, Taiwan (China); Li, Hui-Chun [Department of Biochemistry, Tzu Chi University, Hualien, Taiwan (China); Lee, Shen-Kao; Ma, Hsin-Chieh; Yang, Chee-Hing; Chen, Hung-Ling [Graduate Institute of Medical Biotechnology, Tzu Chi University, Hualien, Taiwan (China); Lo, Shih-Yen, E-mail: losylo@mail.tcu.edu.tw [Graduate Institute of Medical Biotechnology, Tzu Chi University, Hualien, Taiwan (China); Department of Laboratory Medicine, Buddhist Tzu Chi General Hospital, Hualien, Taiwan (China)

2013-05-24

Highlights: •HCV core and F proteins could induce hepatocyte proliferation in the transgenic mice. •β-Catenin signaling pathway was activated by core protein in the transgenic mice. •β-Catenin signaling pathway was activated by myc-F protein in the transgenic mice. •Expression of SMA protein was enhanced by core but not myc-F protein. -- Abstract: The role of the protein encoded by the alternative open reading frame (ARF/F/core+1) of the Hepatitis C virus (HCV) genome in viral pathogenesis remains unknown. The different forms of ARF/F/core+1 protein were labile in cultured cells, a myc-tag fused at the N-terminus of the F protein made it more stable. To determine the role of core and F proteins in HCV pathogenesis, transgenic mice with either protein expression under the control of Albumin promoter were generated. Expression of core protein and F protein with myc tag (myc-F) could be detected by Western blotting analysis in the livers of these mice. The ratio of liver to body weight is increased for both core and myc-F transgenic mice compared to that of wild type mice. Indeed, the proliferating cell nuclear antigen protein, a proliferation marker, was up-regulated in the transgenic mice with core or myc-F protein. Further analyses by microarray and Western blotting suggested that β-catenin signaling pathway was activated by either core or myc-F protein in the transgenic mice. These transgenic mice were further treated with either Diethynitrosamine (a tumor initiator) or Phenobarbital (a tumor promoter). Phenobarbital but not Diethynitrosamine treatment could increase the liver/body weight ratio of these mice. However, no tumor formation was observed in these mice. In conclusion, HCV core and myc-F proteins could induce hepatocyte proliferation in the transgenic mice possibly through β-catenin signaling pathway.

Both core and F proteins of hepatitis C virus could enhance cell proliferation in transgenic mice

International Nuclear Information System (INIS)

Hu, Wen-Ta; Li, Hui-Chun; Lee, Shen-Kao; Ma, Hsin-Chieh; Yang, Chee-Hing; Chen, Hung-Ling; Lo, Shih-Yen

2013-01-01

Highlights: •HCV core and F proteins could induce hepatocyte proliferation in the transgenic mice. •β-Catenin signaling pathway was activated by core protein in the transgenic mice. •β-Catenin signaling pathway was activated by myc-F protein in the transgenic mice. •Expression of SMA protein was enhanced by core but not myc-F protein. -- Abstract: The role of the protein encoded by the alternative open reading frame (ARF/F/core+1) of the Hepatitis C virus (HCV) genome in viral pathogenesis remains unknown. The different forms of ARF/F/core+1 protein were labile in cultured cells, a myc-tag fused at the N-terminus of the F protein made it more stable. To determine the role of core and F proteins in HCV pathogenesis, transgenic mice with either protein expression under the control of Albumin promoter were generated. Expression of core protein and F protein with myc tag (myc-F) could be detected by Western blotting analysis in the livers of these mice. The ratio of liver to body weight is increased for both core and myc-F transgenic mice compared to that of wild type mice. Indeed, the proliferating cell nuclear antigen protein, a proliferation marker, was up-regulated in the transgenic mice with core or myc-F protein. Further analyses by microarray and Western blotting suggested that β-catenin signaling pathway was activated by either core or myc-F protein in the transgenic mice. These transgenic mice were further treated with either Diethynitrosamine (a tumor initiator) or Phenobarbital (a tumor promoter). Phenobarbital but not Diethynitrosamine treatment could increase the liver/body weight ratio of these mice. However, no tumor formation was observed in these mice. In conclusion, HCV core and myc-F proteins could induce hepatocyte proliferation in the transgenic mice possibly through β-catenin signaling pathway

TGF-β mimic proteins form an extended gene family in the murine parasite Heligmosomoides polygyrus.

Science.gov (United States)

Smyth, Danielle J; Harcus, Yvonne; White, Madeleine P J; Gregory, William F; Nahler, Janina; Stephens, Ian; Toke-Bjolgerud, Edward; Hewitson, James P; Ivens, Alasdair; McSorley, Henry J; Maizels, Rick M

2018-04-01

We recently reported the discovery of a new parasite-derived protein that functionally mimics the immunosuppressive cytokine transforming growth factor (TGF)-β. The Heligmosomoides polygyrus TGF-β Mimic (Hp-TGM) shares no homology to any TGF-β family member, however it binds the mammalian TGF-β receptor and induces expression of Foxp3, the canonical transcription factor of both mouse and human regulatory T cells. Hp-TGM consists of five atypical Complement Control Protein (CCP, Pfam 00084) domains, each lacking certain conserved residues and 12-15 amino acids longer than the 60-70 amino acids consensus domain, but with a recognizable 3-cysteine, tryptophan, cysteine motif. We now report on the identification of a family of nine related Hp-TGM homologues represented in the secreted proteome and transcriptome of H. polygyrus. Recombinant proteins from five of the nine new TGM members were tested for TGF-β activity, but only two were functionally active in an MFB-F11 reporter assay, and by the induction of T cell Foxp3 expression. Sequence comparisons reveal that proteins with functional activity are similar or identical to Hp-TGM across the first three CCP domains, but more variable in domains 4 and 5. Inactive proteins diverged in all domains, or lacked some domains entirely. Testing truncated versions of Hp-TGM confirmed that domains 1-3 are essential for full activity in vitro, while domains 4 and 5 are not required. Further studies will elucidate whether these latter domains fulfill other functions in promoting host immune regulation during infection and if the more divergent family members play other roles in immunomodulation. Copyright © 2018. Published by Elsevier Ltd.

Reassessment of the nonlocality of correlation boxes

Energy Technology Data Exchange (ETDEWEB)

Costa, A.P.; Parisio, Fernando, E-mail: parisio@df.ufpe.br

2017-01-15

Correlation boxes are hypothetical systems usually designed to produce the maximal algebraic violation of a Bell inequality, beyond the quantum bound and without superluminal signalling. The fact that these systems show stronger correlations than those presented by maximally entangled quantum states, as the spin singlet, has been regarded as a demonstration that the former are more nonlocal than the latter. By applying an alternative, consistent measure of nonlocality to a family of correlation boxes, we show that this conclusion is not necessarily true. Complementarily, we define a class of systems displaying subquantum correlations which, nevertheless, are more nonlocal than the singlet state, showing that the extent of the numeric violation of an inequality may have little to do with the degree of nonlocality, especially in the case of correlation boxes.

HIPPI: highly accurate protein family classification with ensembles of HMMs

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Nam-phuong Nguyen

2016-11-01

Full Text Available Abstract Background Given a new biological sequence, detecting membership in a known family is a basic step in many bioinformatics analyses, with applications to protein structure and function prediction and metagenomic taxon identification and abundance profiling, among others. Yet family identification of sequences that are distantly related to sequences in public databases or that are fragmentary remains one of the more difficult analytical problems in bioinformatics. Results We present a new technique for family identification called HIPPI (Hierarchical Profile Hidden Markov Models for Protein family Identification. HIPPI uses a novel technique to represent a multiple sequence alignment for a given protein family or superfamily by an ensemble of profile hidden Markov models computed using HMMER. An evaluation of HIPPI on the Pfam database shows that HIPPI has better overall precision and recall than blastp, HMMER, and pipelines based on HHsearch, and maintains good accuracy even for fragmentary query sequences and for protein families with low average pairwise sequence identity, both conditions where other methods degrade in accuracy. Conclusion HIPPI provides accurate protein family identification and is robust to difficult model conditions. Our results, combined with observations from previous studies, show that ensembles of profile Hidden Markov models can better represent multiple sequence alignments than a single profile Hidden Markov model, and thus can improve downstream analyses for various bioinformatic tasks. Further research is needed to determine the best practices for building the ensemble of profile Hidden Markov models. HIPPI is available on GitHub at https://github.com/smirarab/sepp .

[Interconnection between architecture of protein globule and disposition of conformational conservative oligopeptides in proteins from one protein family].

Science.gov (United States)

Batianovskiĭ, A V; Filatov, I V; Namiot, V A; Esipova, N G; Volotovskiĭ, I D

2012-01-01

It was shown that selective interactions between helical segments of macromolecules can realize in globular proteins in the segments characterized by the same periodicities of charge distribution i.e. between conformationally conservative oligopeptides. It was found that in the macromolecules of alpha-helical proteins conformationally conservative oligopeptides are disposed at a distance being characteristic of direct interactions. For representatives of many structural families of alpha-type proteins specific disposition of conformationally conservative segments is observed. This disposition is inherent to a particular structural family. Disposition of conformationally conservative segments is not related to homology of the amino acid sequence but reflects peculiarities of native 3D-architectures of protein globules.

Evolutionary hierarchy of vertebrate-like heterotrimeric G protein families.

Science.gov (United States)

Krishnan, Arunkumar; Mustafa, Arshi; Almén, Markus Sällman; Fredriksson, Robert; Williams, Michael J; Schiöth, Helgi B

2015-10-01

Heterotrimeric G proteins perform a crucial role as molecular switches controlling various cellular responses mediated by G protein-coupled receptor (GPCR) signaling pathway. Recent data have shown that the vertebrate-like G protein families are found across metazoans and their closest unicellular relatives. However, an overall evolutionary hierarchy of vertebrate-like G proteins, including gene family annotations and in particular mapping individual gene gain/loss events across diverse holozoan lineages is still incomplete. Here, with more expanded invertebrate taxon sampling, we have reconstructed phylogenetic trees for each of the G protein classes/families and provide a robust classification and hierarchy of vertebrate-like heterotrimeric G proteins. Our results further extend the evidence that the common ancestor (CA) of holozoans had at least five ancestral Gα genes corresponding to all major vertebrate Gα classes and contain a total of eight genes including two Gβ and one Gγ. Our results also indicate that the GNAI/O-like gene likely duplicated in the last CA of metazoans to give rise to GNAI- and GNAO-like genes, which are conserved across invertebrates. Moreover, homologs of GNB1-4 paralogon- and GNB5 family-like genes are found in most metazoans and that the unicellular holozoans encode two ancestral Gβ genes. Similarly, most bilaterian invertebrates encode two Gγ genes which include a representative of the GNG gene cluster and a putative homolog of GNG13. Interestingly, our results also revealed key evolutionary events such as the Drosophila melanogaster eye specific Gβ subunit that is found conserved in most arthropods and several previously unidentified species specific expansions within Gαi/o, Gαs, Gαq, Gα12/13 classes and the GNB1-4 paralogon. Also, we provide an overall proposed evolutionary scenario on the expansions of all G protein families in vertebrate tetraploidizations. Our robust classification/hierarchy is essential to further

The Drosophila IKK-related kinase (Ik2 and Spindle-F proteins are part of a complex that regulates cytoskeleton organization during oogenesis

Directory of Open Access Journals (Sweden)

Shaanan Boaz

2008-09-01

Full Text Available Abstract Background IkappaB kinases (IKKs regulate the activity of Rel/NF-kappaB transcription factors by targeting their inhibitory partner proteins, IkappaBs, for degradation. The Drosophila genome encodes two members of the IKK family. Whereas the first is a kinase essential for activation of the NF-kappaB pathway, the latter does not act as IkappaB kinase. Instead, recent findings indicate that Ik2 regulates F-actin assembly by mediating the function of nonapoptotic caspases via degradation of DIAP1. Also, it has been suggested that ik2 regulates interactions between the minus ends of the microtubules and the actin-rich cortex in the oocyte. Since spn-F mutants display oocyte defects similar to those of ik2 mutant, we decided to investigate whether Spn-F could be a direct regulatory target of Ik2. Results We found that Ik2 binds physically to Spn-F, biomolecular interaction analysis of Spn-F and Ik2 demonstrating that both proteins bind directly and form a complex. We showed that Ik2 phosphorylates Spn-F and demonstrated that this phosphorylation does not lead to Spn-F degradation. Ik2 is localized to the anterior ring of the oocyte and to punctate structures in the nurse cells together with Spn-F protein, and both proteins are mutually required for their localization. Conclusion We conclude that Ik2 and Spn-F form a complex, which regulates cytoskeleton organization during Drosophila oogenesis and in which Spn-F is the direct regulatory target for Ik2. Interestingly, Ik2 in this complex does not function as a typical IKK in that it does not direct SpnF for degradation following phosphorylation.

Evolution of the DEAD box helicase family in chicken: chickens have no DHX9 ortholog.

Science.gov (United States)

Sato, Haruko; Oshiumi, Hiroyuki; Takaki, Hiromi; Hikono, Hirokazu; Seya, Tsukasa

2015-10-01

Viral RNA represents a pattern molecule that can be recognized by RNA sensors in innate immunity. Humans and mice possess cytoplasmic DNA/RNA sensors for detecting viral replication. There are a number of DEAD (Asp-Glu-Ala-Asp; DExD/H) box-type helicases in mammals, among which retinoic acid-inducible gene 1 (RIG-I) and melanoma differentiation-associated protein 5 (MDA50) are indispensable for RNA sensing; however, they are functionally supported by a number of sensors that directly bind viral RNA or replicative RNA intermediates to convey signals to RIG-I and MDA5. Some DEAD box helicase members recognize DNA irrespective of the origin. These sensors transmit IFN-inducing signals through adaptors, including mitochondrial antiviral signaling. Viral double-stranded RNAs are reportedly sensed by the helicases DDX1, DDX21, DHX36, DHX9, DDX3, DDX41, LGP2 and DDX60, in addition to RIG-I and MDA5, and induce type I IFNs, thereby blocking viral replication. Humans and mice have all nucleic acid sensors listed here. In the RNA sensing system in chicken, it was found in the present study that most DEAD box helicases are conserved; however, DHX9 is genetically deficient in addition to reported RIG-I. Based on the current genome databases, similar DHX9 deficiency was observed in ducks and several other bird species. Because chicken, but not duck, was found to be deficient in RIG-I, the RNA-sensing system of chicken lacks RIG-I and DHX9 and is thus more fragile than that of duck or mammal. DHX9 may generally compensate for the function of RIG-I and deficiency of DHX9 possibly participates in exacerbations of viral infection such as influenza in chickens. © 2015 The Societies and Wiley Publishing Asia Pty Ltd.

Practical analysis of specificity-determining residues in protein families.

Science.gov (United States)

Chagoyen, Mónica; García-Martín, Juan A; Pazos, Florencio

2016-03-01

Determining the residues that are important for the molecular activity of a protein is a topic of broad interest in biomedicine and biotechnology. This knowledge can help understanding the protein's molecular mechanism as well as to fine-tune its natural function eventually with biotechnological or therapeutic implications. Some of the protein residues are essential for the function common to all members of a family of proteins, while others explain the particular specificities of certain subfamilies (like binding on different substrates or cofactors and distinct binding affinities). Owing to the difficulty in experimentally determining them, a number of computational methods were developed to detect these functional residues, generally known as 'specificity-determining positions' (or SDPs), from a collection of homologous protein sequences. These methods are mature enough for being routinely used by molecular biologists in directing experiments aimed at getting insight into the functional specificity of a family of proteins and eventually modifying it. In this review, we summarize some of the recent discoveries achieved through SDP computational identification in a number of relevant protein families, as well as the main approaches and software tools available to perform this type of analysis. © The Author 2015. Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

Comparative evaluation of low-molecular-mass proteins from Mycobacterium tuberculosis identifies members of the ESAT-6 family as immunodominant T-cell antigens

DEFF Research Database (Denmark)

Skjøt, R L; Oettinger, T; Rosenkrands, I

2000-01-01

Culture filtrate from Mycobacterium tuberculosis contains protective antigens of relevance for the generation of a new antituberculosis vaccine. We have identified two previously uncharacterized M. tuberculosis proteins (TB7.3 and TB10.4) from the highly active low-mass fraction of culture filtrate....... The molecules were characterized, mapped in a two-dimensional electrophoresis reference map of short-term culture filtrate, and compared with another recently identified low-mass protein, CFP10 (F. X. Berthet, P. B. Rasmussen, I. Rosenkrands, P. Andersen, and B. Gicquel. Microbiology 144:3195-3203, 1998......), and the well-described ESAT-6 antigen. Genetic analyses demonstrated that TB10.4 as well as CFP10 belongs to the ESAT-6 family of low-mass proteins, whereas TB7.3 is a low-molecular-mass protein outside this family. The proteins were expressed in Escherichia coli, and their immunogenicity was tested...

Cdc20 mediates D-box-dependent degradation of Sp100

International Nuclear Information System (INIS)

Wang, Ran; Li, Ke-min; Zhou, Cai-hong; Xue, Jing-lun; Ji, Chao-neng; Chen, Jin-zhong

2011-01-01

Highlights: ► Cdc20 is a co-activator of APC/C complex. ► Cdc20 recruits Sp100 and mediates its degradation. ► The D-box of Sp100 is required for Cdc20-mediated degradation. ► Sp100 expresses consistently at both the mRNA and protein levels in cell cycle. -- Abstract: Cdc20 is a co-activator of the anaphase-promoting complex/cyclosome (APC/C complex), which recruits substrates at particular phases of the cell cycle and mediates their degradation. Sp100 is a PML-NB scaffold protein, which localizes to nuclear particles during interphase and disperses from them during mitosis, participates in viral resistance, transcriptional regulation, and apoptosis. However, its metabolism during the cell cycle has not yet been fully characterized. We found a putative D-box in Sp100 using the Eukaryotic Linear Motif (ELM) predictor database. The putative D-box of Sp100 was verified by mutational analysis. Overexpression of Cdc20 resulted in decreased levels of both endogenous Sp100 protein and overexpressed Sp100 mRNA in HEK 293 cells. Only an overexpressed D-box deletion mutant of Sp100 accumulated in HEK293 cells that also overexpressed Cdc20. Cdc20 knockdown by cdc20 specific siRNA resulted in increased Sp100 protein levels in cells. Furthermore, we discovered that the Cdc20 mediated degradation of Sp100 is diminished by the proteasome inhibitor MG132, which suggests that the ubiquitination pathway is involved in this process. However, unlike the other Cdc20 substrates, which display oscillating protein levels, the level of Sp100 protein remains constant throughout the cell cycle. Additionally, both overexpression and knockdown of endogenous Sp100 had no effect on the cell cycle. Our results suggested that sp100 is a novel substrate of Cdc20 and it is degraded by the ubiquitination pathway. The intact D-box of Sp100 was necessary for this process. These findings expand our knowledge of both Sp100 and Cdc20 as well as their role in ubiquitination.

BID-F1 and BID-F2 domains of Bartonella henselae effector protein BepF trigger together with BepC the formation of invasome structures.

Science.gov (United States)

Truttmann, Matthias C; Guye, Patrick; Dehio, Christoph

2011-01-01

The gram-negative, zoonotic pathogen Bartonella henselae (Bhe) translocates seven distinct Bartonella effector proteins (Beps) via the VirB/VirD4 type IV secretion system (T4SS) into human cells, thereby interfering with host cell signaling [1], [2]. In particular, the effector protein BepG alone or the combination of effector proteins BepC and BepF trigger massive F-actin rearrangements that lead to the establishment of invasome structures eventually resulting in the internalization of entire Bhe aggregates [2], [3]. In this report, we investigate the molecular function of the effector protein BepF in the eukaryotic host cell. We show that the N-terminal [E/T]PLYAT tyrosine phosphorylation motifs of BepF get phosphorylated upon translocation but do not contribute to invasome-mediated Bhe uptake. In contrast, we found that two of the three BID domains of BepF are capable to trigger invasome formation together with BepC, while a mutation of the WxxxE motif of the BID-F1 domain inhibited its ability to contribute to the formation of invasome structures. Next, we show that BepF function during invasome formation can be replaced by the over-expression of constitutive-active Rho GTPases Rac1 or Cdc42. Finally we demonstrate that BID-F1 and BID-F2 domains promote the formation of filopodia-like extensions in NIH 3T3 and HeLa cells as well as membrane protrusions in HeLa cells, suggesting a role for BepF in Rac1 and Cdc42 activation during the process of invasome formation.

Qualification of box HEPA filters for nuclear applications

International Nuclear Information System (INIS)

Bergman, W.; Larsen, G.; Wilson, K.; Rainer, F.

1995-03-01

We have successfully completed qualification tests on high efficiency particulate air (HEPA) filters that are encapsulated within a box and manufactured by American Air Filters. The qualification tests are required by the American Society of Mechanical Engineers Standard ASME N509 and the U.S. Military Standard MIL-F-51068 for HEPA filters to be used in nuclear applications. The qualification tests specify minimum filter efficiencies following exposure to heated air, overpressure, and rough handling. Prior to this study, no box HEPA filters from any manufacturer had been qualified despite their wide-spread use in Department of Energy (DOE) facilities. Box HEPA filters are not addressed in any of the existing HEPA standards and only briefly discussed in the Nuclear Air Cleaning Handbook

Impact attenuation of protective boxing and taekwondo headgear.

Science.gov (United States)

O'Sullivan, David M; Fife, Gabriel P

2016-11-01

This study aimed to compare the impact attenuation performance of boxing and taekwondo headgear in terms of peak linear and rotational acceleration. To measure the impact attenuation of headgear, a standardized (American Society for Testing and Materials (ASTM) F-2397) martial arts headgear striker was used to impart impacts to a 50th Percentile Male Hybrid III Crash Test Dummy head and neck complex. Two boxing (Adidas and Greenhill) and two taekwondo (Adidas and Nike) headgear, approved by the Association Internationale de Boxe Amateur and the World Taekwondo Federation (WTF), were selected. Each of the selected headgear was fitted to the Hybrid III head and subsequently subjected to five impacts at the front and side with a maximum impact interim time of 60 seconds by the rotating striker at 8 ± 0.3 m/s. Linear and rotational acceleration were recorded at 10,000 Hz. There were significant interactions of the impact location and brand on the rotational acceleration, F(3,40) = 6.7, p < .05. There were significant main effects of both impact location F(1,40) = 9.07, p < .05 and headgear brand F(3,40) = 9.9, p < .05 on the linear acceleration. Pairwise comparisons show significant differences between the front and side for both linear and rotational acceleration. The headgear tested failed the ASTM high impact test requirement to reduce the linear acceleration to below a threshold of 150 g. Further development of headgear to reduce impact linear and rotational acceleration magnitudes should be called for by the relevant sport governing bodies and initiated by headgear manufactures.

Hijacking of the host SCF ubiquitin ligase machinery by plant pathogens

Directory of Open Access Journals (Sweden)

Shimpei eMagori

2011-11-01

Full Text Available The SCF (SKP1-CUL1-F-box protein ubiquitin ligase complex mediates polyubiquitination of proteins targeted for degradation, thereby controlling a plethora of biological processes in eukaryotic cells. Although this ubiquitination machinery is found and functional only in eukaryotes, many non-eukaryotic pathogens also encode F-box proteins, the critical subunits of the SCF complex. Increasing evidence indicates that such non-eukaryotic F-box proteins play an essential role in subverting or exploiting the host ubiquitin/proteasome system for efficient pathogen infection. A recent bioinformatic analysis has identified more than 70 F-box proteins in 22 different bacterial species, suggesting that use of pathogen-encoded F-box effectors in the host cell may be a widespread infection strategy. In this review, we focus on plant pathogen-encoded F-box effectors, such as VirF of Agrobacterium tumefaciens, GALAs of Ralstonia solanacearum, and P0 of Poleroviruses, and discuss the molecular mechanism by which plant pathogens use these factors to manipulate the host cell for their own benefit.

Receptor-interacting protein (RIP) kinase family

OpenAIRE

Zhang, Duanwu; Lin, Juan; Han, Jiahuai

2010-01-01

Receptor-interacting protein (RIP) kinases are a group of threonine/serine protein kinases with a relatively conserved kinase domain but distinct non-kinase regions. A number of different domain structures, such as death and caspase activation and recruitment domain (CARD) domains, were found in different RIP family members, and these domains should be keys in determining the specific function of each RIP kinase. It is known that RIP kinases participate in different biological processes, incl...

The Roles of T-Box Genes in Vertebrate Limb Development.

Science.gov (United States)

Sheeba, C J; Logan, M P O

2017-01-01

Members of the T-box gene family have diverse roles during embryogenesis and many play critical roles in the developing limb. This is exemplified by the fact that, in humans, mutations in T-box genes are associated with several congenital syndromes that include limb defects as part of their characteristic spectrum of abnormalities. T-box genes encode for evolutionary conserved transcription factors that include both transcriptional activators and repressors. The hallmark of T-box gene members is the presence of the eponymous DNA-binding T-box domain. There are 17 mammalian T-box genes, which based on the sequence homology of the T-box domain, are grouped into five subfamilies, namely, T, Tbx1, Tbx2, Tbx6, and Tbr1. At least nine T-box genes are expressed during limb development with distinct and dynamic expression patterns. All four members of Tbx2 subfamily (Tbx2, Tbx3, Tbx4, Tbx5) and three members of Tbx1 (Tbx1, Tbx15, Tbx18), Brachyury (T) and Eomes (Tbr2) are expressed in the developing limb. © 2017 Elsevier Inc. All rights reserved.

Full trans-activation mediated by the immediate-early protein of equine herpesvirus 1 requires a consensus TATA box, but not its cognate binding sequence.

Science.gov (United States)

Kim, Seong K; Shakya, Akhalesh K; O'Callaghan, Dennis J

2016-01-04

The immediate-early protein (IEP) of equine herpesvirus 1 (EHV-1) has extensive homology to the IEP of alphaherpesviruses and possesses domains essential for trans-activation, including an acidic trans-activation domain (TAD) and binding domains for DNA, TFIIB, and TBP. Our data showed that the IEP directly interacted with transcription factor TFIIA, which is known to stabilize the binding of TBP and TFIID to the TATA box of core promoters. When the TATA box of the EICP0 promoter was mutated to a nonfunctional TATA box, IEP-mediated trans-activation was reduced from 22-fold to 7-fold. The IEP trans-activated the viral promoters in a TATA motif-dependent manner. Our previous data showed that the IEP is able to repress its own promoter when the IEP-binding sequence (IEBS) is located within 26-bp from the TATA box. When the IEBS was located at 100 bp upstream of the TATA box, IEP-mediated trans-activation was very similar to that of the minimal IE(nt -89 to +73) promoter lacking the IEBS. As the distance from the IEBS to the TATA box decreased, IEP-mediated trans-activation progressively decreased, indicating that the IEBS located within 100 bp from the TATA box sequence functions as a distance-dependent repressive element. These results indicated that IEP-mediated full trans-activation requires a consensus TATA box of core promoters, but not its binding to the cognate sequence (IEBS). Copyright © 2015 Elsevier B.V. All rights reserved.

Full trans–activation mediated by the immediate–early protein of equine herpesvirus 1 requires a consensus TATA box, but not its cognate binding sequence

Science.gov (United States)

Kim, Seong K.; Shakya, Akhalesh K.; O'Callaghan, Dennis J.

2015-01-01

The immediate-early protein (IEP) of equine herpesvirus 1 (EHV-1) has extensive homology to the IEP of alphaherpesviruses and possesses domains essential for trans-activation, including an acidic trans-activation domain (TAD) and binding domains for DNA, TFIIB, and TBP. Our data showed that the IEP directly interacted with transcription factor TFIIA, which is known to stabilize the binding of TBP and TFIID to the TATA box of core promoters. When the TATA box of the EICP0 promoter was mutated to a nonfunctional TATA box, IEP-mediated trans-activation was reduced from 22-fold to 7-fold. The IEP trans-activated the viral promoters in a TATA motif-dependent manner. Our previous data showed that the IEP is able to repress its own promoter when the IEP-binding sequence (IEBS) is located within 26-bp from the TATA box. When the IEBS was located at 100 bp upstream of the TATA box, IEP-mediated trans-activation was very similar to that of the minimal IE(nt −89 to +73) promoter lacking the IEBS. As the distance from the IEBS to the TATA box decreased, IEP-mediated trans-activation progressively decreased, indicating that the IEBS located within 100 bp from the TATA box sequence functions as a distance-dependent repressive element. These results indicated that IEP-mediated full trans-activation requires a consensus TATA box of core promoters, but not its binding to the cognate sequence (IEBS). PMID:26541315

Two novel heat-soluble protein families abundantly expressed in an anhydrobiotic tardigrade.

Directory of Open Access Journals (Sweden)

Ayami Yamaguchi

Full Text Available Tardigrades are able to tolerate almost complete dehydration by reversibly switching to an ametabolic state. This ability is called anhydrobiosis. In the anhydrobiotic state, tardigrades can withstand various extreme environments including space, but their molecular basis remains largely unknown. Late embryogenesis abundant (LEA proteins are heat-soluble proteins and can prevent protein-aggregation in dehydrated conditions in other anhydrobiotic organisms, but their relevance to tardigrade anhydrobiosis is not clarified. In this study, we focused on the heat-soluble property characteristic of LEA proteins and conducted heat-soluble proteomics using an anhydrobiotic tardigrade. Our heat-soluble proteomics identified five abundant heat-soluble proteins. All of them showed no sequence similarity with LEA proteins and formed two novel protein families with distinct subcellular localizations. We named them Cytoplasmic Abundant Heat Soluble (CAHS and Secretory Abundant Heat Soluble (SAHS protein families, according to their localization. Both protein families were conserved among tardigrades, but not found in other phyla. Although CAHS protein was intrinsically unstructured and SAHS protein was rich in β-structure in the hydrated condition, proteins in both families changed their conformation to an α-helical structure in water-deficient conditions as LEA proteins do. Two conserved repeats of 19-mer motifs in CAHS proteins were capable to form amphiphilic stripes in α-helices, suggesting their roles as molecular shield in water-deficient condition, though charge distribution pattern in α-helices were different between CAHS and LEA proteins. Tardigrades might have evolved novel protein families with a heat-soluble property and this study revealed a novel repertoire of major heat-soluble proteins in these anhydrobiotic animals.

Enhancing the prediction of protein pairings between interacting families using orthology information

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Pazos Florencio

2008-01-01

Full Text Available Abstract Background It has repeatedly been shown that interacting protein families tend to have similar phylogenetic trees. These similarities can be used to predicting the mapping between two families of interacting proteins (i.e. which proteins from one family interact with which members of the other. The correct mapping will be that which maximizes the similarity between the trees. The two families may eventually comprise orthologs and paralogs, if members of the two families are present in more than one organism. This fact can be exploited to restrict the possible mappings, simply by impeding links between proteins of different organisms. We present here an algorithm to predict the mapping between families of interacting proteins which is able to incorporate information regarding orthologues, or any other assignment of proteins to "classes" that may restrict possible mappings. Results For the first time in methods for predicting mappings, we have tested this new approach on a large number of interacting protein domains in order to statistically assess its performance. The method accurately predicts around 80% in the most favourable cases. We also analysed in detail the results of the method for a well defined case of interacting families, the sensor and kinase components of the Ntr-type two-component system, for which up to 98% of the pairings predicted by the method were correct. Conclusion Based on the well established relationship between tree similarity and interactions we developed a method for predicting the mapping between two interacting families using genomic information alone. The program is available through a web interface.

Evolution and Virulence of Influenza A Virus Protein PB1-F2

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Ram P. Kamal

2017-12-01

Full Text Available PB1-F2 is an accessory protein of most human, avian, swine, equine, and canine influenza A viruses (IAVs. Although it is dispensable for virus replication and growth, it plays significant roles in pathogenesis by interfering with the host innate immune response, inducing death in immune and epithelial cells, altering inflammatory responses, and promoting secondary bacterial pneumonia. The effects of PB1-F2 differ between virus strains and host species. This can at least partially be explained by the presence of multiple PB1-F2 sequence variants, including premature stop codons that lead to the expression of truncated PB1-F2 proteins of different lengths and specific virulence-associated residues that enhance susceptibility to bacterial superinfection. Although there has been a tendency for human seasonal IAV to gradually reduce the number of virulence-associated residues, zoonotic IAVs contain a reservoir of PB1-F2 proteins with full length, virulence-associated sequences. Here, we review the molecular mechanisms by which PB1-F2 may affect influenza virulence, and factors associated with the evolution and selection of this protein.

BID-F1 and BID-F2 domains of Bartonella henselae effector protein BepF trigger together with BepC the formation of invasome structures.

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Matthias C Truttmann

Full Text Available The gram-negative, zoonotic pathogen Bartonella henselae (Bhe translocates seven distinct Bartonella effector proteins (Beps via the VirB/VirD4 type IV secretion system (T4SS into human cells, thereby interfering with host cell signaling [1], [2]. In particular, the effector protein BepG alone or the combination of effector proteins BepC and BepF trigger massive F-actin rearrangements that lead to the establishment of invasome structures eventually resulting in the internalization of entire Bhe aggregates [2], [3]. In this report, we investigate the molecular function of the effector protein BepF in the eukaryotic host cell. We show that the N-terminal [E/T]PLYAT tyrosine phosphorylation motifs of BepF get phosphorylated upon translocation but do not contribute to invasome-mediated Bhe uptake. In contrast, we found that two of the three BID domains of BepF are capable to trigger invasome formation together with BepC, while a mutation of the WxxxE motif of the BID-F1 domain inhibited its ability to contribute to the formation of invasome structures. Next, we show that BepF function during invasome formation can be replaced by the over-expression of constitutive-active Rho GTPases Rac1 or Cdc42. Finally we demonstrate that BID-F1 and BID-F2 domains promote the formation of filopodia-like extensions in NIH 3T3 and HeLa cells as well as membrane protrusions in HeLa cells, suggesting a role for BepF in Rac1 and Cdc42 activation during the process of invasome formation.

In vivo immobilization of fusion proteins on bioplastics by the novel tag BioF.

Science.gov (United States)

Moldes, Cristina; García, Pedro; García, José L; Prieto, María A

2004-06-01

A new protein immobilization and purification system has been developed based on the use of polyhydroxyalkanoates (PHAs, or bioplastics), which are biodegradable polymers accumulated as reserve granules in the cytoplasm of certain bacteria. The N-terminal domain of the PhaF phasin (a PHA-granule-associated protein) from Pseudomonas putida GPo1 was used as a polypeptide tag (BioF) to anchor fusion proteins to PHAs. This tag provides a novel way to immobilize proteins in vivo by using bioplastics as supports. The granules carrying the BioF fusion proteins can be isolated by a simple centrifugation step and used directly for some applications. Moreover, when required, a practically pure preparation of the soluble BioF fusion protein can be obtained by a mild detergent treatment of the granule. The efficiency of this system has been demonstrated by constructing two BioF fusion products, including a functional BioF-beta-galactosidase. This is the first example of an active bioplastic consisting of a biodegradable matrix carrying an active enzyme.

Redundancy and divergence in the amyloid precursor protein family.

Science.gov (United States)

Shariati, S Ali M; De Strooper, Bart

2013-06-27

Gene duplication provides genetic material required for functional diversification. An interesting example is the amyloid precursor protein (APP) protein family. The APP gene family has experienced both expansion and contraction during evolution. The three mammalian members have been studied quite extensively in combined knock out models. The underlying assumption is that APP, amyloid precursor like protein 1 and 2 (APLP1, APLP2) are functionally redundant. This assumption is primarily supported by the similarities in biochemical processing of APP and APLPs and on the fact that the different APP genes appear to genetically interact at the level of the phenotype in combined knockout mice. However, unique features in each member of the APP family possibly contribute to specification of their function. In the current review, we discuss the evolution and the biology of the APP protein family with special attention to the distinct properties of each homologue. We propose that the functions of APP, APLP1 and APLP2 have diverged after duplication to contribute distinctly to different neuronal events. Our analysis reveals that APLP2 is significantly diverged from APP and APLP1. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

The impact of F-PEC scale and strategic planning in the family business

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Nazanin Haji Mirza Hossein Yazdi

2018-01-01

Full Text Available Due to the thrive of family businesses in the current economic situation, to solve the problem of the definition of these businesses, in this paper we suggest using a scale that measures the quality of family influence through three dimensions: power, experience, and culture. The family influence on power, experience and culture (F-PEC scale, as well as strategic planning, have been tested precisely using a random sample of more than 211 people from two companies of Golestan and Shemshal industrial group located in Tehran, Iran through using the confirmatory factor analysis technique. The Scale shows a high level of reliability. F-PEC has been used in a number of studies to theoretical development, particularly in terms of the impact of family influence on the individual resources and as a source of competitive advantage. In this research, strategic planning is also considered as a source of competitive advantage and its impact on the success of the family business has been investigated.

Closure of an analytical chemistry glove box in alpha laboratory

International Nuclear Information System (INIS)

Adelfang, P.; Aparicio, G.; Cassaniti, P.

1990-01-01

The works with plutonium are performed in gloves box, operated below atmospheric pressure, to protect the experimenters from this alpha-active material. After 12 years of continual processes, it was necessary the decommissioning of the chemistry glove box in our alpha-laboratory. A great deal of our attention was devoted to the working techniques because of extreme care needed to avoid activity release. The decommissioning includes the following main operations: a) Planning and documentation for the regulatory authority. b) Internal decontamination with surface cleaning and chelating agents. c) Measurement of the remainder internal radioactivity. d) Sealing of the glove ports and nozzles. e) Disconnection of the glove box from the exhaust duct. f) Design and construction of a container for the glove box. g) Transportation of the glove box from alpha-laboratory, to a transitory storage until its final disposal. The above mentioned operations are described in this paper including too: data of personal doses during the operations, characteristics and volumes of radioactive wastes and a description of the instrument used for the measurement of inside glove box activity. (Author) [es

Transcriptional control of the tissue-specific, developmentally regulated osteocalcin gene requires a binding motif for the Msx family of homeodomain proteins.

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Hoffmann, H M; Catron, K M; van Wijnen, A J; McCabe, L R; Lian, J B; Stein, G S; Stein, J L

1994-12-20

The OC box of the rat osteocalcin promoter (nt -99 to -76) is the principal proximal regulatory element contributing to both tissue-specific and developmental control of osteocalcin gene expression. The central motif of the OC box includes a perfect consensus DNA binding site for certain homeodomain proteins. Homeodomain proteins are transcription factors that direct proper development by regulating specific temporal and spatial patterns of gene expression. We therefore addressed the role of the homeodomain binding motif in the activity of the OC promoter. In this study, by the combined application of mutagenesis and site-specific protein recognition analysis, we examined interactions of ROS 17/2.8 osteosarcoma cell nuclear proteins and purified Msx-1 homeodomain protein with the OC box. We detected a series of related specific protein-DNA interactions, a subset of which were inhibited by antibodies directed against the Msx-1 homeodomain but which also recognize the Msx-2 homeodomain. Our results show that the sequence requirements for binding the Msx-1 or Msx-2 homeodomain closely parallel those necessary for osteocalcin gene promoter activity in vivo. This functional relationship was demonstrated by transient expression in ROS 17/2.8 osteosarcoma cells of a series of osteocalcin promoter (nt -1097 to +24)-reporter gene constructs containing mutations within and flanking the homeodomain binding site of the OC box. Northern blot analysis of several bone-related cell types showed that all of the cells expressed msx-1, whereas msx-2 expression was restricted to cells transcribing osteocalcin. Taken together, our results suggest a role for Msx-1 and -2 or related homeodomain proteins in transcription of the osteocalcin gene.

NMR studies of a new family of DNA binding proteins: the THAP proteins

International Nuclear Information System (INIS)

Gervais, Virginie; Campagne, Sébastien; Durand, Jade; Muller, Isabelle; Milon, Alain

2013-01-01

The THAP (THanatos-Associated Protein) domain is an evolutionary conserved C2CH zinc-coordinating domain shared with a large family of cellular factors (THAP proteins). Many members of the THAP family act as transcription factors that control cell proliferation, cell cycle progression, angiogenesis, apoptosis and epigenetic gene silencing. They recognize specific DNA sequences in the promoters of target genes and subsequently recruit effector proteins. Recent structural and functional studies have allowed getting better insight into the nuclear and cellular functions of some THAP members and the molecular mechanisms by which they recognize DNA. The present article reviews recent advances in the knowledge of the THAP domains structures and their interaction with DNA, with a particular focus on NMR. It provides the solution structure of the THAP domain of THAP11, a recently characterized human THAP protein with important functions in transcription and cell growth in colon cancer.

NMR studies of a new family of DNA binding proteins: the THAP proteins

Energy Technology Data Exchange (ETDEWEB)

Gervais, Virginie, E-mail: virginie.gervais@ipbs.fr [IPBS (Institut de Pharmacologie et de Biologie Structurale), CNRS (France); Campagne, Sebastien [ETH Zurich (Switzerland); Durand, Jade; Muller, Isabelle; Milon, Alain, E-mail: alain.milon@ipbs.fr [IPBS (Institut de Pharmacologie et de Biologie Structurale), CNRS (France)

2013-05-15

The THAP (THanatos-Associated Protein) domain is an evolutionary conserved C2CH zinc-coordinating domain shared with a large family of cellular factors (THAP proteins). Many members of the THAP family act as transcription factors that control cell proliferation, cell cycle progression, angiogenesis, apoptosis and epigenetic gene silencing. They recognize specific DNA sequences in the promoters of target genes and subsequently recruit effector proteins. Recent structural and functional studies have allowed getting better insight into the nuclear and cellular functions of some THAP members and the molecular mechanisms by which they recognize DNA. The present article reviews recent advances in the knowledge of the THAP domains structures and their interaction with DNA, with a particular focus on NMR. It provides the solution structure of the THAP domain of THAP11, a recently characterized human THAP protein with important functions in transcription and cell growth in colon cancer.

The Sorcerer II Global Ocean Sampling Expedition: Expanding theUniverse of Protein Families

Energy Technology Data Exchange (ETDEWEB)

Yooseph, Shibu; Sutton, Granger; Rusch, Douglas B.; Halpern,Aaron L.; Williamson, Shannon J.; Remington, Karin; Eisen, Jonathan A.; Heidelberg, Karla B.; Manning, Gerard; Li, Weizhong; Jaroszewski, Lukasz; Cieplak, Piotr; Miller, Christopher S.; Li, Huiying; Mashiyama, Susan T.; Joachimiak, Marcin P.; van Belle, Christopher; Chandonia, John-Marc; Soergel, David A.; Zhai, Yufeng; Natarajan, Kannan; Lee, Shaun; Raphael,Benjamin J.; Bafna, Vineet; Friedman, Robert; Brenner, Steven E.; Godzik,Adam; Eisenberg, David; Dixon, Jack E.; Taylor, Susan S.; Strausberg,Robert L.; Frazier, Marvin; Venter, J.Craig

2006-03-23

Metagenomics projects based on shotgun sequencing of populations of micro-organisms yield insight into protein families. We used sequence similarity clustering to explore proteins with a comprehensive dataset consisting of sequences from available databases together with 6.12 million proteins predicted from an assembly of 7.7 million Global Ocean Sampling (GOS) sequences. The GOS dataset covers nearly all known prokaryotic protein families. A total of 3,995 medium- and large-sized clusters consisting of only GOS sequences are identified, out of which 1,700 have no detectable homology to known families. The GOS-only clusters contain a higher than expected proportion of sequences of viral origin, thus reflecting a poor sampling of viral diversity until now. Protein domain distributions in the GOS dataset and current protein databases show distinct biases. Several protein domains that were previously categorized as kingdom specific are shown to have GOS examples in other kingdoms. About 6,000 sequences (ORFans) from the literature that heretofore lacked similarity to known proteins have matches in the GOS data. The GOS dataset is also used to improve remote homology detection. Overall, besides nearly doubling the number of current proteins, the predicted GOS proteins also add a great deal of diversity to known protein families and shed light on their evolution. These observations are illustrated using several protein families, including phosphatases, proteases, ultraviolet-irradiation DNA damage repair enzymes, glutamine synthetase, and RuBisCO. The diversity added by GOS data has implications for choosing targets for experimental structure characterization as part of structural genomics efforts. Our analysis indicates that new families are being discovered at a rate that is linear or almost linear with the addition of new sequences, implying that we are still far from discovering all protein families in nature.

The Sorcerer II Global Ocean Sampling expedition: expanding the universe of protein families.

Science.gov (United States)

Yooseph, Shibu; Sutton, Granger; Rusch, Douglas B; Halpern, Aaron L; Williamson, Shannon J; Remington, Karin; Eisen, Jonathan A; Heidelberg, Karla B; Manning, Gerard; Li, Weizhong; Jaroszewski, Lukasz; Cieplak, Piotr; Miller, Christopher S; Li, Huiying; Mashiyama, Susan T; Joachimiak, Marcin P; van Belle, Christopher; Chandonia, John-Marc; Soergel, David A; Zhai, Yufeng; Natarajan, Kannan; Lee, Shaun; Raphael, Benjamin J; Bafna, Vineet; Friedman, Robert; Brenner, Steven E; Godzik, Adam; Eisenberg, David; Dixon, Jack E; Taylor, Susan S; Strausberg, Robert L; Frazier, Marvin; Venter, J Craig

2007-03-01

Metagenomics projects based on shotgun sequencing of populations of micro-organisms yield insight into protein families. We used sequence similarity clustering to explore proteins with a comprehensive dataset consisting of sequences from available databases together with 6.12 million proteins predicted from an assembly of 7.7 million Global Ocean Sampling (GOS) sequences. The GOS dataset covers nearly all known prokaryotic protein families. A total of 3,995 medium- and large-sized clusters consisting of only GOS sequences are identified, out of which 1,700 have no detectable homology to known families. The GOS-only clusters contain a higher than expected proportion of sequences of viral origin, thus reflecting a poor sampling of viral diversity until now. Protein domain distributions in the GOS dataset and current protein databases show distinct biases. Several protein domains that were previously categorized as kingdom specific are shown to have GOS examples in other kingdoms. About 6,000 sequences (ORFans) from the literature that heretofore lacked similarity to known proteins have matches in the GOS data. The GOS dataset is also used to improve remote homology detection. Overall, besides nearly doubling the number of current proteins, the predicted GOS proteins also add a great deal of diversity to known protein families and shed light on their evolution. These observations are illustrated using several protein families, including phosphatases, proteases, ultraviolet-irradiation DNA damage repair enzymes, glutamine synthetase, and RuBisCO. The diversity added by GOS data has implications for choosing targets for experimental structure characterization as part of structural genomics efforts. Our analysis indicates that new families are being discovered at a rate that is linear or almost linear with the addition of new sequences, implying that we are still far from discovering all protein families in nature.

The Sorcerer II Global Ocean Sampling expedition: expanding the universe of protein families.

Directory of Open Access Journals (Sweden)

Shibu Yooseph

2007-03-01

Full Text Available Metagenomics projects based on shotgun sequencing of populations of micro-organisms yield insight into protein families. We used sequence similarity clustering to explore proteins with a comprehensive dataset consisting of sequences from available databases together with 6.12 million proteins predicted from an assembly of 7.7 million Global Ocean Sampling (GOS sequences. The GOS dataset covers nearly all known prokaryotic protein families. A total of 3,995 medium- and large-sized clusters consisting of only GOS sequences are identified, out of which 1,700 have no detectable homology to known families. The GOS-only clusters contain a higher than expected proportion of sequences of viral origin, thus reflecting a poor sampling of viral diversity until now. Protein domain distributions in the GOS dataset and current protein databases show distinct biases. Several protein domains that were previously categorized as kingdom specific are shown to have GOS examples in other kingdoms. About 6,000 sequences (ORFans from the literature that heretofore lacked similarity to known proteins have matches in the GOS data. The GOS dataset is also used to improve remote homology detection. Overall, besides nearly doubling the number of current proteins, the predicted GOS proteins also add a great deal of diversity to known protein families and shed light on their evolution. These observations are illustrated using several protein families, including phosphatases, proteases, ultraviolet-irradiation DNA damage repair enzymes, glutamine synthetase, and RuBisCO. The diversity added by GOS data has implications for choosing targets for experimental structure characterization as part of structural genomics efforts. Our analysis indicates that new families are being discovered at a rate that is linear or almost linear with the addition of new sequences, implying that we are still far from discovering all protein families in nature.

MADS goes genomic in conifers: towards determining the ancestral set of MADS-box genes in seed plants.

Science.gov (United States)

Gramzow, Lydia; Weilandt, Lisa; Theißen, Günter

2014-11-01

MADS-box genes comprise a gene family coding for transcription factors. This gene family expanded greatly during land plant evolution such that the number of MADS-box genes ranges from one or two in green algae to around 100 in angiosperms. Given the crucial functions of MADS-box genes for nearly all aspects of plant development, the expansion of this gene family probably contributed to the increasing complexity of plants. However, the expansion of MADS-box genes during one important step of land plant evolution, namely the origin of seed plants, remains poorly understood due to the previous lack of whole-genome data for gymnosperms. The newly available genome sequences of Picea abies, Picea glauca and Pinus taeda were used to identify the complete set of MADS-box genes in these conifers. In addition, MADS-box genes were identified in the growing number of transcriptomes available for gymnosperms. With these datasets, phylogenies were constructed to determine the ancestral set of MADS-box genes of seed plants and to infer the ancestral functions of these genes. Type I MADS-box genes are under-represented in gymnosperms and only a minimum of two Type I MADS-box genes have been present in the most recent common ancestor (MRCA) of seed plants. In contrast, a large number of Type II MADS-box genes were found in gymnosperms. The MRCA of extant seed plants probably possessed at least 11-14 Type II MADS-box genes. In gymnosperms two duplications of Type II MADS-box genes were found, such that the MRCA of extant gymnosperms had at least 14-16 Type II MADS-box genes. The implied ancestral set of MADS-box genes for seed plants shows simplicity for Type I MADS-box genes and remarkable complexity for Type II MADS-box genes in terms of phylogeny and putative functions. The analysis of transcriptome data reveals that gymnosperm MADS-box genes are expressed in a great variety of tissues, indicating diverse roles of MADS-box genes for the development of gymnosperms. This study is

Expression of GIMAP1, a GTPase of the immunity-associated protein family, is not up-regulated in malaria

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Carter Christine

2009-04-01

Full Text Available Abstract Background GIMAP (GTPase of the immunity-associated protein family proteins are a family of putative GTPases believed to be regulators of cell death in lymphomyeloid cells. GIMAP1 was the first reported member of this gene family, identified as a gene up-regulated at the RNA level in the spleens of mice infected with the malarial parasite, Plasmodium chabaudi. Methods A monoclonal antibody against mouse GIMAP1 was developed and was used to analyse the expression of the endogenous protein in tissues of normal mice and in defined sub-populations of cells prepared from lymphoid tissues using flow cytometry. It was also used to assess the expression of GIMAP1 protein after infection and/or immunization of mice with P. chabaudi. Real-time PCR analysis was employed to measure the expression of GIMAP1 for comparison with the protein level analysis. Results GIMAP1 protein expression was detected in all lineages of lymphocytes (T, B, NK, in F4/80+ splenic macrophages and in some lymphoid cell lines. Additional evidence is presented suggesting that the strong expression by mature B cells of GIMAP1 and other GIMAP genes and proteins seen in mice may be a species-dependent characteristic. Unexpectedly, no increase was found in the expression of GIMAP1 in P. chabaudi infected mice at either the mRNA or protein level, and this remained so despite applying a number of variations to the protocol. Conclusion The model of up-regulation of GIMAP1 in response to infection/immunization with P. chabaudi is not a robustly reproducible experimental system. The GIMAP1 protein is widely expressed in lymphoid cells, with an interesting increase in expression in the later stages of B cell development. Alternative approaches will be required to define the functional role of this GTPase in immune cells.

Release of the repressive activity of rice DELLA protein SLR1 by gibberellin does not require SLR1 degradation in the gid2 mutant.

Science.gov (United States)

Ueguchi-Tanaka, Miyako; Hirano, Ko; Hasegawa, Yasuko; Kitano, Hidemi; Matsuoka, Makoto

2008-09-01

The rice (Oryza sativa) DELLA protein SLR1 acts as a repressor of gibberellin (GA) signaling. GA perception by GID1 causes SLR1 protein degradation involving the F-box protein GID2; this triggers GA-associated responses such as shoot elongation and seed germination. In GA-insensitive and GA biosynthesis mutants, SLENDER RICE1 (SLR1) accumulates to high levels, and the severity of dwarfism is usually correlated with the level of SLR1 accumulation. An exception is the GA-insensitive F-box mutant gid2, which shows milder dwarfism than mutants such as gid1 and cps even though it accumulates higher levels of SLR1. The level of SLR1 protein in gid2 was decreased by loss of GID1 function or treatment with a GA biosynthesis inhibitor, and dwarfism was enhanced. Conversely, overproduction of GID1 or treatment with GA(3) increased the SLR1 level in gid2 and reduced dwarfism. These results indicate that derepression of SLR1 repressive activity can be accomplished by GA and GID1 alone and does not require F-box (GID2) function. Evidence for GA signaling without GID2 was also provided by the expression behavior of GA-regulated genes such as GA-20oxidase1, GID1, and SLR1 in the gid2 mutant. Based on these observations, we propose a model for the release of GA suppression that does not require DELLA protein degradation.

The Andalusian Bipolar Family (ABiF) Study: Protocol and sample description.

Science.gov (United States)

Guzman-Parra, Jose; Rivas, Fabio; Strohmaier, Jana; Forstner, Andreas; Streit, Fabian; Auburger, Georg; Propping, Peter; Orozco-Diaz, Guillermo; González, Maria José; Gil-Flores, Susana; Cabaleiro-Fabeiro, Francisco Javier; Del Río-Noriega, Francisco; Perez-Perez, Fermin; Haro-González, Jesus; de Diego-Otero, Yolanda; Romero-Sanchiz, Pablo; Moreno-Küstner, Berta; Cichon, Sven; Nöthen, Markus M; Rietschel, Marcella; Mayoral, Fermin

2017-06-12

Here, we present the first description of the Andalusian Bipolar Family (ABiF) Study. This longitudinal investigation of families from Andalusia, Spain commenced in 1997 with the aim of elucidating the molecular genetic causes of bipolar affective disorder. The cohort has since contributed to a number of key genetic findings, as reported in international journals. However, insight into the genetic underpinnings of the disorder in these families remains limited. In the initial 1997-2003 study phase, 100 multiplex bipolar disorder and other mood disorder families were recruited. The ongoing second phase of the project commenced in 2013, and involves follow-up of a subgroup of the originally recruited families. The aim of the follow-up investigation is to generate: i) longitudinal clinical data; ii) results from detailed neuropsychological assessments; and iii) a more extensive collection of biomaterials for future molecular biological studies. The ABiF Study will thus generate a valuable resource for future investigations into the aetiology of bipolar affective disorder; in particular the causes of high disease loading within multiply affected families. We discuss the value of this approach in terms of new technologies for the identification of high-penetrance genetic factors. These new technologies include exome and whole genome sequencing, and the use of induced pluripotent stem cells or model organisms to determine functional consequences. Copyright © 2017 SEP y SEPB. Publicado por Elsevier España, S.L.U. All rights reserved.

Boxing-acute complications and late sequelae: from concussion to dementia.

Science.gov (United States)

Förstl, Hans; Haass, Christian; Hemmer, Bernhard; Meyer, Bernhard; Halle, Martin

2010-11-01

Boxing has received increased public attention and acceptance in recent years. However, this development has not been accompanied by a critical discussion of the early and late health complications. We selectively review recent studies on the acute, subacute, and chronic neuropsychiatric consequences of boxing. Cerebral concussions ("knock-outs") are the most relevant acute consequence of boxing. The number of reported cases of death in the ring seems to have mildly decreased. Subacute neuropsychological deficits appear to last longer than subjective symptoms. The associated molecular changes demonstrate neuronal and glial injury correlated with the number and severity of blows to the head (altered total tau, beta-amyloid, neurofilament light protein, glial fibrillary acidic protein, and neuron-specific enolase). The risk of a punch-drunk syndrome (boxer's dementia, dementia pugilistica) as a late effect of chronic traumatic brain injury is associated with the duration of a boxer's career and with his earlier stamina. There are similarities (e.g. increased risk with ApoE4-polymorphism, beta-amyloid pathology) and differences (more tau pathology in boxers) compared with Alzheimer's disease. Protective gear has led to a remarkable reduction of risks in amateur boxing. Similar measures can also be used in professional boxing, but may decrease the thrill, which does appeal to many supporters.

Characterization of differential ripening pattern in association with ethylene biosynthesis in the fruits of five naturally occurring banana cultivars and detection of a GCC-box-specific DNA-binding protein.

Science.gov (United States)

Choudhury, Swarup Roy; Roy, Sujit; Saha, Progya Paramita; Singh, Sanjay Kumar; Sengupta, Dibyendu N

2008-07-01

MA-ACS1 and MA-ACO1 are the two major ripening genes in banana and play crucial role in the regulation of ethylene production during ripening. Here, we report a comparative ripening pattern in five different naturally occurring banana cultivars namely Cavendish (AAA), Rasthali (AAB), Kanthali (AB), Poovan (AAB) and Monthan (ABB), which have distinct genome composition. We found a distinct variation in the climacteric ethylene production and in-vivo ACC oxidase activity level during the ripening stages in the five cultivars. We identified the cDNAs for MA-ACS1 and MA-ACO1 from the five cultivars and studied the transcript accumulation patterns of the two genes, which correlated well with the differential timing in the expression of these two genes during ripening. The GCC-box is one of the ethylene-responsive elements (EREs) found in the promoters of many ethylene-inducible genes. We have identified a GCC-box motif (putative ERE) in the promoters of MA-ACS1 and MA-ACO1 in banana cultivars. DNA-protein interaction studies revealed the presence of a GCC-box-specific DNA-binding activity in the fruit nuclear extract and such DNA-binding activity was enhanced following ethylene treatment. South-Western blotting revealed a 25-kDa nuclear protein that binds specifically to GCC-box DNA in the climacteric banana fruit. Together, these results indicate the probable involvement of the GCC-box motif as the cis-acting ERE in the regulation of MA-ACS1 and MA-ACO1 during ripening in banana fruits via binding of specific ERE-binding protein.

Fibrillarin and Nop56 interact before being co-assembled in box C/D snoRNPs

International Nuclear Information System (INIS)

Lechertier, Tanguy; Grob, Alice; Hernandez-Verdun, Daniele; Roussel, Pascal

2009-01-01

Small nucleolar RNAs play crucial roles in ribosome biogenesis. They guide folding, site-specific nucleotide modifications and participate in cleavage of precursor ribosomal RNAs. To better understand how the biogenesis of the box C/D small nucleolar RNPs (snoRNPs) occur in a cellular context, we used a new approach based on the possibility of relocalizing a given nuclear complex by adding an affinity tag for B23 to one component of this complex. We selectively delocalized each core box C/D protein, namely 15.5kD, Nop56, Nop58 and fibrillarin, and analyzed the effect of such changes on other components of the box C/D snoRNPs. We show that modifying the localization and the mobility of core box C/D proteins impairs their association with box C/D snoRNPs. In addition, we demonstrate that fibrillarin and Nop56 directly interact in vivo. This interaction, indispensable for the association of both proteins with the box C/D snoRNPs, does not involve the glycine- and arginine-rich domain or the RNA-binding domain but the alpha-helix domain of fibrillarin. In addition, no RNA seems required to maintain fibrillarin-Nop56 interaction

Cdc20 mediates D-box-dependent degradation of Sp100

Energy Technology Data Exchange (ETDEWEB)

Wang, Ran; Li, Ke-min; Zhou, Cai-hong; Xue, Jing-lun [State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai (China); Ji, Chao-neng, E-mail: Chnji@fudan.edu.cn [State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai (China); Chen, Jin-zhong, E-mail: kingbellchen@fudan.edu.cn [State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai (China)

2011-12-02

Highlights: Black-Right-Pointing-Pointer Cdc20 is a co-activator of APC/C complex. Black-Right-Pointing-Pointer Cdc20 recruits Sp100 and mediates its degradation. Black-Right-Pointing-Pointer The D-box of Sp100 is required for Cdc20-mediated degradation. Black-Right-Pointing-Pointer Sp100 expresses consistently at both the mRNA and protein levels in cell cycle. -- Abstract: Cdc20 is a co-activator of the anaphase-promoting complex/cyclosome (APC/C complex), which recruits substrates at particular phases of the cell cycle and mediates their degradation. Sp100 is a PML-NB scaffold protein, which localizes to nuclear particles during interphase and disperses from them during mitosis, participates in viral resistance, transcriptional regulation, and apoptosis. However, its metabolism during the cell cycle has not yet been fully characterized. We found a putative D-box in Sp100 using the Eukaryotic Linear Motif (ELM) predictor database. The putative D-box of Sp100 was verified by mutational analysis. Overexpression of Cdc20 resulted in decreased levels of both endogenous Sp100 protein and overexpressed Sp100 mRNA in HEK 293 cells. Only an overexpressed D-box deletion mutant of Sp100 accumulated in HEK293 cells that also overexpressed Cdc20. Cdc20 knockdown by cdc20 specific siRNA resulted in increased Sp100 protein levels in cells. Furthermore, we discovered that the Cdc20 mediated degradation of Sp100 is diminished by the proteasome inhibitor MG132, which suggests that the ubiquitination pathway is involved in this process. However, unlike the other Cdc20 substrates, which display oscillating protein levels, the level of Sp100 protein remains constant throughout the cell cycle. Additionally, both overexpression and knockdown of endogenous Sp100 had no effect on the cell cycle. Our results suggested that sp100 is a novel substrate of Cdc20 and it is degraded by the ubiquitination pathway. The intact D-box of Sp100 was necessary for this process. These findings expand

The dengue vector Aedes aegypti contains a functional high mobility group box 1 (HMGB1 protein with a unique regulatory C-terminus.

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Fabio Schneider Ribeiro

Full Text Available The mosquito Aedes aegypti can spread the dengue, chikungunya and yellow fever viruses. Thus, the search for key molecules involved in the mosquito survival represents today a promising vector control strategy. High Mobility Group Box (HMGB proteins are essential nuclear factors that maintain the high-order structure of chromatin, keeping eukaryotic cells viable. Outside the nucleus, secreted HMGB proteins could alert the innate immune system to foreign antigens and trigger the initiation of host defenses. In this work, we cloned and functionally characterized the HMGB1 protein from Aedes aegypti (AaHMGB1. The AaHMGB1 protein typically consists of two HMG-box DNA binding domains and an acidic C-terminus. Interestingly, AaHMGB1 contains a unique alanine/glutamine-rich (AQ-rich C-terminal region that seems to be exclusive of dipteran HMGB proteins. AaHMGB1 is localized to the cell nucleus, mainly associated with heterochromatin. Circular dichroism analyses of AaHMGB1 or the C-terminal truncated proteins revealed α-helical structures. We showed that AaHMGB1 can effectively bind and change the topology of DNA, and that the AQ-rich and the C-terminal acidic regions can modulate its ability to promote DNA supercoiling, as well as its preference to bind supercoiled DNA. AaHMGB1 is phosphorylated by PKA and PKC, but not by CK2. Importantly, phosphorylation of AaHMGB1 by PKA or PKC completely abolishes its DNA bending activity. Thus, our study shows that a functional HMGB1 protein occurs in Aedes aegypt and we provide the first description of a HMGB1 protein containing an AQ-rich regulatory C-terminus.

Nonlinear analysis of sequence symmetry of beta-trefoil family proteins

Energy Technology Data Exchange (ETDEWEB)

Li Mingfeng [Biomolecular Physics and Modeling Group, Department of Physics, Huazhong University of Science and Technology, Wuhan 430074, Hubei (China); Huang Yanzhao [Biomolecular Physics and Modeling Group, Department of Physics, Huazhong University of Science and Technology, Wuhan 430074, Hubei (China); Xu Ruizhen [Biomolecular Physics and Modeling Group, Department of Physics, Huazhong University of Science and Technology, Wuhan 430074, Hubei (China); Xiao Yi [Biomolecular Physics and Modeling Group, Department of Physics, Huazhong University of Science and Technology, Wuhan 430074, Hubei (China)]. E-mail: yxiao@mail.hust.edu.cn

2005-07-01

The tertiary structures of proteins of beta-trefoil family have three-fold quasi-symmetry while their amino acid sequences appear almost at random. In the present paper we show that these amino acid sequences have hidden symmetries in fact and furthermore the degrees of these hidden symmetries are the same as those of their tertiary structures. We shall present a modified recurrence plot to reveal hidden symmetries in protein sequences. Our results can explain the contradiction in sequence-structure relations of proteins of beta-trefoil family.

From particle in a box to PT -symmetric systems via isospectral deformation

OpenAIRE

Cherian, Philip; Abhinav, Kumar; Panigrahi, P. K.

2011-01-01

A family of PT -symmetric complex potentials is obtained, which is isospectral to free particle in an infinite complex box in one dimension (1-D). These are generalizations to the cosec2 (x) potential, isospectral to particle in a real infinite box. In the complex plane, the infinite box is extended parallel to the real axis having a real width, which is found to be an integral multiple of a constant quantum factor, arising due to boundary conditions necessary for maintaining the PT -symmetry...

A Novel Sucrose-Regulatory MADS-Box Transcription Factor GmNMHC5 Promotes Root Development and Nodulation in Soybean (Glycine max [L.] Merr.

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Wei Liu

2015-08-01

Full Text Available The MADS-box protein family includes many transcription factors that have a conserved DNA-binding MADS-box domain. The proteins in this family were originally recognized to play prominent roles in floral development. Recent findings, especially with regard to the regulatory roles of the AGL17 subfamily in root development, have greatly broadened their known functions. In this study, a gene from soybean (Glycine max [L.] Merr., GmNMHC5, was cloned from the Zigongdongdou cultivar and identified as a member of the AGL17 subfamily. Real-time fluorescence quantitative PCR analysis showed that GmNMHC5 was expressed at much higher levels in roots and nodules than in other organs. The activation of expression was first examined in leaves and roots, followed by shoot apexes. GmNMHC5 expression levels rose sharply when the plants were treated under short-day conditions (SD and started to pod, whereas low levels were maintained in non-podding plants under long-day conditions (LD. Furthermore, overexpression of GmNMHC5 in transgenic soybean significantly promoted lateral root development and nodule building. Moreover, GmNMHC5 is upregulated by exogenous sucrose. These results indicate that GmNMHC5 can sense the sucrose signal and plays significant roles in lateral root development and nodule building.

Identification and characterization of the direct interaction between methotrexate (MTX and high-mobility group box 1 (HMGB1 protein.

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Yuki Kuroiwa

Full Text Available BACKGROUND: Methotrexate (MTX is an agent used in chemotherapy of tumors and autoimmune disease including rheumatoid arthritis (RA. In addition, MTX has some anti-inflammatory activity. Although dihydrofolate reductase (DHFR is a well-known target for the anti-tumor effect of MTX, the mode of action for the anti-inflammatory activity of MTX is not fully understood. METHODOLOGY/RESULT: Here, we performed a screening of MTX-binding proteins using T7 phage display with a synthetic biotinylated MTX derivative. We then characterized the interactions using surface plasmon resonance (SPR analysis and electrophoretic mobility shift assay (EMSA. Using a T7 phage display screen, we identified T7 phages that displayed part of high-mobility group box 1 (HMGB1 protein (K86-V175. Binding affinities as well as likely binding sites were characterized using genetically engineered truncated versions of HMGB1 protein (Al G1-K87, Bj: F88-K181, indicating that MTX binds to HMGB1 via two independent sites with a dissociation constants (KD of 0.50±0.03 µM for Al and 0.24 ± 0.01 µM for Bj. Although MTX did not inhibit the binding of HMGB1 to DNA via these domains, HMGB1/RAGE association was impeded in the presence of MTX. These data suggested that binding of MTX to part of the RAGE-binding region (K149-V175 in HMGB1 might be significant for the anti-inflammatory effect of MTX. Indeed, in murine macrophage-like cells (RAW 264.7, TNF-α release and mitogenic activity elicited by specific RAGE stimulation with a truncated monomeric HMGB1 were inhibited in the presence of MTX. CONCLUSIONS/SIGNIFICANCE: These data demonstrate that HMGB1 is a direct binding protein of MTX. Moreover, binding of MTX to RAGE-binding region in HMGB1 inhibited the HMGB1/RAGE interaction at the molecular and cellular levels. These data might explain the molecular basis underlying the mechanism of action for the anti-inflammatory effect of MTX.

Structural insights and ab initio sequencing within the DING proteins family

International Nuclear Information System (INIS)

Elias, Mikael; Liebschner, Dorothee; Gotthard, Guillaume; Chabriere, Eric

2011-01-01

DING proteins constitute a recently discovered protein family that is ubiquitous in eukaryotes. The structural insights and the physiological involvements of these intriguing proteins are hereby deciphered. DING proteins constitute an intriguing family of phosphate-binding proteins that was identified in a wide range of organisms, from prokaryotes and archae to eukaryotes. Despite their seemingly ubiquitous occurrence in eukaryotes, their encoding genes are missing from sequenced genomes. Such a lack has considerably hampered functional studies. In humans, these proteins have been related to several diseases, like atherosclerosis, kidney stones, inflammation processes and HIV inhibition. The human phosphate binding protein is a human representative of the DING family that was serendipitously discovered from human plasma. An original approach was developed to determine ab initio the complete and exact sequence of this 38 kDa protein by utilizing mass spectrometry and X-ray data in tandem. Taking advantage of this first complete eukaryotic DING sequence, a immunohistochemistry study was undertaken to check the presence of DING proteins in various mice tissues, revealing that these proteins are widely expressed. Finally, the structure of a bacterial representative from Pseudomonas fluorescens was solved at sub-angstrom resolution, allowing the molecular mechanism of the phosphate binding in these high-affinity proteins to be elucidated

Structural insights and ab initio sequencing within the DING proteins family

Energy Technology Data Exchange (ETDEWEB)

Elias, Mikael, E-mail: mikael.elias@weizmann.ac.il [Weizmann Institute of Science, Rehovot (Israel); Liebschner, Dorothee [CRM2, Nancy Université (France); Gotthard, Guillaume; Chabriere, Eric [AFMB, Université Aix-Marseille II (France)

2011-01-01

DING proteins constitute a recently discovered protein family that is ubiquitous in eukaryotes. The structural insights and the physiological involvements of these intriguing proteins are hereby deciphered. DING proteins constitute an intriguing family of phosphate-binding proteins that was identified in a wide range of organisms, from prokaryotes and archae to eukaryotes. Despite their seemingly ubiquitous occurrence in eukaryotes, their encoding genes are missing from sequenced genomes. Such a lack has considerably hampered functional studies. In humans, these proteins have been related to several diseases, like atherosclerosis, kidney stones, inflammation processes and HIV inhibition. The human phosphate binding protein is a human representative of the DING family that was serendipitously discovered from human plasma. An original approach was developed to determine ab initio the complete and exact sequence of this 38 kDa protein by utilizing mass spectrometry and X-ray data in tandem. Taking advantage of this first complete eukaryotic DING sequence, a immunohistochemistry study was undertaken to check the presence of DING proteins in various mice tissues, revealing that these proteins are widely expressed. Finally, the structure of a bacterial representative from Pseudomonas fluorescens was solved at sub-angstrom resolution, allowing the molecular mechanism of the phosphate binding in these high-affinity proteins to be elucidated.

A study of airborne chrysotile concentrations associated with handling, unpacking, and repacking boxes of automobile clutch discs.

Science.gov (United States)

Jiang, George C T; Madl, Amy K; Ingmundson, Kelsey J; Murbach, Dana M; Fehling, Kurt A; Paustenbach, Dennis J; Finley, Brent L

2008-06-01

Although automotive friction products (brakes and manual clutches) historically contained chrysotile asbestos, industrial hygiene surveys and epidemiologic studies of auto mechanics have consistently shown that these workers are not at an increased risk of developing asbestos-related diseases. Airborne asbestos levels during brake repair and brake parts handling have been well-characterized, but the potential exposure to airborne asbestos fibers during the handling of clutch parts has not been examined. In this study, breathing zone samples on the lapel of a volunteer worker (n=100) and area samples at bystander (n=50), remote area (n=25), and ambient (n=9) locations collected during the stacking, unpacking, and repacking of boxes of asbestos-containing clutches, and the subsequent cleanup and clothes handling, were analyzed by phase contrast microscopy (PCM) and transmission electron microscopy (TEM). In addition, fiber morphology and size distribution was evaluated using X-ray diffraction, polarized light microscopy, and ISO analytical methods. It was observed that the (1) airborne asbestos concentrations increased with the number of boxes unpacked and repacked, (2) repetitive stacking of unopened boxes of clutches resulted in higher asbestos concentrations than unpacking and repacking the boxes of clutches, (3) cleanup and clothes handling tasks yielded very low asbestos concentrations. Fiber size and morphology analyses showed that amphibole fibers were not detected in the clutches and that the vast majority (>95%) of the airborne chrysotile fibers were less than 20 microm in length. Applying the ratio of asbestos fibers:total fibers (including non-asbestos) as determined by TEM to the PCM results, it was found that 30-min average airborne chrysotile concentrations (PCM adjusted) were 0.026+/-0.004 f/cc or 0.100+/-0.017 f/cc for a worker unpacking and repacking 1 or 2 boxes of clutches, respectively. The 30-min PCM adjusted average airborne asbestos

High mobility group box-1 is phosphorylated by protein kinase C zeta and secreted in colon cancer cells

International Nuclear Information System (INIS)

Lee, Hanna; Park, Minhee; Shin, Nara; Kim, Gamin; Kim, Yun Gi; Shin, Jeon-Soo; Kim, Hoguen

2012-01-01

Highlights: ► Specific enzyme for HMGB1 phosphorylation and its secretion is proposed. ► Inhibition of PKC-ζ leads to significant reduction of the secreted HMGB1. ► Phosphorylation of specific site of HMGB1 redirects its secretion in cancer cells. ► Activation of PKC-ζ in cancers explains the enhanced HMGB1 secretion. -- Abstract: High mobility group box-1 (HMGB1), a nuclear protein, is overexpressed and secreted in cancer cells. Phosphorylation on two different nuclear localization signal regions are known to be important for the nuclear-to-cytoplasmic transport and secretion of HMGB1. However, little is known about the biochemical mechanism of HMGB1 modifications and its subsequent secretion from cancer cells. To identify the specific enzyme and important sites for HMGB1 phosphorylation, we screened the protein kinase C (PKC) family in a colon cancer cell line (HCT116) for HMGB1 binding by pull-down experiments using a 3XFLAG-HMGB1 construct. Strong interactions between atypical PKCs (PKC-ζ, λ, and ι) and cytoplasmic HMGB1 were observed in HCT116 cells. We further identified the most critical PKC isotype that regulates HMGB1 secretion is PKC-ζ by using PKC inhibitors and siRNA experiments. The serine residues at S39, S53 and S181 of HMGB1 were related to enhancing HMGB1 secretion. We also demonstrated overexpression and activation of PKC-ζ in colon cancer tissues. Our findings suggest that PKC-ζ is involved in the phosphorylation of HMGB1, and the phosphorylation of specific serine residues in the nuclear localization signal regions is related to enhanced HMGB1 secretion in colon cancer cells.

The DExH/D protein family database.

Science.gov (United States)

Jankowsky, E; Jankowsky, A

2000-01-01

DExH/D proteins are essential for all aspects of cellular RNA metabolism and processing, in the replication of many viruses and in DNA replication. DExH/D proteins are subject to current biological, biochemical and biophysical research which provides a continuous wealth of data. The DExH/D protein family database compiles this information and makes it available over the WWW (http://www.columbia.edu/ ej67/dbhome.htm ). The database can be fully searched by text based queries, facilitating fast access to specific information about this important class of enzymes.

Targeting functional motifs of a protein family

Science.gov (United States)

Bhadola, Pradeep; Deo, Nivedita

2016-10-01

The structural organization of a protein family is investigated by devising a method based on the random matrix theory (RMT), which uses the physiochemical properties of the amino acid with multiple sequence alignment. A graphical method to represent protein sequences using physiochemical properties is devised that gives a fast, easy, and informative way of comparing the evolutionary distances between protein sequences. A correlation matrix associated with each property is calculated, where the noise reduction and information filtering is done using RMT involving an ensemble of Wishart matrices. The analysis of the eigenvalue statistics of the correlation matrix for the β -lactamase family shows the universal features as observed in the Gaussian orthogonal ensemble (GOE). The property-based approach captures the short- as well as the long-range correlation (approximately following GOE) between the eigenvalues, whereas the previous approach (treating amino acids as characters) gives the usual short-range correlations, while the long-range correlations are the same as that of an uncorrelated series. The distribution of the eigenvector components for the eigenvalues outside the bulk (RMT bound) deviates significantly from RMT observations and contains important information about the system. The information content of each eigenvector of the correlation matrix is quantified by introducing an entropic estimate, which shows that for the β -lactamase family the smallest eigenvectors (low eigenmodes) are highly localized as well as informative. These small eigenvectors when processed gives clusters involving positions that have well-defined biological and structural importance matching with experiments. The approach is crucial for the recognition of structural motifs as shown in β -lactamase (and other families) and selectively identifies the important positions for targets to deactivate (activate) the enzymatic actions.

Ring current shifts in {sup 19}F-NMR of membrane proteins

Energy Technology Data Exchange (ETDEWEB)

Liu, Dongsheng, E-mail: liudsh@shanghaitech.edu.cn; Wüthrich, Kurt, E-mail: kwuthrich@shanghaitech.edu.cn [ShanghaiTech University, iHuman Institute (China)

2016-05-15

Fluorine-19 NMR markers are attractive reporter groups for use in studies of complex biomacromolecular systems, in particular also for studies of function-related conformational equilibria and rate processes in membrane proteins. Advantages of {sup 19}F-NMR probes include high sensitivity of the {sup 19}F chemical shifts to variations in the non-covalent environment. Nonetheless, in studies of G protein-coupled receptors (GPCR) we encountered situations where {sup 19}F chemical shifts were not responsive to conformational changes that had been implicated by other methods. This prompted us to examine possible effects of aromatic ring current fields on the chemical shifts of {sup 19}F-NMR probes used in GPCRs. Analysis of previously reported {sup 19}F-NMR data on the β{sub 2}-adrenergic receptor and mammalian rhodopsin showed that all {sup 19}F-labeling sites which manifested conformational changes are located near aromatic residues. Although ring current effects are small when compared to other known non-covalent effects on {sup 19}F chemical shifts, there is thus an indication that their contributions are significant when studying activation processes in GPCRs, since the observed activation-related {sup 19}F-NMR chemical shifts are comparable in size to the calculated ring current shifts. Considering the impact of ring current shifts may thus be helpful in identifying promising indigenous or engineered labeling sites for future {sup 19}F-NMR studies of GPCR activation, and novel information may be obtained on the nature of conformational rearrangements near the {sup 19}F-labels. It will then also be interesting to see if the presently indicated role of ring current shifts in membrane protein studies with {sup 19}F-NMR markers can be substantiated by a more extensive data base resulting from future studies.

The RB/E2F pathway and regulation of RNA processing

Energy Technology Data Exchange (ETDEWEB)

Ahlander, Joseph [Department of Molecular and Cellular Biology, 1007 East Lowell Street, University of Arizona, Tucson, AZ 85721 (United States); Bosco, Giovanni, E-mail: gbosco@email.arizona.edu [Department of Molecular and Cellular Biology, 1007 East Lowell Street, University of Arizona, Tucson, AZ 85721 (United States)

2009-07-03

The retinoblastoma tumor suppressor protein (RB) is inactivated in a majority of cancers. RB restricts cell proliferation by inhibiting the E2F family of transcription factors. The current model for RB/E2F function describes its role in regulating transcription at gene promoters. Whether the RB or E2F proteins might play a role in gene expression beyond transcription initiation is not well known. This review describes evidence that points to a novel role for the RB/E2F network in the regulation of RNA processing, and we propose a model as a framework for future research. The elucidation of a novel role of RB in RNA processing will have a profound impact on our understanding of the role of this tumor suppressor family in cell and developmental biology.

Sibling rivalry in the E2F family.

Science.gov (United States)

Trimarchi, Jeffrey M; Lees, Jacqueline A

2002-01-01

The E2F transcription factor family determines whether or not a cell will divide by controlling the expression of key cell-cycle regulators. The individual E2Fs can be divided into distinct subgroups that act in direct opposition to one another to promote either cellular proliferation or cell-cycle exit and terminal differentiation. What is the underlying molecular basis of this 'push-me-pull-you' regulation, and what are its biological consequences?

Clustering evolving proteins into homologous families.

Science.gov (United States)

Chan, Cheong Xin; Mahbob, Maisarah; Ragan, Mark A

2013-04-08

Clustering sequences into groups of putative homologs (families) is a critical first step in many areas of comparative biology and bioinformatics. The performance of clustering approaches in delineating biologically meaningful families depends strongly on characteristics of the data, including content bias and degree of divergence. New, highly scalable methods have recently been introduced to cluster the very large datasets being generated by next-generation sequencing technologies. However, there has been little systematic investigation of how characteristics of the data impact the performance of these approaches. Using clusters from a manually curated dataset as reference, we examined the performance of a widely used graph-based Markov clustering algorithm (MCL) and a greedy heuristic approach (UCLUST) in delineating protein families coded by three sets of bacterial genomes of different G+C content. Both MCL and UCLUST generated clusters that are comparable to the reference sets at specific parameter settings, although UCLUST tends to under-cluster compositionally biased sequences (G+C content 33% and 66%). Using simulated data, we sought to assess the individual effects of sequence divergence, rate heterogeneity, and underlying G+C content. Performance decreased with increasing sequence divergence, decreasing among-site rate variation, and increasing G+C bias. Two MCL-based methods recovered the simulated families more accurately than did UCLUST. MCL using local alignment distances is more robust across the investigated range of sequence features than are greedy heuristics using distances based on global alignment. Our results demonstrate that sequence divergence, rate heterogeneity and content bias can individually and in combination affect the accuracy with which MCL and UCLUST can recover homologous protein families. For application to data that are more divergent, and exhibit higher among-site rate variation and/or content bias, MCL may often be the better

Using hierarchical clustering of secreted protein families to classify and rank candidate effectors of rust fungi.

Directory of Open Access Journals (Sweden)

Diane G O Saunders

Full Text Available Rust fungi are obligate biotrophic pathogens that cause considerable damage on crop plants. Puccinia graminis f. sp. tritici, the causal agent of wheat stem rust, and Melampsora larici-populina, the poplar leaf rust pathogen, have strong deleterious impacts on wheat and poplar wood production, respectively. Filamentous pathogens such as rust fungi secrete molecules called disease effectors that act as modulators of host cell physiology and can suppress or trigger host immunity. Current knowledge on effectors from other filamentous plant pathogens can be exploited for the characterisation of effectors in the genome of recently sequenced rust fungi. We designed a comprehensive in silico analysis pipeline to identify the putative effector repertoire from the genome of two plant pathogenic rust fungi. The pipeline is based on the observation that known effector proteins from filamentous pathogens have at least one of the following properties: (i contain a secretion signal, (ii are encoded by in planta induced genes, (iii have similarity to haustorial proteins, (iv are small and cysteine rich, (v contain a known effector motif or a nuclear localization signal, (vi are encoded by genes with long intergenic regions, (vii contain internal repeats, and (viii do not contain PFAM domains, except those associated with pathogenicity. We used Markov clustering and hierarchical clustering to classify protein families of rust pathogens and rank them according to their likelihood of being effectors. Using this approach, we identified eight families of candidate effectors that we consider of high value for functional characterization. This study revealed a diverse set of candidate effectors, including families of haustorial expressed secreted proteins and small cysteine-rich proteins. This comprehensive classification of candidate effectors from these devastating rust pathogens is an initial step towards probing plant germplasm for novel resistance components.

Non-local boxes and their implementation in Minecraft

Science.gov (United States)

Simnacher, Timo Yannick

PR-boxes are binary devices connecting two remote parties satisfying x AND y = a + b mod 2, where x and y denote the binary inputs and a and b are the respective outcomes without signaling. These devices are named after their inventors Sandu Popescu and Daniel Rohrlich and saturate the Clauser-Horne-Shimony-Holt (CHSH) inequality. This Bell-like inequality bounds the correlation that can exist between two remote, non-signaling, classical systems described by local hidden variable theories. Experiments have now convincingly shown that quantum entanglement cannot be explained by local hidden variable theories. Furthermore, the CHSH inequality provides a method to distinguish quantum systems from super-quantum correlations. The correlation between the outputs of the PR-box goes beyond any quantum entanglement. Though PR-boxes would have impressive consequences, as far as we know they are not physically realizable. However, by introducing PR-boxes to Minecraft as part of the redstone system, which simulates the electrical components for binary computing, we can experience the consequences of super-quantum correlations. For instance, Wim van Dam proved that two parties can use a sufficient number of PR-boxes to compute any Boolean function f(x,y) with only one bit of communication.

Roof Box Shape Streamline Adaptation and the Impact towards Fuel Consumption

Directory of Open Access Journals (Sweden)

Abdul Latif M.F.

2017-01-01

Full Text Available The fuel price hike is currently a sensational national issue in Malaysia. Since the rationalization of fuel subsidies many were affected especially the middle income family. Vehicle aerodynamic were directly related to the fuel consumption, were extra frontal area result a higher drag force hence higher fuel consumption. Roof box were among the largest contributor to the extra drag, thus the roof box shape rationalization were prominent to reduce the extra drag. The idea of adopting water drop shape to the roof box design shows prominent result. The roof box has been simulated using MIRA virtual wind tunnel modelling via commercial computational fluid dynamic (CFD package. This streamline shape drastically reduce the drag force by 34% resulting to a 1.7% fuel saving compare to the conventional boxy roof box. This is an effort to reduce the carbon foot print for a sustainable green world.

Rice Dwarf Virus P2 Protein Hijacks Auxin Signaling by Directly Targeting the Rice OsIAA10 Protein, Enhancing Viral Infection and Disease Development.

Directory of Open Access Journals (Sweden)

Lian Jin

2016-09-01

Full Text Available The phytohormone auxin plays critical roles in regulating myriads of plant growth and developmental processes. Microbe infection can disturb auxin signaling resulting in defects in these processes, but the underlying mechanisms are poorly understood. Auxin signaling begins with perception of auxin by a transient co-receptor complex consisting of an F-box transport inhibitor response 1/auxin signaling F-box (TIR1/AFB protein and an auxin/indole-3-acetic acid (Aux/IAA protein. Auxin binding to the co-receptor triggers ubiquitination and 26S proteasome degradation of the Aux/IAA proteins, leading to subsequent events, including expression of auxin-responsive genes. Here we report that Rice dwarf virus (RDV, a devastating pathogen of rice, causes disease symptoms including dwarfing, increased tiller number and short crown roots in infected rice as a result of reduced sensitivity to auxin signaling. The RDV capsid protein P2 binds OsIAA10, blocking the interaction between OsIAA10 and OsTIR1 and inhibiting 26S proteasome-mediated OsIAA10 degradation. Transgenic rice plants overexpressing wild-type or a dominant-negative (degradation-resistant mutant of OsIAA10 phenocopy RDV symptoms are more susceptible to RDV infection; however, knockdown of OsIAA10 enhances the resistance of rice to RDV infection. Our findings reveal a previously unknown mechanism of viral protein reprogramming of a key step in auxin signaling initiation that enhances viral infection and pathogenesis.

Sifting through genomes with iterative-sequence clustering produces a large, phylogenetically diverse protein-family resource.

Science.gov (United States)

Sharpton, Thomas J; Jospin, Guillaume; Wu, Dongying; Langille, Morgan G I; Pollard, Katherine S; Eisen, Jonathan A

2012-10-13

New computational resources are needed to manage the increasing volume of biological data from genome sequencing projects. One fundamental challenge is the ability to maintain a complete and current catalog of protein diversity. We developed a new approach for the identification of protein families that focuses on the rapid discovery of homologous protein sequences. We implemented fully automated and high-throughput procedures to de novo cluster proteins into families based upon global alignment similarity. Our approach employs an iterative clustering strategy in which homologs of known families are sifted out of the search for new families. The resulting reduction in computational complexity enables us to rapidly identify novel protein families found in new genomes and to perform efficient, automated updates that keep pace with genome sequencing. We refer to protein families identified through this approach as "Sifting Families," or SFams. Our analysis of ~10.5 million protein sequences from 2,928 genomes identified 436,360 SFams, many of which are not represented in other protein family databases. We validated the quality of SFam clustering through statistical as well as network topology-based analyses. We describe the rapid identification of SFams and demonstrate how they can be used to annotate genomes and metagenomes. The SFam database catalogs protein-family quality metrics, multiple sequence alignments, hidden Markov models, and phylogenetic trees. Our source code and database are publicly available and will be subject to frequent updates (http://edhar.genomecenter.ucdavis.edu/sifting_families/).

[Research progress on cathepsin F of parasitic helminths].

Science.gov (United States)

Qu, Zi-Gang; Fu, Bao-Quan

2013-10-01

Cathepsin F is an important member of papain-like subfamily in cysteine protease family. Cathepsin F of helminth parasites can hydrolyze the specific substrate, degrade host protein such as hemoglobin for nutrition, and be involved in invasion into host tissue. Therefore, cathepsin F serves as a potential target for parasitic disease immunodiagnosis, vaccine design and anti-parasite drug screening. This article reviews the structural characteristics and mechanisms of cathepsin F, and research advances on cathepsin F of parasitic helminths.

The nuclear IκB family of proteins controls gene regulation and immune homeostasis.

Science.gov (United States)

MaruYama, Takashi

2015-10-01

The inhibitory IκB family of proteins is subdivided into two groups based on protein localization in the cytoplasm or in the nucleus. These proteins interact with NF-κB, a major transcription factor regulating the expression of many inflammatory cytokines, by modulating its transcriptional activity. However, nuclear IκB family proteins not only interact with NF-κB to change its transcriptional activity, but they also bind to chromatin and control gene expression. This review provides an overview of nuclear IκB family proteins and their role in immune homeostasis. Copyright © 2015 Elsevier B.V. All rights reserved.

FAM26F: An Enigmatic Protein Having a Complex Role in the Immune System.

Science.gov (United States)

Malik, Uzma; Javed, Aneela

2016-09-19

Mammalian immune system is a complex amalgam of diverse cellular and noncellular components such as cytokines, receptors and co-receptors. FAM26F (family with sequence similarity 26, member F) is a recently identified tetraspanin-like membrane glycoprotein which is predicted to make homophilic interactions and potential synapses between several immune cells including CD4 + , CD8 + , NK, dendritic cells and macrophages. Various whole transcriptome analyses have demonstrated the differential expression of FAM26F in several bacterial, viral and parasitic infections, in certain pathophysiological conditions such as liver and heart transplantation, and in various cancers. The complete understanding of transcriptional regulation of FAM26F is in its infancy however it is up regulated by various stimulants such as polyI:C, LPS, INF gamma and TNF alpha, and via various proposed pathways including TLR3, TLR4 IFN-β and Dectin-1. These pathways can merge in STAT1 activation. The synergistic expression of FAM26F on both NK-cells and myeloid dendritic cells is required to activate NK-cells against tumors via its cytoplasmic tail, thus emphasizing therapeutic potential of FAM26F for NK sensitive tumors. Current review provides a comprehensive basis to propose that FAM26F expression level is at least a hallmark for IFN-γ-lead immune responses and thus can proficiently be regarded as an early diagnostic marker. Future investigation dissecting the role of FAM26F in activation of various immune cell populations in local amplification by cell-cell contact is crucial to provide the missing link imperative for elucidating the relevance of this protein in immune responses.

Molecular and functional characterisation of E2F-5, a new member of the E2F family

NARCIS (Netherlands)

Buck, V.; Allen, K.E.; Sørensen, T.; Bybee, A.; Hijmans, E.M.; Voorhoeve, P.M.; Bernards, R.A.; Thangue, N.B. La

1995-01-01

The transcription factor DRTF1/E2F is implicated in the control of cellular proliferation due to its interaction with key regulators of cell cycle progression, such as the retinoblastoma tumour suppressor gene product and related pocket proteins, cyclins and cyclin-dependent kinases. DRTF1/E2F DNA

Using Förster-Resonance Energy Transfer to Measure Protein Interactions Between Bcl-2 Family Proteins on Mitochondrial Membranes.

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Pogmore, Justin P; Pemberton, James M; Chi, Xiaoke; Andrews, David W

2016-01-01

The Bcl-2 family of proteins regulates the process of mitochondrial outer membrane permeabilization, causing the release of cytochrome c and committing a cell to apoptosis. The majority of the functional interactions between these proteins occur at, on, or within the mitochondrial outer membrane, complicating structural studies of the proteins and complexes. As a result most in vitro studies of these protein-protein interactions use truncated proteins and/or detergents which can cause artificial interactions. Herein, we describe a detergent-free, fluorescence-based, in vitro technique to study binding between full-length recombinant Bcl-2 family proteins, particularly cleaved BID (cBID) and BCL-XL, on the membranes of purified mitochondria.

Role of the MAGUK protein family in synapse formation and function.

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Oliva, Carlos; Escobedo, Pía; Astorga, César; Molina, Claudia; Sierralta, Jimena

2012-01-01

Synaptic function is crucially dependent on the spatial organization of the presynaptic and postsynaptic apparatuses and the juxtaposition of both membrane compartments. This precise arrangement is achieved by a protein network at the submembrane region of each cell that is built around scaffold proteins. The membrane-associated guanylate kinase (MAGUK) family of proteins is a widely expressed and well-conserved group of proteins that plays an essential role in the formation and regulation of this scaffolding. Here, we review general features of this protein family, focusing on the discs large and calcium/calmodulin-dependent serine protein kinase subfamilies of MAGUKs in the formation, function, and plasticity of synapses. Copyright © 2011 Wiley Periodicals, Inc.

Potential proteins targeted by let-7f-5p in HeLa cells.

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Wang, Yu; Chen, Xiujuan; Zhang, Yi; Song, Jiandong

2017-07-24

MicroRNAs are a class of small, endogenous, non-coding RNAs mediating posttranscriptional gene silencing. The current authors hypothesized that let-7f-5p is likely involved in cell invasion and proliferation by regulating the expression of target genes. The current study combined let-7f-5p with iTRAQ to assess its effect on gene expression in HeLa cells. Results indicated that 164 proteins were expressed at different levels in HeLa cells overexpressing let-7f-5p and negative controls and that 172 proteins were expressed at different levels in let-7f-5p-silenced HeLa cells and negative controls. Results indicated that let-7f-5p may suppress insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) in HeLa cells.

Higher accumulation of F1-V fusion recombinant protein in plants after induction of protein body formation.

Science.gov (United States)

Alvarez, M Lucrecia; Topal, Emel; Martin, Federico; Cardineau, Guy A

2010-01-01

Improving foreign protein accumulation is crucial for enhancing the commercial success of plant-based production systems since product yields have a major influence on process economics. Cereal grain evolved to store large amounts of proteins in tightly organized aggregates. In maize, gamma-Zein is the major storage protein synthesized by the rough endoplasmic reticulum (ER) and stored in specialized organelles called protein bodies (PB). Zera (gamma-Zein ER-accumulating domain) is the N-terminal proline-rich domain of gamma-zein that is sufficient to induce the assembly of PB formation. Fusion of the Zera domain to proteins of interest results in assembly of dense PB-like, ER-derived organelles, containing high concentration of recombinant protein. Our main goal was to increase recombinant protein accumulation in plants in order to enhance the efficiency of orally-delivered plant-made vaccines. It is well known that oral vaccination requires substantially higher doses than parental formulations. As a part of a project to develop a plant-made plague vaccine, we expressed our model antigen, the Yersinia pestis F1-V antigen fusion protein, with and without a fused Zera domain. We demonstrated that Zera-F1-V protein accumulation was at least 3x higher than F1-V alone when expressed in three different host plant systems: Ncotiana benthamiana, Medicago sativa (alfalfa) and Nicotiana tabacum NT1 cells. We confirmed the feasibility of using Zera technology to induce protein body formation in non-seed tissues. Zera expression and accumulation did not affect plant development and growth. These results confirmed the potential exploitation of Zera technology to substantially increase the accumulation of value-added proteins in plants.

Identification and characterization of cytosolic Hansenula polymorpha proteins belonging to the Hsp70 protein family

NARCIS (Netherlands)

Titorenko, Vladimir I.; Evers, Melchior E.; Diesel, Andre; Samyn, Bart; Beeumen, Josef van; Roggenkamp, Rainer; Kiel, Jan A.K.W.; Klei, Ida J. van der; Veenhuis, Marten

We have isolated two members of the Hsp70 protein family from the yeast Hansenula polymorpha using affinity chromatography. Both proteins were located in the cytoplasm. One of these, designated Hsp72, was inducible in nature (e.g. by heat shock). The second protein (designated Hsc74) was

Chitinase family GH18: evolutionary insights from the genomic history of a diverse protein family

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Aronson Nathan N

2007-06-01

Full Text Available Abstract Background Chitinases (EC.3.2.1.14 hydrolyze the β-1,4-linkages in chitin, an abundant N-acetyl-β-D-glucosamine polysaccharide that is a structural component of protective biological matrices such as insect exoskeletons and fungal cell walls. The glycoside hydrolase 18 (GH18 family of chitinases is an ancient gene family widely expressed in archea, prokaryotes and eukaryotes. Mammals are not known to synthesize chitin or metabolize it as a nutrient, yet the human genome encodes eight GH18 family members. Some GH18 proteins lack an essential catalytic glutamic acid and are likely to act as lectins rather than as enzymes. This study used comparative genomic analysis to address the evolutionary history of the GH18 multiprotein family, from early eukaryotes to mammals, in an effort to understand the forces that shaped the human genome content of chitinase related proteins. Results Gene duplication and loss according to a birth-and-death model of evolution is a feature of the evolutionary history of the GH18 family. The current human family likely originated from ancient genes present at the time of the bilaterian expansion (approx. 550 mya. The family expanded in the chitinous protostomes C. elegans and D. melanogaster, declined in early deuterostomes as chitin synthesis disappeared, and expanded again in late deuterostomes with a significant increase in gene number after the avian/mammalian split. Conclusion This comprehensive genomic study of animal GH18 proteins reveals three major phylogenetic groups in the family: chitobiases, chitinases/chitolectins, and stabilin-1 interacting chitolectins. Only the chitinase/chitolectin group is associated with expansion in late deuterostomes. Finding that the human GH18 gene family is closely linked to the human major histocompatibility complex paralogon on chromosome 1, together with the recent association of GH18 chitinase activity with Th2 cell inflammation, suggests that its late expansion

Sifting through genomes with iterative-sequence clustering produces a large, phylogenetically diverse protein-family resource

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Sharpton Thomas J

2012-10-01

Full Text Available Abstract Background New computational resources are needed to manage the increasing volume of biological data from genome sequencing projects. One fundamental challenge is the ability to maintain a complete and current catalog of protein diversity. We developed a new approach for the identification of protein families that focuses on the rapid discovery of homologous protein sequences. Results We implemented fully automated and high-throughput procedures to de novo cluster proteins into families based upon global alignment similarity. Our approach employs an iterative clustering strategy in which homologs of known families are sifted out of the search for new families. The resulting reduction in computational complexity enables us to rapidly identify novel protein families found in new genomes and to perform efficient, automated updates that keep pace with genome sequencing. We refer to protein families identified through this approach as “Sifting Families,” or SFams. Our analysis of ~10.5 million protein sequences from 2,928 genomes identified 436,360 SFams, many of which are not represented in other protein family databases. We validated the quality of SFam clustering through statistical as well as network topology–based analyses. Conclusions We describe the rapid identification of SFams and demonstrate how they can be used to annotate genomes and metagenomes. The SFam database catalogs protein-family quality metrics, multiple sequence alignments, hidden Markov models, and phylogenetic trees. Our source code and database are publicly available and will be subject to frequent updates (http://edhar.genomecenter.ucdavis.edu/sifting_families/.

Developmental expression of Drosophila Wiskott-Aldrich Syndrome family proteins

Science.gov (United States)

Rodriguez-Mesa, Evelyn; Abreu-Blanco, Maria Teresa; Rosales-Nieves, Alicia E.; Parkhurst, Susan M.

2012-01-01

Background Wiskott-Aldrich Syndrome (WASP) family proteins participate in many cellular processes involving rearrangements of the actin cytoskeleton. To the date, four WASP subfamily members have been described in Drosophila: Wash, WASp, SCAR, and Whamy. Wash, WASp, and SCAR are essential during early Drosophila development where they function in orchestrating cytoplasmic events including membrane-cytoskeleton interactions. A mutant for Whamy has not yet been reported. Results We generated monoclonal antibodies that are specific to Drosophila Wash, WASp, SCAR, and Whamy, and use these to describe their spatial and temporal localization patterns. Consistent with the importance of WASP family proteins in flies, we find that Wash, WASp, SCAR, and Whamy are dynamically expressed throughout oogenesis and embryogenesis. For example, we find that Wash accumulates at the oocyte cortex. WASp is highly expressed in the PNS, while SCAR is the most abundantly expressed in the CNS. Whamy exhibits an asymmetric subcellular localization that overlaps with mitochondria and is highly expressed in muscle. Conclusion All four WASP family members show specific expression patterns, some of which reflect their previously known roles and others revealing new potential functions. The monoclonal antibodies developed offer valuable new tools to investigate how WASP family proteins regulate actin cytoskeleton dynamics. PMID:22275148

Hemoadsorption of high-mobility-group box 1 using a porous polymethylmethacrylate fiber in a swine acute liver failure model.

Science.gov (United States)

Amemiya, Ryusuke; Shinoda, Masahiro; Yamada, Masayuki; Ueno, Yoshiyuki; Shimada, Kaoru; Fujieda, Hiroaki; Yagi, Hiroshi; Mizota, Takamasa; Nishiyama, Ryo; Oshima, Go; Yamada, Shingo; Matsubara, Kentaro; Abe, Yuta; Hibi, Taizo; Kitago, Minoru; Obara, Hideaki; Itano, Osamu; Kitagawa, Yuko

2018-04-01

High-mobility-group box chromosomal protein 1 has been identified as an important mediator of various kinds of acute and chronic inflammation. In this study, we aimed to develop a column that effectively adsorbs high-mobility-group box chromosomal protein 1 by altering the pore size of the fiber. First, we produced three types of porous polymethylmethacrylate fiber by altering the concentration of polymethylmethacrylate dissolved in dimethylsulfoxide. We then selected a fiber based on the results of an in vitro incubation test of high-mobility-group box chromosomal protein 1 adsorption. Using the selected fiber, we constructed a new column and tested its high-mobility-group box chromosomal protein 1 adsorption capacity during 4-h extracorporeal hemoperfusion in a swine acute liver failure model. Electron microscope observation showed that the three types of fibers had different pore sizes on the surface and in cross section, which were dependent on the concentration of polymethylmethacrylate. In the in vitro incubation test, fiber with moderate-sized pores demonstrated the highest adsorption capacity. In the in vivo hemoperfusion study, the ratio of the high-mobility-group box chromosomal protein 1 concentration at the outlet versus the inlet of the column was significantly lower with the new column than with the control column during 4-h extracorporeal hemoperfusion. The normalized plasma level of high-mobility-group box chromosomal protein 1 at 12 h after the completion of hemoperfusion was significantly lower with the new column than with the control column. The newly developed polymethylmethacrylate column adsorbs high-mobility-group box chromosomal protein 1 during hemoperfusion in swine ALF model.

BC-Box Motif-Mediated Neuronal Differentiation of Somatic Stem Cells

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Hiroshi Kanno

2018-02-01

Full Text Available Von Hippel-Lindau tumor suppressor protein (pVHL functions to induce neuronal differentiation of neural stem/progenitor cells (NSCs and skin-derived precursors (SKPs. Here we identified a neuronal differentiation domain (NDD in pVHL. Neuronal differentiation of SKPs was induced by intracellular delivery of a peptide composed of the amino-acid sequences encoded by the NDD. Neuronal differentiation mediated by the NDD was caused by the binding between it and elongin C followed by Janus kinase-2 (JAK2 ubiquitination of JAK2 and inhibition of the JAK2/the signal transducer and activator of transcription-3(STAT3 pathway. The NDD in pVHL contained the BC-box motif ((A,P,S,TLXXX (A,C XXX(A,I,L,V corresponding to the binding site of elongin C. Therefore, we proposed that other BC-box proteins might also contain an NDD; and subsequently also identified in them an NDD containing the amino-acid sequence encoded by the BC-box motif in BC-box proteins. Furthermore, we showed that different NDD peptide-delivered cells differentiated into different kinds of neuron-like cells. That is, dopaminergic neuron-like cells, cholinergic neuron-like cells, GABAnergic neuron-like cells or rhodopsin-positive neuron-like cells were induced by different NDD peptides. These novel findings might contribute to the development of a new method for promoting neuronal differentiation and shed further light on the mechanism of neuronal differentiation of somatic stem cells.

The Puf family of RNA-binding proteins in plants: phylogeny, structural modeling, activity and subcellular localization

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Tam Michael WC

2010-03-01

Full Text Available Abstract Background Puf proteins have important roles in controlling gene expression at the post-transcriptional level by promoting RNA decay and repressing translation. The Pumilio homology domain (PUM-HD is a conserved region within Puf proteins that binds to RNA with sequence specificity. Although Puf proteins have been well characterized in animal and fungal systems, little is known about the structural and functional characteristics of Puf-like proteins in plants. Results The Arabidopsis and rice genomes code for 26 and 19 Puf-like proteins, respectively, each possessing eight or fewer Puf repeats in their PUM-HD. Key amino acids in the PUM-HD of several of these proteins are conserved with those of animal and fungal homologs, whereas other plant Puf proteins demonstrate extensive variability in these amino acids. Three-dimensional modeling revealed that the predicted structure of this domain in plant Puf proteins provides a suitable surface for binding RNA. Electrophoretic gel mobility shift experiments showed that the Arabidopsis AtPum2 PUM-HD binds with high affinity to BoxB of the Drosophila Nanos Response Element I (NRE1 RNA, whereas a point mutation in the core of the NRE1 resulted in a significant reduction in binding affinity. Transient expression of several of the Arabidopsis Puf proteins as fluorescent protein fusions revealed a dynamic, punctate cytoplasmic pattern of localization for most of these proteins. The presence of predicted nuclear export signals and accumulation of AtPuf proteins in the nucleus after treatment of cells with leptomycin B demonstrated that shuttling of these proteins between the cytosol and nucleus is common among these proteins. In addition to the cytoplasmically enriched AtPum proteins, two AtPum proteins showed nuclear targeting with enrichment in the nucleolus. Conclusions The Puf family of RNA-binding proteins in plants consists of a greater number of members than any other model species studied to

A Review on Structures and Functions of Bcl-2 Family Proteins from Homo sapiens.

Science.gov (United States)

Sivakumar, Dakshinamurthy; Sivaraman, Thirunavukkarasu

2016-01-01

Cancer cells evade apoptosis, which is regulated by proteins of Bcl-2 family in the intrinsic pathways. Numerous experimental three-dimensional (3D) structures of the apoptotic proteins and the proteins bound with small chemical molecules/peptides/proteins have been reported in the literature. In this review article, the 3D structures of the Bcl-2 family proteins from Homo sapiens and as well complex structures of the anti-apoptotic proteins bound with small molecular inhibitors reported in the literature to date have been comprehensively listed out and described in detail. Moreover, the molecular mechanisms by which the Bcl-2 family proteins modulate the apoptotic processes and strategies for designing antagonists to anti-apoptotic proteins have been concisely discussed.

A Deg-protease family protein in marine Synechococcus is involved in outer membrane protein organization

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Rhona Kayra Stuart

2014-06-01

Full Text Available Deg-family proteases are a periplasm-associated group of proteins that are known to be involved in envelope stress responses and are found in most microorganisms. Orthologous genes SYNW2176 (in strain WH8102 and sync_2523 (strain CC9311 are predicted members of the Deg-protease family and are among the few genes induced by copper stress in both open ocean and coastal marine Synechococcus strains. In contrast to the lack of a phenotype in a similar knockout in Synechocystis PCC6803, a SYNW2176 knockout mutant in strain WH8102 was much more resistant to copper than the wild-type. The mutant also exhibited a significantly altered outer membrane protein composition which may contribute to copper resistance, longer lag phase after transfer, low-level consistent alkaline phosphatase activity, and an inability to induce high alkaline phosphatase activity in response to phosphate stress. This phenotype suggests a protein-quality-control role for SYNW2176, the absence of which leads to a constitutively activated stress response. Deg-protease family proteins in this ecologically important cyanobacterial group thus help to determine outer membrane responses to both nutrients and toxins.

Evolution and Origin of HRS, a Protein Interacting with Merlin, the Neurofibromatosis 2 Gene Product

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Leonid V. Omelyanchuk

2009-10-01

Full Text Available Hepatocyte growth factor receptor tyrosine kinase substrate (HRS is an endosomal protein required for trafficking receptor tyrosine kinases from the early endosome to the lysosome. HRS interacts with Merlin, the Neurofibromatosis 2 (NF2 gene product, and this interaction may be important for Merlin’s tumor suppressor activity. Understanding the evolution, origin, and structure of HRS may provide new insight into Merlin function. We show that HRS homologs are present across a wide range of Metazoa with the yeast Vps27 protein as their most distant ancestor. The phylogenetic tree of the HRS family coincides with species evolution and divergence, suggesting a unique function for HRS. Sequence alignment shows that various protein domains of HRS, including the VHS domain, the FYVE domain, the UIM domain, and the clathrin-binding domain, are conserved from yeast to multicellular organisms. The evolutionary transition from unicellular to multicellular organisms was accompanied by the appearance of a binding site for Merlin, which emerges in the early Metazoa after its separation from flatworms. In addition to the region responsible for growth suppression, the Merlin-binding and STAM-binding domains of HRS are conserved among multicellular organisms. The residue equivalent to tyrosine-377, which is phosphorylated in the human HRS protein, is highly conserved throughout the HRS family. Three additional conserved boxes lacking assigned functions are found in the HRS proteins of Metazoa. While boxes 1 and 3 may constitute the Eps-15- and Snx1-binding sites, respectively, box 2, containing the residue equivalent to tyrosine-377, is likely to be important for HRS phosphorylation. While several functional domains are conserved throughout the HRS family, the STAM-binding, Merlin-binding, and growth suppression domains evolved in the early Metazoa around the time the Merlin protein emerged. As these domains appear during the transition to multicellularity

Identification of novel putative-binding proteins for cellular prion protein and a specific interaction with the STIP1 homology and U-Box-containing protein 1

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Gimenez, Ana Paula Lappas; Richter, Larissa Morato Luciani; Atherino, Mariana Campos; Beirão, Breno Castello Branco; Fávaro, Celso; Costa, Michele Dietrich Moura; Zanata, Silvio Marques; Malnic, Bettina; Mercadante, Adriana Frohlich

2015-01-01

ABSTRACT Prion diseases involve the conversion of the endogenous cellular prion protein, PrPC, into a misfolded infectious isoform, PrPSc. Several functions have been attributed to PrPC, and its role has also been investigated in the olfactory system. PrPC is expressed in both the olfactory bulb (OB) and olfactory epithelium (OE) and the nasal cavity is an important route of transmission of diseases caused by prions. Moreover, Prnp−/− mice showed impaired behavior in olfactory tests. Given the high PrPC expression in OE and its putative role in olfaction, we screened a mouse OE cDNA library to identify novel PrPC-binding partners. Ten different putative PrPC ligands were identified, which were involved in functions such as cellular proliferation and apoptosis, cytoskeleton and vesicle transport, ubiquitination of proteins, stress response, and other physiological processes. In vitro binding assays confirmed the interaction of PrPC with STIP1 homology and U-Box containing protein 1 (Stub1) and are reported here for the first time. Stub1 is a co-chaperone with ubiquitin E3-ligase activity, which is associated with neurodegenerative diseases characterized by protein misfolding and aggregation. Physiological and pathological implications of PrPC-Stub1 interaction are under investigation. The PrPC-binding proteins identified here are not exclusive to the OE, suggesting that these interactions may occur in other tissues and play general biological roles. These data corroborate the proposal that PrPC is part of a multiprotein complex that modulates several cellular functions and provide a platform for further studies on the physiological and pathological roles of prion protein. PMID:26237451

Characterization of the Pichia pastoris protein-O-mannosyltransferase gene family.

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Juergen H Nett

Full Text Available The methylotrophic yeast, Pichiapastoris, is an important organism used for the production of therapeutic proteins. However, the presence of fungal-like glycans, either N-linked or O-linked, can elicit an immune response or enable the expressed protein to bind to mannose receptors, thus reducing their efficacy. Previously we have reported the elimination of β-linked glycans in this organism. In the current report we have focused on reducing the O-linked mannose content of proteins produced in P. pastoris, thereby reducing the potential to bind to mannose receptors. The initial step in the synthesis of O-linked glycans in P. pastoris is the transfer of mannose from dolichol-phosphomannose to a target protein in the yeast secretory pathway by members of the protein-O-mannosyltransferase (PMT family. In this report we identify and characterize the members of the P. pastoris PMT family. Like Candida albicans, P. pastoris has five PMT genes. Based on sequence homology, these PMTs can be grouped into three sub-families, with both PMT1 and PMT2 sub-families possessing two members each (PMT1 and PMT5, and PMT2 and PMT6, respectively. The remaining sub-family, PMT4, has only one member (PMT4. Through gene knockouts we show that PMT1 and PMT2 each play a significant role in O-glycosylation. Both, by gene knockouts and the use of Pmt inhibitors we were able to significantly reduce not only the degree of O-mannosylation, but also the chain-length of these glycans. Taken together, this reduction of O-glycosylation represents an important step forward in developing the P. pastoris platform as a suitable system for the production of therapeutic glycoproteins.

Structure of the d-alanylgriseoluteic acid biosynthetic protein EhpF, an atypical member of the ANL superfamily of adenylating enzymes

International Nuclear Information System (INIS)

Bera, Asim K.; Atanasova, Vesna; Gamage, Swarna; Robinson, Howard; Parsons, James F.

2010-01-01

The structure of EhpF from P. agglomerans has been solved alone and in complex with phenazine-1,6-dicarboxylate. Apo EhpF was solved and refined in two different space groups at 1.95 and 2.3 Å resolution and the EhpF–phenazine-1,6-dicarboxylate complex structure was determined at 2.8 Å resolution. The structure of EhpF, a 41 kDa protein that functions in the biosynthetic pathway leading to the broad-spectrum antimicrobial compound d-alanylgriseoluteic acid (AGA), is reported. A cluster of approximately 16 genes, including ehpF, located on a 200 kbp plasmid native to certain strains of Pantoea agglomerans encodes the proteins that are required for the conversion of chorismic acid to AGA. Phenazine-1,6-dicarboxylate has been identified as an intermediate in AGA biosynthesis and deletion of ehpF results in accumulation of this compound in vivo. The crystallographic data presented here reveal that EhpF is an atypical member of the acyl-CoA synthase or ANL superfamily of adenylating enzymes. These enzymes typically catalyze two-step reactions involving adenylation of a carboxylate substrate followed by transfer of the substrate from AMP to coenzyme A or another phosphopantetheine. EhpF is distinguished by the absence of the C-terminal domain that is characteristic of enzymes from this family and is involved in phosphopantetheine binding and in the second half of the canonical two-step reaction that is typically observed. Based on the structure of EhpF and a bioinformatic analysis, it is proposed that EhpF and EhpG convert phenazine-1,6-dicarboxylate to 6-formylphenazine-1-carboxylate via an adenylyl intermediate

Genetics Home Reference: familial candidiasis

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... Facebook Twitter Home Health Conditions Familial candidiasis Familial candidiasis Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Familial candidiasis is an inherited tendency to develop infections caused ...

Virtual Box

DEFF Research Database (Denmark)

Davis, Hilary; Skov, Mikael B.; Stougaard, Malthe

2007-01-01

. This paper reports on the design, implementation and initial evaluation of Virtual Box. Virtual Box attempts to create a physical and engaging context in order to support reciprocal interactions with expressive content. An implemented version of Virtual Box is evaluated in a location-aware environment...

High mobility group box-1 is phosphorylated by protein kinase C zeta and secreted in colon cancer cells

Energy Technology Data Exchange (ETDEWEB)

Lee, Hanna; Park, Minhee; Shin, Nara; Kim, Gamin [Department of Pathology, Yonsei University College of Medicine, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul (Korea, Republic of); Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul (Korea, Republic of); Kim, Yun Gi [Department of Internal Medicine, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-744 (Korea, Republic of); Shin, Jeon-Soo [Department of Microbiology, Yonsei University College of Medicine, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul (Korea, Republic of); Kim, Hoguen, E-mail: hkyonsei@yuhs.ac [Department of Pathology, Yonsei University College of Medicine, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul (Korea, Republic of); Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul (Korea, Republic of)

2012-07-27

Highlights: Black-Right-Pointing-Pointer Specific enzyme for HMGB1 phosphorylation and its secretion is proposed. Black-Right-Pointing-Pointer Inhibition of PKC-{zeta} leads to significant reduction of the secreted HMGB1. Black-Right-Pointing-Pointer Phosphorylation of specific site of HMGB1 redirects its secretion in cancer cells. Black-Right-Pointing-Pointer Activation of PKC-{zeta} in cancers explains the enhanced HMGB1 secretion. -- Abstract: High mobility group box-1 (HMGB1), a nuclear protein, is overexpressed and secreted in cancer cells. Phosphorylation on two different nuclear localization signal regions are known to be important for the nuclear-to-cytoplasmic transport and secretion of HMGB1. However, little is known about the biochemical mechanism of HMGB1 modifications and its subsequent secretion from cancer cells. To identify the specific enzyme and important sites for HMGB1 phosphorylation, we screened the protein kinase C (PKC) family in a colon cancer cell line (HCT116) for HMGB1 binding by pull-down experiments using a 3XFLAG-HMGB1 construct. Strong interactions between atypical PKCs (PKC-{zeta}, {lambda}, and {iota}) and cytoplasmic HMGB1 were observed in HCT116 cells. We further identified the most critical PKC isotype that regulates HMGB1 secretion is PKC-{zeta} by using PKC inhibitors and siRNA experiments. The serine residues at S39, S53 and S181 of HMGB1 were related to enhancing HMGB1 secretion. We also demonstrated overexpression and activation of PKC-{zeta} in colon cancer tissues. Our findings suggest that PKC-{zeta} is involved in the phosphorylation of HMGB1, and the phosphorylation of specific serine residues in the nuclear localization signal regions is related to enhanced HMGB1 secretion in colon cancer cells.

Caracterização do microclima dos diferentes layouts de caixas no transporte de ovos férteis Characterization of microclimate in different layout of boxes during transport of fertile eggs

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Aérica C. Nazareno

2013-03-01

Full Text Available Quando as condições microclimáticas e de transporte são combinadas inadequadamente, os componentes dos ovos férteis podem ser danificados. Objetivou-se, com este trabalho, caracterizar e analisar a dependência espacial dos perfis microclimáticos no transporte de ovos férteis em diferentes layouts de caixas. A pesquisa foi conduzida na empresa integradora avícola, em Mogi Mirim, SP, por meio do acompanhamento de cinco carregamentos. Utilizou-se um caminhão climatizado do tipo baú, com capacidade para 592 caixas de ovos e se avaliaram dois layouts da carga (caixas superiores e inferiores de 9 pilhas com seis caixas de ovos férteis. Um microprocessador foi instalado dentro das caixas de ovos, totalizando 18 microprocessadores. As variáveis meteorológicas analisadas foram: temperatura (ºC umidade relativa (% e entalpia específica (kJ kg-1 ar seco. O delineamento experimental adotado foi o inteiramente casualizado com parcelas subdividas e realizada a análise geoestatística (krigagem ordinária. No layout das caixas superiores foram registrados valores elevados de temperatura e baixos valores de umidade relativa e entalpia específica. As regiões de piores condições microclimáticas nos dois layouts se situaram nas partes central e traseira do caminhão, durante o transporte dos ovos férteis.The fertile egg components may be damaged when the microclimatic conditions and transportation characteristics are combined inadequately. Thus, the aim of this study was to characterize and to assess the spatial dependence of microclimatic profiles during transport of fertile eggs with different layout of boxes. A trial was conducted in a commercial poultry industry in Mogi Mirim in the State of São Paulo, Brazil, through monitoring five loads of hatching eggs. An air conditioned truck was used, with maximum capacity of 592 egg boxes. Two layout of boxes were assessed in the present study. One data logger was installed inside the egg boxes

The toxicity of NaF on BmN cells and a comparative proteomics approach to identify protein expression changes in cells under NaF-stress

International Nuclear Information System (INIS)

Chen, Liang; Chen, Huiqing; Yao, Chun; Chang, Cheng; Xia, Hengchuan; Zhang, Chunxia; Zhou, Yang; Yao, Qin; Chen, Keping

2015-01-01

Highlights: • On the cellular level, we identified IC 50 of NaF on BmN cell by flow cytometry. • High concentration of NaF gives effect on BmN cell morphology. • Five significantly differential proteins were identified by two-dimensional electrophoresis and mass spectrometry. • ALDH2 and WPH were up-regulated, while CRT and SCF were down-regulated, providing new information for metabolic pathway of fluoride. - Abstract: Fluorides negatively affect the development of organisms and are a threat to human health and environmental safety. In this study, Bombyx mori N cell line (BmN) were used to explore effects of NaF on insect cells. We found that 8 h (hrs) culture with high concentration of NaF (≥1 mM) induced significantly morphological changes. Dose-response curves of 72 h continuously cultured BmN treated with NaF showed that the half inhibitory concentration (IC 50 ) value was 56.60 μM. Treatment of BmN with 100 and 300 μM of NaF induced apoptosis and necrosis. 2-D electrophoresis of whole cell extracted from BmN showed that treatment with 300 μM NaF up-regulated 32 proteins and down-regulated 11 proteins when compared with controls. We identified 5 different proteins by MALDI-TOF MS, and 4 of them were identified for the first time, including 2 up-regulated proteins (mitochondrial aldehyde dehydrogenase ALDH2 and prohibitin protein WPH) and 2 down-regulated proteins (calreticulin precursor CRT and DNA supercoiling factor SCF). These observations were further confirmed by fluorescence quantitative PCR. Together, our data suggest that these target proteins could be regarded as targets influenced by NaF and also provide clues for studies on the response metabolism pathway under NaF stress

The toxicity of NaF on BmN cells and a comparative proteomics approach to identify protein expression changes in cells under NaF-stress

Energy Technology Data Exchange (ETDEWEB)

Chen, Liang; Chen, Huiqing [Institute of Life Sciences, Jiangsu University, Zhenjiang, Jiangsu 212013 (China); Yao, Chun [Department of Stomatology, Zhenjiang First People’s Hospital, Zhenjiang, Jiangsu 212013 (China); Chang, Cheng; Xia, Hengchuan; Zhang, Chunxia; Zhou, Yang; Yao, Qin [Institute of Life Sciences, Jiangsu University, Zhenjiang, Jiangsu 212013 (China); Chen, Keping, E-mail: kpchen@ujs.edu.cn [Institute of Life Sciences, Jiangsu University, Zhenjiang, Jiangsu 212013 (China)

2015-04-09

Highlights: • On the cellular level, we identified IC{sub 50} of NaF on BmN cell by flow cytometry. • High concentration of NaF gives effect on BmN cell morphology. • Five significantly differential proteins were identified by two-dimensional electrophoresis and mass spectrometry. • ALDH2 and WPH were up-regulated, while CRT and SCF were down-regulated, providing new information for metabolic pathway of fluoride. - Abstract: Fluorides negatively affect the development of organisms and are a threat to human health and environmental safety. In this study, Bombyx mori N cell line (BmN) were used to explore effects of NaF on insect cells. We found that 8 h (hrs) culture with high concentration of NaF (≥1 mM) induced significantly morphological changes. Dose-response curves of 72 h continuously cultured BmN treated with NaF showed that the half inhibitory concentration (IC{sub 50}) value was 56.60 μM. Treatment of BmN with 100 and 300 μM of NaF induced apoptosis and necrosis. 2-D electrophoresis of whole cell extracted from BmN showed that treatment with 300 μM NaF up-regulated 32 proteins and down-regulated 11 proteins when compared with controls. We identified 5 different proteins by MALDI-TOF MS, and 4 of them were identified for the first time, including 2 up-regulated proteins (mitochondrial aldehyde dehydrogenase ALDH2 and prohibitin protein WPH) and 2 down-regulated proteins (calreticulin precursor CRT and DNA supercoiling factor SCF). These observations were further confirmed by fluorescence quantitative PCR. Together, our data suggest that these target proteins could be regarded as targets influenced by NaF and also provide clues for studies on the response metabolism pathway under NaF stress.

Protein-protein docking with F(2Dock 2.0 and GB-rerank.

Directory of Open Access Journals (Sweden)

Rezaul Chowdhury

Full Text Available Computational simulation of protein-protein docking can expedite the process of molecular modeling and drug discovery. This paper reports on our new F(2 Dock protocol which improves the state of the art in initial stage rigid body exhaustive docking search, scoring and ranking by introducing improvements in the shape-complementarity and electrostatics affinity functions, a new knowledge-based interface propensity term with FFT formulation, a set of novel knowledge-based filters and finally a solvation energy (GBSA based reranking technique. Our algorithms are based on highly efficient data structures including the dynamic packing grids and octrees which significantly speed up the computations and also provide guaranteed bounds on approximation error.The improved affinity functions show superior performance compared to their traditional counterparts in finding correct docking poses at higher ranks. We found that the new filters and the GBSA based reranking individually and in combination significantly improve the accuracy of docking predictions with only minor increase in computation time. We compared F(2 Dock 2.0 with ZDock 3.0.2 and found improvements over it, specifically among 176 complexes in ZLab Benchmark 4.0, F(2 Dock 2.0 finds a near-native solution as the top prediction for 22 complexes; where ZDock 3.0.2 does so for 13 complexes. F(2 Dock 2.0 finds a near-native solution within the top 1000 predictions for 106 complexes as opposed to 104 complexes for ZDock 3.0.2. However, there are 17 and 15 complexes where F(2 Dock 2.0 finds a solution but ZDock 3.0.2 does not and vice versa; which indicates that the two docking protocols can also complement each other.The docking protocol has been implemented as a server with a graphical client (TexMol which allows the user to manage multiple docking jobs, and visualize the docked poses and interfaces. Both the server and client are available for download. Server: http://www.cs.utexas.edu/~bajaj/cvc/software/f

Protein-Protein Docking with F2Dock 2.0 and GB-Rerank

Science.gov (United States)

Chowdhury, Rezaul; Rasheed, Muhibur; Keidel, Donald; Moussalem, Maysam; Olson, Arthur; Sanner, Michel; Bajaj, Chandrajit

2013-01-01

Motivation Computational simulation of protein-protein docking can expedite the process of molecular modeling and drug discovery. This paper reports on our new F2 Dock protocol which improves the state of the art in initial stage rigid body exhaustive docking search, scoring and ranking by introducing improvements in the shape-complementarity and electrostatics affinity functions, a new knowledge-based interface propensity term with FFT formulation, a set of novel knowledge-based filters and finally a solvation energy (GBSA) based reranking technique. Our algorithms are based on highly efficient data structures including the dynamic packing grids and octrees which significantly speed up the computations and also provide guaranteed bounds on approximation error. Results The improved affinity functions show superior performance compared to their traditional counterparts in finding correct docking poses at higher ranks. We found that the new filters and the GBSA based reranking individually and in combination significantly improve the accuracy of docking predictions with only minor increase in computation time. We compared F2 Dock 2.0 with ZDock 3.0.2 and found improvements over it, specifically among 176 complexes in ZLab Benchmark 4.0, F2 Dock 2.0 finds a near-native solution as the top prediction for 22 complexes; where ZDock 3.0.2 does so for 13 complexes. F2 Dock 2.0 finds a near-native solution within the top 1000 predictions for 106 complexes as opposed to 104 complexes for ZDock 3.0.2. However, there are 17 and 15 complexes where F2 Dock 2.0 finds a solution but ZDock 3.0.2 does not and vice versa; which indicates that the two docking protocols can also complement each other. Availability The docking protocol has been implemented as a server with a graphical client (TexMol) which allows the user to manage multiple docking jobs, and visualize the docked poses and interfaces. Both the server and client are available for download. Server: http://www.cs.utexas.edu/~bajaj/cvc/software/f

The centromeric/nucleolar chromatin protein ZFP-37 may function to specify neuronal nuclear domains

NARCIS (Netherlands)

E. Payen; T. Verkerk (Ton); D. Michalovich (David); S.D. Dreyer; A. Winterpacht; B. Lee (Brendan); C.I. de Zeeuw (Chris); N.J. Galjart (Niels); F.G. Grosveld (Frank)

1998-01-01

textabstractMurine ZFP-37 is a member of the large family of C2H2 type zinc finger proteins. It is characterized by a truncated NH2-terminal Kruppel-associated box and is thought to play a role in transcriptional regulation. During development Zfp-37 mRNA is most

A family of fluoride-specific ion channels with dual-topology architecture.

Science.gov (United States)

Stockbridge, Randy B; Robertson, Janice L; Kolmakova-Partensky, Ludmila; Miller, Christopher

2013-08-27

Fluoride ion, ubiquitous in soil, water, and marine environments, is a chronic threat to microorganisms. Many prokaryotes, archea, unicellular eukaryotes, and plants use a recently discovered family of F(-) exporter proteins to lower cytoplasmic F(-) levels to counteract the anion's toxicity. We show here that these 'Fluc' proteins, purified and reconstituted in liposomes and planar phospholipid bilayers, form constitutively open anion channels with extreme selectivity for F(-) over Cl(-). The active channel is a dimer of identical or homologous subunits arranged in antiparallel transmembrane orientation. This dual-topology assembly has not previously been seen in ion channels but is known in multidrug transporters of the SMR family, and is suggestive of an evolutionary antecedent of the inverted repeats found within the subunits of many membrane transport proteins. DOI:http://dx.doi.org/10.7554/eLife.01084.001.

Timing of APC/C substrate degradation is determined by fzy/fzr specificity of destruction boxes

OpenAIRE

Zur, Amit; Brandeis, Michael

2002-01-01

The anaphase promoting complex/cyclosome (APC/C), activated by fzy and fzr, degrades cell cycle proteins that carry RXXL or KEN destruction boxes (d-boxes). APC/C substrates regulate sequential events and must be degraded in the correct order during mitosis and G1. We studied how d-boxes determine APC/Cfzy/APC/Cfzr specificity and degradation timing. Cyclin B1 has an RXXL box and is degraded by both APC/Cfzy and APC/Cfzr; fzy has a KEN box and is degraded by APC/Cfzr only. We characterized th...

Induction of DNA synthesis and apoptosis are separable functions of E2F-1

DEFF Research Database (Denmark)

Phillips, A C; Bates, S; Ryan, K M

1997-01-01

The family of E2F transcription factors have an essential role in mediating cell cycle progression, and recently, one of the E2F protein family, E2F-1, has been shown to participate in the induction of apoptosis. Cooperation between E2F and the p53 tumor suppressor protein in this apoptotic...... response had led to the suggestion that cell cycle progression induced by E2F-1 expression provides an apoptotic signal when placed in conflict with an arrest to cell cycle progression, such as provided by p53. We show here that although apoptosis is clearly enhanced by p53, E2F-1 can induce significant...... apoptosis in the absence of p53. Furthermore, this apoptotic function of E2F-1 is separable from the ability to accelerate entry into DNA synthesis. Analysis of E2F-1 mutants indicates that although DNA-binding is required, transcriptional transactivation is not necessary for the induction of apoptosis by E...

Structural and functional annotation of human FAM26F: A multifaceted protein having a critical role in the immune system.

Science.gov (United States)

Malik, Uzma; Javed, Aneela; Ali, Amjad; Asghar, Kashif

2017-01-15

Human immune system is a complex amalgam of a greatly diverse ensemble comprising of various cellular and non-cellular components, including proteins. FAM26F (family with sequence similarity 26, member F) is a relatively recently identified gene reported to play important role in diverse immune responses. Numerous studies have reported FAM26F to be differentially expressed in several viral, bacterial and parasitic infections, in certain pathophysiological conditions such as heart and liver transplantation, and in several cancers. FAM26F has also been found to be upregulated by various stimulants such as polyI:C, LPS, INF gamma and TNF alpha, and via various anticipated pathways including TLR3, TLR4 IFN-β and Dectin-1. Moreover, the synergistic expression of FAM26F on both NK-cells and myeloid dendritic cells is required to activate NK-cells against tumors via its cytoplasmic tail, thus emphasizing the therapeutic potential of FAM26F for NK sensitive tumors. Although a considerable amount of evidence is present regarding the potential role of FAM26F in immune modulation, the exact function and modulatory pathways of this gene are yet to be elucidated. We aimed to completely characterize FAM26F in order to apprehend its function and role in the immune responses. The results revealed human FAM26F to be located at chromosomal position 6q22.1. FAM26F mRNA contains 1141bp coding region encoding a 315 amino acid long, stable protein that has been well-conserved throughout evolution. It is a signal peptide deprived transmembrane protein that is secreted through non-classical pathway. The presence of a single well-conserved Ca_hom_mod domain indicated FAM26F to be a cation channel involved in the transport of molecules. A potential N-glycosylation and 14 phosphorylation sites were also predicted, along with four interacting partners of FAM26F. The secondary and tertiary structures of FAM26F were determined. Moreover, the presence of an immunoglobulin-like fold in FAM26F

Betabaculovirus F proteins showed different efficiencies when rescuing the infectivity of gp64-null Autographa californica nucleopolyhedrovirus

NARCIS (Netherlands)

Yin, F.; Wang, M.; Ying, T.; Deng, F.; Vlak, J.M.; Hu, Z.; Wang, H.

2013-01-01

The Agrotis segetum granulovirus (AgseGV) F protein was previously identified as the first betabaculovirus F protein with functional homology to Autographa californica nucleopolyhedrovirus (AcMNPV) GP64. In the current study, F proteins from Xestia c-nigrum granulovirus (XecnGV), Cydia pomonella

Motif III in superfamily 2 "helicases" helps convert the binding energy of ATP into a high-affinity RNA binding site in the yeast DEAD-box protein Ded1.

Science.gov (United States)

Banroques, Josette; Doère, Monique; Dreyfus, Marc; Linder, Patrick; Tanner, N Kyle

2010-03-05

Motif III in the putative helicases of superfamily 2 is highly conserved in both its sequence and its structural context. It typically consists of the sequence alcohol-alanine-alcohol (S/T-A-S/T). Historically, it was thought to link ATPase activity with a "helicase" strand displacement activity that disrupts RNA or DNA duplexes. DEAD-box proteins constitute the largest family of superfamily 2; they are RNA-dependent ATPases and ATP-dependent RNA binding proteins that, in some cases, are able to disrupt short RNA duplexes. We made mutations of motif III (S-A-T) in the yeast DEAD-box protein Ded1 and analyzed in vivo phenotypes and in vitro properties. Moreover, we made a tertiary model of Ded1 based on the solved structure of Vasa. We used Ded1 because it has relatively high ATPase and RNA binding activities; it is able to displace moderately stable duplexes at a large excess of substrate. We find that the alanine and the threonine in the second and third positions of motif III are more important than the serine, but that mutations of all three residues have strong phenotypes. We purified the wild-type and various mutants expressed in Escherichia coli. We found that motif III mutations affect the RNA-dependent hydrolysis of ATP (k(cat)), but not the affinity for ATP (K(m)). Moreover, mutations alter and reduce the affinity for single-stranded RNA and subsequently reduce the ability to disrupt duplexes. We obtained intragenic suppressors of the S-A-C mutant that compensate for the mutation by enhancing the affinity for ATP and RNA. We conclude that motif III and the binding energy of gamma-PO(4) of ATP are used to coordinate motifs I, II, and VI and the two RecA-like domains to create a high-affinity single-stranded RNA binding site. It also may help activate the beta,gamma-phosphoanhydride bond of ATP. (c) 2009 Elsevier Ltd. All rights reserved.

Structure of Escherichia coli RutC, a member of the YjgF family and putative aminoacrylate peracid reductase of the rut operon

Energy Technology Data Exchange (ETDEWEB)

Knapik, Aleksandra Alicja; Petkowski, Janusz Jurand; Otwinowski, Zbyszek; Cymborowski, Marcin Tadeusz; Cooper, David Robert; Chruszcz, Maksymilian; Krajewska, Wanda Malgorzata; Minor, Wladek

2014-10-02

RutC is the third enzyme in the Escherichia coli rut pathway of uracil degradation. RutC belongs to the highly conserved YjgF family of proteins. The structure of the RutC protein was determined and refined to 1.95 Å resolution. This crystal belonged to space group P21212 and contained six molecules in the asymmetric unit. The structure was solved by SAD phasing and was refined to an Rwork of 19.3% (Rfree = 21.7%). Moreover, the final model revealed that this protein has a Bacillus chorismate mutase-like fold and forms a homotrimer with a hydrophobic cavity in the center of the structure and ligand-binding clefts between two subunits. A likely function for RutC is the reduction of peroxy-aminoacrylate to aminoacrylate as a part of a detoxification process.

A Theoretical Framework for Association Studies in F2 Family Pools Using Allele Frequencies from Genotyping-By-Sequencing

DEFF Research Database (Denmark)

Janss, Luc L; Ashraf, Bilal H; Greve-Pedersen, Morten

a sequencing approach to obtain Single Nucleotide Polymorphisms (SNPs) frequencies is considered here. In this work we develop the theoretical framework to perform association studies using allele frequencies from such F2 family pools. We show that expected allele frequencies in the F2 families will have...

Identification of a novel IL-1 cytokine family member in teleost fish.

Science.gov (United States)

Wang, Tiehui; Bird, Steve; Koussounadis, Antonis; Holland, Jason W; Carrington, Allison; Zou, Jun; Secombes, Christopher J

2009-07-15

A novel IL-1 family member (nIL-1F) has been discovered in fish, adding a further member to this cytokine family. The unique gene organization of nIL-1F, together with its location in the genome and low homology to known family members, suggests that this molecule is not homologous to known IL-1F. Nevertheless, it contains a predicted C-terminal beta-trefoil structure, an IL-1F signature region within the final exon, a potential IL-1 converting enzyme cut site, and its expression level is clearly increased following infection, or stimulation of macrophages with LPS or IL-1beta. A thrombin cut site is also present and may have functional relevance. The C-terminal recombinant protein antagonized the effects of rainbow trout rIL-1beta on inflammatory gene expression in a trout macrophage cell line, suggesting it is an IL-1beta antagonist. Modeling studies confirmed that nIL-1F has the potential to bind to the trout IL-1RI receptor protein, and may be a novel IL-1 receptor antagonist.

Correlation between centromere protein-F autoantibodies and cancer analyzed by enzyme-linked immunosorbent assay

DEFF Research Database (Denmark)

Welner, Simon; Trier, Nicole Hartwig; Morten Frisch, Morten

2013-01-01

Centromere protein-F (CENP-F) is a large nuclear protein of 367 kDa, which is involved in multiple mitosis-related events such as proper assembly of the kinetochores, stabilization of heterochromatin, chromosome alignment and mitotic checkpoint signaling. Several studies have shown a correlation...

UK114, a YjgF/Yer057p/UK114 family protein highly conserved from bacteria to mammals, is localized in rat liver peroxisomes

International Nuclear Information System (INIS)

Antonenkov, Vasily D.; Ohlmeier, Steffen; Sormunen, Raija T.; Hiltunen, J. Kalervo

2007-01-01

Mammalian UK114 belongs to a highly conserved family of proteins with unknown functions. Although it is believed that UK114 is a cytosolic or mitochondrial protein there is no detailed study of its intracellular localization. Using analytical subcellular fractionation, electron microscopic colloidal gold technique, and two-dimensional gel electrophoresis of peroxisomal matrix proteins combined with mass spectrometric analysis we show here that a large portion of UK114 is present in rat liver peroxisomes. The peroxisomal UK114 is a soluble matrix protein and it is not inducible by the peroxisomal proliferator clofibrate. The data predict involvement of UK114 in peroxisomal metabolism

Design, construction and performance evaluation of aBox type solar cooker with a glazing wiper mechanism

Directory of Open Access Journals (Sweden)

Zeleke Ademe

2018-01-01

Full Text Available This research work describes the performance evaluation of a double-glazed box-type solar oven with three reflectors and with a vapor wiper mechanism fabricated using locally available materials. The box cooker has external box dimensions of 600 mm × 600 mm × 250 mm and pyramidal internal box dimensions of 460 mm × 460 mm top face and 300 mm × 300 mm bottom face with depth of 150 mm. The thermal performance was tested as per the ASAE International Test procedure and Bureau of Indian Standards (BIS for testing the thermal performance of a box-type solar cooker. The obtained test results after employing required calculations were figures of merit F1 = 0.123 Km2/W, F2 = 0.540, the standard cooking power P50 = 36 W and the cumulative efficiency to be 22%, whereas with the application of the wiper mechanism, it was found that F1 = 0.123, F2 = 0.827, the standard cooking power (P50 = 51 W, and the cumulative efficiency to be 31.4%. The standard boiling time of 1.43 kg of water was calculated to be 53.54 and 88.84 minutes for the cooker with and without the application of wiper mechanism respectively. The thermal distribution of the cooker was modeled using interior box geometry as a boundary condition with ANSYS 15.0. The temperature distribution inside the box was simulated and the maximum wall temperature was found to be 139 ℃. This was lower than the experimental results by 22 ℃. The method of modeling and simulation of the cooker with and without a wiper mechanism is similar except for the variation of the transmittance of the glass due to shading of vapor which can be deducted from the cumulative efficiency for the latter case. The results show that using the vapor wiper mechanism increases the cumulative efficiency by 9.4% and reduces the boiling time by 35.3 minutes. Finally, the techno-economic analysis shows that the cooker with a vapor wiper mechanism has a good reliability for outdoor cooking of food and is economically feasible.

Induction of S-phase entry by E2F transcription factors depends on their nuclear localization

DEFF Research Database (Denmark)

Müller, H; Moroni, M C; Vigo, E

1997-01-01

The E2F transcription factors are essential for regulating the correct timing of activation of several genes whose products are implicated in cell proliferation and DNA replication. The E2Fs are targets for negative regulation by the retinoblastoma protein family, which includes pRB, p107, and p130......, and they are in a pathway that is frequently found altered in human cancers. There are five members of the E2F family, and they can be divided into two functional subgroups. Whereas, upon overexpression, E2F-1, -2, and -3 induce S phase in quiescent fibroblasts and override G1 arrests mediated by the p16INK4A tumor...... suppressor protein or neutralizing antibodies to cyclin D1, E2F-4 and -5 do not. Using E2F-1 and E2F-4 as representatives of the two subgroups, we showed here, by constructing a set of chimeric proteins, that the amino terminus of E2F-1 is sufficient to confer S-phase-inducing potential as well...

Crystallization and preliminary X-ray diffraction studies of choline-binding protein F from Streptococcus pneumoniae

Energy Technology Data Exchange (ETDEWEB)

Molina, Rafael [Grupo de Cristalografía Macromolecular y Biología Estructural, Instituto Química Física Rocasolano, CSIC, Serrano 119, 28006 Madrid (Spain); González, Ana; Moscoso, Miriam; García, Pedro [Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain); Stelter, Meike; Kahn, Richard [Institut de Biologie Structurale J.-P. Ebel CEA CNRS UJF, Laboratoire de Cristallographie Macromoléculaire, 41 Rue Jules Horowitz, 38027 Grenoble CEDEX 1 (France); Hermoso, Juan A., E-mail: xjuan@iqfr.csic.es [Grupo de Cristalografía Macromolecular y Biología Estructural, Instituto Química Física Rocasolano, CSIC, Serrano 119, 28006 Madrid (Spain)

2007-09-01

The modular choline-binding protein F (CbpF) from S. pneumoniae has been crystallized by the hanging-drop vapour-diffusion method. A SAD data set from a gadolinium-complex derivative has been collected to 2.1 Å resolution. Choline-binding protein F (CbpF) is a modular protein that is bound to the pneumococcal cell wall through noncovalent interactions with choline moieties of the bacterial teichoic and lipoteichoic acids. Despite being one of the more abundant proteins on the surface, along with the murein hydrolases LytA, LytB, LytC and Pce, its function is still unknown. CbpF has been crystallized using the hanging-drop vapour-diffusion method at 291 K. Diffraction-quality orthorhombic crystals belong to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 49.13, b = 114.94, c = 75.69 Å. A SAD data set from a Gd-HPDO3A-derivatized CbpF crystal was collected to 2.1 Å resolution at the gadolinium L{sub III} absorption edge using synchrotron radiation.

Crystallization and preliminary X-ray diffraction studies of choline-binding protein F from Streptococcus pneumoniae

International Nuclear Information System (INIS)

Molina, Rafael; González, Ana; Moscoso, Miriam; García, Pedro; Stelter, Meike; Kahn, Richard; Hermoso, Juan A.

2007-01-01

The modular choline-binding protein F (CbpF) from S. pneumoniae has been crystallized by the hanging-drop vapour-diffusion method. A SAD data set from a gadolinium-complex derivative has been collected to 2.1 Å resolution. Choline-binding protein F (CbpF) is a modular protein that is bound to the pneumococcal cell wall through noncovalent interactions with choline moieties of the bacterial teichoic and lipoteichoic acids. Despite being one of the more abundant proteins on the surface, along with the murein hydrolases LytA, LytB, LytC and Pce, its function is still unknown. CbpF has been crystallized using the hanging-drop vapour-diffusion method at 291 K. Diffraction-quality orthorhombic crystals belong to space group P2 1 2 1 2, with unit-cell parameters a = 49.13, b = 114.94, c = 75.69 Å. A SAD data set from a Gd-HPDO3A-derivatized CbpF crystal was collected to 2.1 Å resolution at the gadolinium L III absorption edge using synchrotron radiation

Cartilage acidic protein 1, a new member of the beta-propeller protein family with amyloid propensity.

Science.gov (United States)

Anjos, Liliana; Morgado, Isabel; Guerreiro, Marta; Cardoso, João C R; Melo, Eduardo P; Power, Deborah M

2017-02-01

Cartilage acidic protein1 (CRTAC1) is an extracellular matrix protein of chondrogenic tissue in humans and its presence in bacteria indicate it is of ancient origin. Structural modeling of piscine CRTAC1 reveals it belongs to the large family of beta-propeller proteins that in mammals have been associated with diseases, including amyloid diseases such as Alzheimer's. In order to characterize the structure/function evolution of this new member of the beta-propeller family we exploited the unique characteristics of piscine duplicate genes Crtac1a and Crtac1b and compared their structural and biochemical modifications with human recombinant CRTAC1. We demonstrate that CRTAC1 has a beta-propeller structure that has been conserved during evolution and easily forms high molecular weight thermo-stable aggregates. We reveal for the first time the propensity of CRTAC1 to form amyloid-like structures, and hypothesize that the aggregating property of CRTAC1 may be related to its disease-association. We further contribute to the general understating of CRTAC1's and beta-propeller family evolution and function. Proteins 2017; 85:242-255. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Correlation-induced suppression of decoherence in capacitively coupled Cooper-pair boxes

Science.gov (United States)

Hu, Xuedong; You, J. Q.; Nori, Franco

2005-03-01

Charge fluctuations from gate bias and background traps severely limit the performance of a charge qubit in a Cooper-pair box (CPB). Here we discuss an encoding approachootnotetextJ.Q. You, X.Hu, and F. Nori, cond-mat/0407423. to control the decoherence effects of these charge fluctuations using two strongly capacitively coupled CPBs. This coupled-box system has a low-decoherence subspace of two states, for which we calculate the dephasing and relaxation rates using a master equation approach. Our results show that the inter-box Coulomb correlation can significantly suppress decoherence of this two-level system by reducing the strength of the system-environment interaction, making it a promising candidate as a logical qubit, encoded using two CPBs.

fLPS: Fast discovery of compositional biases for the protein universe.

Science.gov (United States)

Harrison, Paul M

2017-11-13

Proteins often contain regions that are compositionally biased (CB), i.e., they are made from a small subset of amino-acid residue types. These CB regions can be functionally important, e.g., the prion-forming and prion-like regions that are rich in asparagine and glutamine residues. Here I report a new program fLPS that can rapidly annotate CB regions. It discovers both single-residue and multiple-residue biases. It works through a process of probability minimization. First, contigs are constructed for each amino-acid type out of sequence windows with a low degree of bias; second, these contigs are searched exhaustively for low-probability subsequences (LPSs); third, such LPSs are iteratively assessed for merger into possible multiple-residue biases. At each of these stages, efficiency measures are taken to avoid or delay probability calculations unless/until they are necessary. On a current desktop workstation, the fLPS algorithm can annotate the biased regions of the yeast proteome (>5700 sequences) in 65 million sequences) in as little as ~1 h, which is >2 times faster than the commonly used program SEG, using default parameters. fLPS discovers both shorter CB regions (of the sort that are often termed 'low-complexity sequence'), and milder biases that may only be detectable over long tracts of sequence. fLPS can readily handle very large protein data sets, such as might come from metagenomics projects. It is useful in searching for proteins with similar CB regions, and for making functional inferences about CB regions for a protein of interest. The fLPS package is available from: http://biology.mcgill.ca/faculty/harrison/flps.html , or https://github.com/pmharrison/flps , or is a supplement to this article.

Mitochondrial Band-7 family proteins: scaffolds for respiratory chain assembly?

Directory of Open Access Journals (Sweden)

Bernadette eGehl

2014-04-01

Full Text Available The band-7 protein family comprises a diverse set of membrane-bound proteins characterised by the presence of a conserved domain. The exact function of this band-7 domain remains elusive, but examples from animal and bacterial stomatin-type proteins demonstrate binding to lipids and the ability to assemble into membrane-bound oligomers that form putative scaffolds. Some members, such as prohibitins and human stomatin-like protein 2 (HsSLP2, localise to the mitochondrial inner membrane where they function in cristae formation and hyperfusion. In Arabidopsis, the band-7 protein family has diversified and includes plant-specific members. Mitochondrial-localised members include prohibitins (AtPHBs and two stomatin-like proteins (AtSLP1 and -2. Studies into PHB function in plants have demonstrated an involvement in root meristem proliferation and putative scaffold formation for mAAA proteases, but it remains unknown how these roles are achieved at the molecular level. In this minireview we summarise the current status of band-7 protein functions in Arabidopsis, and speculate how the mitochondrial members might recruit specific lipids to form microdomains that could shape the organisation and functioning of the respiratory chain.

Molecular genetic analysis of the F11 gene in 14 Turkish patients with factor XI deficiency: identification of novel and recurrent mutations and their inheritance within families.

Science.gov (United States)

Colakoglu, Seyma; Bayhan, Turan; Tavil, Betül; Keskin, Ebru Yılmaz; Cakir, Volkan; Gümrük, Fatma; Çetin, Mualla; Aytaç, Selin; Berber, Ergul

2018-01-01

Factor XI (FXI) deficiency is an autosomal bleeding disease associated with genetic defects in the F11 gene which cause decreased FXI levels or impaired FXI function. An increasing number of mutations has been reported in the FXI mutation database, most of which affect the serine protease domain of the protein. FXI is a heterogeneous disorder associated with a variable bleeding tendency and a variety of causative F11 gene mutations. The molecular basis of FXI deficiency in 14 patients from ten unrelated families in Turkey was analysed to establish genotype-phenotype correlations and inheritance of the mutations in the patients' families. Fourteen index cases with a diagnosis of FXI deficiency and family members of these patients were enrolled into the study. The patients' F11 genes were amplified by polymerase chain reaction and subjected to direct DNA sequencing analysis. The findings were analysed statistically using bivariate correlations, Pearson's correlation coefficient and the nonparametric Mann-Whitney test. Direct DNA sequencing analysis of the F11 genes revealed that all of the 14 patients had a F11 gene mutation. Eight different mutations were identified in the apple 1, apple 2 or serine protease domains, except one which was a splice site mutation. Six of the mutations were recurrent. Two of the mutations were novel missense mutations, p.Val522Gly and p.Cys581Arg, within the catalytic domain. The p.Trp519Stop mutation was observed in two families whereas all the other mutations were specific to a single family. Identification of mutations confirmed the genetic heterogeneity of FXI deficiency. Most of the patients with mutations did not have any bleeding complications, whereas some had severe bleeding symptoms. Genetic screening for F11 gene mutations is important to decrease the mortality and morbidity rate associated with FXI deficiency, which can be life-threatening if bleeding occurs in tissues with high fibrinolytic activity.

Characterization of the porcine FBX07 gene: the first step towards generation of a pig model for Parkinsonian pyramidal syndrome

DEFF Research Database (Denmark)

Larsen, Knud; Bendixen, Christian

2012-01-01

Parkinsonian pyramidal syndrome, also named pallido-pyramidal syndrome (PKPS), is the combination of early-onset progressive Parkinsonism with pyramidal tract signs. FBXO7, an F-box protein, is a component of modular E3 ubiquitin protein ligases called SCFs (SKP1, cullin, F-box proteins), which...

The V279F polymorphism might change protein character and ...

African Journals Online (AJOL)

Widodo

2017-08-18

Aug 18, 2017 ... family [1,2] that can degrade oxidized lipids, presumably having .... with aquadest (9 ul), then 90 μL coating buffer was added, then vortexed. .... the 279 F variant is protective, while some express it as an inductor of AMI [19,20 ...

Orphan Nuclear Receptor NR4A1 Binds a Novel Protein Interaction Site on Anti-apoptotic B Cell Lymphoma Gene 2 Family Proteins.

Science.gov (United States)

Godoi, Paulo H C; Wilkie-Grantham, Rachel P; Hishiki, Asami; Sano, Renata; Matsuzawa, Yasuko; Yanagi, Hiroko; Munte, Claudia E; Chen, Ya; Yao, Yong; Marassi, Francesca M; Kalbitzer, Hans R; Matsuzawa, Shu-Ichi; Reed, John C

2016-07-01

B cell lymphoma gene 2 (Bcl-2) family proteins are key regulators of programmed cell death and important targets for drug discovery. Pro-apoptotic and anti-apoptotic Bcl-2 family proteins reciprocally modulate their activities in large part through protein interactions involving a motif known as BH3 (Bcl-2 homology 3). Nur77 is an orphan member of the nuclear receptor family that lacks a BH3 domain but nevertheless binds certain anti-apoptotic Bcl-2 family proteins (Bcl-2, Bfl-1, and Bcl-B), modulating their effects on apoptosis and autophagy. We used a combination of NMR spectroscopy-based methods, mutagenesis, and functional studies to define the interaction site of a Nur77 peptide on anti-apoptotic Bcl-2 family proteins and reveal a novel interaction surface. Nur77 binds adjacent to the BH3 peptide-binding crevice, suggesting the possibility of cross-talk between these discrete binding sites. Mutagenesis of residues lining the identified interaction site on Bcl-B negated the interaction with Nur77 protein in cells and prevented Nur77-mediated modulation of apoptosis and autophagy. The findings establish a new protein interaction site with the potential to modulate the apoptosis and autophagy mechanisms governed by Bcl-2 family proteins. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

46 CFR 111.81-1 - Outlet boxes and junction boxes; general.

Science.gov (United States)

2010-10-01

... fixture, wiring device, or similar item, including each separately installed connection and junction box... used. (d) As appropriate, each outlet-box or junction-box installation must meet the following...

Identification of a novel centrosomal protein CrpF46 involved in cell cycle progression and mitosis

International Nuclear Information System (INIS)

Wei Yi; Shen Enzhi; Zhao Na; Liu Qian; Fan Jinling; Marc, Jan; Wang Yongchao; Sun Le; Liang Qianjin

2008-01-01

A novel centrosome-related protein Crp F46 was detected using a serum F46 from a patient suffering from progressive systemic sclerosis. We identified the protein by immunoprecipitation and Western blotting followed by tandem mass spectrometry sequencing. The protein Crp F46 has an apparent molecular mass of ∼ 60 kDa, is highly homologous to a 527 amino acid sequence of the C-terminal portion of the protein Golgin-245, and appears to be a splice variant of Golgin-245. Immunofluorescence microscopy of synchronized HeLa cells labeled with an anti-Crp F46 monoclonal antibody revealed that Crp F46 localized exclusively to the centrosome during interphase, although it dispersed throughout the cytoplasm at the onset of mitosis. Domain analysis using Crp F46 fragments in GFP-expression vectors transformed into HeLa cells revealed that centrosomal targeting is conferred by a C-terminal coiled-coil domain. Antisense Crp F46 knockdown inhibited cell growth and proliferation and the cell cycle typically stalled at S phase. The knockdown also resulted in the formation of poly-centrosomal and multinucleate cells, which finally became apoptotic. These results suggest that Crp F46 is a novel centrosome-related protein that associates with the centrosome in a cell cycle-dependent manner and is involved in the progression of the cell cycle and M phase mechanism

Genetics Home Reference: familial atrial fibrillation

Science.gov (United States)

... Twitter Home Health Conditions Familial atrial fibrillation Familial atrial fibrillation Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Familial atrial fibrillation is an inherited abnormality of the heart's normal ...

Analysis of E2F factors during epidermal differentiation.

Science.gov (United States)

Chang, Wing Y; Dagnino, Lina

2005-01-01

The multigene E2F family of transcription factors is central in the control of cell cycle progression. The expression and activity of E2F proteins is tightly regulated transcriptionally and posttranslationally as a function of the proliferation and differentiation status of the cell. In this chapter, we review protocols designed to determine E2F mRNA abundance in tissues by in situ hybridization techniques. The ability to culture primary epidermal keratinocytes and maintain them as either undifferentiated or terminally differentiated cells allows the biochemical and molecular characterization of changes in E2F expression and activity. Thus, we also discuss in detail methods to analyze E2F protein abundance by immunoblot and their ability to bind DNA in cultured cells using electrophoretic mobility shift assays.

A novel family of plant nuclear envelope-associated proteins.

Science.gov (United States)

Pawar, Vidya; Poulet, Axel; Détourné, Gwénaëlle; Tatout, Christophe; Vanrobays, Emmanuel; Evans, David E; Graumann, Katja

2016-10-01

This paper describes the characterisation of a new family of higher plant nuclear envelope-associated proteins (NEAPs) that interact with other proteins of the nuclear envelope. In the model plant Arabidopsis thaliana, the family consists of three genes expressed ubiquitously (AtNEAP1-3) and a pseudogene (AtNEAP4). NEAPs consist of extensive coiled-coil domains, followed by a nuclear localisation signal and a C-terminal predicted transmembrane domain. Domain deletion mutants confirm the presence of a functional nuclear localisation signal and transmembrane domain. AtNEAP proteins localise to the nuclear periphery as part of stable protein complexes, are able to form homo- and heteromers, and interact with the SUN domain proteins AtSUN1 and AtSUN2, involved in the linker of nucleoskeleton and cytoskeleton (LINC) complex. An A. thaliana cDNA library screen identified a putative transcription factor called AtbZIP18 as a novel interactor of AtNEAP1, which suggest a connection between NEAP and chromatin. An Atneap1 Atneap3 double-knockout mutant showed reduced root growth, and altered nuclear morphology and chromatin structure. Thus AtNEAPs are suggested as inner nuclear membrane-anchored coiled-coil proteins with roles in maintaining nuclear morphology and chromatin structure. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Improving Your Data Transformations: Applying the Box-Cox Transformation

Directory of Open Access Journals (Sweden)

Jason W. Osborne

2010-10-01

Full Text Available Many of us in the social sciences deal with data that do not conform to assumptions of normality and/or homoscedasticity/homogeneity of variance. Some research has shown that parametric tests (e.g., multiple regression, ANOVA can be robust to modest violations of these assumptions. Yet the reality is that almost all analyses (even nonparametric tests benefit from improved the normality of variables, particularly where substantial non-normality is present. While many are familiar with select traditional transformations (e.g., square root, log, inverse for improving normality, the Box-Cox transformation (Box & Cox, 1964 represents a family of power transformations that incorporates and extends the traditional options to help researchers easily find the optimal normalizing transformation for each variable. As such, Box-Cox represents a potential best practice where normalizing data or equalizing variance is desired. This paper briefly presents an overview of traditional normalizing transformations and how Box-Cox incorporates, extends, and improves on these traditional approaches to normalizing data. Examples of applications are presented, and details of how to automate and use this technique in SPSS and SAS are included.

FAM20: an evolutionarily conserved family of secreted proteins expressed in hematopoietic cells

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Cobos Everardo

2005-01-01

Full Text Available Abstract Background Hematopoiesis is a complex developmental process controlled by a large number of factors that regulate stem cell renewal, lineage commitment and differentiation. Secreted proteins, including the hematopoietic growth factors, play critical roles in these processes and have important biological and clinical significance. We have employed representational difference analysis to identify genes that are differentially expressed during experimentally induced myeloid differentiation in the murine EML hematopoietic stem cell line. Results One identified clone encoded a previously unidentified protein of 541 amino acids that contains an amino terminal signal sequence but no other characterized domains. This protein is a member of family of related proteins that has been named family with sequence similarity 20 (FAM20 with three members (FAM20A, FAM20B and FAM20C in mammals. Evolutionary comparisons revealed the existence of a single FAM20 gene in the simple vertebrate Ciona intestinalis and the invertebrate worm Caenorhabditis elegans and two genes in two insect species, Drosophila melanogaster and Anopheles gambiae. Six FAM20 family members were identified in the genome of the pufferfish, Fugu rubripes and five members in the zebrafish, Danio rerio. The mouse Fam20a protein was ectopically expressed in a mammalian cell line and found to be a bona fide secreted protein and efficient secretion was dependent on the integrity of the signal sequence. Expression analysis revealed that the Fam20a gene was indeed differentially expressed during hematopoietic differentiation and that the other two family members (Fam20b and Fam20c were also expressed during hematcpoiesis but that their mRNA levels did not vary significantly. Likewise FAM20A was expressed in more limited set of human tissues than the other two family members. Conclusions The FAM20 family represents a new family of secreted proteins with potential functions in regulating

Msx1 and Msx2 are functional interacting partners of T-box factors in the regulation of Connexin43.

Science.gov (United States)

Boogerd, Kees-Jan; Wong, L Y Elaine; Christoffels, Vincent M; Klarenbeek, Meinke; Ruijter, Jan M; Moorman, Antoon F M; Barnett, Phil

2008-06-01

T-box factors Tbx2 and Tbx3 play key roles in the development of the cardiac conduction system, atrioventricular canal, and outflow tract of the heart. They regulate the gap-junction-encoding gene Connexin43 (Cx43) and other genes critical for heart development and function. Discovering protein partners of Tbx2 and Tbx3 will shed light on the mechanisms by which these factors regulate these gene programs. Employing an yeast 2-hybrid screen and subsequent in vitro pull-down experiments we demonstrate that muscle segment homeobox genes Msx1 and Msx2 are able to bind the cardiac T-box proteins Tbx2, Tbx3, and Tbx5. This interaction, as that of the related Nkx2.5 protein, is supported by the T-box and homeodomain alone. Overlapping spatiotemporal expression patterns of Msx1 and Msx2 together with the T-box genes during cardiac development in mouse and chicken underscore the biological significance of this interaction. We demonstrate that Msx proteins together with Tbx2 and Tbx3 suppress Cx43 promoter activity and down regulate Cx43 gene activity in a rat heart-derived cell line. Using chromatin immunoprecipitation analysis we demonstrate that Msx1 can bind the Cx43 promoter at a conserved binding site located in close proximity to a previously defined T-box binding site, and that the activity of Msx proteins on this promoter appears dependent in the presence of Tbx3. Msx1 and Msx2 can function in concert with the T-box proteins to suppress Cx43 and other working myocardial genes.

Horizontal transfer, not duplication, drives the expansion of protein families in prokaryotes.

Directory of Open Access Journals (Sweden)

Todd J Treangen

2011-01-01

Full Text Available Gene duplication followed by neo- or sub-functionalization deeply impacts the evolution of protein families and is regarded as the main source of adaptive functional novelty in eukaryotes. While there is ample evidence of adaptive gene duplication in prokaryotes, it is not clear whether duplication outweighs the contribution of horizontal gene transfer in the expansion of protein families. We analyzed closely related prokaryote strains or species with small genomes (Helicobacter, Neisseria, Streptococcus, Sulfolobus, average-sized genomes (Bacillus, Enterobacteriaceae, and large genomes (Pseudomonas, Bradyrhizobiaceae to untangle the effects of duplication and horizontal transfer. After removing the effects of transposable elements and phages, we show that the vast majority of expansions of protein families are due to transfer, even among large genomes. Transferred genes--xenologs--persist longer in prokaryotic lineages possibly due to a higher/longer adaptive role. On the other hand, duplicated genes--paralogs--are expressed more, and, when persistent, they evolve slower. This suggests that gene transfer and gene duplication have very different roles in shaping the evolution of biological systems: transfer allows the acquisition of new functions and duplication leads to higher gene dosage. Accordingly, we show that paralogs share most protein-protein interactions and genetic regulators, whereas xenologs share very few of them. Prokaryotes invented most of life's biochemical diversity. Therefore, the study of the evolution of biology systems should explicitly account for the predominant role of horizontal gene transfer in the diversification of protein families.

Ubiquitin-Mediated Regulation of Endocytosis by Proteins of the Arrestin Family

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Michel Becuwe

2012-01-01

Full Text Available In metazoans, proteins of the arrestin family are key players of G-protein-coupled receptors (GPCRS signaling and trafficking. Following stimulation, activated receptors are phosphorylated, thus allowing the binding of arrestins and hence an “arrest” of receptor signaling. Arrestins act by uncoupling receptors from G proteins and contribute to the recruitment of endocytic proteins, such as clathrin, to direct receptor trafficking into the endocytic pathway. Arrestins also serve as adaptor proteins by promoting the recruitment of ubiquitin ligases and participate in the agonist-induced ubiquitylation of receptors, known to have impact on their subcellular localization and stability. Recently, the arrestin family has expanded following the discovery of arrestin-related proteins in other eukaryotes such as yeasts or fungi. Surprisingly, most of these proteins are also involved in the ubiquitylation and endocytosis of plasma membrane proteins, thus suggesting that the role of arrestins as ubiquitin ligase adaptors is at the core of these proteins' functions. Importantly, arrestins are themselves ubiquitylated, and this modification is crucial for their function. In this paper, we discuss recent data on the intricate connections between arrestins and the ubiquitin pathway in the control of endocytosis.

Structural basis for substrate placement by an archaeal box C/D ribonucleoprotein particle.

Science.gov (United States)

Xue, Song; Wang, Ruiying; Yang, Fangping; Terns, Rebecca M; Terns, Michael P; Zhang, Xinxin; Maxwell, E Stuart; Li, Hong

2010-09-24

Box C/D small nucleolar and Cajal body ribonucleoprotein particles (sno/scaRNPs) direct site-specific 2'-O-methylation of ribosomal and spliceosomal RNAs and are critical for gene expression. Here we report crystal structures of an archaeal box C/D RNP containing three core proteins (fibrillarin, Nop56/58, and L7Ae) and a half-mer box C/D guide RNA paired with a substrate RNA. The structure reveals a guide-substrate RNA duplex orientation imposed by a composite protein surface and the conserved GAEK motif of Nop56/58. Molecular modeling supports a dual C/D RNP structure that closely mimics that recently visualized by electron microscopy. The substrate-bound dual RNP model predicts an asymmetric protein distribution between the RNP that binds and methylates the substrate RNA. The predicted asymmetric nature of the holoenzyme is consistent with previous biochemical data on RNP assembly and provides a simple solution for accommodating base-pairing between the C/D guide RNA and large ribosomal and spliceosomal substrate RNAs. Copyright © 2010 Elsevier Inc. All rights reserved.

Disorder and function: a review of the dehydrin protein family

Directory of Open Access Journals (Sweden)

Steffen P Graether

2014-10-01

Full Text Available Dehydration proteins (dehydrins are group 2 members of the late embryogenesis abundant (LEA protein family. The protein architecture of dehydrins can be described by the presence of three types of conserved sequence motifs that have been named the K-, Y- and S-segments. By definition, a dehydrin must contain at least one copy of the lysine-rich K-segment. Abiotic stresses such as drought, cold, and salinity cause the upregulation of dehydrin mRNA and protein levels. Despite the large body of genetic and protein evidence of the importance of these proteins in stress response, the in vivo protective mechanism is not fully known. In vitro experimental evidence from biochemical assays and localization experiments suggest multiple roles for dehydrins, including membrane protection, cryoprotection of enzymes, and protection from reactive oxygen species. Membrane binding by dehydrins is likely to be as a peripheral membrane protein, since the protein sequences are highly hydrophilic and contain many charged amino acids. Because of this, dehydrins in solution are intrinsically disordered proteins, that is, they have no well-defined secondary or tertiary structure. Despite their disorder, dehydrins have been shown to gain structure when bound to ligands such as membranes, and to possibly change their oligomeric state when bound to ions. We review what is currently known about dehydrin sequences and their structures, and examine the various ligands that have been shown to bind to this family of proteins.

An estimated 5% of new protein structures solved today represent a new Pfam family

International Nuclear Information System (INIS)

Mistry, Jaina; Kloppmann, Edda; Rost, Burkhard; Punta, Marco

2013-01-01

This study uses the Pfam database to show that the sequence redundancy of protein structures deposited in the PDB is increasing. The possible reasons behind this trend are discussed. High-resolution structural knowledge is key to understanding how proteins function at the molecular level. The number of entries in the Protein Data Bank (PDB), the repository of all publicly available protein structures, continues to increase, with more than 8000 structures released in 2012 alone. The authors of this article have studied how structural coverage of the protein-sequence space has changed over time by monitoring the number of Pfam families that acquired their first representative structure each year from 1976 to 2012. Twenty years ago, for every 100 new PDB entries released, an estimated 20 Pfam families acquired their first structure. By 2012, this decreased to only about five families per 100 structures. The reasons behind the slower pace at which previously uncharacterized families are being structurally covered were investigated. It was found that although more than 50% of current Pfam families are still without a structural representative, this set is enriched in families that are small, functionally uncharacterized or rich in problem features such as intrinsically disordered and transmembrane regions. While these are important constraints, the reasons why it may not yet be time to give up the pursuit of a targeted but more comprehensive structural coverage of the protein-sequence space are discussed

Distinct mechanisms of nuclear accumulation regulate the functional consequence of E2F transcription factors

NARCIS (Netherlands)

Allen, K.E.; Luna, S. de la; Kerkhoven, R.M.; Bernards, R.A.; Thangue, N.B. La

1997-01-01

Transcription factor E2F plays an important role in coordinating and integrating early cell cycle progression with the transcription apparatus. It is known that physiological E2F arises when a member of two families of proteins, E2F and DP, interact as E2F/DP heterodimers and that transcriptional

The Role of the E2F Transcription Factor Family in UV-Induced Apoptosis

Directory of Open Access Journals (Sweden)

Orla Gannon

2011-12-01

Full Text Available The E2F transcription factor family is traditionally associated with cell cycle control. However, recent data has shown that activating E2Fs (E2F1-3a are potent activators of apoptosis. In contrast, the recently cloned inhibitory E2Fs (E2F7 and 8 appear to antagonize E2F-induced cell death. In this review we will discuss (i the potential role of E2Fs in UV-induced cell death and (ii the implications of this to the development of UV-induced cutaneous malignancies.

Expansion of banana (Musa acuminata) gene families involved in ethylene biosynthesis and signalling after lineage-specific whole-genome duplications.

Science.gov (United States)

Jourda, Cyril; Cardi, Céline; Mbéguié-A-Mbéguié, Didier; Bocs, Stéphanie; Garsmeur, Olivier; D'Hont, Angélique; Yahiaoui, Nabila

2014-05-01

Whole-genome duplications (WGDs) are widespread in plants, and three lineage-specific WGDs occurred in the banana (Musa acuminata) genome. Here, we analysed the impact of WGDs on the evolution of banana gene families involved in ethylene biosynthesis and signalling, a key pathway for banana fruit ripening. Banana ethylene pathway genes were identified using comparative genomics approaches and their duplication modes and expression profiles were analysed. Seven out of 10 banana ethylene gene families evolved through WGD and four of them (1-aminocyclopropane-1-carboxylate synthase (ACS), ethylene-insensitive 3-like (EIL), ethylene-insensitive 3-binding F-box (EBF) and ethylene response factor (ERF)) were preferentially retained. Banana orthologues of AtEIN3 and AtEIL1, two major genes for ethylene signalling in Arabidopsis, were particularly expanded. This expansion was paralleled by that of EBF genes which are responsible for control of EIL protein levels. Gene expression profiles in banana fruits suggested functional redundancy for several MaEBF and MaEIL genes derived from WGD and subfunctionalization for some of them. We propose that EIL and EBF genes were co-retained after WGD in banana to maintain balanced control of EIL protein levels and thus avoid detrimental effects of constitutive ethylene signalling. In the course of evolution, subfunctionalization was favoured to promote finer control of ethylene signalling. © 2014 CIRAD New Phytologist © 2014 New Phytologist Trust.

Identification of Heat Shock Protein families and J-protein types by incorporating Dipeptide Composition into Chou's general PseAAC.

Science.gov (United States)

Ahmad, Saeed; Kabir, Muhammad; Hayat, Maqsood

2015-11-01

Heat Shock Proteins (HSPs) are the substantial ingredients for cell growth and viability, which are found in all living organisms. HSPs manage the process of folding and unfolding of proteins, the quality of newly synthesized proteins and protecting cellular homeostatic processes from environmental stress. On the basis of functionality, HSPs are categorized into six major families namely: (i) HSP20 or sHSP (ii) HSP40 or J-proteins types (iii) HSP60 or GroEL/ES (iv) HSP70 (v) HSP90 and (vi) HSP100. Identification of HSPs family and sub-family through conventional approaches is expensive and laborious. It is therefore, highly desired to establish an automatic, robust and accurate computational method for prediction of HSPs quickly and reliably. Regard, a computational model is developed for the prediction of HSPs family. In this model, protein sequences are formulated using three discrete methods namely: Split Amino Acid Composition, Pseudo Amino Acid Composition, and Dipeptide Composition. Several learning algorithms are utilized to choice the best one for high throughput computational model. Leave one out test is applied to assess the performance of the proposed model. The empirical results showed that support vector machine achieved quite promising results using Dipeptide Composition feature space. The predicted outcomes of proposed model are 90.7% accuracy for HSPs dataset and 97.04% accuracy for J-protein types, which are higher than existing methods in the literature so far. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Channel box

International Nuclear Information System (INIS)

Tanabe, Akira.

1993-01-01

In a channel box of a BWR type reactor, protruding pads are disposed in axial position on the lateral side of a channel box opposing to a control rod and facing the outer side portion of the control rod in a reactor core loaded state. In the initial loading stage of fuel assemblies, channel fasteners and spacer pads are abutted against each other in the upper portion between the channel boxes sandwiching the control rod therebetween. Further, in the lower portion, a gap as a channel for the movement of the control rod is ensured by the support of fuel support metals. If the channel box is bent toward the control rod along with reactor operation, the pads are abutted against each other to always ensure the gap through which the control rod can move easily. Further, when the pads are brought into contact with each other, the bending deformation of the channel box is corrected by urging to each other. Thus, the control rod can always be moved smoothly to attain reactor safety operation. (N.H.)

Phosphorylation of protein synthesis initiation factor 2 (elF-2) in the yeast Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Romero, D.P.

1986-01-01

Initiation Factor 2 (elF-2) in the yeast Saccharomyces cerevisiae is comprised of 3 subunits. The control of protein synthesis in mammalian cells have been shown to involve the phosphorylation of the small (alpha) subunit by a specific protein kinase. Phosphorylation results in an inhibition of protein synthesis. In order to determine whether or not an analogous system is operative in yeast, the phosphorylation state of the alpha subunit of elF-2 in Saccharomyces was determined during various growth and nongrowth conditions. Cells were radiolabelled with 32 P and 35 S, and the whole cell lysates were analyzed by two dimensional gel electrophoresis. These experiments revealed that the smallest subunit (alpha, M/sub r/ = 31,000) is a phosphoprotein in vivo under a variety of growth and nongrowth conditions. This is in direct contrast to the pattern exhibited in mammalian cells. The fact that the small subunit of elF-2 in yeast is phosphorylated under a variety of physiological conditions indicates that such a covalent modification is important for some aspects of elF-2 function. In order to investigate this problem further, a protein kinase that specifically labels the alpha subunit of elF-2 in vitro was isolated. The kinase is not autophosphorylating, utilizes ATP as a phosphate donor, phosphorylates an exogenous protein, casein, modifies serine residues in elF-2, is cyclic nucleotide-independent, and is strongly inhibited by heparin

Transcriptome analysis reveals the same 17 S-locus F-box genes in two haplotypes of the self-incompatibility locus of Petunia inflata.

Science.gov (United States)

Williams, Justin S; Der, Joshua P; dePamphilis, Claude W; Kao, Teh-Hui

2014-07-01

Petunia possesses self-incompatibility, by which pistils reject self-pollen but accept non-self-pollen for fertilization. Self-/non-self-recognition between pollen and pistil is regulated by the pistil-specific S-RNase gene and by multiple pollen-specific S-locus F-box (SLF) genes. To date, 10 SLF genes have been identified by various methods, and seven have been shown to be involved in pollen specificity. For a given S-haplotype, each SLF interacts with a subset of its non-self S-RNases, and an as yet unknown number of SLFs are thought to collectively mediate ubiquitination and degradation of all non-self S-RNases to allow cross-compatible pollination. To identify a complete suite of SLF genes of P. inflata, we used a de novo RNA-seq approach to analyze the pollen transcriptomes of S2-haplotype and S3-haplotype, as well as the leaf transcriptome of the S3S3 genotype. We searched for genes that fit several criteria established from the properties of the known SLF genes and identified the same seven new SLF genes in S2-haplotype and S3-haplotype, suggesting that a total of 17 SLF genes constitute pollen specificity in each S-haplotype. This finding lays the foundation for understanding how multiple SLF genes evolved and the biochemical basis for differential interactions between SLF proteins and S-RNases. © 2014 American Society of Plant Biologists. All rights reserved.

Ankyrin repeat and SOCS box containing protein 4 (Asb-4 colocalizes with insulin receptor substrate 4 (IRS4 in the hypothalamic neurons and mediates IRS4 degradation

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Xia Zefeng

2011-09-01

Full Text Available Abstract Background The arcuate nucleus of the hypothalamus regulates food intake. Ankyrin repeat and SOCS box containing protein 4 (Asb-4 is expressed in neuropeptide Y and proopiomelanocortin (POMC neurons in the arcuate nucleus, target neurons in the regulation of food intake and metabolism by insulin and leptin. However, the target protein(s of Asb-4 in these neurons remains unknown. Insulin receptor substrate 4 (IRS4 is an adaptor molecule involved in the signal transduction by both insulin and leptin. In the present study we examined the colocalization and interaction of Asb-4 with IRS4 and the involvement of Asb-4 in insulin signaling. Results In situ hybridization showed that the expression pattern of Asb-4 was consistent with that of IRS4 in the rat brain. Double in situ hybridization showed that IRS4 colocalized with Asb-4, and both Asb-4 and IRS4 mRNA were expressed in proopiomelanocortin (POMC and neuropeptide Y (NPY neurons within the arcuate nucleus of the hypothalamus. In HEK293 cells co-transfected with Myc-tagged Asb-4 and Flag-tagged IRS4, Asb-4 co-immunoprecipitated with IRS4; In these cells endogenous IRS4 also co-immunoprecipitated with transfected Myc-Asb-4; Furthermore, Asb-4 co-immunoprecipitated with IRS4 in rat hypothalamic extracts. In HEK293 cells over expression of Asb-4 decreased IRS4 protein levels and deletion of the SOCS box abolished this effect. Asb-4 increased the ubiquitination of IRS4; Deletion of SOCS box abolished this effect. Expression of Asb-4 decreased both basal and insulin-stimulated phosphorylation of AKT at Thr308. Conclusions These data demonstrated that Asb-4 co-localizes and interacts with IRS4 in hypothalamic neurons. The interaction of Asb-4 with IRS4 in cell lines mediates the degradation of IRS4 and decreases insulin signaling.

Embryonal Fyn-associated substrate (EFS) and CASS4: The lesser-known CAS protein family members.

Science.gov (United States)

Deneka, Alexander; Korobeynikov, Vladislav; Golemis, Erica A

2015-10-01

The CAS (Crk-associated substrate) adaptor protein family consists of four members: CASS1/BCAR1/p130Cas, CASS2/NEDD9/HEF1/Cas-L, CASS3/EFS/Sin and CASS4/HEPL. While CAS proteins lack enzymatic activity, they contain specific recognition and binding sites for assembly of larger signaling complexes that are essential for cell proliferation, survival, migration, and other processes. All family members are intermediates in integrin-dependent signaling pathways mediated at focal adhesions, and associate with FAK and SRC family kinases to activate downstream effectors regulating the actin cytoskeleton. Most studies of CAS proteins to date have been focused on the first two members, BCAR1 and NEDD9, with altered expression of these proteins now appreciated as influencing disease development and prognosis for cancer and other serious pathological conditions. For these family members, additional mechanisms of action have been defined in receptor tyrosine kinase (RTK) signaling, estrogen receptor signaling or cell cycle progression, involving discrete partner proteins such as SHC, NSP proteins, or AURKA. By contrast, EFS and CASS4 have been less studied, although structure-function analyses indicate they conserve many elements with the better-known family members. Intriguingly, a number of recent studies have implicated these proteins in immune system function, and the pathogenesis of developmental disorders, autoimmune disorders including Crohn's disease, Alzheimer's disease, cancer and other diseases. In this review, we summarize the current understanding of EFS and CASS4 protein function in the context of the larger CAS family group. Copyright © 2015 Elsevier B.V. All rights reserved.

Genome-wide comparative in silico analysis of the RNA helicase gene family in Zea mays and Glycine max: a comparison with Arabidopsis and Oryza sativa.

Science.gov (United States)

Xu, Ruirui; Zhang, Shizhong; Huang, Jinguang; Zheng, Chengchao

2013-01-01

RNA helicases are enzymes that are thought to unwind double-stranded RNA molecules in an energy-dependent fashion through the hydrolysis of NTP. RNA helicases are associated with all processes involving RNA molecules, including nuclear transcription, editing, splicing, ribosome biogenesis, RNA export, and organelle gene expression. The involvement of RNA helicase in response to stress and in plant growth and development has been reported previously. While their importance in Arabidopsis and Oryza sativa has been partially studied, the function of RNA helicase proteins is poorly understood in Zea mays and Glycine max. In this study, we identified a total of RNA helicase genes in Arabidopsis and other crop species genome by genome-wide comparative in silico analysis. We classified the RNA helicase genes into three subfamilies according to the structural features of the motif II region, such as DEAD-box, DEAH-box and DExD/H-box, and different species showed different patterns of alternative splicing. Secondly, chromosome location analysis showed that the RNA helicase protein genes were distributed across all chromosomes with different densities in the four species. Thirdly, phylogenetic tree analyses identified the relevant homologs of DEAD-box, DEAH-box and DExD/H-box RNA helicase proteins in each of the four species. Fourthly, microarray expression data showed that many of these predicted RNA helicase genes were expressed in different developmental stages and different tissues under normal growth conditions. Finally, real-time quantitative PCR analysis showed that the expression levels of 10 genes in Arabidopsis and 13 genes in Zea mays were in close agreement with the microarray expression data. To our knowledge, this is the first report of a comparative genome-wide analysis of the RNA helicase gene family in Arabidopsis, Oryza sativa, Zea mays and Glycine max. This study provides valuable information for understanding the classification and putative functions of

Evaluation of cyclooxygenase protein expression in traumatized versus normal tissues from eastern box turtles (Terrapene carolina carolina).

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Royal, Lillian W; Lascelles, B Duncan X; Lewbart, Gregory A; Correa, Maria T; Jones, Samuel L

2012-06-01

This pilot study was designed to determine whether cyclooxygenase (COX)-1, COX-2, or both are expressed in normal turtle tissues and whether level of expression changes when tissue becomes inflamed. Five eastern box turtles, Terrapene carolina carolina, that either died or were euthanatized due to disease or injuries were used for this work. Tissues were obtained from the five turtles. Western blot analysis was used to evaluate tissues for COX-1 and COX-2 proteins. Densiometric analysis was used to compare Western blot bands within each turtle. COX-1 and COX-2 were found in the liver, kidney, grossly normal muscle, and grossly traumatized (inflamed) muscle of all study turtles. In all cases, COX-1 and COX-2 proteins were increased in traumatized muscle over grossly normal nontraumatized muscle. The highest levels of COX-1 and COX-2 proteins were found in kidney and liver. There was no statistical difference between the amount of COX-1 protein in liver and kidney, but traumatized muscle compared with grossly normal muscle had significantly greater COX-1 but not COX 2 protein concentrations. There was no statistical difference between the amount of COX-2 protein in liver and kidney. Traumatized muscle expressed nonstatistically significant greater amounts of COX-2 compared with grossly normal muscle. COX-1 and COX-2 proteins are expressed in turtle tissues, and both isoforms are upregulated during inflammation of muscle tissue. Traditional nonsteroidal anti-inflammatory drugs (NSAIDs) that block both COX isoforms might be more efficacious than COX-2-selective drugs. This work suggests that NSAIDs should be evaluated for potential liver and kidney toxicity in turtles.

Diversity and dispersal of a ubiquitous protein family: acyl-CoA dehydrogenases.

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Shen, Yao-Qing; Lang, B Franz; Burger, Gertraud

2009-09-01

Acyl-CoA dehydrogenases (ACADs), which are key enzymes in fatty acid and amino acid catabolism, form a large, pan-taxonomic protein family with at least 13 distinct subfamilies. Yet most reported ACAD members have no subfamily assigned, and little is known about the taxonomic distribution and evolution of the subfamilies. In completely sequenced genomes from approximately 210 species (eukaryotes, bacteria and archaea), we detect ACAD subfamilies by rigorous ortholog identification combining sequence similarity search with phylogeny. We then construct taxonomic subfamily-distribution profiles and build phylogenetic trees with orthologous proteins. Subfamily profiles provide unparalleled insight into the organisms' energy sources based on genome sequence alone and further predict enzyme substrate specificity, thus generating explicit working hypotheses for targeted biochemical experimentation. Eukaryotic ACAD subfamilies are traditionally considered as mitochondrial proteins, but we found evidence that in fungi one subfamily is located in peroxisomes and participates in a distinct beta-oxidation pathway. Finally, we discern horizontal transfer, duplication, loss and secondary acquisition of ACAD genes during evolution of this family. Through these unorthodox expansion strategies, the ACAD family is proficient in utilizing a large range of fatty acids and amino acids-strategies that could have shaped the evolutionary history of many other ancient protein families.

2',3-dihydroxy-5-methoxybiphenyl suppresses fMLP-induced superoxide anion production and cathepsin G release by targeting the β-subunit of G-protein in human neutrophils.

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Liao, Hsiang-Ruei; Chen, Ih-Sheng; Liu, Fu-Chao; Lin, Shinn-Zhi; Tseng, Ching-Ping

2018-06-15

This study investigates the effect and the underlying mechanism of 2',3-dihydroxy-5-methoxybiphenyl (RIR-2), a lignan extracted from the roots of Rhaphiolepis indica (L.) Lindl. ex Ker var. tashiroi Hayata ex Matsum. & Hayata (Rosaceae), on N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced respiratory burst and cathepsin G in human neutrophils. Signaling pathways regulated by RIR-2 which modulated fMLP-induced respiratory burst were evaluated by an interaction between β subunit of G-protein (Gβ) with downstream signaling induced by fMLP and by immunoblotting analysis of the downstream targets of Gβ-protein. RIR-2 inhibited fMLP-induced superoxide anion production (IC 50 :2.57 ± 0.22 μM), cathepsin G release (IC 50 :18.72 ± 3.76 μM) and migration in a concentration dependent manner. RIR-2 specifically suppresses fMLP-induced Src family kinases phosphorylation by inhibiting the interaction between Gβ-protein with Src kinases without inhibiting Src kinases activities, therefore, RIR-2 attenuated the downstream targets of Src kinase, such as phosphorylation of Raf/ERK, AKT, P38, PLCγ2, PKC and translocation Tec, p47 ph ° x and P40 ph ° x from the cytosol to the inner leaflet of the plasma membrane. Furthermore, RIR-2 attenuated fMLP-induced intracellular calcium mobilization by inhibiting the interaction between Gβ-protein with PLCβ2. RIR-2 was not a competitive or allosteric antagonist of fMLP. On the contrary, phorbol 12-myristate 13-acetate (PMA)-induced phosphorylation of Src, AKT, P38, PKC and membrane localization of p47 ph ° x and P40 ph ° x remained unaffected. RIR-2 specifically modulates fMLP-mediated neutrophil superoxide anion production and cathepsin G release by inhibiting the interaction between Gβ-protein with downstream signaling which subsequently interferes with the activation of intracellular calcium, PLCγ2, AKT, p38, PKC, ERK, p47 ph ° x and p40 phox . Copyright © 2018 Elsevier B.V. All rights reserved.

Structure-function correlations of pulmonary surfactant protein SP-B and the saposin-like family of proteins.

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Olmeda, Bárbara; García-Álvarez, Begoña; Pérez-Gil, Jesús

2013-03-01

Pulmonary surfactant is a lipid-protein complex secreted by the respiratory epithelium of mammalian lungs, which plays an essential role in stabilising the alveolar surface and so reducing the work of breathing. The surfactant protein SP-B is part of this complex, and is strictly required for the assembly of pulmonary surfactant and its extracellular development to form stable surface-active films at the air-liquid alveolar interface, making the lack of SP-B incompatible with life. In spite of its physiological importance, a model for the structure and the mechanism of action of SP-B is still needed. The sequence of SP-B is homologous to that of the saposin-like family of proteins, which are membrane-interacting polypeptides with apparently diverging activities, from the co-lipase action of saposins to facilitate the degradation of sphingolipids in the lysosomes to the cytolytic actions of some antibiotic proteins, such as NK-lysin and granulysin or the amoebapore of Entamoeba histolytica. Numerous studies on the interactions of these proteins with membranes have still not explained how a similar sequence and a potentially related fold can sustain such apparently different activities. In the present review, we have summarised the most relevant features of the structure, lipid-protein and protein-protein interactions of SP-B and the saposin-like family of proteins, as a basis to propose an integrated model and a common mechanistic framework of the apparent functional versatility of the saposin fold.

Characterization of dopaminergic dysfunction in familial progressive supranuclear palsy: an 18F-dopa PET study

International Nuclear Information System (INIS)

Tai, Y.F.; Ahsan, R.L.; Pavese, N.; Brooks, D.J.; Piccini, P.; Yebenes de, J.G.

2007-01-01

We analyzed 18 F-dopa PET data from 11 members of kindreds with familial progressive supranuclear palsy (PSP) to characterize their cerebral dopaminergic dysfunction. Three clinically-affected PSP patients showed reduced 18 F-dopa uptake in the striatum, orbitofrontal cortex and amygdala. One asymptomatic subject exhibited progressive putamen dopaminergic dysfunction. 60 % of subjects with abnormal 18 F-dopa scans developed PSP subsequently. This is the first in vivo documentation of cortical dopaminergic deficiency in PSP. Reduced striatal 18 F-dopa uptake in susceptible relatives may predict later clinical disease. (author)

Evolution of the MAGUK protein gene family in premetazoan lineages

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Ruiz-Trillo Iñaki

2010-04-01

Full Text Available Abstract Background Cell-to-cell communication is a key process in multicellular organisms. In multicellular animals, scaffolding proteins belonging to the family of membrane-associated guanylate kinases (MAGUK are involved in the regulation and formation of cell junctions. These MAGUK proteins were believed to be exclusive to Metazoa. However, a MAGUK gene was recently identified in an EST survey of Capsaspora owczarzaki, an unicellular organism that branches off near the metazoan clade. To further investigate the evolutionary history of MAGUK, we have undertook a broader search for this gene family using available genomic sequences of different opisthokont taxa. Results Our survey and phylogenetic analyses show that MAGUK proteins are present not only in Metazoa, but also in the choanoflagellate Monosiga brevicollis and in the protist Capsaspora owczarzaki. However, MAGUKs are absent from fungi, amoebozoans or any other eukaryote. The repertoire of MAGUKs in Placozoa and eumetazoan taxa (Cnidaria + Bilateria is quite similar, except for one class that is missing in Trichoplax, while Porifera have a simpler MAGUK repertoire. However, Vertebrata have undergone several independent duplications and exhibit two exclusive MAGUK classes. Three different MAGUK types are found in both M. brevicollis and C. owczarzaki: DLG, MPP and MAGI. Furthermore, M. brevicollis has suffered a lineage-specific diversification. Conclusions The diversification of the MAGUK protein gene family occurred, most probably, prior to the divergence between Metazoa+choanoflagellates and the Capsaspora+Ministeria clade. A MAGI-like, a DLG-like, and a MPP-like ancestral genes were already present in the unicellular ancestor of Metazoa, and new gene members have been incorporated through metazoan evolution within two major periods, one before the sponge-eumetazoan split and another within the vertebrate lineage. Moreover, choanoflagellates have suffered an independent MAGUK

Evolutionary plasticity of plasma membrane interaction in DREPP family proteins.

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Vosolsobě, Stanislav; Petrášek, Jan; Schwarzerová, Kateřina

2017-05-01

The plant-specific DREPP protein family comprises proteins that were shown to regulate the actin and microtubular cytoskeleton in a calcium-dependent manner. Our phylogenetic analysis showed that DREPPs first appeared in ferns and that DREPPs have a rapid and plastic evolutionary history in plants. Arabidopsis DREPP paralogues called AtMDP25/PCaP1 and AtMAP18/PCaP2 are N-myristoylated, which has been reported as a key factor in plasma membrane localization. Here we show that N-myristoylation is neither conserved nor ancestral for the DREPP family. Instead, by using confocal microscopy and a new method for quantitative evaluation of protein membrane localization, we show that DREPPs rely on two mechanisms ensuring their plasma membrane localization. These include N-myristoylation and electrostatic interaction of a polybasic amino acid cluster. We propose that various plasma membrane association mechanisms resulting from the evolutionary plasticity of DREPPs are important for refining plasma membrane interaction of these signalling proteins under various conditions and in various cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Two Chimeric Regulators of G-protein Signaling (RGS) Proteins Differentially Modulate Soybean Heterotrimeric G-protein Cycle*

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Roy Choudhury, Swarup; Westfall, Corey S.; Laborde, John P.; Bisht, Naveen C.; Jez, Joseph M.; Pandey, Sona

2012-01-01

Heterotrimeric G-proteins and the regulator of G-protein signaling (RGS) proteins, which accelerate the inherent GTPase activity of Gα proteins, are common in animals and encoded by large gene families; however, in plants G-protein signaling is thought to be more limited in scope. For example, Arabidopsis thaliana contains one Gα, one Gβ, three Gγ, and one RGS protein. Recent examination of the Glycine max (soybean) genome reveals a larger set of G-protein-related genes and raises the possibility of more intricate G-protein networks than previously observed in plants. Stopped-flow analysis of GTP-binding and GDP/GTP exchange for the four soybean Gα proteins (GmGα1–4) reveals differences in their kinetic properties. The soybean genome encodes two chimeric RGS proteins with an N-terminal seven transmembrane domain and a C-terminal RGS box. Both GmRGS interact with each of the four GmGα and regulate their GTPase activity. The GTPase-accelerating activities of GmRGS1 and -2 differ for each GmGα, suggesting more than one possible rate of the G-protein cycle initiated by each of the Gα proteins. The differential effects of GmRGS1 and GmRGS2 on GmGα1–4 result from a single valine versus alanine difference. The emerging picture suggests complex regulation of the G-protein cycle in soybean and in other plants with expanded G-protein networks. PMID:22474294

Air tight electrical box

Energy Technology Data Exchange (ETDEWEB)

Pringle, C.G.

1990-08-14

An air-impervious electrical box to facilitate air sealing a house comprises an integral, rigid box body having a continuous flange, integral with the body, circumscribing and outwardly extending from the sides of the body. This flange is rearwardly positioned behind the front edges of the sides of the body a predetermined distance so that the electrical box may be secured to framing by nailing through the flange. Drywall is then secured to the frame on top of and adjecent to the flange. Such box eliminates the necessity for solid backing and minimizes passage of air through the box and space between the drywall and the box.

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

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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

Identification and characterization of NF-YB family genes in tung tree.

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Yang, Susu; Wang, Yangdong; Yin, Hengfu; Guo, Haobo; Gao, Ming; Zhu, Huiping; Chen, Yicun

2015-12-01

The NF-YB transcription factor gene family encodes a subunit of the CCAAT box-binding factor (CBF), a highly conserved trimeric activator that strongly binds to the CCAAT box promoter element. Studies on model plants have shown that NF-YB proteins participate in important developmental and physiological processes, but little is known about NF-YB proteins in trees. Here, we identified seven NF-YB transcription factor-encoding genes in Vernicia fordii, an important oilseed tree in China. A phylogenetic analysis separated the genes into two groups; non-LEC1 type (VfNF-YB1, 5, 7, 9, 11, 13) and LEC1-type (VfNF-YB 14). A gene structure analysis showed that VfNF-YB 5 has three introns and the other genes have no introns. The seven VfNF-YB sequences contain highly conserved domains, a disordered region at the N terminus, and two long helix structures at the C terminus. Phylogenetic analyses showed that VfNF-YB family genes are highly homologous to GmNF-YB genes, and many of them are closely related to functionally characterized NF-YBs. In expression analyses of various tissues (root, stem, leaf, and kernel) and the root during pathogen infection, VfNF-YB1, 5, and 11 were dominantly expressed in kernels, and VfNF-YB7 and 9 were expressed only in the root. Different VfNF-YB family genes showed different responses to pathogen infection, suggesting that they play different roles in the pathogen response. Together, these findings represent the first extensive evaluation of the NF-YB family in tung tree and provide a foundation for dissecting the functions of VfNF-YB genes in seed development, stress adaption, fatty acid synthesis, and pathogen response.

Structural and functional analyses of the putrescine binding protein PotF from Xanthomonas citri

International Nuclear Information System (INIS)

Santana, L.D.F.; Balan, A.

2012-01-01

Full text: The focus of our group is to determinate the role of ABC transporters in the physiology and growth of Xanthomonas citri, a phytopathogenic bacteria that infects citrus plants causing significant losses for the economy. One of the ABC transporters identified in the X. citri genome and that was showed to be active during the infection in Citrus sinensis plants was the putrescine transporter. This transporter consists of two internal membrane proteins PotG and PotH that form a pore, a cytoplasmic protein that gives energy for the transport and the periplasmic-binding protein PotF, which is responsible for the affinity and specificity of the system. Its function is associated to the microbial carcinogenesis, biofilm formation, escape from phagolysosomes, bacteriocin production, toxin activity and protection from oxidative and acid stress. In this work, we show for the first time, the expression, purification, functional and structural analyses of the X. citri PotF protein. The PotF was expressed from Escherichia coli cells strain Arctic, as a 40 kDa soluble protein, after induction of IPTG for twenty four hours at thirteen deg C. Using immobilized metal affinity chromatography for purification, the protein was eluted in the fractions with 10-500 mM of imidazole. To test the folding and cability to bind putrescine, spectroscopic analyses were performed using circular dichroism and intrinsic fluorescence. The data showed that PotF suffers conformational changes in presence of ligands and in different pH, suggesting a possible interaction with the tested ligand. Moreover, based on bioinformatics studies and molecular modeling analyses, we showed that X. citri PotF is highly conserved when compared to orthologs present in other bacteria, including the residues that form the ligand-binding site. The production of PotF in a soluble and stable form will allow us to start the crystallization trials in attempt to solve its structure. (author)

Structural and functional analyses of the putrescine binding protein PotF from Xanthomonas citri

Energy Technology Data Exchange (ETDEWEB)

Santana, L.D.F.; Balan, A. [Laboratorio Nacional de Biociencias - LNBIO, Campinas, SP (Brazil)

2012-07-01

Full text: The focus of our group is to determinate the role of ABC transporters in the physiology and growth of Xanthomonas citri, a phytopathogenic bacteria that infects citrus plants causing significant losses for the economy. One of the ABC transporters identified in the X. citri genome and that was showed to be active during the infection in Citrus sinensis plants was the putrescine transporter. This transporter consists of two internal membrane proteins PotG and PotH that form a pore, a cytoplasmic protein that gives energy for the transport and the periplasmic-binding protein PotF, which is responsible for the affinity and specificity of the system. Its function is associated to the microbial carcinogenesis, biofilm formation, escape from phagolysosomes, bacteriocin production, toxin activity and protection from oxidative and acid stress. In this work, we show for the first time, the expression, purification, functional and structural analyses of the X. citri PotF protein. The PotF was expressed from Escherichia coli cells strain Arctic, as a 40 kDa soluble protein, after induction of IPTG for twenty four hours at thirteen deg C. Using immobilized metal affinity chromatography for purification, the protein was eluted in the fractions with 10-500 mM of imidazole. To test the folding and cability to bind putrescine, spectroscopic analyses were performed using circular dichroism and intrinsic fluorescence. The data showed that PotF suffers conformational changes in presence of ligands and in different pH, suggesting a possible interaction with the tested ligand. Moreover, based on bioinformatics studies and molecular modeling analyses, we showed that X. citri PotF is highly conserved when compared to orthologs present in other bacteria, including the residues that form the ligand-binding site. The production of PotF in a soluble and stable form will allow us to start the crystallization trials in attempt to solve its structure. (author)

Protein-observed (19)F-NMR for fragment screening, affinity quantification and druggability assessment.

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Gee, Clifford T; Arntson, Keith E; Urick, Andrew K; Mishra, Neeraj K; Hawk, Laura M L; Wisniewski, Andrea J; Pomerantz, William C K

2016-08-01

NMR spectroscopy can be used to quantify the binding affinity between proteins and low-complexity molecules, termed 'fragments'; this versatile screening approach allows researchers to assess the druggability of new protein targets. Protein-observed (19)F-NMR (PrOF NMR) using (19)F-labeled amino acids generates relatively simple spectra that are able to provide dynamic structural information toward understanding protein folding and function. Changes in these spectra upon the addition of fragment molecules can be observed and quantified. This protocol describes the sequence-selective labeling of three proteins (the first bromodomains of Brd4 and BrdT, and the KIX domain of the CREB-binding protein) using commercially available fluorinated aromatic amino acids and fluorinated precursors as example applications of the method developed by our research group. Fragment-screening approaches are discussed, as well as Kd determination, ligand-efficiency calculations and druggability assessment, i.e., the ability to target these proteins using small-molecule ligands. Experiment times on the order of a few minutes and the simplicity of the NMR spectra obtained make this approach well-suited to the investigation of small- to medium-sized proteins, as well as the screening of multiple proteins in the same experiment.

Upregulation of human heme oxygenase gene expression by Ets-family proteins.

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Deramaudt, B M; Remy, P; Abraham, N G

1999-03-01

Overexpression of human heme oxygenase-1 has been shown to have the potential to promote EC proliferation and angiogenesis. Since Ets-family proteins have been shown to play an important role in angiogenesis, we investigated the presence of ETS binding sites (EBS), GGAA/T, and ETS protein contributing to human HO-1 gene expression. Several chloramphenicol acetyltransferase constructs were examined in order to analyze the effect of ETS family proteins on the transduction of HO-1 in Xenopus oocytes and in microvessel endothelial cells. Heme oxygenase promoter activity was up-regulated by FLI-1ERGETS-1 protein(s). Chloramphenicol acetyltransferase (CAT) assays demonstrated that the promoter region (-1500 to +19) contains positive and negative control elements and that all three members of the ETS protein family were responsible for the up-regulation of HHO-1. Electrophoretic mobility shift assays (EMSA), performed with nuclear extracts from endothelial cells overexpressing HHO-1 gene, and specific HHO-1 oligonucleotides probes containing putative EBS resulted in a specific and marked bandshift. Synergistic binding was observed in EMSA between AP-1 on the one hand, FLI-1, ERG, and ETS-1 protein on the other. Moreover, 5'-deletion analysis demonstrated the existence of a negative control element of HHO-1 expression located between positions -1500 and -120 on the HHO-1 promoter. The presence of regulatory sequences for transcription factors such as ETS-1, FLI-1, or ERG, whose activity is associated with cell proliferation, endothelial cell differentiation, and matrix metalloproteinase transduction, may be an indication of the important role that HO-1 may play in coronary collateral circulation, tumor growth, angiogenesis, and hemoglobin-induced endothelial cell injuries.

Influenza virus PB1-F2 protein induces cell death through mitochondrial ANT3 and VDAC1.

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Dmitriy Zamarin

2005-09-01

Full Text Available The influenza virus PB1-F2 is an 87-amino acid mitochondrial protein that previously has been shown to induce cell death, although the mechanism of apoptosis induction has remained unclear. In the process of characterizing its mechanism of action we found that the viral PB1-F2 protein sensitizes cells to apoptotic stimuli such as tumor necrosis factor alpha, as demonstrated by increased cleavage of caspase 3 substrates in PB1-F2-expressing cells. Moreover, treatment of purified mouse liver mitochondria with recombinant PB1-F2 protein resulted in cytochrome c release, loss of the mitochondrial membrane potential, and enhancement of tBid-induced mitochondrial permeabilization, suggesting a possible mechanism for the observed cellular sensitization to apoptosis. Using glutathione-S-transferase pulldowns with subsequent mass spectrometric analysis, we identified the mitochondrial interactors of the PB1-F2 protein and showed that the viral protein uniquely interacts with the inner mitochondrial membrane adenine nucleotide translocator 3 and the outer mitochondrial membrane voltage-dependent anion channel 1, both of which are implicated in the mitochondrial permeability transition during apoptosis. Consistent with this interaction, blockers of the permeability transition pore complex (PTPC inhibited PB1-F2-induced mitochondrial permeabilization. Based on our findings, we propose a model whereby the proapoptotic PB1-F2 protein acts through the mitochondrial PTPC and may play a role in the down-regulation of the host immune response to infection.

Phylogeny of the Vitamin K 2,3-Epoxide Reductase (VKOR) Family and Evolutionary Relationship to the Disulfide Bond Formation Protein B (DsbB) Family.

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Bevans, Carville G; Krettler, Christoph; Reinhart, Christoph; Watzka, Matthias; Oldenburg, Johannes

2015-07-29

In humans and other vertebrate animals, vitamin K 2,3-epoxide reductase (VKOR) family enzymes are the gatekeepers between nutritionally acquired K vitamins and the vitamin K cycle responsible for posttranslational modifications that confer biological activity upon vitamin K-dependent proteins with crucial roles in hemostasis, bone development and homeostasis, hormonal carbohydrate regulation and fertility. We report a phylogenetic analysis of the VKOR family that identifies five major clades. Combined phylogenetic and site-specific conservation analyses point to clade-specific similarities and differences in structure and function. We discovered a single-site determinant uniquely identifying VKOR homologs belonging to human pathogenic, obligate intracellular prokaryotes and protists. Building on previous work by Sevier et al. (Protein Science 14:1630), we analyzed structural data from both VKOR and prokaryotic disulfide bond formation protein B (DsbB) families and hypothesize an ancient evolutionary relationship between the two families where one family arose from the other through a gene duplication/deletion event. This has resulted in circular permutation of primary sequence threading through the four-helical bundle protein folds of both families. This is the first report of circular permutation relating distant a-helical membrane protein sequences and folds. In conclusion, we suggest a chronology for the evolution of the five extant VKOR clades.