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

PubMed

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

2009-06-23

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

Genome-Wide Identification and Expression of Xenopus F-Box Family of Proteins.

PubMed

Saritas-Yildirim, Banu; Pliner, Hannah A; Ochoa, Angelica; Silva, Elena M

2015-01-01

Protein degradation via the multistep ubiquitin/26S proteasome pathway is a rapid way to alter the protein profile and drive cell processes and developmental changes. Many key regulators of embryonic development are targeted for degradation by E3 ubiquitin ligases. The most studied family of E3 ubiquitin ligases is the SCF ubiquitin ligases, which use F-box adaptor proteins to recognize and recruit target proteins. Here, we used a bioinformatics screen and phylogenetic analysis to identify and annotate the family of F-box proteins in the Xenopus tropicalis genome. To shed light on the function of the F-box proteins, we analyzed expression of F-box genes during early stages of Xenopus development. Many F-box genes are broadly expressed with expression domains localized to diverse tissues including brain, spinal cord, eye, neural crest derivatives, somites, kidneys, and heart. All together, our genome-wide identification and expression profiling of the Xenopus F-box family of proteins provide a foundation for future research aimed to identify the precise role of F-box dependent E3 ubiquitin ligases and their targets in the regulatory circuits of development.

Regulating the ethylene response of a plant by modulation of F-box proteins

DOEpatents

Guo, Hongwei [Beijing, CN; Ecker, Joseph R [Carlsbad, CA

2014-01-07

The relationship between F-box proteins and proteins invovled in the ethylene response in plants is described. In particular, F-box proteins may bind to proteins involved in the ethylene response and target them for degradation by the ubiquitin/proteasome pathway. The transcription factor EIN3 is a key transcription factor mediating ethylne-regulated gene expression and morphological responses. EIN3 is degraded through a ubiquitin/proteasome pathway mediated by F-box proteins EBF1 and EBF2. The link between F-box proteins and the ethylene response is a key step in modulating or regulating the response of a plant to ethylene. Described herein are transgenic plants having an altered sensitivity to ethylene, and methods for making transgenic plant haing an althered sensitivity to ethylene by modulating the level of activity of F-box proteins. Methods of altering the ethylene response in a plant by modulating the activity or expression of an F-box protein are described. Also described are methods of identifying compounds that modulate the ethylene response in plants by modulating the level of F-box protein expression or activity.

Efficient ASK-assisted system for expression and purification of plant F-box proteins.

PubMed

Li, Haiou; Yao, Ruifeng; Ma, Sui; Hu, Shuai; Li, Suhua; Wang, Yupei; Yan, Chun; Xie, Daoxin; Yan, Jianbin

2017-11-01

Ubiquitin-mediated protein degradation plays an essential role in plant growth and development as well as responses to environmental and endogenous signals. F-box protein is one of the key components of the SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase complex, which recruit specific substrate proteins for subsequent ubiquitination and 26S proteasome-mediated degradation to regulate developmental processes and signaling networks. However, it is not easy to obtain purified F-box proteins with high activity due to their unstable protein structures. Here, we found that Arabidopsis SKP-like proteins (ASKs) can significantly improve soluble expression of F-box proteins and maintain their bioactivity. We established an efficient ASK-assisted method to express and purify plant F-box proteins. The method meets a broad range of criteria required for the biochemical analysis or protein crystallization of plant F-box proteins. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Transfer of Ho Endonuclease and Ufo1 to the Proteasome by the UbL-UbA Shuttle Protein, Ddi1, Analysed by Complex Formation In Vitro

PubMed Central

Voloshin, Olga; Bakhrat, Anya; Herrmann, Sharon; Raveh, Dina

2012-01-01

The F-box protein, Ufo1, recruits Ho endonuclease to the SCFUfo1 complex for ubiquitylation. Both ubiquitylated Ho and Ufo1 are transferred by the UbL-UbA protein, Ddi1, to the 19S Regulatory Particle (RP) of the proteasome for degradation. The Ddi1-UbL domain binds Rpn1 of the 19S RP, the Ddi1-UbA domain binds ubiquitin chains on the degradation substrate. Here we used complex reconstitution in vitro to identify stages in the transfer of Ho and Ufo1 from the SCFUfo1 complex to the proteasome. We report SCFUfo1 complex at the proteasome formed in the presence of Ho. Subsequently Ddi1 is recruited to this complex by interaction between the Ddi1-UbL domain and Ufo1. The core of Ddi1 binds both Ufo1 and Rpn1; this interaction confers specificity of SCFUfo1 for Ddi1. The substrate-shield model predicts that Ho would protect Ufo1 from degradation and we find that Ddi1 binds Ho, Ufo1, and Rpn1 simultaneously forming a complex for transfer of Ho to the 19S RP. In contrast, in the absence of Ho, Rpn1 displaces Ufo1 from Ddi1 indicating a higher affinity of the Ddi1-UbL for the 19S RP. However, at high Rpn1 levels there is synergistic binding of Ufo1 to Ddi1 that is dependent on the Ddi1-UbA domain. Our interpretation is that in the absence of substrate, the Ddi1-UbL binds Rpn1 while the Ddi1-UbA binds ubiquitin chains on Ufo1. This would promote degradation of Ufo1 and disassembly of SCFUfo1 complexes. PMID:22815701

F-box proteins involved in cancer-associated drug resistance.

PubMed

Gong, Jian; Zhou, Yuqian; Liu, Deliang; Huo, Jirong

2018-06-01

The ubiquitin proteasome system (UPS) regulated human biological processes through the appropriate and efficient proteolysis of cellular proteins. F-box proteins are the vital components of SKP1-CUL1-FBP (SCF)-type E3 ubiquitin ligases that determine substrate specificity. As F-box proteins have the ability to control the degradation of several crucial protein targets associated with drug resistance, the dysregulation of these proteins may lead to induction of chemoresistance in cancer cells. Chemotherapy is one of the most conventional therapeutic approaches of treatment of patients with cancer. However, its exclusive application in clinical settings is restricted due to the development of chemoresistance, which typically results treatment failure. Therefore, overcoming drug resistance is considered as one of the most critical issues that researchers and clinician associated with oncology face. The present review serves to provide a comprehensive overview of F-box proteins and their possible targets as well as their correlation with the chemoresistance and chemosensitization of cancer cells. The article also presents an integrated representation of the complex regulatory mechanisms responsible for chemoresistance, which may lay the foundation to explore sensible candidate drugs for therapeutic intervention.

Functional redundancy and/or ongoing pseudogenization among F-box protein genes expressed in Arabidopsis male gametophyte.

PubMed

Ikram, Sobia; Durandet, Monique; Vesa, Simona; Pereira, Serge; Guerche, Philippe; Bonhomme, Sandrine

2014-06-01

F-box protein genes family is one of the largest gene families in plants, with almost 700 predicted genes in the model plant Arabidopsis. F-box proteins are key components of the ubiquitin proteasome system that allows targeted protein degradation. Transcriptome analyses indicate that half of these F-box protein genes are found expressed in microspore and/or pollen, i.e., during male gametogenesis. To assess the role of F-box protein genes during this crucial developmental step, we selected 34 F-box protein genes recorded as highly and specifically expressed in pollen and isolated corresponding insertion mutants. We checked the expression level of each selected gene by RT-PCR and confirmed pollen expression for 25 genes, but specific expression for only 10 of the 34 F-box protein genes. In addition, we tested the expression level of selected F-box protein genes in 24 mutant lines and showed that 11 of them were null mutants. Transmission analysis of the mutations to the progeny showed that none of the single mutations was gametophytic lethal. These unaffected transmission efficiencies suggested leaky mutations or functional redundancy among F-box protein genes. Cytological observation of the gametophytes in the mutants confirmed these results. Combinations of mutations in F-box protein genes from the same subfamily did not lead to transmission defect either, further highlighting functional redundancy and/or a high proportion of pseudogenes among these F-box protein genes.

Regulating the ethylene response of a plant by modulation of F-box proteins

DOEpatents

Guo, Hongwei [Beijing, CN; Ecker, Joseph R [Carlsbad, CA

2011-03-08

The invention relates to transgenic plants having reduced sensitivity to ethylene as a result of having a recombinant nucleic acid encoding an F-box protein that interacts with a EIN3 involved in an ethylene response of plants, and a method of producing a transgenic plant with reduced ethylene sensitivity by transforming the plant with a nucleic acid sequence encoding an F-box protein. The inventions also relates to methods of altering the ethylene response in a plant by modulating the activity or expression of an F-box protein.

F-Box Protein FBX92 Affects Leaf Size in Arabidopsis thaliana

PubMed Central

Baute, Joke; Polyn, Stefanie; De Block, Jolien; Blomme, Jonas; Van Lijsebettens, Mieke

2017-01-01

F-box proteins are part of one of the largest families of regulatory proteins that play important roles in protein degradation. In plants, F-box proteins are functionally very diverse, and only a small subset has been characterized in detail. Here, we identified a novel F-box protein FBX92 as a repressor of leaf growth in Arabidopsis. Overexpression of AtFBX92 resulted in plants with smaller leaves than the wild type, whereas plants with reduced levels of AtFBX92 showed, in contrast, increased leaf growth by stimulating cell proliferation. Detailed cellular analysis suggested that AtFBX92 specifically affects the rate of cell division during early leaf development. This is supported by the increased expression levels of several cell cycle genes in plants with reduced AtFBX92 levels. Surprisingly, overexpression of the maize homologous gene ZmFBX92 in maize had no effect on plant growth, whereas ectopic expression in Arabidopsis increased leaf growth. Expression of a truncated form of AtFBX92 showed that the contrasting effects of ZmFBX92 and AtFBX92 gain of function in Arabidopsis are due to the absence of the F-box-associated domain in the ZmFBX92 gene. Our work reveals an additional player in the complex network that determines leaf size and lays the foundation for identifying putative substrates. PMID:28340173

F-Box Protein FBX92 Affects Leaf Size in Arabidopsis thaliana.

PubMed

Baute, Joke; Polyn, Stefanie; De Block, Jolien; Blomme, Jonas; Van Lijsebettens, Mieke; Inzé, Dirk

2017-05-01

F-box proteins are part of one of the largest families of regulatory proteins that play important roles in protein degradation. In plants, F-box proteins are functionally very diverse, and only a small subset has been characterized in detail. Here, we identified a novel F-box protein FBX92 as a repressor of leaf growth in Arabidopsis. Overexpression of AtFBX92 resulted in plants with smaller leaves than the wild type, whereas plants with reduced levels of AtFBX92 showed, in contrast, increased leaf growth by stimulating cell proliferation. Detailed cellular analysis suggested that AtFBX92 specifically affects the rate of cell division during early leaf development. This is supported by the increased expression levels of several cell cycle genes in plants with reduced AtFBX92 levels. Surprisingly, overexpression of the maize homologous gene ZmFBX92 in maize had no effect on plant growth, whereas ectopic expression in Arabidopsis increased leaf growth. Expression of a truncated form of AtFBX92 showed that the contrasting effects of ZmFBX92 and AtFBX92 gain of function in Arabidopsis are due to the absence of the F-box-associated domain in the ZmFBX92 gene. Our work reveals an additional player in the complex network that determines leaf size and lays the foundation for identifying putative substrates. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

Deregulation of F-box proteins and its consequence on cancer development, progression and metastasis

PubMed Central

Heo, Jinho; Eki, Rebeka; Abbas, Tarek

2015-01-01

F-box proteins are substrate receptors of the SCF (SKP1-Cullin 1-F-box protein) E3 ubiquitin ligase that play important roles in a number of physiological processes and activities. Through their ability to assemble distinct E3 ubiquitin ligases and target key regulators of cellular activities for ubiquitylation and degradation, this versatile group of proteins is able to regulate the abundance of cellular proteins whose deregulated expression or activity contributes to disease. In this review, we describe the important roles of select F-box proteins in regulating cellular activities, the perturbation of which contributes to the initiation and progression of a number of human malignancies. PMID:26432751

Regulating the ethylene response of a plant by modulation of F-box proteins

DOEpatents

Guo, Hongwei; Ecker, Joseph R.

2010-02-02

The invention relates to transgenic plants having reduced sensitivity to ethylene as a result of having a recombinant nucleic acid encoding a F-box protein, and a method of producing a transgenic plant with reduced ethylene sensitivity by transforming the plant with a nucleic acid sequence encoding a F-box protein.

The ASK1 gene regulates development and interacts with the UFO gene to control floral organ identity in Arabidopsis.

PubMed

Zhao, D; Yang, M; Solava, J; Ma, H

1999-09-01

Normal flower development likely requires both specific and general regulators. We have isolated an Arabidopsis mutant ask1-1 (for -Arabidopsis skp1-like1-1), which exhibits defects in both vegetative and reproductive development. In the ask1-1mutant, rosette leaf growth is reduced, resulting in smaller than normal rosette leaves, and internodes in the floral stem are shorter than normal. Examination of cell sizes in these organs indicates that cell expansion is normal in the mutant, but cell number is reduced. In the mutant, the numbers of petals and stamens are reduced, and many flowers have one or more petals with a reduced size. In addition, all mutant flowers have short stamen filaments. Furthermore, petal/stamen chimeric organs are found in many flowers. These results indicate that the ASK1 gene affects the size of vegetative and floral organs. The ask1 floral phenotype resembles somewhat that of the Arabidopsis ufo mutants in that both genes affect whorls 2 and 3. We therefore tested for possible interactions between ASK1 and UFO by analyzing the phenotypes of ufo-2 ask1-1 double mutant plants. In these plants, vegetative development is similar to that of the ask1-1 single mutant, whereas the floral defects are more severe than those in either single mutant. Interior to the first whorl, the double mutant flowers have more sepals or sepal-like organs than are found in ufo-2, and less petals than ask1-1. Our results suggest that ASK1 interacts with UFO to control floral organ identity in whorls 2 and 3. This is very intriguing because ASK1 is very similar in sequence to the yeast SKP1 protein and UFO contains an F-box, a motif known to interact with SKP1 in yeast. Although the precise mechanism of ASK1 and UFO action is unknown, our results support the hypothesis that these two proteins physically interact in vivo. Copyright 1999 Wiley-Liss, Inc.

Genome-wide identification and characterisation of F-box family in maize.

PubMed

Jia, Fengjuan; Wu, Bingjiang; Li, Hui; Huang, Jinguang; Zheng, Chengchao

2013-11-01

F-box-containing proteins, as the key components of the protein degradation machinery, are widely distributed in higher plants and are considered as one of the largest known families of regulatory proteins. The F-box protein family plays a crucial role in plant growth and development and in response to biotic and abiotic stresses. However, systematic analysis of the F-box family in maize (Zea mays) has not been reported yet. In this paper, we identified and characterised the maize F-box genes in a genome-wide scale, including phylogenetic analysis, chromosome distribution, gene structure, promoter analysis and gene expression profiles. A total of 359 F-box genes were identified and divided into 15 subgroups by phylogenetic analysis. The F-box domain was relatively conserved, whereas additional motifs outside the F-box domain may indicate the functional diversification of maize F-box genes. These genes were unevenly distributed in ten maize chromosomes, suggesting that they expanded in the maize genome because of tandem and segmental duplication events. The expression profiles suggested that the maize F-box genes had temporal and spatial expression patterns. Putative cis-acting regulatory DNA elements involved in abiotic stresses were observed in maize F-box gene promoters. The gene expression profiles under abiotic stresses also suggested that some genes participated in stress responsive pathways. Furthermore, ten genes were chosen for quantitative real-time PCR analysis under drought stress and the results were consistent with the microarray data. This study has produced a comparative genomics analysis of the maize ZmFBX gene family that can be used in further studies to uncover their roles in maize growth and development.

Atrogin-1, a muscle-specific F-box protein highly expressed during muscle atrophy

NASA Technical Reports Server (NTRS)

Gomes, M. D.; Lecker, S. H.; Jagoe, R. T.; Navon, A.; Goldberg, A. L.

2001-01-01

Muscle wasting is a debilitating consequence of fasting, inactivity, cancer, and other systemic diseases that results primarily from accelerated protein degradation by the ubiquitin-proteasome pathway. To identify key factors in this process, we have used cDNA microarrays to compare normal and atrophying muscles and found a unique gene fragment that is induced more than ninefold in muscles of fasted mice. We cloned this gene, which is expressed specifically in striated muscles. Because this mRNA also markedly increases in muscles atrophying because of diabetes, cancer, and renal failure, we named it atrogin-1. It contains a functional F-box domain that binds to Skp1 and thereby to Roc1 and Cul1, the other components of SCF-type Ub-protein ligases (E3s), as well as a nuclear localization sequence and PDZ-binding domain. On fasting, atrogin-1 mRNA levels increase specifically in skeletal muscle and before atrophy occurs. Atrogin-1 is one of the few examples of an F-box protein or Ub-protein ligase (E3) expressed in a tissue-specific manner and appears to be a critical component in the enhanced proteolysis leading to muscle atrophy in diverse diseases.

Analysis of CFB, a cytokinin-responsive gene of Arabidopsis thaliana encoding a novel F-box protein regulating sterol biosynthesis.

PubMed

Brenner, Wolfram G; Leuendorf, Jan Erik; Cortleven, Anne; Martin, Laetitia B B; Schaller, Hubert; Schmülling, Thomas

2017-05-17

Protein degradation by the ubiquitin-26S proteasome pathway is important for the regulation of cellular processes, but the function of most F-box proteins relevant to substrate recognition is unknown. We describe the analysis of the gene Cytokinin-induced F-box encoding (CFB, AT3G44326), identified in a meta-analysis of cytokinin-related transcriptome studies as one of the most robust cytokinin response genes. F-box domain-dependent interaction with the E3 ubiquitin ligase complex component ASK1 classifies CFB as a functional F-box protein. Apart from F-box and transmembrane domains, CFB contains no known functional domains. CFB is expressed in all plant tissues, predominantly in root tissue. A ProCFB:GFP-GUS fusion gene showed strongest expression in the lateral root cap and during lateral root formation. CFB-GFP fusion proteins were mainly localized in the nucleus and the cytosol but also at the plasma membrane. cfb mutants had no discernible phenotype, but CFB overexpressing plants showed several defects, such as a white upper inflorescence stem, similar to the hypomorphic cycloartenol synthase mutant cas1-1. Both CFB overexpressing plants and cas1-1 mutants accumulated the CAS1 substrate 2,3-oxidosqualene in the white stem tissue, the latter even more after cytokinin treatment, indicating impairment of CAS1 function. This suggests that CFB may link cytokinin and the sterol biosynthesis pathway. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

The COP9 signalosome interacts with SCF UFO and participates in Arabidopsis flower development.

PubMed

Wang, Xiping; Feng, Suhua; Nakayama, Naomi; Crosby, W L; Irish, Vivian; Deng, Xing Wang; Wei, Ning

2003-05-01

The COP9 signalosome (CSN) is involved in multiple developmental processes. It interacts with SCF ubiquitin ligases and deconjugates Nedd8/Rub1 from cullins (deneddylation). CSN is highly expressed in Arabidopsis floral tissues. To investigate the role of CSN in flower development, we examined the expression pattern of CSN in developing flowers. We report here that two csn1 partially deficient Arabidopsis strains exhibit aberrant development of floral organs, decline of APETALA3 (AP3) expression, and low fertility in addition to defects in shoot and inflorescence meristems. We show that UNUSUAL FLORAL ORGANS (UFO) forms a SCF(UFO) complex, which is associated with CSN in vivo. Genetic interaction analysis indicates that CSN is necessary for the gain-of-function activity of the F-box protein UFO in AP3 activation and in floral organ transformation. Compared with the previously reported csn5 antisense and csn1 null mutants, partial deficiency of CSN1 causes a reduction in the level of CUL1 in the mutant flowers without an obvious defect in CUL1 deneddylation. We conclude that CSN is an essential regulator of Arabidopsis flower development and suggest that CSN regulates Arabidopsis flower development in part by modulating SCF(UFO)-mediated AP3 activation.

F-BOX proteins in cancer cachexia and muscle wasting: emerging regulators and therapeutic opportunities

PubMed Central

Sukari, Ammar; Muqbil, Irfana; Mohammad, Ramzi M.; Philip, Philip A.; Azmi, Asfar S.

2016-01-01

Cancer cachexia is a debilitating metabolic syndrome accounting for fatigue, an impairment of normal activities, loss of muscle mass associated with body weight loss eventually leading to death in majority of patients with advanced disease. Cachexia patients undergoing skeletal muscle atrophy show consistent activation of the SCF ubiquitin ligase (F-BOX) family member Atrogin-1 (also known as MAFBx/FBXO32) alongside the activation of the muscle ring finger protein1 (MuRF1). Other lesser known F-BOX family members are also emerging as key players supporting muscle wasting pathways. Recent work highlights a spectrum of different cancer signaling mechanisms impacting F-BOX family members that feed forward muscle atrophy related genes during cachexia. These novel players provide unique opportunities to block cachexia induced skeletal muscle atrophy by therapeutically targeting the SCF protein ligases. Conversely, strategies that induce the production of proteins may be helpful to counter the effects of these F-BOX proteins. Through this review, we bring forward some novel targets that promote atrogin-1 signaling in cachexia and muscle wasting and highlight newer therapeutic opportunities that can help in the better management of patients with this devastating and fatal disorder. PMID:26804424

A combined photoelectron spectroscopy and relativistic ab initio studies of the electronic structures of UFO and UFO(-).

PubMed

Roy, Soumendra K; Jian, Tian; Lopez, Gary V; Li, Wei-Li; Su, Jing; Bross, David H; Peterson, Kirk A; Wang, Lai-Sheng; Li, Jun

2016-02-28

The observation of the gaseous UFO(-) anion is reported, which is investigated using photoelectron spectroscopy and relativisitic ab initio calculations. Two strong photoelectron bands are observed at low binding energies due to electron detachment from the U-7sσ orbital. Numerous weak detachment bands are also observed due to the strongly correlated U-5f electrons. The electron affinity of UFO is measured to be 1.27(3) eV. High-level relativistic quantum chemical calculations have been carried out on the ground state and many low-lying excited states of UFO to help interpret the photoelectron spectra and understand the electronic structure of UFO. The ground state of UFO(-) is linear with an O-U-F structure and a (3)H4 spectral term derived from a U 7sσ(2)5fφ(1)5fδ(1) electron configuration, whereas the ground state of neutral UFO has a (4)H(7/2) spectral term with a U 7sσ(1)5fφ(1)5fδ(1) electron configuration. Strong electron correlation effects are found in both the anionic and neutral electronic configurations. In the UFO neutral, a high density of electronic states with strong configuration mixing is observed in most of the scalar relativistic and spin-orbit coupled states. The strong electron correlation, state mixing, and spin-orbit coupling of the electronic states make the excited states of UFO very challenging for accurate quantum chemical calculations.

A combined photoelectron spectroscopy and relativistic ab initio studies of the electronic structures of UFO and UFO-

NASA Astrophysics Data System (ADS)

Roy, Soumendra K.; Jian, Tian; Lopez, Gary V.; Li, Wei-Li; Su, Jing; Bross, David H.; Peterson, Kirk A.; Wang, Lai-Sheng; Li, Jun

2016-02-01

The observation of the gaseous UFO- anion is reported, which is investigated using photoelectron spectroscopy and relativisitic ab initio calculations. Two strong photoelectron bands are observed at low binding energies due to electron detachment from the U-7sσ orbital. Numerous weak detachment bands are also observed due to the strongly correlated U-5f electrons. The electron affinity of UFO is measured to be 1.27(3) eV. High-level relativistic quantum chemical calculations have been carried out on the ground state and many low-lying excited states of UFO to help interpret the photoelectron spectra and understand the electronic structure of UFO. The ground state of UFO- is linear with an O-U-F structure and a 3H4 spectral term derived from a U 7sσ25fφ15fδ1 electron configuration, whereas the ground state of neutral UFO has a 4H7/2 spectral term with a U 7sσ15fφ15fδ1 electron configuration. Strong electron correlation effects are found in both the anionic and neutral electronic configurations. In the UFO neutral, a high density of electronic states with strong configuration mixing is observed in most of the scalar relativistic and spin-orbit coupled states. The strong electron correlation, state mixing, and spin-orbit coupling of the electronic states make the excited states of UFO very challenging for accurate quantum chemical calculations.

A New Set of ESTs from Chickpea (Cicer arietinum L.) Embryo Reveals Two Novel F-Box Genes, CarF-box_PP2 and CarF-box_LysM, with Potential Roles in Seed Development

PubMed Central

Gupta, Shefali; Garg, Vanika; Bhatia, Sabhyata

2015-01-01

Considering the economic importance of chickpea (C. arietinum L.) seeds, it is important to understand the mechanisms underlying seed development for which a cDNA library was constructed from 6 day old chickpea embryos. A total of 8,186 ESTs were obtained from which 4,048 high quality ESTs were assembled into 1,480 unigenes that majorly encoded genes involved in various metabolic and regulatory pathways. Of these, 95 ESTs were found to be involved in ubiquitination related protein degradation pathways and 12 ESTs coded specifically for putative F-box proteins. Differential transcript accumulation of these putative F-box genes was observed in chickpea tissues as evidenced by quantitative real-time PCR. Further, to explore the role of F-box proteins in chickpea seed development, two F-box genes were selected for molecular characterization. These were named as CarF-box_PP2 and CarF-box_LysM depending on their C-terminal domains, PP2 and LysM, respectively. Their highly conserved structures led us to predict their target substrates. Subcellular localization experiment revealed that CarF-box_PP2 was localized in the cytoplasm and CarF-box_LysM was localized in the nucleus. We demonstrated their physical interactions with SKP1 protein, which validated that they function as F-box proteins in the formation of SCF complexes. Sequence analysis of their promoter regions revealed certain seed specific cis-acting elements that may be regulating their preferential transcript accumulation in the seed. Overall, the study helped in expanding the EST database of chickpea, which was further used to identify two novel F-box genes having a potential role in seed development. PMID:25803812

F-BOX proteins in cancer cachexia and muscle wasting: Emerging regulators and therapeutic opportunities.

PubMed

Sukari, Ammar; Muqbil, Irfana; Mohammad, Ramzi M; Philip, Philip A; Azmi, Asfar S

2016-02-01

Cancer cachexia is a debilitating metabolic syndrome accounting for fatigue, an impairment of normal activities, loss of muscle mass associated with body weight loss eventually leading to death in majority of patients with advanced disease. Cachexia patients undergoing skeletal muscle atrophy show consistent activation of the SCF ubiquitin ligase (F-BOX) family member Atrogin-1 (also known as MAFBx/FBXO32) alongside the activation of the muscle ring finger protein1 (MuRF1). Other lesser known F-BOX family members are also emerging as key players supporting muscle wasting pathways. Recent work highlights a spectrum of different cancer signaling mechanisms impacting F-BOX family members that feed forward muscle atrophy related genes during cachexia. These novel players provide unique opportunities to block cachexia induced skeletal muscle atrophy by therapeutically targeting the SCF protein ligases. Conversely, strategies that induce the production of proteins may be helpful to counter the effects of these F-BOX proteins. Through this review, we bring forward some novel targets that promote atrogin-1 signaling in cachexia and muscle wasting and highlight newer therapeutic opportunities that can help in the better management of patients with this devastating and fatal disorder. Copyright © 2016 Elsevier Ltd. All rights reserved.

UFO in the Arabidopsis inflorescence apex is required for floral-meristem identity and bract suppression.

PubMed

Hepworth, Shelley R; Klenz, Jennifer E; Haughn, George W

2006-03-01

The UNUSUAL FLORAL ORGANS (UFO) gene of Arabidopsis encodes an F-box protein required for the determination of floral-organ and floral-meristem identity. Mutation of UFO leads to dramatic changes in floral-organ type which are well-characterized whereas inflorescence defects are more subtle and less understood. These defects include an increase in the number of secondary inflorescences, nodes that alternate between forming flowers and secondary inflorescences, and nodes in which a single flower is subtended by a bract. Here, we show how inflorescence defects correlate with the abnormal development of floral primordia and establish a temporal requirement for UFO in this process. At the inflorescence apex of ufo mutants, newly formed primordia are initially bract-like. Expression of the floral-meristem identity genes LFY and AP1 are confined to a relatively small adaxial region of these primordia with expression of the bract-identity marker FIL observed in cells that comprise the balance of the primordia. Proliferation of cells in the adaxial region of these early primordia is delayed by several nodes such that primordia appear "chimeric" at several nodes, having visible floral and bract components. However, by late stage 2 of floral development, growth of the bract generally ceases and is overtaken by development of the floral primordium. This abnormal pattern of floral meristem development is not rescued by expression of UFO from the AP1 promoter, indicating that UFO is required prior to AP1 activation for normal development of floral primordia. We propose that UFO and LFY are jointly required in the inflorescence meristem to both promote floral meristem development and inhibit, in a non-cell autonomous manner, growth of the bract.

The F-box family genes as key elements in response to salt, heavy mental, and drought stresses in Medicago truncatula.

PubMed

Song, Jian Bo; Wang, Yan Xiang; Li, Hai Bo; Li, Bo Wen; Zhou, Zhao Sheng; Gao, Shuai; Yang, Zhi Min

2015-07-01

F-box protein is a subunit of Skp1-Rbx1-Cul1-F-box protein (SCF) complex with typically conserved F-box motifs of approximately 40 amino acids and is one of the largest protein families in eukaryotes. F-box proteins play critical roles in selective and specific protein degradation through the 26S proteasome. In this study, we bioinformatically identified 972 putative F-box proteins from Medicago truncatula genome. Our analysis showed that in addition to the conserved motif, the F-box proteins have several other functional domains in their C-terminal regions (e.g., LRRs, Kelch, FBA, and PP2), some of which were found to be M. truncatula species-specific. By phylogenetic analysis of the F-box motifs, these proteins can be classified into three major families, and each family can be further grouped into more subgroups. Analysis of the genomic distribution of F-box genes on M. truncatula chromosomes revealed that the evolutional expansion of F-box genes in M. truncatula was probably due to localized gene duplications. To investigate the possible response of the F-box genes to abiotic stresses, both publicly available and customer-prepared microarrays were analyzed. Most of the F-box protein genes can be responding to salt and heavy metal stresses. Real-time PCR analysis confirmed that some of the F-box protein genes containing heat, drought, salicylic acid, and abscisic acid responsive cis-elements were able to respond to the abiotic stresses.

F-box-like domain in the polerovirus protein P0 is required for silencing suppressor function

PubMed Central

Pazhouhandeh, Maghsoud; Dieterle, Monika; Marrocco, Katia; Lechner, Esther; Berry, Bassam; Brault, Véronique; Hemmer, Odile; Kretsch, Thomas; Richards, Kenneth E.; Genschik, Pascal; Ziegler-Graff, Véronique

2006-01-01

Plants employ small RNA-mediated posttranscriptional gene silencing as a virus defense mechanism. In response, plant viruses encode proteins that can suppress RNA silencing, but the mode of action of most such proteins is poorly understood. Here, we show that the silencing suppressor protein P0 of two Arabidopsis-infecting poleroviruses interacts by means of a conserved minimal F-box motif with Arabidopsis thaliana orthologs of S-phase kinase-related protein 1 (SKP1), a component of the SCF family of ubiquitin E3 ligases. Point mutations in the F-box-like motif abolished the P0–SKP1 ortholog interaction, diminished virus pathogenicity, and inhibited the silencing suppressor activity of P0. Knockdown of expression of a SKP1 ortholog in Nicotiana benthamiana rendered the plants resistant to polerovirus infection. Together, the results support a model in which P0 acts as an F-box protein that targets an essential component of the host posttranscriptional gene silencing machinery. PMID:16446454

A Highly Expressed High-Molecular-Weight S-Layer Complex of Pelosinus sp. Strain UFO1 Binds Uranium

PubMed Central

Thorgersen, Michael P.; Lancaster, W. Andrew; Rajeev, Lara; Ge, Xiaoxuan; Vaccaro, Brian J.; Poole, Farris L.; Arkin, Adam P.; Mukhopadhyay, Aindrila

2016-01-01

ABSTRACT Cell suspensions of Pelosinus sp. strain UFO1 were previously shown, using spectroscopic analysis, to sequester uranium as U(IV) complexed with carboxyl and phosphoryl group ligands on proteins. The goal of our present study was to characterize the proteins involved in uranium binding. Virtually all of the uranium in UFO1 cells was associated with a heterodimeric protein, which was termed the uranium-binding complex (UBC). The UBC was composed of two S-layer domain proteins encoded by UFO1_4202 and UFO1_4203. Samples of UBC purified from the membrane fraction contained 3.3 U atoms/heterodimer, but significant amounts of phosphate were not detected. The UBC had an estimated molecular mass by gel filtration chromatography of 15 MDa, and it was proposed to contain 150 heterodimers (UFO1_4203 and UFO1_4202) and about 500 uranium atoms. The UBC was also the dominant extracellular protein, but when purified from the growth medium, it contained only 0.3 U atoms/heterodimer. The two genes encoding the UBC were among the most highly expressed genes within the UFO1 genome, and their expressions were unchanged by the presence or absence of uranium. Therefore, the UBC appears to be constitutively expressed and is the first line of defense against uranium, including by secretion into the extracellular medium. Although S-layer proteins were previously shown to bind U(VI), here we showed that U(IV) binds to S-layer proteins, we identified the proteins involved, and we quantitated the amount of uranium bound. IMPORTANCE Widespread uranium contamination from industrial sources poses hazards to human health and to the environment. Herein, we identified a highly abundant uranium-binding complex (UBC) from Pelosinus sp. strain UFO1. The complex makes up the primary protein component of the S-layer of strain UFO1 and binds 3.3 atoms of U(IV) per heterodimer. While other bacteria have been shown to bind U(VI) on their S-layer, we demonstrate here an example of U(IV) bound by an

A Highly Expressed High-Molecular-Weight S-Layer Complex of Pelosinus sp. Strain UFO1 Binds Uranium

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

Thorgersen, Michael P.; Lancaster, W. Andrew; Rajeev, Lara

Cell suspensions of Pelosinus sp. strain UFO1 were previously shown, using spectroscopic analysis, to sequester uranium as U(IV) complexed with carboxyl and phosphoryl group ligands on proteins. The goal of our present study was to characterize the proteins involved in uranium binding. Virtually all of the uranium in UFO1 cells was associated with a heterodimeric protein, which was termed the uranium-binding complex (UBC). The UBC was composed of two S-layer domain proteins encoded by UFO1_4202 and UFO1_4203. Samples of UBC purified from the membrane fraction contained 3.3 U atoms/heterodimer, but significant amounts of phosphate were not detected. The UBC hadmore » an estimated molecular mass by gel filtration chromatography of 15 MDa, and it was proposed to contain 150 heterodimers (UFO1_4203 and UFO1_4202) and about 500 uranium atoms. The UBC was also the dominant extracellular protein, but when purified from the growth medium, it contained only 0.3 U atoms/heterodimer. The two genes encoding the UBC were among the most highly expressed genes within the UFO1 genome, and their expressions were unchanged by the presence or absence of uranium. Therefore, the UBC appears to be constitutively expressed and is the first line of defense against uranium, including by secretion into the extracellular medium. Although S-layer proteins were previously shown to bind U(VI), here we showed that U(IV) binds to S-layer proteins, we identified the proteins involved, and we quantitated the amount of uranium bound. Widespread uranium contamination from industrial sources poses hazards to human health and to the environment. Here in this paper, we identified a highly abundant uranium-binding complex (UBC) from Pelosinus sp. strain UFO1. The complex makes up the primary protein component of the S-layer of strain UFO1 and binds 3.3 atoms of U(IV) per heterodimer. Finally, while other bacteria have been shown to bind U(VI) on their S-layer, we demonstrate here an example of U

A Highly Expressed High-Molecular-Weight S-Layer Complex of Pelosinus sp. Strain UFO1 Binds Uranium.

PubMed

Thorgersen, Michael P; Lancaster, W Andrew; Rajeev, Lara; Ge, Xiaoxuan; Vaccaro, Brian J; Poole, Farris L; Arkin, Adam P; Mukhopadhyay, Aindrila; Adams, Michael W W

2017-02-15

Cell suspensions of Pelosinus sp. strain UFO1 were previously shown, using spectroscopic analysis, to sequester uranium as U(IV) complexed with carboxyl and phosphoryl group ligands on proteins. The goal of our present study was to characterize the proteins involved in uranium binding. Virtually all of the uranium in UFO1 cells was associated with a heterodimeric protein, which was termed the uranium-binding complex (UBC). The UBC was composed of two S-layer domain proteins encoded by UFO1_4202 and UFO1_4203. Samples of UBC purified from the membrane fraction contained 3.3 U atoms/heterodimer, but significant amounts of phosphate were not detected. The UBC had an estimated molecular mass by gel filtration chromatography of 15 MDa, and it was proposed to contain 150 heterodimers (UFO1_4203 and UFO1_4202) and about 500 uranium atoms. The UBC was also the dominant extracellular protein, but when purified from the growth medium, it contained only 0.3 U atoms/heterodimer. The two genes encoding the UBC were among the most highly expressed genes within the UFO1 genome, and their expressions were unchanged by the presence or absence of uranium. Therefore, the UBC appears to be constitutively expressed and is the first line of defense against uranium, including by secretion into the extracellular medium. Although S-layer proteins were previously shown to bind U(VI), here we showed that U(IV) binds to S-layer proteins, we identified the proteins involved, and we quantitated the amount of uranium bound. Widespread uranium contamination from industrial sources poses hazards to human health and to the environment. Herein, we identified a highly abundant uranium-binding complex (UBC) from Pelosinus sp. strain UFO1. The complex makes up the primary protein component of the S-layer of strain UFO1 and binds 3.3 atoms of U(IV) per heterodimer. While other bacteria have been shown to bind U(VI) on their S-layer, we demonstrate here an example of U(IV) bound by an S-layer complex

A proteomic screen reveals the mitochondrial outer membrane protein Mdm34p as an essential target of the F-box protein Mdm30p.

PubMed

Ota, Kazuhisa; Kito, Keiji; Okada, Satoshi; Ito, Takashi

2008-10-01

Ubiquitination plays various critical roles in eukaryotic cellular regulation and is mediated by a cascade of enzymes including ubiquitin protein ligase (E3). The Skp1-Cullin-F-box protein complex comprises the largest E3 family, in each member of which a unique F-box protein binds its targets to define substrate specificity. Although genome sequencing uncovers a growing number of F-box proteins, most of them have remained as "orphans" because of the difficulties in identification of their substrates. To address this issue, we tested a quantitative proteomic approach by combining the stable isotope labeling by amino acids in cell culture (SILAC), parallel affinity purification (PAP) that we had developed for efficient enrichment of ubiquitinated proteins, and mass spectrometry (MS). We applied this SILAC-PAP-MS approach to compare ubiquitinated proteins between yeast cells with and without over-expressed Mdm30p, an F-box protein implicated in mitochondrial morphology. Consequently, we identified the mitochondrial outer membrane protein Mdm34p as a target of Mdm30p. Furthermore, we found that mitochondrial defects induced by deletion of MDM30 are not only recapitulated by a mutant Mdm34p defective in interaction with Mdm30p but alleviated by ubiquitination-mimicking forms of Mdm34p. These results indicate that Mdm34p is a physiologically important target of Mdm30p.

Genome-wide survey and expression analysis of F-box genes in chickpea.

PubMed

Gupta, Shefali; Garg, Vanika; Kant, Chandra; Bhatia, Sabhyata

2015-02-13

The F-box genes constitute one of the largest gene families in plants involved in degradation of cellular proteins. F-box proteins can recognize a wide array of substrates and regulate many important biological processes such as embryogenesis, floral development, plant growth and development, biotic and abiotic stress, hormonal responses and senescence, among others. However, little is known about the F-box genes in the important legume crop, chickpea. The available draft genome sequence of chickpea allowed us to conduct a genome-wide survey of the F-box gene family in chickpea. A total of 285 F-box genes were identified in chickpea which were classified based on their C-terminal domain structures into 10 subfamilies. Thirteen putative novel motifs were also identified in F-box proteins with no known functional domain at their C-termini. The F-box genes were physically mapped on the 8 chickpea chromosomes and duplication events were investigated which revealed that the F-box gene family expanded largely due to tandem duplications. Phylogenetic analysis classified the chickpea F-box genes into 9 clusters. Also, maximum syntenic relationship was observed with soybean followed by Medicago truncatula, Lotus japonicus and Arabidopsis. Digital expression analysis of F-box genes in various chickpea tissues as well as under abiotic stress conditions utilizing the available chickpea transcriptome data revealed differential expression patterns with several F-box genes specifically expressing in each tissue, few of which were validated by using quantitative real-time PCR. The genome-wide analysis of chickpea F-box genes provides new opportunities for characterization of candidate F-box genes and elucidation of their function in growth, development and stress responses for utilization in chickpea improvement.

F-box proteins Pof3 and Pof1 regulate Wee1 degradation and mitotic entry in fission yeast.

PubMed

Qiu, Cui; Yi, Yuan-Yuan; Lucena, Rafael; Wu, Meng-Juan; Sun, Jia-Hao; Wang, Xi; Jin, Quan-Wen; Wang, Yamei

2018-02-02

The key cyclin-dependent kinase Cdk1 (Cdc2) promotes irreversible mitotic entry, mainly by activating the phosphatase Cdc25 while suppressing the tyrosine kinase Wee1. Wee1 needs to be downregulated at the onset of mitosis to ensure rapid activation of Cdk1. In human somatic cells, one mechanism of suppressing Wee1 activity is mediated by ubiquitylation-dependent proteolysis through the Skp1/Cul1/F-box protein (SCF) ubiquitin E3 ligase complex. This mechanism is believed to be conserved from yeasts to humans. So far, the best-characterized human F-box proteins involved in recognition of Wee1 are β-TrCP (BTRCP) and Tome-1 (CDCA3). Although fission yeast Wee1 was the first identified member of its conserved kinase family, the F-box proteins involved in recognition and ubiquitylation of Wee1 have not been identified in this organism. In this study, our screen using Wee1- Renilla luciferase as the reporter revealed that two F-box proteins, Pof1 and Pof3, are required for downregulating Wee1 and are possibly responsible for recruiting Wee1 to SCF. Our genetic analyses supported a functional relevance between Pof1 and Pof3 and the rate of mitotic entry, and Pof3 might play a major role in this process. © 2018. Published by The Company of Biologists Ltd.

NSs Virulence Factor of Rift Valley Fever Virus Engages the F-Box Proteins FBXW11 and β-TRCP1 To Degrade the Antiviral Protein Kinase PKR.

PubMed

Kainulainen, Markus; Lau, Simone; Samuel, Charles E; Hornung, Veit; Weber, Friedemann

2016-07-01

Rift Valley fever virus (RVFV, family Bunyaviridae, genus Phlebovirus) is a relevant pathogen of both humans and livestock in Africa. The nonstructural protein NSs is a major virulence factor known to suppress the type I interferon (IFN) response by inhibiting host cell transcription and by proteasomal degradation of a major antiviral IFN effector, the translation-inhibiting protein kinase PKR. Here, we identified components of the modular SCF (Skp1, Cul1, F-box protein)-type E3 ubiquitin ligases as mediators of PKR destruction by NSs. Small interfering RNAs (siRNAs) against the conserved SCF subunit Skp1 protected PKR from NSs-mediated degradation. Consequently, RVFV replication was severely reduced in Skp1-depleted cells when PKR was present. SCF complexes have a variable F-box protein subunit that determines substrate specificity for ubiquitination. We performed an siRNA screen for all (about 70) human F-box proteins and found FBXW11 to be involved in PKR degradation. The partial stabilization of PKR by FBXW11 depletion upregulated PKR autophosphorylation and phosphorylation of the PKR substrate eIF2α and caused a shutoff of host cell protein synthesis in RVFV-infected cells. To maximally protect PKR from the action of NSs, knockdown of structurally and functionally related FBXW1 (also known as β-TRCP1), in addition to FBXW11 deletion, was necessary. Consequently, NSs was found to interact with both FBXW11 and β-TRCP1. Thus, NSs eliminates the antiviral kinase PKR by recruitment of SCF-type E3 ubiquitin ligases containing FBXW11 and β-TRCP1 as substrate recognition subunits. This antagonism of PKR by NSs is essential for efficient RVFV replication in mammalian cells. Rift Valley fever virus is a pathogen of humans and animals that has the potential to spread from Africa and the Arabian Peninsula to other regions. A major virulence mechanism is the proteasomal degradation of the antiviral kinase PKR by the viral protein NSs. Here, we demonstrate that NSs

NSs Virulence Factor of Rift Valley Fever Virus Engages the F-Box Proteins FBXW11 and β-TRCP1 To Degrade the Antiviral Protein Kinase PKR

PubMed Central

Kainulainen, Markus; Lau, Simone; Samuel, Charles E.; Hornung, Veit

2016-01-01

ABSTRACT Rift Valley fever virus (RVFV, family Bunyaviridae, genus Phlebovirus) is a relevant pathogen of both humans and livestock in Africa. The nonstructural protein NSs is a major virulence factor known to suppress the type I interferon (IFN) response by inhibiting host cell transcription and by proteasomal degradation of a major antiviral IFN effector, the translation-inhibiting protein kinase PKR. Here, we identified components of the modular SCF (Skp1, Cul1, F-box protein)-type E3 ubiquitin ligases as mediators of PKR destruction by NSs. Small interfering RNAs (siRNAs) against the conserved SCF subunit Skp1 protected PKR from NSs-mediated degradation. Consequently, RVFV replication was severely reduced in Skp1-depleted cells when PKR was present. SCF complexes have a variable F-box protein subunit that determines substrate specificity for ubiquitination. We performed an siRNA screen for all (about 70) human F-box proteins and found FBXW11 to be involved in PKR degradation. The partial stabilization of PKR by FBXW11 depletion upregulated PKR autophosphorylation and phosphorylation of the PKR substrate eIF2α and caused a shutoff of host cell protein synthesis in RVFV-infected cells. To maximally protect PKR from the action of NSs, knockdown of structurally and functionally related FBXW1 (also known as β-TRCP1), in addition to FBXW11 deletion, was necessary. Consequently, NSs was found to interact with both FBXW11 and β-TRCP1. Thus, NSs eliminates the antiviral kinase PKR by recruitment of SCF-type E3 ubiquitin ligases containing FBXW11 and β-TRCP1 as substrate recognition subunits. This antagonism of PKR by NSs is essential for efficient RVFV replication in mammalian cells. IMPORTANCE Rift Valley fever virus is a pathogen of humans and animals that has the potential to spread from Africa and the Arabian Peninsula to other regions. A major virulence mechanism is the proteasomal degradation of the antiviral kinase PKR by the viral protein NSs. Here, we

An Activated Form of UFO Alters Leaf Development and Produces Ectopic Floral and Inflorescence Meristems

PubMed Central

Risseeuw, Eddy; Venglat, Prakash; Xiang, Daoquan; Komendant, Kristina; Daskalchuk, Tim; Babic, Vivijan; Crosby, William; Datla, Raju

2013-01-01

Plants are unique in their ability to continuously produce new meristems and organ primordia. In Arabidopsis, the transcription factor LEAFY (LFY) functions as a master regulator of a gene network that is important for floral meristem and organ specification. UNUSUAL FLORAL ORGANS (UFO) is a co-activator of LEAFY and is required for proper activation of APETALA3 in the floral meristem during the specification of stamens and petals. The ufo mutants display defects in other parts of the flower and the inflorescence, suggestive of additional roles. Here we show that the normal determinacy of the developing Arabidopsis leaves is affected by the expression of a gain-of-function UFO fusion protein with the VP16 transcriptional activator domain. In these lines, the rosette and cauline leaf primordia exhibit reiterated serration, and upon flowering produce ectopic meristems that develop into flowers, bract leaves and inflorescences. These striking phenotypes reveal that developing leaves maintain the competency to initiate flower and inflorescence programs. Furthermore, the gain-of-function phenotypes are dependent on LFY and the SEPALLATA (SEP) MADS-box transcription factors, indicative of their functional interactions with UFO. The findings of this study also suggest that UFO promotes the establishment of the lateral meristems and primordia in the peripheral zone of the apical and floral meristems by enhancing the activity of LFY. These novel phenotypes along with the mutant phenotypes of UFO orthologs in other plant species suggest a broader function for UFO in plants. PMID:24376756

An activated form of UFO alters leaf development and produces ectopic floral and inflorescence meristems.

PubMed

Risseeuw, Eddy; Venglat, Prakash; Xiang, Daoquan; Komendant, Kristina; Daskalchuk, Tim; Babic, Vivijan; Crosby, William; Datla, Raju

2013-01-01

Plants are unique in their ability to continuously produce new meristems and organ primordia. In Arabidopsis, the transcription factor LEAFY (LFY) functions as a master regulator of a gene network that is important for floral meristem and organ specification. UNUSUAL FLORAL ORGANS (UFO) is a co-activator of LEAFY and is required for proper activation of APETALA3 in the floral meristem during the specification of stamens and petals. The ufo mutants display defects in other parts of the flower and the inflorescence, suggestive of additional roles. Here we show that the normal determinacy of the developing Arabidopsis leaves is affected by the expression of a gain-of-function UFO fusion protein with the VP16 transcriptional activator domain. In these lines, the rosette and cauline leaf primordia exhibit reiterated serration, and upon flowering produce ectopic meristems that develop into flowers, bract leaves and inflorescences. These striking phenotypes reveal that developing leaves maintain the competency to initiate flower and inflorescence programs. Furthermore, the gain-of-function phenotypes are dependent on LFY and the SEPALLATA (SEP) MADS-box transcription factors, indicative of their functional interactions with UFO. The findings of this study also suggest that UFO promotes the establishment of the lateral meristems and primordia in the peripheral zone of the apical and floral meristems by enhancing the activity of LFY. These novel phenotypes along with the mutant phenotypes of UFO orthologs in other plant species suggest a broader function for UFO in plants.

The involvement of wheat F-box protein gene TaFBA1 in the oxidative stress tolerance of plants.

PubMed

Zhou, Shu-Mei; Kong, Xiang-Zhu; Kang, Han-Han; Sun, Xiu-Dong; Wang, Wei

2015-01-01

As one of the largest gene families, F-box domain proteins have been found to play important roles in abiotic stress responses via the ubiquitin pathway. TaFBA1 encodes a homologous F-box protein contained in E3 ubiquitin ligases. In our previous study, we found that the overexpression of TaFBA1 enhanced drought tolerance in transgenic plants. To investigate the mechanisms involved, in this study, we investigated the tolerance of the transgenic plants to oxidative stress. Methyl viologen was used to induce oxidative stress conditions. Real-time PCR and western blot analysis revealed that TaFBA1 expression was up-regulated by oxidative stress treatments. Under oxidative stress conditions, the transgenic tobacco plants showed a higher germination rate, higher root length and less growth inhibition than wild type (WT). The enhanced oxidative stress tolerance of the transgenic plants was also indicated by lower reactive oxygen species (ROS) accumulation, malondialdehyde (MDA) content and cell membrane damage under oxidative stress compared with WT. Higher activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POD), were observed in the transgenic plants than those in WT, which may be related to the upregulated expression of some antioxidant genes via the overexpression of TaFBA1. In others, some stress responsive elements were found in the promoter region of TaFBA1, and TaFBA1 was located in the nucleus, cytoplasm and plasma membrane. These results suggest that TaFBA1 plays an important role in the oxidative stress tolerance of plants. This is important for understanding the functions of F-box proteins in plants' tolerance to multiple stress conditions.

The Involvement of Wheat F-Box Protein Gene TaFBA1 in the Oxidative Stress Tolerance of Plants

PubMed Central

Zhou, Shu-Mei; Kong, Xiang-Zhu; Kang, Han-Han; Sun, Xiu-Dong; Wang, Wei

2015-01-01

As one of the largest gene families, F-box domain proteins have been found to play important roles in abiotic stress responses via the ubiquitin pathway. TaFBA1 encodes a homologous F-box protein contained in E3 ubiquitin ligases. In our previous study, we found that the overexpression of TaFBA1 enhanced drought tolerance in transgenic plants. To investigate the mechanisms involved, in this study, we investigated the tolerance of the transgenic plants to oxidative stress. Methyl viologen was used to induce oxidative stress conditions. Real-time PCR and western blot analysis revealed that TaFBA1 expression was up-regulated by oxidative stress treatments. Under oxidative stress conditions, the transgenic tobacco plants showed a higher germination rate, higher root length and less growth inhibition than wild type (WT). The enhanced oxidative stress tolerance of the transgenic plants was also indicated by lower reactive oxygen species (ROS) accumulation, malondialdehyde (MDA) content and cell membrane damage under oxidative stress compared with WT. Higher activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POD), were observed in the transgenic plants than those in WT, which may be related to the upregulated expression of some antioxidant genes via the overexpression of TaFBA1. In others, some stress responsive elements were found in the promoter region of TaFBA1, and TaFBA1 was located in the nucleus, cytoplasm and plasma membrane. These results suggest that TaFBA1 plays an important role in the oxidative stress tolerance of plants. This is important for understanding the functions of F-box proteins in plants’ tolerance to multiple stress conditions. PMID:25906259

Stringent Control of NFE2L3 (Nuclear Factor, Erythroid 2-Like 3; NRF3) Protein Degradation by FBW7 (F-box/WD Repeat-containing Protein 7) and Glycogen Synthase Kinase 3 (GSK3)*

PubMed Central

Kannan, Meenakshi B.; Dodard-Friedman, Isadore; Blank, Volker

2015-01-01

The NFE2L3 transcription factor has been implicated in various cellular processes, including carcinogenesis, stress response, differentiation, and inflammation. Previously it has been shown that NFE2L3 has a rapid turnover and is stabilized by proteasomal inhibitors. The mechanisms regulating the degradation of this protein have not been investigated. Here we report ubiquitination of NFE2L3 and demonstrate that F-box/WD repeat-containing protein 7 (FBW7 or FBWX7), a component of Skp1, Cullin 1, F-box containing complex (SCF)-type E3 ligase, is the E3 ligase mediating the degradation of NFE2L3. We showed that FBW7 interacts with NFE2L3 and that dimerization of FBW7 is required for the degradation of the transcription factor. We also demonstrate that the kinase glycogen synthase kinase 3 (GSK3) mediates the FBW7-dependent ubiquitination of NFE2L3. We show phosphorylation of NFE2L3 by GSK3 and its significance in the regulation of NFE2L3 by the tumor suppressor FBW7. FBW7 abrogated NFE2L3-mediated repression of the NAD(P)H:quinone oxidoreductase 1 (NQO1) gene antioxidant response element (ARE). Our findings reveal FBW7 and GSK3 as novel regulators of the NFE2L3 transcription factor and a potential mechanism by which FBW7 might regulate detoxification and the cellular response to stress. PMID:26306035

Linking F-box protein 7 and parkin to neuronal degeneration in Parkinson's disease (PD).

PubMed

Zhou, Zhi Dong; Sathiyamoorthy, Sushmitha; Angeles, Dario C; Tan, Eng King

2016-04-18

Mutations of F-box protein 7 (FBXO7) and Parkin, two proteins in ubiquitin-proteasome system (UPS), are both implicated in pathogenesis of dopamine (DA) neuron degeneration in Parkinson's disease (PD). Parkin is a HECT/RING hybrid ligase that physically receives ubiquitin on its catalytic centre and passes ubiquitin onto its substrates, whereas FBXO7 is an adaptor protein in Skp-Cullin-F-box (SCF) SCF(FBXO7) ubiquitin E3 ligase complex to recognize substrates and mediate substrates ubiquitination by SCF(FBXO7) E3 ligase. Here, we discuss the overlapping pathophysiologic mechanisms and clinical features linking Parkin and FBXO7 with autosomal recessive PD. Both proteins play an important role in neuroprotective mitophagy to clear away impaired mitochondria. Parkin can be recruited to impaired mitochondria whereas cellular stress can promote FBXO7 mitochondrial translocation. PD-linked FBXO7 can recruit Parkin into damaged mitochondria and facilitate its aggregation. WT FBXO7, but not PD-linked FBXO7 mutants can rescue DA neuron degeneration in Parkin null Drosophila. A better understanding of the common pathophysiologic mechanisms of these two proteins could unravel specific pathways for targeted therapy in PD.

Evolution of the F-Box Gene Family in Euarchontoglires: Gene Number Variation and Selection Patterns

PubMed Central

Wang, Ailan; Fu, Mingchuan; Jiang, Xiaoqian; Mao, Yuanhui; Li, Xiangchen; Tao, Shiheng

2014-01-01

F-box proteins are substrate adaptors used by the SKP1–CUL1–F-box protein (SCF) complex, a type of E3 ubiquitin ligase complex in the ubiquitin proteasome system (UPS). SCF-mediated ubiquitylation regulates proteolysis of hundreds of cellular proteins involved in key signaling and disease systems. However, our knowledge of the evolution of the F-box gene family in Euarchontoglires is limited. In the present study, 559 F-box genes and nine related pseudogenes were identified in eight genomes. Lineage-specific gene gain and loss events occurred during the evolution of Euarchontoglires, resulting in varying F-box gene numbers ranging from 66 to 81 among the eight species. Both tandem duplication and retrotransposition were found to have contributed to the increase of F-box gene number, whereas mutation in the F-box domain was the main mechanism responsible for reduction in the number of F-box genes, resulting in a balance of expansion and contraction in the F-box gene family. Thus, the Euarchontoglire F-box gene family evolved under a birth-and-death model. Signatures of positive selection were detected in substrate-recognizing domains of multiple F-box proteins, and adaptive changes played a role in evolution of the Euarchontoglire F-box gene family. In addition, single nucleotide polymorphism (SNP) distributions were found to be highly non-random among different regions of F-box genes in 1092 human individuals, with domain regions having a significantly lower number of non-synonymous SNPs. PMID:24727786

Mutation in Torenia fournieri Lind. UFO homolog confers loss of TfLFY interaction and results in a petal to sepal transformation.

PubMed

Sasaki, Katsutomo; Yamaguchi, Hiroyasu; Aida, Ryutaro; Shikata, Masahito; Abe, Tomoko; Ohtsubo, Norihiro

2012-09-01

We identified a Torenia fournieri Lind. mutant (no. 252) that exhibited a sepaloid phenotype in which the second whorls were changed to sepal-like organs. This mutant had no stamens, and the floral organs consisted of sepals and carpels. Although the expression of a torenia class B MADS-box gene, GLOBOSA (TfGLO), was abolished in the 252 mutant, no mutation of TfGLO was found. Among torenia homologs such as APETALA1 (AP1), LEAFY (LFY), and UNUSUAL FLORAL ORGANS (UFO), which regulate expression of class B genes in Arabidopsis, only accumulation of the TfUFO transcript was diminished in the 252 mutant. Furthermore, a missense mutation was found in the coding region of the mutant TfUFO. Intact TfUFO complemented the mutant phenotype whereas mutated TfUFO did not; in addition, the transgenic phenotype of TfUFO-knockdown torenias coincided with the mutant phenotype. Yeast two-hybrid analysis revealed that the mutated TfUFO lost its ability to interact with TfLFY protein. In situ hybridization analysis indicated that the transcripts of TfUFO and TfLFY were partially accumulated in the same region. These results clearly demonstrate that the defect in TfUFO caused the sepaloid phenotype in the 252 mutant due to the loss of interaction with TfLFY. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

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

PubMed

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.

F-box protein FBXL2 targets cyclin D2 for ubiquitination and degradation to inhibit leukemic cell proliferation

PubMed Central

Chen, Bill B.; Glasser, Jennifer R.; Coon, Tiffany A.; Zou, Chunbin; Miller, Hannah L.; Fenton, Moon; McDyer, John F.; Boyiadzis, Michael

2012-01-01

Hematologic maligancies exhibit a growth advantage by up-regulation of components within the molecular apparatus involved in cell-cycle progression. The SCF (Skip-Cullin1-F-box protein) E3 ligase family provides homeostatic feedback control of cell division by mediating ubiquitination and degradation of cell-cycle proteins. By screening several previously undescribed E3 ligase components, we describe the behavior of a relatively new SCF subunit, termed FBXL2, that ubiquitinates and destabilizes cyclin D2 protein leading to G0 phase arrest and apoptosis in leukemic and B-lymphoblastoid cell lines. FBXL2 expression was strongly suppressed, and yet cyclin D2 protein levels were robustly expressed in acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) patient samples. Depletion of endogenous FBXL2 stabilized cyclin D2 levels, whereas ectopically expressed FBXL2 decreased cyclin D2 lifespan. FBXL2 did not bind a phosphodegron within its substrate, which is typical of other F-box proteins, but uniquely targeted a calmodulin-binding signature within cyclin D2 to facilitate its polyubiquitination. Calmodulin competes with the F-box protein for access to this motif where it bound and protected cyclin D2 from FBXL2. Calmodulin reversed FBXL2-induced G0 phase arrest and attenuated FBXL2-induced apoptosis of lymphoblastoid cells. These results suggest an antiproliferative effect of SCFFBXL2 in lymphoproliferative malignancies. PMID:22323446

Molecular dynamics simulations elucidate the mode of protein recognition by Skp1 and the F-box domain in the SCF complex.

PubMed

Chandra Dantu, Sarath; Nathubhai Kachariya, Nitin; Kumar, Ashutosh

2016-01-01

Polyubiquitination of the target protein by a ubiquitin transferring machinery is key to various cellular processes. E3 ligase Skp1-Cul1-F-box (SCF) is one such complex which plays crucial role in substrate recognition and transfer of the ubiquitin molecule. Previous computational studies have focused on S-phase kinase-associated protein 2 (Skp2), cullin, and RING-finger proteins of this complex, but the roles of the adapter protein Skp1 and F-box domain of Skp2 have not been determined. Using sub-microsecond molecular dynamics simulations of full-length Skp1, unbound Skp2, Skp2-Cks1 (Cks1: Cyclin-dependent kinases regulatory subunit 1), Skp1-Skp2, and Skp1-Skp2-Cks1 complexes, we have elucidated the function of Skp1 and the F-box domain of Skp2. We found that the L16 loop of Skp1, which was deleted in previous X-ray crystallography studies, can offer additional stability to the ternary complex via its interactions with the C-terminal tail of Skp2. Moreover, Skp1 helices H6, H7, and H8 display vivid conformational flexibility when not bound to Skp2, suggesting that these helices can recognize and lock the F-box proteins. Furthermore, we observed that the F-box domain could rotate (5°-129°), and that the binding partner determined the degree of conformational flexibility. Finally, Skp1 and Skp2 were found to execute a domain motion in Skp1-Skp2 and Skp1-Skp2-Cks1 complexes that could decrease the distance between ubiquitination site of the substrate and the ubiquitin molecule by 3 nm. Thus, we propose that both the F-box domain of Skp2 and Skp1-Skp2 domain motions displaying preferential conformational control can together facilitate polyubiquitination of a wide variety of substrates. © 2015 Wiley Periodicals, Inc.

KELCH F-BOX protein positively influences Arabidopsis seed germination by targeting PHYTOCHROME-INTERACTING FACTOR1

USDA-ARS?s Scientific Manuscript database

Seeds employ sensory systems that assess various environmental cues over time to maximize the successful transition from embryo to seedling. Here, we show that the Arabidopsis F-Box protein Cold Temperature-Germinating (CTG)-10, identified by activation tagging, is a positive regulator during this p...

The stomatin-like protein SLP-1 and Cdk2 interact with the F-Box protein Fbw7-γ.

PubMed

Zhang, Wei; MacDonald, Elizabeth M; Koepp, Deanna M

2012-01-01

Control of cellular proliferation is critical to cell viability. The F-box protein Fbw7 (hAgo/hCdc4/FBXW7) functions as a specificity factor for the Skp1-Cul1-F-box protein (SCF) ubiquitin ligase complex and targets several proteins required for cellular proliferation for ubiquitin-mediated destruction. Fbw7 exists as three splice variants but the mechanistic role of each is not entirely clear. We examined the regulation of the Fbw7-γ isoform, which has been implicated in the degradation of c-Myc. We show here that Fbw7-γ is an unstable protein and that its turnover is proteasome-dependent in transformed cells. Using a two-hybrid screen, we identified a novel interaction partner, SLP-1, which binds the N-terminal domain of Fbw7-γ. Overexpression of SLP-1 inhibits the degradation of Fbw7-γ, suggesting that this interaction can happen in vivo. When Fbw7-γ is stabilized by overexpression of SLP-1, c-Myc protein abundance decreases, suggesting that the SCF(Fbw7-γ) complex maintains activity. We demonstrate that Cdk2 also binds the N-terminal domain of Fbw7-γ as well as SLP-1. Interestingly, co-expression of Cdk2 and SLP-1 does not inhibit Fbw7-γ degradation, suggesting that Cdk2 and SLP-1 may have opposing functions.

CRISPR/Cas9-mediated knockout of PiSSK1 reveals essential role of S-locus F-box protein-containing SCF complexes in recognition of non-self S-RNases during cross-compatible pollination in self-incompatible Petunia inflata.

PubMed

Sun, Linhan; Kao, Teh-Hui

2018-06-01

Function of Petunia PiSSK1. Self-incompatibility (SI), an inbreeding-preventing mechanism, is regulated in Petunia inflata by the polymorphic S-locus, which houses multiple pollen-specific S-locus F-box (SLF) genes and a single pistil-specific S-RNase gene. S 2 -haplotype and S 3 -haplotype possess the same 17 polymorphic SLF genes (named SLF1 to SLF17), and each SLF protein produced in pollen is assembled into an SCF (Skp1-Cullin1-F-box) E3 ubiquitin ligase complex. A complete suite of SLF proteins is thought to collectively interact with all non-self S-RNases to mediate their ubiquitination and degradation by the 26S proteasome, allowing cross-compatible pollination. For each SCF SLF complex, the Cullin1 subunit (named PiCUL1-P) and Skp1 subunit (named PiSSK1), like the F-box protein subunits (SLFs), are pollen-specific, raising the possibility that they also evolved specifically to function in SI. Here we used CRISPR/Cas9-meditated genome editing to generate frame-shift indel mutations in PiSSK1 and examined the SI behavior of a T 0 plant (S 2 S 3 ) with biallelic mutations in the pollen genome and two progeny plants (S 2 S 2 ) each homozygous for one of the indel alleles and not carrying the Cas9-containing T-DNA. Their pollen was completely incompatible with pistils of seven otherwise-compatible S-genotypes, but fully compatible with pistils of an S 3 S 3 transgenic plant in which production of S 3 -RNase was completely suppressed by an antisense S 3 -RNase gene, and with pistils of immature flower buds, which produce little S-RNase. These results suggest that PiSSK1 specifically functions in SI and support the hypothesis that SLF-containing SCF complexes are essential for compatible pollination.

S locus-linked F-box genes expressed in anthers of Hordeum bulbosum.

PubMed

Kakeda, Katsuyuki

2009-09-01

Diploid Hordeum bulbosum (a wild relative of cultivated barley) exhibits a two-locus self-incompatibility (SI) system gametophytically controlled by the unlinked multiallelic loci S and Z. This unique SI system is observed in the grasses (Poaceae) including the tribe Triticeae. This paper describes the identification and characterization of two F-box genes cosegregating with the S locus in H. bulbosum, named Hordeum S locus-linked F-box 1 (HSLF1) and HSLF2, which were derived from an S (3) haplotype-specific clone (HAS175) obtained by previous AMF (AFLP-based mRNA fingerprinting) analysis. Sequence analysis showed that both genes encode similar F-box proteins with a C-terminal leucine-rich repeat (LRR) domain, which are distinct from S locus (or S haplotype-specific) F-box protein (SLF/SFB), a class of F-box proteins identified as the pollen S determinant in S-RNase-based gametophytic SI systems. A number of homologous F-box genes with an LRR domain were found in the rice genome, although the functions of the gene family are unknown. One allele of the HSLF1 gene (HSLF1-S (3)) was expressed specifically in mature anthers, whereas no expression was detected from the other two alleles examined. Although the degree of sequence polymorphism among the three HSLF1 alleles was low, a frameshift mutation was found in one of the unexpressed alleles. The HSLF2 gene showed a low level of expression with no tissue specificity as well as little sequence polymorphism among the three alleles. The multiplicity of S locus-linked F-box genes is discussed in comparison with those found in the S-RNase-based SI system.

Overexpression of VpEIFP1, a novel F-box/Kelch-repeat protein from wild Chinese Vitis pseudoreticulata, confers higher tolerance to powdery mildew by inducing thioredoxin z proteolysis.

PubMed

Wang, Jie; Yao, Wenkong; Wang, Lei; Ma, Fuli; Tong, Weihuo; Wang, Chen; Bao, Rui; Jiang, Changyue; Yang, Yazhou; Zhang, Jianxia; Xu, Yan; Wang, Xiping; Zhang, Chaohong; Wang, Yuejin

2017-10-01

An F-box protein (VpEIFP1) induced by Erysiphe necator was isolated from Vitis pseudoreticulata, a wild Chinese grapevine species naturally resistant to powdery mildew (PM). It contains an F-box domain and two Kelch-repeat motifs. Expression profiles indicate the VpEIFP1 is strongly induced at both transcriptional and translational levels by PM infection. A subcellular localisation assay showed that VpEIFP1 is predominantly located in the nucleus and cytoplasm. Overexpression of VpEIFP1 accelerated the accumulation of hydrogen peroxide (H 2 O 2 ) and up-regulated the expressions of ICS2, NPR1 and PR1 involved in defence responses, resulting in suppression of PM germination and growth. As an F-box protein, VpEIFP1 interacts with thioredoxin z (VpTrxz) in the yeast-two-hybrid (Y2H) assay and in the bimolecular fluorescence complementation (BiFC) assay. Decreased amounts of VpTrxz protein in transgenic grapevine leaves overexpressing VpEIFP1 were restored by proteasome inhibitor MG132, implying that VpEIFP1 mediated VpTrxz for degradation through the SCF VpEIFP1 (Skp1-Cullin-F-box) E3 ubiquitin ligase complex. The RNA interference line of VpTrxz showed increased H 2 O 2 accumulation following PM inoculation. We propose VpEIFP1 positively modulates the grapevine defence response to PM by inducing the degradation of VpTrxz via the ubiquitin/26S proteasome system. Copyright © 2017 Elsevier B.V. All rights reserved.

An anther development F-box (ADF) protein regulated by tapetum degeneration retardation (TDR) controls rice anther development.

PubMed

Li, Li; Li, Yixing; Song, Shufeng; Deng, Huafeng; Li, Na; Fu, Xiqin; Chen, Guanghui; Yuan, Longping

2015-01-01

In this study, we reported that a F-box protein, OsADF, as one of the direct targets of TDR , plays a critical role in rice tapetum cell development and pollen formation. The tapetum, the innermost sporophytic tissue of anther, plays an important supportive role in male reproduction in flowering plants. After meiosis, tapetal cells undergo programmed cell death (PCD) and provide nutrients for pollen development. Previously we showed that tapetum degeneration retardation (TDR), a basic helix-loop-helix transcription factor, can trigger tapetal PCD and control pollen wall development during anther development. However, the comprehensive regulatory network of TDR remains to be investigated. In this study, we cloned and characterized a panicle-specific expression F-box protein, anther development F-box (OsADF). By qRT-PCR and RNA in situ hybridization, we further confirmed that OsADF expressed specially in tapetal cells from stage 9 to stage 12 during anther development. In consistent with this specific expression pattern, the RNAi transgenic lines of OsADF exhibited abnormal tapetal degeneration and aborted microspores development, which eventually grew pollens with reduced fertility. Furthermore, we demonstrated that the TDR, a key regulator in controlling rice anther development, could regulate directly the expression of OsADF by binding to E-box motifs of its promoter. Therefore, this work highlighted the possible regulatory role of TDR, which regulates tapetal cell development and pollen formation via triggering the possible ADF-mediated proteolysis pathway.

Skp1 Independent Function of Cdc53/Cul1 in F-box Protein Homeostasis.

PubMed

Mathur, Radhika; Yen, James L; Kaiser, Peter

2015-12-01

Abundance of substrate receptor subunits of Cullin-RING ubiquitin ligases (CRLs) is tightly controlled to maintain the full repertoire of CRLs. Unbalanced levels can lead to sequestration of CRL core components by a few overabundant substrate receptors. Numerous diseases, including cancer, have been associated with misregulation of substrate receptor components, particularly for the largest class of CRLs, the SCF ligases. One relevant mechanism that controls abundance of their substrate receptors, the F-box proteins, is autocatalytic ubiquitylation by intact SCF complex followed by proteasome-mediated degradation. Here we describe an additional pathway for regulation of F-box proteins on the example of yeast Met30. This ubiquitylation and degradation pathway acts on Met30 that is dissociated from Skp1. Unexpectedly, this pathway required the cullin component Cdc53/Cul1 but was independent of the other central SCF component Skp1. We demonstrated that this non-canonical degradation pathway is critical for chromosome stability and effective defense against heavy metal stress. More importantly, our results assign important biological functions to a sub-complex of cullin-RING ligases that comprises Cdc53/Rbx1/Cdc34, but is independent of Skp1.

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

PubMed

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.

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

PubMed

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.

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

PubMed

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-box genes: Genome-wide expansion, evolution and their contribution to pollen growth in pear (Pyrus bretschneideri).

PubMed

Wang, Guo-Ming; Yin, Hao; Qiao, Xin; Tan, Xu; Gu, Chao; Wang, Bao-Hua; Cheng, Rui; Wang, Ying-Zhen; Zhang, Shao-Ling

2016-12-01

F-box gene family, as one of the largest gene families in plants, plays crucial roles in regulating plant development, reproduction, cellular protein degradation and responses to biotic and abiotic stresses. However, comprehensive analysis of the F-box gene family in pear (Pyrus bretschneideri Rehd.) and other Rosaceae species has not been reported yet. Herein, we identified a total of 226 full-length F-box genes in pear for the first time. And these genes were further divided into various subgroups based on specific domains and phylogenetic analysis. Intriguingly, we observed that whole-genome duplication and dispersed duplication have a major contribution to F-box family expansion. Furthermore, the dynamic evolution for different modes of gene duplication was dissected. Interestingly, we found that dispersed and tandem duplicate have been evolving at a high rate. In addition, we found that F-box genes exhibited functional specificity based on GO analysis, and most of the F-box genes were significantly enriched in the protein binding (GO: 0005515) term, supporting that F-box genes might play a critical role for gene regulation in pear. Transcriptome and digital expression profiles revealed that F-box genes are involved in the development of multiple pear tissues. Overall, these results will set stage for elaborating the biological role of F-box genes in pear and other plants. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Cofactor-dependent specificity of a DEAD-box protein.

PubMed

Young, Crystal L; Khoshnevis, Sohail; Karbstein, Katrin

2013-07-16

DEAD-box proteins, a large class of RNA-dependent ATPases, regulate all aspects of gene expression and RNA metabolism. They can facilitate dissociation of RNA duplexes and remodeling of RNA-protein complexes, serve as ATP-dependent RNA-binding proteins, or even anneal duplexes. These proteins have highly conserved sequence elements that are contained within two RecA-like domains; consequently, their structures are nearly identical. Furthermore, crystal structures of DEAD-box proteins with bound RNA reveal interactions exclusively between the protein and the RNA backbone. Together, these findings suggest that DEAD-box proteins interact with their substrates in a nonspecific manner, which is confirmed in biochemical experiments. Nevertheless, this contrasts with the need to target these enzymes to specific substrates in vivo. Using the DEAD-box protein Rok1 and its cofactor Rrp5, which both function during maturation of the small ribosomal subunit, we show here that Rrp5 provides specificity to the otherwise nonspecific biochemical activities of the Rok1 DEAD-domain. This finding could reconcile the need for specific substrate binding of some DEAD-box proteins with their nonspecific binding surface and expands the potential roles of cofactors to specificity factors. Identification of helicase cofactors and their RNA substrates could therefore help define the undescribed roles of the 19 DEAD-box proteins that function in ribosome assembly.

UFO: a web server for ultra-fast functional profiling of whole genome protein sequences.

PubMed

Meinicke, Peter

2009-09-02

Functional profiling is a key technique to characterize and compare the functional potential of entire genomes. The estimation of profiles according to an assignment of sequences to functional categories is a computationally expensive task because it requires the comparison of all protein sequences from a genome with a usually large database of annotated sequences or sequence families. Based on machine learning techniques for Pfam domain detection, the UFO web server for ultra-fast functional profiling allows researchers to process large protein sequence collections instantaneously. Besides the frequencies of Pfam and GO categories, the user also obtains the sequence specific assignments to Pfam domain families. In addition, a comparison with existing genomes provides dissimilarity scores with respect to 821 reference proteomes. Considering the underlying UFO domain detection, the results on 206 test genomes indicate a high sensitivity of the approach. In comparison with current state-of-the-art HMMs, the runtime measurements show a considerable speed up in the range of four orders of magnitude. For an average size prokaryotic genome, the computation of a functional profile together with its comparison typically requires about 10 seconds of processing time. For the first time the UFO web server makes it possible to get a quick overview on the functional inventory of newly sequenced organisms. The genome scale comparison with a large number of precomputed profiles allows a first guess about functionally related organisms. The service is freely available and does not require user registration or specification of a valid email address.

UFO's in the Classroom?

ERIC Educational Resources Information Center

Coleman, William

1980-01-01

This article is in response to the Scientific-Study of Unidentified Flying Objects (UFOs) by the University of Colorado which stated that miseducation was taking place in schools that allowed children to read UFO books. The author encourages the aerospace teacher to allow UFO studies to take place. (SA)

Down-regulation of kelch domain-containing F-box protein in Arabidopsis enhances the production of (poly)phenols and tolerance to ultraviolet radiation

DOE PAGES

Zhang, Xuebin; Liu, Chang -Jun; Gou, Mingyue; ...

2014-12-01

Phenylpropanoid biosynthesis in plants engenders myriad phenolics with diverse biological functions. Phenylalanine ammonia-lyase (PAL) is the first committed enzyme in the pathway, directing primary metabolic flux into a phenylpropanoid branch. Previously, we demonstrated that the Arabidopsis Kelch-domain containing F-box proteins, AtKFB01, -20, and -50, function as the negative regulators controlling phenylpropanoid biosynthesis via mediating PAL’s ubiquitination and subsequent degradation. Here, we reveal that Arabidopsis KFB39, a close homolog of AtKFB50, also interacts physically with PAL isozymes and modulates PALs' stability and activity. Disturbing the expression of KFB39 reciprocally affects the accumulation/deposition of a set of phenylpropanoid end products, suggesting thatmore » KFB39 is an additional post-translational regulator responsible for the turnover of PAL and negatively controlling phenylpropanoid biosynthesis. Furthermore, we discover that exposure of Arabidopsis to UV-B radiation suppresses the expression of all four KFB genes while inducing the transcription of PAL isogenes; these data suggest that Arabidopsis consolidates both transcriptional and post-translational regulation mechanisms to maximize its responses to UV stress. Simultaneous down-regulation of all four identified KFBs significantly enhances the production of (poly)phenols and the plant’s tolerance to UV irradiation. This study offers a biotechnological approach for engineering the production of useful phenolic chemicals and for increasing a plant’s resistance to environmental stress.« less

O2 sensing-associated glycosylation exposes the F-box-combining site of the Dictyostelium Skp1 subunit in E3 ubiquitin ligases.

PubMed

Sheikh, M Osman; Thieker, David; Chalmers, Gordon; Schafer, Christopher M; Ishihara, Mayumi; Azadi, Parastoo; Woods, Robert J; Glushka, John N; Bendiak, Brad; Prestegard, James H; West, Christopher M

2017-11-17

Skp1 is a conserved protein linking cullin-1 to F-box proteins in SCF ( S kp1/ C ullin-1/ F -box protein) E3 ubiquitin ligases, which modify protein substrates with polyubiquitin chains that typically target them for 26S proteasome-mediated degradation. In Dictyostelium (a social amoeba), Toxoplasma gondii (the agent for human toxoplasmosis), and other protists, Skp1 is regulated by a unique pentasaccharide attached to hydroxylated Pro-143 within its C-terminal F-box-binding domain. Prolyl hydroxylation of Skp1 contributes to O 2 -dependent Dictyostelium development, but full glycosylation at that position is required for optimal O 2 sensing. Previous studies have shown that the glycan promotes organization of the F-box-binding region in Skp1 and aids in Skp1's association with F-box proteins. Here, NMR and MS approaches were used to determine the glycan structure, and then a combination of NMR and molecular dynamics simulations were employed to characterize the impact of the glycan on the conformation and motions of the intrinsically flexible F-box-binding domain of Skp1. Molecular dynamics trajectories of glycosylated Skp1 whose calculated monosaccharide relaxation kinetics and rotational correlation times agreed with the NMR data indicated that the glycan interacts with the loop connecting two α-helices of the F-box-combining site. In these trajectories, the helices separated from one another to create a more accessible and dynamic F-box interface. These results offer an unprecedented view of how a glycan modification influences a disordered region of a full-length protein. The increased sampling of an open Skp1 conformation can explain how glycosylation enhances interactions with F-box proteins in cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

MEIOTIC F-BOX Is Essential for Male Meiotic DNA Double-Strand Break Repair in Rice[OPEN

PubMed Central

Wang, Chong; Yu, Junping; Zong, Jie; Lu, Pingli

2016-01-01

F-box proteins constitute a large superfamily in plants and play important roles in controlling many biological processes, but the roles of F-box proteins in male meiosis in plants remain unclear. Here, we identify the rice (Oryza sativa) F-box gene MEIOTIC F-BOX (MOF), which is essential for male meiotic progression. MOF belongs to the FBX subfamily and is predominantly active during leptotene to pachytene of prophase I. mof meiocytes display disrupted telomere bouquet formation, impaired pairing and synapsis of homologous chromosomes, and arrested meiocytes at late prophase I, followed by apoptosis. Although normal, programmed double-stranded DNA breaks (DSBs) form in mof mutants, foci of the phosphorylated histone variant γH2AX, a marker for DSBs, persist in the mutant, indicating that many of the DSBs remained unrepaired. The recruitment of Completion of meiosis I (COM1) and Radiation sensitive51C (RAD51C) to DSBs is severely compromised in mutant meiocytes, indicating that MOF is crucial for DSB end-processing and repair. Further analyses showed that MOF could physically interact with the rice SKP1-like Protein1 (OSK1), indicating that MOF functions as a component of the SCF E3 ligase to regulate meiotic progression in rice. Thus, this study reveals the essential role of an F-box protein in plant meiosis and provides helpful information for elucidating the roles of the ubiquitin proteasome system in plant meiotic progression. PMID:27436711

Genome-Wide Analyses of the Soybean F-Box Gene Family in Response to Salt Stress

PubMed Central

Jia, Qi; Xiao, Zhi-Xia; Wong, Fuk-Ling; Sun, Song; Liang, Kang-Jing; Lam, Hon-Ming

2017-01-01

The F-box family is one of the largest gene families in plants that regulate diverse life processes, including salt responses. However, the knowledge of the soybean F-box genes and their roles in salt tolerance remains limited. Here, we conducted a genome-wide survey of the soybean F-box family, and their expression analysis in response to salinity via in silico analysis of online RNA-sequencing (RNA-seq) data and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) to predict their potential functions. A total of 725 potential F-box proteins encoded by 509 genes were identified and classified into 9 subfamilies. The gene structures, conserved domains and chromosomal distributions were characterized. There are 76 pairs of duplicate genes identified, including genome-wide segmental and tandem duplication events, which lead to the expansion of the number of F-box genes. The in silico expression analysis showed that these genes would be involved in diverse developmental functions and play an important role in salt response. Our qRT-PCR analysis confirmed 12 salt-responding F-box genes. Overall, our results provide useful information on soybean F-box genes, especially their potential roles in salt tolerance. PMID:28417911

Genome-Wide Analyses of the Soybean F-Box Gene Family in Response to Salt Stress.

PubMed

Jia, Qi; Xiao, Zhi-Xia; Wong, Fuk-Ling; Sun, Song; Liang, Kang-Jing; Lam, Hon-Ming

2017-04-12

The F-box family is one of the largest gene families in plants that regulate diverse life processes, including salt responses. However, the knowledge of the soybean F-box genes and their roles in salt tolerance remains limited. Here, we conducted a genome-wide survey of the soybean F-box family, and their expression analysis in response to salinity via in silico analysis of online RNA-sequencing (RNA-seq) data and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) to predict their potential functions. A total of 725 potential F-box proteins encoded by 509 genes were identified and classified into 9 subfamilies. The gene structures, conserved domains and chromosomal distributions were characterized. There are 76 pairs of duplicate genes identified, including genome-wide segmental and tandem duplication events, which lead to the expansion of the number of F-box genes. The in silico expression analysis showed that these genes would be involved in diverse developmental functions and play an important role in salt response. Our qRT-PCR analysis confirmed 12 salt-responding F-box genes. Overall, our results provide useful information on soybean F-box genes, especially their potential roles in salt tolerance.

The murine ufo receptor: molecular cloning, chromosomal localization and in situ expression analysis.

PubMed

Faust, M; Ebensperger, C; Schulz, A S; Schleithoff, L; Hameister, H; Bartram, C R; Janssen, J W

1992-07-01

We have cloned the mouse homologue of the ufo oncogene. It encodes a novel tyrosine kinase receptor characterized by a unique extracellular domain containing two immunoglobulin-like and two fibronectin type III repeats. Comparison of the predicted ufo amino acid sequences of mouse and man revealed an overall identity of 87.6%. The ufo locus maps to mouse chromosome 7A3-B1 and thereby extends the known conserved linkage group between mouse chromosome 7 and human chromosome 19. RNA in situ hybridization analysis established the onset of specific ufo expression in the late embryogenesis at day 12.5 post coitum (p.c.) and localized ufo transcription to distinct substructures of a broad spectrum of developing tissues (e.g. subepidermal cells of the skin, mesenchymal cells of the periosteum). In adult animals ufo is expressed in cells forming organ capsules as well as in connective tissue structures. ufo may function as a signal transducer between specific cell types of mesodermal origin.

Hepatic F-Box Protein FBXW7 Maintains Glucose Homeostasis Through Degradation of Fetuin-A.

PubMed

Zhao, Jiejie; Xiong, Xuelian; Li, Yao; Liu, Xing; Wang, Tao; Zhang, Hong; Jiao, Yang; Jiang, Jingjing; Zhang, Huijie; Tang, Qiqun; Gao, Xin; Li, Xuejun; Lu, Yan; Liu, Bin; Hu, Cheng; Li, Xiaoying

2018-05-01

Type 2 diabetes mellitus (T2DM) has become one of the most serious and long-term threats to human health. However, the molecular mechanism that links obesity to insulin resistance remains largely unknown. Here, we show that F-box and WD repeat domain-containing 7 (FBXW7), an E3 ubiquitin protein ligase, is markedly downregulated in the liver of two obese mouse models and obese human subjects. We further identify a functional low-frequency human FBXW7 coding variant (p.Ala204Thr) in the Chinese population, which is associated with elevated blood glucose and T2DM risk. Notably, mice with liver-specific knockout of FBXW7 develop hyperglycemia, glucose intolerance, and insulin resistance even on a normal chow diet. Conversely, overexpression of FBXW7 in the liver not only prevents the development of high-fat diet-induced insulin resistance but also attenuates the disease signature of obese mice. Mechanistically, FBXW7 directly binds to hepatokine fetuin-A to induce its ubiquitination and subsequent proteasomal degradation, comprising an important mechanism maintaining glucose homeostasis. Thus, we provide evidence showing a beneficial role of FBXW7 in glucose homeostasis. © 2018 by the American Diabetes Association.

Hsp27 and F-box protein β-TrCP promote degradation of mRNA decay factor AUF1.

PubMed

Li, Mei-Ling; Defren, Jennifer; Brewer, Gary

2013-06-01

Activation of the mitogen-activated protein (MAP) pathway kinases p38 and MK2 induces phosphorylation of the chaperone Hsp27 and stabilization of mRNAs containing AU-rich elements (AREs) (ARE-mRNAs). Likewise, expression of phosphomimetic mutant forms of Hsp27 also stabilizes ARE-mRNAs. It appears to perform this function by promoting degradation of the ARE-mRNA decay factor AUF1 by proteasomes. In this study, we examined the molecular mechanism linking Hsp27 phosphorylation to AUF1 degradation by proteasomes. AUF1 is a target of β-TrCP, the substrate recognition subunit of the E3 ubiquitin ligase Skp1-cullin-F-box protein complex, SCF(β-TrCP). Depletion of β-TrCP stabilized AUF1. In contrast, overexpression of β-TrCP enhanced ubiquitination and degradation of AUF1 and led to stabilization of reporter mRNAs containing cytokine AREs. Enhanced AUF1 degradation required expression of phosphomimetic mutant forms of both Hsp27 and AUF1. Our results suggest that a signaling axis composed of p38 MAP kinase-MK2-Hsp27-β-TrCP may promote AUF1 degradation by proteasomes and stabilization of cytokine ARE-mRNAs.

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

PubMed

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.

Down-Regulation of Kelch Domain-Containing F-Box Protein in Arabidopsis Enhances the Production of (Poly)phenols and Tolerance to Ultraviolet Radiation1[OPEN

PubMed Central

Zhang, Xuebin; Gou, Mingyue; Guo, Chunrong; Yang, Huijun; Liu, Chang-Jun

2015-01-01

Phenylpropanoid biosynthesis in plants engenders myriad phenolics with diverse biological functions. Phenylalanine ammonia-lyase (PAL) is the first committed enzyme in the pathway, directing primary metabolic flux into a phenylpropanoid branch. Previously, we demonstrated that the Arabidopsis (Arabidopsis thaliana) Kelch domain-containing F-box proteins, AtKFB01, AtKFB20, and AtKFB50, function as the negative regulators controlling phenylpropanoid biosynthesis via mediating PAL’s ubiquitination and subsequent degradation. Here, we reveal that Arabidopsis KFB39, a close homolog of AtKFB50, also interacts physically with PAL isozymes and modulates PAL stability and activity. Disturbing the expression of KFB39 reciprocally affects the accumulation/deposition of a set of phenylpropanoid end products, suggesting that KFB39 is an additional posttranslational regulator responsible for the turnover of PAL and negatively controlling phenylpropanoid biosynthesis. Furthermore, we discover that exposure of Arabidopsis to ultraviolet (UV)-B radiation suppresses the expression of all four KFB genes while inducing the transcription of PAL isogenes; these data suggest that Arabidopsis consolidates both transcriptional and posttranslational regulation mechanisms to maximize its responses to UV light stress. Simultaneous down-regulation of all four identified KFBs significantly enhances the production of (poly)phenols and the plant’s tolerance to UV irradiation. This study offers a biotechnological approach for engineering the production of useful phenolic chemicals and for increasing a plant’s resistance to environmental stress. PMID:25502410

Polyubiquitination of the B-cell translocation gene 1 and 2 proteins is promoted by the SCF ubiquitin ligase complex containing βTrCP.

PubMed

Sasajima, Hitoshi; Nakagawa, Koji; Kashiwayanagi, Makoto; Yokosawa, Hideyoshi

2012-01-01

B-cell translocation gene 1 and 2 (BTG1 and BTG2) are members of the BTG/Tob antiproliferative protein family, which is able to regulate the cell cycle and cell proliferation. We previously reported that BTG1, BTG2, Tob, and Tob2 are degraded via the ubiquitin-proteasome pathway. In this study, we investigated the mechanism of polyubiquitination of BTG1 and BTG2. Since the Skp1-Cdc53/Cullin 1-F-box protein (SCF) complex functions as one of the major ubiquitin ligases for cell cycle regulation, we first examined interactions between BTG proteins and components of the SCF complex, and found that BTG1 and BTG2 were capable of interacting with the SCF complex containing Cullin-1 (a scaffold protein) and Skp1 (a linker protein). As the SCF complex can ubiquitinate various target proteins by substituting different F-box proteins as subunits that recognize different target proteins, we next examined which F-box proteins could bind the two BTG proteins, and found that Skp2, β-transducin repeat-containing protein 1 (βTrCP1), and βTrCP2 were able to associate with both BTG1 and BTG2. Furthermore, we obtained evidence showing that βTrCP1 enhanced the polyubiquitination of both BTG1 and BTG2 more efficiently than Skp2 did, and that an F-box truncated mutant of βTrCP1 had a dominant negative effect on this polyubiquitination. Thus, we propose that BTG1 and BTG2 are subjected to polyubiquitination, more efficiently when it is mediated by SCFβTrCP than by SCFSkp2.

Differential expression of the ufo/axl oncogene in human leukemia-lymphoma cell lines.

PubMed

Challier, C; Uphoff, C C; Janssen, J W; Drexler, H G

1996-05-01

The ufo protein (also termed axl) is a member of a new family of receptor tyrosine kinases and is encoded by a transforming gene that was initially isolated from primary human myeloid leukemia cells by DNA-mediated transformation of NIH/3T3 cells. The ligand, Gas6, a protein S-related molecule lacking any known function yet, has recently been identified. We report the expression pattern of ufo mRNA in a panel of 76 human continuous leukemia-lymphoma cell lines. The gene was not expressed in cell lines derived from lymphoid malignancies (n=28), but transcription was seen in 3/11 myeloid, 0/6 monocytic, 9/13 erythroid and 11/18 megakaryocytic cell lines. Several cell lines were treated with phorbol ester leading to significant upregulation of the ufo message in constitutively positive cells. An apparent ufo mRNA overexpression was not found in any of the positive leukemia cell lines, but was identified in the drug-resistant subclones of the cervix carcinoma cell line HeLa. Southern blot analysis of restriction enzyme-digested genomic DNA did not provide evidence for gene amplification, but the HeLa subclones showed banding patterns suggestive of gene rearrangement. Two main ufo mRNA bands of 3.2 and 5.0 kb were identified; no differences in the half-lives (t1/2 = 2.5 h) of these two mRNA species could be identified. In summary, ufo, representing a novel type of receptor tyrosine kinase, is expressed solely in myeloid and erythro-megakaryocytic leukemias but not in lymphoid malignancies. These and previous data suggest an involvement of the ufo receptor tyrosine kinase in normal and malignant myelopoiesis; however, its exact role, if any, and mode of operation in leukemogenesis remains to be determined.

Unilateral incompatibility gene ui1.1 encodes an S-locus F-box protein expressed in pollen of Solanum species.

PubMed

Li, Wentao; Chetelat, Roger T

2015-04-07

Unilateral interspecific incompatibility (UI) is a postpollination, prezygotic reproductive barrier that prevents hybridization between related species when the female parent is self-incompatible (SI) and the male parent is self-compatible (SC). In tomato and related Solanum species, two genes, ui1.1 and ui6.1, are required for pollen compatibility on pistils of SI species or hybrids. We previously showed that ui6.1 encodes a Cullin1 (CUL1) protein. Here we report that ui1.1 encodes an S-locus F-box (SLF) protein. The ui1.1 gene was mapped to a 0.43-cM, 43.2-Mbp interval at the S-locus on chromosome 1, but positional cloning was hampered by low recombination frequency. We hypothesized that ui1.1 encodes an SLF protein(s) that interacts with CUL1 and Skp1 proteins to form an SCF-type (Skp1, Cullin1, F-box) ubiquitin E3 ligase complex. We identified 23 SLF genes in the S. pennellii genome, of which 19 were also represented in cultivated tomato (S. lycopersicum). Data from recombination events, expression analysis, and sequence annotation highlighted 11 S. pennellii genes as candidates. Genetic transformations demonstrated that one of these, SpSLF-23, is sufficient for ui1.1 function. A survey of cultivated and wild tomato species identified SLF-23 orthologs in each of the SI species, but not in the SC species S. lycopersicum, S. cheesmaniae, and S. galapagense, pollen of which lacks ui1.1 function. These results demonstrate that pollen compatibility in UI is mediated by protein degradation through the ubiquitin-proteasome pathway, a mechanism related to that which controls pollen recognition in SI.

Intracellular signaling of the Ufo/Axl receptor tyrosine kinase is mediated mainly by a multi-substrate docking-site.

PubMed

Braunger, J; Schleithoff, L; Schulz, A S; Kessler, H; Lammers, R; Ullrich, A; Bartram, C R; Janssen, J W

1997-06-05

Ufo/Axl belongs to a new family of receptor tyrosine kinases with an extracellular structure similar to that of neural cell adhesion molecules. In order to elucidate intracellular signaling, the cytoplasmic moiety of Ufo/Axl was used to screen an expression library according to the CORT (cloning of receptor targets) method. Three putative Ufo substrates were identified: phospholipase Cgamma1 (PLCgamma), as well as p85alpha and p85beta subunits of phosphatidylinositol 3'-kinase (PI3-kinase). Subsequently, chimeric EGFR/Ufo receptors consisting of the extracellular domains of the epidermal growth factor receptor (EGFR) and the transmembrane and intracellular moiety of Ufo were engineered. Using different far-Western blot analyses and coimmunoprecipitation assays, receptor binding of PLCgamma and p85 proteins as well as GRB2, c-src and lck was examined in vitro and in vivo. Competitive inhibition of substrate binding and mutagenesis experiments with EGFR/Ufo constructs revealed C-terminal tyrosine 821 (EILpYVNMDEG) as a docking site for multiple effectors, namely PLCgamma, p85 proteins, GRB2, c-src and lck. Tyrosine 779 (DGLpYALMSRC) demonstrated an additional, but lower binding affinity for the p85 proteins in vitro. In addition, binding of PLCgamma occurred through tyrosine 866 (AGRpYVLCPST). Moreover, our in vivo data indicate that further direct or indirect binding sites for PLCgamma, GRB2, c-src and lck on the human Ufo receptor may exist.

Investigation of UFO defect on DUV CAR and BARC process

NASA Astrophysics Data System (ADS)

Yet, Siew Ing; Ko, Bong Sang; Lee, Soo Man; May, Mike

2004-05-01

Photo process defect reduction is one of the most important factors to improve the process stability and yield in sub-0.18um DUV process. In this paper, a new approach to minimize the Deep-UV (DUV) Chemically Amplified Resist (CAR) and Bottom Anti-Reflective Coating (BARC) induced defect known as UFO (UnidentiFied Object) defect will be introduced. These defects have mild surface topography difference on BARC; it only exists on the wide exposed area where there is no photoresist pattern. In this test, Nikon KrF Stepper & Scanner and TEL Clean track were used. Investigation was carried out on the defect formulation on both Acetal and ESCAP type of photoresist while elemental analysis was done by Atomic Force Microscope (AFM) & Auger Electron Spectroscopy (AES). Result indicated that both BARC and photoresist induce this UFO defect; total defect quantity is related with Post Exposure Bake (PEB) condition. Based on the elemental analysis and process-split test, we can conclude that this defect is caused by lack of acid amount and low diffusivity which is related to PAG (Photo Acid Generator) and TAG (Thermal Acid Generator) in KrF photoresist and BARC material. By optimizing photoresist bake condition, this UFO defect as well as other related defect such as Satellite defect could be eliminated.

SVP-like MADS-box protein from Carya cathayensis forms higher-order complexes.

PubMed

Wang, Jingjing; Hou, Chuanming; Huang, Jianqin; Wang, Zhengjia; Xu, Yingwu

2015-03-01

To properly regulate plant flowering time and construct floral pattern, MADS-domain containing transcription factors must form multimers including homo- and hetero-dimers. They are also active in forming hetero-higher-order complexes with three to five different molecules. However, it is not well known if a MADS-box protein can also form homo-higher-order complex. In this study a biochemical approach is utilized to provide insight into the complex formation for an SVP-like MADS-box protein cloned from hickory. The results indicated that the protein is a heterogeneous higher-order complex with the peak population containing over 20 monomers. Y2H verified the protein to form homo-complex in yeast cells. Western blot of the hickory floral bud sample revealed that the protein exists in higher-order polymers in native. Deletion assays indicated that the flexible C-terminal residues are mainly responsible for the higher-order polymer formation and the heterogeneity. Current results provide direct biochemical evidences for an active MADS-box protein to be a high order complex, much higher than a quartermeric polymer. Analysis suggests that a MADS-box subset may be able to self-assemble into large complexes, and thereby differentiate one subfamily from the other in a higher-order structural manner. Present result is a valuable supplement to the action of mechanism for MADS-box proteins in plant development. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

The Arabidopsis COP9 SIGNALOSOME INTERACTING F-BOX KELCH 1 protein forms an SCF ubiquitin ligase and regulates hypocotyl elongation.

PubMed

Franciosini, Anna; Lombardi, Benedetta; Iafrate, Silvia; Pecce, Valeria; Mele, Giovanni; Lupacchini, Leonardo; Rinaldi, Gianmarco; Kondou, Youichi; Gusmaroli, Giuliana; Aki, Shiori; Tsuge, Tomohiko; Deng, Xing-Wang; Matsui, Minami; Vittorioso, Paola; Costantino, Paolo; Serino, Giovanna

2013-09-01

The regulation of protein turnover by the ubiquitin proteasome system (UPS) is a major posttranslational mechanism in eukaryotes. One of the key components of the UPS, the COP9 signalosome (CSN), regulates 'cullin-ring' E3 ubiquitin ligases. In plants, CSN participates in diverse cellular and developmental processes, ranging from light signaling to cell cycle control. In this work, we isolated a new plant-specific CSN-interacting F-box protein, which we denominated CFK1 (COP9 INTERACTING F-BOX KELCH 1). We show that, in Arabidopsis thaliana, CFK1 is a component of a functional ubiquitin ligase complex. We also show that CFK1 stability is regulated by CSN and by proteasome-dependent proteolysis, and that light induces accumulation of the CFK1 transcript in the hypocotyl. Analysis of CFK1 knockdown, mutant, and overexpressing seedlings indicates that CFK1 promotes hypocotyl elongation by increasing cell size. Reduction of CSN levels enhances the short hypocotyl phenotype of CFK1-depleted seedlings, while complete loss of CSN activity suppresses the long-hypocotyl phenotype of CFK1-overexpressing seedlings. We propose that CFK1 (and its regulation by CSN) is a novel component of the cellular mechanisms controlling hypocotyl elongation.

Inhibition of SCF ubiquitin ligases by engineered ubiquitin variants that target the Cul1 binding site on the Skp1–F-box interface

DOE PAGES

Gorelik, Maryna; Orlicky, Stephen; Sartori, Maria A.; ...

2016-03-14

Skp1–Cul1–F-box (SCF) E3 ligases play key roles in multiple cellular processes through ubiquitination and subsequent degradation of substrate proteins. Although Skp1 and Cul1 are invariant components of all SCF complexes, the 69 different human F-box proteins are variable substrate binding modules that determine specificity. SCF E3 ligases are activated in many cancers and inhibitors could have therapeutic potential. Here, we used phage display to develop specific ubiquitin-based inhibitors against two F-box proteins, Fbw7 and Fbw11. Unexpectedly, the ubiquitin variants bind at the interface of Skp1 and F-box proteins and inhibit ligase activity by preventing Cul1 binding to the same surface.more » Using structure-based design and phage display, we modified the initial inhibitors to generate broad-spectrum inhibitors that targeted many SCF ligases, or conversely, a highly specific inhibitor that discriminated between even the close homologs Fbw11 and Fbw1. We propose that most F-box proteins can be targeted by this approach for basic research and for potential cancer therapies.« less

UV and arsenate toxicity: a specific and sensitive yeast bioluminescence assay.

PubMed

Bakhrat, Anya; Eltzov, Evgeni; Finkelstein, Yishay; Marks, Robert S; Raveh, Dina

2011-06-01

We describe a Saccharomyces cerevisiae bioluminescence assay for UV and arsenate in which bacterial luciferase genes are regulated by the promoter of the yeast gene, UFO1. UFO1 encodes the F-box subunit of the Skp1–Cdc53–F-box protein ubiquitin ligase complex and is induced by DNA damage and by arsenate. We engineered the UFO1 promoter into an existing yeast bioreporter that employs human genes for detection of steroid hormone-disrupting compounds in water bodies. Our analysis indicates that use of an endogenous yeast promoter in different mutant backgrounds allows discrimination between different environmental signals. The UFO1-engineered yeast give a robust bioluminescence response to UVB and can be used for evaluating UV protective sunscreens. They are also effective in detecting extremely low concentrations of arsenate, particularly in pdr5Δ mutants that lack a mechanism to extrude toxic chemicals; however, they do not respond to cadmium or mercury. Combined use of endogenous yeast promoter elements and mutants of stress response pathways may facilitate development of high-specificity yeast bioreporters able to discriminate between closely related chemicals present together in the environment.

A new regulatory pathway of mRNA export by an F-box protein, Mdm30.

PubMed

Durairaj, Geetha; Lahudkar, Shweta; Bhaumik, Sukesh R

2014-02-01

Mdm30, an F-box protein in yeast, has been recently shown to promote mRNA export. However, it remains unknown how Mdm30 facilitates mRNA export. Here, we show that Mdm30 targets the Sub2 component of the TREX (Transcription/Export) complex for ubiquitylation and subsequent proteasomal degradation. Such a targeted degradation of Sub2 enhances the recruitment of the mRNA export adaptor, Yra1, to the active genes to promote mRNA export. Together, these results elucidate that Mdm30 promotes mRNA export by lowering Sub2's stability and consequently enhancing Yra1 recruitment, thus illuminating new regulatory mechanisms of mRNA export by Mdm30.

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

PubMed

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.

UFO Literature for the Serious Ufologist.

ERIC Educational Resources Information Center

Eberhart, George M.

1980-01-01

Discusses the differences between the serious ufologist and the interested novice, reviews essential reference works and journals dealing with UFOs, and addresses problems that libraries have in collecting UFO literature. (FM)

Genetically engineered mouse models for functional studies of SKP1-CUL1-F-box-protein (SCF) E3 ubiquitin ligases.

PubMed

Zhou, Weihua; Wei, Wenyi; Sun, Yi

2013-05-01

The SCF (SKP1 (S-phase-kinase-associated protein 1), Cullin-1, F-box protein) E3 ubiquitin ligases, the founding member of Cullin-RING ligases (CRLs), are the largest family of E3 ubiquitin ligases in mammals. Each individual SCF E3 ligase consists of one adaptor protein SKP1, one scaffold protein cullin-1 (the first family member of the eight cullins), one F-box protein out of 69 family members, and one out of two RING (Really Interesting New Gene) family proteins RBX1/ROC1 or RBX2/ROC2/SAG/RNF7. Various combinations of these four components construct a large number of SCF E3s that promote the degradation of many key regulatory proteins in cell-context, temporally, and spatially dependent manners, thus controlling precisely numerous important cellular processes, including cell cycle progression, apoptosis, gene transcription, signal transduction, DNA replication, maintenance of genome integrity, and tumorigenesis. To understand how the SCF E3 ligases regulate these cellular processes and embryonic development under in vivo physiological conditions, a number of mouse models with transgenic (Tg) expression or targeted deletion of components of SCF have been established and characterized. In this review, we will provide a brief introduction to the ubiquitin-proteasome system (UPS) and the SCF E3 ubiquitin ligases, followed by a comprehensive overview on the existing Tg and knockout (KO) mouse models of the SCF E3s, and discuss the role of each component in mouse embryogenesis, cell proliferation, apoptosis, carcinogenesis, as well as other pathogenic processes associated with human diseases. We will end with a brief discussion on the future directions of this research area and the potential applications of the knowledge gained to more effective therapeutic interventions of human diseases.

Hypothesis on the nature of atmospheric UFOs

NASA Astrophysics Data System (ADS)

Mukharev, L. A.

1991-08-01

A hypothesis is developed according to which the atmospheric UFO phenomenon has an electromagnetic nature. It is suggested that an atmospheric UFO is an agglomeration of charged atmospheric dust within which there exists a slowly damped electromagnetic field. This field is considered to be the source of the observed optical effects and the motive force of the UFO.

Bimolecular fluorescence complementation studies support an in vivo interaction between the F-BOX protein COLD TEMPERATURE GERMINATING10 and PHYTOCHROME INTERACTING FACTOR1

USDA-ARS?s Scientific Manuscript database

The Arabidopsis thaliana F-BOX protein COLD TEMPERATURE GERMINATING10 (CTG10) was identified from an activation tagged mutant screen as causing seeds to complete germination faster than wild type at 10°C when its expression is increased (Salaita et al. 2005. J. Exp. Bot. 56: 2059). Our unpublished d...

A large complement of the predicted Arabidopsis ARM repeat proteins are members of the U-box E3 ubiquitin ligase family.

PubMed

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.

Close encounters: an examination of UFO experiences.

PubMed

Spanos, N P; Cross, P A; Dickson, K; DuBreuil, S C

1993-11-01

Ss who reported UFO experiences were divided into those whose experiences were nonintense (e.g., seeing lights and shapes in the sky) and those whose experiences were intense (e.g., seeing and communicating with aliens or missing time). On a battery of objective tests Ss in these 2 groups did not score as more psychopathological, less intelligent, or more fantasy prone and hypnotizable than a community comparison group or a student comparison group. However, Ss in the UFO groups believed more strongly in space alien visitation than did comparison Ss. The UFO experiences of Ss in the intense group were more frequently sleep-related than the experiences of Ss in the nonintense group. Among the combined UFO Ss, intensity of UFO experiences correlated significantly with inventories that assessed proneness toward fantasy and unusual sensory experiences. Implications are discussed.

First UFO Incident for Our Country

DTIC Science & Technology

1983-09-09

researchers abroad still have obtained nothing rela- tive to this kind of UFO or aliens . Nor do they include in their large collection of photos of flying...FTD-ID(RS)T-1019-83 M FOREIGN TECHNOLOGY DIVISION FIRST UFO INCIDENT FOR OUR COUNTRY by Long Ruyi :DOO OCT 71983 Li. -Approved for public release...extracted from the best quality copy available. ’* CO • -.i o ° - .- - - - - • - ° • • . • • ° ° I . . . ... . .- , . • - - ° , . FIRST UFO INCIDENT

The B-Box Domain Protein BBX21 Promotes Photomorphogenesis.

PubMed

Xu, Dongqing; Jiang, Yan; Li, Jian; Holm, Magnus; Deng, Xing Wang

2018-03-01

B-box-containing (BBX) proteins play critical roles in a variety of cellular and developmental processes in plants. BBX21 (also known as SALT TOLERANCE HOMOLOG2), which contains two B-box domains in tandem at the N terminus, has been previously demonstrated as a key component involved in the COP1-HY5 signaling hub. However, the exact molecular and physiological roles of B-box domains in BBX21 are largely unclear. Here, we found that structurally disruption of the second B-box domain, but not the first one, in BBX21 completely abolishes its biological and physiological activity in conferring hyperphotomorphogenetic phenotype in Arabidopsis ( Arabidopsis thaliana ). Intact B-box domains in BBX21 are not required for interaction with COP1 and its degradation by COP1 via the 26S proteasome system. However, disruption of the second B-box of BBX21 nearly impairs its ability for binding of T/G-box within the HY5 promoter both in vitro and in vivo, as well as controlling HY5 and HY5-regulated gene expression in Arabidopsis seedlings. Taken together, this study provides a mechanistic framework in which BBX21 directly binds to the T/G-box present in the HY5 promoter possibly through its second B-box domain, which in turn controls HY5 and HY5-regulated gene expression to promote photomorphogenesis. © 2018 American Society of Plant Biologists. All Rights Reserved.

Apple F-Box Protein MdMAX2 Regulates Plant Photomorphogenesis and Stress Response.

PubMed

An, Jian-Ping; Li, Rui; Qu, Feng-Jia; You, Chun-Xiang; Wang, Xiao-Fei; Hao, Yu-Jin

2016-01-01

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.

Recent Developments in the Scientific Study of UFO's

ERIC Educational Resources Information Center

Salisbury, Frank B.

1975-01-01

Reviews the interest of the last few years, both inside and outside the scientific community, in unidentified flying objects (UFO), placing special emphasis on the extraterrestrial intelligence hypothesis. Cites numerous examples of UFO sightings and urges that the investigation of UFO's proceed in a scientific manner, despite skeptical public…

Patterning of inflorescences and flowers by the F-Box protein DOUBLE TOP and the LEAFY homolog ABERRANT LEAF AND FLOWER of petunia.

PubMed

Souer, Erik; Rebocho, Alexandra B; Bliek, Mattijs; Kusters, Elske; de Bruin, Robert A M; Koes, Ronald

2008-08-01

Angiosperms display a wide variety of inflorescence architectures differing in the positions where flowers or branches arise. The expression of floral meristem identity (FMI) genes determines when and where flowers are formed. In Arabidopsis thaliana, this is regulated via transcription of LEAFY (LFY), which encodes a transcription factor that promotes FMI. We found that this is regulated in petunia (Petunia hybrida) via transcription of a distinct gene, DOUBLE TOP (DOT), a homolog of UNUSUAL FLORAL ORGANS (UFO) from Arabidopsis. Mutation of DOT or its tomato (Solanum lycopersicum) homolog ANANTHA abolishes FMI. Ubiquitous expression of DOT or UFO in petunia causes very early flowering and transforms the inflorescence into a solitary flower and leaves into petals. Ectopic expression of DOT or UFO together with LFY or its homolog ABERRANT LEAF AND FLOWER (ALF) in petunia seedlings activates genes required for identity or outgrowth of organ primordia. DOT interacts physically with ALF, suggesting that it activates ALF by a posttranslational mechanism. Our findings suggest a wider role than previously thought for DOT and UFO in the patterning of flowers and indicate that the different roles of LFY and UFO homologs in the spatiotemporal control of floral identity in distinct species result from their divergent expression patterns.

Hydrothermal synthesis of (C6N2H14)2(UVI2UIVO4F12), a mixed-valent one-dimensional uranium oxyfluoride.

PubMed

Allen, S; Barlow, S; Halasyamani, P S; Mosselmans, J F; O'Hare, D; Walker, S M; Walton, R I

2000-08-21

A new hybrid organic-inorganic mixed-valent uranium oxyfluoride, (C6N2H14)2(U3O4F12), UFO-17, has been synthesized under hydrothermal conditions using uranium dioxide as the uranium source, hydrofluoric acid as mineralizer, and 1,4-diazabicyclo[2.2.2]octane as template. The single-crystal X-ray structure was determined. Crystals of UFO-17 belonged to the orthorhombic space group Cmcm (no. 63), with a = 14.2660(15) A, b = 24.5130(10) A, c = 7.201(2) A, and Z = 4. The structure reveals parallel uranium-containing chains of two types: one type is composed of edge-sharing UO2F5 units; the other has a backbone of edge-sharing UF8 units, each sharing an edge with a pendant UO2F5 unit. Bond-valence calculations suggest the UF8 groups contain UIV, while the UO2F5 groups contain UVI. EXAFS data give results consistent with the single-crystal X-ray structure determination, while comparison of the uranium LIII-edge XANES of UFO-17 with that of related UIV and UVI compounds supports the oxidation-state assignment. Variable-temperature magnetic susceptibility measurements on UFO-17 and a range of related hybrid organic-inorganic uranium(IV) and uranium(VI) fluorides and oxyfluorides further support the formulation of UFO-17 as a mixed-valent UIV/UVI compound.

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

PubMed

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.

Virulence Factor NSs of Rift Valley Fever Virus Recruits the F-Box Protein FBXO3 To Degrade Subunit p62 of General Transcription Factor TFIIH

PubMed Central

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

2014-01-01

ABSTRACT 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. IMPORTANCE 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. PMID:24403578

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

PubMed Central

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

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.

PubMed

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.

Regulation of Aspergillus nidulans CreA-Mediated Catabolite Repression by the F-Box Proteins Fbx23 and Fbx47.

PubMed

de Assis, Leandro José; Ulas, Mevlut; Ries, Laure Nicolas Annick; El Ramli, Nadia Ali Mohamed; Sarikaya-Bayram, Ozlem; Braus, Gerhard H; Bayram, Ozgur; Goldman, Gustavo Henrique

2018-06-19

The attachment of one or more ubiquitin molecules by SCF ( S kp- C ullin- F -box) complexes to protein substrates targets them for subsequent degradation by the 26S proteasome, allowing the control of numerous cellular processes. Glucose-mediated signaling and subsequent carbon catabolite repression (CCR) are processes relying on the functional regulation of target proteins, ultimately controlling the utilization of this carbon source. In the filamentous fungus Aspergillus nidulans , CCR is mediated by the transcription factor CreA, which modulates the expression of genes encoding biotechnologically relevant enzymes. Although CreA-mediated repression of target genes has been extensively studied, less is known about the regulatory pathways governing CCR and this work aimed at further unravelling these events. The Fbx23 F-box protein was identified as being involved in CCR and the Δ fbx23 mutant presented impaired xylanase production under repressing (glucose) and derepressing (xylan) conditions. Mass spectrometry showed that Fbx23 is part of an SCF ubiquitin ligase complex that is bridged via the GskA protein kinase to the CreA-SsnF-RcoA repressor complex, resulting in the degradation of the latter under derepressing conditions. Upon the addition of glucose, CreA dissociates from the ubiquitin ligase complex and is transported into the nucleus. Furthermore, casein kinase is important for CreA function during glucose signaling, although the exact role of phosphorylation in CCR remains to be determined. In summary, this study unraveled novel mechanistic details underlying CreA-mediated CCR and provided a solid basis for studying additional factors involved in carbon source utilization which could prove useful for biotechnological applications. IMPORTANCE The production of biofuels from plant biomass has gained interest in recent years as an environmentally friendly alternative to production from petroleum-based energy sources. Filamentous fungi, which naturally

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

PubMed

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.

High mobility group box (HMGB) proteins of Plasmodium falciparum: DNA binding proteins with pro-inflammatory activity.

PubMed

Kumar, Krishan; Singal, Ankita; Rizvi, M Moshahid A; Chauhan, Virander S

2008-06-01

High mobility group box chromosomal protein 1 (HMGB1), known as an abundant, non-histone architectural chromosomal protein, is highly conserved across different species. Homologues of HMGB1 were identified and cloned from malaria parasite, Plasmodium falciparum. Sequence analyses showed that the P. falciparum HMGB1 (PfHMGB1) exhibits 45, 23 and 18%, while PfHMGB2 shares 42, 21 and 17% homology with Saccharomyces cerevisiae, human and mouse HMG box proteins respectively. Parasite PfHMGB1and PfHMGB2 proteins contain one HMG Box domain similar to B-Box of mammalian HMGB1. Electrophoretic Mobility Shift Assay (EMSA) showed that recombinant PfHMGB1 and PfHMGB2 bind to DNA. Immunofluorescence Assay using specific antibodies revealed that these proteins are expressed abundantly in the ring stage nuclei. Significant levels of PfHMGB1 and PfHMGB2 were also present in the parasite cytosol at trophozoite and schizont stages. Both, PfHMGB1 and PfHMGB2 were found to be potent inducers of pro-inflammatory cytokines such as TNFalpha from mouse peritoneal macrophages as analyzed by both reverse transcription PCR and by ELISA. These results suggest that secreted PfHMGB1 and PfHMGB2 may be responsible for eliciting/ triggering host inflammatory immune responses associated with malaria infection.

UFO (UnFold Operator) computer program abstract

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

Kissel, L.; Biggs, F.

UFO (UnFold Operator) is an interactive user-oriented computer program designed to solve a wide range of problems commonly encountered in physical measurements. This document provides a summary of the capabilities of version 3A of UFO.

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

PubMed

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.

Vaccinia Virus C9 Ankyrin Repeat/F-Box Protein Is a Newly Identified Antagonist of the Type I Interferon-Induced Antiviral State.

PubMed

Liu, Ruikang; Moss, Bernard

2018-05-01

Type I interferons (IFNs) induce expression of more than 300 cellular genes that provide protection against viruses and other pathogens. For survival, viruses evolved defenses to prevent the IFN response or counteract the IFN-induced antiviral state. However, because viruses and cells coevolved, the dynamic relationship between virus and host is difficult to discern. In the present study, we demonstrated that vaccinia virus with a large deletion near the left end of the genome had a diminished ability to replicate in cells that had been pretreated with beta interferon (IFN-β), suggesting that one or more of the missing 17 open reading frames (ORFs) encode an antagonist of the IFN-induced antiviral state. By systematically deleting groups of ORFs and then individual ORFs, the C9L gene was shown to be required for IFN resistance. Replication of the C9L deletion mutant (vΔC9) was impaired in human cells that had been pretreated with IFN-β. Expression of viral early genes occurred, but subsequent events, including genome uncoating, genome replication, and postreplicative gene expression, were inhibited. Expression of the C9 protein occurred prior to genome replication, consistent with an early role in counteracting the IFN-induced antiviral state. C9 contains six ankyrin repeat motifs and a near C-terminal F-box. Mass spectrometry and immunoblotting identified host proteins that copurified with a functional epitope-tagged C9. The most abundant proteins were components of the SCF (CUL1, SKP1, F-box) and signalosome/deneddylation complexes, which interact with each other, suggesting a possible role in proteolysis of one or more interferon-induced proteins. IMPORTANCE Poxviruses comprise a family of large DNA viruses that replicate in the cytoplasm of vertebrate and insect hosts and cause human and zoonotic diseases. In most cases the primary infection is moderated by innate immune defenses. Vertebrates, including fish, amphibians, reptiles, birds, and mammals, all

The Dengue Vector Aedes aegypti Contains a Functional High Mobility Group Box 1 (HMGB1) Protein with a Unique Regulatory C-Terminus

PubMed Central

Ribeiro, Fabio Schneider; de Abreu da Silva, Isabel Caetano; Carneiro, Vitor Coutinho; Belgrano, Fabrício dos Santos; Mohana-Borges, Ronaldo; de Andrade Rosa, Ivone; Benchimol, Marlene; Souza, Nathalia Rocha Quintino; Mesquita, Rafael Dias; Sorgine, Marcos Henrique Ferreira; Gazos-Lopes, Felipe; Vicentino, Amanda Roberta Revoredo; Wu, Wenjie; de Moraes Maciel, Renata; da Silva-Neto, Mario Alberto Cardoso; Fantappié, Marcelo Rosado

2012-01-01

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. PMID:22802955

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

PubMed Central

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

2016-01-01

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 SCFFBXW11 E3 ligase. We further show that disrupting the assembly of the SCFFBXW11-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 SCFFBXW11-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 SCFFBXW11 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. PMID

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.

PubMed

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.

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

DOE PAGES

Mudhasani, Rajini; Tran, Julie P.; Retterer, Cary; ...

2016-02-02

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 anmore » alternate binding site to the SCFFBXW11 E3 ligase. We further show that disrupting the assembly of the SCFFBXW11-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 SCFFBXW11-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 SCFFBXW11 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.« less

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

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

Mudhasani, Rajini; Tran, Julie P.; Retterer, Cary

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 anmore » alternate binding site to the SCFFBXW11 E3 ligase. We further show that disrupting the assembly of the SCFFBXW11-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 SCFFBXW11-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 SCFFBXW11 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.« less

UFO (UnFold Operator) default data format

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

Kissel, L.; Biggs, F.; Marking, T.R.

The default format for the storage of x,y data for use with the UFO code is described. The format assumes that the data stored in a file is a matrix of values; two columns of this matrix are selected to define a function of the form y = f(x). This format is specifically designed to allow for easy importation of data obtained from other sources, or easy entry of data using a text editor, with a minimum of reformatting. This format is flexible and extensible through the use of inline directives stored in the optional header of the file. Amore » special extension of the format implements encoded data which significantly reduces the storage required as compared wth the unencoded form. UFO supports several extensions to the file specification that implement execute-time operations, such as, transformation of the x and/or y values, selection of specific columns of the matrix for association with the x and y values, input of data directly from other formats (e.g., DAMP and PFF), and a simple type of library-structured file format. Several examples of the use of the format are given.« less

Untrained Forward Observer (UFO) Translator for Call for Fire

DTIC Science & Technology

2013-09-01

release; distribution is unlimited UNTRAINED FORWARD OBSERVER ( UFO ) TRANSLATOR FOR CALL FOR FIRE by Regan R. King September 2013 Thesis...DATE September 2013 3. REPORT TYPE AND DATES COVERED Master’s Thesis 4. TITLE AND SUBTITLE UNTRAINED FORWARD OBSERVER ( UFO ) TRANSLATOR FOR CALL...application, which we call the Untrained Forward Observer ( UFO ) Translator, capable of assisting untrained observers in performing Call for Fire by asking a

Role of SKP1-CUL1-F-Box-Protein (SCF) E3 Ubiquitin Ligases in Skin Cancer

PubMed Central

Xie, Chuan-Ming; Wei, Wenyi; Sun, Yi

2013-01-01

Many biological processes such as cell proliferation, differentiation, and cell death depend precisely on the timely synthesis and degradation of key regulatory proteins. While protein synthesis can be regulated at multiple levels, protein degradation is mainly controlled by the ubiquitin—proteasome system (UPS), which consists of two distinct steps: (1) ubiquitylation of targeted protein by E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme and E3 ubiquitin ligase, and (2) subsequent degradation by the 26S proteasome. Among all E3 ubiquitin ligases, the SCF (SKP1-CUL1-F-box protein) E3 ligases are the largest family and are responsible for the turnover of many key regulatory proteins. Aberrant regulation of SCF E3 ligases is associated with various human diseases, such as cancers, including skin cancer. In this review, we provide a comprehensive overview of all currently published data to define a promoting role of SCF E3 ligases in the development of skin cancer. The future directions in this area of research are also discussed with an ultimate goal to develop small molecule inhibitors of SCF E3 ligases as a novel approach for the treatment of human skin cancer. Furthermore, altered components or substrates of SCF E3 ligases may also be developed as the biomarkers for early diagnosis or predicting prognosis. PMID:23522382

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

PubMed

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.

The role of two F-box proteins, SLEEPY1 and SNEEZY, in arabidopsis GA signaling

USDA-ARS?s Scientific Manuscript database

The F-box gene SLY1 is a positive regulator of gibberellin (GA) signaling and loss of SLY1 results in GA-insensitive phenotypes including dwarfism, reduced fertility, delayed flowering, and increased seed dormancy. These sly1 phenotypes can be partially rescued by overexpression of the SLY1 homolog...

The rice F-box protein KISS ME DEADLY2 functions as a negative regulator of cytokinin signalling.

PubMed

Kim, Hyo Jung; Kieber, Joseph J; Schaller, G Eric

2013-01-01

Cytokinins are plant hormones that play critical roles in growth and development. We recently determined that the transcriptional response to cytokinin of Arabidopsis is modulated by the KISS ME DEADLY (KMD) family of F-box proteins. Here we demonstrate a conserved function for a member of the rice KMD family. Ectopic overexpression of OsKMD2 in Arabidopsis results in decreased cytokinin sensitivity based on a hypocotyl growth response assay, the decrease in sensitivity correlating with a decrease in the levels of the transcriptional regulator AtARR12. Furthermore, OsKMD2 directly interacts with AtARR12 based on yeast two-hybrid and co-immunoprecipitation assays. These results indicate that both monocots and dicots employ a similar KMD-dependent mechanism to regulate the transcriptional response to cytokinin.

UFO: an Arabidopsis gene involved in both floral meristem and floral organ development.

PubMed

Levin, J Z; Meyerowitz, E M

1995-05-01

We describe the role of the UNUSUAL FLORAL ORGANS (UFO) gene in Arabidopsis floral development based on a genetic and molecular characterization of the phenotypes of nine ufo alleles. UFO is required for the proper identity of the floral meristem and acts in three different aspects of the process that distinguishes flowers from shoots. UFO is involved in establishing the whorled pattern of floral organs, controlling the determinacy of the floral meristem, and activating the APETALA3 and PISTILLATA genes required for petal and stamen identity. In many respects, UFO acts in a manner similar to LEAFY, but the ufo mutant phenotype also suggests an additional role for UFO in defining boundaries within the floral primordia or controlling cell proliferation during floral organ growth. Finally, genetic interactions that prevent flower formation and lead to the generation of filamentous structures implicate UFO as a member of a new, large, and diverse class of genes in Arabidopsis necessary for flower formation.

F-box protein FBXW7 inhibits cancer metastasis in a non-cell-autonomous manner

PubMed Central

Yumimoto, Kanae; Akiyoshi, Sayuri; Ueo, Hiroki; Sagara, Yasuaki; Onoyama, Ichiro; Ueo, Hiroaki; Ohno, Shinji; Mori, Masaki; Mimori, Koshi; Nakayama, Keiichi I.

2015-01-01

The gene encoding F-box protein FBXW7 is frequently mutated in many human cancers. Although most previous studies have focused on the tumor-suppressive capacity of FBXW7 in tumor cells themselves, we determined that FBXW7 in the host microenvironment also suppresses cancer metastasis. Deletion of Fbxw7 in murine BM-derived stromal cells induced accumulation of NOTCH and consequent transcriptional activation of Ccl2. FBXW7-deficient mice exhibited increased serum levels of the chemokine CCL2, which resulted in the recruitment of both monocytic myeloid-derived suppressor cells and macrophages, thereby promoting metastatic tumor growth. Administration of a CCL2 receptor antagonist blocked the enhancement of metastasis in FBXW7-deficient mice. Furthermore, in human breast cancer patients, FBXW7 expression in peripheral blood was associated with serum CCL2 concentration and disease prognosis. Together, these results suggest that FBXW7 antagonizes cancer development in not only a cell-autonomous manner, but also a non-cell-autonomous manner, and that modulation of the FBXW7/NOTCH/CCL2 axis may provide a potential approach to suppression of cancer metastasis. PMID:25555218

A DNA-binding protein from Candida albicans that binds to the RPG box of Saccharomyces cerevisiae and the telomeric repeat sequence of C. albicans.

PubMed

Ishii, N; Yamamoto, M; Lahm, H W; Iizumi, S; Yoshihara, F; Nakayama, H; Arisawa, M; Aoki, Y

1997-02-01

Electromobility shift assays with a DNA probe containing the Saccharomyces cerevisiae ENO1 RPG box identified a specific DNA-binding protein in total protein extracts of Candida albicans. The protein, named Rbf1p (RPG-box-binding protein 1), bound to other S. cerevisiae RPG boxes, although the nucleotide recognition profile was not completely the same as that of S. cerevisiae Rap 1p (repressor-activator protein 1), an RPG-box-binding protein. The repetitive sequence of the C. albicans chromosomal telomere also competed with RPG-box binding to Rbf1p. For further analysis, we purified Rbf1p 57,600-fold from C. albicans total protein extracts, raised mAbs against the purified protein and immunologically cloned the gene, whose ORF specified a protein of 527 aa. The bacterially expressed protein showed RPG-box-binding activity with the same profile as that of the purified one. The Rbf1p, containing two glutamine-rich regions that are found in many transcription factors, showed transcriptional activation capability in S. cerevisiae and was predominantly observed in nuclei. These results suggest that Rbf1p is a transcription factor with telomere-binding activity in C. albicans.

BAL QSOs AND EXTREME UFOs: THE EDDINGTON CONNECTION

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

Zubovas, Kastytis; King, Andrew, E-mail: kastytis.zubovas@ftmc.lt

We suggest a common physical origin connecting the fast, highly ionized winds (UFOs) seen in nearby active galactic nuclei (AGNs), and the slower and less ionized winds of broad absorption line (BAL) QSOs. The primary difference is the mass-loss rate in the wind, which is ultimately determined by the rate at which mass is fed toward the central supermassive black hole (SMBH) on large scales. This is below the Eddington accretion rate in most UFOs, and slightly super-Eddington in extreme UFOs such as PG1211+143, but ranges up to {approx}10-50 times this in BAL QSOs. For UFOs this implies black holemore » accretion rates and wind mass-loss rates which are at most comparable to Eddington, giving fast, highly ionized winds. In contrast, BAL QSO black holes have mildly super-Eddington accretion rates, and drive winds whose mass-loss rates are significantly super-Eddington, and so are slower and less ionized. This picture correctly predicts the velocities and ionization states of the observed winds, including the recently discovered one in SDSS J1106+1939. We suggest that luminous AGNs may evolve through a sequence from BAL QSO through LoBAL to UFO-producing Seyfert or quasar as their Eddington factors drop during the decay of a bright accretion event. LoBALs correspond to a short-lived stage in which the AGN radiation pressure largely evacuates the ionization cone, but before the large-scale accretion rate has dropped to the Eddington value. We show that sub-Eddington wind rates would produce an M-{sigma} relation lying above that observed. We conclude that significant SMBH mass growth must occur in super-Eddington phases, either as BAL QSOs, extreme UFOs, or obscured from direct observation.« less

BAL QSOs and Extreme UFOs: The Eddington Connection

NASA Astrophysics Data System (ADS)

Zubovas, Kastytis; King, Andrew

2013-05-01

We suggest a common physical origin connecting the fast, highly ionized winds (UFOs) seen in nearby active galactic nuclei (AGNs), and the slower and less ionized winds of broad absorption line (BAL) QSOs. The primary difference is the mass-loss rate in the wind, which is ultimately determined by the rate at which mass is fed toward the central supermassive black hole (SMBH) on large scales. This is below the Eddington accretion rate in most UFOs, and slightly super-Eddington in extreme UFOs such as PG1211+143, but ranges up to ~10-50 times this in BAL QSOs. For UFOs this implies black hole accretion rates and wind mass-loss rates which are at most comparable to Eddington, giving fast, highly ionized winds. In contrast, BAL QSO black holes have mildly super-Eddington accretion rates, and drive winds whose mass-loss rates are significantly super-Eddington, and so are slower and less ionized. This picture correctly predicts the velocities and ionization states of the observed winds, including the recently discovered one in SDSS J1106+1939. We suggest that luminous AGNs may evolve through a sequence from BAL QSO through LoBAL to UFO-producing Seyfert or quasar as their Eddington factors drop during the decay of a bright accretion event. LoBALs correspond to a short-lived stage in which the AGN radiation pressure largely evacuates the ionization cone, but before the large-scale accretion rate has dropped to the Eddington value. We show that sub-Eddington wind rates would produce an M-σ relation lying above that observed. We conclude that significant SMBH mass growth must occur in super-Eddington phases, either as BAL QSOs, extreme UFOs, or obscured from direct observation.

Separable roles of UFO during floral development revealed by conditional restoration of gene function.

PubMed

Laufs, Patrick; Coen, Enrico; Kronenberger, Jocelyne; Traas, Jan; Doonan, John

2003-02-01

The UNUSUAL FLORAL ORGANS (UFO) gene is required for several aspects of floral development in Arabidopsis including specification of organ identity in the second and third whorls and the proper pattern of primordium initiation in the inner three whorls. UFO is expressed in a dynamic pattern during the early phases of flower development. Here we dissect the role of UFO by ubiquitously expressing it in ufo loss-of-function flowers at different developmental stages and for various durations using an ethanol-inducible expression system. The previously known functions of UFO could be separated and related to its expression at specific stages of development. We show that a 24- to 48-hour period of UFO expression from floral stage 2, before any floral organs are visible, is sufficient to restore normal petal and stamen development. The earliest requirement for UFO is during stage 2, when the endogenous UFO gene is transiently expressed in the centre of the wild-type flower and is required to specify the initiation patterns of petal, stamen and carpel primordia. Petal and stamen identity is determined during stages 2 or 3, when UFO is normally expressed in the presumptive second and third whorl. Although endogenous UFO expression is absent from the stamen whorl from stage 4 onwards, stamen identity can be restored by UFO activation up to stage 6. We also observed floral phenotypes not observed in loss-of-function or constitutive gain-of-function backgrounds, revealing additional roles of UFO in outgrowth of petal primordia.

Flight and abduction in witchcraft and UFO lore.

PubMed

Musgrave, J B; Houran, J

2000-04-01

The lore surrounding the mythical Witches' Sabbat and contemporary reports of UFO abductions share three main characteristics: the use of masks, the appearance of "Men in Black," and references to flight and abduction. We review these three commonalities with particular focus on the aspect of flight and abduction. We argue that narratives of the Witches' Sabbat and UFO abductions share the same basic structure, common symbolism, and serve the same psychological needs of providing a coherent explanation for anomalous (ambiguous) experiences while simultaneously giving the experient a sense of freedom, release, and escape from the self. This pattern of similarities suggests the possibility that UFO abductions are a modern version of tales of flight to the Sabbat.

UFO - The Universal FEYNRULES Output

NASA Astrophysics Data System (ADS)

Degrande, Céline; Duhr, Claude; Fuks, Benjamin; Grellscheid, David; Mattelaer, Olivier; Reiter, Thomas

2012-06-01

We present a new model format for automatized matrix-element generators, the so-called Universal FEYNRULES Output (UFO). The format is universal in the sense that it features compatibility with more than one single generator and is designed to be flexible, modular and agnostic of any assumption such as the number of particles or the color and Lorentz structures appearing in the interaction vertices. Unlike other model formats where text files need to be parsed, the information on the model is encoded into a PYTHON module that can easily be linked to other computer codes. We then describe an interface for the MATHEMATICA package FEYNRULES that allows for an automatic output of models in the UFO format.

Crystal structure of a DEAD box protein from the hyperthermophile Methanococcus jannaschii

PubMed Central

Story, Randall M.; Li, Hong; Abelson, John N.

2001-01-01

We have determined the structure of a DEAD box putative RNA helicase from the hyperthermophile Methanococcus jannaschii. Like other helicases, the protein contains two α/β domains, each with a recA-like topology. Unlike other helicases, the protein exists as a dimer in the crystal. Through an interaction that resembles the dimer interface of insulin, the amino-terminal domain's 7-strand β-sheet is extended to 14 strands across the two molecules. Motifs conserved in the DEAD box family cluster in the cleft between domains, and many of their functions can be deduced by mutational data and by comparison with other helicase structures. Several lines of evidence suggest that motif III Ser-Ala-Thr may be involved in binding RNA. PMID:11171974

UFO (UnFold Operator) user guide

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

Kissel, L.; Biggs, F.; Marking, T.R.

UFO is a collection of interactive utility programs for estimating unknown functions of one variable using a wide-ranging class of information as input, for miscellaneous data-analysis applications, for performing feasibility studies, and for supplementing our other software. Inverse problems, which include spectral unfolds, inverse heat-transfer problems, time-domain deconvolution, and unusual or difficult curve-fit problems, are classes of applications for which UFO is well suited. Extensive use of B-splines and (X,Y)-datasets is made to represent functions. The (X,Y)-dataset representation is unique in that it is not restricted to equally-spaced data. This feature is used, for example, in a table-generating algorithm thatmore » evaluates a function to a user-specified interpolation accuracy while minimizing the number of points stored in the corresponding dataset. UFO offers a variety of miscellaneous data-analysis options such as plotting, comparing, transforming, scaling, integrating; and adding, subtracting, multiplying, and dividing functions together. These options are often needed as intermediate steps in analyzing and solving difficult inverse problems, but they also find frequent use in other applications. Statistical options are available to calculate goodness-of-fit to measurements, specify error bands on solutions, give confidence limits on calculated quantities, and to point out the statistical consequences of operations such as smoothing. UFO is designed to do feasibility studies on a variety of engineering measurements. It is also tailored to supplement our Test Analysis and Design codes, SRAD Test-Data Archive software, and Digital Signal Analysis routines.« less

Space Station Freedom solar array containment box mechanisms

NASA Technical Reports Server (NTRS)

Johnson, Mark E.; Haugen, Bert; Anderson, Grant

1994-01-01

Space Station Freedom will feature six large solar arrays, called solar array wings, built by Lockheed Missiles & Space Company under contract to Rockwell International, Rocketdyne Division. Solar cells are mounted on flexible substrate panels which are hinged together to form a 'blanket.' Each wing is comprised of two blankets supported by a central mast, producing approximately 32 kW of power at beginning-of-life. During launch, the blankets are fan-folded and compressed to 1.5 percent of their deployed length into containment boxes. This paper describes the main containment box mechanisms designed to protect, deploy, and retract the solar array blankets: the latch, blanket restraint, tension, and guidewire mechanisms.

Evaluation of Containment Boxes as a Fire Mitigation Method in Elevated Oxygen Conditions

NASA Technical Reports Server (NTRS)

Juarez, Alfredo; Harper, Susana; Perez, Horacio

2016-01-01

NASA performed testing to evaluate the efficacy of fire containment boxes without forced ventilation. Configurational flammability testing was performed on a simulation avionics box replicating critical design features and filled with materials possessing representative flammability characteristics. This paper discusses the box's ability, under simulated end-use conditions, to inhibit the propagation of combustion to surrounding materials. Analysis was also performed to evaluate the potential for the fire containment box to serve as an overheat/ignition source to temperature sensitive equipment (such as items with lithium-ion batteries). Unrealistically severe combustion scenarios were used as a means to better understand the fire containment mechanism. These scenarios were achieved by utilizing materials/fuels not typically used in space vehicles due to flammability concerns. Oxygen depletion, during combustion within the fire containment boxes, drove self-extinguishment and proved an effective method of fire containment

The regulation of the Z- and G-box containing promoters by light signaling components, SPA1 and MYC2, in Arabidopsis.

PubMed

Gangappa, Sreeramaiah N; Maurya, Jay P; Yadav, Vandana; Chattopadhyay, Sudip

2013-01-01

Although many transcription factors and regulatory proteins have been identified and functionally characterized in light signaling pathways, photoperception to transcription remains largely fragmented. The Z-box is one of the LREs (Light responsive elements) that plays important role in the regulation of transcription during light-controlled Arabidopsis seedling development. The involvement of photoreceptors in the modulation of the activity of the Z-box containing promoters has been demonstrated. However, the role of downstream signaling components such as SPA1 and MYC2/ZBF1, which are functionally interrelated, remains unknown. In this study, we have investigated the regulation of the Z-box containing synthetic and native promoters by SPA1 and MYC2 by using stable transgenic lines. Our studies suggest that SPA1 negatively regulates the expression of CAB1 native promoter. MYC2 negatively regulates the activity of Z- and/or G-box containing synthetic as well as native promoters irrespective of light quality. Moreover, MYC2 negatively regulates the expression of Z/G-NOS101-GUS even in the darkness. Furthermore, analyses of tissue specific expression in adult plants suggest that MYC2 strongly regulates the activity of Z- and G-box containing promoters specifically in leaves and stems. In roots, whereas MYC2 positively regulates the activity of the Z-box containing synthetic promoter, it does not seem to control the activity of the G-box containing promoters. Taken together, these results provide insights into SPA1- and MYC2-mediated transcriptional regulation of the Z- and G-box containing promoters in light signaling pathways.

F-box protein MAX2 has dual roles in karrikin and strigolactone signaling in Arabidopsis thaliana

PubMed Central

Nelson, David C.; Scaffidi, Adrian; Dun, Elizabeth A.; Waters, Mark T.; Flematti, Gavin R.; Dixon, Kingsley W.; Beveridge, Christine A.; Ghisalberti, Emilio L.; Smith, Steven M.

2011-01-01

Smoke is an important abiotic cue for plant regeneration in postfire landscapes. Karrikins are a class of compounds discovered in smoke that promote seed germination and influence early development of many plants by an unknown mechanism. A genetic screen for karrikin-insensitive mutants in Arabidopsis thaliana revealed that karrikin signaling requires the F-box protein MAX2, which also mediates responses to the structurally-related strigolactone family of phytohormones. Karrikins and the synthetic strigolactone GR24 trigger similar effects on seed germination, seedling photomorphogenesis, and expression of a small set of genes during these developmental stages. Karrikins also repress MAX4 and IAA1 transcripts, which show negative feedback regulation by strigolactone. We demonstrate that all of these common responses are abolished in max2 mutants. Unlike strigolactones, however, karrikins do not inhibit shoot branching in Arabidopsis or pea, indicating that plants can distinguish between these signals. These results suggest that a MAX2-dependent signal transduction mechanism was adapted to mediate responses to two chemical cues with distinct roles in plant ecology and development. PMID:21555559

F-box protein MAX2 has dual roles in karrikin and strigolactone signaling in Arabidopsis thaliana.

PubMed

Nelson, David C; Scaffidi, Adrian; Dun, Elizabeth A; Waters, Mark T; Flematti, Gavin R; Dixon, Kingsley W; Beveridge, Christine A; Ghisalberti, Emilio L; Smith, Steven M

2011-05-24

Smoke is an important abiotic cue for plant regeneration in postfire landscapes. Karrikins are a class of compounds discovered in smoke that promote seed germination and influence early development of many plants by an unknown mechanism. A genetic screen for karrikin-insensitive mutants in Arabidopsis thaliana revealed that karrikin signaling requires the F-box protein MAX2, which also mediates responses to the structurally-related strigolactone family of phytohormones. Karrikins and the synthetic strigolactone GR24 trigger similar effects on seed germination, seedling photomorphogenesis, and expression of a small set of genes during these developmental stages. Karrikins also repress MAX4 and IAA1 transcripts, which show negative feedback regulation by strigolactone. We demonstrate that all of these common responses are abolished in max2 mutants. Unlike strigolactones, however, karrikins do not inhibit shoot branching in Arabidopsis or pea, indicating that plants can distinguish between these signals. These results suggest that a MAX2-dependent signal transduction mechanism was adapted to mediate responses to two chemical cues with distinct roles in plant ecology and development.

Regulation of flower development in Arabidopsis by SCF complexes.

PubMed

Ni, Weimin; Xie, Daoxin; Hobbie, Lawrence; Feng, Baomin; Zhao, Dazhong; Akkara, Joseph; Ma, Hong

2004-04-01

SCF complexes are the largest and best studied family of E3 ubiquitin protein ligases that facilitate the ubiquitylation of proteins targeted for degradation. The SCF core components Skp1, Cul1, and Rbx1 serve in multiple SCF complexes involving different substrate-specific F-box proteins that are involved in diverse processes including cell cycle and development. In Arabidopsis, mutations in the F-box gene UNUSUAL FLORAL ORGANS (UFO) result in a number of defects in flower development. However, functions of the core components Cul1 and Rbx1 in flower development are poorly understood. In this study we analyzed floral phenotypes caused by altering function of Cul1 or Rbx1, as well as the effects of mutations in ASK1 and ASK2. Plants homozygous for a point mutation in the AtCUL1 gene showed reduced floral organ number and several defects in each of the four whorls. Similarly, plants with reduced AtRbx1 expression due to RNA interference also exhibited floral morphological defects. In addition, compared to the ask1 mutant, plants homozygous for ask1 and heterozygous for ask2 displayed enhanced reduction of B function, as well as other novel defects of flower development, including carpelloid sepals and an inhibition of petal development. Genetic analyses demonstrate that AGAMOUS (AG) is required for the novel phenotypes observed in the first and second whorls. Furthermore, the genetic interaction between UFO and AtCUL1 supports the idea that UFO regulates multiple aspects of flower development as a part of SCF complexes. These results suggest that SCF complexes regulate several aspects of floral development in Arabidopsis.

A receptor tyrosine kinase, UFO/Axl, and other genes isolated by a modified differential display PCR are overexpressed in metastatic prostatic carcinoma cell line DU145.

PubMed

Jacob, A N; Kalapurakal, J; Davidson, W R; Kandpal, G; Dunson, N; Prashar, Y; Kandpal, R P

1999-01-01

We have used a modified differential display PCR protocol for isolating 3' restriction fragments of cDNAs specifically expressed or overexpressed in metastatic prostate carcinoma cell line DU145. Several cDNA fragments were identified that matched to milk fat globule protein, UFO/Axl, a receptor tyrosine kinase, human homologue of a Xenopus maternal transcript, laminin and laminin receptor, human carcinoma-associated antigen, and some expressed sequence tags. The transcript for milk fat globule protein, a marker protein shown to be overexpressed in breast tumors, was elevated in DU145 cells. The expression of UFO/Axl, a receptor tyrosine kinase, was considerably higher in DU145 cells as compared to normal prostate cells and prostatic carcinoma cell line PC-3. The overexpression of UFO oncogene in DU145 cells is discussed in the context of prostate cancer metastasis.

Functional and Structural Insights of the Zinc-Finger HIT protein family members Involved in Box C/D snoRNP Biogenesis.

PubMed

Bragantini, Benoit; Tiotiu, Decebal; Rothé, Benjamin; Saliou, Jean-Michel; Marty, Hélène; Cianférani, Sarah; Charpentier, Bruno; Quinternet, Marc; Manival, Xavier

2016-06-05

Zf–HIT family members share the zf–HIT domain (ZHD), which is characterized by a fold in “treble-clef” through interleaved CCCC and CCHC ZnF motifs that both bind a zinc atom. Six proteins containing ZHD are present in human and three in yeast proteome, all belonging to multimodular RNA/protein complexes involved in gene regulation, chromatin remodeling, and snoRNP assembly. An interesting characteristic of the cellular complexes that ensure these functions is the presence of the RuvBL1/2/Rvb1/2 ATPases closely linked with zf–HIT proteins. Human ZNHIT6/BCD1 and its counterpart in yeast Bcd1p were previously characterized as assembly factors of the box C/D snoRNPs. Our data reveal that the ZHD of Bcd1p is necessary but not sufficient for yeast growth and that the motif has no direct RNA-binding capacity but helps Bcd1p maintain the box C/D snoRNAs level in steady state. However, we demonstrated that Bcd1p interacts nonspecifically with RNAs depending on their length. Interestingly, the ZHD of Bcd1p is functionally interchangeable with that of Hit1p, another box C/D snoRNP assembly factor belonging to the zf–HIT family. This prompted us to use NMR to solve the 3D structures of ZHD from yeast Bcd1p and Hit1p to highlight the structural similarity in the zf–HIT family. We identified structural features associated with the requirement of Hit1p and Bcd1p ZHD for cell growth and box C/D snoRNA stability under heat stress. Altogether, our data suggest an important role of ZHD could be to maintain functional folding to the rest of the protein, especially under heat stress conditions.

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

PubMed Central

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

F-box protein DOR functions as a novel inhibitory factor for abscisic acid-induced stomatal closure under drought stress in Arabidopsis,.

PubMed

Zhang, Yu'e; Xu, Wenying; Li, Zhonghui; Deng, Xing Wang; Wu, Weihua; Xue, Yongbiao

2008-12-01

Guard cells, which form stoma in leaf epidermis, sense and integrate environmental signals to modulate stomatal aperture in response to diverse conditions. Under drought stress, plants synthesize abscisic acid (ABA), which in turn induces a rapid closing of stoma, to prevent water loss by transpiration. However, many aspects of the molecular mechanism for ABA-mediated stomatal closure are still not understood. Here, we report a novel negative regulator of guard cell ABA signaling, DOR, in Arabidopsis (Arabidopsis thaliana). The DOR gene encodes a putative F-box protein, a member of the S-locus F-box-like family related to AhSLF-S(2) and specifically interacting with ASK14 and CUL1. A null mutation in DOR resulted in a hypersensitive ABA response of stomatal closing and a substantial increase of drought tolerance; in contrast, the transgenic plants overexpressing DOR were more susceptible to the drought stress. DOR is strongly expressed in guard cells and suppressed by ABA treatment, suggesting a negative feedback loop of DOR in ABA responses. Double-mutant analyses of dor with ABA-insensitive mutant abi1-1 showed that abi1-1 is epistatic to dor, but no apparent change of phospholipase Dalpha1 was detected between the wild type and dor. Affymetrix GeneChip analysis showed that DOR likely regulates ABA biosynthesis under drought stress. Taken together, our results demonstrate that DOR acts independent of phospholipase Dalpha1 in an ABA signaling pathway to inhibit the ABA-induced stomatal closure under drought stress.

Geophysical variables and behavior: XXIII. Relations between UFO reports within the Uinta Basin and local seismicity.

PubMed

Persinger, M A; Derr, J S

1985-02-01

A strong temporal correlation was found between the numbers of reports of UFOs (unidentified flying objects) and nearby seismic activity within the Uinta Basin for the year 1967. The numbers of UFO reports per month during this classic UFO flap were correlated 0.80 with the sum of the earthquake magnitudes per month for events within 150 km of the report area. Numbers of UFO reports were not correlated significantly with earthquake activity at distances greater than 150 km but less than 250 km away. The strongest correlation occurred between UFO reports and nearby seismic activity within the same month but not for previous or consequent months. Close scrutiny of daily shifts in epicenters and reports of UFOs indicate that they occurred when the locus of successive epicenters shifted across the area. These analyses were interpreted as support for the existence of strain fields whose movements generate natural phenomena that are reported as UFOs.

Molecular characterization of atrogin-1/F-box protein-32 (FBXO32) and F-box protein 25 (FBXO25) in rainbow trout (Oncorhynchus mykiss); expression across tissues in response to feed deprivation

USDA-ARS?s Scientific Manuscript database

The characteristic increase in protein catabolism during muscle atrophy is largely the result of an increase in E3 ubiquitin ligase expression, specifically that of atrogin-1, or FBXO32, which functions to polyubiquitinate proteins. In rainbow trout, the cDNA sequences of two E3 ubiquitin ligase F-...

Ultra-fast outflows (aka UFOs) from AGNs and QSOs

NASA Astrophysics Data System (ADS)

Cappi, M.; Tombesi, F.; Giustini, M.

During the last decade, strong observational evidence has been accumulated for the existence of massive, high velocity winds/outflows (aka Ultra Fast Outflows, UFOs) in nearby AGNs and in more distant quasars. Here we briefly review some of the most recent developments in this field and discuss the relevance of UFOs for both understanding the physics of accretion disk winds in AGNs, and for quantifying the global amount of AGN feedback on the surrounding medium.

Ctf4 Is a Hub in the Eukaryotic Replisome that Links Multiple CIP-Box Proteins to the CMG Helicase.

PubMed

Villa, Fabrizio; Simon, Aline C; Ortiz Bazan, Maria Angeles; Kilkenny, Mairi L; Wirthensohn, David; Wightman, Mel; Matak-Vinkovíc, Dijana; Pellegrini, Luca; Labib, Karim

2016-08-04

Replisome assembly at eukaryotic replication forks connects the DNA helicase to DNA polymerases and many other factors. The helicase binds the leading-strand polymerase directly, but is connected to the Pol α lagging-strand polymerase by the trimeric adaptor Ctf4. Here, we identify new Ctf4 partners in addition to Pol α and helicase, all of which contain a "Ctf4-interacting-peptide" or CIP-box. Crystallographic analysis classifies CIP-boxes into two related groups that target different sites on Ctf4. Mutations in the CIP-box motifs of the Dna2 nuclease or the rDNA-associated protein Tof2 do not perturb DNA synthesis genome-wide, but instead lead to a dramatic shortening of chromosome 12 that contains the large array of rDNA repeats. Our data reveal unexpected complexity of Ctf4 function, as a hub that connects multiple accessory factors to the replisome. Most strikingly, Ctf4-dependent recruitment of CIP-box proteins couples other processes to DNA synthesis, including rDNA copy-number regulation. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Measuring Helicase Inhibition of the DEAD-box Protein Dbp2 by Yra1

PubMed Central

Ma, Wai Kit; Tran, Elizabeth J.

2016-01-01

Despite the highly conserved helicase core, individual DEAD-box proteins are specialized in diverse RNA metabolic processes. One mechanism that determines DEAD-box protein specificity is enzymatic regulation by other protein cofactors. In this chapter, we describe a protocol for purifying the Saccharomyces cerevisiae DEAD-box RNA helicase Dbp2 and RNA-binding protein Yra1 and subsequent analysis of helicase regulation. The experiments described here can be adapted to RNA helicase and purified co-factor. PMID:25579587

Functional roles and efficiencies of the thioredoxin boxes of calcium-binding proteins 1 and 2 in protein folding.

PubMed Central

Kramer, B; Ferrari, D M; Klappa, P; Pöhlmann, N; Söling, H D

2001-01-01

The rat luminal endoplasmic-recticulum calcium-binding proteins 1 and 2 (CaBP1 and CaBP2 respectively) are members of the protein disulphide-isomerase (PDI) family. They contain two and three thioredoxin boxes (Cys-Gly-His-Cys) respectively and, like PDI, may be involved in the folding of nascent proteins. We demonstrate here that CaBP1, similar to PDI and CaBP2, can complement the lethal phenotype of the disrupted Saccharomyces cerevisiae PDI gene, provided that the natural C-terminal Lys-Asp-Glu-Leu sequence is replaced by His-Asp-Glu-Leu. Both the in vitro RNase AIII-re-activation assays and in vivo pro-(carboxypeptidase Y) processing assays using CaBP1 and CaBP2 thioredoxin (trx)-box mutants revealed that, whereas the three trx boxes in CaBP2 seem to be functionally equivalent, the first trx box of CaBP1 is significantly more active than the second trx box. Furthermore, only about 65% re-activation of denatured reduced RNase AIII could be obtained with CaBP1 or CaBP2 compared with PDI, and the yield of PDI-catalysed reactions was significantly reduced in the presence of either CaBP1 or CaBP2. In contrast with PDI, neither CaBP1 nor CaBP2 could catalyse the renaturation of denatured glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is a redox-independent process, and neither protein had any effect on the PDI-catalysed refolding of GAPDH. Furthermore, although PDI can bind peptides via its b' domain, a property it shares with PDIp, the pancreas-specific PDI homologue, and although PDI can bind malfolded proteins such as 'scrambled' ribonuclease, no such interactions could be detected for CaBP2. We conclude that: (1) both CaBP2 and CaBP1 lack peptide-binding activity for GAPDH attributed to the C-terminal region of the a' domain of PDI; (2) CaBP2 lacks the general peptide-binding activity attributed to the b' domain of PDI; (3) interaction of CaBP2 with substrate (RNase AIII) is different from that of PDI and substrate; and (4) both CaBP2 and CaBP1 may

Molecular and functional characterization of single-box high-mobility group B (HMGB) chromosomal protein from Aedes aegypti.

PubMed

de Abreu da Silva, Isabel Caetano; Vicentino, Amanda Roberta Revoredo; Dos Santos, Renata Coutinho; da Fonseca, Rodrigo Nunes; de Mendonça Amarante, Anderson; Carneiro, Vitor Coutinho; de Amorim Pinto, Marcia; Aguilera, Estefania Anahi; Mohana-Borges, Ronaldo; Bisch, Paulo Mascarello; da Silva-Neto, Mario Alberto Cardoso; Fantappié, Marcelo Rosado

2018-05-30

High-mobility group B (HMGB) proteins have highly conserved, unique DNA-binding domains, HMG boxes, that can bind non-B-type DNA structures, such as bent, kinked and unwound structures, with high affinity. HMGB proteins also promote DNA bending, looping and unwinding. In this study, we determined the role of the Aedes aegypti single HMG-box domain protein AaHMGB; characterized its structure, spatiotemporal expression levels, subcellular localization, and nucleic acid binding activities; and compared these properties with those of its double-HMG-box counterpart protein, AaHMGB1. Via qRT-PCR, we showed that AaHMGB is expressed at much higher levels than AaHMGB1 throughout mosquito development. In situ hybridization results suggested a role for AaHMGB and AaHMGB1 during embryogenesis. Immunolocalization in the midgut revealed that AaHMGB is exclusively nuclear. Circular dichroism and fluorescence spectroscopy analyses showed that AaHMGB exhibits common features of α-helical structures and is more stably folded than AaHMGB1, likely due to the presence of one or two HMG boxes. Using several DNA substrates or single-stranded RNAs as probes, we observed significant differences between AaHMGB and AaHMGB1 in terms of their binding patterns, activity and/or specificity. Importantly, we showed that the phosphorylation of AaHMGB plays a critical role in its DNA-binding activity. Our study provides additional insight into the roles of single- versus double-HMG-box-containing proteins in nucleic acid interactions for better understanding of mosquito development, physiology and homeostasis. Copyright © 2017. Published by Elsevier B.V.

RNA polymerase II components and Rrn7 form a preinitiation complex on the HomolD box to promote ribosomal protein gene expression in Schizosaccharomyces pombe.

PubMed

Montes, Matías; Moreira-Ramos, Sandra; Rojas, Diego A; Urbina, Fabiola; Käufer, Norbert F; Maldonado, Edio

2017-02-01

In Schizosaccharomyces pombe, ribosomal protein gene (RPG) promoters contain a TATA box analog, the HomolD box, which is bound by the Rrn7 protein. Despite the importance of ribosome biogenesis for cell survival, the mechanisms underlying RPG transcription remain unknown. In this study, we found that components of the RNA polymerase II (RNAPII) system, consisting of the initiation or general transcription factors (GTFs) TFIIA, IIB, IIE, TATA-binding protein (TBP) and the RNAPII holoenzyme, interacted directly with Rrn7 in vitro, and were able to form a preinitiation complex (PIC) on the HomolD box. PIC complex formation follows an ordered pathway on these promoters. The GTFs and RNAPII can also be cross-linked to HomolD-containing promoters in vivo. In an in vitro reconstituted transcription system, RNAPII components and Rrn7 were necessary for HomolD-directed transcription. The Mediator complex was required for basal transcription from those promoters in whole cell extract (WCE). The Med17 subunit of Mediator also can be cross-linked to the promoter region of HomolD-containing promoters in vivo, suggesting the presence of the Mediator complex on HomolD box-containing promoters. Together, these data show that components of the RNAPII machinery and Rrn7 participate in the PIC assembly on the HomolD box, thereby directing RPG transcription. © 2017 Federation of European Biochemical Societies.

Role of indirect readout mechanism in TATA box binding protein-DNA interaction.

PubMed

Mondal, Manas; Choudhury, Devapriya; Chakrabarti, Jaydeb; Bhattacharyya, Dhananjay

2015-03-01

Gene expression generally initiates from recognition of TATA-box binding protein (TBP) to the minor groove of DNA of TATA box sequence where the DNA structure is significantly different from B-DNA. We have carried out molecular dynamics simulation studies of TBP-DNA system to understand how the DNA structure alters for efficient binding. We observed rigid nature of the protein while the DNA of TATA box sequence has an inherent flexibility in terms of bending and minor groove widening. The bending analysis of the free DNA and the TBP bound DNA systems indicate presence of some similar structures. Principal coordinate ordination analysis also indicates some structural features of the protein bound and free DNA are similar. Thus we suggest that the DNA of TATA box sequence regularly oscillates between several alternate structures and the one suitable for TBP binding is induced further by the protein for proper complex formation.

DEAD-box Helicases as Integrators of RNA, Nucleotide and Protein Binding

PubMed Central

Putnam, Andrea A.

2013-01-01

DEAD-box helicases perform diverse cellular functions in virtually all steps of RNA metabolism from Bacteria to Humans. Although DEAD-box helicases share a highly conserved core domain, the enzymes catalyze a wide range of biochemical reactions. In addition to the well established RNA unwinding and corresponding ATPase activities, DEAD-box helicases promote duplex formation and displace proteins from RNA. They can also function as assembly platforms for larger ribonucleoprotein complexes, and as metabolite sensors. This review aims to provide a perspective on the diverse biochemical features of DEAD-box helicases and connections to structural information. We discuss these data in the context of a model that views the enzymes as integrators of RNA, nucleotide, and protein binding. PMID:23416748

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

PubMed

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.

Packing Boxes into Multiple Containers Using Genetic Algorithm

NASA Astrophysics Data System (ADS)

Menghani, Deepak; Guha, Anirban

2016-07-01

Container loading problems have been studied extensively in the literature and various analytical, heuristic and metaheuristic methods have been proposed. This paper presents two different variants of a genetic algorithm framework for the three-dimensional container loading problem for optimally loading boxes into multiple containers with constraints. The algorithms are designed so that it is easy to incorporate various constraints found in real life problems. The algorithms are tested on data of standard test cases from literature and are found to compare well with the benchmark algorithms in terms of utilization of containers. This, along with the ability to easily incorporate a wide range of practical constraints, makes them attractive for implementation in real life scenarios.

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

PubMed

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.

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

PubMed

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

Verification of unfold error estimates in the UFO code

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

Fehl, D.L.; Biggs, F.

Spectral unfolding is an inverse mathematical operation which attempts to obtain spectral source information from a set of tabulated response functions and data measurements. Several unfold algorithms have appeared over the past 30 years; among them is the UFO (UnFold Operator) code. In addition to an unfolded spectrum, UFO also estimates the unfold uncertainty (error) induced by running the code in a Monte Carlo fashion with prescribed data distributions (Gaussian deviates). In the problem studied, data were simulated from an arbitrarily chosen blackbody spectrum (10 keV) and a set of overlapping response functions. The data were assumed to have anmore » imprecision of 5% (standard deviation). 100 random data sets were generated. The built-in estimate of unfold uncertainty agreed with the Monte Carlo estimate to within the statistical resolution of this relatively small sample size (95% confidence level). A possible 10% bias between the two methods was unresolved. The Monte Carlo technique is also useful in underdetemined problems, for which the error matrix method does not apply. UFO has been applied to the diagnosis of low energy x rays emitted by Z-Pinch and ion-beam driven hohlraums.« less

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

PubMed

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.

Ultra-fast outflows (aka UFOs) in AGNs and their relevance for feedback

NASA Astrophysics Data System (ADS)

Cappi, Massimo; Tombesi, F.; Giustini, M.; Dadina, M.; Braito, V.; Kaastra, J.; Reeves, J.; Chartas, G.; Gaspari, M.; Vignali, C.; Gofford, J.; Lanzuisi, G.

2012-09-01

During the last decade, several observational evidences have been accumulated for the existence of massive, high velocity winds/outflows (aka UFOs) in nearby AGNs and, possibly, distant quasars. I will review here such evidences, present some of the latest results in this field, and discuss the relevance of UFOs for both understanding the physics of accretion/ejection flows on supermassive black holes, and for quantifying the amount of AGN feedback.

Identification and expression analysis of the SQUAMOSA promoter-binding protein (SBP)-box gene family in Prunus mume.

PubMed

Xu, Zongda; Sun, Lidan; Zhou, Yuzhen; Yang, Weiru; Cheng, Tangren; Wang, Jia; Zhang, Qixiang

2015-10-01

SQUAMOSA promoter-binding protein (SBP)-box family genes encode plant-specific transcription factors that play crucial roles in plant development, especially flower and fruit development. However, little information on this gene family is available for Prunus mume, an ornamental and fruit tree widely cultivated in East Asia. To explore the evolution of SBP-box genes in Prunus and explore their functions in flower and fruit development, we performed a genome-wide analysis of the SBP-box gene family in P. mume. Fifteen SBP-box genes were identified, and 11 of them contained an miR156 target site. Phylogenetic and comprehensive bioinformatics analyses revealed that different groups of SBP-box genes have undergone different evolutionary processes and varied in their length, structure, and motif composition. Purifying selection has been the main selective constraint on both paralogous and orthologous SBP-box genes. In addition, the sequences of orthologous SBP-box genes did not diverge widely after the split of P. mume and Prunus persica. Expression analysis of P. mume SBP-box genes revealed their diverse spatiotemporal expression patterns. Three duplicated SBP-box genes may have undergone subfunctionalization in Prunus. Most of the SBP-box genes showed high transcript levels in flower buds and young fruit. The four miR156-nontargeted genes were upregulated during fruit ripening. Together, these results provide information about the evolution of SBP-box genes in Prunus. The expression analysis lays the foundation for further research on the functions of SBP-box genes in P. mume and other Prunus species, especially during flower and fruit development.

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

PubMed

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.

Discovery of a novel oocyte-specific Krüppel-associated box domain-containing zinc finger protein required for early embryogenesis in cattle.

PubMed

Hand, Jacqelyn M; Zhang, Kun; Wang, Lei; Koganti, Prasanthi P; Mastrantoni, Kristen; Rajput, Sandeep K; Ashry, Mohamed; Smith, George W; Yao, Jianbo

2017-04-01

Zinc finger (ZNF) transcription factors interact with DNA through zinc finger motifs and play important roles in a variety of cellular functions including cell growth, proliferation, development, apoptosis, and intracellular signal transduction. One-third of ZNF proteins in metazoans contain a highly conserved N-terminal motif known as the Krüppel-associated box (KRAB) domain, which acts as a potent, DNA-binding dependent transcriptional repression module. Analysis of RNA-Seq data generated from a bovine oocyte cDNA library identified a novel transcript, which encodes a KRAB-containing ZNF transcription factor (named ZNFO). Characterization of ZNFO mRNA expression revealed that it is exclusively expressed in bovine oocytes and early embryos. A GFP reporter assay demonstrated that ZNFO protein localizes specifically to the nucleus, supporting its role in transcriptional regulation. To test the role of ZNFO in early embryonic development, zygotes were generated by in vitro maturation and fertilization of oocytes, and injected with small interfering RNA (siRNA) designed to knockdown ZNFO. Cleavage rates were not affected by ZNFO siRNA injection. However, embryonic development to 8- to 16-cell stage and blastocyst stage was significantly reduced relative to the uninjected and negative control siRNA-injected embryos. Further, interaction of ZNFO with the highly conserved co-factor, KRAB-associated protein-1 (KAP1), was demonstrated, and evidence supporting transcriptional repression by ZNFO was demonstrated using a GAL4-luciferase reporter system. Results of described studies demonstrate that ZNFO is a maternally-derived oocyte-specific nuclear factor required for early embryonic development in cattle, presumably functioning by repressing transcription. Copyright © 2017 Elsevier B.V. All rights reserved.

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

PubMed Central

Stamatopoulou, Vassiliki; Apostolidi, Maria; Li, Shuang; Lamprinou, Katerina; Papakyriakou, Athanasios

2017-01-01

Abstract 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. PMID:28973457

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

PubMed Central

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

Characterization of an AGAMOUS-like MADS box protein, a probable constituent of flowering and fruit ripening regulatory system in banana.

PubMed

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.

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

PubMed

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.

Yet another UFO in the X-ray spectrum of a high-z lensed QSO

NASA Astrophysics Data System (ADS)

Dadina, M.; Vignali, C.; Cappi, M.; Lanzuisi, G.; Ponti, G.; Torresi, E.; De Marco, B.; Chartas, G.; Giustini, M.

2018-02-01

Aim. Ultra-fast outflows (UFO) appear to be common in local active galactic nuclei (AGN) and may be powerful enough (Ėkin ≥ 1% of Lbol) to effectively quench the star formation in their host galaxies. To test feedback models based on AGN outflows, it is mandatory to investigate UFOs near the peak of AGN activity, that is, at high-z where only a few studies are available to date. Methods: UFOs produce Fe resonant absorption lines measured above ≈7 keV. The most critical problem in detecting such features in distant objects is the difficulty in obtaining X-ray data with sufficient signal-to-noise. We therefore selected a distant QSO that gravitational lensing made bright enough for these purposes, the z = 2.64 QSO MG J0414+0534, and observed it with XMM-Newton for ≈78 ks. Results: The X-ray spectrum of MG J0414+0534 is complex and shows signatures of cold absorption (NH ≈ 4 × 1022 cm-2) and of the presence of an iron emission line (E ≈ 6.4 keV, EW = 95 ± 53 eV) consistent with it originating in the cold absorber. Our main result, however, is the robust detection (more than 5σ) of an absorption line at Eint ≈ 9.2 keV (Eobs ≈ 2.5 keV observer frame). If interpreted as due to FeXXVI, it implies gas outflowing at vout ≈ 0.3c. To our knowledge, this is the first detection of an UFO in a radio-loud quasar at z ≥ 1.5. We estimated that the UFO mechanical output is Ėkin ≈ 2.5Lbol with ṗout/ṗrad ≈ 17 indicating that it is capable of installing significant feedback between the super-massive black hole and the bulge of the host galaxy. We argue that this also suggests a magnetic driving origin of the UFO.

Transcriptional Regulation of Fruit Ripening by Tomato FRUITFULL Homologs and Associated MADS Box Proteins[W

PubMed Central

Fujisawa, Masaki; Shima, Yoko; Nakagawa, Hiroyuki; Kitagawa, Mamiko; Kimbara, Junji; Nakano, Toshitsugu; Kasumi, Takafumi; Ito, Yasuhiro

2014-01-01

The tomato (Solanum lycopersicum) MADS box FRUITFULL homologs FUL1 and FUL2 act as key ripening regulators and interact with the master regulator MADS box protein RIPENING INHIBITOR (RIN). Here, we report the large-scale identification of direct targets of FUL1 and FUL2 by transcriptome analysis of FUL1/FUL2 suppressed fruits and chromatin immunoprecipitation coupled with microarray analysis (ChIP-chip) targeting tomato gene promoters. The ChIP-chip and transcriptome analysis identified FUL1/FUL2 target genes that contain at least one genomic region bound by FUL1 or FUL2 (regions that occur mainly in their promoters) and exhibit FUL1/FUL2-dependent expression during ripening. These analyses identified 860 direct FUL1 targets and 878 direct FUL2 targets; this set of genes includes both direct targets of RIN and nontargets of RIN. Functional classification of the FUL1/FUL2 targets revealed that these FUL homologs function in many biological processes via the regulation of ripening-related gene expression, both in cooperation with and independent of RIN. Our in vitro assay showed that the FUL homologs, RIN, and tomato AGAMOUS-LIKE1 form DNA binding complexes, suggesting that tetramer complexes of these MADS box proteins are mainly responsible for the regulation of ripening. PMID:24415769

Recognition of a wide-range of S-RNases by S locus F-box like 2, a general-inhibitor candidate in the Prunus-specific S-RNase-based self-incompatibility system.

PubMed

Matsumoto, Daiki; Tao, Ryutaro

2016-07-01

Many species in the Rosaceae, the Solanaceae, and the Plantaginaceae exhibit S-RNase-based gametophytic self-incompatibility (GSI). This system comprises S-ribonucleases (S-RNases) as the pistil S determinant and a single or multiple F-box proteins as the pollen S determinants. In Prunus, pollen specificity is determined by a single S haplotype-specific F-box protein (SFB). The results of several studies suggested that SFB exerts cognate S-RNase cytotoxicity, and a hypothetical general inhibitor (GI) is assumed to detoxify S-RNases in non-specific manner unless it is affected by SFB. Although the identity of the GI is unknown, phylogenetic and evolutionary analyses have indicated that S locus F-box like 1-3 (or S locus F-box with low allelic sequence polymorphism 1-3; SLFL1-3), which are encoded by a region of the Prunus genome linked to the S locus, are good GI candidates. Here, we examined the biochemical characteristics of SLFL1-3 to determine whether they have appropriate GI characteristics. Pull-down assays and quantitative expression analyses indicated that Prunus avium SLFL1-3 mainly formed a canonical SCF complex with PavSSK1 and PavCul1A. Binding assays with PavS(1,3,4,6)-RNases showed that PavSLFL1, PavSLFL2, and PavSLFL3 bound to PavS(3)-RNase, all PavS-RNases tested, and none of the PavS-RNases tested, respectively. Together, these results suggested that SLFL2 has the appropriate characteristics to be the GI in sweet cherry pollen, while SLFL1 may redundantly work with SLFL2 to detoxify all S-RNases. We discuss the possible roles of SLFL1-3 as the GI in the Prunus-specific S-RNase-based GSI mechanism.

Magical Boxes

ERIC Educational Resources Information Center

Costello, Judith

2005-01-01

Students get excited when they realize that they can transform a flat sheet of paper into a box. By using different sizes of paper, they can make different sizes of boxes and put a box inside a box, inside a box. These magical boxes within boxes can contain unwanted emotions or special treasures. The project described in this article incorporates…

Imprecision and Uncertainty in the UFO Database Model.

ERIC Educational Resources Information Center

Van Gyseghem, Nancy; De Caluwe, Rita

1998-01-01

Discusses how imprecision and uncertainty are dealt with in the UFO (Uncertainty and Fuzziness in an Object-oriented) database model. Such information is expressed by means of possibility distributions, and modeled by means of the proposed concept of "role objects." The role objects model uncertain, tentative information about objects,…

PopF1 and PopF2, Two Proteins Secreted by the Type III Protein Secretion System of Ralstonia solanacearum, Are Translocators Belonging to the HrpF/NopX Family†

PubMed Central

Meyer, Damien; Cunnac, Sébastien; Guéneron, Mareva; Declercq, Céline; Van Gijsegem, Frédérique; Lauber, Emmanuelle; Boucher, Christian; Arlat, Matthieu

2006-01-01

Ralstonia solanacearum GMI1000 is a gram-negative plant pathogen which contains an hrp gene cluster which codes for a type III protein secretion system (TTSS). We identified two novel Hrp-secreted proteins, called PopF1 and PopF2, which display similarity to one another and to putative TTSS translocators, HrpF and NopX, from Xanthomonas spp. and rhizobia, respectively. They also show similarities with TTSS translocators of the YopB family from animal-pathogenic bacteria. Both popF1 and popF2 belong to the HrpB regulon and are required for the interaction with plants, but PopF1 seems to play a more important role in virulence and hypersensitive response (HR) elicitation than PopF2 under our experimental conditions. PopF1 and PopF2 are not necessary for the secretion of effector proteins, but they are required for the translocation of AvrA avirulence protein into tobacco cells. We conclude that PopF1 and PopF2 are type III translocators belonging to the HrpF/NopX family. The hrpF gene of Xanthomonas campestris pv. campestris partially restored HR-inducing ability to popF1 popF2 mutants of R. solanacearum, suggesting that translocators of R. solanacearum and Xanthomonas are functionally conserved. Finally, R. solanacearum strain UW551, which does not belong to the same phylotype as GMI1000, also possesses two putative translocator proteins. However, although one of these proteins is clearly related to PopF1 and PopF2, the other seems to be different and related to NopX proteins, thus showing that translocators might be variable in R. solanacearum. PMID:16788199

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

PubMed Central

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 that CRL4Cdt2-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 CRL4Cdt2 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 CRL4Cdt2 pathway in the switch of PCNA partners on DNA damage. PMID:24423875

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

PubMed

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.

High-Throughput Genetic Identification of Functionally Important Regions of the Yeast DEAD-Box Protein Mss116p

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

Mohr, Georg; Del Campo, Mark; Turner, Kathryn G.

The Saccharomyces cerevisiae DEAD-box protein Mss116p is a general RNA chaperone that functions in splicing mitochondrial group I and group II introns. Recent X-ray crystal structures of Mss116p in complex with ATP analogs and single-stranded RNA show that the helicase core induces a bend in the bound RNA, as in other DEAD-box proteins, while a C-terminal extension (CTE) induces a second bend, resulting in RNA crimping. Here, we illuminate these structures by using high-throughput genetic selections, unigenic evolution, and analyses of in vivo splicing activity to comprehensively identify functionally important regions and permissible amino acid substitutions throughout Mss116p. The functionallymore » important regions include those containing conserved sequence motifs involved in ATP and RNA binding or interdomain interactions, as well as previously unidentified regions, including surface loops that may function in protein-protein interactions. The genetic selections recapitulate major features of the conserved helicase motifs seen in other DEAD-box proteins but also show surprising variations, including multiple novel variants of motif III (SAT). Patterns of amino acid substitutions indicate that the RNA bend induced by the helicase core depends on ionic and hydrogen-bonding interactions with the bound RNA; identify a subset of critically interacting residues; and indicate that the bend induced by the CTE results primarily from a steric block. Finally, we identified two conserved regions - one the previously noted post II region in the helicase core and the other in the CTE - that may help displace or sequester the opposite RNA strand during RNA unwinding.« less

[STUDY OF PROTECTIVE ACTIVITY OF PROTEIN-CONTAINING ANTIGENS OF STREPTOCOCCUS PNEUMONIAE IN A HETEROLOGOUS SYSTEM].

PubMed

Vorobiev, D S; Semenova, I B; Volokh, Yu V; Romanenko, E E; Baturo, A P; Mikhailova, N A

2015-01-01

Study protective activity of protein-containing antigens of pneumococcus, obtained from serotypes 6B, 10A, 14, 19F, 23F and 36R, against infection with heterologous strains of S. pneumoniae. S. pneumoniae strains of serotypes 3, 6B, 10A, 14, 19F, 23F and 36R, obtained from the collection of pneumococcus strains of Mechnikov RIVS, were used in the study. Protein-containing antigens of S. pneumoniae were isolated by acetone precipitations of supernatant fraction of culture medium. Protective activity of preparations of protein-containing antigens of pneumococcus as studied in experiments of active protection of BALb/c line mice. The data obtained give evidence, that protein-containing antigens of pneumococcus, isolated from serotypes 6B, 10A, 14, 19F and 23F, effectively protect animals from subsequent infection with a heterologous S. pneumoniae strain of serotype 3 No. 11/56. Protection was noted at a level from 80 to 100% (p ≤ 0.05). Similar protective effect was detected in another experiment in a group of mice, immunized with preparations of protein-containing antigens of pneumococcus, obtained from serotypes 6B and 36R, against infection with a heterologous S. pneumoniae strain of serotype 3 No. 11/56. Protection was noted at a level of 90% (p ≤ 0.05). The results of the experiments carried out allow to assume, that the main role in formation of cross-protection in experiments in animals is played by pneumococcus, proteins, that are a part of the studied preparations, and not polysaccharide antigens.

Unidentified Flying Objects (UFOs). LC Science Tracer Bullet.

ERIC Educational Resources Information Center

Library of Congress, Washington, DC. Science and Technology Div.

This guide lists information sources dealing with unidentified flying objects (UFOs). Not meant to be a comprehensive bibliography, this compilation is designed--as the name of the series implies--to put the reader "on target." Included are: (1) subject headings used by the Library of Congress, under which publications on this subject…

Physcomitrella patens MAX2 characterization suggests an ancient role for this F-box protein in photomorphogenesis rather than strigolactone signalling.

PubMed

Lopez-Obando, Mauricio; de Villiers, Ruan; Hoffmann, Beate; Ma, Linnan; de Saint Germain, Alexandre; Kossmann, Jens; Coudert, Yoan; Harrison, C Jill; Rameau, Catherine; Hills, Paul; Bonhomme, Sandrine

2018-05-21

Strigolactones (SLs) are key hormonal regulators of flowering plant development and are widely distributed amongst streptophytes. In Arabidopsis, SLs signal via the F-box protein MORE AXILLARY GROWTH2 (MAX2), affecting multiple aspects of development including shoot branching, root architecture and drought tolerance. Previous characterization of a Physcomitrella patens moss mutant with defective SL synthesis supports an ancient role for SLs in land plants, but the origin and evolution of signalling pathway components are unknown. Here we investigate the function of a moss homologue of MAX2, PpMAX2, and characterize its role in SL signalling pathway evolution by genetic analysis. We report that the moss Ppmax2 mutant shows very distinct phenotypes from the moss SL-deficient mutant. In addition, the Ppmax2 mutant remains sensitive to SLs, showing a clear transcriptional SL response in dark conditions, and the response to red light is also altered. These data suggest divergent evolutionary trajectories for SL signalling pathway evolution in mosses and vascular plants. In P. patens, the primary roles for MAX2 are in photomorphogenesis and moss early development rather than in SL response, which may require other, as yet unidentified, factors. © 2018 INRA New Phytologist © 2018 New Phytologist Trust.

Interleukin-1 homologues IL-1F7b and IL-18 contain functional mRNA instability elements within the coding region responsive to lipopolysaccharide

PubMed Central

2004-01-01

IL-1F7b, a novel homologue of the IL-1 (interleukin 1) family, was discovered by computational cloning. We demonstrated that IL-1F7b shares critical amino acid residues with IL-18 and binds to the IL-18-binding protein enhancing its ability to inhibit IL-18-induced interferon-γ. We also showed that low levels of IL-1F7b are constitutively present intracellularly in human blood monocytes. In this study, we demonstrate that similar to IL-18, both mRNA and intracellular protein expression of IL-1F7b are up-regulated by LPS (lipopolysaccharide) in human monocytes. In stable transfectants of murine RAW264.7 macrophage cells, there was no IL-1F7b protein expression despite a highly active CMV promoter. We found that IL-1F7b-specific mRNA was rapidly degraded in transfected cells, via a 3′-UTR (untranslated region)-independent control of IL-1F7b transcript stability. After LPS stimulation, there was a rapid transient increase in IL-1F7b-specific mRNA and concomitant protein levels. Using sequence alignment, we found a conserved ten-nucleotide homology box within the open reading frame of IL-F7b, which is flanking the coding region instability elements of some selective genes. In-frame deletion of downstream exon 5 from the full-length IL-1F7b cDNA markedly increased the levels of IL-1F7b mRNA. A similar coding region element is located in IL-18. When transfected into RAW264.7 macrophages, IL-18 mRNA was also unstable unless treated with LPS. These results indicate that both IL-1F7b and IL-18 mRNA contain functional instability determinants within their coding region, which influence mRNA decay as a novel mechanism to regulate the expression of IL-1 family members. PMID:15046617

Compound Heterozygosity for Y Box Proteins Causes Sterility Due to Loss of Translational Repression

PubMed Central

Sharma, Manju; Dearth, Andrea; Smith, Benjamin; Braun, Robert E.

2015-01-01

The Y-box proteins YBX2 and YBX3 bind RNA and DNA and are required for metazoan development and fertility. However, possible functional redundancy between YBX2 and YBX3 has prevented elucidation of their molecular function as RNA masking proteins and identification of their target RNAs. To investigate possible functional redundancy between YBX2 and YBX3, we attempted to construct Ybx2 -/- ;Ybx3 -/- double mutants using a previously reported Ybx2 -/- model and a newly generated global Ybx3 -/- model. Loss of YBX3 resulted in reduced male fertility and defects in spermatid differentiation. However, homozygous double mutants could not be generated as haploinsufficiency of both Ybx2 and Ybx3 caused sterility characterized by extensive defects in spermatid differentiation. RNA sequence analysis of mRNP and polysome occupancy in single and compound Ybx2/3 heterozygotes revealed loss of translational repression almost exclusively in the compound Ybx2/3 heterozygotes. RNAseq analysis also demonstrated that Y-box protein dose-dependent loss of translational regulation was inversely correlated with the presence of a Y box recognition target sequence, suggesting that Y box proteins bind RNA hierarchically to modulate translation in a range of targets. PMID:26646932

Apple EIN3 BINDING F-box 1 inhibits the activity of three apple EIN3-like transcription factors

PubMed Central

Tacken, Emma J.; Ireland, Hilary S.; Wang, Yen-Yi; Putterill, Jo; Schaffer, Robert J.

2012-01-01

Background and aims Fruit ripening in Malus× domestica (apple) is controlled by ethylene. Work in model species has shown that following the detection of ethylene, the ETHYLENE INSENSITIVE 3 (EIN3) transcription factor is stabilized, leading to an increase in transcript accumulation of ethylene-responsive genes, such as POLYGALACTURONASE1 (PG1). In the absence of ethylene, the EIN3 BINDING F-box (EBF) proteins rapidly degrade EIN3 via the ubiquitination/SCF (Skp, Cullin, F-Box) proteasome pathway. In this study, we aim to identify and characterize the apple EBF genes, and test their activity against apple EIN3-like proteins (EILs). Methodology The apple genome sequence was mined for EBF-like genes. The expression of EBF-like genes was measured during fruit development. Using a transient assay in Nicotiana benthamiana leaves, the activity of three apple EILs was tested against the PG1 promoter, with and without ethylene and EBF1. Principal results Four EBF-like genes in apple were identified and grouped into two sub-clades. Sub-clade I genes had constant expression over fruit development while sub-clade II genes increased in expression at ripening. EBF1 was shown to reduce the transactivation of the apple PG1 promoter by the EIL1, EIL2 and EIL3 transcription factors in the presence of ethylene. Conclusions The apple EBF1 gene identified here is likely to be a functionally conserved EBF orthologue, modulating EIL activity in apples. The activity of EBF1 suggests that it is not specific to a single EIL, instead acting as a global regulator of apple EIL transcription factors. PMID:23585922

Downregulation of Forkhead box F1 gene expression in the pulmonary vasculature of nitrofen-induced congenital diaphragmatic hernia.

PubMed

Zimmer, J; Takahashi, T; Hofmann, A D; Puri, Prem

2016-12-01

High mortality and morbidity in infants born with congenital diaphragmatic hernia (CDH) are attributed to pulmonary hypoplasia and pulmonary hypertension (PH). Forkhead box (Fox) transcription factors are known to be crucial for cell proliferation and homeostasis. FoxF1 is essential for lung morphogenesis, vascular development, and endothelial proliferation. Mutations in FoxF1 and also the Fox family member FoxC2 have been identified in neonates with PH. In human and experimental models of arterial PH, the Fox protein FoxO1 was found to be downregulated. We hypothesized that Fox expression is altered in the lungs of the nitrofen-induced CDH rat model and investigated the expression of FoxF1, FoxC2, and FoxO1. Following ethical approval (Rec 913b), time-pregnant Sprague-Dawley rats received nitrofen or vehicle on gestational day (D9). Fetuses were sacrificed on D21, inspected for CDH and divided into CDH (n = 11) and control group (n = 11). Gene expression of FoxF1, FoxC2, and FoxO1 was evaluated with qRT-PCR. Detected alterations of mRNA levels were subsequently assessed on the protein level by performing western blot analysis and laser scanning confocal microscopy. The relative mRNA level of FoxF1 was significantly downregulated in CDH lungs compared to controls (FoxF1 CDH 1.047 ± 0.108, FoxF1 Ctrl 1.419 ± 0.01, p = 0.014). Relative mRNA levels of FoxC2 and FoxO1 were not found to be altered between the experimental groups (FoxC2 CDH 30.74 ± 8.925, FoxC2 Ctrl 27.408 ± 7.487, p = 0.776; FoxO1 CDH 0.011 ± 0.002, FoxO1 Ctrl 0.011 ± 0.001, p = 0.809). On the protein level, western blotting demonstrated a reduced pulmonary protein expression of FoxF1 in CDH lungs. Confocal microscopy showed a markedly diminished expression of FoxF1 in the pulmonary vasculature of CDH lungs compared to controls. Our study demonstrates a strikingly reduced expression of FoxF1 in the pulmonary vasculature of nitrofen-induced CDH. Altered FoxF1 gene expression

Searching for the UV counterpart of the extraordinary X-ray UFO in the NLSy1 IRAS17020+4544

NASA Astrophysics Data System (ADS)

Krongold, Yair

2017-08-01

We recently reported the first unambiguous discovery in high resolution X-ray data of an ultra fast outflow (UFO) with velocity .1c. This wind, in Narrow Line Seyfert 1 galaxy IRAS17020+4544, represents so far the most compelling detection of an UFO, with many different absorption lines that give rise to very high significance detections. The charge states that form the wind clearly indicate a large range of ionization states in the gas, and significant absorption by Ly alpha, C IV, Si IV and N V (among other ions) is expected in the UV band. The goal of our proposed program is to observe an characterize the best X-ray detected UFO in the UV. These observations are crucial to study in great detail the UFO phenomenon, and understand its nature and its relation to the narrow absorption line low velocity systems. Only through detection of Ly alpha absorption in the UV data, measurements of the metallicity of these winds will be possible. The proposed program will help guide new theoretical models of UFOs origins, beyond the simple actual picture that predicts only very high ionization Fe absorption. UV data are required to understand the wind nature and launching mechanism (whether due to radiation pressure via line or continuum opacity, or magnetic forces). Fully characterizing the wind properties will put stronger constraints in the mass outflow and kinetic outflow rates of these systems, as well as in their geometry. Such estimates will give a much clearer picture of UFOs feedback potential, and will provide clues on the feedback mode in action (e.g. energy conserving vs. momentum conserving).

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

PubMed

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.

Mutations in the F-box gene SNEEZY result in decreased arabidopsis GA signaling

USDA-ARS?s Scientific Manuscript database

We previously reported that the SLEEPY1 (SLY1) homolog, F-box gene SNEEZY/SLEEPY2 (SNE/SLY2), can partly replace SLY1 in gibberellin (GA) hormone signaling through interaction with DELLAs RGA and GAI. To determine whether SNE normally functions in GA signaling, we characterized the phenotypes of tw...

Identification and Characterization of Components of a Putative Petunia S-Locus F-Box–Containing E3 Ligase Complex Involved in S-RNase–Based Self-Incompatibility[W

PubMed Central

Hua, Zhihua; Kao, Teh-hui

2006-01-01

Petunia inflata S-locus F-box (Pi SLF) is thought to function as a typical F-box protein in ubiquitin-mediated protein degradation and, along with Skp1, Cullin-1, and Rbx1, could compose an SCF complex mediating the degradation of nonself S-RNase but not self S-RNase. We isolated three P. inflata Skp1s (Pi SK1, -2, and -3), two Cullin-1s (Pi CUL1-C and -G), and an Rbx1 (Pi RBX1) cDNAs and found that Pi CUL1-G did not interact with Pi RBX1 and that none of the three Pi SKs interacted with Pi SLF2. We also isolated a RING-HC protein, S-RNase Binding Protein1 (Pi SBP1), almost identical to Petunia hybrida SBP1, which interacts with Pi SLFs, S-RNases, Pi CUL1-G, and an E2 ubiquitin-conjugating enzyme, suggesting that Pi CUL1-G, SBP1, and SLF may be components of a novel E3 ligase complex, with Pi SBP1 playing the roles of Skp1 and Rbx1. S-RNases interact more with nonself Pi SLFs than with self Pi SLFs, and Pi SLFs also interact more with nonself S-RNases than with self S-RNases. Bacterially expressed S1-, S2-, and S3-RNases are degraded by the 26S proteasomal pathway in a cell-free system, albeit not in an S-allele–specific manner. Native glycosylated S3-RNase is not degraded to any significant extent; however, deglycosylated S3-RNase is degraded as efficiently as the bacterially expressed S-RNases. Finally, S-RNases are ubiquitinated in pollen tube extracts, but whether this is mediated by the Pi SLF–containing E3 complex is unknown. PMID:17028207

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

PubMed

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.

Titration of DnaA protein by oriC DnaA-boxes increases dnaA gene expression in Escherichia coli.

PubMed Central

Hansen, F G; Koefoed, S; Sørensen, L; Atlung, T

1987-01-01

Binding of the DnaA protein to its binding sites, the DnaA-boxes (TTATCCACA), was measured by a simple physiological approach. The presence of extra DnaA-boxes in growing cells leads to a derepression of dnaA gene expression, measured as beta-galactosidase activity of a dnaA-lacZ fusion polypeptide. Different DnaA-boxes caused different degrees of derepression indicating that the DnaA protein requires sequences in addition to the DnaA-box for efficient binding. The DnaA-boxes in oriC might act cooperatively in binding of the DnaA protein. The derepressed levels of DnaA protein obtained in a strain carrying an oriC+-pBR322 chimera were very high and sufficient to activate oriC on the chimeric plasmid, which was maintained at a copy number more than three times that of pBR322. PMID:3034578

Expression analysis of genes encoding double B-box zinc finger proteins in maize.

PubMed

Li, Wenlan; Wang, Jingchao; Sun, Qi; Li, Wencai; Yu, Yanli; Zhao, Meng; Meng, Zhaodong

2017-11-01

The B-box proteins play key roles in plant development. The double B-box (DBB) family is one of the subfamily of the B-box family, with two B-box domains and without a CCT domain. In this study, 12 maize double B-box genes (ZmDBBs) were identified through a genome-wide survey. Phylogenetic analysis of DBB proteins from maize, rice, Sorghum bicolor, Arabidopsis, and poplar classified them into five major clades. Gene duplication analysis indicated that segmental duplications made a large contribution to the expansion of ZmDBBs. Furthermore, a large number of cis-acting regulatory elements related to plant development, response to light and phytohormone were identified in the promoter regions of the ZmDBB genes. The expression patterns of the ZmDBB genes in various tissues and different developmental stages demonstrated that ZmDBBs might play essential roles in plant development, and some ZmDBB genes might have unique function in specific developmental stages. In addition, several ZmDBB genes showed diurnal expression pattern. The expression levels of some ZmDBB genes changed significantly under light/dark treatment conditions and phytohormone treatments, implying that they might participate in light signaling pathway and hormone signaling. Our results will provide new information to better understand the complexity of the DBB gene family in maize.

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.

PubMed

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.

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

PubMed Central

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. PMID:26529408

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

PubMed

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.

Unusual features of fibrillarin cDNA and gene structure in Euglena gracilis: evolutionary conservation of core proteins and structural predictions for methylation-guide box C/D snoRNPs throughout the domain Eucarya.

PubMed

Russell, Anthony G; Watanabe, Yoh-ichi; Charette, J Michael; Gray, Michael W

2005-01-01

Box C/D ribonucleoprotein (RNP) particles mediate O2'-methylation of rRNA and other cellular RNA species. In higher eukaryotic taxa, these RNPs are more complex than their archaeal counterparts, containing four core protein components (Snu13p, Nop56p, Nop58p and fibrillarin) compared with three in Archaea. This increase in complexity raises questions about the evolutionary emergence of the eukaryote-specific proteins and structural conservation in these RNPs throughout the eukaryotic domain. In protists, the primarily unicellular organisms comprising the bulk of eukaryotic diversity, the protein composition of box C/D RNPs has not yet been extensively explored. This study describes the complete gene, cDNA and protein sequences of the fibrillarin homolog from the protozoon Euglena gracilis, the first such information to be obtained for a nucleolus-localized protein in this organism. The E.gracilis fibrillarin gene contains a mixture of intron types exhibiting markedly different sizes. In contrast to most other E.gracilis mRNAs characterized to date, the fibrillarin mRNA lacks a spliced leader (SL) sequence. The predicted fibrillarin protein sequence itself is unusual in that it contains a glycine-lysine (GK)-rich domain at its N-terminus rather than the glycine-arginine-rich (GAR) domain found in most other eukaryotic fibrillarins. In an evolutionarily diverse collection of protists that includes E.gracilis, we have also identified putative homologs of the other core protein components of box C/D RNPs, thereby providing evidence that the protein composition seen in the higher eukaryotic complexes was established very early in eukaryotic cell evolution.

Evaluation of genetic and metabolic role of SKIP11 in Arabidopsis thaliana

NASA Astrophysics Data System (ADS)

Hassan, Muhammad Naeem ul; Ismail, Ismanizan

2015-09-01

Most of the regulatory proteins are degraded by 26S proteasome complex, only when they are tagged by Ubiquitin. A complex of four proteins, SKP1-Cullin-Ring box-F box (SCF) catalyses the final step to link the Ubiquitin tag with the target proteins. SCF complex interacts with the target proteins through F-box proteins, which confer the overall substrate specificity to the complex. F-box proteins, one of the largest family of proteins in plants have an N-terminal F-box domain and variable C-terminal domains, like leucine-rich repeat, WD-40 repeat and the kelch-repeat domains. In this study, we analysed the role of SKIP11, a kelch containing F-box protein (KFB) from Arabidopsis thaliana, by using reverse genetics strategy. The results show that SKIP11 is involved in the down-regulation of oxylipin pathway, possibly through the degradation of enzymes or/ and the regulatory factors of the pathway.

Comparative structural analysis of human DEAD-box RNA helicases.

PubMed

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

PubMed Central

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

2010-01-01

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. PMID:20941364

The VirD2 pilot protein of Agrobacterium-transferred DNA interacts with the TATA box-binding protein and a nuclear protein kinase in plants

PubMed Central

Bakó, László; Umeda, Masaaki; Tiburcio, Antonio F.; Schell, Jeff; Koncz, Csaba

2003-01-01

The bacterial virulence protein VirD2 plays an important role in nuclear import and chromosomal integration of Agrobacterium-transferred DNA in fungal, plant, animal, and human cells. Here we show that in nuclei of alfalfa cells, VirD2 interacts with and is phosphorylated by CAK2Ms, a conserved plant ortholog of cyclin-dependent kinase-activating kinases. CAK2Ms binds to and phosphorylates the C-terminal regulatory domain of RNA polymerase II largest subunit, which can recruit the TATA box-binding protein. VirD2 is found in tight association with the TATA box-binding protein in vivo. These results indicate that recognition of VirD2 is mediated by widely conserved nuclear factors in eukaryotes. PMID:12900506

A MADS Box Protein Interacts with a Mating-Type Protein and Is Required for Fruiting Body Development in the Homothallic Ascomycete Sordaria macrospora

PubMed Central

Nolting, Nicole; Pöggeler, Stefanie

2006-01-01

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 Δmcm1 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. PMID:16835449

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

PubMed

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.

A soybean MADS-box protein modulates floral organ numbers, petal identity and sterility

PubMed Central

2014-01-01

Background The MADS-box transcription factors play fundamental roles in reproductive developmental control. Although the roles of many plant MADS-box proteins have been extensively studied, there are almost no functional studies of them in soybean, an important protein and oil crop in the world. In addition, the MADS-box protein orthologs may have species-specific functions. Controlling male fertility is an important goal in plant hybrid breeding but is difficult in some crops like soybean. The morphological structure of soybean flowers prevents the cross-pollination. Understanding the molecular mechanisms for floral development will aid in engineering new sterile materials that could be applied in hybrid breeding programs in soybean. Result Through microarray analysis, a flower-enriched gene in soybean was selected and designated as GmMADS28. GmMADS28 belongs to AGL9/SEP subfamily of MADS-box proteins, localized in nucleus and showed specific expression patterns in floral meristems as well as stamen and petal primordia. Expression of GmMADS28 in the stamens and petals of a soybean mutant NJS-10Hfs whose stamens are converted into petals was higher than in those of wild-type plants. Constitutive expression of GmMADS28 in tobacco promoted early flowering and converted stamens and sepals to petals. Interestingly, transgenic plants increased the numbers of sepal, petal and stamen from five to six and exhibited male sterility due to the shortened and curly filaments and the failure of pollen release from the anthers. The ectopic expression of GmMADS28 was found to be sufficient to activate expression of tobacco homologs of SOC1, LEAFY, AGL8/FUL, and DEF. In addition, we observed the interactions of GmMADS28 with soybean homologs of SOC1, AP1, and AGL8/FUL proteins. Conclusion In this study, we observed the roles of GmMADS28 in the regulation of floral organ number and petal identity. Compared to other plant AGL9/SEP proteins, GmMADS28 specifically regulates floral

A soybean MADS-box protein modulates floral organ numbers, petal identity and sterility.

PubMed

Huang, Fang; Xu, Guangli; Chi, Yingjun; Liu, Haicui; Xue, Qian; Zhao, Tuanjie; Gai, Junyi; Yu, Deyue

2014-04-02

The MADS-box transcription factors play fundamental roles in reproductive developmental control. Although the roles of many plant MADS-box proteins have been extensively studied, there are almost no functional studies of them in soybean, an important protein and oil crop in the world. In addition, the MADS-box protein orthologs may have species-specific functions. Controlling male fertility is an important goal in plant hybrid breeding but is difficult in some crops like soybean. The morphological structure of soybean flowers prevents the cross-pollination. Understanding the molecular mechanisms for floral development will aid in engineering new sterile materials that could be applied in hybrid breeding programs in soybean. Through microarray analysis, a flower-enriched gene in soybean was selected and designated as GmMADS28. GmMADS28 belongs to AGL9/SEP subfamily of MADS-box proteins, localized in nucleus and showed specific expression patterns in floral meristems as well as stamen and petal primordia. Expression of GmMADS28 in the stamens and petals of a soybean mutant NJS-10Hfs whose stamens are converted into petals was higher than in those of wild-type plants. Constitutive expression of GmMADS28 in tobacco promoted early flowering and converted stamens and sepals to petals. Interestingly, transgenic plants increased the numbers of sepal, petal and stamen from five to six and exhibited male sterility due to the shortened and curly filaments and the failure of pollen release from the anthers. The ectopic expression of GmMADS28 was found to be sufficient to activate expression of tobacco homologs of SOC1, LEAFY, AGL8/FUL, and DEF. In addition, we observed the interactions of GmMADS28 with soybean homologs of SOC1, AP1, and AGL8/FUL proteins. In this study, we observed the roles of GmMADS28 in the regulation of floral organ number and petal identity. Compared to other plant AGL9/SEP proteins, GmMADS28 specifically regulates floral organ number, filament length and

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

PubMed

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.

Plant polycistronic precursors containing non-homologous microRNAs target transcripts encoding functionally related proteins

PubMed Central

2009-01-01

Background MicroRNAs (miRNAs) are endogenous single-stranded small RNAs that regulate the expression of specific mRNAs involved in diverse biological processes. In plants, miRNAs are generally encoded as a single species in independent transcriptional units, referred to as MIRNA genes, in contrast to animal miRNAs, which are frequently clustered. Results We performed a comparative genomic analysis in three model plants (rice, poplar and Arabidopsis) and characterized miRNA clusters containing two to eight miRNA species. These clusters usually encode miRNAs of the same family and certain share a common evolutionary origin across monocot and dicot lineages. In addition, we identified miRNA clusters harboring miRNAs with unrelated sequences that are usually not evolutionarily conserved. Strikingly, non-homologous miRNAs from the same cluster were predicted to target transcripts encoding related proteins. At least four Arabidopsis non-homologous clusters were expressed as single transcriptional units. Overexpression of one of these polycistronic precursors, producing Ath-miR859 and Ath-miR774, led to the DCL1-dependent accumulation of both miRNAs and down-regulation of their different mRNA targets encoding F-box proteins. Conclusions In addition to polycistronic precursors carrying related miRNAs, plants also contain precursors allowing coordinated expression of non-homologous miRNAs to co-regulate functionally related target transcripts. This mechanism paves the way for using polycistronic MIRNA precursors as a new molecular tool for plant biologists to simultaneously control the expression of different genes. PMID:19951405

Self-Incompatibility in Petunia inflata: The Relationship between a Self-Incompatibility Locus F-Box Protein and Its Non-Self S-RNases[W

PubMed Central

Sun, Penglin; Kao, Teh-hui

2013-01-01

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. PMID:23444333

The Dtk receptor tyrosine kinase, which binds protein S, is expressed during hematopoiesis.

PubMed

Crosier, P S; Freeman, S A; Orlic, D; Bodine, D M; Crosier, K E

1996-02-01

Dtk (Tyro 3/Sky/Rse/Brt/Tif) belongs to a recently recognized subfamily of receptor tyrosine kinases that also includes Ufo (Axl/Ark) and Mer (Eyk). Ligands for Dtk and Ufo have been identified as protein S and the related molecule Gas6, respectively. This study examined expression of Dtk during ontogeny of the hematopoietic system and compared the pattern of expression with that of Ufo. Both receptors were abundantly expressed in differentiating embryonic stem cells, yolk sac blood islands, para-aortic splanchnopleural mesoderm, fractionated AA4+ fetal liver cells, and fetal thymus from day 14 until birth. Although Ufo was expressed at moderate levels in adult bone marrow, expression of Dtk in this tissue was barely detectable. In adult bone marrow subpopulations fractionated using counterflow centrifugal elutriation, immunomagnetic bead selection for lineage-depletion and FACS sorting for c-kit expression, very low levels of Dtk and/or Ufo were detected in some cell fractions. These results suggest that Dtk and Ufo are likely to be involved in the regulation of hematopoiesis, particularly during the embryonic stages of blood cell development.

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

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

Sánchez-Giraldo, R.; Acosta-Reyes, F. J.; Malarkey, C. S.

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 ismore » 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.« less

The DEAD-box Protein Dbp2 Functions with the RNA-binding Protein Yra1 to Promote mRNP Assembly

PubMed Central

Ma, Wai Kit; Cloutier, Sara C.; Tran, Elizabeth J.

2013-01-01

Eukaryotic gene expression involves numerous biochemical steps that are dependent on RNA structure and ribonucleoprotein (RNP) complex formation. The DEAD-box class of RNA helicases plays fundamental roles in formation of RNA and RNP structure in every aspect of RNA metabolism. In an effort to explore the diversity of biological roles for DEAD-box proteins, our laboratory recently demonstrated that the DEAD-box protein Dbp2 associates with actively transcribing genes and is required for normal gene expression in Saccharomyces cerevisiae. We now provide evidence that Dbp2 interacts genetically and physically with the mRNA export factor Yra1. In addition, we find that Dbp2 is required for in vivo assembly of mRNA-binding proteins Yra1, Nab2 and Mex67 onto poly(A)+ RNA. Strikingly, we also show that Dbp2 is an efficient RNA helicase in vitro and that Yra1 decreases the efficiency of ATP-dependent duplex unwinding. We provide a model whereby mRNP assembly requires Dbp2 unwinding activity and once the mRNP is properly assembled, inhibition by Yra1 prevents further rearrangements. Both Yra1 and Dbp2 are conserved in multicellular eukaryotes suggesting that this constitutes a broadly conserved mechanism for stepwise assembly of mature mRNPs in the nucleus. PMID:23721653

The Emerging Roles of Forkhead Box (FOX) Proteins in Osteosarcoma

PubMed Central

Zhang, Wentao; Duan, Ning; Song, Tao; Li, Zhong; Zhang, Caiguo; Chen, Xun

2017-01-01

Osteosarcoma is the most common bone cancer primarily occurring in children and young adults. Over the past few years, the deregulation of a superfamily transcription factors, known as forkhead box (FOX) proteins, has been demonstrated to contribute to the pathogenesis of osteosarcoma. Molecular mechanism studies have demonstrated that FOX family proteins participate in a variety of signaling pathways and that their expression can be regulated by multiple factors. The dysfunction of FOX genes can alter osteosarcoma cell differentiation, metastasis and progression. In this review, we summarized the evidence that FOX genes play direct or indirect roles in the development and progression of osteosarcoma, and evaluated the emerging role of FOX proteins as targets for therapeutic intervention. PMID:28775781

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

PubMed Central

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

2017-01-01

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

Pax1, a member of the paired box-containing class of developmental control genes, is mapped to human chromosome 20p11.2 by in situ hybridization (ISH and FISH).

PubMed

Schnittger, S; Rao, V V; Deutsch, U; Gruss, P; Balling, R; Hansmann, I

1992-11-01

Pax-1, a member of a murine multigene family, belongs to the paired box-containing class of developmental control genes first identified in Drosophila. The Pax-1 gene encodes a sequence-specific DNA-binding protein with transcriptional activating properties and has been found to be mutated in the autosomal recessive mutation undulated (un) on mouse chromosome 2 with vertebral anomalies along the entire rostrocaudal axis. By radioactive in situ hybridization (ISH) using a fragment from the murine Pax-1 paired box that is almost identical to the respective sequences from the cognate human gene HuP48 and fluorescence in situ hybridization (FISH) using a complete mouse Pax-1 cDNA, we have assigned the human homologue of murine Pax-1, the PAX1 locus, to chromosome 20p. The map position of PAX1 after FISH (FL-pter value of 0.34 +/- 0.04) corresponds to band p11.2. These results confirm the exceptional homology between human chromosome 20 and the distal segment of mouse chromosome 2, extending from bands F to G, and add PAX1 to the group of genes on 20p like PTPA, PRNP, SCG1, BMP2A, which are located in proximity on both chromosomes.

F2Dock: Fast Fourier Protein-Protein Docking

PubMed Central

Bajaj, Chandrajit; Chowdhury, Rezaul; Siddavanahalli, Vinay

2009-01-01

The functions of proteins is often realized through their mutual interactions. Determining a relative transformation for a pair of proteins and their conformations which form a stable complex, reproducible in nature, is known as docking. It is an important step in drug design, structure determination and understanding function and structure relationships. In this paper we extend our non-uniform fast Fourier transform docking algorithm to include an adaptive search phase (both translational and rotational) and thereby speed up its execution. We have also implemented a multithreaded version of the adaptive docking algorithm for even faster execution on multicore machines. We call this protein-protein docking code F2Dock (F2 = Fast Fourier). We have calibrated F2Dock based on an extensive experimental study on a list of benchmark complexes and conclude that F2Dock works very well in practice. Though all docking results reported in this paper use shape complementarity and Coulombic potential based scores only, F2Dock is structured to incorporate Lennard-Jones potential and re-ranking docking solutions based on desolvation energy. PMID:21071796

Max-E47, a Designed Minimalist Protein that Targets the E-Box DNA Site In Vivo and In Vitro

PubMed Central

Xu, Jing; Chen, Gang; De Jong, Antonia T.; Shahravan, S. Hesam; Shin, Jumi A.

2009-01-01

Max-E47 is a designed hybrid protein comprising the Max DNA-binding basic region and E47 HLH dimerization subdomain. In the yeast one-hybrid system (Y1H), Max-E47 shows strong transcriptional activation from the E-box site, 5'-CACGTG, targeted by the Myc/Max/Mad network of transcription factors; two mutants, Max-E47Y and Max-E47YF, activate more weakly from the E-box in the Y1H. Quantitative fluorescence anisotropy titrations to gain free energies of protein:DNA binding gave low nM Kd values for the native MaxbHLHZ, Max-E47, and the Y and YF mutants binding to the E-box site (14 nM, 15 nM, 9 nM, and 6 nM, respectively), with no detectable binding to a nonspecific control duplex. Because these minimalist, E-box-binding hybrids have no activation domain and no interactions with the c-MycbHLHZ, as shown by the yeast two-hybrid assay, they can potentially serve as dominant-negative inhibitors that suppress activation of E-box-responsive genes targeted by transcription factors including the c-Myc/Max complex. As proof-of-principle, we used our modified Y1H, which allows direct competition between two proteins vying for a DNA target, to show that Max-E47 effectively outcompetes the native MaxbHLHZ for the E-box; weaker competition is observed from the two mutants, consistent with Y1H results. These hybrids provide a minimalist scaffold for further exploration of the relationship between protein structure and DNA-binding function and may have applications as protein therapeutics or biochemical probes capable of targeting the E-box site. PMID:19449889

Molecular and functional characterization of two drought-induced zinc finger proteins, ZmZnF1 and ZmZnF2 from maize kernels

USDA-ARS?s Scientific Manuscript database

We have isolated two cDNA clones encoding Zinc Finger proteins, designated as ZmZnF1 and ZmZnF2, from water-stressed maize kernels. Sequence analyses indicates that ZmZnF1 is homologous to the A20/AN1-type zinc finger protein and contains the zinc finger motif of Cx2–Cx10–CxCx4Cx2Hx5HxC. Whereas ZmZ...

Gene transfer of high-mobility group box 1 box-A domain in a rat acute liver failure model.

PubMed

Tanaka, Masayuki; Shinoda, Masahiro; Takayanagi, Atsushi; Oshima, Go; Nishiyama, Ryo; Fukuda, Kazumasa; Yagi, Hiroshi; Hayashida, Tetsu; Masugi, Yohei; Suda, Koichi; Yamada, Shingo; Miyasho, Taku; Hibi, Taizo; Abe, Yuta; Kitago, Minoru; Obara, Hideaki; Itano, Osamu; Takeuchi, Hiroya; Sakamoto, Michiie; Tanabe, Minoru; Maruyama, Ikuro; Kitagawa, Yuko

2015-04-01

High-mobility group box 1 (HMGB1) has recently been identified as an important mediator of various kinds of acute and chronic inflammation. The protein encoded by the box-A domain of the HMGB1 gene is known to act as a competitive inhibitor of HMGB1. In this study, we investigated whether box-A gene transfer results in box-A protein production in rats and assessed therapeutic efficacy in vivo using an acute liver failure (ALF) model. Three types of adenovirus vectors were constructed-a wild type and two mutants-and a mutant vector was then selected based on the secretion from HeLa cells. The secreted protein was subjected to a tumor necrosis factor (TNF) production inhibition test in vitro. The vector was injected via the portal vein in healthy Wistar rats to confirm box-A protein production in the liver. The vector was then injected via the portal vein in rats with ALF. Western blot analysis showed enhanced expression of box-A protein in HeLa cells transfected with one of the mutant vectors. The culture supernatant from HeLa cells transfected with the vector inhibited TNF-α production from macrophages. Expression of box-A protein was confirmed in the transfected liver at 72 h after transfection. Transfected rats showed decreased hepatic enzymes, plasma HMGB1, and hepatic TNF-α messenger RNA levels, and histologic findings and survival were significantly improved. HMGB1 box-A gene transfer results in box-A protein production in the liver and appears to have a beneficial effect on ALF in rats. Copyright © 2015 Elsevier Inc. All rights reserved.

SARAH 3.2: Dirac gauginos, UFO output, and more

NASA Astrophysics Data System (ADS)

Staub, Florian

2013-07-01

SARAH is a Mathematica package optimized for the fast, efficient and precise study of supersymmetric models beyond the MSSM: a new model can be defined in a short form and all vertices are derived. This allows SARAH to create model files for FeynArts/FormCalc, CalcHep/CompHep and WHIZARD/O'Mega. The newest version of SARAH now provides the possibility to create model files in the UFO format which is supported by MadGraph 5, MadAnalysis 5, GoSam, and soon by Herwig++. Furthermore, SARAH also calculates the mass matrices, RGEs and 1-loop corrections to the mass spectrum. This information is used to write source code for SPheno in order to create a precision spectrum generator for the given model. This spectrum-generator-generator functionality as well as the output of WHIZARD and CalcHep model files has seen further improvement in this version. Also models including Dirac gauginos are supported with the new version of SARAH, and additional checks for the consistency of the implementation of new models have been created. Program summaryProgram title:SARAH Catalogue identifier: AEIB_v2_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AEIB_v2_0.html Program obtainable from: CPC Program Library, Queen's University, Belfast, N. Ireland Licensing provisions: Standard CPC licence, http://cpc.cs.qub.ac.uk/licence/licence.html No. of lines in distributed program, including test data, etc.: 3 22 411 No. of bytes in distributed program, including test data, etc.: 3 629 206 Distribution format: tar.gz Programming language: Mathematica. Computer: All for which Mathematica is available. Operating system: All for which Mathematica is available. Classification: 11.1, 11.6. Catalogue identifier of previous version: AEIB_v1_0 Journal reference of previous version: Comput. Phys. Comm. 182 (2011) 808 Does the new version supersede the previous version?: Yes, the new version includes all known features of the previous version but also provides the new features mentioned below

GPS-ARM: Computational Analysis of the APC/C Recognition Motif by Predicting D-Boxes and KEN-Boxes

PubMed Central

Ren, Jian; Cao, Jun; Zhou, Yanhong; Yang, Qing; Xue, Yu

2012-01-01

Anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase incorporated with Cdh1 and/or Cdc20 recognizes and interacts with specific substrates, and faithfully orchestrates the proper cell cycle events by targeting proteins for proteasomal degradation. Experimental identification of APC/C substrates is largely dependent on the discovery of APC/C recognition motifs, e.g., the D-box and KEN-box. Although a number of either stringent or loosely defined motifs proposed, these motif patterns are only of limited use due to their insufficient powers of prediction. We report the development of a novel GPS-ARM software package which is useful for the prediction of D-boxes and KEN-boxes in proteins. Using experimentally identified D-boxes and KEN-boxes as the training data sets, a previously developed GPS (Group-based Prediction System) algorithm was adopted. By extensive evaluation and comparison, the GPS-ARM performance was found to be much better than the one using simple motifs. With this powerful tool, we predicted 4,841 potential D-boxes in 3,832 proteins and 1,632 potential KEN-boxes in 1,403 proteins from H. sapiens, while further statistical analysis suggested that both the D-box and KEN-box proteins are involved in a broad spectrum of biological processes beyond the cell cycle. In addition, with the co-localization information, we predicted hundreds of mitosis-specific APC/C substrates with high confidence. As the first computational tool for the prediction of APC/C-mediated degradation, GPS-ARM is a useful tool for information to be used in further experimental investigations. The GPS-ARM is freely accessible for academic researchers at: http://arm.biocuckoo.org. PMID:22479614

Hubble Spies a UFO

NASA Image and Video Library

2017-12-08

The Hubble Space Telescope has spotted a UFO — well, the UFO Galaxy, to be precise. NGC 2683 is a spiral galaxy seen almost edge-on, giving it the shape of a classic science fiction spaceship. This is why the astronomers at the Astronaut Memorial Planetarium and Observatory gave it this attention-grabbing nickname. While a bird’s eye view lets us see the detailed structure of a galaxy (such as this Hubble image of a barred spiral), a side-on view has its own perks. In particular, it gives astronomers a great opportunity to see the delicate dusty lanes of the spiral arms silhouetted against the golden haze of the galaxy’s core. In addition, brilliant clusters of young blue stars shine scattered throughout the disc, mapping the galaxy’s star-forming regions. Perhaps surprisingly, side-on views of galaxies like this one do not prevent astronomers from deducing their structures. Studies of the properties of the light coming from NGC 2683 suggest that this is a barred spiral galaxy, even though the angle we see it at does not let us see this directly. NGC 2683, discovered on 5 February 1788 by the famous astronomer William Herschel, lies in the Northern constellation of Lynx. A constellation named not because of its resemblance to the feline animal, but because it is fairly faint, requiring the “sensitive eyes of a cat” to discern it. And when you manage to get a look at it, you’ll find treasures like this, making it well worth the effort. This image is produced from two adjacent fields observed in visible and infrared light by Hubble’s Advanced Camera for Surveys. A narrow strip which appears slightly blurred and crosses most the image horizontally is a result of a gap between Hubble’s detectors. This strip has been patched using images from observations of the galaxy made by ground-based telescopes, which show significantly less detail. The field of view is approximately 6.5 by 3.3 arcminutes. Credit: ESA/Hubble & NASA NASA image use policy. NASA

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

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

Yu, Jungeun; Shin, Bongjin; Park, Eui-Soon

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 andmore » 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.« less

A novel member of the SAF (scaffold attachment factor)-box protein family inhibits gene expression and induces apoptosis

PubMed Central

Chan, Ching Wan; Lee, Youn-Bok; Uney, James; Flynn, Andrea; Tobias, Jonathan H.; Norman, Michael

2007-01-01

The SLTM [SAF (scaffold attachment factor)-like transcription modulator] protein contains a SAF-box DNA-binding motif and an RNA-binding domain, and shares an overall identity of 34% with SAFB1 {scaffold attachment factor-B1; also known as SAF-B (scaffold attachment factor B), HET [heat-shock protein 27 ERE (oestrogen response element) and TATA-box-binding protein] or HAP (heterogeneous nuclear ribonucleoprotein A1-interacting protein)}. Here, we show that SLTM is localized to the cell nucleus, but excluded from nucleoli, and to a large extent it co-localizes with SAFB1. In the nucleus, SLTM has a punctate distribution and it does not co-localize with SR (serine/arginine) proteins. Overexpression of SAFB1 has been shown to exert a number of inhibitory effects, including suppression of oestrogen signalling. Although SLTM also suppressed the ability of oestrogen to activate a reporter gene in MCF-7 breast-cancer cells, inhibition of a constitutively active β-galactosidase gene suggested that this was primarily the consequence of a generalized inhibitory effect on transcription. Measurement of RNA synthesis, which showed a particularly marked inhibition of [3H]uridine incorporation into mRNA, supported this conclusion. In addition, analysis of cell-cycle parameters, chromatin condensation and cytochrome c release showed that SLTM induced apoptosis in a range of cultured cell lines. Thus the inhibitory effects of SLTM on gene expression appear to result from generalized down-regulation of mRNA synthesis and initiation of apoptosis consequent upon overexpressing the protein. While indicating a crucial role for SLTM in cellular function, these results also emphasize the need for caution when interpreting phenotypic changes associated with manipulation of protein expression levels. PMID:17630952

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

PubMed

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.

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

PubMed

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

2018-03-01

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

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

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

Yunus, Abdul S.; Jackson, Trent P.; Crisafi, Katherine

2010-01-20

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 themore » 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

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

PubMed

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.

UFO: A THREE-DIMENSIONAL NEUTRON DIFFUSION CODE FOR THE IBM 704

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

Auerbach, E.H.; Jewett, J.P.; Ketchum, M.A.

A description of UFO, a code for the solution of the fewgroup neutron diffusion equation in three-dimensional Cartesian coordinates on the IBM 704, is given. An accelerated Liebmann flux iteration scheme is used, and optimum parameters can be calculated by the code whenever they are required. The theory and operation of the program are discussed. (auth)

The divergently transcribed genes encoding yeast ribosomal proteins L46 and S24 are activated by shared RPG-boxes.

PubMed Central

Kraakman, L S; Mager, W H; Maurer, K T; Nieuwint, R T; Planta, R J

1989-01-01

Transcription of the majority of the ribosomal protein (rp) genes in yeast is activated through common cis-acting elements, designated RPG-boxes. These elements have been shown to act as specific binding sites for the protein factor TUF/RAP1/GRF1 in vitro. Two such elements occur in the intergenic region separating the divergently transcribed genes encoding L46 and S24. To investigate whether the two RPG-boxes mediate transcription activation of both the L46 and S24 gene, two experimental strategies were followed: cloning of the respective genes on multicopy vectors and construction of fusion genes. Cloning of the L46 + S24 gene including the intergenic region in a multicopy yeast vector indicated that both genes are transcriptionally active. Using constructs in which only the S24 or the L46 gene is present, with or without the intergenic region, we obtained evidence that the intergenic region is indispensable for transcription activation of either gene. To demarcate the element(s) responsible for this activation, fusions of the intergenic region in either orientation to the galK reporter gene were made. Northern analysis of the levels of hybrid mRNA demonstrated that the intergenic region can serve as an heterologous promoter when it is in the 'S24-orientation'. Surprisingly, however, when fused in the reverse orientation the intergenic region did hardly confer transcription activity on the fusion gene. Furthermore, a 274 bp FnuDII-FnuDII fragment from the intergenic region that contains the RPG-boxes, could replace the naturally occurring upstream activation site (UASrpg) of the L25 rp-gene only when inserted in the 'S24-orientation'. Removal of 15 bp from the FnuDII fragment appeared to be sufficient to obtain transcription activation in the 'L46 orientation' as well. Analysis of a construct in which the RPG-boxes were selectively deleted from the promoter region of the L46 gene indicated that the RPG-boxes are needed for efficient transcriptional activation of

The divergently transcribed genes encoding yeast ribosomal proteins L46 and S24 are activated by shared RPG-boxes.

PubMed

Kraakman, L S; Mager, W H; Maurer, K T; Nieuwint, R T; Planta, R J

1989-12-11

Transcription of the majority of the ribosomal protein (rp) genes in yeast is activated through common cis-acting elements, designated RPG-boxes. These elements have been shown to act as specific binding sites for the protein factor TUF/RAP1/GRF1 in vitro. Two such elements occur in the intergenic region separating the divergently transcribed genes encoding L46 and S24. To investigate whether the two RPG-boxes mediate transcription activation of both the L46 and S24 gene, two experimental strategies were followed: cloning of the respective genes on multicopy vectors and construction of fusion genes. Cloning of the L46 + S24 gene including the intergenic region in a multicopy yeast vector indicated that both genes are transcriptionally active. Using constructs in which only the S24 or the L46 gene is present, with or without the intergenic region, we obtained evidence that the intergenic region is indispensable for transcription activation of either gene. To demarcate the element(s) responsible for this activation, fusions of the intergenic region in either orientation to the galK reporter gene were made. Northern analysis of the levels of hybrid mRNA demonstrated that the intergenic region can serve as an heterologous promoter when it is in the 'S24-orientation'. Surprisingly, however, when fused in the reverse orientation the intergenic region did hardly confer transcription activity on the fusion gene. Furthermore, a 274 bp FnuDII-FnuDII fragment from the intergenic region that contains the RPG-boxes, could replace the naturally occurring upstream activation site (UASrpg) of the L25 rp-gene only when inserted in the 'S24-orientation'. Removal of 15 bp from the FnuDII fragment appeared to be sufficient to obtain transcription activation in the 'L46 orientation' as well. Analysis of a construct in which the RPG-boxes were selectively deleted from the promoter region of the L46 gene indicated that the RPG-boxes are needed for efficient transcriptional activation of

High Mobility Group Box 1 Protein as an Auxiliary Biomarker for Dengue Diagnosis

PubMed Central

Allonso, Diego; Vázquez, Susana; Guzmán, Maria G.; Mohana-Borges, Ronaldo

2013-01-01

Despite the availability of many methods for rapid and early diagnosis of dengue, there is still a need to develop new approaches that not only combine low cost, specificity, and sensitivity, but also are capable of accurately detecting secondary infection in the early stages of the disease. We report the potential of the high mobility group box 1 protein as an auxiliary biomarker for early dengue diagnosis. We tested a 205-sample serum panel that included negative and positive samples from primary and secondary dengue cases, as well as samples from patients with dengue-like symptoms. We observed that high mobility group box 1 protein was generally detected only in dengue-positive samples for persons with primary and secondary infections. These results highlight the possibility of using this endogenous molecule as an auxiliary biomarker to aid in dengue detection and improve current methods for early diagnosis of dengue. PMID:23269659

Identification of SFBB-containing canonical and noncanonical SCF complexes in pollen of apple (Malus × domestica).

PubMed

Minamikawa, Mai F; Koyano, Ruriko; Kikuchi, Shinji; Koba, Takato; Sassa, Hidenori

2014-01-01

Gametophytic self-incompatibility (GSI) of Rosaceae, Solanaceae and Plantaginaceae is controlled by a single polymorphic S locus. The S locus contains at least two genes, S-RNase and F-box protein encoding gene SLF/SFB/SFBB that control pistil and pollen specificity, respectively. Generally, the F-box protein forms an E3 ligase complex, SCF complex with Skp1, Cullin1 (CUL1) and Rbx1, however, in Petunia inflata, SBP1 (S-RNase binding protein1) was reported to play the role of Skp1 and Rbx1, and form an SCFSLF-like complex for ubiquitination of non-self S-RNases. On the other hand, in Petunia hybrida and Petunia inflata of Solanaceae, Prunus avium and Pyrus bretschneideri of Rosaceae, SSK1 (SLF-interacting Skp1-like protein1) is considered to form the SCFSLF/SFB complex. Here, we isolated pollen-expressed apple homologs of SSK1 and CUL1, and named MdSSK1, MdCUL1A and MdCUL1B. MdSSK1 was preferentially expressed in pollen, but weakly in other organs analyzed, while, MdCUL1A and MdCUL1B were almost equally expressed in all the organs analyzed. MdSSK1 transcript abundance was significantly (>100 times) higher than that of MdSBP1. In vitro binding assays showed that MdSSK1 and MdSBP1 interacted with MdSFBB1-S9 and MdCUL1, and MdSFBB1-S9 interacted more strongly with MdSSK1 than with MdSBP1. The results suggest that both MdSSK1-containing SCFSFBB1 and MdSBP1-containing SCFSFBB1-like complexes function in pollen of apple, and the former plays a major role.

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

PubMed

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.

Development of a Flexible Seal for a 60 psi Cryogenic Pressure Box

NASA Technical Reports Server (NTRS)

Glass, David E.

1998-01-01

A cryogenic pressure box test facility has been designed and fabricated for use at NASA Langley Research Center (LaRC) to subject 5 ft x 6 ft curved panels to cryogenic temperatures and biaxial tensile loads. The cryogenic pressure box is capable of testing curved panels down to -423 F (20 K) with 54 psig maximum pressure. The key challenge in the design and fabrication of the pressure box was the development of a seal that could remain flexible at -423 F and contain 60 psi gaseous helium as the pressurization gas. A C-shaped seal was developed using a Gore-tex woven fabric. Mechanical testing of the fabric at room and elevated temperature, liquid nitrogen temperature, and liquid helium temperature demonstrated the strength and creep resistance of the material over the desired operating range. A small scale cryogenic pressure box was used to test prototype seals at cryogenic temperatures up to 60 psi. Preliminary tests indicated that excessive leakage was present through the seal. As a result, an aluminized mylar liner was placed inside the Gore-tex seal to reduce leakage through the seal. The final seal configuration resulted in minimal pressure loss during seal testing.

Neurochemical aftermath of amateur boxing.

PubMed

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

The KNOXI Transcription Factor SHOOT MERISTEMLESS Regulates Floral Fate in Arabidopsis.

PubMed

Roth, Ohad; Alvarez, John; Levy, Matan; Bowman, John L; Ori, Naomi; Shani, Eilon

2018-05-09

Plants have evolved a unique and conserved developmental program that enables the conversion of leaves into floral organs. Elegant genetic and molecular work has identified key regulators of flower meristem identity. However, further understanding of flower meristem specification has been hampered by redundancy and by pleiotropic effects. The KNOXI transcription factor SHOOT MERISTEMLESS (STM) is a well-characterized regulator of shoot apical meristem maintenance. Arabidopsis thaliana stm loss-of-function mutants arrest shortly after germination, and therefore the knowledge on later roles of STM in later processes, including flower development, is limited. Here, we uncover a role for STM in the specification of flower meristem identity. Silencing STM in the APETALA1 (AP1) expression domain in the ap1-4 mutant background resulted in a leafy-flower phenotype, and an intermediate stm-2 allele enhanced the flower meristem identity phenotype of ap1-4. Transcriptional profiling of STM perturbation suggested that STM activity affects multiple floral fate genes, among them the F-Box protein-encoding gene UNUSUAL FLORAL ORGANS (UFO). In agreement with this notion, stm-2 enhanced the ufo-2 floral fate phenotype, and ectopic UFO expression rescued the leafy flowers in genetic backgrounds with compromised AP1 and STM activities. This work suggests a genetic mechanism that underlies the activity of STM in the specification of flower meristem identity. © 2018 American Society of Plant Biologists. All rights reserved.

Multiple interactions amongst floral homeotic MADS box proteins.

PubMed Central

Davies, B; Egea-Cortines, M; de Andrade Silva, E; Saedler, H; Sommer, H

1996-01-01

Most known floral homeotic genes belong to the MADS box family and their products act in combination to specify floral organ identity by an unknown mechanism. We have used a yeast two-hybrid system to investigate the network of interactions between the Antirrhinum organ identity gene products. Selective heterodimerization is observed between MADS box factors. Exclusive interactions are detected between two factors, DEFICIENS (DEF) and GLOBOSA (GLO), previously known to heterodimerize and control development of petals and stamens. In contrast, a third factor, PLENA (PLE), which is required for reproductive organ development, can interact with the products of MADS box genes expressed at early, intermediate and late stages. We also demonstrate that heterodimerization of DEF and GLO requires the K box, a domain not found in non-plant MADS box factors, indicating that the plant MADS box factors may have different criteria for interaction. The association of PLENA and the temporally intermediate MADS box factors suggests that part of their function in mediating between the meristem and organ identity genes is accomplished through direct interaction. These data reveal an unexpectedly complex network of interactions between the factors controlling flower development and have implications for the determination of organ identity. Images PMID:8861961

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

PubMed

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

Cloning, characterization, and expression analysis of the DEAD-box family genes, Fc-vasa and Fc-PL10a, in Chinese shrimp ( Fenneropenaeus chinensis)

NASA Astrophysics Data System (ADS)

Zhou, Qianru; Shao, Mingyu; Qin, Zhenkui; Kyoung, Ho Kang; Zhang, Zhifeng

2010-01-01

RNA helicases of the DEAD-box and related families are involved in various cellular processes including DNA replication, DNA repair, and RNA processing. However, the function of DEAD-box proteins in aquaculture species is poorly understood at molecular level. We obtained the full-length cDNA sequences of two genes encoding helicase-related proteins, Fc-vasa and Fc-PL10a, from the testes of Chinese shrimp, Fenneropenaeus chinensis. The two predicted amino acid sequences contain all the conserved motifs characterized by the DEAD-box family and several RGG repeats in the N-terminal regions. Homology and phylogenetic analyses indicate that they belong to the vasa and PL10 subfamilies. The three-dimensional structures of the two proteins were predicted with a homology modeling approach. Both core proteins consist of two tandem RecA-like domains similar to those of the DEAD-box RNA helicase. Using reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR we found that Fc-vasa was expressed specifically in the adult gonads. Transcription decreased in the ovary but increased in the testis during gonadal development. Fc-PL10a expression was widely distributed in the tissues we examined. Using in situ hybridization, we demonstrated that the Fc-vasa transcript is localized to the cytoplasm of the spermatogonia and oocytes. Thus, our results suggest that Fc-vasa plays an important role in germ-line development, and has utility as a germ cell lineage marker which will help to generate new insight into the origin and differentiation of germ cells as well as the regulation of reproduction in F. chinensis.

A truncated F-box protein confers the dwarfism in cucumber

USDA-ARS?s Scientific Manuscript database

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 ...

rse, a novel receptor-type tyrosine kinase with homology to Axl/Ufo, is expressed at high levels in the brain.

PubMed

Mark, M R; Scadden, D T; Wang, Z; Gu, Q; Goddard, A; Godowski, P J

1994-04-08

We have isolated cDNA clones that encode the human and murine forms of a novel receptor-type tyrosine kinase termed Rse. Sequence analysis indicates that human Rse contains 890 amino acids, with an extracellular region composed of two immunoglobulin-like domains followed by two fibronectin type III domains. Murine Rse contains 880 amino acids and shares 90% amino acid identity with its human counterpart. Rse is structurally similar to the receptor-type tyrosine kinase Axl/Ufo, and the two proteins have 35 and 63% sequence identity in their extracellular and intracellular domains, respectively. To study the synthesis and activation of this putative receptor-type tyrosine kinase, we constructed a version of Rse (termed gD-Rse, where gD represents glycoprotein D) that contains an NH2-terminal epitope tag. NIH3T3 cells were engineered to express gD-Rse, which could be detected at the cell surface by fluorescence-activated cell sorting. Moreover, gD-Rse was rapidly phosphorylated on tyrosine residues upon incubation of the cells with an antibody directed against the epitope tag, suggesting that rse encodes an active tyrosine kinase. In the human tissues we examined, the highest level of expression of rse mRNA was observed in the brain; rse mRNA was also detected in the premegakaryocytopoietic cell lines CMK11-5 and Dami. The gene for rse was localized to human chromosome 15.

49 CFR 178.515 - Standards for reconstituted wood boxes.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Standards for reconstituted wood boxes. 178.515... 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...

Phenomenology of the Higgs effective Lagrangian via F eynR ules

NASA Astrophysics Data System (ADS)

Alloul, Adam; Fuks, Benjamin; Sanz, Verónica

2014-04-01

The Higgs discovery and the lack of any other hint for new physics favor a description of non-standard Higgs physics in terms of an effective field theory. We present an implementation of a general Higgs effective Lagrangian containing operators up to dimension six in the framework of F eynR ules and provide details on the translation between the mass and interaction bases, in particular for three- and four-point interaction vertices involving Higgs and gauge bosons. We illustrate the strengths of this implementation by using the UFO interface of F eynR ules capable to generate model files that can be understood by the M adG raph 5 event generator and that have the specificity to contain all interaction vertices, without any restriction on the number of external legs or on the complexity of the Lorentz structures. We then investigate several new physics effects in total rates and differential distributions for different Higgs production modes, including gluon fusion, associated production with a gauge boson and di-Higgs production. We finally study contact interactions of gauge and Higgs bosons to fermions.

The importance of RSV F protein conformation in VLPs in stimulation of neutralizing antibody titers in mice previously infected with RSV.

PubMed

Cullen, Lori M; Schmidt, Madelyn R; Morrison, Trudy G

2017-12-02

Respiratory syncytial virus (RSV) is a significant respiratory pathogen but no vaccine is available. RSV infections present 2 major, unique problems. First, humans can experience repeated infections caused by the same virus sero-group indicating that protective memory responses to RSV infection are defective. Second, most people have been infected with RSV by age 5. Immune responses to these infections, while poorly protective, could impact the effectiveness of a vaccine. The goal of this study was to assess the generation of protective immune responses in mice previously infected with RSV by virus-like particle (VLP) vaccine candidates containing a stabilized pre-fusion form of the RSV F protein or a stabilized post-fusion F protein. We report that a single immunization of RSV-experienced animals with a stabilized pre-fusion F protein VLP stimulated high titers of neutralizing antibody while a single injection of a post-fusion F protein VLP or a second RSV infection only weakly stimulated neutralizing antibody titers. These results suggest that prior RSV infection can induce neutralizing antibody memory responses, which can be activated by pre-F protein VLPs but not by post-F protein VLPs or a subsequent infection. Thus the F protein conformation has a major impact on enhancing production of neutralizing antibodies in RSV-experienced animals. Furthermore, although both VLPs contained the same RSV G protein, the pre-F VLP stimulated significantly higher titers of total anti-G protein IgG than the post-F VLP in both naïve and RSV-experienced animals. Thus the F protein conformation also influences anti-G protein responses.

The Tomato Transcription Factor Pti4 Regulates Defense-Related Gene Expression via GCC Box and Non-GCC Box cis ElementsW⃞

PubMed Central

Chakravarthy, Suma; Tuori, Robert P.; D'Ascenzo, Mark D.; Fobert, Pierre R.; Després, Charles; Martin, Gregory B.

2003-01-01

The tomato transcription factor Pti4, an ethylene-responsive factor (ERF), interacts physically with the disease resistance protein Pto and binds the GCC box cis element that is present in the promoters of many pathogenesis-related (PR) genes. We reported previously that Arabidopsis plants expressing Pti4 constitutively express several GCC box–containing PR genes and show reduced disease symptoms compared with wild-type plants after inoculation with Pseudomonas syringae pv tomato or Erysiphe orontii. To gain insight into how genome-wide gene expression is affected by Pti4, we used serial analysis of gene expression (SAGE) to compare transcripts in wild-type and Pti4-expressing Arabidopsis plants. SAGE provided quantitative measurements of >20,000 transcripts and identified the 50 most highly expressed genes in Arabidopsis vegetative tissues. Comparison of the profiles from wild-type and Pti4-expressing Arabidopsis plants revealed 78 differentially abundant transcripts encoding defense-related proteins, protein kinases, ribosomal proteins, transporters, and two transcription factors (TFs). Many of the genes identified were expressed differentially in wild-type Arabidopsis during infection by Pseudomonas syringae pv tomato, supporting a role for them in defense-related processes. Unexpectedly, the promoters of most Pti4-regulated genes did not have a GCC box. Chromatin immunoprecipitation experiments confirmed that Pti4 binds in vivo to promoters lacking this cis element. Potential binding sites for ERF, MYB, and GBF TFs were present in statistically significantly increased numbers in promoters regulated by Pti4. Thus, Pti4 appears to regulate gene expression directly by binding the GCC box and possibly a non-GCC box element and indirectly by either activating the expression of TF genes or interacting physically with other TFs. PMID:14630974

Transcriptional regulation of cellular ageing by the CCAAT box-binding factor CBF/NF-Y.

PubMed

Matuoka, Koozi; Chen, Kuang Yu

2002-09-01

Cellular ageing is a systematic process affecting the entirety of cell structure and function. Since changes in gene expression are extensive and global during ageing, involvement of general transcription regulators in the phenomenon is likely. Here, we focus on NF-Y, the major CCAAT box-binding factor, which exerts differential regulation on a wide variety of genes through its interaction with the CCAAT box present in as many as 25% of the eukaryotic genes. When a cell ages, senescing signals arise, typically through DNA damage due to oxidative stress or telomere shortening, and are transduced to proteins such as p53, retinoblastoma protein, and phosphatidylinositol 3-kinase. Among them, activated p53 family proteins suppress the function of NF-Y and thereby downregulate a set of cell cycle-related genes, including E2F1, which further leads to downregulation of E2F-regulated genes and cell cycle arrest. The p53 family also induces other ageing phenotypes such as morphological alterations and senescence-associated beta-galactosidase (SA-gal) presumably by upregulation of some genes through NF-Y suppression. In fact, the activities of NF-Y and E2F decrease during ageing and a dominant negative NF-YA induces SA-gal. Based on these observations, NF-Y appears to play an important role in the process of cellular ageing.

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

PubMed Central

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

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

ERIC Educational Resources Information Center

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…

SRY-box-containing Gene 2 Regulation of Nuclear Receptor Tailless (Tlx) Transcription in Adult Neural Stem Cells*

PubMed Central

Shimozaki, Koji; Zhang, Chun-Li; Suh, Hoonkyo; Denli, Ahmet M.; Evans, Ronald M.; Gage, Fred H.

2012-01-01

Adult neurogenesis is maintained by self-renewable neural stem cells (NSCs). Their activity is regulated by multiple signaling pathways and key transcription factors. However, it has been unclear whether these factors interplay with each other at the molecular level. Here we show that SRY-box-containing gene 2 (Sox2) and nuclear receptor tailless (TLX) form a molecular network in adult NSCs. We observed that both Sox2 and TLX proteins bind to the upstream region of Tlx gene. Sox2 positively regulates Tlx expression, whereas the binding of TLX to its own promoter suppresses its transcriptional activity in luciferase reporter assays. Such TLX-mediated suppression can be antagonized by overexpressing wild-type Sox2 but not a mutant lacking the transcriptional activation domain. Furthermore, through regions involved in DNA-binding activity, Sox2 and TLX physically interact to form a complex on DNAs that contain a consensus binding site for TLX. Finally, depletion of Sox2 revealed the potential negative feedback loop of TLX expression that is antagonized by Sox2 in adult NSCs. These data suggest that Sox2 plays an important role in Tlx transcription in cultured adult NSCs. PMID:22194602

SRY-box-containing gene 2 regulation of nuclear receptor tailless (Tlx) transcription in adult neural stem cells.

PubMed

Shimozaki, Koji; Zhang, Chun-Li; Suh, Hoonkyo; Denli, Ahmet M; Evans, Ronald M; Gage, Fred H

2012-02-17

Adult neurogenesis is maintained by self-renewable neural stem cells (NSCs). Their activity is regulated by multiple signaling pathways and key transcription factors. However, it has been unclear whether these factors interplay with each other at the molecular level. Here we show that SRY-box-containing gene 2 (Sox2) and nuclear receptor tailless (TLX) form a molecular network in adult NSCs. We observed that both Sox2 and TLX proteins bind to the upstream region of Tlx gene. Sox2 positively regulates Tlx expression, whereas the binding of TLX to its own promoter suppresses its transcriptional activity in luciferase reporter assays. Such TLX-mediated suppression can be antagonized by overexpressing wild-type Sox2 but not a mutant lacking the transcriptional activation domain. Furthermore, through regions involved in DNA-binding activity, Sox2 and TLX physically interact to form a complex on DNAs that contain a consensus binding site for TLX. Finally, depletion of Sox2 revealed the potential negative feedback loop of TLX expression that is antagonized by Sox2 in adult NSCs. These data suggest that Sox2 plays an important role in Tlx transcription in cultured adult NSCs.

Geophysical variables and behavior: XXVI. A response to Rutkowski's critique of the tectonic strain hypothesis for UFO phenomena.

PubMed

Persinger, M A

1985-04-01

The tectonic strain hypothesis for many reports of UFOs (UFORs), primarily odd luminosities and metallic-looking phenomena, has been criticized on the basis of inadequate data. This reply begins with the distinction between the empirical basis for the association between UFORs and seismic activity, the hypothesis, and laboratory experiments. It is emphasized that criticisms of data should be based upon empirical criteria rather than value judgments about scientific credibility. Multivariate and bivariate analyses have indicated systematic relationships between UFORs and earthquake measures within several different areas and for different historical periods. However, the physical mechanisms for the generation of individual UFO events and their relationship to UFORs require closer examination.

The importance of RSV F protein conformation in VLPs in stimulation of neutralizing antibody titers in mice previously infected with RSV

PubMed Central

Cullen, Lori M.; Schmidt, Madelyn R.; Morrison, Trudy G.

2017-01-01

ABSTRACT Respiratory syncytial virus (RSV) is a significant respiratory pathogen but no vaccine is available. RSV infections present 2 major, unique problems. First, humans can experience repeated infections caused by the same virus sero-group indicating that protective memory responses to RSV infection are defective. Second, most people have been infected with RSV by age 5. Immune responses to these infections, while poorly protective, could impact the effectiveness of a vaccine. The goal of this study was to assess the generation of protective immune responses in mice previously infected with RSV by virus-like particle (VLP) vaccine candidates containing a stabilized pre-fusion form of the RSV F protein or a stabilized post-fusion F protein. We report that a single immunization of RSV-experienced animals with a stabilized pre-fusion F protein VLP stimulated high titers of neutralizing antibody while a single injection of a post-fusion F protein VLP or a second RSV infection only weakly stimulated neutralizing antibody titers. These results suggest that prior RSV infection can induce neutralizing antibody memory responses, which can be activated by pre-F protein VLPs but not by post-F protein VLPs or a subsequent infection. Thus the F protein conformation has a major impact on enhancing production of neutralizing antibodies in RSV-experienced animals. Furthermore, although both VLPs contained the same RSV G protein, the pre-F VLP stimulated significantly higher titers of total anti-G protein IgG than the post-F VLP in both naïve and RSV-experienced animals. Thus the F protein conformation also influences anti-G protein responses. PMID:28604155

Proteomic Identification of Putative MicroRNA394 Target Genes in Arabidopsis thaliana Identifies Major Latex Protein Family Members Critical for Normal Development.

PubMed

Litholdo, Celso G; Parker, Benjamin L; Eamens, Andrew L; Larsen, Martin R; Cordwell, Stuart J; Waterhouse, Peter M

2016-06-01

Expression of the F-Box protein Leaf Curling Responsiveness (LCR) is regulated by microRNA, miR394, and alterations to this interplay in Arabidopsis thaliana produce defects in leaf polarity and shoot apical meristem organization. Although the miR394-LCR node has been documented in Arabidopsis, the identification of proteins targeted by LCR F-box itself has proven problematic. Here, a proteomic analysis of shoot apices from plants with altered LCR levels identified a member of the Latex Protein (MLP) family gene as a potential LCR F-box target. Bioinformatic and molecular analyses also suggested that other MLP family members are likely to be targets for this post-translational regulation. Direct interaction between LCR F-Box and MLP423 was validated. Additional MLP members had reduction in protein accumulation, in varying degrees, mediated by LCR F-Box. Transgenic Arabidopsis lines, in which MLP28 expression was reduced through an artificial miRNA technology, displayed severe developmental defects, including changes in leaf patterning and morphology, shoot apex defects, and eventual premature death. These phenotypic characteristics resemble those of Arabidopsis plants modified to over-express LCR Taken together, the results demonstrate that MLPs are driven to degradation by LCR, and indicate that MLP gene family is target of miR394-LCR regulatory node, representing potential targets for directly post-translational regulation mediated by LCR F-Box. In addition, MLP28 family member is associated with the LCR regulation that is critical for normal Arabidopsis development. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Proteomic Identification of Putative MicroRNA394 Target Genes in Arabidopsis thaliana Identifies Major Latex Protein Family Members Critical for Normal Development*

PubMed Central

Litholdo, Celso G.; Parker, Benjamin L.; Eamens, Andrew L.; Larsen, Martin R.; Cordwell, Stuart J.; Waterhouse, Peter M.

2016-01-01

Expression of the F-Box protein Leaf Curling Responsiveness (LCR) is regulated by microRNA, miR394, and alterations to this interplay in Arabidopsis thaliana produce defects in leaf polarity and shoot apical meristem organization. Although the miR394-LCR node has been documented in Arabidopsis, the identification of proteins targeted by LCR F-box itself has proven problematic. Here, a proteomic analysis of shoot apices from plants with altered LCR levels identified a member of the Latex Protein (MLP) family gene as a potential LCR F-box target. Bioinformatic and molecular analyses also suggested that other MLP family members are likely to be targets for this post-translational regulation. Direct interaction between LCR F-Box and MLP423 was validated. Additional MLP members had reduction in protein accumulation, in varying degrees, mediated by LCR F-Box. Transgenic Arabidopsis lines, in which MLP28 expression was reduced through an artificial miRNA technology, displayed severe developmental defects, including changes in leaf patterning and morphology, shoot apex defects, and eventual premature death. These phenotypic characteristics resemble those of Arabidopsis plants modified to over-express LCR. Taken together, the results demonstrate that MLPs are driven to degradation by LCR, and indicate that MLP gene family is target of miR394-LCR regulatory node, representing potential targets for directly post-translational regulation mediated by LCR F-Box. In addition, MLP28 family member is associated with the LCR regulation that is critical for normal Arabidopsis development. PMID:27067051

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

PubMed

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.

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

PubMed

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.

The mouse neuronal cell surface protein F3: a phosphatidylinositol- anchored member of the immunoglobulin superfamily related to chicken contactin

PubMed Central

1989-01-01

Several members of the Ig superfamily are expressed on neural cells where they participate in surface interactions between cell bodies and processes. Their Ig domains are more closely related to each other than to Ig variable and constant domains and have been grouped into the C2 set. Here, we report the cloning and characterization of another member of this group, the mouse neuronal cell surface antigen F3. The F3 cDNA sequence contains an open reading frame that could encode a 1,020-amino acid protein consisting of a signal sequence, six Ig-like domains of the C2 type, a long premembrane region containing two segments that exhibit sequence similarity to fibronectin type III repeats and a moderately hydrophobic COOH-terminal sequence. The protein does not contain a typical transmembrane segment but appears to be attached to the membrane by a phosphatidylinositol anchor. Antibodies against the F3 protein recognize a prominent 135-kD protein in mouse brain. In fetal brain cultures, they stain the neuronal cell surface and, in cultures maintained in chemically defined medium, most prominently neurites and neurite bundles. The mouse f3 gene maps to band F of chromosome 15. The gene transcripts detected in the brain by F3 cDNA probes are developmentally regulated, the highest amounts being expressed between 1 and 2 wk after birth. The F3 nucleotide and deduced amino acid sequence show striking similarity to the recently published sequence of the chicken neuronal cell surface protein contactin. However, there are important differences between the two molecules. In contrast to F3, contactin has a transmembrane and a cytoplasmic domain. Whereas contactin is insoluble in nonionic detergent and is tightly associated with the cytoskeleton, about equal amounts of F3 distribute between buffer-soluble, nonionic detergent-soluble, and detergent- insoluble fractions. Among other neural cell surface proteins, F3 most resembles the neuronal cell adhesion protein L1, with 25% amino

The F0F1-ATP Synthase Complex Contains Novel Subunits and Is Essential for Procyclic Trypanosoma brucei

PubMed Central

Zíková, Alena; Schnaufer, Achim; Dalley, Rachel A.; Panigrahi, Aswini K.; Stuart, Kenneth D.

2009-01-01

The mitochondrial F0F1 ATP synthase is an essential multi-subunit protein complex in the vast majority of eukaryotes but little is known about its composition and role in Trypanosoma brucei, an early diverged eukaryotic pathogen. We purified the F0F1 ATP synthase by a combination of affinity purification, immunoprecipitation and blue-native gel electrophoresis and characterized its composition and function. We identified 22 proteins of which five are related to F1 subunits, three to F0 subunits, and 14 which have no obvious homology to proteins outside the kinetoplastids. RNAi silencing of expression of the F1 α subunit or either of the two novel proteins showed that they are each essential for the viability of procyclic (insect stage) cells and are important for the structural integrity of the F0F1-ATP synthase complex. We also observed a dramatic decrease in ATP production by oxidative phosphorylation after silencing expression of each of these proteins while substrate phosphorylation was not severely affected. Our procyclic T. brucei cells were sensitive to the ATP synthase inhibitor oligomycin even in the presence of glucose contrary to earlier reports. Hence, the two novel proteins appear essential for the structural organization of the functional complex and regulation of mitochondrial energy generation in these organisms is more complicated than previously thought. PMID:19436713

Blends of cysteine-containing proteins

NASA Astrophysics Data System (ADS)

Barone, Justin

2005-03-01

Many agricultural wastes are made of proteins such as keratin, lactalbumin, gluten, and albumin. These proteins contain the amino acid cysteine. Cysteine allows for the formation of inter-and intra-molecular sulfur-sulfur bonds. Correlations are made between the properties of films made from the proteins and the amino acid sequence. Blends of cysteine-containing proteins show possible synergies in physical properties at intermediate concentrations. FT-IR spectroscopy shows increased hydrogen bonding at intermediate concentrations suggesting that this contributes to increased physical properties. DSC shows limited miscibility and the formation of new crystalline phases in the blends suggesting that this too contributes.

Characterization of the Transcriptional Activators SalA and SyrF, Which Are Required for Syringomycin and Syringopeptin Production by Pseudomonas syringae pv. syringae

PubMed Central

Wang, Nian; Lu, Shi-En; Records, Angela R.; Gross, Dennis C.

2006-01-01

Production of the phytotoxins syringomycin and syringopeptin by Pseudomonas syringae pv. syringae is controlled by the regulatory genes salA and syrF. Analysis with 70-mer oligonucleotide microarrays established that the syr-syp genes responsible for synthesis and secretion of syringomycin and syringopeptin belong to the SyrF regulon. Vector pMEKm12 was successfully used to express both SalA and SyrF proteins fused to a maltose-binding protein (MBP) in Escherichia coli and P. syringae pv. syringae. Both the MBP-SalA and MBP-SyrF fusion proteins were purified by maltose affinity chromatography. Gel shift analysis revealed that the purified MBP-SyrF, but not the MBP-SalA fusion protein, bound to a 262-bp fragment of the syrB1 promoter region containing the syr-syp box. Purified MBP-SalA caused a shift of a 324-bp band containing the putative syrF promoter. Gel filtration analysis and cross-linking experiments indicated that both SalA and SyrF form homodimers in vitro. Overexpression of the N-terminal regions of SalA and SyrF resulted in decreased syringomycin production by strain B301D and reduced levels of β-glucuronidase activities of the sypA::uidA and syrB1::uidA reporters by 59% to 74%. The effect of SalA on the expression of the syr-syp genes is mediated by SyrF, which activates the syr-syp genes by directly binding to the promoter regions. Both SalA and SyrF resemble other LuxR family proteins in dimerization and interaction with promoter regions of target genes. PMID:16621822

[An electron microscopy study of the structure of polyelectrolyte microcapsules containing protein and containing no protein].

PubMed

Kazakova, L I; Dubrovskiĭ, A V; Moshkov, D A; Shabarchina, L I; Sukhorukov, B I

2007-01-01

Electron micrographs of ultrathin sections of polyelectrolyte microparticles containing protein and free from protein for the formation of which CaCO3 spherulites served as a core basis have been obtained and analyzed. Polyelectrolyte microparticles with the number of alternately layered polyelectrolyte layers of polystyrene sulfonate and polyallylamine from 6 to 11 have been studied. It follows from the data obtained that protein-free polyelectrolyte particles having the dimensions 4.5-5 mm are formations of an intricate internal organization, which consist of a set of threadlike and closed nanoelements of polyelectrolyte nature with a thickness of 20-30 nm. The particles containing six to eight polyelectrolyte layers lack the external envelope; therefore, they were called polyelectrolyte microspherulites. With the number of layers nine and more, when a polyelectrolyte envelope appears on the surface, they transfer into polyelectrolyte microcapsules. It was found that, in a protein-containing polyelectrolyte microcapsule, as distinct from protein-free polyelectrolyte microspherulite and microcapsule, polyelectrolytes are located only in the nearsurface layer, and the external spatially organized envelope restricts the internal volume filled with protein solution. As the number of polyelectrolyte layers increases, the thickness of the envelope increases. The reasons for such substantial differences in the structures of polyelectrolyte microcapsules formed on protein-containing and protein-free CaCO3 spherulite are discussed.

A Unique HMG-Box Domain of Mouse Maelstrom Binds Structured RNA but Not Double Stranded DNA

PubMed Central

Genzor, Pavol; Bortvin, Alex

2015-01-01

Piwi-interacting piRNAs are a major and essential class of small RNAs in the animal germ cells with a prominent role in transposon control. Efficient piRNA biogenesis and function require a cohort of proteins conserved throughout the animal kingdom. Here we studied Maelstrom (MAEL), which is essential for piRNA biogenesis and germ cell differentiation in flies and mice. MAEL contains a high mobility group (HMG)-box domain and a Maelstrom-specific domain with a presumptive RNase H-fold. We employed a combination of sequence analyses, structural and biochemical approaches to evaluate and compare nucleic acid binding of mouse MAEL HMG-box to that of canonical HMG-box domain proteins (SRY and HMGB1a). MAEL HMG-box failed to bind double-stranded (ds)DNA but bound to structured RNA. We also identified important roles of a novel cluster of arginine residues in MAEL HMG-box in these interactions. Cumulatively, our results suggest that the MAEL HMG-box domain may contribute to MAEL function in selective processing of retrotransposon RNA into piRNAs. In this regard, a cellular role of MAEL HMG-box domain is reminiscent of that of HMGB1 as a sentinel of immunogenic nucleic acids in the innate immune response. PMID:25807393

Synergistic effects of ATP and RNA binding to human DEAD-box protein DDX1.

PubMed

Kellner, Julian N; Reinstein, Jochen; Meinhart, Anton

2015-03-11

RNA helicases of the DEAD-box protein family form the largest group of helicases. The human DEAD-box protein 1 (DDX1) plays an important role in tRNA and mRNA processing, is involved in tumor progression and is also hijacked by several virus families such as HIV-1 for replication and nuclear export. Although important in many cellular processes, the mechanism of DDX1's enzymatic function is unknown. We have performed equilibrium titrations and transient kinetics to determine affinities for nucleotides and RNA. We find an exceptional tight binding of DDX1 to adenosine diphosphate (ADP), one of the strongest affinities observed for DEAD-box helicases. ADP binds tighter by three orders of magnitude when compared to adenosine triphosphate (ATP), arresting the enzyme in a potential dead-end ADP conformation under physiological conditions. We thus suggest that a nucleotide exchange factor leads to DDX1 recycling. Furthermore, we find a strong cooperativity in binding of RNA and ATP to DDX1 that is also reflected in ATP hydrolysis. We present a model in which either ATP or RNA binding alone can partially shift the equilibrium from an 'open' to a 'closed'-state; this shift appears to be not further pronounced substantially even in the presence of both RNA and ATP as the low rate of ATP hydrolysis does not change. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

PubMed Central

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

2012-01-01

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

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

PubMed

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

2012-08-01

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

49 CFR 178.515 - Standards for reconstituted wood boxes.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 49 Transportation 3 2012-10-01 2012-10-01 false Standards for reconstituted wood boxes. 178.515... PACKAGINGS Non-bulk Performance-Oriented Packaging Standards § 178.515 Standards for reconstituted wood boxes. (a) The identification code for a reconstituted wood box is 4F. (b) Construction requirements for...

49 CFR 178.515 - Standards for reconstituted wood boxes.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Standards for reconstituted wood boxes. 178.515... PACKAGINGS Non-bulk Performance-Oriented Packaging Standards § 178.515 Standards for reconstituted wood boxes. (a) The identification code for a reconstituted wood box is 4F. (b) Construction requirements for...

49 CFR 178.515 - Standards for reconstituted wood boxes.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 49 Transportation 3 2014-10-01 2014-10-01 false Standards for reconstituted wood boxes. 178.515... PACKAGINGS Non-bulk Performance-Oriented Packaging Standards § 178.515 Standards for reconstituted wood boxes. (a) The identification code for a reconstituted wood box is 4F. (b) Construction requirements for...

49 CFR 178.515 - Standards for reconstituted wood boxes.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 3 2011-10-01 2011-10-01 false Standards for reconstituted wood boxes. 178.515... PACKAGINGS Non-bulk Performance-Oriented Packaging Standards § 178.515 Standards for reconstituted wood boxes. (a) The identification code for a reconstituted wood box is 4F. (b) Construction requirements for...

Spliced X-box Binding Protein 1 Couples the Unfolded Protein Response to Hexosamine Biosynthetic Pathway

PubMed Central

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.

2014-01-01

SUMMARY The hexosamine biosynthetic pathway (HBP) generates UDP-GlcNAc (uridine diphosphate N-acetylglucosamine) 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. PMID:24630721

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

PubMed

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.

The plant G box promoter sequence activates transcription in Saccharomyces cerevisiae and is bound in vitro by a yeast activity similar to GBF, the plant G box binding factor.

PubMed Central

Donald, R G; Schindler, U; Batschauer, A; Cashmore, A R

1990-01-01

G box and I box sequences of the Arabidopsis thaliana ribulose-bisphosphate-1,5-carboxylase small subunit (RBCS) promoter are required for expression mediated by the Arabidopsis rbcS-1A promoter in transgenic tobacco plants and are bound in vitro by factors from plant nuclear extracts termed GBF and GA-1, respectively. We show here that a -390 to -60 rbcS-1A promoter fragment containing the G box and two I boxes activates transcription from a truncated iso-1-cytochrome c (CYC1) gene promoter in Saccharomyces cerevisiae. Mutagenesis of either the rbcS-1A G box or both I box sequences eliminated the expression mediated by this fragment. When polymerized, I box oligonucleotides were also capable of enhancing expression from the truncated CYC1 promoter. Single-copy G box sequences from the Arabidopsis rbcS-1A, Arabidopsis Adh and tomato rbcS-3A promoters were more potent activators and were used in mobility shift assays to identify a DNA binding activity in yeast functionally similar to GBF. In methylation interference experiments, the binding specificity of the yeast protein was indistinguishable from that obtained with plant nuclear extracts. Images Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:2161333

Myopodin is an F-actin bundling protein with multiple independent actin-binding regions.

PubMed

Linnemann, Anja; Vakeel, Padmanabhan; Bezerra, Eduardo; Orfanos, Zacharias; Djinović-Carugo, Kristina; van der Ven, Peter F M; Kirfel, Gregor; Fürst, Dieter O

2013-02-01

The assembly of striated muscle myofibrils is a multistep process in which a variety of proteins is involved. One of the first and most important steps in myofibrillogenesis is the arrangement of thin myofilaments into ordered I-Z-I brushes, requiring the coordinated activity of numerous actin binding proteins. The early expression of myopodin prior to sarcomeric α-actinin, as well as its binding to actin, α-actinin and filamin indicate an important role for this protein in actin cytoskeleton remodelling with the precise function of myopodin in this process yet remaining to be resolved. While myopodin was previously described as a protein capable of cross-linking actin filaments into thick bundles upon transient transfections, it has remained unclear whether myopodin alone is capable of bundling actin, or if additional proteins are involved. We have therefore investigated the in vitro actin binding properties of myopodin. High speed cosedimentation assays with skeletal muscle actin confirmed direct binding of myopodin to F-actin and showed that this interaction is mediated by at least two independent actin binding sites, found in all myopodin isoforms identified to date. Furthermore, low-speed cosedimentation assays revealed that not only full length myopodin, but also the fragment containing only the second binding site, bundles microfilaments in the absence of accessory proteins. Ultrastructural analysis demonstrated that this bundling activity resembled that of α-actinin. Biochemical experiments revealed that bundling was not achieved by myopodin's ability to dimerize, indicating the presence of two individual F-actin binding sites within the second binding segment. Thus full length myopodin contains at least three F-actin binding sites. These data provide further understanding of the mechanisms by which myopodin contributes to actin reorganization during myofibril assembly.

Media Impact on Fright Reactions and Belief in UFOs: The Potential Role of Mental Imagery.

ERIC Educational Resources Information Center

Sparks, Glenn G.; And Others

1995-01-01

Explores the potential role of mental imagery for media effects in emotional responses to frightening mass media, and in the effects of the media on beliefs in UFOs. Finds that individual differences in vividness of mental imagery may play a crucial role in moderating both types of media impact. (SR)

Chlorovirus Skp1-binding ankyrin repeat protein interplay and mimicry of cellular ubiquitin ligase machinery.

PubMed

Noel, Eric A; Kang, Ming; Adamec, Jiri; Van Etten, James L; Oyler, George A

2014-12-01

The ubiquitin-proteasome system is targeted by many viruses that have evolved strategies to redirect host ubiquitination machinery. Members of the genus Chlorovirus are proposed to share an ancestral lineage with a broader group of related viruses, nucleo-cytoplasmic large DNA viruses (NCLDV). Chloroviruses encode an Skp1 homolog and ankyrin repeat (ANK) proteins. Several chlorovirus-encoded ANK repeats contain C-terminal domains characteristic of cellular F-boxes or related NCLDV chordopox PRANC (pox protein repeats of ankyrin at C-terminal) domains. These observations suggested that this unique combination of Skp1 and ANK repeat proteins might form complexes analogous to the cellular Skp1-Cul1-F-box (SCF) ubiquitin ligase complex. We identified two ANK proteins from the prototypic chlorovirus Paramecium bursaria chlorella virus-1 (PBCV-1) that functioned as binding partners for the virus-encoded Skp1, proteins A682L and A607R. These ANK proteins had a C-terminal Skp1 interactional motif that functioned similarly to cellular F-box domains. A C-terminal motif of ANK protein A682L binds Skp1 proteins from widely divergent species. Yeast two-hybrid analyses using serial domain deletion constructs confirmed the C-terminal localization of the Skp1 interactional motif in PBCV-1 A682L. ANK protein A607R represents an ANK family with one member present in all 41 sequenced chloroviruses. A comprehensive phylogenetic analysis of these related ANK and viral Skp1 proteins suggested partnered function tailored to the host alga or common ancestral heritage. Here, we show protein-protein interaction between corresponding family clusters of virus-encoded ANK and Skp1 proteins from three chlorovirus types. Collectively, our results indicate that chloroviruses have evolved complementing Skp1 and ANK proteins that mimic cellular SCF-associated proteins. Viruses have evolved ways to direct ubiquitination events in order to create environments conducive to their replication. As

SYVN1, NEDD8, and FBXO2 Proteins Regulate ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Ubiquitin-mediated Proteasomal Degradation.

PubMed

Ramachandran, Shyam; Osterhaus, Samantha R; Parekh, Kalpaj R; Jacobi, Ashley M; Behlke, Mark A; McCray, Paul B

2016-12-02

We previously reported that delivery of a microRNA-138 mimic or siRNA against SIN3A to cultured cystic fibrosis (ΔF508/ΔF508) airway epithelia partially restored ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR)-mediated cAMP-stimulated Cl - conductance. We hypothesized that dissecting this microRNA-138/SIN3A-regulated gene network would identify individual proteins contributing to the rescue of ΔF508-CFTR function. Among the genes in the network, we rigorously validated candidates using functional CFTR maturation and electrolyte transport assays in polarized airway epithelia. We found that depletion of the ubiquitin ligase SYVN1, the ubiquitin/proteasome system regulator NEDD8, or the F-box protein FBXO2 partially restored ΔF508-CFTR-mediated Cl - transport in primary cultures of human cystic fibrosis airway epithelia. Moreover, knockdown of SYVN1, NEDD8, or FBXO2 in combination with corrector compound 18 further potentiated rescue of ΔF508-CFTR-mediated Cl - conductance. This study provides new knowledge of the CFTR biosynthetic pathway. It suggests that SYVN1 and FBXO2 represent two distinct multiprotein complexes that may degrade ΔF508-CFTR in airway epithelia and identifies a new role for NEDD8 in regulating ΔF508-CFTR ubiquitination. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

A flexible system to capture sample vials in a storage box - the box vial scanner.

PubMed

Nowakowski, Steven E; Kressin, Kenneth R; Deick, Steven D

2009-01-01

Tracking sample vials in a research environment is a critical task and doing so efficiently can have a large impact on productivity, especially in high volume laboratories. There are several challenges to automating the capture process, including the variety of containers used to store samples. We developed a fast and robust system to capture the location of sample vials being placed in storage that allows the laboratories the flexibility to use sample containers of varying dimensions. With a single scan, this device captures the box identifier, the vial identifier and the location of each vial within a freezer storage box. The sample vials are tracked through a barcode label affixed to the cap while the boxes are tracked by a barcode label on the side of the box. Scanning units are placed at the point of use and forward data to a sever application for processing the scanned data. Scanning units consist of an industrial barcode reader mounted in a fixture positioning the box for scanning and providing lighting during the scan. The server application transforms the scan data into a list of storage locations holding vial identifiers. The list is then transferred to the laboratory database. The box vial scanner captures the IDs and location information for an entire box of sample vials into the laboratory database in a single scan. The system accommodates a wide variety of vials sizes by inserting risers under the sample box and a variety of storage box layouts are supported via the processing algorithm on the server.

Subcellular pigment distribution is altered under far-red light acclimation in cyanobacteria that contain chlorophyll f.

PubMed

Majumder, Erica L-W; Wolf, Benjamin M; Liu, Haijun; Berg, R Howard; Timlin, Jerilyn A; Chen, Min; Blankenship, Robert E

2017-11-01

Far-Red Light (FRL) acclimation is a process that has been observed in cyanobacteria and algae that can grow solely on light above 700 nm. The acclimation to FRL results in rearrangement and synthesis of new pigments and pigment-protein complexes. In this study, cyanobacteria containing chlorophyll f, Synechococcus sp. PCC 7335 and Halomicronema hongdechloris, were imaged as live cells with confocal microscopy. H. hongdechloris was further studied with hyperspectral confocal fluorescence microscopy (HCFM) and freeze-substituted thin-section transmission electron microscopy (TEM). Under FRL, phycocyanin-containing complexes and chlorophyll-containing complexes were determined to be physically separated and the synthesis of red-form phycobilisome and Chl f was increased. The timing of these responses was observed. The heterogeneity and eco-physiological response of the cells was noted. Additionally, a gliding motility for H. hongdechloris is reported.

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

PubMed Central

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

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

PubMed Central

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

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

PubMed

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 <1 s, and of the whole current TrEMBL database (>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.

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

PubMed Central

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

1996-01-01

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

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

PubMed

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.

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

PubMed

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.

Expression of chimeric ras protein with OmpF signal peptide in Escherichia coli: localization of OmpF fusion protein in the inner membrane.

PubMed

Yamamoto, T; Okawa, N; Endo, T; Kaji, A

1991-08-01

The ras gene was fused with the DNA sequence of OmpF signal peptide or with the DNA sequence of OmpF signal peptide plus the amino terminal portion of the OmpF gene. They were placed in plasmids together with the bacteriophage lambda PL promoter. These plasmids were introduced into Escherichia coli strain K-12 and the OmpF signal peptide fusion proteins were expressed. These fusion proteins were identified as 29.0 and 30.0 kDa proteins. However, processed products of these proteins were not found in the extract. The fusion proteins were localized mostly in the cytoplasm and the inner membrane, but none of them was secreted into the periplasmic space. On the other hand, the ras protein alone was found in the cytoplasm and not in the inner membrane. Viable counts of E. coli harbouring these plasmids decreased when these fused proteins were induced. Induction of the ras protein alone did not harm cells. These observations suggest that insertion of the heterologous proteins into the inner membrane may cause the bactericidal effect.

Protein F, a fibronectin-binding protein, is an adhesin of the group A streptococcus Streptococcus pyogenes.

PubMed

Hanski, E; Caparon, M

1992-07-01

Binding to fibronectin has been suggested to play an important role in adherence of the group A streptococcus Streptococcus pyrogenes to host epithelial cells; however, the identity of the streptococcal fibronectin receptor has been elusive. Here we demonstrate that the fibronectin-binding property of S. pyogenes is mediated by protein F, a bacterial surface protein that binds fibronectin at high affinity. The gene encoding protein F (prtF) produced a functional fibronectin-binding protein in Escherichia coli. Insertional mutagenesis of the cloned gene generated a mutation that resulted in the loss of fibronectin-binding activity. When this mutation was introduced into the S. pyrogenes chromosome by homologous recombination with the wild-type allele, the resulting strains no longer produced protein F and lost their ability to bind fibronectin. The mutation could be complemented by prtF introduced on a plasmid. Mutants lacking protein F had a much lower capacity to adhere to respiratory epithelial cells. These results demonstrate that protein F is an important adhesin of S. pyogenes.

Glove box for water pit applications

DOEpatents

Mills, William C [Richland, WA; Rabe, Richard A [North Fork, ID

2005-01-18

A glove box assembly that includes a glove box enclosure attached to a longitudinally extending hollow tube having an entranceway, wherein the portion of the tube is in a liquid environment. An elevator member is provided for raising an object that is introduced into the hollow tube from the liquid environment to a gas environment inside the glove box enclosure while maintaining total containment.

Geophysical variables and behavior: LIII. Epidemiological considerations for incidence of cancer and depression in areas of frequent UFO reports.

PubMed

Persinger, M A

1988-12-01

Luminous phenomena and anomalous physical forces have been hypothesized to be generated by focal tectonic strain fields that precede earthquakes. If these geophysical processes exist, then their spatial and temporal density should be greatest during periods of protracted, localized UFO reports; they might be used as dosimetric indicators. Contemporary epidemiological data concerning the health risks of power frequency electromagnetic fields and radon gas levels (expected correlates of certain tectonic strain fields), suggest that increased incidence (odds ratios greater 1:3) of brain tumors and leukemia should be evident within "flap" areas. In addition the frequency of variants of temporal lobe lability, psychological depression and posttraumatic stress should be significantly elevated. UFO field investigators, because they have repeated, intermittent close proximity to these fields, are considered to be a particularly high risk population for these disorders.

LaSota fusion (F) cleavage motif-mediated fusion activity is affected by other regions of the F protein from different genotype Newcastle disease virus in a chimeric virus: implication for virulence attenuation.

PubMed

Kim, Shin-Hee; Xiao, Sa; Collins, Peter L; Samal, Siba K

2016-06-01

The cleavage site sequence of the fusion (F) protein contributes to a wide range of virulence of Newcastle disease virus (NDV). In this study, we identified other important amino acid sequences of the F protein that affect cleavage and modulation of fusion. We generated chimeric Beaudette C (BC) viruses containing the cleavage site sequence of avirulent strain LaSota (Las-Fc) together with various regions of the F protein of another virulent strain AKO. We found that the F1 subunit is important for cleavage inhibition. Further dissection of the F1 subunit showed that replacement of four amino acids in the BC/Las-Fc protein with their AKO counterparts (T341S, M384I, T385A and I386L) resulted in an increase in fusion and replication in vitro. In contrast, the mutation N403D greatly reduced cleavage and viral replication, and affected protein conformation. These findings will be useful in developing improved live NDV vaccines and vaccine vectors.

Novel functions of prototype foamy virus Gag glycine- arginine-rich boxes in reverse transcription and particle morphogenesis.

PubMed

Müllers, Erik; Uhlig, Tobias; Stirnnagel, Kristin; Fiebig, Uwe; Zentgraf, Hanswalter; Lindemann, Dirk

2011-02-01

Prototype foamy virus (PFV) Gag lacks the characteristic orthoretroviral Cys-His motifs that are essential for various steps of the orthoretroviral replication cycle, such as RNA packaging, reverse transcription, infectivity, integration, and viral assembly. Instead, it contains three glycine-arginine-rich boxes (GR boxes) in its C terminus that putatively represent a functional equivalent. We used a four-plasmid replication-deficient PFV vector system, with uncoupled RNA genome packaging and structural protein translation, to analyze the effects of deletion and various substitution mutations within each GR box on particle release, particle-associated protein composition, RNA packaging, DNA content, infectivity, particle morphology, and intracellular localization. The degree of viral particle release by all mutants was similar to that of the wild type. Only minimal effects on Pol encapsidation, exogenous reverse transcriptase (RT) activity, and genomic viral RNA packaging were observed. In contrast, particle-associated DNA content and infectivity were drastically reduced for all deletion mutants and were undetectable for all alanine substitution mutants. Furthermore, GR box I mutants had significant changes in particle morphology, and GR box II mutants lacked the typical nuclear localization pattern of PFV Gag. Finally, it could be shown that GR boxes I and III, but not GR box II, can functionally complement each other. It therefore appears that, similar to the orthoretroviral Cys-His motifs, the PFV Gag GR boxes are important for RNA encapsidation, genome reverse transcription, and virion infectivity as well as for particle morphogenesis.

HbMADS4, a MADS-box Transcription Factor from Hevea brasiliensis, Negatively Regulates HbSRPP.

PubMed

Li, Hui-Liang; Wei, Li-Ran; Guo, Dong; Wang, Ying; Zhu, Jia-Hong; Chen, Xiong-Ting; Peng, Shi-Qing

2016-01-01

In plants MADS-box transcription factors (TFs) play important roles in growth and development. However, no plant MADS-box gene has been identified to have a function related to secondary metabolites regulation. Here, a MADS-box TF gene, designated as HbMADS4 , was isolated from Hevea brasiliensis by the yeast one-hybrid experiment to screen the latex cDNA library using the promoter of the gene encoding H. brasiliensis small rubber particle protein (HbSRPP) as bait. HbMADS4 was 984-bp containing 633-bp open reading frame encoding a deduced protein of 230 amino acid residues with a typical conserved MADS-box motif at the N terminus. HbMADS4 was preferentially expressed in the latex, but little expression was detected in the leaves, flowers, and roots. Its expression was inducible by methyl jasmonate and ethylene. Furthermore, transient over-expression and over-expression of HbMADS4 in transgenic tobacco plants significantly suppressed the activity of the HbSRP promoter. Altogether, it is proposed that HbMADS4 is a negative regulator of HbSRPP which participates in the biosynthesis of natural rubber.

Fis protein induced λF-DNA bending observed by single-pair fluorescence resonance energy transfer

NASA Astrophysics Data System (ADS)

Chi-Cheng, Fu; Wunshain, Fann; Yuan Hanna, S.

2006-03-01

Fis, a site-specific DNA binding protein, regulates many biological processes including recombination, transcription, and replication in E.coli. Fis induced DNA bending plays an important role in regulating these functions and bending angle range from ˜50 to 95 dependent on the DNA sequence. For instance, the average bending angle of λF-DNA (26 bp, 8.8nm long, contained λF binding site on the center) measured by gel mobility shift assays was ˜ 94 . But the traditional method cannot provide information about the dynamics and the angle distribution. In this study, λF-DNA was labeled with donor (Alexa Fluor 546) and acceptor (Alexa Fluor 647) dyes on its two 5' ends and the donor-acceptor distances were measured using single-pair fluorescence resonance energy transfer (sp-FRET) with and without the present of Fis protein. Combing with structure information of Fis-DNA complex, the sp-FRET results are used to estimate the protein induced DNA bending angle distribution and dynamics.

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

PubMed

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.

Identifying the substrate proteins of U-box E3s E4B and CHIP by orthogonal ubiquitin transfer.

PubMed

Bhuripanyo, Karan; Wang, Yiyang; Liu, Xianpeng; Zhou, Li; Liu, Ruochuan; Duong, Duc; Zhao, Bo; Bi, Yingtao; Zhou, Han; Chen, Geng; Seyfried, Nicholas T; Chazin, Walter J; Kiyokawa, Hiroaki; Yin, Jun

2018-01-01

E3 ubiquitin (UB) ligases E4B and carboxyl terminus of Hsc70-interacting protein (CHIP) use a common U-box motif to transfer UB from E1 and E2 enzymes to their substrate proteins and regulate diverse cellular processes. To profile their ubiquitination targets in the cell, we used phage display to engineer E2-E4B and E2-CHIP pairs that were free of cross-reactivity with the native UB transfer cascades. We then used the engineered E2-E3 pairs to construct "orthogonal UB transfer (OUT)" cascades so that a mutant UB (xUB) could be exclusively used by the engineered E4B or CHIP to label their substrate proteins. Purification of xUB-conjugated proteins followed by proteomics analysis enabled the identification of hundreds of potential substrates of E4B and CHIP in human embryonic kidney 293 cells. Kinase MAPK3 (mitogen-activated protein kinase 3), methyltransferase PRMT1 (protein arginine N -methyltransferase 1), and phosphatase PPP3CA (protein phosphatase 3 catalytic subunit alpha) were identified as the shared substrates of the two E3s. Phosphatase PGAM5 (phosphoglycerate mutase 5) and deubiquitinase OTUB1 (ovarian tumor domain containing ubiquitin aldehyde binding protein 1) were confirmed as E4B substrates, and β-catenin and CDK4 (cyclin-dependent kinase 4) were confirmed as CHIP substrates. On the basis of the CHIP-CDK4 circuit identified by OUT, we revealed that CHIP signals CDK4 degradation in response to endoplasmic reticulum stress.

Identifying the substrate proteins of U-box E3s E4B and CHIP by orthogonal ubiquitin transfer

PubMed Central

Bhuripanyo, Karan; Wang, Yiyang; Liu, Xianpeng; Zhou, Li; Liu, Ruochuan; Duong, Duc; Zhao, Bo; Bi, Yingtao; Zhou, Han; Chen, Geng; Seyfried, Nicholas T.; Chazin, Walter J.; Kiyokawa, Hiroaki; Yin, Jun

2018-01-01

E3 ubiquitin (UB) ligases E4B and carboxyl terminus of Hsc70-interacting protein (CHIP) use a common U-box motif to transfer UB from E1 and E2 enzymes to their substrate proteins and regulate diverse cellular processes. To profile their ubiquitination targets in the cell, we used phage display to engineer E2-E4B and E2-CHIP pairs that were free of cross-reactivity with the native UB transfer cascades. We then used the engineered E2-E3 pairs to construct “orthogonal UB transfer (OUT)” cascades so that a mutant UB (xUB) could be exclusively used by the engineered E4B or CHIP to label their substrate proteins. Purification of xUB-conjugated proteins followed by proteomics analysis enabled the identification of hundreds of potential substrates of E4B and CHIP in human embryonic kidney 293 cells. Kinase MAPK3 (mitogen-activated protein kinase 3), methyltransferase PRMT1 (protein arginine N-methyltransferase 1), and phosphatase PPP3CA (protein phosphatase 3 catalytic subunit alpha) were identified as the shared substrates of the two E3s. Phosphatase PGAM5 (phosphoglycerate mutase 5) and deubiquitinase OTUB1 (ovarian tumor domain containing ubiquitin aldehyde binding protein 1) were confirmed as E4B substrates, and β-catenin and CDK4 (cyclin-dependent kinase 4) were confirmed as CHIP substrates. On the basis of the CHIP-CDK4 circuit identified by OUT, we revealed that CHIP signals CDK4 degradation in response to endoplasmic reticulum stress. PMID:29326975

Geophysical variables and behavior: LIII. Epidemiological considerations for incidence of cancer and depression in areas of frequent UFO reports

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

Persinger, M.A.

Luminous phenomena and anomalous physical forces have been hypothesized to be generated by focal tectonic strain fields that precede earthquakes. If these geophysical processes exist, then their spatial and temporal density should be greatest during periods of protracted, localized UFO reports; they might be used as dosimetric indicators. Contemporary epidemiological data concerning the health risks of power frequency electromagnetic fields and radon gas levels (expected correlates of certain tectonic strain fields), suggest that increased incidence (odds ratios greater 1:3) of brain tumors and leukemia should be evident within flap areas. In addition the frequency of variants of temporal lobe lability,more » psychological depression and posttraumatic stress should be significantly elevated. UFO field investigators, because they have repeated, intermittent close proximity to these fields, are considered to be a particularly high risk population for these disorders. 22 references.« less

Fauna using nest boxes in four timber types in eastern Texas

Treesearch

Richard N. Conner; Daniel Saenz; D. Craig Rudolph

1995-01-01

Occupancy of 240 nest boxes in pure pine, pine-hardwood, upland hardwood, and bottomland hardwood forests (60 boxes in each forest type) were monitored for six years on the Stephen F. Austin Experimental Forest, Nacogdoches County in eastern Texas. Three boxes were placed at twenty sites in each forest type. Initially, each site had a box with 3.2, 4.7, or 5.7 cm...

A calmodulin-binding/CGCG box DNA-binding protein family involved in multiple signaling pathways in plants

NASA Technical Reports Server (NTRS)

Yang, Tianbao; Poovaiah, B. W.

2002-01-01

We reported earlier that the tobacco early ethylene-responsive gene NtER1 encodes a calmodulin-binding protein (Yang, T., and Poovaiah, B. W. (2000) J. Biol. Chem. 275, 38467-38473). Here we demonstrate that there is one NtER1 homolog as well as five related genes in Arabidopsis. These six genes are rapidly and differentially induced by environmental signals such as temperature extremes, UVB, salt, and wounding; hormones such as ethylene and abscisic acid; and signal molecules such as methyl jasmonate, H(2)O(2), and salicylic acid. Hence, they were designated as AtSR1-6 (Arabidopsis thaliana signal-responsive genes). Ca(2+)/calmodulin binds to all AtSRs, and their calmodulin-binding regions are located on a conserved basic amphiphilic alpha-helical motif in the C terminus. AtSR1 targets the nucleus and specifically recognizes a novel 6-bp CGCG box (A/C/G)CGCG(G/T/C). The multiple CGCG cis-elements are found in promoters of genes such as those involved in ethylene signaling, abscisic acid signaling, and light signal perception. The DNA-binding domain in AtSR1 is located on the N-terminal 146 bp where all AtSR1-related proteins share high similarity but have no similarity to other known DNA-binding proteins. The calmodulin-binding nuclear proteins isolated from wounded leaves exhibit specific CGCG box DNA binding activities. These results suggest that the AtSR gene family encodes a family of calmodulin-binding/DNA-binding proteins involved in multiple signal transduction pathways in plants.

Optimization of Paclitaxel Containing pH-Sensitive Liposomes By 3 Factor, 3 Level Box-Behnken Design.

PubMed

Rane, Smita; Prabhakar, Bala

2013-07-01

The aim of this study was to investigate the combined influence of 3 independent variables in the preparation of paclitaxel containing pH-sensitive liposomes. A 3 factor, 3 levels Box-Behnken design was used to derive a second order polynomial equation and construct contour plots to predict responses. The independent variables selected were molar ratio phosphatidylcholine:diolylphosphatidylethanolamine (X1), molar concentration of cholesterylhemisuccinate (X2), and amount of drug (X3). Fifteen batches were prepared by thin film hydration method and evaluated for percent drug entrapment, vesicle size, and pH sensitivity. The transformed values of the independent variables and the percent drug entrapment were subjected to multiple regression to establish full model second order polynomial equation. F was calculated to confirm the omission of insignificant terms from the full model equation to derive a reduced model polynomial equation to predict the dependent variables. Contour plots were constructed to show the effects of X1, X2, and X3 on the percent drug entrapment. A model was validated for accurate prediction of the percent drug entrapment by performing checkpoint analysis. The computer optimization process and contour plots predicted the levels of independent variables X1, X2, and X3 (0.99, -0.06, 0, respectively), for maximized response of percent drug entrapment with constraints on vesicle size and pH sensitivity.

Working your SOCS off: The role of ASB10 and protein degradation pathways in glaucoma.

PubMed

Keller, Kate E; Wirtz, Mary K

2017-05-01

Evidence is accumulating to suggest that mutations in the Ankyrin and SOCS Box-containing protein-10 (ASB10) gene are associated with glaucoma. Since its identification in a large Oregon family with primary open-angle glaucoma (POAG), ASB10 variants have been associated with disease in US, German and Pakistani cohorts. ASB10 is a member of the ASB family of proteins, which have a common structure including a unique N-terminus, a variable number of central ankyrin (ANK) repeat domains and a suppressor of cytokine signaling (SOCS) box at the C-terminus. Mutations in ASB10 are distributed throughout the entire length of the gene including the two alternatively spliced variants of exon 1. A homozygous mutation in a Pakistani individual with POAG, which lies in the center of the SOCS box, is associated with a particularly severe form of the disease. Like other SOCS box-containing proteins, ASB10 functions in ubiquitin-mediated degradation pathways. The ANK repeats bind to proteins destined for degradation. The SOCS box recruits ubiquitin ligase proteins to form a complex to transfer ubiquitin to a substrate bound to the ANK repeats. The ubiquitin-tagged protein then enters either the proteasomal degradation pathway or the autophagic-lysosomal pathway. The choice of pathway appears to be dependent on which lysine residues are used to build polyubiquitin chains. However, these reciprocal pathways work in tandem to degrade proteins because inhibition of one pathway increases degradation via the other pathway. In this publication, we will review the literature that supports identification of ASB10 as a glaucoma-associated gene and the current knowledge of the function of the ASB10 protein. In addition, we present new data that indicates ASB10 expression is up-regulated by the inflammatory cytokines tumor necrosis factor-α and interleukin-1α. Finally, we will describe the emerging role of other SOCS box-containing proteins in protein degradation pathways in ocular cells

Coilin association with Box C/D scaRNA suggests a direct role for the Cajal body marker protein in scaRNP biogenesis

PubMed Central

Enwerem, Isioma I.; Velma, Venkatramreddy; Broome, Hanna J.; Kuna, Marija; Begum, Rowshan A.; Hebert, Michael D.

2014-01-01

ABSTRACT Spliceosomal small nuclear ribonucleoproteins (snRNPs) are enriched in the Cajal body (CB). Guide RNAs, known as small Cajal body-specific RNAs (scaRNAs), direct modification of the small nuclear RNA (snRNA) component of the snRNP. The protein WRAP53 binds a sequence motif (the CAB box) found in many scaRNAs and the RNA component of telomerase (hTR) and targets these RNAs to the CB. We have previously reported that coilin, the CB marker protein, associates with certain non-coding RNAs. For a more comprehensive examination of the RNAs associated with coilin, we have sequenced the RNA isolated from coilin immunocomplexes. A striking preferential association of coilin with the box C/D scaRNAs 2 and 9, which lack a CAB box, was observed. This association varied by treatment condition and WRAP53 knockdown. In contrast, reduction of WRAP53 did not alter the level of coilin association with hTR. Additional studies showed that coilin degrades/processes scaRNA 2 and 9, associates with active telomerase and can influence telomerase activity. These findings suggest that coilin plays a novel role in the biogenesis of box C/D scaRNPs and telomerase. PMID:24659245

The kinetochore proteins CENP-E and CENP-F directly and specifically interact with distinct BUB mitotic checkpoint Ser/Thr kinases.

PubMed

Ciossani, Giuseppe; Overlack, Katharina; Petrovic, Arsen; Huis In 't Veld, Pim J; Koerner, Carolin; Wohlgemuth, Sabine; Maffini, Stefano; Musacchio, Andrea

2018-05-10

The segregation of chromosomes during cell division relies on the function of the kinetochores, protein complexes that physically connect chromosomes with microtubules of the spindle. The metazoan proteins, centromere protein E (CENP-E) and CENP-F, are components of a fibrous layer of mitotic kinetochores named the corona. Several of their features suggest that CENP-E and CENP-F are paralogs: they are very large (comprising approximately 2700 and 3200 residues, respectively), contain abundant predicted coiled-coil structures, are C-terminally prenylated, and are endowed with microtubule-binding sites at their termini. Moreover, CENP-E contains an ATP-hydrolyzing motor domain that promotes microtubule plus end-directed motion. Here, we show that both CENP-E and CENP-F are recruited to mitotic kinetochores independently of the main corona constituent, the Rod-Zwilch-ZW10 (RZZ) complex. We identified specific interactions of CENP-F and CENP-E with budding uninhibited by benzimidazole 1 (BUB1) and BUB1-related (BUBR1) mitotic checkpoint Ser/Thr kinases, respectively, paralogous proteins involved in mitotic checkpoint control and chromosome alignment. Whereas BUBR1 was dispensable for kinetochore localization of CENP-E, BUB1 was stringently required for CENP-F localization. Through biochemical reconstitution, we demonstrated that the CENP-E-BUBR1 and CENP-F-BUB1 interactions are direct and require similar determinants, a dimeric coiled-coil in CENP-E or CENP-F and a kinase domain in BUBR1 or BUB1. Our findings are consistent with the existence of structurally similar BUB1-CENP-F and BUBR1-CENP-E complexes, supporting the notion that CENP-E and CENP-F are evolutionarily related. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of FBXO25-interacting Proteins Using an Integrated Proteomics Approach

PubMed Central

Teixeira, Felipe R.; Yokoo, Sami; Gartner, Carlos G.; Manfiolli, Adriana O.; Baqui, Munira M. A.; Assmann, Eliana M.; Maragno, Ana Leticia G. C.; Yu, Huijun; de Lanerolle, Primal; Kobarg, Jörg; Gygi, Steven P.; Gomes, Marcelo D.

2011-01-01

FBXO25 is one of 68 human F-box proteins that serve as specificity factors for a family of ubiquitin ligases composed of Skp1, Rbx1, Cullin1 and F-box protein (SCF1) that are involved in targeting proteins for destruction across the ubiquitin proteasome system. We recently reported that the FBXO25 protein accumulates in novel subnuclear structures named FBXO25-associated nuclear domains (FANDs). Combining two-step affinity purification followed by mass spectrometry with a classical two-hybrid screen, we identified 132 novel potential FBXO25 interacting partners. One of the identified proteins, β-actin, physically interacts through its N-terminus with FBXO25 and is enriched in the FBXO25 nuclear compartments. Inhibitors of actin polymerization promote a significant disruption of FANDs, indicating that they are compartments influenced by the organizational state of actin in the nucleus. Furthermore, FBXO25 antibodies interfered with RNA polymerase II transcription in vitro. Our results open new perspectives for the understanding of this novel compartment and its nuclear functions. PMID:20473970

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

PubMed

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.

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

PubMed

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.

A Network of HMG-box Transcription Factors Regulates Sexual Cycle in the Fungus Podospora anserina

PubMed Central

Ait Benkhali, Jinane; Coppin, Evelyne; Brun, Sylvain; Peraza-Reyes, Leonardo; Martin, Tom; Dixelius, Christina; Lazar, Noureddine; van Tilbeurgh, Herman; Debuchy, Robert

2013-01-01

High-mobility group (HMG) B proteins are eukaryotic DNA-binding proteins characterized by the HMG-box functional motif. These transcription factors play a pivotal role in global genomic functions and in the control of genes involved in specific developmental or metabolic pathways. The filamentous ascomycete Podospora anserina contains 12 HMG-box genes. Of these, four have been previously characterized; three are mating-type genes that control fertilization and development of the fruit-body, whereas the last one encodes a factor involved in mitochondrial DNA stability. Systematic deletion analysis of the eight remaining uncharacterized HMG-box genes indicated that none were essential for viability, but that seven were involved in the sexual cycle. Two HMG-box genes display striking features. PaHMG5, an ortholog of SpSte11 from Schizosaccharomyces pombe, is a pivotal activator of mating-type genes in P. anserina, whereas PaHMG9 is a repressor of several phenomena specific to the stationary phase, most notably hyphal anastomoses. Transcriptional analyses of HMG-box genes in HMG-box deletion strains indicated that PaHMG5 is at the hub of a network of several HMG-box factors that regulate mating-type genes and mating-type target genes. Genetic analyses revealed that this network also controls fertility genes that are not regulated by mating-type transcription factors. This study points to the critical role of HMG-box members in sexual reproduction in fungi, as 11 out of 12 members were involved in the sexual cycle in P. anserina. PaHMG5 and SpSte11 are conserved transcriptional regulators of mating-type genes, although P. anserina and S. pombe diverged 550 million years ago. Two HMG-box genes, SOX9 and its upstream regulator SRY, also play an important role in sex determination in mammals. The P. anserina and S. pombe mating-type genes and their upstream regulatory factor form a module of HMG-box genes analogous to the SRY/SOX9 module, revealing a commonality of sex

The DEAD-box helicase eIF4A: paradigm or the odd one out?

PubMed

Andreou, Alexandra Z; Klostermeier, Dagmar

2013-01-01

DEAD-box helicases catalyze the ATP-dependent unwinding of RNA duplexes. They share a helicase core formed by two RecA-like domains that carries a set of conserved motifs contributing to ATP binding and hydrolysis, RNA binding and duplex unwinding. The translation initiation factor eIF4A is the founding member of the DEAD-box protein family, and one of the few examples of DEAD-box proteins that consist of a helicase core only. It is an RNA-stimulated ATPase and a non-processive helicase that unwinds short RNA duplexes. In the catalytic cycle, a series of conformational changes couples the nucleotide cycle to RNA unwinding. eIF4A has been considered a paradigm for DEAD-box proteins, and studies of its function have revealed the governing principles underlying the DEAD-box helicase mechanism. However, as an isolated helicase core, eIF4A is rather the exception, not the rule. Most helicase modules in other DEAD-box proteins are modified, some by insertions into the RecA-like domains, and the majority by N- and C-terminal appendages. While the basic catalytic function resides within the helicase core, its modulation by insertions, additional domains or a network of interaction partners generates the diversity of DEAD-box protein functions in the cell. This review summarizes the current knowledge on eIF4A and its regulation, and discusses to what extent eIF4A serves as a model DEAD-box protein.

A genetic screen for modifiers of UFO meristem activity identifies three novel FUSED FLORAL ORGANS genes required for early flower development in Arabidopsis.

PubMed

Levin, J Z; Fletcher, J C; Chen, X; Meyerowitz, E M

1998-06-01

In a screen to identify novel genes required for early Arabidopsis flower development, we isolated four independent mutations that enhance the Ufo phenotype toward the production of filamentous structures in place of flowers. The mutants fall into three complementation groups, which we have termed FUSED FLORAL ORGANS (FFO) loci. ffo mutants have specific defects in floral organ separation and/or positioning; thus, the FFO genes identify components of a boundary formation mechanism(s) acting between developing floral organ primordia. FFO1 and FFO3 have specific functions in cauline leaf/stem separation and in first- and third-whorl floral organ separation, with FFO3 likely acting to establish and FFO1 to maintain floral organ boundaries. FFO2 acts at early floral stages to regulate floral organ number and positioning and to control organ separation within and between whorls. Plants doubly mutant for two ffo alleles display additive phenotypes, indicating that the FFO genes may act in separate pathways. Plants doubly mutant for an ffo gene and for ufo, lfy, or clv3 reveal that the FFO genes play roles related to those of UFO and LFY in floral meristem initiation and that FFO2 and FFO3 may act to control cell proliferation late in inflorescence development.

A genetic screen for modifiers of UFO meristem activity identifies three novel FUSED FLORAL ORGANS genes required for early flower development in Arabidopsis.

PubMed Central

Levin, J Z; Fletcher, J C; Chen, X; Meyerowitz, E M

1998-01-01

In a screen to identify novel genes required for early Arabidopsis flower development, we isolated four independent mutations that enhance the Ufo phenotype toward the production of filamentous structures in place of flowers. The mutants fall into three complementation groups, which we have termed FUSED FLORAL ORGANS (FFO) loci. ffo mutants have specific defects in floral organ separation and/or positioning; thus, the FFO genes identify components of a boundary formation mechanism(s) acting between developing floral organ primordia. FFO1 and FFO3 have specific functions in cauline leaf/stem separation and in first- and third-whorl floral organ separation, with FFO3 likely acting to establish and FFO1 to maintain floral organ boundaries. FFO2 acts at early floral stages to regulate floral organ number and positioning and to control organ separation within and between whorls. Plants doubly mutant for two ffo alleles display additive phenotypes, indicating that the FFO genes may act in separate pathways. Plants doubly mutant for an ffo gene and for ufo, lfy, or clv3 reveal that the FFO genes play roles related to those of UFO and LFY in floral meristem initiation and that FFO2 and FFO3 may act to control cell proliferation late in inflorescence development. PMID:9611175

The Strictly Aerobic Yeast Yarrowia lipolytica Tolerates Loss of a Mitochondrial DNA-Packaging Protein

PubMed Central

Bakkaiova, Jana; Arata, Kosuke; Matsunobu, Miki; Ono, Bungo; Aoki, Tomoyo; Lajdova, Dana; Nebohacova, Martina; Nosek, Jozef; Miyakawa, Isamu

2014-01-01

Mitochondrial DNA (mtDNA) is highly compacted into DNA-protein structures termed mitochondrial nucleoids (mt-nucleoids). The key mt-nucleoid components responsible for mtDNA condensation are HMG box-containing proteins such as mammalian mitochondrial transcription factor A (TFAM) and Abf2p of the yeast Saccharomyces cerevisiae. To gain insight into the function and organization of mt-nucleoids in strictly aerobic organisms, we initiated studies of these DNA-protein structures in Yarrowia lipolytica. We identified a principal component of mt-nucleoids in this yeast and termed it YlMhb1p (Y. lipolytica mitochondrial HMG box-containing protein 1). YlMhb1p contains two putative HMG boxes contributing both to DNA binding and to its ability to compact mtDNA in vitro. Phenotypic analysis of a Δmhb1 strain lacking YlMhb1p resulted in three interesting findings. First, although the mutant exhibits clear differences in mt-nucleoids accompanied by a large decrease in the mtDNA copy number and the number of mtDNA-derived transcripts, its respiratory characteristics and growth under most of the conditions tested are indistinguishable from those of the wild-type strain. Second, our results indicate that a potential imbalance between subunits of the respiratory chain encoded separately by nuclear DNA and mtDNA is prevented at a (post)translational level. Third, we found that mtDNA in the Δmhb1 strain is more prone to mutations, indicating that mtHMG box-containing proteins protect the mitochondrial genome against mutagenic events. PMID:24972935

Crystal structure of human PCNA in complex with the PIP box of DVC1

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

Wang, Yong; University of Chinese Academy of Sciences, 19A Yuquan Road, Shijingshan District, Beijing 100049; Xu, Min

2016-05-27

In higher eukaryotes, DVC1 (SPRTN, Spartan or C1orf124) is implicated in the translesion synthesis (TLS) pathway. DVC1 localizes to sites of DNA damage, binds to the proliferating cell nuclear antigen (PCNA) via its conserved PCNA-interacting motif (PIP box), and associates with ubiquitin selective segregase p97 and other factors, thus regulating translesion synthesis polymerases. Here, we report the crystal structure of human PCNA in complex with a peptide ({sup 321}SNSHQNVLSNYFPRVS{sup 336}) derived from human DVC1 that contains a unique YF type PIP box. Structural analysis reveals the detailed PIP box-PCNA interaction. Interestingly, substitution of Y331 with Phe severely reduces its PCNAmore » binding affinity. These findings offer new insights into the determinants of PIP box for PCNA binding. -- Highlights: •Crystal structure of PCNA in complex with DVC1{sup PIP} peptide was determined. •The Y331{sup P7}F mutation severely impairs DVC1's PCNA binding affinity. •The intramolecular hydrogen bond N326−Y331 in the 3{sub 10} helix affects DVC1's PCNA binding affinity.« less

Alternate Reading Frame Protein (F Protein) of Hepatitis C Virus: Paradoxical Effects of Activation and Apoptosis on Human Dendritic Cells Lead to Stimulation of T Cells

PubMed Central

Samrat, Subodh Kumar; Li, Wen; Singh, Shakti; Kumar, Rakesh; Agrawal, Babita

2014-01-01

Hepatitis C virus (HCV) leads to chronic infection in the majority of infected individuals due to lack, failure, or inefficiency of generated adaptive immune responses. In a minority of patients, acute infection is followed by viral clearance. The immune correlates of viral clearance are not clear yet but have been extensively investigated, suggesting that multispecific and multifunctional cellular immunity is involved. The generation of cellular immunity is highly dependent upon how antigen presenting cells (APCs) process and present various viral antigens. Various structural and non-structural HCV proteins derived from the open reading frame (ORF) have been implicated in modulation of dendritic cells (DCs) and APCs. Besides the major ORF proteins, the HCV core region also encodes an alternate reading frame protein (ARFP or F), whose function in viral pathogenesis is not clear. In the current studies, we sought to determine the role of HCV-derived ARFP in modulating dendritic cells and stimulation of T cell responses. Recombinant adenovirus vectors containing F or core protein derived from HCV (genotype 1a) were prepared and used to endogenously express these proteins in dendritic cells. We made an intriguing observation that endogenous expression of F protein in human DCs leads to contrasting effects on activation and apoptosis of DCs, allowing activated DCs to efficiently internalize apoptotic DCs. These in turn result in efficient ability of DCs to process and present antigen and to prime and stimulate F protein derived peptide-specific T cells from HCV-naive individuals. Taken together, our findings suggest important aspects of F protein in modulating DC function and stimulating T cell responses in humans. PMID:24475147

Innovative production of fungal pulp from Trametes versicolor and its application in a fungal paper box containing clove oil.

PubMed

Srikaew, Benyapa; Matan, Narumol; Aewsiri, Tanong

2017-09-01

The aims of this study were to develop fungal pulp from Trametes versicolor (white-rot fungi) and apply it with clove oil in a paper box to inhibit mold growth on the surface of peanuts. Broken rice media with different sugar solutions (2-10% w w -1 ) were prepared and then inoculated with T. versicolor mycelium at amounts from 0.5 to 1.5% w w -1 . Fungal pulp and commercial paper (50 g) at different ratios (100:0, 70:30, 50:50, 30:70 and 0:100) were mixed and prepared before being placed into a stainless box (5 cm long by 5 cm wide). For the antimicrobial activity against Aspergillus flavus on peanuts, a paper box was incorporated with 2.5, 5 and 7.5% w w -1 of clove oil, eugenol, caryophyllene, and a combination of eugenol and caryophyllene at ratios of 7:1, 4:4, and 1:7. Results indicated that the highest fungal pulp biomass of T. versicolor in broken rice media was found when using 6% sugar with 1% mycelium inoculums. Fungal pulp and commercial paper at the ratio of 70:30 produced the highest value of hardness. The paper box containing clove oil at 7.5% w w -1 inhibited A. flavus on peanuts for at least 28 days while the control had mold growth within 3 days. Combining eugenol and caryophyllene, the main components of clove oil, at the ratio of 7:1 (7.5% w w -1 ) in the paper box should be a key factor to inhibit A. flavus during storage.

The RecF protein antagonizes RecX function via direct interaction

PubMed Central

Lusetti, Shelley L.; Hobbs, Michael D.; Stohl, Elizabeth A.; Chitteni-Pattu, Sindhu; Inman, Ross B.; Seifert, H. Steven; Cox, Michael M.

2014-01-01

Summary The RecX protein inhibits RecA filament extension leading to net filament disassembly. The RecF protein physically interacts with the RecX protein and protects RecA from the inhibitory effects of RecX. In vitro, efficient RecA filament formation onto SSB-coated circular single-stranded DNA in the presence of RecX occurs only when all of the RecFOR proteins are present. The RecOR proteins contribute only to RecA filament nucleation onto SSB-coated single-stranded DNA and are unable to counter the inhibitory effects of RecX on RecA filaments. RecF protein uniquely supports substantial RecA filament extension in the presence of RecX. In vivo, RecF protein counters a RecX-mediated inhibition of plasmid recombination. Thus, a significant positive contribution of RecF to RecA filament assembly is to antagonize the effects of the negative modulator, RecX, specifically during the extension phase. PMID:16387652

Comparative Analysis of the 15.5kD Box C/D snoRNP Core Protein in the Primitive Eukaryote Giardia lamblia Reveals Unique Structural and Functional Features

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

Biswas, Shyamasri; Buhrman, Greg; Gagnon, Keith

2012-07-11

Box C/D ribonucleoproteins (RNP) guide the 2'-O-methylation of targeted nucleotides in archaeal and eukaryotic rRNAs. The archaeal L7Ae and eukaryotic 15.5kD box C/D RNP core protein homologues initiate RNP assembly by recognizing kink-turn (K-turn) motifs. The crystal structure of the 15.5kD core protein from the primitive eukaryote Giardia lamblia is described here to a resolution of 1.8 {angstrom}. The Giardia 15.5kD protein exhibits the typical {alpha}-{beta}-{alpha} sandwich fold exhibited by both archaeal L7Ae and eukaryotic 15.5kD proteins. Characteristic of eukaryotic homologues, the Giardia 15.5kD protein binds the K-turn motif but not the variant K-loop motif. The highly conserved residues ofmore » loop 9, critical for RNA binding, also exhibit conformations similar to those of the human 15.5kD protein when bound to the K-turn motif. However, comparative sequence analysis indicated a distinct evolutionary position between Archaea and Eukarya. Indeed, assessment of the Giardia 15.5kD protein in denaturing experiments demonstrated an intermediate stability in protein structure when compared with that of the eukaryotic mouse 15.5kD and archaeal Methanocaldococcus jannaschii L7Ae proteins. Most notable was the ability of the Giardia 15.5kD protein to assemble in vitro a catalytically active chimeric box C/D RNP utilizing the archaeal M. jannaschii Nop56/58 and fibrillarin core proteins. In contrast, a catalytically competent chimeric RNP could not be assembled using the mouse 15.5kD protein. Collectively, these analyses suggest that the G. lamblia 15.5kD protein occupies a unique position in the evolution of this box C/D RNP core protein retaining structural and functional features characteristic of both archaeal L7Ae and higher eukaryotic 15.5kD homologues.« less

The Crystal Structure of the Escherichia coli Autoinducer-2 Processing Protein LsrF

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

Diaz, Z.; Xavier, K; Miller, S

2009-01-01

Many bacteria produce and respond to the quorum sensing signal autoinducer-2 (AI-2). Escherichia coli and Salmonella typhimurium are among the species with the lsr operon, an operon containing AI-2 transport and processing genes that are up regulated in response to AI-2. One of the Lsr proteins, LsrF, has been implicated in processing the phosphorylated form of AI-2. Here, we present the structure of LsrF, unliganded and in complex with two phospho-AI-2 analogues, ribose-5-phosphate and ribulose-5-phosphate. The crystal structure shows that LsrF is a decamer of (??)8-barrels that exhibit a previously unseen N-terminal domain swap and have high structural homology withmore » aldolases that process phosphorylated sugars. Ligand binding sites and key catalytic residues are structurally conserved, strongly implicating LsrF as a class I aldolase.« less

Copper Causes Regiospecific Formation of C4F8-Containing Six-Membered Rings and their Defluorination/Aromatization to C4F4-Containing Rings in Triphenylene/1,4-C4F8I2 Reactions

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

Rippy, Kerry C.; Bukovsky, Eric V.; Clikeman, Tyler T.

The presence of Cu in reactions of triphenylene (TRPH) and 1,4-C4F8I2 at 360 °C led to regiospecific substitution of TRPH ortho C(β) atoms to form C4F8-containing rings, completely suppressing substitution on C(α) atoms. In addition, Cu caused selective reductive-defluorination/aromatization (RD/A) to form C4F4- containing aromatic rings. Without Cu, the reactions of TRPH and 1,4- C4F8I2 were not regiospecific and no RD/A was observed. These results, supported by DFT calculations, are the first examples of Cupromoted (i) regiospecific perfluoroannulation, (ii) preparative C–F activation, and (iii) RD/A. HPLC-purified products were characterized by X-ray diffraction, low-temperature PES, and 1H/19F NMR.

Nucleoplasmic Nup98 controls gene expression by regulating a DExH/D-box protein.

PubMed

Capitanio, Juliana S; Montpetit, Ben; Wozniak, Richard W

2018-01-01

The nucleoporin Nup98 has been linked to the regulation of transcription and RNA metabolism, 1-3 but the mechanisms by which Nup98 contributes to these processes remains largely undefined. Recently, we uncovered interactions between Nup98 and several DExH/D-box proteins (DBPs), a protein family well-known for modulating gene expression and RNA metabolism. 4-6 Analysis of Nup98 and one of these DBPs, DHX9, showed that they directly interact, their association is facilitated by RNA, and Nup98 binding stimulates DHX9 ATPase activity. 7 Furthermore, these proteins were dependent on one another for their proper association with a subset of gene loci to control transcription and modulate mRNA splicing. 7 On the basis of these observations, we proposed that Nup98 functions to regulate DHX9 activity within the nucleoplasm. 7 Since Nup98 is associated with several DBPs, regulation of DHX9 by Nup98 may represent a paradigm for understanding how Nup98, and possibly other FG-Nup proteins, could direct the diverse cellular activities of multiple DBPs.

SEPALLATA3: the 'glue' for MADS box transcription factor complex formation

PubMed Central

Immink, Richard GH; Tonaco, Isabella AN; de Folter, Stefan; Shchennikova, Anna; van Dijk, Aalt DJ; Busscher-Lange, Jacqueline; Borst, Jan W; Angenent, Gerco C

2009-01-01

Background Plant MADS box proteins play important roles in a plethora of developmental processes. In order to regulate specific sets of target genes, MADS box proteins dimerize and are thought to assemble into multimeric complexes. In this study a large-scale yeast three-hybrid screen is utilized to provide insight into the higher-order complex formation capacity of the Arabidopsis MADS box family. SEPALLATA3 (SEP3) has been shown to mediate complex formation and, therefore, special attention is paid to this factor in this study. Results In total, 106 multimeric complexes were identified; in more than half of these at least one SEP protein was present. Besides the known complexes involved in determining floral organ identity, various complexes consisting of combinations of proteins known to play a role in floral organ identity specification, and flowering time determination were discovered. The capacity to form this latter type of complex suggests that homeotic factors play essential roles in down-regulation of the MADS box genes involved in floral timing in the flower via negative auto-regulatory loops. Furthermore, various novel complexes were identified that may be important for the direct regulation of the floral transition process. A subsequent detailed analysis of the APETALA3, PISTILLATA, and SEP3 proteins in living plant cells suggests the formation of a multimeric complex in vivo. Conclusions Overall, these results provide strong indications that higher-order complex formation is a general and essential molecular mechanism for plant MADS box protein functioning and attribute a pivotal role to the SEP3 'glue' protein in mediating multimerization. PMID:19243611

ABC-F Proteins Mediate Antibiotic Resistance through Ribosomal Protection.

PubMed

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.

Identification of a Skp1-like protein interacting with SFB, the pollen S determinant of the gametophytic self-incompatibility in Prunus.

PubMed

Matsumoto, Daiki; Yamane, Hisayo; Abe, Kazuyuki; Tao, Ryutaro

2012-07-01

Many species in Rosaceae, Solanaceae, and Plantaginaceae exhibit S-RNase-based self-incompatibility (SI). In this system, the pistil and pollen specificities are determined by S-RNase and the S locus F-box protein, respectively. The pollen S determinant F-box protein in Prunus (Rosaceae) is referred to by two different terms, SFB (for S-haplotype-specific F-box protein) and SLF (for S locus F box), whereas it is called SLF in Solanaceae and Plantaginaceae. Prunus SFB is thought to be a molecule indispensable for its cognate S-RNase to exert cytotoxicity and to arrest pollen tube growth in incompatible reactions. Although recent studies have demonstrated the molecular function of SCF(SLF) in the SI reaction of Solanaceae and Plantaginaceae, how SFB participates in the Prunus SI mechanism remains to be elucidated. Here we report the identification of sweet cherry (Prunus avium) SFB (PavSFB)-interacting Skp1-like1 (PavSSK1) using a yeast (Saccharomyces cerevisiae) two-hybrid screening against the pollen cDNA library. Phylogenetic analysis showed that PavSSK1 belongs to the same clade as Antirrhinum hispanicum SLF-interacting Skp1-like1 and Petunia hybrida SLF-interacting Skp1-like1 (PhSSK1). In yeast, PavSSK1 interacted not only with PavSFBs from different S haplotypes and Cullin1-likes (PavCul1s), but also with S-locus F-box-likes. A pull-down assay confirmed the interactions between PavSSK1 and PavSFB and between PavSSK1 and PavCul1s. These results collectively indicate that PavSSK1 could be a functional component of the SCF complex and that PavSFB may function as a component of the SCF complex. We discuss the molecular function of PavSFB in self-/nonself-recognition in the gametophytic SI of Prunus.

Seed storage protein gene promoters contain conserved DNA motifs in Brassicaceae, Fabaceae and Poaceae

PubMed Central

Fauteux, François; Strömvik, Martina V

2009-01-01

Background Accurate computational identification of cis-regulatory motifs is difficult, particularly in eukaryotic promoters, which typically contain multiple short and degenerate DNA sequences bound by several interacting factors. Enrichment in combinations of rare motifs in the promoter sequence of functionally or evolutionarily related genes among several species is an indicator of conserved transcriptional regulatory mechanisms. This provides a basis for the computational identification of cis-regulatory motifs. Results We have used a discriminative seeding DNA motif discovery algorithm for an in-depth analysis of 54 seed storage protein (SSP) gene promoters from three plant families, namely Brassicaceae (mustards), Fabaceae (legumes) and Poaceae (grasses) using backgrounds based on complete sets of promoters from a representative species in each family, namely Arabidopsis (Arabidopsis thaliana (L.) Heynh.), soybean (Glycine max (L.) Merr.) and rice (Oryza sativa L.) respectively. We have identified three conserved motifs (two RY-like and one ACGT-like) in Brassicaceae and Fabaceae SSP gene promoters that are similar to experimentally characterized seed-specific cis-regulatory elements. Fabaceae SSP gene promoter sequences are also enriched in a novel, seed-specific E2Fb-like motif. Conserved motifs identified in Poaceae SSP gene promoters include a GCN4-like motif, two prolamin-box-like motifs and an Skn-1-like motif. Evidence of the presence of a variant of the TATA-box is found in the SSP gene promoters from the three plant families. Motifs discovered in SSP gene promoters were used to score whole-genome sets of promoters from Arabidopsis, soybean and rice. The highest-scoring promoters are associated with genes coding for different subunits or precursors of seed storage proteins. Conclusion Seed storage protein gene promoter motifs are conserved in diverse species, and different plant families are characterized by a distinct combination of conserved motifs

Surface Density of the Hendra G Protein Modulates Hendra F Protein-Promoted Membrane Fusion: Role for Hendra G Protein Trafficking and Degradation

PubMed Central

Whitman, Shannon D.; Dutch, Rebecca Ellis

2007-01-01

Hendra virus, like most paramyxoviruses, requires both a fusion (F) and attachment (G) protein for promotion of cell-cell fusion. Recent studies determined that Hendra F is proteolytically processed by the cellular protease cathepsin L after endocytosis. This unique cathepsin L processing results in a small percentage of Hendra F on the cell surface. To determine how the surface densities of the two Hendra glycoproteins affect fusion promotion, we performed experiments that varied the levels of glycoproteins expressed in transfected cells. Using two different fusion assays, we found a marked increase in fusion when expression of the Hendra G protein was increased, with a 1:1 molar ratio of Hendra F:G on the cell surface resulting in optimal membrane fusion. Our results also showed that Hendra G protein levels are modulated by both more rapid protein turnover and slower protein trafficking than is seen for Hendra F. PMID:17328935

Utilizing protein-lean coproducts from corn containing recombinant pharmaceutical proteins for ethanol production.

PubMed

Paraman, Ilankovan; Moeller, Lorena; Scott, M Paul; Wang, Kan; Glatz, Charles E; Johnson, Lawrence A

2010-10-13

Protein-lean fractions of corn (maize) containing recombinant (r) pharmaceutical proteins were evaluated as a potential feedstock to produce fuel ethanol. The levels of residual r-proteins in the coproduct, distillers dry grains with solubles (DDGS), were determined. Transgenic corn lines containing recombinant green fluorescence protein (r-GFP) and a recombinant subunit vaccine of Escherichia coli enterotoxin (r-LTB), primarily expressed in endosperm, and another two corn lines containing recombinant human collagen (r-CIα1) and r-GFP, primarily expressed in germ, were used as model systems. The kernels were either ground and used for fermentation or dry fractionated to recover germ-rich fractions prior to grinding for fermentation. The finished beers of whole ground kernels and r-protein-spent endosperm solids contained 127-139 and 138-155 g/L ethanol concentrations, respectively. The ethanol levels did not differ among transgenic and normal corn feedstocks, indicating the residual r-proteins did not negatively affect ethanol production. r-Protein extraction and germ removal also did not negatively affect fermentation of the remaining mass. Most r-proteins were inactivated during the mashing process used to prepare corn for fermentation. No functionally active r-GFP or r-LTB proteins were found after fermentation of the r-protein-spent solids; however, a small quantity of residual r-CIα1 was detected in DDGS, indicating that the safety of DDGS produced from transgenic grain for r-protein production needs to be evaluated for each event. Protease treatment during fermentation completely hydrolyzed the residual r-CIα1, and no residual r-proteins were detectable in DDGS.

EVM005: an ectromelia-encoded protein with dual roles in NF-κB inhibition and virulence.

PubMed

van Buuren, Nicholas; Burles, Kristin; Schriewer, Jill; Mehta, Ninad; Parker, Scott; Buller, R Mark; Barry, Michele

2014-08-01

Poxviruses contain large dsDNA genomes encoding numerous open reading frames that manipulate cellular signalling pathways and interfere with the host immune response. The NF-κB signalling cascade is an important mediator of innate immunity and inflammation, and is tightly regulated by ubiquitination at several key points. A critical step in NF-κB activation is the ubiquitination and degradation of the inhibitor of kappaB (IκBα), by the cellular SCFβ-TRCP ubiquitin ligase complex. We show here that upon stimulation with TNFα or IL-1β, Orthopoxvirus-infected cells displayed an accumulation of phosphorylated IκBα, indicating that NF-κB activation was inhibited during poxvirus infection. Ectromelia virus is the causative agent of lethal mousepox, a natural disease that is fatal in mice. Previously, we identified a family of four ectromelia virus genes (EVM002, EVM005, EVM154 and EVM165) that contain N-terminal ankyrin repeats and C-terminal F-box domains that interact with the cellular SCF ubiquitin ligase complex. Since degradation of IκBα is catalyzed by the SCFβ-TRCP ubiquitin ligase, we investigated the role of the ectromelia virus ankyrin/F-box protein, EVM005, in the regulation of NF-κB. Expression of Flag-EVM005 inhibited both TNFα- and IL-1β-stimulated IκBα degradation and p65 nuclear translocation. Inhibition of the NF-κB pathway by EVM005 was dependent on the F-box domain, and interaction with the SCF complex. Additionally, ectromelia virus devoid of EVM005 was shown to inhibit NF-κB activation, despite lacking the EVM005 open reading frame. Finally, ectromelia virus devoid of EVM005 was attenuated in both A/NCR and C57BL/6 mouse models, indicating that EVM005 is required for virulence and immune regulation in vivo.

EVM005: An Ectromelia-Encoded Protein with Dual Roles in NF-κB Inhibition and Virulence

PubMed Central

Schriewer, Jill; Mehta, Ninad; Parker, Scott; Buller, R. Mark; Barry, Michele

2014-01-01

Poxviruses contain large dsDNA genomes encoding numerous open reading frames that manipulate cellular signalling pathways and interfere with the host immune response. The NF-κB signalling cascade is an important mediator of innate immunity and inflammation, and is tightly regulated by ubiquitination at several key points. A critical step in NF-κB activation is the ubiquitination and degradation of the inhibitor of kappaB (IκBα), by the cellular SCFβ-TRCP ubiquitin ligase complex. We show here that upon stimulation with TNFα or IL-1β, Orthopoxvirus-infected cells displayed an accumulation of phosphorylated IκBα, indicating that NF-κB activation was inhibited during poxvirus infection. Ectromelia virus is the causative agent of lethal mousepox, a natural disease that is fatal in mice. Previously, we identified a family of four ectromelia virus genes (EVM002, EVM005, EVM154 and EVM165) that contain N-terminal ankyrin repeats and C-terminal F-box domains that interact with the cellular SCF ubiquitin ligase complex. Since degradation of IκBα is catalyzed by the SCFβ-TRCP ubiquitin ligase, we investigated the role of the ectromelia virus ankyrin/F-box protein, EVM005, in the regulation of NF-κB. Expression of Flag-EVM005 inhibited both TNFα- and IL-1β-stimulated IκBα degradation and p65 nuclear translocation. Inhibition of the NF-κB pathway by EVM005 was dependent on the F-box domain, and interaction with the SCF complex. Additionally, ectromelia virus devoid of EVM005 was shown to inhibit NF-κB activation, despite lacking the EVM005 open reading frame. Finally, ectromelia virus devoid of EVM005 was attenuated in both A/NCR and C57BL/6 mouse models, indicating that EVM005 is required for virulence and immune regulation in vivo. PMID:25122471

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

PubMed

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.

UFOs and Physical Sciences

NASA Astrophysics Data System (ADS)

Teodorani, M.

1999-03-01

The psycho-sociological reasons why the academic science is not willing to face operatively and officially a hard problem such as the 'UFO phenomenon', are introduced in the ambit of an episthemological discussion. It is shown how such a phenomenon, due to his peculiar nature, might impose a drastic revision of the laws of physics which are commonly accepted. It is demonstrated how a strict application of the measurement methods which are normally adopted by physics can permit to obtain relevant quantitative results, whatever they are. As an example of such a procedure, the anomalous light phenomenology which reoccurs in the Hessdalen valley in Norway is presented, by showing that it represents so far the ideal worldly physical laboratory for the study of luminous phenomena in the low atmosphere. After describing the multiform morphologic and dynamic characteristics of the luminous component of the phenomenon as they are deduced from visual and photographic reports, the results coming out from the magnetometric, radiometric and radar monitoring operations which were carried out by Project Hessdalen in 1984, are presented in detail. Subsequently, the postumous analysis carried out by the author is shown, by pointing out the clear cohexistence of the magnetic and the optical phenomenology and the apparent correlation of the magnetic component of the phenomenon with maxima of solar activity. In a subsequent phase, the most credited physical theories, which have been proposed so far in order to interpret the phenomenon, are described and discussed, together with 'non-canonical hypotheses'. Finally, it is pointed out how the physical parameters which are expected to be measured with the proper instrumented equipment and aimed tactics and strategies, resemble strictly the methodology which is normally used during astrophysical observations.

Structure of the Newcastle disease virus F protein in the post-fusion conformation

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

Swanson, Kurt; Wen, Xiaolin; Leser, George P.

2010-11-17

The paramyxovirus F protein is a class I viral membrane fusion protein which undergoes a significant refolding transition during virus entry. Previous studies of the Newcastle disease virus, human parainfluenza virus 3 and parainfluenza virus 5 F proteins revealed differences in the pre- and post-fusion structures. The NDV Queensland (Q) F structure lacked structural elements observed in the other two structures, which are key to the refolding and fusogenic activity of F. Here we present the NDV Australia-Victoria (AV) F protein post-fusion structure and provide EM evidence for its folding to a pre-fusion form. The NDV AV F structure containsmore » heptad repeat elements missing in the previous NDV Q F structure, forming a post-fusion six-helix bundle (6HB) similar to the post-fusion hPIV3 F structure. Electrostatic and temperature factor analysis of the F structures points to regions of these proteins that may be functionally important in their membrane fusion activity.« less

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

PubMed

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.

Purification of proteins from solutions containing residual host cell proteins via preparative crystallization.

PubMed

Hekmat, Dariusch; Breitschwerdt, Peter; Weuster-Botz, Dirk

2015-09-01

To investigate quantitatively and reproducibly a scalable, preparative crystallization method in novel stirred tanks using three different protein solutions containing residual microbial host cell proteins (HCP). Lysozyme from solutions being spiked with up to 15% host cell proteins (HCP) (corresponding to 176,500 ppm) was crystallized within a 2.4-4.6 h at 93.7% yield using NaCl and glycerol. Lipase was crystallized under comparable conditions using NaCl and a mixture of two polyethylene glycols (PEG). Enhanced green fluorescent protein (eGFP) was overexpressed in E. coli yielding a solution containing 23% target protein. Residual HCP content after pre-treatment was 7-16%. eGFP was crystallized from these solutions within 1.75-4 h at 88.7% step yield using ethanol and the same mixture of two PEG as in the case of lipase. HCP contained in the solvent channels of the protein crystals could be removed by diffusive washing yielding final purities at or above 99%. Preparative crystallization can be carried out with fast kinetics and high yields from solutions containing residual impurities and may represent an attractive alternative purification method compared to preparative chromatography, especially at large production scales.

Spectroscopic studies on peptides and proteins with cysteine-containing heme regulatory motifs (HRM).

PubMed

Schubert, Erik; Florin, Nicole; Duthie, Fraser; Henning Brewitz, H; Kühl, Toni; Imhof, Diana; Hagelueken, Gregor; Schiemann, Olav

2015-07-01

The role of heme as a cofactor in enzymatic reactions has been studied for a long time and in great detail. Recently it was discovered that heme can also serve as a signalling molecule in cells but so far only few examples of this regulation have been studied. In order to discover new potentially heme-regulated proteins, we screened protein sequence databases for bacterial proteins that contain sequence features like a Cysteine-Proline (CP) motif, which is known for its heme-binding propensity. Based on this search we synthesized a series of these potential heme regulatory motifs (HRMs). We used cw EPR spectroscopy to investigate whether these sequences do indeed bind to heme and if the spin state of heme is changed upon interaction with the peptides. The corresponding proteins of two potential HRMs, FeoB and GlpF, were expressed and purified and their interaction with heme was studied by cw EPR and UV-Visible (UV-Vis) spectroscopy. Copyright © 2015 Elsevier Inc. All rights reserved.

Protein crystal growth results from shuttle flight 51-F

NASA Technical Reports Server (NTRS)

Bugg, C. E.

1985-01-01

The protein crystal growth (PCG) experiments run on 51-F were analyzed. It was found that: (1) sample stability is increased over that observed during the experiments on flight 51-D; (2) the dialysis experiments produced lysozyme crystals that were significantly larger than those obtained in our identical ground-based studies; (3) temperature fluctuations apparently caused problems during the crystallization experiments on 51-F; (4) it is indicated that teflon tape stabilizes droplets on the syringe tips; (5) samples survived during the reentry and landing in glass tips that were not stoppered with plungers; (6) from the ground-based studies, it was expected that equilibration should be complete within 2 to 4 days for all of these vapor-diffusion experiments, thus it appears that the vapor diffusion rates are somewhat slower under microgravity conditions; (7) drop tethering was highly successful, all four of the tethered drops were stable, even though they contained MPD solutions; (8) the PCG experiments on 51-F were done to assess the hardware and experimental procedures that are developed for future flights, when temperature control will be available. Lysozyme crystals obtained by microdialysis are considerably larger than those obtained on the ground, using the identical apparatus and procedures.

Analysis of a Soluble (UreD:UreF:UreG)2 Accessory Protein Complex and its Interactions with Klebsiella aerogenes Urease by Mass Spectrometry

PubMed Central

Farrugia, Mark A.; Han, Linjie; Zhong, Yueyang; Boer, Jodi L.; Ruotolo, Brandon T.; Hausinger, Robert P.

2013-01-01

Maturation of the nickel-containing urease of Klebsiella aerogenes is facilitated by the UreD, UreF, and UreG accessory proteins along with the UreE metallo-chaperone. A fusion of the maltose binding protein and UreD (MBP-UreD) was co-isolated with UreF and UreG in a soluble complex possessing a (MBP-UreD:UreF:UreG)2 quaternary structure. Within this complex a UreF:UreF interaction was identified by chemical cross-linking of the amino termini of its two UreF protomers, as shown by mass spectrometry of tryptic peptides. A pre-activation complex was formed by the interaction of (MBP-UreD:UreF:UreG)2 and urease. Mass spectrometry of intact protein species revealed a pathway for synthesis of the urease pre-activation complex in which individual hetero-trimer units of the (MBP-UreD:UreF:UreG)2 complex bind to urease. Together, these data provide important new insights into the structures of protein complexes associated with urease activation. PMID:23797863

Analysis of a Soluble (UreD:UreF:UreG)2 Accessory Protein Complex and Its Interactions with Klebsiella aerogenes Urease by Mass Spectrometry

NASA Astrophysics Data System (ADS)

Farrugia, Mark A.; Han, Linjie; Zhong, Yueyang; Boer, Jodi L.; Ruotolo, Brandon T.; Hausinger, Robert P.

2013-09-01

Maturation of the nickel-containing urease of Klebsiella aerogenes is facilitated by the UreD, UreF, and UreG accessory proteins along with the UreE metallo-chaperone. A fusion of the maltose binding protein and UreD (MBP-UreD) was co-isolated with UreF and UreG in a soluble complex possessing a (MBP-UreD:UreF:UreG)2 quaternary structure. Within this complex a UreF:UreF interaction was identified by chemical cross-linking of the amino termini of its two UreF protomers, as shown by mass spectrometry of tryptic peptides. A pre-activation complex was formed by the interaction of (MBP-UreD:UreF:UreG)2 and urease. Mass spectrometry of intact protein species revealed a pathway for synthesis of the urease pre-activation complex in which individual hetero-trimer units of the (MBP-UreD:UreF:UreG)2 complex bind to urease. Together, these data provide important new insights into the structures of protein complexes associated with urease activation.

Regulatory T Cell and Forkhead Box Protein 3 as Modulators of Immune Homeostasis

PubMed Central

Pereira, Leonn Mendes Soares; Gomes, Samara Tatielle Monteiro; Ishak, Ricardo; Vallinoto, Antonio Carlos Rosário

2017-01-01

The transcription factor forkhead box protein 3 (FOXP3) is an essential molecular marker of regulatory T cell (Treg) development in different microenvironments. Tregs are cells specialized in the suppression of inadequate immune responses and the maintenance of homeostatic tolerance. Studies have addressed and elucidated the role played by FOXP3 and Treg in countless autoimmune and infectious diseases as well as in more specific cases, such as cancer. Within this context, the present article reviews aspects of the immunoregulatory profile of FOXP3 and Treg in the management of immune homeostasis, including issues relating to pathology as well as immune tolerance. PMID:28603524

Identification of a Skp1-Like Protein Interacting with SFB, the Pollen S Determinant of the Gametophytic Self-Incompatibility in Prunus1[W

PubMed Central

Matsumoto, Daiki; Yamane, Hisayo; Abe, Kazuyuki; Tao, Ryutaro

2012-01-01

Many species in Rosaceae, Solanaceae, and Plantaginaceae exhibit S-RNase-based self-incompatibility (SI). In this system, the pistil and pollen specificities are determined by S-RNase and the S locus F-box protein, respectively. The pollen S determinant F-box protein in Prunus (Rosaceae) is referred to by two different terms, SFB (for S-haplotype-specific F-box protein) and SLF (for S locus F box), whereas it is called SLF in Solanaceae and Plantaginaceae. Prunus SFB is thought to be a molecule indispensable for its cognate S-RNase to exert cytotoxicity and to arrest pollen tube growth in incompatible reactions. Although recent studies have demonstrated the molecular function of SCFSLF in the SI reaction of Solanaceae and Plantaginaceae, how SFB participates in the Prunus SI mechanism remains to be elucidated. Here we report the identification of sweet cherry (Prunus avium) SFB (PavSFB)-interacting Skp1-like1 (PavSSK1) using a yeast (Saccharomyces cerevisiae) two-hybrid screening against the pollen cDNA library. Phylogenetic analysis showed that PavSSK1 belongs to the same clade as Antirrhinum hispanicum SLF-interacting Skp1-like1 and Petunia hybrida SLF-interacting Skp1-like1 (PhSSK1). In yeast, PavSSK1 interacted not only with PavSFBs from different S haplotypes and Cullin1-likes (PavCul1s), but also with S-locus F-box-likes. A pull-down assay confirmed the interactions between PavSSK1 and PavSFB and between PavSSK1 and PavCul1s. These results collectively indicate that PavSSK1 could be a functional component of the SCF complex and that PavSFB may function as a component of the SCF complex. We discuss the molecular function of PavSFB in self-/nonself-recognition in the gametophytic SI of Prunus. PMID:22548785

Phenotypic analysis of newly isolated short-lifespan Neurospora crassa mutant deficient in a high mobility group box protein.

PubMed

Yoshihara, Ryouhei; Li, ZhengHao; Ishimori, Keisuke; Kuwabara, Kazuki; Hatakeyama, Shin; Tanaka, Shuuitsu

2017-08-01

To elucidate genetic mechanisms affecting the lifespan of the filamentous fungus Neurospora crassa, we attempted to identify a gene of which a defect causes a short-lifespan. By screening a Neurospora knockout library, provided by the Fungal Genetics Stock Center at Kansas State University, several KO strains with a short-lifespan were isolated. FGSC#11693 is one of these, which shows similar phenotypes to known Neurospora short-lifespan mutants as follows: 1) hyphal growth ceases after about 2weeks of cultivation, despite that of the wild-type continuing for over 2years, 2) viability of conidia is lower than that of the wild-type, and 3) high sensitivity to mutagens such as methyl methanesulfonate, ultraviolet radiation, and hydroxyl urea is exhibited. The NCU number of the knocked-out gene in the KO strain is NCU02695, and recovery from the short-lifespan and mutagen sensitivity was achieved by the introduction of this gene from the wild-type. The putative amino acid sequence of the knocked-out gene contains two high mobility group box domains and a mitochondrial localization signal is found at the N-terminal of this sequence. Upon analyzing the subcellular localization of the gene product fused with GFP, GFP signals were detected in mitochondria. From these observations, the gene and KO strain were named mitochondrial high mobility group box protein 1 (MHG1) and mhg1 KO strain, respectively. The amount of mtDNA relative to the nuclear amount was lower in the mhg1 KO strain than in the wild-type. mtDNA aberration was also observed in the mhg1 KO strain. These results suggest that the MHG1 protein plays an important role in the maintenance of mitochondrial DNA, and mitochondrial abnormality caused by mtDNA aberration is responsible for the short-lifespan of the mhg1 KO strain. Copyright © 2017 Elsevier Inc. All rights reserved.

Primary structure and nuclear localization of a murine homeodomain protein.

PubMed Central

Kessel, M; Schulze, F; Fibi, M; Gruss, P

1987-01-01

The murine homeobox Hox 1.1 (m6) is the first of a cluster of six boxes on chromosome 6. Using probes and synthetic peptides derived from the Hox 1.1 sequence, we were able to isolate cDNAs and antibodies that allowed us to characterize the product of this homeobox-containing gene. From the open reading frame on the cDNA clone B21, a protein could be predicted, made up of 229 amino acids and having a calculated molecular weight of 25,740. A unique feature of this protein is that it has 15 glutamic acid residues as its carboxyl terminus, which gives it a very hydrophilic and acidic carboxyl terminal structure, most probably folding onto an alpha-helix. A second domain of six amino acids is present on the Hox 1.1 protein, which is conserved in other homeodomain proteins. Antibodies generated against synthetic peptides from the homeobox region were used in the immunoblotting procedure and revealed a major protein band of Mr 31,000 in extracts from 3T3 cells and F9 teratocarcinoma cells induced by retinoic acid and cAMP. The nuclear location of the protein was established by immunofluorescence. The presence of this protein in F9 cell nuclei is in faithful accordance with the kinetics established for the 2.4-kilobase Hox 1.1 transcript during differentiation into parietal endoderm cells. Images PMID:2885847

Benefits of a Space-Based Group System Architecture

DTIC Science & Technology

2015-06-01

Relay Satellite TRL Technology Readiness Level TT&C Telemetry, Tracking, and Control UFO UHF Follow-On xv ACKNOWLEDGMENTS I would like to...replacement with more advanced systems. An example of this addition was adding UHF Follow-On ( UFO ) satellite F11, as a gap filler between the UFO

Expression of a tetraheme protein, Desulfovibrio vulgaris Miyazaki F cytochrome c(3), in Shewanella oneidensis MR-1

NASA Technical Reports Server (NTRS)

Ozawa, K.; Tsapin, A. I.; Nealson, K. H.; Cusanovich, M. A.; Akutsu, H.

2000-01-01

Cytochrome c(3) from Desulfovibrio vulgaris Miyazaki F was successfully expressed in the facultative aerobe Shewanella oneidensis MR-1 under anaerobic, microaerophilic, and aerobic conditions, with yields of 0.3 to 0.5 mg of cytochrome/g of cells. A derivative of the broad-host-range plasmid pRK415 containing the cytochrome c(3) gene from D. vulgaris Miyazaki F was used for transformation of S. oneidensis MR-1, resulting in the production of protein product that was indistinguishable from that produced by D. vulgaris Miyazaki F, except for the presence of one extra alanine residue at the N terminus.

Bromodomain-containing Protein 4 Activates Voltage-gated Sodium Channel 1.7 Transcription in Dorsal Root Ganglia Neurons to Mediate Thermal Hyperalgesia in Rats.

PubMed

Hsieh, Ming-Chun; Ho, Yu-Cheng; Lai, Cheng-Yuan; Wang, Hsueh-Hsiao; Lee, An-Sheng; Cheng, Jen-Kun; Chau, Yat-Pang; Peng, Hsien-Yu

2017-11-01

Bromodomain-containing protein 4 binds acetylated promoter histones and promotes transcription; however, the role of bromodomain-containing protein 4 in inflammatory hyperalgesia remains unclear. Male Sprague-Dawley rats received hind paw injections of complete Freund's adjuvant to induce hyperalgesia. The dorsal root ganglia were examined to detect changes in bromodomain-containing protein 4 expression and the activation of genes involved in the expression of voltage-gated sodium channel 1.7, which is a key pain-related ion channel. The intraplantar complete Freund's adjuvant injections resulted in thermal hyperalgesia (4.0 ± 1.5 s; n = 7). The immunohistochemistry and immunoblotting results demonstrated an increase in the bromodomain-containing protein 4-expressing dorsal root ganglia neurons (3.78 ± 0.38 fold; n = 7) and bromodomain-containing protein 4 protein levels (2.62 ± 0.39 fold; n = 6). After the complete Freund's adjuvant injection, histone H3 protein acetylation was enhanced in the voltage-gated sodium channel 1.7 promoter, and cyclin-dependent kinase 9 and phosphorylation of RNA polymerase II were recruited to this area. Furthermore, the voltage-gated sodium channel 1.7-mediated currents were enhanced in neurons of the complete Freund's adjuvant rats (55 ± 11 vs. 19 ± 9 pA/pF; n = 4 to 6 neurons). Using bromodomain-containing protein 4-targeted antisense small interfering RNA to the complete Freund's adjuvant-treated rats, the authors demonstrated a reduction in the expression of bromodomain-containing protein 4 (0.68 ± 0.16 fold; n = 7), a reduction in thermal hyperalgesia (7.5 ± 1.5 s; n = 7), and a reduction in the increased voltage-gated sodium channel 1.7 currents (21 ± 4 pA/pF; n = 4 to 6 neurons). Complete Freund's adjuvant triggers enhanced bromodomain-containing protein 4 expression, ultimately leading to the enhanced excitability of nociceptive neurons and thermal hyperalgesia. This effect is

Performance oriented packaging testing of nine Mk 3 Mod 0 signal containers in PPP-B-621 wood box for packing group II solid hazardous materials. Final report

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

Libbert, K.J.

1992-10-01

A PPP-B-621 wood box containing nine Mk 3 Mod 0 Signal containers was tested for conformance to Performance Oriented Packaging criteria established by Code of Federal Regulations Title 49 CFR. The container was tested with a gross weight of 123.3 pounds (56 kilograms) and met all requirements.

Bacterial DNA segregation dynamics mediated by the polymerizing protein ParF.

PubMed

Barillà, Daniela; Rosenberg, Mark F; Nobbmann, Ulf; Hayes, Finbarr

2005-04-06

Prokaryotic DNA segregation most commonly involves members of the Walker-type ParA superfamily. Here we show that the ParF partition protein specified by the TP228 plasmid is a ParA ATPase that assembles into extensive filaments in vitro. Polymerization is potentiated by ATP binding and does not require nucleotide hydrolysis. Analysis of mutations in conserved residues of the Walker A motif established a functional coupling between filament dynamics and DNA partitioning. The partner partition protein ParG plays two separable roles in the ParF polymerization process. ParF is unrelated to prokaryotic polymerizing proteins of the actin or tubulin families, but is a homologue of the MinD cell division protein, which also assembles into filaments. The ultrastructures of the ParF and MinD polymers are remarkably similar. This points to an evolutionary parallel between DNA segregation and cytokinesis in prokaryotic cells, and reveals a potential molecular mechanism for plasmid and chromosome segregation mediated by the ubiquitous ParA-type proteins.

Molecular characterization and expression analysis of Triticum aestivum squamosa-promoter binding protein-box genes involved in ear development.

PubMed

Zhang, Bin; Liu, Xia; Zhao, Guangyao; Mao, Xinguo; Li, Ang; Jing, Ruilian

2014-06-01

Wheat (Triticum aestivum L.) is one of the most important crops in the world. Squamosa-promoter binding protein (SBP)-box genes play a critical role in regulating flower and fruit development. In this study, 10 novel SBP-box genes (TaSPL genes) were isolated from wheat ((Triticum aestivum L.) cultivar Yanzhan 4110). Phylogenetic analysis classified the TaSPL genes into five groups (G1-G5). The motif combinations and expression patterns of the TaSPL genes varied among the five groups with each having own distinctive characteristics: TaSPL20/21 in G1 and TaSPL17 in G2 mainly expressed in the shoot apical meristem and the young ear, and their expression levels responded to development of the ear; TaSPL6/15 belonging to G3 were upregulated and TaSPL1/23 in G4 were downregulated during grain development; the gene in G5 (TaSPL3) expressed constitutively. Thus, the consistency of the phylogenetic analysis, motif compositions, and expression patterns of the TaSPL genes revealed specific gene structures and functions. On the other hand, the diverse gene structures and different expression patterns suggested that wheat SBP-box genes have a wide range of functions. The results also suggest a potential role for wheat SBP-box genes in ear development. This study provides a significant beginning of functional analysis of SBP-box genes in wheat. © 2014 The Authors. Journal of Integrative Plant Biology Published by Wiley Publishing Asia Pty Ltd on behalf of Institute of Botany, Chinese Academy of Sciences.

A potent transrepression domain in the retinoblastoma protein induces a cell cycle arrest when bound to E2F sites.

PubMed Central

Sellers, W R; Rodgers, J W; Kaelin, W G

1995-01-01

An intact T/E1A-binding domain (the pocket) is necessary, but not sufficient, for the retinoblastoma protein (RB) to bind to DNA-protein complexes containing E2F and for RB to induce a G1/S block. Indirect evidence suggests that the binding of RB to E2F may, in addition to inhibiting E2F transactivation function, generate a complex capable of functioning as a transrepressor. Here we show that a chimera in which the E2F1 transactivation domain was replaced with the RB pocket could, in a DNA-binding and pocket-dependent manner, mimic the ability of RB to repress transcription and induce a cell cycle arrest. In contrast, a transdominant negative E2F1 mutant that is capable of blocking E2F-dependent transactivation did not. Fusion of the RB pocket to a heterologous DNA-binding domain unrelated to E2F likewise generated a transrepressor protein when scored against a suitable reporter. These results suggest that growth suppression by RB is due, at least in part, to transrepression mediated by the pocket domain bound to certain promoters via E2F. Images Fig. 4 Fig. 5 PMID:8524800

Reduction of erosion by protein-containing toothpastes.

PubMed

Jager, D H J; Vissink, A; Timmer, C J; Bronkhorst, E; Vieira, A M; Huysmans, M C D N J M

2013-01-01

To assess the effect of protein-containing toothpastes on the progression of dental erosion in situ (with pellicle) and in vitro (without pellicle). A combined split-mouth (extraoral water or toothpaste brushing) and crossover (type of toothpaste) setup was used. Two protein-containing (high/low concentrations of colostrum) and one nonprotein (placebo) toothpaste were investigated. Sixteen volunteers wore intraoral appliances containing 2 human enamel samples on 3 afternoons for pellicle growth during 90 min. One enamel sample was brushed for 5 s with one of the three toothpastes and subsequently exposed to a slurry of the corresponding toothpaste for 2 min. The other sample was exposed to water. Both samples were subsequently exposed to citric acid (extraorally). Loss of calcium and inorganic phosphate were determined. The same sequence of exposures was applied to 16 enamel samples in an in vitro setup without pellicle. With the in situ-formed pellicle, all toothpastes significantly reduced calcium loss compared to water brushing, although no significant differences were found among toothpastes (p = 0.073). For the loss of phosphate, a significant reduction could be found with the use of the high-protein toothpaste compared to the nonprotein toothpaste. Overall there were only slight differences between the toothpastes. Toothpaste effects were less clear in the in vitro experiment. The addition of proteins to toothpaste shows some promise for the prevention of erosion. Further research is needed to investigate the performance of the protein-containing toothpastes in longer in situ studies with regard to erosive wear. Copyright © 2012 S. Karger AG, Basel.

The Box Turtle: Room with a View on Species Decline.

ERIC Educational Resources Information Center

Belzer, Bill; Steisslinger, Mary Beth

1999-01-01

Surveys salient aspects of eastern box-turtle natural history. Explores the societal and ecological factors that have contributed to the decline of the box-turtle population. Contains 18 references. (WRM)

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

PubMed

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.

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.

PubMed

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.

Surface chemistry of photoluminescent F8BT conjugated polymer nanoparticles determines protein corona formation and internalization by phagocytic cells.

PubMed

Ahmad Khanbeigi, Raha; Abelha, Thais Fedatto; Woods, Arcadia; Rastoin, Olivia; Harvey, Richard D; Jones, Marie-Christine; Forbes, Ben; Green, Mark A; Collins, Helen; Dailey, Lea Ann

2015-03-09

Conjugated polymer nanoparticles are being developed for a variety of diagnostic and theranostic applications. The conjugated polymer, F8BT, a polyfluorene derivative, was used as a model system to examine the biological behavior of conjugated polymer nanoparticle formulations stabilized with ionic (sodium dodecyl sulfate; F8BT-SDS; ∼207 nm; -31 mV) and nonionic (pegylated 12-hydroxystearate; F8BT-PEG; ∼175 nm; -5 mV) surfactants, and compared with polystyrene nanoparticles of a similar size (PS200; ∼217 nm; -40 mV). F8BT nanoparticles were as hydrophobic as PS200 (hydrophobic interaction chromatography index value: 0.96) and showed evidence of protein corona formation after incubation with serum-containing medium; however, unlike polystyrene, F8BT nanoparticles did not enrich specific proteins onto the nanoparticle surface. J774A.1 macrophage cells internalized approximately ∼20% and ∼60% of the F8BT-SDS and PS200 delivered dose (calculated by the ISDD model) in serum-supplemented and serum-free conditions, respectively, while cell association of F8BT-PEG was minimal (<5% of the delivered dose). F8BT-PEG, however, was more cytotoxic (IC50 4.5 μg cm(-2)) than F8BT-SDS or PS200. The study results highlight that F8BT surface chemistry influences the composition of the protein corona, while the properties of the conjugated polymer nanoparticle surfactant stabilizer used determine particle internalization and biocompatibility profile.

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

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

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

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

The Unique Morgue Ubiquitination Protein Is Conserved in a Diverse but Restricted Set of Invertebrates

PubMed Central

Zhou, Ying; Carpenter, Zachary W.; Brennan, Gregory

2009-01-01

Drosophila Morgue is a unique ubiquitination protein that facilitates programmed cell death and associates with DIAP1, a critical cell death inhibitor with E3 ubiquitin ligase activity. Morgue possesses a unique combination of functional domains typically associated with distinct types of ubiquitination enzymes. This includes an F box characteristic of the substrate-binding subunit in Skp, Cullin, and F box (SCF)-type ubiquitin E3 ligase complexes and a variant ubiquitin E2 conjugase domain where the active site cysteine is replaced by a glycine. Morgue also contains a single C4-type zinc finger motif. This architecture suggests potentially novel ubiquitination activities for Morgue. In this study, we address the evolutionary origins of this distinctive protein utilizing a combination of bioinformatics and molecular biology approaches. We find that Morgue exhibits widespread but restricted phylogenetic distribution among metazoans. Morgue proteins were identified in a wide range of Protostome phyla, including Arthropoda, Annelida, Mollusca, Nematoda, and Platyhelminthes. However, with one potential exception, Morgue was not detected in Deuterostomes, including Chordates, Hemichordates, or Echinoderms. Morgue was also not found in Ctenophora, Cnidaria, Placozoa, or Porifera. Characterization of Morgue sequences within specific animal lineages suggests that gene deletion or acquisition has occurred during divergence of nematodes and that at least one arachnid expresses an atypical form of Morgue consisting only of the variant E2 conjugase domain. Analysis of the organization of several morgue genes suggests that exon-shuffling events have contributed to the evolution of the Morgue protein. These results suggest that Morgue mediates conserved and distinctive ubiquitination functions in specific cell death pathways. PMID:19602541

A Conserved Region between the Heptad Repeats of Paramyxovirus Fusion Proteins is Critical for Proper F Protein Folding†

PubMed Central

Gardner, Amanda E.; Martin, Kimberly L.; Dutch, Rebecca E.

2008-01-01

Paramyxoviruses are a diverse family which utilizes a fusion (F) protein to enter cells via fusion of the viral lipid bilayer with a target cell membrane. Although certain regions of F are known to play critical roles in membrane fusion, the function of much of the protein remains unclear. Sequence alignment of a set of paramyxovirus F proteins and analysis utilizing Block Maker identified a region of conserved amino acid sequence in a large domain between the heptad repeats of F1, designated CBF1. We employed site-directed mutagenesis to analyze the function of completely conserved residues of CBF1 in both the simian virus 5 (SV5) and Hendra virus F proteins. The majority of CBF1 point mutants were deficient in homotrimer formation, proteolytic processing, and transport to the cell surface. For some SV5 F mutants, proteolytic cleavage and surface expression could be restored by expression at 30°C, and varying levels of fusion promotion were observed at this temperature. In addition, the mutant SV5 F V402A displayed a hyperfusogenic phenotype at both 30°C and 37°C, indicating this mutation allows for efficient fusion with only an extremely small amount of cleaved, active protein. The recently published prefusogenic structure of PIV5/SV5 F [Yin, H.S., et al. (2006) Nature 439, 38–44] indicates that residues within and flanking CBF1 interact with the fusion peptide domain. Together, these data suggest that CBF1-fusion peptide interactions are critical for the initial folding of paramyxovirus F proteins from across this important viral family, and can also modulate subsequent membrane fusion promotion. PMID:17417875

The Polerovirus silencing suppressor P0 targets ARGONAUTE proteins for degradation.

PubMed

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.

Interaction of Prevotella intermedia Strain 17 Leucine-Rich Repeat Domain Protein AdpF with Eukaryotic Cells Promotes Bacterial Internalization

PubMed Central

Sengupta, Dipanwita; Kang, Dae-Joong; Anaya-Bergman, Cecilia; Wyant, Tiana; Ghosh, Arnab K.; Miyazaki, Hiroshi

2014-01-01

Prevotella intermedia is an oral bacterium implicated in a variety of oral diseases. Although internalization of this bacterium by nonphagocytic host cells is well established, the molecular players mediating the process are not well known. Here, the properties of a leucine-rich repeat (LRR) domain protein, designated AdpF, are described. This protein contains a leucine-rich region composed of 663 amino acid residues, and molecular modeling shows that it folds into a classical curved solenoid structure. The cell surface localization of recombinant AdpF (rAdpF) was confirmed by electron and confocal microscopy analyses. The recombinant form of this protein bound fibronectin in a dose-dependent manner. Furthermore, the protein was internalized by host cells, with the majority of the process accomplished within 30 min. The internalization of rAdpF was inhibited by nystatin, cytochalasin, latrunculin, nocodazole, and wortmannin, indicating that microtubules, microfilaments, and signal transduction are required for the invasion. It is noteworthy that preincubation of eukaryotic cells with AdpF increased P. intermedia 17 internalization by 5- and 10-fold for HeLa and NIH 3T3 fibroblast cell lines, respectively. The addition of the rAdpF protein was also very effective in inducing bacterial internalization into the oral epithelial cell line HN4, as well as into primary cells, including human oral keratinocytes (HOKs) and human umbilical vein endothelial cells (HUVECs). Finally, cells exposed to P. intermedia 17 internalized the bacteria more readily upon reinfection. Taken together, our data demonstrate that rAdpF plays a role in the internalization of P. intermedia 17 by a variety of host cells. PMID:24711565

Interaction of Prevotella intermedia strain 17 leucine-rich repeat domain protein AdpF with eukaryotic cells promotes bacterial internalization.

PubMed

Sengupta, Dipanwita; Kang, Dae-Joong; Anaya-Bergman, Cecilia; Wyant, Tiana; Ghosh, Arnab K; Miyazaki, Hiroshi; Lewis, Janina P

2014-06-01

Prevotella intermedia is an oral bacterium implicated in a variety of oral diseases. Although internalization of this bacterium by nonphagocytic host cells is well established, the molecular players mediating the process are not well known. Here, the properties of a leucine-rich repeat (LRR) domain protein, designated AdpF, are described. This protein contains a leucine-rich region composed of 663 amino acid residues, and molecular modeling shows that it folds into a classical curved solenoid structure. The cell surface localization of recombinant AdpF (rAdpF) was confirmed by electron and confocal microscopy analyses. The recombinant form of this protein bound fibronectin in a dose-dependent manner. Furthermore, the protein was internalized by host cells, with the majority of the process accomplished within 30 min. The internalization of rAdpF was inhibited by nystatin, cytochalasin, latrunculin, nocodazole, and wortmannin, indicating that microtubules, microfilaments, and signal transduction are required for the invasion. It is noteworthy that preincubation of eukaryotic cells with AdpF increased P. intermedia 17 internalization by 5- and 10-fold for HeLa and NIH 3T3 fibroblast cell lines, respectively. The addition of the rAdpF protein was also very effective in inducing bacterial internalization into the oral epithelial cell line HN4, as well as into primary cells, including human oral keratinocytes (HOKs) and human umbilical vein endothelial cells (HUVECs). Finally, cells exposed to P. intermedia 17 internalized the bacteria more readily upon reinfection. Taken together, our data demonstrate that rAdpF plays a role in the internalization of P. intermedia 17 by a variety of host cells.

The DEAH-box helicase Dhr1 dissociates U3 from the pre-rRNA to promote formation of the central pseudoknot.

PubMed

Sardana, Richa; Liu, Xin; Granneman, Sander; Zhu, Jieyi; Gill, Michael; Papoulas, Ophelia; Marcotte, Edward M; Tollervey, David; Correll, Carl C; Johnson, Arlen W

2015-02-01

In eukaryotes, the highly conserved U3 small nucleolar RNA (snoRNA) base-pairs to multiple sites in the pre-ribosomal RNA (pre-rRNA) to promote early cleavage and folding events. Binding of the U3 box A region to the pre-rRNA is mutually exclusive with folding of the central pseudoknot (CPK), a universally conserved rRNA structure of the small ribosomal subunit essential for protein synthesis. Here, we report that the DEAH-box helicase Dhr1 (Ecm16) is responsible for displacing U3. An active site mutant of Dhr1 blocked release of U3 from the pre-ribosome, thereby trapping a pre-40S particle. This particle had not yet achieved its mature structure because it contained U3, pre-rRNA, and a number of early-acting ribosome synthesis factors but noticeably lacked ribosomal proteins (r-proteins) that surround the CPK. Dhr1 was cross-linked in vivo to the pre-rRNA and to U3 sequences flanking regions that base-pair to the pre-rRNA including those that form the CPK. Point mutations in the box A region of U3 suppressed a cold-sensitive mutation of Dhr1, strongly indicating that U3 is an in vivo substrate of Dhr1. To support the conclusions derived from in vivo analysis we showed that Dhr1 unwinds U3-18S duplexes in vitro by using a mechanism reminiscent of DEAD box proteins.

The DEAH-box Helicase Dhr1 Dissociates U3 from the Pre-rRNA to Promote Formation of the Central Pseudoknot

PubMed Central

Granneman, Sander; Zhu, Jieyi; Gill, Michael; Papoulas, Ophelia; Marcotte, Edward M.; Tollervey, David; Correll, Carl C.; Johnson, Arlen W.

2015-01-01

In eukaryotes, the highly conserved U3 small nucleolar RNA (snoRNA) base-pairs to multiple sites in the pre-ribosomal RNA (pre-rRNA) to promote early cleavage and folding events. Binding of the U3 box A region to the pre-rRNA is mutually exclusive with folding of the central pseudoknot (CPK), a universally conserved rRNA structure of the small ribosomal subunit essential for protein synthesis. Here, we report that the DEAH-box helicase Dhr1 (Ecm16) is responsible for displacing U3. An active site mutant of Dhr1 blocked release of U3 from the pre-ribosome, thereby trapping a pre-40S particle. This particle had not yet achieved its mature structure because it contained U3, pre-rRNA, and a number of early-acting ribosome synthesis factors but noticeably lacked ribosomal proteins (r-proteins) that surround the CPK. Dhr1 was cross-linked in vivo to the pre-rRNA and to U3 sequences flanking regions that base-pair to the pre-rRNA including those that form the CPK. Point mutations in the box A region of U3 suppressed a cold-sensitive mutation of Dhr1, strongly indicating that U3 is an in vivo substrate of Dhr1. To support the conclusions derived from in vivo analysis we showed that Dhr1 unwinds U3-18S duplexes in vitro by using a mechanism reminiscent of DEAD box proteins. PMID:25710520

Characterization of two genes encoding leucine-rich repeat-containing proteins in grass carp Ctenopharyngodon idellus.

PubMed

Chang, M X; Nie, P; Xie, H X; Sun, B J; Gao, Q

2005-01-01

The cDNAs and genes of two different types of leucine-rich repeat-containing proteins from grass carp (Ctenopharyngodon idellus) were cloned. Homology search revealed that the two genes, designated as GC-GARP and GC-LRG, have 37% and 32% deduced amino-acid sequence similarities with human glycoprotein A repetitions predominant precursor (GARP) and leucine-rich alpha2-glycoprotein (LRG), respectively. The cDNAs of GC-GARP and GC-LRG encoded 664 and 339 amino acid residues, respectively. GC-GARP and GC-LRG contain many distinct structural and/or functional motifs of the leucine-rich repeat (LRR) subfamily, such as multiple conserved 11-residue segments with the consensus sequence LxxLxLxxN/CxL (x can be any amino acid). The genes GC-GARP and GC-LRG consist of two exons, with 4,782 bp and 2,119 bp in total length, respectively. The first exon of each gene contains a small 5'-untranslated region and partial open reading frame. The putative promoter region of GC-GARP was found to contain transcription factor binding sites for GATA-1, IRF4, Oct-1, IRF-7, IRF-1, AP1, GATA-box and NFAT, and the promoter region of GC-LRG for MYC-MAX, MEIS1, ISRE, IK3, HOXA9 and C/EBP alpha. Phylogenetic analysis showed that GC-GARP and mammalian GARPs were clustered into one branch, while GC-LRG and mammalian LRGs were in another branch. The GC-GARP gene was only detected in head kidney, and GC-LRG in the liver, spleen and heart in the copepod (Sinergasilus major)-infected grass carp, indicating the induction of gene expression by the parasite infection. The results obtained in the present study provide insight into the structure of fish LRR genes, and further study should be carried out to understand the importance of LRR proteins in host-pathogen interactions.

Survey of rice proteins interacting with OsFCA and OsFY proteins which are homologous to the Arabidopsis flowering time proteins, FCA and FY.

PubMed

Jang, Yun Hee; Park, Hyo-Young; Kim, Soon-Kap; Lee, Jeong Hwan; Suh, Mi Chung; Chung, Young Soo; Paek, Kyung-Hee; Kim, Jeong-Kook

2009-08-01

The FCA protein is involved in controlling flowering time and plays more general roles in RNA-mediated chromatin silencing in Arabidopsis. It contains two RNA-binding domains and a WW domain. The FCA protein interacts with FY, a polyadenylation factor, via its WW domain. We previously characterized a rice gene, OsFCA, which was homologous to FCA. Here, we found that the OsFCA protein could interact through its WW domain with the following proteins: OsFY, a protein containing a CID domain present in RNA-processing factors such as Pcf11 and Nrd1; a protein similar to splicing factor SF1; a protein similar to FUSE splicing factor; and OsMADS8. The FY protein is associated with the 3' end processing machinery in Arabidopsis. Thus, we examined interactions between OsFY and the rice homologs (OsCstF-50, -64 and -77) of the AtCstF-50, -64 and -77 proteins. We found that OsFY could bind OsCstF50, whereas the OsCstF77 protein could bridge the interaction between OsCstF50 and OsCstF64. Taken together, our data suggest that OsFCA could interact with several proteins other than OsFY through its WW domain and may play several roles in rice.

The pollen of metaphor: Box, cage, and trap as containment in the eighteenth century.