A novel TBP-TAF complex on RNA polymerase II-transcribed snRNA genes.

Science.gov (United States)

Zaborowska, Justyna; Taylor, Alice; Roeder, Robert G; Murphy, Shona

2012-01-01

Initiation of transcription of most human genes transcribed by RNA polymerase II (RNAP II) requires the formation of a preinitiation complex comprising TFIIA, B, D, E, F, H and RNAP II. The general transcription factor TFIID is composed of the TATA-binding protein and up to 13 TBP-associated factors. During transcription of snRNA genes, RNAP II does not appear to make the transition to long-range productive elongation, as happens during transcription of protein-coding genes. In addition, recognition of the snRNA gene-type specific 3' box RNA processing element requires initiation from an snRNA gene promoter. These characteristics may, at least in part, be driven by factors recruited to the promoter. For example, differences in the complement of TAFs might result in differential recruitment of elongation and RNA processing factors. As precedent, it already has been shown that the promoters of some protein-coding genes do not recruit all the TAFs found in TFIID. Although TAF5 has been shown to be associated with RNAP II-transcribed snRNA genes, the full complement of TAFs associated with these genes has remained unclear. Here we show, using a ChIP and siRNA-mediated approach, that the TBP/TAF complex on snRNA genes differs from that found on protein-coding genes. Interestingly, the largest TAF, TAF1, and the core TAFs, TAF10 and TAF4, are not detected on snRNA genes. We propose that this snRNA gene-specific TAF subset plays a key role in gene type-specific control of expression.

c-Jun binds the N terminus of human TAF(II)250 to derepress RNA polymerase II transcription in vitro.

Science.gov (United States)

Lively, T N; Ferguson, H A; Galasinski, S K; Seto, A G; Goodrich, J A

2001-07-06

c-Jun is an oncoprotein that activates transcription of many genes involved in cell growth and proliferation. We studied the mechanism of transcriptional activation by human c-Jun in a human RNA polymerase II transcription system composed of highly purified recombinant and native transcription factors. Transcriptional activation by c-Jun depends on the TATA-binding protein (TBP)-associated factor (TAF) subunits of transcription factor IID (TFIID). Protein-protein interaction assays revealed that c-Jun binds with high specificity to the largest subunit of human TFIID, TAF(II)250. The region of TAF(II)250 bound by c-Jun lies in the N-terminal 163 amino acids. This same region of TAF(II)250 binds to TBP and represses its interaction with TATA boxes, thereby decreasing DNA binding by TFIID. We hypothesized that c-Jun is capable of derepressing the effect of the TAF(II)250 N terminus on TFIID-driven transcription. In support of this hypothesis, we found that c-Jun increased levels of TFIID-driven transcription in vitro when added at high concentrations to a DNA template lacking activator protein 1 (AP-1) sites. Moreover, c-Jun blocked the repression of TBP DNA binding caused by the N terminus of TAF(II)250. In addition to revealing a mechanism by which c-Jun activates transcription, our studies provide the first evidence that an activator can bind directly to the N terminus of TAF(II)250 to derepress RNA polymerase II transcription in vitro.

Lactoferrin binding protein B - a bi-functional bacterial receptor protein.

Directory of Open Access Journals (Sweden)

Nicholas K H Ostan

2017-03-01

Full Text Available Lactoferrin binding protein B (LbpB is a bi-lobed outer membrane-bound lipoprotein that comprises part of the lactoferrin (Lf receptor complex in Neisseria meningitidis and other Gram-negative pathogens. Recent studies have demonstrated that LbpB plays a role in protecting the bacteria from cationic antimicrobial peptides due to large regions rich in anionic residues in the C-terminal lobe. Relative to its homolog, transferrin-binding protein B (TbpB, there currently is little evidence for its role in iron acquisition and relatively little structural and biophysical information on its interaction with Lf. In this study, a combination of crosslinking and deuterium exchange coupled to mass spectrometry, information-driven computational docking, bio-layer interferometry, and site-directed mutagenesis was used to probe LbpB:hLf complexes. The formation of a 1:1 complex of iron-loaded Lf and LbpB involves an interaction between the Lf C-lobe and LbpB N-lobe, comparable to TbpB, consistent with a potential role in iron acquisition. The Lf N-lobe is also capable of binding to negatively charged regions of the LbpB C-lobe and possibly other sites such that a variety of higher order complexes are formed. Our results are consistent with LbpB serving dual roles focused primarily on iron acquisition when exposed to limited levels of iron-loaded Lf on the mucosal surface and effectively binding apo Lf when exposed to high levels at sites of inflammation.

TRF2 and the evolution of the bilateria.

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Duttke, Sascha H C; Doolittle, Russell F; Wang, Yuan-Liang; Kadonaga, James T

2014-10-01

The development of a complex body plan requires a diversity of regulatory networks. Here we consider the concept of TATA-box-binding protein (TBP) family proteins as "system factors" that each supports a distinct set of transcriptional programs. For instance, TBP activates TATA-box-dependent core promoters, whereas TBP-related factor 2 (TRF2) activates TATA-less core promoters that are dependent on a TCT or downstream core promoter element (DPE) motif. These findings led us to investigate the evolution of TRF2. TBP occurs in Archaea and eukaryotes, but TRF2 evolved prior to the emergence of the bilateria and subsequent to the evolutionary split between bilaterians and nonbilaterian animals. Unlike TBP, TRF2 does not bind to the TATA box and could thus function as a new system factor that is largely independent of TBP. We postulate that this TRF2-based system served as the foundation for new transcriptional programs, such as those involved in triploblasty and body plan development, that facilitated the evolution of bilateria. © 2014 Duttke et al.; Published by Cold Spring Harbor Laboratory Press.

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

Science.gov (United States)

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.

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

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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.

[open quotes]Cryptic[close quotes] repeating triplets of purines and pyrimidines (cRRY(i)) are frequent and polymorphic: Analysis of coding cRRY(i) in the proopiomelanocortin (POMC) and TATA-binding protein (TBP) genes

Energy Technology Data Exchange (ETDEWEB)

Gostout, B.; Qiang Liu; Sommer, S.S. (Mayo Clinic/Foundation, Rochester, MN (United States))

1993-06-01

Triplets of the form of purine, purine, pyrimidine (RRY(i)) are enhanced in frequency in the genomes of primates, rodents, and bacteria. Some RRY(i) are [open quotes]cryptic[close quotes] repeats (cRRY(i)) in which no one tandem run of a trinucleotide predominates. A search of human GenBank sequence revealed that the sequences of cRRY(i) are highly nonrandom. Three randomly chosen human cRRY(i) were sequenced in search of polymorphic alleles. Multiple polymorphic alleles were found in cRRY(i) in the coding regions of the genes for proopiomelanocortin (POMC) and TATA-binding protein (TBP). The highly polymorphic TBP cRRY(i) was characterized in detail. Direct sequencing of 157 unrelated human alleles demonstrated the presence of 20 different alleles which resulted in 29--40 consecutive glutamines in the amino-terminal region of TBP. These alleles are differently distributed among the races. PCR was used to screen 1,846 additional alleles in order to characterize more fully the range of variation in the population. Three additional alleles were discovered, but there was no example of a substantial sequence amplification as is seen in the repeat sequences associated with X-linked spinal and bulbar muscular atrophy, myotonic dystrophy, or the fragile-X syndrome. The structure of the TBP cRRY(i) is conserved in the five monkey species examined. In the chimpanzee, examination of four individuals revealed that the cRRY(i) was highly polymorphic, but the pattern of polymorphism differed from that in humans. The TBP cRRY(i) displays both similarities with and differences from the previously described RRY(i) in the coding sequence of the androgen receptor. The data suggest how simple tandem repeats could evolve from cryptic repeats. 18 refs., 3 figs., 6 tabs.

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

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

2017-09-29

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

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

Science.gov (United States)

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

2008-07-01

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

TAF4 nucleates a core subcomplex of TFIID and mediates activated transcription from a TATA-less promoter

OpenAIRE

Wright, Kevin J.; Marr, Michael T.; Tjian, Robert

2006-01-01

Activator-dependent recruitment of TFIID initiates formation of the transcriptional preinitiation complex. TFIID binds core promoter DNA elements and directs the assembly of other general transcription factors, leading to binding of RNA polymerase II and activation of RNA synthesis. How TATA box-binding protein (TBP) and the TBP-associated factors (TAFs) are assembled into a functional TFIID complex with promoter recognition and coactivator activities in vivo remains unknown. Here, we use RNA...

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

Science.gov (United States)

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.

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

DEFF Research Database (Denmark)

Kjaerulff, S; Dooijes, D; Clevers, H

1997-01-01

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

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

Science.gov (United States)

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

2018-01-01

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

Trastuzumab-binding peptide display by Tobacco mosaic virus

International Nuclear Information System (INIS)

Frolova, Olga Y.; Petrunia, Igor V.; Komarova, Tatiana V.; Kosorukov, Vyacheslav S.; Sheval, Eugene V.; Gleba, Yuri Y.; Dorokhov, Yuri L.

2010-01-01

Human epidermal growth factor receptor-2 (HER2/neu) is a target for the humanized monoclonal antibody trastuzumab. Recently, trastuzumab-binding peptides (TBP) of HER2/neu that inhibit proliferation of breast cancer cells were identified. We have now studied conditions of efficient assembly in vivo of Tobacco mosaic virus (TMV)-based particles displaying TBP on its surface. The system is based on an Agrobacterium-mediated co-delivery of binary vectors encoding TMV RNA and coat protein (CP) with TBP in its C-terminal extension into plant leaves. We show how the fusion of amino acid substituted TBP (sTBP) to CP via a flexible peptide linker can improve the manufacturability of recombinant TMV (rTMV). We also reveal that rTMV particles with exposed sTBP retained trastuzumab-binding capacity but lost an anti-HER2/neu immunogenic scaffold function. Mouse antibodies against rTMV did not recognize HER2/neu on surface of human SK-BR-3 cells.

Activation of silenced cytokine gene promoters by the synergistic effect of TBP-TALE and VP64-TALE activators.

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Anthony, Kim; More, Abhijit; Zhang, Xiaoliu

2014-01-01

Recent work has shown that the combinatorial use of multiple TALE activators can selectively activate certain cellular genes in inaccessible chromatin regions. In this study, we aimed to interrogate the activation potential of TALEs upon transcriptionally silenced immune genes in the context of non-immune cells. We designed a unique strategy, in which a single TALE fused to the TATA-box binding protein (TBP-TALE) is coupled with multiple VP64-TALE activators. We found that our strategy is significantly more potent than multiple TALE activators alone in activating expression of IL-2 and GM-CSF in diverse cell origins in which both genes are otherwise completely silenced. Chromatin analysis revealed that the gene activation was due in part to displacement of a distinctly positioned nucleosome. These studies provide a novel epigenetic mechanism for artificial gene induction and have important implications for targeted cancer immunotherapy, DNA vaccine development, as well as rational design of TALE activators.

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

Science.gov (United States)

Iost, Isabelle; Bizebard, Thierry; Dreyfus, Marc

2013-08-01

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

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

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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

Sequence-specific DNA binding by MYC/MAX to low-affinity non-E-box motifs.

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Michael Allevato

Full Text Available The MYC oncoprotein regulates transcription of a large fraction of the genome as an obligatory heterodimer with the transcription factor MAX. The MYC:MAX heterodimer and MAX:MAX homodimer (hereafter MYC/MAX bind Enhancer box (E-box DNA elements (CANNTG and have the greatest affinity for the canonical MYC E-box (CME CACGTG. However, MYC:MAX also recognizes E-box variants and was reported to bind DNA in a "non-specific" fashion in vitro and in vivo. Here, in order to identify potential additional non-canonical binding sites for MYC/MAX, we employed high throughput in vitro protein-binding microarrays, along with electrophoretic mobility-shift assays and bioinformatic analyses of MYC-bound genomic loci in vivo. We identified all hexameric motifs preferentially bound by MYC/MAX in vitro, which include the low-affinity non-E-box sequence AACGTT, and found that the vast majority (87% of MYC-bound genomic sites in a human B cell line contain at least one of the top 21 motifs bound by MYC:MAX in vitro. We further show that high MYC/MAX concentrations are needed for specific binding to the low-affinity sequence AACGTT in vitro and that elevated MYC levels in vivo more markedly increase the occupancy of AACGTT sites relative to CME sites, especially at distal intergenic and intragenic loci. Hence, MYC binds diverse DNA motifs with a broad range of affinities in a sequence-specific and dose-dependent manner, suggesting that MYC overexpression has more selective effects on the tumor transcriptome than previously thought.

Rapid quantification of TBP and TBP degradation product ratios by FTIR-ATR

International Nuclear Information System (INIS)

Gillens, A.R.; Powell, B.A.; Clemson University, Clemson, SC

2013-01-01

Tri-n-butyl phosphate (TBP) is the key complexant within the plutonium and uranium reduction extraction process used to extract uranium and plutonium from used nuclear fuel. During reprocessing TBP degrades to dibutyl phosphate (DBP), butyl acid phosphate (MBP), butanol, and phosphoric acid over time. A method for rapidly monitoring TBP degradation is needed for the support of nuclear forensics. Therefore, a Fourier transform infrared spectrometry-attenuated total reflectance (FTIR-ATR) technique was developed to determine approximate peak intensity ratios of TBP and its degradation products. The technique was developed by combining variable concentrations of TBP, DBP, and MBP to simulate TBP degradation. This method is achieved by analyzing selected peak positions and peak intensity ratios of TBP and DBP at different stages of degradation. The developed technique was tested on TBP samples degraded with nitric acid. In mock degradation samples, the 1,235 cm -1 peak position shifts to 1,220 cm -1 as the concentration of TBP decreases and DBP increases. Peak intensity ratios of TBP positions at 1,279 and 1,020 cm -1 relative to DBP positions at 909 and 1,003 cm -1 demonstrate an increasing trend as the concentration of DBP increases. The same peak intensity ratios were used to analyze DBP relative to MBP whereas a decreasing trend is seen with increasing DBP concentrations. The technique developed from this study may be used as a tool to determine TBP degradation in nuclear reprocessing via a rapid FTIR-ATR measurement without gas chromatography analysis. (author)

The structural basis of transferrin sequestration by transferrin-binding protein B

Energy Technology Data Exchange (ETDEWEB)

Calmettes, Charles; Alcantara, Joenel; Yu, Rong-Hua; Schryvers, Anthony B.; Moraes, Trevor F. (Toronto); (Calgary)

2012-03-28

Neisseria meningitidis, the causative agent of bacterial meningitis, acquires the essential element iron from the host glycoprotein transferrin during infection through a surface transferrin receptor system composed of proteins TbpA and TbpB. Here we present the crystal structures of TbpB from N. meningitidis in its apo form and in complex with human transferrin. The structure reveals how TbpB sequesters and initiates iron release from human transferrin.

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

Science.gov (United States)

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

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

Directory of Open Access Journals (Sweden)

N V Bobkova

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

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

Science.gov (United States)

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

2010-03-05

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

Reference: 439 [Arabidopsis Phenome Database[Archive

Lifescience Database Archive (English)

Full Text Available or IID (TFIID) complex. Overexpression of atTAF10 under the control of the 35S promoter in Arabidopsis impro...is TATA box-binding protein (TBP)-associated factor 10 (atTAF10), which constitutes the transcriptional fact

Indole and synthetic derivative activate chaperone expression to reduce polyQ aggregation in SCA17 neuronal cell and slice culture models

Directory of Open Access Journals (Sweden)

Kung PJ

2014-10-01

Full Text Available Pin-Jui Kung,1,* Yu-Chen Tao,1,* Ho-Chiang Hsu,1 Wan-Ling Chen,1 Te-Hsien Lin,1 Donala Janreddy,2 Ching-Fa Yao,2 Kuo-Hsuan Chang,3 Jung-Yaw Lin,1 Ming-Tsan Su,1 Chung-Hsin Wu,1 Guey-Jen Lee-Chen,1 Hsiu-Mei Hsieh-Li1 1Department of Life Science, 2Department of Chemistry, National Taiwan Normal University, Taipei, Taiwan; 3Department of Neurology, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taipei, Taiwan *These authors contributed equally to this work Abstract: In spinocerebellar ataxia type 17 (SCA17, the expansion of a translated CAG repeat in the TATA box binding protein (TBP gene results in a long polyglutamine (polyQ tract in the TBP protein, leading to intracellular accumulation of aggregated TBP and cell death. The molecular chaperones act in preventing protein aggregation to ameliorate downstream harmful events. In this study, we used Tet-On SH-SY5Y cells with inducible SCA17 TBP/Q79-green fluorescent protein (GFP expression to test indole and synthetic derivative NC001-8 for neuroprotection. We found that indole and NC001-8 up-regulated chaperone expression to reduce polyQ aggregation in neuronal differentiated TBP/Q79 cells. The effects on promoting neurite outgrowth and on reduction of aggregation on Purkinje cells were also confirmed with cerebellar primary and slice cultures of SCA17 transgenic mice. Our results demonstrate how indole and derivative NC001-8 reduce polyQ aggregation to support their therapeutic potentials in SCA17 treatment. Keywords: spinocerebellar ataxia type 17, TATA box binding protein, polyQ aggregation, indole and derivative, therapeutics

Mechanism of selective recruitment of RNA polymerases II and III to snRNA gene promoters.

Science.gov (United States)

Dergai, Oleksandr; Cousin, Pascal; Gouge, Jerome; Satia, Karishma; Praz, Viviane; Kuhlman, Tracy; Lhôte, Philippe; Vannini, Alessandro; Hernandez, Nouria

2018-05-01

RNA polymerase II (Pol II) small nuclear RNA (snRNA) promoters and type 3 Pol III promoters have highly similar structures; both contain an interchangeable enhancer and "proximal sequence element" (PSE), which recruits the SNAP complex (SNAPc). The main distinguishing feature is the presence, in the type 3 promoters only, of a TATA box, which determines Pol III specificity. To understand the mechanism by which the absence or presence of a TATA box results in specific Pol recruitment, we examined how SNAPc and general transcription factors required for Pol II or Pol III transcription of SNAPc-dependent genes (i.e., TATA-box-binding protein [TBP], TFIIB, and TFIIA for Pol II transcription and TBP and BRF2 for Pol III transcription) assemble to ensure specific Pol recruitment. TFIIB and BRF2 could each, in a mutually exclusive fashion, be recruited to SNAPc. In contrast, TBP-TFIIB and TBP-BRF2 complexes were not recruited unless a TATA box was present, which allowed selective and efficient recruitment of the TBP-BRF2 complex. Thus, TBP both prevented BRF2 recruitment to Pol II promoters and enhanced BRF2 recruitment to Pol III promoters. On Pol II promoters, TBP recruitment was separate from TFIIB recruitment and enhanced by TFIIA. Our results provide a model for specific Pol recruitment at SNAPc-dependent promoters. © 2018 Dergai et al.; Published by Cold Spring Harbor Laboratory Press.

Targeting TBP-associated factors in ovarian cancer

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Jennifer R Ribeiro

2014-03-01

Full Text Available As ovarian tumors progress, they undergo a process of dedifferentiation, allowing adaptive changes in growth and morphology that promote metastasis and chemoresistance. Herein, we outline a hypothesis that TATA-box binding protein (TBP associated factors (TAFs, which compose the RNA Polymerase II initiation factor, TFIID, contribute to regulation of dedifferentiation states in ovarian cancer. Numerous studies demonstrate that TAFs regulate differentiation and proliferation states; their expression is typically high in pluripotent cells and reduced upon differentiation. Strikingly, TAF2 exhibits copy number increases or mRNA overexpression in 73% of high grade serous ovarian cancers (HGSC. At the biochemical level, TAF2 directs TFIID to TATA-less promoters by contact with an Initiator element, which may lead to the deregulation of the transcriptional output of these tumor cells. TAF4, which is altered in 66% of HGSC, is crucial for the stability of the TFIID complex and helps drive dedifferentiation of mouse embryonic fibroblasts to induced pluripotent stem cells. Its ovary-enriched paralog, TAF4B, is altered in 26% of HGSC. Here, we show that Taf4b mRNA correlates with Cyclin D2 mRNA expression in human granulosa cell tumors. TAF4B may also contribute to regulation of tumor microenvironment due to its estrogen-responsiveness and ability to act as a cofactor for NFκB. Conversely, TAF9, a cofactor for p53 in regulating apoptosis, may act as a tumor suppressor in ovarian cancer, since it is downregulated or deleted in 98% of HGSC. We conclude that a greater understanding of mechanisms of transcriptional regulation that execute signals from oncogenic signaling cascades is needed in order to expand our understanding of the etiology and progression of ovarian cancer, and most importantly to identify novel targets for therapeutic intervention.

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

Science.gov (United States)

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

2008-08-01

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

TAF9b (Formerly TAF9L) Is a Bona Fide TAF That Has Unique and Overlapping Roles with TAF9

OpenAIRE

Frontini, Mattia; Soutoglou, Evi; Argentini, Manuela; Bole-Feysot, Christine; Jost, Bernard; Scheer, Elisabeth; Tora, Làszlò

2005-01-01

TFIID plays a key role in transcription initiation of RNA polymerase II preinitiation complex assembly. TFIID is comprised of the TATA box binding protein (TBP) and 14 TBP-associated factors (TAFs). A second set of transcriptional regulatory multiprotein complexes containing TAFs has been described (called SAGA, TFTC, STAGA, and PCAF/GCN5). Using matrix-assisted laser desorption ionization mass spectrometry, we identified a novel TFTC subunit, human TAF9Like, encoded by a TAF9 paralogue gene....

Investigation into radiolysis of tbp labelled with 32P in the 30%tbp-diluent-HNO3 systems

International Nuclear Information System (INIS)

Novak, M.; Novak, Z.; Rokhon', A.

1975-01-01

Phosphorus-containing compounds, prepared by radiolysis of the TBP-diluent-HNO 3 system, are determined quantitatively. Effects of the HNO 3 concentration and the diluent type upon the degree of the TBP decomposition in the system under investigation were determined as well. To separate the TBP decomposition products the thin layer chromatography method was used, and for the quantitative determination TBP, labelled with 32 P was used. As a result of TBP radiolysis except dibutyl phosphate and monobutylphosphate other compounds are prepared, which contain phosphorus in a molecule, besides, their quantity depends on the nitric acid concentration and the diluent type (n-dodecan, carbon tetrachloride, mesitylen), together with which TBP was irradiated. The prepared compounds are grouped in the aqueous and organic phases

Rapid detection and purification of sequence specific DNA binding proteins using magnetic separation

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TIJANA SAVIC

2006-02-01

Full Text Available In this paper, a method for the rapid identification and purification of sequence specific DNA binding proteins based on magnetic separation is presented. This method was applied to confirm the binding of the human recombinant USF1 protein to its putative binding site (E-box within the human SOX3 protomer. It has been shown that biotinylated DNA attached to streptavidin magnetic particles specifically binds the USF1 protein in the presence of competitor DNA. It has also been demonstrated that the protein could be successfully eluted from the beads, in high yield and with restored DNA binding activity. The advantage of these procedures is that they could be applied for the identification and purification of any high-affinity sequence-specific DNA binding protein with only minor modifications.

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

Science.gov (United States)

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.

Main: 1QN6 [RPSD[Archive

Lifescience Database Archive (English)

Full Text Available itiation Factor Tfiid-1; Chain: A, B; Synonym: Tata-Box Factor 1, Tata Sequence-Binding Protein 1, Tbp1; Eng...ineered: Yes; Molecule: Dna (5'-D(*Gp*Cp*Tp*Ap*Tp*Ap*Ap*Tp*Ap*Gp*Gp*Gp*Cp*A)-3'); Chai...n: C, E; Molecule: Dna (5'-D(*Tp*Gp*Cp*Cp*Cp*Tp*Ap*Tp*Tp*Ap*Tp*Ap*Gp*C)-3'); Chain: D, F Tata Box-Bindi

Main: 1QNB [RPSD[Archive

Lifescience Database Archive (English)

Full Text Available itiation Factor Tfiid-1; Chain: A, B; Synonym: Tata-Box Factor 1, Tata Sequence-Binding Protein 1, Tbp1; Eng...ineered: Yes; Molecule: Dna (5'-D(*Gp*Cp*Tp*Ap*Tp*Ap*Ap*Ap*Tp*Gp*Gp*Gp*Cp*A)-3'); Chai...n: C, E; Molecule: Dna (5'-D(*Tp*Gp*Cp*Cp*Cp*Ap*Tp*Tp*Tp*Ap*Tp*Ap*Gp*C)-3'); Chain: D, F Tata Box-Bindi

Main: 1QN5 [RPSD[Archive

Lifescience Database Archive (English)

Full Text Available itiation Factor Tfiid-1; Chain: A, B; Synonym: Tata-Box Factor 1, Tata Sequence-Binding Protein 1, Tbp1; Eng...ineered: Yes; Molecule: Dna (5'-D(*Gp*Cp*Tp*Ap*Tp*Ap*Ap*Gp*Ap*Gp*Gp*Gp*Cp*A)-3'); Chai...n: C, E; Molecule: Dna (5'-D(*Tp*Gp*Cp*Cp*Cp*Tp*Cp*Tp*Tp*Ap*Tp*Ap*Gp*C)-3'); Chain: D, F Tata Box-Bindi

Main: 1QNA [RPSD[Archive

Lifescience Database Archive (English)

Full Text Available itiation Factor Tfiid-1; Chain: A, B; Synonym: Tata-Box Factor 1, Tata Sequence-Binding Protein 1, Tbp1; Eng...ineered: Yes; Molecule: Dna (5'-D(*Gp*Cp*Tp*Tp*Tp*Ap*Ap*Ap*Ap*Gp*Gp*Gp*Cp*A)-3'); Chai...n: C, E; Molecule: Dna (5'-D(*Tp*Gp*Cp*Cp*Cp*Tp*Tp*Tp*Tp*Ap*Ap*Ap*Gp*C)-3'); Chain: D, F Tata Box-Bindi

Arabidopsis AtbHLH112 regulates the expression of genes involved in abiotic stress tolerance by binding to their E-box and GCG-box motifs.

Science.gov (United States)

Liu, Yujia; Ji, Xiaoyu; Nie, Xianguang; Qu, Min; Zheng, Lei; Tan, Zilong; Zhao, Huimin; Huo, Lin; Liu, Shengnan; Zhang, Bing; Wang, Yucheng

2015-08-01

Plant basic helix-loop-helix (bHLH) transcription factors play essential roles in abiotic stress tolerance. However, most bHLHs have not been functionally characterized. Here, we characterized the functional role of a bHLH transcription factor from Arabidopsis, AtbHLH112, in response to abiotic stress. AtbHLH112 is a nuclear-localized protein, and its nuclear localization is induced by salt, drought and abscisic acid (ABA). In addition, AtbHLH112 serves as a transcriptional activator, with the activation domain located at its N-terminus. In addition to binding to the E-box motifs of stress-responsive genes, AtbHLH112 binds to a novel motif with the sequence 'GG[GT]CC[GT][GA][TA]C' (GCG-box). Gain- and loss-of-function analyses showed that the transcript level of AtbHLH112 is positively correlated with salt and drought tolerance. AtbHLH112 mediates stress tolerance by increasing the expression of P5CS genes and reducing the expression of P5CDH and ProDH genes to increase proline levels. AtbHLH112 also increases the expression of POD and SOD genes to improve reactive oxygen species (ROS) scavenging ability. We present a model suggesting that AtbHLH112 is a transcriptional activator that regulates the expression of genes via binding to their GCG- or E-boxes to mediate physiological responses, including proline biosynthesis and ROS scavenging pathways, to enhance stress tolerance. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

The integrated endoplasmic reticulum stress response in Leishmania amazonensis macrophage infection: the role of X-box binding protein 1 transcription factor.

Science.gov (United States)

Dias-Teixeira, Karina Luiza; Calegari-Silva, Teresa Cristina; dos Santos, Guilherme R R M; Vitorino Dos Santos, José; Lima, Carolina; Medina, Jorge Mansur; Aktas, Bertal Huseyin; Lopes, Ulisses G

2016-04-01

Endoplasmic reticulum (ER) stress triggers the integrated ER-stress response (IERSR) that ensures cellular survival of ER stress and represents a primordial form of innate immunity. We investigated the role of IERSR duringLeishmania amazonensisinfection. Treatment of RAW 264.7 infected macrophages with the ER stress-inducing agent thapsigargin (TG; 1 μM) increasedL. amazonensisinfectivity in an IFN1-α receptor (IFNAR)-dependent manner. In Western blot assays, we showed thatL. amazonensisactivates the inositol-requiring enzyme (IRE1)/ X-box binding protein (XBP)-1-splicing arms of the IERSR in host cells. In chromatin immunoprecipitation (ChIP) assays, we showed an increased occupancy of enhancer and promoter sequences for theIfnbgene by XBP1 in infected RAW 264.7 cells. Knocking down XBP1 expression by transducing RAW 264.7 cells with the short hairpin XBP1 lentiviral vector significantly reduced the parasite proliferation associated with impaired translocation of phosphorylated IFN regulatory transcription factor (IRF)-3 to the nucleus and a decrease in IFN1-β expression. Knocking down XBP1 expression also increased NO concentration, as determined by Griess reaction and reduced the expression of antioxidant genes, such as heme oxygenase (HO)-1, that protect parasites from oxidative stress. We conclude thatL. amazonensisactivation of XBP1 plays a critical role in infection by protecting the parasites from oxidative stress and increasing IFN1-β expression.-Dias-Teixeira, K. L., Calegari-Silva, T. C., Dos Santos, G. R. R. M., Vitorino dos Santos, J., Lima, C., Medina, J. M., Aktas, B. H., Lopes, U. G. The integrated endoplasmic reticulum stress response inLeishmania amazonensismacrophage infection: the role of X-box binding protein 1 transcription factor. © FASEB.

DNA-modified electrodes fabricated using copper-free click chemistry for enhanced protein detection.

Science.gov (United States)

Furst, Ariel L; Hill, Michael G; Barton, Jacqueline K

2013-12-31

A method of DNA monolayer formation has been developed using copper-free click chemistry that yields enhanced surface homogeneity and enables variation in the amount of DNA assembled; extremely low-density DNA monolayers, with as little as 5% of the monolayer being DNA, have been formed. These DNA-modified electrodes (DMEs) were characterized visually, with AFM, and electrochemically, and were found to facilitate DNA-mediated reduction of a distally bound redox probe. These low-density monolayers were found to be more homogeneous than traditional thiol-modified DNA monolayers, with greater helix accessibility through an increased surface area-to-volume ratio. Protein binding efficiency of the transcriptional activator TATA-binding protein (TBP) was also investigated on these surfaces and compared to that on DNA monolayers formed with standard thiol-modified DNA. Our low-density monolayers were found to be extremely sensitive to TBP binding, with a signal decrease in excess of 75% for 150 nM protein. This protein was detectable at 4 nM, on the order of its dissociation constant, with our low-density monolayers. The improved DNA helix accessibility and sensitivity of our low-density DNA monolayers to TBP binding reflects the general utility of this method of DNA monolayer formation for DNA-based electrochemical sensor development.

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

Science.gov (United States)

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

TAF(II)250: a transcription toolbox.

Science.gov (United States)

Wassarman, D A; Sauer, F

2001-08-01

Activation of RNA-polymerase-II-dependent transcription involves conversion of signals provided by gene-specific activator proteins into the synthesis of messenger RNA. This conversion requires dynamic structural changes in chromatin and assembly of general transcription factors (GTFs) and RNA polymerase II at core promoter sequence elements surrounding the transcription start site of genes. One hallmark of transcriptional activation is the interaction of DNA-bound activators with coactivators such as the TATA-box binding protein (TBP)-associated factors (TAF(II)s) within the GTF TFIID. TAF(II)250 possesses a variety of activities that are likely to contribute to the initial steps of RNA polymerase II transcription. TAF(II)250 is a scaffold for assembly of other TAF(II)s and TBP into TFIID, TAF(II)250 binds activators to recruit TFIID to particular promoters, TAF(II)250 regulates binding of TBP to DNA, TAF(II)250 binds core promoter initiator elements, TAF(II)250 binds acetylated lysine residues in core histones, and TAF(II)250 possesses protein kinase, ubiquitin-activating/conjugating and acetylase activities that modify histones and GTFs. We speculate that these activities achieve two goals--(1) they aid in positioning and stabilizing TFIID at particular promoters, and (2) they alter chromatin structure at the promoter to allow assembly of GTFs--and we propose a model for how TAF(II)250 converts activation signals into active transcription.

Influence of the diluent on the radiolytic degradation of TBP in TBP systems, 30% (V/V) - diluent-nitric acid

International Nuclear Information System (INIS)

Rubenich, M.N.

1976-03-01

The influence of the diluent on the degradation of TBP was studied by a gas chromatographic technique. The results obtained have shown that the aromatic diluents decrease markedly the HDBP production in the radiolysis of TBP, while n-dodecane, which is being used as diluent, promotes this radiolysis. However, the influence of the diluent become not too significant on the total (radiolysis + hydrolysis) solutions containing nitric acid. In view of foreseeing applications of aromatic diluents or their mixtures with aliphatic diluents on nuclear fuel reprocessing, it would be advisable to carry out more research on the system TBP/diluent, particularly on the kinetics of the hydrolysis of TBP and the influence of the diluent on the TBP degradation under conditions similar to those verified in the Purex Process [pt

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

Science.gov (United States)

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.

Induction of fibroblast growth factor 21 does not require activation of the hepatic X-box binding protein 1 in mice

Directory of Open Access Journals (Sweden)

Shantel Olivares

2017-12-01

Full Text Available Objective: Fibroblast growth factor 21 (FGF21, a key regulator of the metabolic response to fasting, is highly induced by endoplasmic reticulum (ER stress. The X-box binding protein 1 (Xbp1 is one of several ER stress proteins that has been shown to directly activate the FGF21 promoter. We aimed to determine whether hepatic Xbp1 is required for induction of hepatic FGF21 in vivo. Methods: Mice bearing a hepatocyte-specific deletion of Xbp1 (Xbp1LKO were subjected to fasting, pharmacologic ER stress, or a ketogenic diet, all potent stimuli of Fgf21 expression. Results: Hepatocyte-specific Xbp1 knockout mice demonstrated normal induction of FGF21 in response to fasting or pharmacologic ER stress and enhanced induction of FGF21 in response to a ketogenic diet. Consistent with preserved induction of FGF21, Xbp1LKO mice exhibited normal induction of FGF21 target genes and normal ketogenesis in response to fasting or a ketogenic diet. Conclusion: Hepatic Xbp1 is not required for induction of FGF21 under physiologic or pathophysiologic conditions in vivo. Keywords: Unfolded protein response, Endoplasmic reticulum stress, Fasting, Fatty acid oxidation, Ketogenic diet

Determination of tributyl phosphate (TBP) by precision densimetry : TBP-varsol-HNO3 system

International Nuclear Information System (INIS)

Matsuda, H.T.; Araujo, B.F. de; Araujo, J.A. de

1980-01-01

A simple and rapid method for TBP direct determination is presented, based on precision densimetry, aiming to control solvent concentration in the TBP-Varsol system during the reprocessing of irradiated uranium. The method comprises the determination of the density of liquids or gases by electronic measurement of the variation in the frequency (f) or period (T = 1/f) of a glass oscillator containing the liquid or the gas. (C.L.B.) [pt

Recovery Ce from Ce - TBP Used Oxalic Acid

International Nuclear Information System (INIS)

Purwani, MV; Subagiono, R.; Suyanti

2007-01-01

Recovery or stripping Ce from Ce - TBP product of monazite sand used oxalic acid. Ce - TBP as organic phase and oxalic acid as aqueous phase and as strong precipitant compound to precipitate metal element. The stripping product as Ce - oxalic precipitate. The influence parameter were percentage of oxalic acid, volume ratio of Ce-TBP with oxalic acid, time and rate of stripping. At stripping of 25 ml Ce - TBP used oxalic acid, the optimum condition were achieve at using 5% oxalic acid, volume ratio of Ce - TBP : 5% oxalic acid = 1 : 1, time of stripping 7.5 minute and rate of stripping 150 rpm. At the optimum condition was obtained the recovery efficiency was 100%. (author)

Study of TBP stability limits in pyrolysis, radiolysis and hydrolysis

International Nuclear Information System (INIS)

Azzouz, A.; Attou, M.

1985-10-01

The present investigations deal with the determination of the stability limits of different degradation phenomena during TBP process achievement, its purification, storage or in radioelements extraction. The results were obtained using a simple and quick analysis method based on TBP:HC104 complex formation. The TBP is considered stable (when its degradation rate is less than 0,5% Vol.) for temperature less than 100 0 C and time contact value of about 2 hours. It was depicted that TBP has an appreciable stability under the following conditions: NaOH 0,1 N/TBP=0,5 ml/ml and time contact <4 hours; POC13/TBP=0,02 ml/ml and time contact <1 hour; concentrated acid/TBP=0,025 ml/ml and time contact <1 h. Radiation cumulative effect seems to be the main precursor of TBP degradation. However, its stability remains appreciable under certain conditions. Practically, in all the cases, TBP decomposition is mainly tied to C-O bond breakdown

Determination of tributyl phosphate (TBP) by precision densimetry : TBP-varsol-HNO/sub 3/ system

Energy Technology Data Exchange (ETDEWEB)

Matsuda, H T; Araujo, B.F. de; Araujo, J.A. de [Instituto de Pesquisas Energeticas e Nucleares, Sao Paulo (Brazil). Centro de Engenharia Quimica

1980-01-01

A simple and rapid method for TBP direct determination is presented, based on precision densimetry, aiming to control solvent concentration in the TBP-Varsol system during the reprocessing of irradiated uranium. The method comprises the determination of the density of liquids or gases by electronic measurement of the variation in the frequency (f) or period (T = 1/f) of a glass oscillator containing the liquid or the gas.

[Roles of Y box-binding protein 1 in SK-BR-3 breast cancer proliferation].

Science.gov (United States)

Shi, Jianhong; Lü, Xinrui; Wang, Bing; Daudan, Lin; Yanan, Wang; Yuhui, Bu; Zhenfeng, Ma

2014-09-30

To explore the roles of Y box-binding protein 1 (YB-1) in breast cancer cell proliferation. Twenty cases of surgical removal of breast cancer tissue (diagnosed with invasive ductal carcinoma, stage II, by postoperative paraffin pathology) and normal breast tissues adjacent to carcinoma were collected during June 2013 to August 2013.Quantitative real-time PCR (qRT-PCR) was performed to detect the YB1 mRNA levels. Cultured mammary epithelial cells (HBL-100) and breast cancer cells (MCF7, MDA-MB-231 & SK-BR-3 cells) were harvested and qRT-PCR was performed to detect the YB1 mRNA levels.SK-BR-3 cells were stimulated with various concentrations of PDGF-BB and YB1 expression levels were detected by qRT-PCR. Down-regulation or over-expression of YB1 by si-YB1 or Ad-GFP-YB1 was detected in SK-BR-3 cells. And MTS cell proliferation assay kit was used to detect cell proliferation. YB1 mRNA levels were significantly higher in breast cancer tissues and MDA-MB-231 and SK-BR-3 breast cancer cell lines than that in adjacent normal breast tissues and HBL-100 mammary epithelial cells respectively (P BR-3 cells in a dose-dependent manner. A down-regulation of endogenous YB1 decreases and an over-expression of exogenous YB1 promotes the proliferation activity in SK-BR-3 cells.

Functional characterization of the ER stress induced X-box-binding protein-1 (Xbp-1 in the porcine system

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Jin Dong-Il

2011-05-01

Full Text Available Abstract Background The unfolded protein response (UPR is an evolutionary conserved adaptive reaction for increasing cell survival under endoplasmic reticulum (ER stress conditions. X-box-binding protein-1 (Xbp1 is a key transcription factor of UPR that activates genes involved in protein folding, secretion, and degradation to restore ER function. The UPR induced by ER stress was extensively studied in diseases linked to protein misfolding and aggregations. However, in the porcine system, genes in the UPR pathway were not investigated. In this study, we isolated and characterized the porcine Xbp1 (pXbp1 gene in ER stress using porcine embryonic fibroblast (PEF cells and porcine organs. ER stress was induced by the treatment of tunicamycin and cell viability was investigated by the MTT assay. For cloning and analyzing the expression pattern of pXbp1, RT-PCR analysis and Western blot were used. Knock-down of pXbp1 was performed by the siRNA-mediated gene silencing. Results We found that the pXbp1 mRNA was the subject of the IRE1α-mediated unconventional splicing by ER stress. Knock-down of pXbp1 enhanced ER stress-mediated cell death in PEF cells. In adult organs, pXbp1 mRNA and protein were expressed and the spliced forms were detected. Conclusions It was first found that the UPR mechanisms and the function of pXbp1 in the porcine system. These results indicate that pXbp1 plays an important role during the ER stress response like other animal systems and open a new opportunity for examining the UPR pathway in the porcine model system.

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

Science.gov (United States)

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

2007-01-01

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

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

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

1994-12-20

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

Mass transfer model for two-layer TBP oxidation reactions

International Nuclear Information System (INIS)

Laurinat, J.E.

1994-01-01

To prove that two-layer, TBP-nitric acid mixtures can be safely stored in the canyon evaporators, it must be demonstrated that a runaway reaction between TBP and nitric acid will not occur. Previous bench-scale experiments showed that, at typical evaporator temperatures, this reaction is endothermic and therefore cannot run away, due to the loss of heat from evaporation of water in the organic layer. However, the reaction would be exothermic and could run away if the small amount of water in the organic layer evaporates before the nitric acid in this layer is consumed by the reaction. Provided that there is enough water in the aqueous layer, this would occur if the organic layer is sufficiently thick so that the rate of loss of water by evaporation exceeds the rate of replenishment due to mixing with the aqueous layer. This report presents measurements of mass transfer rates for the mixing of water and butanol in two-layer, TBP-aqueous mixtures, where the top layer is primarily TBP and the bottom layer is comprised of water or aqueous salt solution. Mass transfer coefficients are derived for use in the modeling of two-layer TBP-nitric acid oxidation experiments. Three cases were investigated: (1) transfer of water into the TBP layer with sparging of both the aqueous and TBP layers, (2) transfer of water into the TBP layer with sparging of just the TBP layer, and (3) transfer of butanol into the aqueous layer with sparging of both layers. The TBP layer was comprised of 99% pure TBP (spiked with butanol for the butanol transfer experiments), and the aqueous layer was comprised of either water or an aluminum nitrate solution. The liquid layers were air sparged to simulate the mixing due to the evolution of gases generated by oxidation reactions. A plastic tube and a glass frit sparger were used to provide different size bubbles. Rates of mass transfer were measured using infrared spectrophotometers provided by SRTC/Analytical Development

Drosophila TRF2 and TAF9 regulate lipid droplet size and phospholipid fatty acid composition.

Science.gov (United States)

Fan, Wei; Lam, Sin Man; Xin, Jingxue; Yang, Xiao; Liu, Zhonghua; Liu, Yuan; Wang, Yong; Shui, Guanghou; Huang, Xun

2017-03-01

The general transcription factor TBP (TATA-box binding protein) and its associated factors (TAFs) together form the TFIID complex, which directs transcription initiation. Through RNAi and mutant analysis, we identified a specific TBP family protein, TRF2, and a set of TAFs that regulate lipid droplet (LD) size in the Drosophila larval fat body. Among the three Drosophila TBP genes, trf2, tbp and trf1, only loss of function of trf2 results in increased LD size. Moreover, TRF2 and TAF9 regulate fatty acid composition of several classes of phospholipids. Through RNA profiling, we found that TRF2 and TAF9 affects the transcription of a common set of genes, including peroxisomal fatty acid β-oxidation-related genes that affect phospholipid fatty acid composition. We also found that knockdown of several TRF2 and TAF9 target genes results in large LDs, a phenotype which is similar to that of trf2 mutants. Together, these findings provide new insights into the specific role of the general transcription machinery in lipid homeostasis.

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

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Andrea Ciammaruconi

2008-01-01

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

Retinoid-binding proteins: similar protein architectures bind similar ligands via completely different ways.

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Yu-Ru Zhang

Full Text Available BACKGROUND: Retinoids are a class of compounds that are chemically related to vitamin A, which is an essential nutrient that plays a key role in vision, cell growth and differentiation. In vivo, retinoids must bind with specific proteins to perform their necessary functions. Plasma retinol-binding protein (RBP and epididymal retinoic acid binding protein (ERABP carry retinoids in bodily fluids, while cellular retinol-binding proteins (CRBPs and cellular retinoic acid-binding proteins (CRABPs carry retinoids within cells. Interestingly, although all of these transport proteins possess similar structures, the modes of binding for the different retinoid ligands with their carrier proteins are different. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we analyzed the various retinoid transport mechanisms using structure and sequence comparisons, binding site analyses and molecular dynamics simulations. Our results show that in the same family of proteins and subcellular location, the orientation of a retinoid molecule within a binding protein is same, whereas when different families of proteins are considered, the orientation of the bound retinoid is completely different. In addition, none of the amino acid residues involved in ligand binding is conserved between the transport proteins. However, for each specific binding protein, the amino acids involved in the ligand binding are conserved. The results of this study allow us to propose a possible transport model for retinoids. CONCLUSIONS/SIGNIFICANCE: Our results reveal the differences in the binding modes between the different retinoid-binding proteins.

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

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

2010-01-01

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

The transcriptional activator GAL4-VP16 regulates the intra ...

Indian Academy of Sciences (India)

Activator also reduced the TBP dimer levels both in vitro and in vivo, suggesting the dimer may be a direct target of transcriptional activators. The transcriptional activator facilitated the dimer to monomer transition and activated monomers further to help TBP bind even the weaker TATA boxes stably. The overall stimulatory ...

DNA binding specificity of the basic-helix-loop-helix protein MASH-1.

Science.gov (United States)

Meierhan, D; el-Ariss, C; Neuenschwander, M; Sieber, M; Stackhouse, J F; Allemann, R K

1995-09-05

Despite the high degree of sequence similarity in their basic-helix-loop-helix (BHLH) domains, MASH-1 and MyoD are involved in different biological processes. In order to define possible differences between the DNA binding specificities of these two proteins, we investigated the DNA binding properties of MASH-1 by circular dichroism spectroscopy and by electrophoretic mobility shift assays (EMSA). Upon binding to DNA, the BHLH domain of MASH-1 underwent a conformational change from a mainly unfolded to a largely alpha-helical form, and surprisingly, this change was independent of the specific DNA sequence. The same conformational transition could be induced by the addition of 20% 2,2,2-trifluoroethanol. The apparent dissociation constants (KD) of the complexes of full-length MASH-1 with various oligonucleotides were determined from half-saturation points in EMSAs. MASH-1 bound as a dimer to DNA sequences containing an E-box with high affinity KD = 1.4-4.1 x 10(-14) M2). However, the specificity of DNA binding was low. The dissociation constant for the complex between MASH-1 and the highest affinity E-box sequence (KD = 1.4 x 10(-14) M2) was only a factor of 10 smaller than for completely unrelated DNA sequences (KD = approximately 1 x 10(-13) M2). The DNA binding specificity of MASH-1 was not significantly increased by the formation of an heterodimer with the ubiquitous E12 protein. MASH-1 and MyoD displayed similar binding site preferences, suggesting that their different target gene specificities cannot be explained solely by differential DNA binding. An explanation for these findings is provided on the basis of the known crystal structure of the BHLH domain of MyoD.

Y-box Binding Protein-1 Enhances Oncogenic Transforming Growth Factor β Signaling in Breast Cancer Cells via Triggering Phospho-Activation of Smad2.

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Stope, Matthias B; Weiss, Martin; Koensgen, Dominique; Popp, Simone L; Joffroy, Christian; Mustea, Alexander; Buck, Miriam B; Knabbe, Cornelius

2017-12-01

Transforming growth factor β (TGFβ) plays a role in diverse oncogenic pathways including cell proliferation and cell motility and is regulated by the pleiotropic factor Y-box binding protein-1 (YB-1). In breast cancer, Sma/Mad related protein 2 (Smad2) represents the most common downstream transducer in TGFβ signaling. Here, YB-1's impact on Smad2 phospho-activation was characterized by incubation of the breast cancer cell line MCF-7 with or without TGFβ1 in the absence or presence of overexpressed YB-1 protein. The phospho-status of Smad2 was assessed via western blotting. Analysis of MCF-7 cells revealed no induction of total Smad2 neither in the presence of TGFβ1, nor during YB-1 overexpression. In contrast, incubation with TGFβ1 led to an increase of phosphorylated Smad2 forms which was significantly amplified by simultaneously overexpressed YB-1 (2.8±0.2-fold). Oncogenic YB-1 indirectly enhances TGFβ signaling cascades via Smad2 phospho-activation and may represent a promising factor for future diagnosis and therapy of breast cancer. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

The dsRNA binding protein RDE-4 interacts with RDE-1, DCR-1, and a DExH-box helicase to direct RNAi in C. elegans.

Science.gov (United States)

Tabara, Hiroaki; Yigit, Erbay; Siomi, Haruhiko; Mello, Craig C

2002-06-28

Double-stranded (ds) RNA induces potent gene silencing, termed RNA interference (RNAi). At an early step in RNAi, an RNaseIII-related enzyme, Dicer (DCR-1), processes long-trigger dsRNA into small interfering RNAs (siRNAs). DCR-1 is also required for processing endogenous regulatory RNAs called miRNAs, but how DCR-1 recognizes its endogenous and foreign substrates is not yet understood. Here we show that the C. elegans RNAi pathway gene, rde-4, encodes a dsRNA binding protein that interacts during RNAi with RNA identical to the trigger dsRNA. RDE-4 protein also interacts in vivo with DCR-1, RDE-1, and a conserved DExH-box helicase. Our findings suggest a model in which RDE-4 and RDE-1 function together to detect and retain foreign dsRNA and to present this dsRNA to DCR-1 for processing.

Non-canonical TAF complexes regulate active promoters in human embryonic stem cells.

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Maston, Glenn A; Zhu, Lihua Julie; Chamberlain, Lynn; Lin, Ling; Fang, Minggang; Green, Michael R

2012-11-13

The general transcription factor TFIID comprises the TATA-box-binding protein (TBP) and approximately 14 TBP-associated factors (TAFs). Here we find, unexpectedly, that undifferentiated human embryonic stem cells (hESCs) contain only six TAFs (TAFs 2, 3, 5, 6, 7 and 11), whereas following differentiation all TAFs are expressed. Directed and global chromatin immunoprecipitation analyses reveal an unprecedented promoter occupancy pattern: most active genes are bound by only TAFs 3 and 5 along with TBP, whereas the remaining active genes are bound by TBP and all six hESC TAFs. Consistent with these results, hESCs contain a previously undescribed complex comprising TAFs 2, 6, 7, 11 and TBP. Altering the composition of hESC TAFs, either by depleting TAFs that are present or ectopically expressing TAFs that are absent, results in misregulated expression of pluripotency genes and induction of differentiation. Thus, the selective expression and use of TAFs underlies the ability of hESCs to self-renew.DOI:http://dx.doi.org/10.7554/eLife.00068.001.

Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR

DEFF Research Database (Denmark)

Hillig, Ann-Britt Nygaard; Jørgensen, Claus Bøttcher; Cirera, Susanna

2007-01-01

-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethylbilane synthase (HMBS), hypoxanthine phosphoribosyltransferase I (HPRT I), ribosomal protein L4 (RPL4), succinate dehydrogenase complex subunit A (SDHA), TATA box binding protein (TPB) and tyrosine 3-monooxygenase/tryptophan 5......-monooxygenase activation protein zeta polypeptide (YWHAZ). The stability of these reference genes in different pig tissues was investigated using the geNorm application. The range of expression stability in the genes analysed was (from the most stable to the least stable): ACTB/RPL4, TBP, HPRT, HMBS, YWHAZ...

Competitive protein binding assay

International Nuclear Information System (INIS)

Kaneko, Toshio; Oka, Hiroshi

1975-01-01

The measurement of cyclic GMP (cGMP) by competitive protein binding assay was described and discussed. The principle of binding assay was represented briefly. Procedures of our method by binding protein consisted of preparation of cGMP binding protein, selection of 3 H-cyclic GMP on market, and measurement procedures. In our method, binding protein was isolated from the chrysalis of silk worm. This method was discussed from the points of incubation medium, specificity of binding protein, the separation of bound cGMP from free cGMP, and treatment of tissue from which cGMP was extracted. cGMP existing in the tissue was only one tenth or one scores of cGMP, and in addition, cGMP competed with cGMP in binding with binding protein. Therefore, Murad's technique was applied to the isolation of cGMP. This method provided the measurement with sufficient accuracy; the contamination by cAMP was within several per cent. (Kanao, N.)

Comparative structural analysis of human DEAD-box RNA helicases.

Science.gov (United States)

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

2010-09-30

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

Comparative structural analysis of human DEAD-box RNA helicases.

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Patrick Schütz

2010-09-01

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

Nonnuclear hydrometallurgical applications of TBP

International Nuclear Information System (INIS)

Ritcey, G.M.; Pouskouleli, G.

1987-01-01

This chapter on the nonnuclear solvent extraction applications of tri-n-butyl phosphate (TBP) has been assembled from information in the analytical, chemical, and metallurgical literature. There are actually few operating plants, or, in fact, process flowsheets designed for the use of TBP in the extraction of nonnuclear metals and other materials. Perhaps one of the main reasons for this is that TBP operates through a solvating mechanism of extraction, and, therefore, extraction is highly dependent upon, the ionic strength of the anion. Many developments in process metallurgy utilizing solvent extraction involve extraction from a sulfate medium. Very few plants have gone on-stream employing chloride or nitrate systems, even though TBP is an excellent extractant from aqueous chloride or nitrate media. It is very likely that in the future we shall see many hydrometallurgical processes developed and plants designed in which the aqueous medium is chloride. With this in mind, we have included analytical and chemical techniques as possible guides to future design trends. The chapter includes most of the elements in the Periodic Table and covers all the various groups in the table. The chemistry of metals within the same group is, of course, quite similar, and this is evident in the comparison of extraction properties. For the convenience of the reader we have put the various metals into groups found within the Periodic Table, or in other appropriate groups that show chemical relationships. The literature surveyed in this chapter covers the period 1966 to 1984

IR and UV spectroscopic analysis of TBP complexes

International Nuclear Information System (INIS)

Azzouz, A.; Berrak, A.; Seridi, L.; Attou, M.

1985-06-01

The complexity of TBP molecule and the limited number of references stimulated the elaboration of this report. The spectroscopic of TBP and its complexes in the IR and UV fields permitted to elucidate or to confirm certain aspects concerning the solvation phenomenum. In IR spectroscopy, the stretching band of the P→O bond only is characteristic of the complex formed. The position of this band gives sufficient information about the kind and the stability of a complex. The TBP electronic spectra are characterized by two bands (200-220 nm) 1 and (268-290 nm) 2 whose intensity ratio (2/1) is about 0,13. The solvent nature seems to influence the positions of these bands and that of the inflexion point. The band 2 disappears when the TBP is complexed and the position and the intensity of the band 1 depend upon the complex nature

The immunosuppressives FK 506 and cyclosporin A inhibit the generation of protein factors binding to the two purine boxes of the interleukin 2 enhancer.

Science.gov (United States)

Brabletz, T; Pietrowski, I; Serfling, E

1991-01-11

Like Cyclosporin A (CsA), the macrolide FK 506 is a potent immunosuppressive that inhibits early steps of T cell activation, including the synthesis of Interleukin 2 (II-2) and numerous other lymphokines. The block of II-2 synthesis occurs at the transcriptional level. At concentrations that block T cell activation, FK 506 and CsA inhibit the proto-enhancer activity of Purine boxes of the II-2 promoter and the generation of lymphocyte-specific factors binding to the Purine boxes. Under the same conditions, the DNA binding of other II-2 enhancer factors remains unaffected by both compounds. These results support the view that FK 506 and CsA, which both inhibit the activity of peptidylprolyl cis/trans isomerases, suppress T cell activation by a similar, if not identical mechanism.

The negatively charged regions of lactoferrin binding protein B, an adaptation against anti-microbial peptides.

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Ari Morgenthau

Full Text Available Lactoferrin binding protein B (LbpB is a bi-lobed membrane bound lipoprotein that is part of the lactoferrin receptor complex in a variety of Gram-negative pathogens. Despite high sequence diversity among LbpBs from various strains and species, a cluster of negatively charged amino acids is invariably present in the protein's C-terminal lobe in all species except Moraxella bovis. The function of LbpB in iron acquisition has yet to be experimentally demonstrated, whereas in vitro studies have shown that LbpB confers protection against lactoferricin, a short cationic antimicrobial peptide released from the N- terminus of lactoferrin. In this study we demonstrate that the negatively charged regions can be removed from the Neisseria meningitidis LbpB without compromising stability, and this results in the inability of LbpB to protect against the bactericidal effects of lactoferricin. The release of LbpB from the cell surface by the autotransporter NalP reduces the protection against lactoferricin in the in vitro killing assay, attributed to removal of LbpB during washing steps, but is unlikely to have a similar impact in vivo. The protective effect of the negatively charged polysaccharide capsule in the killing assay was less than the protection conferred by LbpB, suggesting that LbpB plays a major role in protection against cationic antimicrobial peptides in vivo. The selective release of LbpB by NalP has been proposed to be a mechanism for evading the adaptive immune response, by reducing the antibody binding to the cell surface, but may also provide insights into the primary function of LbpB in vivo. Although TbpB and LbpB have been shown to be major targets of the human immune response, the selective release of LbpB suggests that unlike TbpB, LbpB may not be essential for iron acquisition, but important for protection against cationic antimicrobial peptides.

Sequence characteristics required for cooperative binding and efficient in vivo titration of the replication initiator protein DnaA in E. coli

DEFF Research Database (Denmark)

Hansen, Flemming G.; Christensen, Bjarke Bak; Atlung, Tove

2007-01-01

Plasmids carrying the mioC promoter region, which contains two DnaA boxes, R5 and R6 with one misfit to the consensus TT(A)/(T)TNCACA, are as efficient in in vivo titration of the DnaA protein as plasmids carrying a replication-inactivated oriC region with its eight DnaA boxes. Three additional Dna......A boxes around the promoter proximal R5 DnaA box were identified and shown by mutational analysis to be necessary for the cooperative binding of DnaA required for titration. These four DnaA boxes are located in the same orientation and with a spacing of two or three base-pairs. The cooperative binding...... was eliminated by insertion of half a helical turn between any of the DnaA boxes. Titration strongly depends on the presence and orientation of the promoter distal R6 DnaA box located 104 bp upstream of the R5 box as well as neighbouring sequences downstream of R6. Titration depends on the integrity of a 43 bp...

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

Energy Technology Data Exchange (ETDEWEB)

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

2015-06-30

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

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

International Nuclear Information System (INIS)

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

2015-01-01

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

Body Temperature Cycles Control Rhythmic Alternative Splicing in Mammals.

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Preußner, Marco; Goldammer, Gesine; Neumann, Alexander; Haltenhof, Tom; Rautenstrauch, Pia; Müller-McNicoll, Michaela; Heyd, Florian

2017-08-03

The core body temperature of all mammals oscillates with the time of the day. However, direct molecular consequences of small, physiological changes in body temperature remain largely elusive. Here we show that body temperature cycles drive rhythmic SR protein phosphorylation to control an alternative splicing (AS) program. A temperature change of 1°C is sufficient to induce a concerted splicing switch in a large group of functionally related genes, rendering this splicing-based thermometer much more sensitive than previously described temperature-sensing mechanisms. AS of two exons in the 5' UTR of the TATA-box binding protein (Tbp) highlights the general impact of this mechanism, as it results in rhythmic TBP protein levels with implications for global gene expression in vivo. Together our data establish body temperature-driven AS as a core clock-independent oscillator in mammalian peripheral clocks. Copyright © 2017 Elsevier Inc. All rights reserved.

Lead-Binding Proteins: A Review

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Harvey C. Gonick

2011-01-01

Full Text Available Lead-binding proteins are a series of low molecular weight proteins, analogous to metallothionein, which segregate lead in a nontoxic form in several organs (kidney, brain, lung, liver, erythrocyte. Whether the lead-binding proteins in every organ are identical or different remains to be determined. In the erythrocyte, delta-aminolevulinic acid dehydratase (ALAD isoforms have commanded the greatest attention as proteins and enzymes that are both inhibitable and inducible by lead. ALAD-2, although it binds lead to a greater degree than ALAD-1, appears to bind lead in a less toxic form. What may be of greater significance is that a low molecular weight lead-binding protein, approximately 10 kDa, appears in the erythrocyte once blood lead exceeds 39 μg/dL and eventually surpasses the lead-binding capacity of ALAD. In brain and kidney of environmentally exposed humans and animals, a cytoplasmic lead-binding protein has been identified as thymosin β4, a 5 kDa protein. In kidney, but not brain, another lead-binding protein has been identified as acyl-CoA binding protein, a 9 kDa protein. Each of these proteins, when coincubated with liver ALAD and titrated with lead, diminishes the inhibition of ALAD by lead, verifying their ability to segregate lead in a nontoxic form.

Insights into the quality of DnaA boxes and their cooperativity

DEFF Research Database (Denmark)

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

2006-01-01

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

Y-box-binding protein 1 interacts with hepatitis C virus NS3/4A and influences the equilibrium between viral RNA replication and infectious particle production.

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Chatel-Chaix, Laurent; Melançon, Pierre; Racine, Marie-Ève; Baril, Martin; Lamarre, Daniel

2011-11-01

The hepatitis C virus (HCV) NS3/4A protein has several essential roles in the virus life cycle, most probably through dynamic interactions with host factors. To discover cellular cofactors that are co-opted by HCV for its replication, we elucidated the NS3/4A interactome using mass spectrometry and identified Y-box-binding protein 1 (YB-1) as an interacting partner of NS3/4A protein and HCV genomic RNA. Importantly, silencing YB-1 expression decreased viral RNA replication and severely impaired the propagation of the infectious HCV molecular clone JFH-1. Immunofluorescence studies further revealed a drastic HCV-dependent redistribution of YB-1 to the surface of the lipid droplets, an important organelle for HCV assembly. Core and NS3 protein-dependent polyprotein maturation were shown to be required for YB-1 relocalization. Unexpectedly, YB-1 knockdown cells showed the increased production of viral infectious particles while HCV RNA replication was impaired. Our data support that HCV hijacks YB-1-containing ribonucleoparticles and that YB-1-NS3/4A-HCV RNA complexes regulate the equilibrium between HCV RNA replication and viral particle production.

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

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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.

Large Polyglutamine Repeats Cause Muscle Degeneration in SCA17 Mice

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Shanshan Huang

2015-10-01

Full Text Available In polyglutamine (polyQ diseases, large polyQ repeats cause juvenile cases with different symptoms than those of adult-onset patients, who carry smaller expanded polyQ repeats. The mechanisms behind the differential pathology mediated by different polyQ repeat lengths remain unknown. By studying knockin mouse models of spinal cerebellar ataxia-17 (SCA17, we found that a large polyQ (105 glutamines in the TATA-box-binding protein (TBP preferentially causes muscle degeneration and reduces the expression of muscle-specific genes. Direct expression of TBP with different polyQ repeats in mouse muscle revealed that muscle degeneration is mediated only by the large polyQ repeats. Different polyQ repeats differentially alter TBP’s interaction with neuronal and muscle-specific transcription factors. As a result, the large polyQ repeat decreases the association of MyoD with TBP and DNA promoters. Our findings suggest that specific alterations in protein interactions by large polyQ repeats may account for the unique pathology in juvenile polyQ diseases.

Large Polyglutamine Repeats Cause Muscle Degeneration in SCA17 Mice

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Huang, Shanshan; Yang, Su; Guo, Jifeng; Yan, Sen; Gaertig, Marta A.; Li, Shihua; Li, Xiao-Jiang

2015-01-01

SUMMARY In polyglutamine (polyQ) diseases, large polyQ repeats cause juvenile cases with different symptoms than adult-onset patients, who carry smaller expanded polyQ repeats. The mechanisms behind the differential pathology mediated by different polyQ repeat lengths remain unknown. By studying knock-in mouse models of spinal cerebellar ataxia-17 (SCA17), we found that a large polyQ (105 glutamines) in the TATA box-binding protein (TBP) preferentially causes muscle degeneration and reduces the expression of muscle-specific genes. Direct expression of TBP with different polyQ repeats in mouse muscle revealed that muscle degeneration is mediated only by the large polyQ repeats. Different polyQ repeats differentially alter TBP’s interaction with neuronal and muscle-specific transcription factors. As a result, the large polyQ repeat decreases the association of MyoD with TBP and DNA promoters. Our findings suggest that specific alterations in protein interactions by large polyQ repeats may account for the unique pathology in juvenile polyQ diseases. PMID:26387956

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

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Fabio Schneider Ribeiro

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

Safe handling of TBP and nitrates in the nuclear process industry

International Nuclear Information System (INIS)

Hyder, M.L.

1994-07-01

A laboratory and literature study was made of the reactions of tri-n-butyl phosphate (TBP) with nitric acid and nitrates. Its goal was to establish safe conditions for solvent extraction processes involving these chemicals. The damaging explosions at the Tomsk-7 PUREX plant in Russia graphically illustrated the potential hazard involved in such operations. The study has involved a review of prior and contemporary experiments, and new experiments to answer particular questions about these reactions. TBP extracts nitric acid and some metal nitrates from aqueous solutions. The resulting liquid contains both oxidant and reductant, and can react exothermically if heated sufficiently. Safe handling of these potentially reactive materials involves not only limiting the heat generated by the chemical reaction, but also providing adequate heat removal and venting. Specifically, the following recommendations are made to ensure safety: (1) tanks in which TBP-nitrate complexes are or may be present should be adequately vented to avoid pressurization. Data are supplied as a basis for adequacy; (2) chemically degraded TBP, or TBP that has sat a long time in the presence of acids or radiation, should be purified before use in solvent extraction; (3) evaporators in which TBP might be introduced should be operated at a controlled temperature, and their TBP content should be limited; (4) evaporator bottoms that may contain TBP should be cooled under conditions that ensure heat removal. Finally, process design should consider the potential for such reactions, and operators should be made aware of this potential, so that it is considered during training and process operation

Interfacial tension in systems involving TBP in dodecane, nitric acid, uranyl nitrate and water

International Nuclear Information System (INIS)

Kolarik, Z.; Pipkin, N.

1982-08-01

The interfacial tension was measured at 25 0 C in the systems TBP - n-dodecane/nitric acid - water and TBP - n-dodecane/nitric acid - uranyl nitrate - water. Empirical equations describing the interfacial tension as a function of the concentration of TBP in the starting organic phase and of uranium-(VI) and nitric acid in the equilibrium aqueous phase were suggested. In the absence of uranium (VI), the interfacial tension can also be correlated with the concentration of water in the equilibrium organic phase. Free TBP, hydrated or nonhydrated, and hydrated TBP solvates of nitric acid are interfacially active. Anhydrous TBP solvates of nitric acid and the TBP solvate of uranyl nitrate, which neither is hydrated, do not exhibit any visible interfacial activity. (orig.) [de

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

DEFF Research Database (Denmark)

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

2008-01-01

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

Arg156 in the AP2-domain exhibits the highest binding activity among the 20 individuals to the GCC box in BnaERF-B3-hy15, a mutant ERF transcription factor from Brassica napus

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Jing Zhuang

2016-10-01

Full Text Available To develop mutants of the ERF factor with more binding activities to the GCC box, we performed in vitro directed evolution by using DNA shuffling and screened mutants through yeast one-hybrid assay. Here, a series of mutants were obtained and used to reveal key amino acids that induce changes in the DNA binding activity of the BnaERF-B3 protein. With the BnaERF-B3-hy15 as the template, we produced 12 mutants which host individual mutation of potential key residues. We found that amino acid 156 is the key site, and the other 18 mutants host the 18 corresponding individual amino acid residues at site 156. Among the 20 individuals comprising WT (Gly156, Mu3 (Arg156, and 18 mutants with other 18 amino acid residues, Arg156 in the AP2-domain is the amino acid residue with the highest binding activity to the GCC box. The structure of the α-helix in the AP2-domain affects the binding activity. Other residues within AP2-domain modulated binding activity of ERF protein, suggesting that these positions are important for binding activity. Comparison of the mutant and wild-type transcription factors revealed the relationship of protein function and sequence modification. Our result provides a potential useful resource for understanding the trans-activation of ERF proteins.

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

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

2008-06-01

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

Hybrids of the bHLH and bZIP protein motifs display different DNA-binding activities in vivo vs. in vitro.

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Hiu-Kwan Chow

Full Text Available Minimalist hybrids comprising the DNA-binding domain of bHLH/PAS (basic-helix-loop-helix/Per-Arnt-Sim protein Arnt fused to the leucine zipper (LZ dimerization domain from bZIP (basic region-leucine zipper protein C/EBP were designed to bind the E-box DNA site, CACGTG, targeted by bHLHZ (basic-helix-loop-helix-zipper proteins Myc and Max, as well as the Arnt homodimer. The bHLHZ-like structure of ArntbHLH-C/EBP comprises the Arnt bHLH domain fused to the C/EBP LZ: i.e. swap of the 330 aa PAS domain for the 29 aa LZ. In the yeast one-hybrid assay (Y1H, transcriptional activation from the E-box was strong by ArntbHLH-C/EBP, and undetectable for the truncated ArntbHLH (PAS removed, as detected via readout from the HIS3 and lacZ reporters. In contrast, fluorescence anisotropy titrations showed affinities for the E-box with ArntbHLH-C/EBP and ArntbHLH comparable to other transcription factors (K(d 148.9 nM and 40.2 nM, respectively, but only under select conditions that maintained folded protein. Although in vivo yeast results and in vitro spectroscopic studies for ArntbHLH-C/EBP targeting the E-box correlate well, the same does not hold for ArntbHLH. As circular dichroism confirms that ArntbHLH-C/EBP is a much more strongly alpha-helical structure than ArntbHLH, we conclude that the nonfunctional ArntbHLH in the Y1H must be due to misfolding, leading to the false negative that this protein is incapable of targeting the E-box. Many experiments, including protein design and selections from large libraries, depend on protein domains remaining well-behaved in the nonnative experimental environment, especially small motifs like the bHLH (60-70 aa. Interestingly, a short helical LZ can serve as a folding- and/or solubility-enhancing tag, an important device given the focus of current research on exploration of vast networks of biomolecular interactions.

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

Science.gov (United States)

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

2017-07-19

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

Nopaline-type Ti plasmid of Agrobacterium encodes a VirF-like functional F-box protein.

Science.gov (United States)

Lacroix, Benoît; Citovsky, Vitaly

2015-11-20

During Agrobacterium-mediated genetic transformation of plants, several bacterial virulence (Vir) proteins are translocated into the host cell to facilitate infection. One of the most important of such translocated factors is VirF, an F-box protein produced by octopine strains of Agrobacterium, which presumably facilitates proteasomal uncoating of the invading T-DNA from its associated proteins. The presence of VirF also is thought to be involved in differences in host specificity between octopine and nopaline strains of Agrobacterium, with the current dogma being that no functional VirF is encoded by nopaline strains. Here, we show that a protein with homology to octopine VirF is encoded by the Ti plasmid of the nopaline C58 strain of Agrobacterium. This protein, C58VirF, possesses the hallmarks of functional F-box proteins: it contains an active F-box domain and specifically interacts, via its F-box domain, with SKP1-like (ASK) protein components of the plant ubiquitin/proteasome system. Thus, our data suggest that nopaline strains of Agrobacterium have evolved to encode a functional F-box protein VirF.

YB1/p32, a nuclear Y-box binding protein 1, is a novel regulator of myoblast differentiation that interacts with Msx1 homeoprotein

Energy Technology Data Exchange (ETDEWEB)

Song, Young Joon [Department of Biological Sciences, College of Natural Science, Inha University, 253 Yonghyun-dong, Nam-Gu, Incheon, Korea, 402-751 (Korea, Republic of); Lee, Hansol, E-mail: hlee@inha.ac.kr [Department of Biological Sciences, College of Natural Science, Inha University, 253 Yonghyun-dong, Nam-Gu, Incheon, Korea, 402-751 (Korea, Republic of)

2010-02-15

Precisely controlled cellular differentiation is essential for the proper development of vertebrate embryo and deregulated differentiation is a major cause of many human congenital diseases as well as cancer. Msx1 is a member of the homeoprotein family implicated in these processes, which inhibits the differentiation of skeletal muscle and other cell types, presumably by regulating transcription of target genes through interaction with other cellular factors. We presently show that YB1/p32, a nuclear Y-box binding protein 1, interacts with Msx1 homeoprotein and functions as a regulator of C2C12 myoblast differentiation. We demonstrate that YB1/p32 functionally interacts with Msx1 through its N-terminal region and colocalizes with Msx1 at the nuclear periphery. Moreover, we find that YB1/p32 is competent for inhibition of C2C12 myoblast differentiation, which is correlated with its activity as a negative regulator of MyoD gene expression and binding to the MyoD core enhancer region (CER). Furthermore, YB1/p32 cooperates with Msx1 in transcriptional repression and knocking down the expression of endogenous YB1 attenuates the effects of Msx1. Taken together, our study has uncovered a new function of YB1/p32, a regulator of skeletal muscle differentiation.

YB1/p32, a nuclear Y-box binding protein 1, is a novel regulator of myoblast differentiation that interacts with Msx1 homeoprotein

International Nuclear Information System (INIS)

Song, Young Joon; Lee, Hansol

2010-01-01

Precisely controlled cellular differentiation is essential for the proper development of vertebrate embryo and deregulated differentiation is a major cause of many human congenital diseases as well as cancer. Msx1 is a member of the homeoprotein family implicated in these processes, which inhibits the differentiation of skeletal muscle and other cell types, presumably by regulating transcription of target genes through interaction with other cellular factors. We presently show that YB1/p32, a nuclear Y-box binding protein 1, interacts with Msx1 homeoprotein and functions as a regulator of C2C12 myoblast differentiation. We demonstrate that YB1/p32 functionally interacts with Msx1 through its N-terminal region and colocalizes with Msx1 at the nuclear periphery. Moreover, we find that YB1/p32 is competent for inhibition of C2C12 myoblast differentiation, which is correlated with its activity as a negative regulator of MyoD gene expression and binding to the MyoD core enhancer region (CER). Furthermore, YB1/p32 cooperates with Msx1 in transcriptional repression and knocking down the expression of endogenous YB1 attenuates the effects of Msx1. Taken together, our study has uncovered a new function of YB1/p32, a regulator of skeletal muscle differentiation.

Imparting albumin-binding affinity to a human protein by mimicking the contact surface of a bacterial binding protein.

Science.gov (United States)

Oshiro, Satoshi; Honda, Shinya

2014-04-18

Attachment of a bacterial albumin-binding protein module is an attractive strategy for extending the plasma residence time of protein therapeutics. However, a protein fused with such a bacterial module could induce unfavorable immune reactions. To address this, we designed an alternative binding protein by imparting albumin-binding affinity to a human protein using molecular surface grafting. The result was a series of human-derived 6 helix-bundle proteins, one of which specifically binds to human serum albumin (HSA) with adequate affinity (KD = 100 nM). The proteins were designed by transferring key binding residues of a bacterial albumin-binding module, Finegoldia magna protein G-related albumin-binding domain (GA) module, onto the human protein scaffold. Despite 13-15 mutations, the designed proteins maintain the original secondary structure by virtue of careful grafting based on structural informatics. Competitive binding assays and thermodynamic analyses of the best binders show that the binding mode resembles that of the GA module, suggesting that the contacting surface of the GA module is mimicked well on the designed protein. These results indicate that the designed protein may act as an alternative low-risk binding module to HSA. Furthermore, molecular surface grafting in combination with structural informatics is an effective approach for avoiding deleterious mutations on a target protein and for imparting the binding function of one protein onto another.

Resolution of the mixture TBP-H/sub 2/ MPB-HDB-PH/sub 3/PO/sub 4/. Application to the systems UO/sub 2/(NO/sub 3/)/sub 2/-TBP, Th(NO/sub 3/)/sub 4/-TBP and ZrO(NO/sub 3/)/sub 2/-TBP

Energy Technology Data Exchange (ETDEWEB)

Pires, M A.F.; Abrao, A [Instituto de Pesquisas Energeticas e Nucleares, Sao Paulo (Brazil). Centro de Engenharia Quimica

1980-01-01

Tributylphosphate (TBP) is presently the most used extractant for uranium, thorium and zirconium technologies, both for the purification of these elements from their concentrates and for the recovery of heavy metals in the reprocessing of irradiated fuels. Nevertheless, in the presence of phosphoric acid, some degradation products are formed, such as: di-n-butylphosphoric acid (HDBP), monobutylphosphoric ancd (H/sub 2/MBP) and ortophosphoric acid. The accumulation of these degradation products in the organic phase of the liquid-liquid extraction processes is highly undesirable. In order to avoid this problem, the separation of the mixture HDBP, H/sub 2/MBP and H/sub 3/PO/sub 4/ in TBP-dilutent or TBP-diluent-uranyl, thorium of zirconyl nitrate was then studied. Several separation techniques were investigated: adsorption using a cationic resin saturated with U(VI), chromatography in macroporous polymers, chromatography in alumina columns. Refractive index and condutimetric measurements were carried out to follow up the separation processes. The best results were obtained in an alumina column.

Determination of the trace TBP in industrial feed liquid by gas chromatography

International Nuclear Information System (INIS)

Gao Yuehua; Jiang Junqing; Mu Ling; Yang Songtao

2012-01-01

The determination of the trace TBP in kerosene of the industrial feed liquid by gas chromatography is studied in the paper. It first takes the purification treatment for the kerosene containing trace TBP. The plutonium is removed by 0.2 mol/L ferrous sulfamate-1 mol/L nitric acid using the back-extraction. The uranium and the nitric acid in the organic phase are removed by the deionized water. The impurity which affect the measurement of the TBP and is harmful to the gas chromatograph are eliminated. Then the content of the TBP of the organic phase can be determined by gas chromatography. Results show that the measuring range of the content of the TBP is 0.02% ∼ 2%. The precision of the method is better than 5% and the recovery is between 95%∼106%. (authors)

Determination of dibutylphosphate and monobutylphosphate in TBP by ion chromatography

International Nuclear Information System (INIS)

Siva Kumar, B.; Vijayalakshmi, S.; Sankaran, K.; Ganesan, V.

2012-01-01

Tri-n-butyl phosphate (TBP) is used as solvent in the PUREX (Plutonium Uranium Refining by Extraction) process of nuclear fuel reprocessing. TBP undergoes chemical and radiological degradation to give DBP and MBP which in turn extracts the heavy metal such as U, Pu thereby affecting the performance of the extraction process. Analytical method using ion chromatography (IC) was developed for the determination of dibutyl phosphate (DBP) and monobutyl phosphate (MBP) in TBP. In this method, DBP and MBP were extracted from tri-n-butyl phosphate using carbonate-bicarbonate mixture of eluent composition and the aqueous phase was analyzed using suppressed ion chromatography employing carbonate as eluent. Standardization of extraction was carried out by standard addition studies. The detection limits for both DBP and MBP are found to be in sub ppm level. This method was applied to the analysis of TBP supplied by different suppliers

Inhibition of Y-box binding protein-1 slows the growth of glioblastoma multiforme and sensitizes to temozolomide independent O6-methylguanine-DNA methyltransferase.

Science.gov (United States)

Gao, Yuanyuan; Fotovati, Abbas; Lee, Cathy; Wang, Michelle; Cote, Gilbert; Guns, Emma; Toyota, Brian; Faury, Damien; Jabado, Nada; Dunn, Sandra E

2009-12-01

Glioblastoma multiforme (GBM) is an aggressive type of brain tumor where 5 years. In adults, GBM is the most common type of brain tumor. It is rarer in children, where it constitutes approximately 15% of all brain tumors diagnosed. These tumors are often invasive, making surgical resection difficult. Further, they can be refractory to current therapies such as temozolomide. The current dogma is that temozolomide resistance rests on the expression of O6-methylguanine-DNA methyltransferase (MGMT) because it cleaves methylated DNA adducts formed by the drug. Our laboratory recently reported that another drug resistance gene known as the Y-box binding protein-1 (YB-1) is highly expressed in primary GBM but not in normal brain tissues based on the evaluation of primary tumors. We therefore questioned whether GBM depend on YB-1 for growth and/or response to temozolomide. Herein, we report that YB-1 inhibition reduced tumor cell invasion and growth in monolayer as well as in soft agar. Moreover, blocking this protein ultimately delayed tumor onset in mice. Importantly, inhibiting YB-1 enhanced temozolomide sensitivity in a manner that was independent of MGMT in models of adult and pediatric GBM. In conclusion, inhibiting YB-1 may be a novel way to improve the treatment of GBM.

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

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Bastian Jöhnk

2016-09-01

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

Extraction of Eu(III) and Th(IV) with TBP in n-dodecane in the presence of nitric acid and the thermodynamic activity of the TBP in the system

International Nuclear Information System (INIS)

Santos, F.S.M. dos.

1983-02-01

A qualitative description of the behavior of distribution coefficients of nitric acid, Th(IV) and Eu(III) separately in the system HNO 3 -H 2 O-TBP-n-dodecane and the effect of the temperature in the distribution coefficient of Th(IV) are presented. The distribution coefficient of Eu(III) between aqueous solutions nitric acid and solutions of TBP in n-dodecane are measured. Since the composition of the extracted Eu(III) complex is known, the theoretical course of the dependency of the distribution coefficient on the TBP concentration can be predicted. Deviations of experimental dependencies from theoretical ones give them information about the thermodynamic activity of TBP in the organic phase. A mathematical description for the distribution coefficients of Eu(III) and HNO 3 in the mentioned system was sugested. This description will make possible an avaliation of the behavior of the activity coefficients of TBP in the organic phase. (A.R.H.) [pt

The architecture of mammalian ribosomal protein promoters

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Perry Robert P

2005-02-01

Full Text Available Abstract Background Mammalian ribosomes contain 79 different proteins encoded by widely scattered single copy genes. Coordinate expression of these genes at transcriptional and post-transcriptional levels is required to ensure a roughly equimolar accumulation of ribosomal proteins. To date, detailed studies of only a very few ribosomal protein (rp promoters have been made. To elucidate the general features of rp promoter architecture, I made a detailed sequence comparison of the promoter regions of the entire set of orthologous human and mouse rp genes. Results A striking evolutionarily conserved feature of most rp genes is the separation by an intron of the sequences involved in transcriptional and translational regulation from the sequences with protein encoding function. Another conserved feature is the polypyrimidine initiator, which conforms to the consensus (Y2C+1TY(T2(Y3. At least 60 % of the rp promoters contain a largely conserved TATA box or A/T-rich motif, which should theoretically have TBP-binding capability. A remarkably high proportion of the promoters contain conserved binding sites for transcription factors that were previously implicated in rp gene expression, namely upstream GABP and Sp1 sites and downstream YY1 sites. Over 80 % of human and mouse rp genes contain a transposable element residue within 900 bp of 5' flanking sequence; very little sequence identity between human and mouse orthologues was evident more than 200 bp upstream of the transcriptional start point. Conclusions This analysis has provided some valuable insights into the general architecture of mammalian rp promoters and has identified parameters that might coordinately regulate the transcriptional activity of certain subsets of rp genes.

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

Science.gov (United States)

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.

Analytical resolution of the mixture TBP-HDBP-H2MBP-H3PO4

International Nuclear Information System (INIS)

Pires, M.A.F.

1983-01-01

Several schemes for the separation of dibutylphosphoric acid (HDBP), main degradation product of tributylphosphate (TBP), in TBP/diluent, TBP/diluent-uranyl nitrate and TBP/diluent-thorium nitrate mixture were studied. For the resolution of HDBP-TBP/diluent-heavy metal nitrates (U-VI,Th-IV) systems, techniques such as: in exchange chromatography, ion chromatography using common ion exchangers and chromatographic separation with alumina column were investigated. For the identification, determination and analytical resolution following up the several systems studied, techniques such as refraction index measurement, electrical conductivity measurement, molecular absorption spectrophotometry, gas chromatography and ion chromatography, were applied. The separation of HDBP component was achieved using an alumina column where it was adsorbed from the TBP/diluent-uranyl nitrate and selectively eluted. Several modifications of this procedure for samples from the Uranium Purification Pilot Plant at Instituto de Pesquisas Energeticas e Nucleares (Sao Paulo, Brazil) were made. Special emphasis was given to the determination of HDBP using the ion chromatography technique. HDBP along with any monobutylphosphate acid (H 2 MBP) and phosphoric acid (H 3 PO 4 ) were stripped from the organic phase into dilute sodium hydroxide. HDBP is separated from H 2 MBP and H 3 PO 4 by ion chromatography and determined by its peak height. The determination of degradation products from TBP in TBP/diluent-uranyl nitrate and TBP/diluent-thorium nitrate systems was then performed. The detection limit for dibutylphosphate is 1.0μg HDBP/ml of analyte solution. (Author) [pt

Computational analysis of phosphopeptide binding to the polo-box domain of the mitotic kinase PLK1 using molecular dynamics simulation.

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David J Huggins

2010-08-01

Full Text Available The Polo-Like Kinase 1 (PLK1 acts as a central regulator of mitosis and is over-expressed in a wide range of human tumours where high levels of expression correlate with a poor prognosis. PLK1 comprises two structural elements, a kinase domain and a polo-box domain (PBD. The PBD binds phosphorylated substrates to control substrate phosphorylation by the kinase domain. Although the PBD preferentially binds to phosphopeptides, it has a relatively broad sequence specificity in comparison with other phosphopeptide binding domains. We analysed the molecular determinants of recognition by performing molecular dynamics simulations of the PBD with one of its natural substrates, CDC25c. Predicted binding free energies were calculated using a molecular mechanics, Poisson-Boltzmann surface area approach. We calculated the per-residue contributions to the binding free energy change, showing that the phosphothreonine residue and the mainchain account for the vast majority of the interaction energy. This explains the very broad sequence specificity with respect to other sidechain residues. Finally, we considered the key role of bridging water molecules at the binding interface. We employed inhomogeneous fluid solvation theory to consider the free energy of water molecules on the protein surface with respect to bulk water molecules. Such an analysis highlights binding hotspots created by elimination of water molecules from hydrophobic surfaces. It also predicts that a number of water molecules are stabilized by the presence of the charged phosphate group, and that this will have a significant effect on the binding affinity. Our findings suggest a molecular rationale for the promiscuous binding of the PBD and highlight a role for bridging water molecules at the interface. We expect that this method of analysis will be very useful for probing other protein surfaces to identify binding hotspots for natural binding partners and small molecule inhibitors.

Cytosine methylation does not affect binding of transcription factor Sp1

International Nuclear Information System (INIS)

Harrington, M.A.; Jones, P.A.; Imagawa, M.; Karin, M.

1988-01-01

DNA methylation may be a component of a multilevel control mechanism that regulates eukaryotic gene expression. The authors used synthetic oligonucleotides to investigate the effect of cytosine methylation on the binding of the transcription factor Sp1 to its target sequence (a G+C-rich sequence known as a GC box). Concatemers of double-stranded 14-mers containing a GC box successfully competed with the human metallothionein IIA promoter for binding to Sp1 in DNase I protection experiments. The presence of 5-methylcytosine in the CpG sequence of the GC box did not influence Sp1 binding. The result was confirmed using double-stranded 20-mers containing 16 base pairs of complementary sequence. Electrophoretic gel retardation analysis of annealed 28-mers containing a GC box incubated with an Sp1-containing HeLa cell nuclear extract demonstrated the formation of DNA-protein complexes; formation of these complexes was not inhibited when an oligomer without a GC box was used as a competitor. Once again, the presence of a 5-methylcytosine residue in the GC box did not influence the binding of the protein to DNA. The results therefore preclude a direct effect of cytosine methylation on Sp1-DNA interactions

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

Science.gov (United States)

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.

Mass transfer model for two-layer TBP oxidation reactions: Revision 1

International Nuclear Information System (INIS)

Laurinat, J.E.

1994-01-01

To prove that two-layer, TBP-nitric acid mixtures can be safely stored in the Canyon evaporators, it must be demonstrated that a runaway reaction between TBP and nitric acid will not occur. Previous bench-scale experiments showed that, at typical evaporator temperatures, this reaction is endothermic and therefore cannot run away, due to the loss of heat from evaporation of water in the organic layer. However, the reaction would be exothermic and could run away if the small amount of water in the organic layer evaporates before the nitric acid in this layer is consumed by the reaction. Provided that there is enough water in the aqueous layer, this would occur if the organic layer is sufficiently thick so that the rate of loss of water by evaporation exceeds the rate of replenishment due to mixing with the aqueous layer. Bubbles containing reaction products enhance the rate of transfer of water from the aqueous layer to the organic layer. These bubbles are generated by the oxidation of TBP and its reaction products in the organic layer and by the oxidation of butanol in the aqueous layer. Butanol is formed by the hydrolysis of TBP in the organic layer. For aqueous-layer bubbling to occur, butanol must transfer into the aqueous layer. Consequently, the rate of oxidation and bubble generation in the aqueous layer strongly depends on the rate of transfer of butanol from the organic to the aqueous layer. This report presents measurements of mass transfer rates for the mixing of water and butanol in two-layer, TBP-aqueous mixtures, where the top layer is primarily TBP and the bottom layer is comprised of water or aqueous salt solution. Mass transfer coefficients are derived for use in the modeling of two-layer TBP-nitric acid oxidation experiments

A hierarchical analysis of transcriptome alterations in intrauterine growth restriction (IUGR) reveals common pathophysiological pathways in mammals.

Science.gov (United States)

Buffat, C; Mondon, F; Rigourd, V; Boubred, F; Bessières, B; Fayol, L; Feuerstein, J-M; Gamerre, M; Jammes, H; Rebourcet, R; Miralles, F; Courbières, B; Basire, A; Dignat-Georges, F; Carbonne, B; Simeoni, U; Vaiman, D

2007-11-01

Intra-uterine growth restriction (IUGR) is a frequent disease, affecting up to 10% of human pregnancies and responsible for increased perinatal morbidity and mortality. Moreover, low birth weight is an important cause of the metabolic syndrome in the adult. Protein depletion during the gestation of rat females has been widely used as a model for human IUGR. By transcriptome analysis of control and protein-deprived rat placentas, we were able to identify 2543 transcripts modified more than 2.5 fold (1347 induced and 1196 repressed). Automatic functional classification enabled us to identify clusters of induced genes affecting chromosome structure, transcription, intracellular transport, protein modifications and apoptosis. In particular, we suggest the existence of a complex balance regulating apoptosis. Among repressed genes, we noted several groups of genes involved in immunity, signalling and degradation of noxious chemicals. These observations suggest that IUGR placentas have a decreased resistance to external aggression. The promoters of the most induced and most repressed genes were contrasted for their composition in putative transcription factor binding sites. There was an over-representation of Zn finger (ZNF) proteins and Pdx1 (pancreatic and duodenal homeobox protein 1) putative binding sites. Consistently, Pdx1 and a high proportion of ZNF genes were induced at the transcriptional level. A similar analysis of ZNF promoters showed an increased presence of putative binding sites for the Tata box binding protein (Tbp). Consistently again, we showed that the Tbp and TBP-associated factors (Tafs) were up-regulated in IUGR placentas. Also, samples of human IUGR and control placentas showed that human orthologous ZNFs and PDX1 were transcriptionally induced, especially in non-vascular IUGR. Immunohistochemistry revealed increased expression of PDX1 in IUGR human placentas. In conclusion, our approach permitted the proposition of hypotheses on a hierarchy of

Adipocyte spliced form of X-box-binding protein 1 promotes adiponectin multimerization and systemic glucose homeostasis

NARCIS (Netherlands)

Sha, H.; Yang, L.; Liu, M.; Xia, S.; Liu, Y.; Liu, F.; Kersten, A.H.; Qi, L.

2014-01-01

The physiological role of the spliced form of X-box–binding protein 1 (XBP1s), a key transcription factor of the endoplasmic reticulum (ER) stress response, in adipose tissue remains largely unknown. In this study, we show that overexpression of XBP1s promotes adiponectin multimerization in

Resolution of TBP-H/sub 2/MBP-HDBP-H/sub 3/PO/sub 4/. Application to UO/sub 2/(NO/sub 3/)/sub 2/-TBP, Th(NO/sub 3/)/sub 4/-TBP, and ZrO(NO/sub 3/)/sub 2/-TBP systems. [Separation of dibutyl phosphoric acid, monobutylphosphoric acid and orthophosphoric acid from tri-butylphosphate

Energy Technology Data Exchange (ETDEWEB)

Pires, M A.F.; Abrao, A

1981-04-01

Several schemes for the separation of dibutylphosphoric acid (HDBP), monobutylphosphoric acid (H/sub 2/MBP) and orthophosphoric acid (H/sub 3/PO/sub 4/) as hydrolytic and radiolytic degradation products from tri-n-butylphosphate (TBP) were studied. For the resolution of a HDBP, H/sub 2/MPB and H/sub 3/PO/sub 4/ mixture in TBP-diluent, or in TBP-diluent-heavy metal nitrate (U-VI, Th-IV or Zr-IV), techniques such as ion exchange chromatography, ion chromatography and separation onto a chromatographic alumina column were investigated. For the identification, determination and analytical resolution following up for the several systems studied, techniques such as refraction index measurement, electrical conductivity measurement, molecular spectrophotometry and gas chromatography were applied. Special emphasis was given to the separation using alumina column where the HDBP acid was retained and eluted selectively for its separation from TBP-varsol-uranyl nitrate mixtures. This analytical procedure was applied to the samples coming from the Uranium Purification Pilot Plant in operation at the Centro de Engenharia Quimica (IPEN).

The occurrence of gibberellin-binding protein(s) in pea

Energy Technology Data Exchange (ETDEWEB)

Liu, Z.H.

1988-01-01

In vitro gibberellin (GA) binding properties of a cytosol fraction from epicotyls of dwarf pea (Pisum sativum L. cv. Progress No. 9) and tall pea (Pisum sativum L. cv. Alaska) were investigated using ({sup 3}H)GA{sub 4} in a DEAE filter paper assay at 0-3 C. The binding obtained is saturable, reversible, and temperature labile in dwarf pea, and has a half-life of dissociation of 5-6 min. By varying the concentration of ({sup 3}H)GA{sub 4} in the incubation medium the Kd was estimated to be 120-140 nM in dwarf pea and 70 nM in tall pea. The number of binding sites (n) was estimated to be 0.66 and 0.43 pmole mg{sup {minus}1} soluble protein in dwarf pea and in tall pea, respectively. In competition binding assays, biologically active GAs, such as GA{sub 3} and GA{sub 4} could reduce the level of ({sup 3}H)GA{sub 4} binding much more than the biologically inactive GA{sub 4} methyl ester and epi-GA{sub 4}. Changes in gibberellin-binding protein(s) were studied during seed germination. While the Kd of the binding protein(s) for ({sup 3}H)GA{sub 4} remained the same, there was a marked increase in the number of binding sites from 24 h soaked seed to 8-day old seedlings. Also, the Kd and the number of binding sites in the GA-responsive apical part and in the nonresponsive basal part in the epicotyl were similar. The effect of light on gibberellin-binding protein in dwarf pea was also studied. The GA-binding protein in dwarf pea was partially purified by gel filtration and ion exchange chromatography.

Predicting protein-binding RNA nucleotides with consideration of binding partners.

Science.gov (United States)

Tuvshinjargal, Narankhuu; Lee, Wook; Park, Byungkyu; Han, Kyungsook

2015-06-01

In recent years several computational methods have been developed to predict RNA-binding sites in protein. Most of these methods do not consider interacting partners of a protein, so they predict the same RNA-binding sites for a given protein sequence even if the protein binds to different RNAs. Unlike the problem of predicting RNA-binding sites in protein, the problem of predicting protein-binding sites in RNA has received little attention mainly because it is much more difficult and shows a lower accuracy on average. In our previous study, we developed a method that predicts protein-binding nucleotides from an RNA sequence. In an effort to improve the prediction accuracy and usefulness of the previous method, we developed a new method that uses both RNA and protein sequence data. In this study, we identified effective features of RNA and protein molecules and developed a new support vector machine (SVM) model to predict protein-binding nucleotides from RNA and protein sequence data. The new model that used both protein and RNA sequence data achieved a sensitivity of 86.5%, a specificity of 86.2%, a positive predictive value (PPV) of 72.6%, a negative predictive value (NPV) of 93.8% and Matthews correlation coefficient (MCC) of 0.69 in a 10-fold cross validation; it achieved a sensitivity of 58.8%, a specificity of 87.4%, a PPV of 65.1%, a NPV of 84.2% and MCC of 0.48 in independent testing. For comparative purpose, we built another prediction model that used RNA sequence data alone and ran it on the same dataset. In a 10 fold-cross validation it achieved a sensitivity of 85.7%, a specificity of 80.5%, a PPV of 67.7%, a NPV of 92.2% and MCC of 0.63; in independent testing it achieved a sensitivity of 67.7%, a specificity of 78.8%, a PPV of 57.6%, a NPV of 85.2% and MCC of 0.45. In both cross-validations and independent testing, the new model that used both RNA and protein sequences showed a better performance than the model that used RNA sequence data alone in

Determination of TBP in nitric solutions of process

International Nuclear Information System (INIS)

Kuada, T.A.; Carvalho, E.I. de; Araujo, I. da C. de; Cohen, V.H.

1988-01-01

Heavy organic phases have been observed on some occasions during TBP extraction process. These products, described as red oils, were considered as the main cause for process failures, specially in evaporators and concentrators. In view of safety aspects it is necessary to control organic concentration in product and waste solutions. The proposed method involves the organic removal by chloroform as a first step, followed by purification onto a silica gel column. The results are given from analysing TBP and its degradation product (DBP) by gas chromatography. (author) [pt

Fragment-based quantum mechanical calculation of protein-protein binding affinities.

Science.gov (United States)

Wang, Yaqian; Liu, Jinfeng; Li, Jinjin; He, Xiao

2018-04-29

The electrostatically embedded generalized molecular fractionation with conjugate caps (EE-GMFCC) method has been successfully utilized for efficient linear-scaling quantum mechanical (QM) calculation of protein energies. In this work, we applied the EE-GMFCC method for calculation of binding affinity of Endonuclease colicin-immunity protein complex. The binding free energy changes between the wild-type and mutants of the complex calculated by EE-GMFCC are in good agreement with experimental results. The correlation coefficient (R) between the predicted binding energy changes and experimental values is 0.906 at the B3LYP/6-31G*-D level, based on the snapshot whose binding affinity is closest to the average result from the molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) calculation. The inclusion of the QM effects is important for accurate prediction of protein-protein binding affinities. Moreover, the self-consistent calculation of PB solvation energy is required for accurate calculations of protein-protein binding free energies. This study demonstrates that the EE-GMFCC method is capable of providing reliable prediction of relative binding affinities for protein-protein complexes. © 2018 Wiley Periodicals, Inc. © 2018 Wiley Periodicals, Inc.

The Negatively Charged Regions of Lactoferrin Binding Protein B, an Adaptation against Anti-Microbial Peptides

Science.gov (United States)

Morgenthau, Ari; Beddek, Amanda; Schryvers, Anthony B.

2014-01-01

Lactoferrin binding protein B (LbpB) is a bi-lobed membrane bound lipoprotein that is part of the lactoferrin receptor complex in a variety of Gram-negative pathogens. Despite high sequence diversity among LbpBs from various strains and species, a cluster of negatively charged amino acids is invariably present in the protein’s C-terminal lobe in all species except Moraxella bovis. The function of LbpB in iron acquisition has yet to be experimentally demonstrated, whereas in vitro studies have shown that LbpB confers protection against lactoferricin, a short cationic antimicrobial peptide released from the N- terminus of lactoferrin. In this study we demonstrate that the negatively charged regions can be removed from the Neisseria meningitidis LbpB without compromising stability, and this results in the inability of LbpB to protect against the bactericidal effects of lactoferricin. The release of LbpB from the cell surface by the autotransporter NalP reduces the protection against lactoferricin in the in vitro killing assay, attributed to removal of LbpB during washing steps, but is unlikely to have a similar impact in vivo. The protective effect of the negatively charged polysaccharide capsule in the killing assay was less than the protection conferred by LbpB, suggesting that LbpB plays a major role in protection against cationic antimicrobial peptides in vivo. The selective release of LbpB by NalP has been proposed to be a mechanism for evading the adaptive immune response, by reducing the antibody binding to the cell surface, but may also provide insights into the primary function of LbpB in vivo. Although TbpB and LbpB have been shown to be major targets of the human immune response, the selective release of LbpB suggests that unlike TbpB, LbpB may not be essential for iron acquisition, but important for protection against cationic antimicrobial peptides. PMID:24465982

Hydrothermal decomposition of TBP and fixation of its decomposed residue by HHP technique

International Nuclear Information System (INIS)

Yamasaki, N.; Fujiki, M.; Nishioka, M.; Ioku, K.; Yanagisawa, K.; Kozai, N.; Muraoka, S.

1991-01-01

The tributyl phosphate (TBP) used for the fuel reprocessing by Purex process is discharged as spent solvent because of the chemical decomposition and the damage due to radiation. Alkaline hydrothermal treatment in oxygen which is the reaction in a closed system is effective for the decomposition of TBP as it can transform organic materials to stable inorganic ions. Hydrothermal hot pressing technique has been applied to the immobilization of various radioactive wastes. This work deals with the continuous treatment process for the decomposition of TBP waste and the immobilization of its decomposed residue under hydrothermal condition. These processes are outlined. The experiment and the results are reported. TBP was completely decomposed above 200degC, and COD value showed the maximum at 250degC. The reaction process consists of two steps of the hydrolysis of TBP and the oxidation of the formed organic material. (K.I.)

Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

Energy Technology Data Exchange (ETDEWEB)

Gangi Setty, Thanuja [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Cho, Christine [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Govindappa, Sowmya [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Apicella, Michael A. [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Ramaswamy, S., E-mail: ramas@instem.res.in [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India)

2014-07-01

Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.

Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

International Nuclear Information System (INIS)

Gangi Setty, Thanuja; Cho, Christine; Govindappa, Sowmya; Apicella, Michael A.; Ramaswamy, S.

2014-01-01

Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states

Identification of functional domains of the IR2 protein of equine herpesvirus 1 required for inhibition of viral gene expression and replication

International Nuclear Information System (INIS)

Kim, Seong K.; Kim, Seongman; Dai Gan; Zhang Yunfei; Ahn, Byung C.; O'Callaghan, Dennis J.

2011-01-01

The equine herpesvirus 1 (EHV-1) negative regulatory IR2 protein (IR2P), an early 1,165-amino acid (aa) truncated form of the 1487-aa immediate-early protein (IEP), lacks the trans-activation domain essential for IEP activation functions but retains domains for binding DNA, TFIIB, and TBP and the nuclear localization signal. IR2P mutants of the N-terminal region which lack either DNA-binding activity or TFIIB-binding activity were unable to down-regulate EHV-1 promoters. In EHV-1-infected cells expressing full-length IR2P, transcription and protein expression of viral regulatory IE, early EICP0, IR4, and UL5, and late ETIF genes were dramatically inhibited. Viral DNA levels were reduced to 2.1% of control infected cells, but were vey weakly affected in cells that express the N-terminal 706 residues of IR2P. These results suggest that IR2P function requires the two N-terminal domains for binding DNA and TFIIB as well as the C-terminal residues 707 to 1116 containing the TBP-binding domain. - Highlights: → We examine the functional domains of IR2P that mediates negative regulation. → IR2P inhibits at the transcriptional level. → DNA-binding mutant or TFIIB-binding mutant fails to inhibit. → C-terminal aa 707 to 1116 are required for full inhibition. → Inhibition requires the DNA-binding domain, TFIIB-binding domain, and C-terminus.

Radiation damage to DNA-binding proteins

International Nuclear Information System (INIS)

Culard, G.; Eon, S.; DeVuyst, G.; Charlier, M.; Spotheim-Maurizot, M.

2003-01-01

The DNA-binding properties of proteins are strongly affected upon irradiation. The tetrameric lactose repressor (a dimer of dimers) losses its ability to bind operator DNA as soon as at least two damages per protomer of each dimer occur. The monomeric MC1 protein losses its ability to bind DNA in two steps : i) at low doses only the specific binding is abolished, whereas the non-specific one is still possible; ii) at high doses all binding vanishes. Moreover, the DNA bending induced by MC1 binding is less pronounced for a protein that underwent the low dose irradiation. When the entire DNA-protein complexes are irradiated, the observed disruption of the complexes is mainly due to the damage of the proteins and not to that of DNA. The doses necessary for complex disruption are higher than those inactivating the free protein. This difference, larger for MC1 than for lactose repressor, is due to the protection of the protein by the bound DNA. The oxidation of the protein side chains that are accessible to the radiation-induced hydroxyl radicals seems to represent the inactivating damage

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

Directory of Open Access Journals (Sweden)

Hiroshi Kanno

2018-02-01

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

Prognostic implications of the nuclear localization of Y-box-binding protein-1 and CXCR4 expression in ovarian cancer: their correlation with activated Akt, LRP/MVP and P-glycoprotein expression.

Science.gov (United States)

Oda, Yoshinao; Ohishi, Yoshihiro; Basaki, Yuji; Kobayashi, Hiroaki; Hirakawa, Toshio; Wake, Norio; Ono, Mayumi; Nishio, Kazuto; Kuwano, Michihiko; Tsuneyoshi, Masazumi

2007-07-01

The nuclear localization of Y-box-binding protein-1 (YB-1) is known to be a poor prognostic factor in several human malignancies, including ovarian carcinoma. Following on from our basic study dealing with microarray analyses of YB-1-associated gene expression in ovarian cancer cells, we examined whether nuclear localization of YB-1 is associated with the expression of CXCR4, a vault protein named lung resistance-related vault protein (LRP/MVP), phosphorylated Akt (p-Akt) or P-glycoprotein (P-gp) in human ovarian carcinoma. Fifty-three surgically resected ovarian carcinomas treated with paclitaxel and carboplatin were examined immunohistochemically for nuclear YB-1 expression and intrinsic expression of p-Akt, P-gp, LRP/MVP and CXCR4. Nuclear expression of YB-1 demonstrated significant correlation with p-Akt, P-gp and LRP expression, but no relationship with CXCR4 expression. By multivariate analysis, only YB-1 nuclear expression and CXCR4 expression were independent prognostic factors with regard to overall survival. These results indicate that YB-1 nuclear expression and CXCR4 expression are important prognostic factors in ovarian carcinoma.

The structure of lactoferrin-binding protein B from Neisseria meningitidis suggests roles in iron acquisition and neutralization of host defences

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Brooks, Cory L.; Arutyunova, Elena; Lemieux, M. Joanne

2014-01-01

Pathogens have evolved a range of mechanisms to acquire iron from the host during infection. Several Gram-negative pathogens including members of the genera Neisseria and Moraxella have evolved two-component systems that can extract iron from the host glycoproteins lactoferrin and transferrin. The homologous iron-transport systems consist of a membrane-bound transporter and an accessory lipoprotein. While the mechanism behind iron acquisition from transferrin is well understood, relatively little is known regarding how iron is extracted from lactoferrin. Here, the crystal structure of the N-terminal domain (N-lobe) of the accessory lipoprotein lactoferrin-binding protein B (LbpB) from the pathogen Neisseria meningitidis is reported. The structure is highly homologous to the previously determined structures of the accessory lipoprotein transferrin-binding protein B (TbpB) and LbpB from the bovine pathogen Moraxella bovis. Docking the LbpB structure with lactoferrin reveals extensive binding interactions with the N1 subdomain of lactoferrin. The nature of the interaction precludes apolactoferrin from binding LbpB, ensuring the specificity of iron-loaded lactoferrin. The specificity of LbpB safeguards proper delivery of iron-bound lactoferrin to the transporter lactoferrin-binding protein A (LbpA). The structure also reveals a possible secondary role for LbpB in protecting the bacteria from host defences. Following proteolytic digestion of lactoferrin, a cationic peptide derived from the N-terminus is released. This peptide, called lactoferricin, exhibits potent antimicrobial effects. The docked model of LbpB with lactoferrin reveals that LbpB interacts extensively with the N-terminal lactoferricin region. This may provide a venue for preventing the production of the peptide by proteolysis, or directly sequestering the peptide, protecting the bacteria from the toxic effects of lactoferricin. PMID:25286931

A study on the safety of TBP(150A) with forming analysis and strength test

International Nuclear Information System (INIS)

Lee, Sung Ho; Kim, Hei Song

2008-01-01

For this study, the forming analyses and body strength tests of TBP were performed on the main pipe size 150A(KS D3507, KS D 3576 10S). The branched pipe sizes utilized were 25A, 32A, 40A, 50A, 65A, 80A, 100A and 125A. A general FEM program, ABAQUS, was used as the forming analyses method of TBP. Using the results, the strength of TBP was then tested in order to determine the safety of TBP when the working pressure was applied. The results indicate that TBP may be safely used in water-based fire protection pipe systems in terms of the strength

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

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Yuki Kuroiwa

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

Identification of histone H4-like TAF in Schizosaccharomyces pombe as a protein that interacts with WD repeat-containing TAF

OpenAIRE

Mitsuzawa, Hiroshi; Ishihama, Akira

2002-01-01

The general transcription factor TFIID consists of the TATA-binding protein (TBP) and multiple TBP-associated factors (TAFs). We previously identified two distinct WD repeat-containing TAFs, spTAF72 and spTAF73, in the fission yeast Schizosaccharomyces pombe. Here we report the identification of another S.pombe TAF, spTAF50, which is the S.pombe homolog of histone H4-like TAFs such as human TAF80, Drosophila TAF60 and Saccharomyces cerevisiae TAF60. spTAF50 was identified in a two-hybrid scre...

Quantify Water Extraction by TBP/Dodecane via Molecular Dynamics Simulations

International Nuclear Information System (INIS)

Khomami, Bamin; Cui, Shengting; De Almeida, Valmor F.

2013-01-01

The purpose of this project is to quantify the interfacial transport of water into the most prevalent nuclear reprocessing solvent extractant mixture, namely tri-butyl- phosphate (TBP) and dodecane, via massively parallel molecular dynamics simulations on the most powerful machines available for open research. Specifically, we will accomplish this objective by evolving the water/TBP/dodecane system up to 1 ms elapsed time, and validate the simulation results by direct comparison with experimentally measured water solubility in the organic phase. The significance of this effort is to demonstrate for the first time that the combination of emerging simulation tools and state-of-the-art supercomputers can provide quantitative information on par to experimental measurements for solvent extraction systems of relevance to the nuclear fuel cycle. Results: Initially, the isolated single component, and single phase systems were studied followed by the two-phase, multicomponent counterpart. Specifically, the systems we studied were: pure TBP; pure n-dodecane; TBP/n-dodecane mixture; and the complete extraction system: water-TBP/n-dodecane two phase system to gain deep insight into the water extraction process. We have completely achieved our goal of simulating the molecular extraction of water molecules into the TBP/n-dodecane mixture up to the saturation point, and obtained favorable comparison with experimental data. Many insights into fundamental molecular level processes and physics were obtained from the process. Most importantly, we found that the dipole moment of the extracting agent is crucially important in affecting the interface roughness and the extraction rate of water molecules into the organic phase. In addition, we have identified shortcomings in the existing OPLS-AA force field potential for long-chain alkanes. The significance of this force field is that it is supposed to be optimized for molecular liquid simulations. We found that it failed for dodecane and

A structural classification of substrate-binding proteins

NARCIS (Netherlands)

Berntsson, Ronnie P. -A.; Smits, Sander H. J.; Schmitt, Lutz; Slotboom, Dirk-Jan; Poolman, Bert

2010-01-01

Substrate-binding proteins (SBP) are associated with a wide variety of protein complexes. The proteins are part of ATP-binding cassette transporters for substrate uptake, ion gradient driven transporters, DNA-binding proteins, as well as channels and receptors from both pro-and eukaryotes. A wealth

Elucidating the evolutionary conserved DNA-binding specificities of WRKY transcription factors by molecular dynamics and in vitro binding assays

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Brand, Luise H.; Fischer, Nina M.; Harter, Klaus; Kohlbacher, Oliver; Wanke, Dierk

2013-01-01

WRKY transcription factors constitute a large protein family in plants that is involved in the regulation of developmental processes and responses to biotic or abiotic stimuli. The question arises how stimulus-specific responses are mediated given that the highly conserved WRKY DNA-binding domain (DBD) exclusively recognizes the ‘TTGACY’ W-box consensus. We speculated that the W-box consensus might be more degenerate and yet undetected differences in the W-box consensus of WRKYs of different evolutionary descent exist. The phylogenetic analysis of WRKY DBDs suggests that they evolved from an ancestral group IIc-like WRKY early in the eukaryote lineage. A direct descent of group IIc WRKYs supports a monophyletic origin of all other group II and III WRKYs from group I by loss of an N-terminal DBD. Group I WRKYs are of paraphyletic descent and evolved multiple times independently. By homology modeling, molecular dynamics simulations and in vitro DNA–protein interaction-enzyme-linked immunosorbent assay with AtWRKY50 (IIc), AtWRKY33 (I) and AtWRKY11 (IId) DBDs, we revealed differences in DNA-binding specificities. Our data imply that other components are essentially required besides the W-box-specific binding to DNA to facilitate a stimulus-specific WRKY function. PMID:23975197

Multiple protonation equilibria in electrostatics of protein-protein binding.

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Piłat, Zofia; Antosiewicz, Jan M

2008-11-27

All proteins contain groups capable of exchanging protons with their environment. We present here an approach, based on a rigorous thermodynamic cycle and the partition functions for energy levels characterizing protonation states of the associating proteins and their complex, to compute the electrostatic pH-dependent contribution to the free energy of protein-protein binding. The computed electrostatic binding free energies include the pH of the solution as the variable of state, mutual "polarization" of associating proteins reflected as changes in the distribution of their protonation states upon binding and fluctuations between available protonation states. The only fixed property of both proteins is the conformation; the structure of the monomers is kept in the same conformation as they have in the complex structure. As a reference, we use the electrostatic binding free energies obtained from the traditional Poisson-Boltzmann model, computed for a single macromolecular conformation fixed in a given protonation state, appropriate for given solution conditions. The new approach was tested for 12 protein-protein complexes. It is shown that explicit inclusion of protonation degrees of freedom might lead to a substantially different estimation of the electrostatic contribution to the binding free energy than that based on the traditional Poisson-Boltzmann model. This has important implications for the balancing of different contributions to the energetics of protein-protein binding and other related problems, for example, the choice of protein models for Brownian dynamics simulations of their association. Our procedure can be generalized to include conformational degrees of freedom by combining it with molecular dynamics simulations at constant pH. Unfortunately, in practice, a prohibitive factor is an enormous requirement for computer time and power. However, there may be some hope for solving this problem by combining existing constant pH molecular dynamics

UV-induced DNA-binding proteins in human cells

International Nuclear Information System (INIS)

Glazer, P.M.; Greggio, N.A.; Metherall, J.E.; Summers, W.C.

1989-01-01

To investigate the response of human cells to DNA-damaging agents such as UV irradiation, the authors examined nuclear protein extracts of UV-irradiated HeLa cells for the presence of DNA-binding proteins. Electrophoretically separated proteins were transferred to a nitrocellulose filter that was subsequently immersed in a binding solution containing radioactively labeled DNA probes. Several DNA-binding proteins were induced in HeLa cells after UV irradiation. These included proteins that bind predominantly double-stranded DNA and proteins that bind both double-stranded and single-stranded DNA. The binding proteins were induced in a dose-dependent manner by UV light. Following a dose of 12 J/m 2 , the binding proteins in the nuclear extracts increased over time to a peak in the range of 18 hr after irradiation. Experiments with metabolic inhibitors (cycloheximide and actinomycin D) revealed that de novo synthesis of these proteins is not required for induction of the binding activities, suggesting that the induction is mediated by protein modification

Transcription initiation complex structures elucidate DNA opening.

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Plaschka, C; Hantsche, M; Dienemann, C; Burzinski, C; Plitzko, J; Cramer, P

2016-05-19

Transcription of eukaryotic protein-coding genes begins with assembly of the RNA polymerase (Pol) II initiation complex and promoter DNA opening. Here we report cryo-electron microscopy (cryo-EM) structures of yeast initiation complexes containing closed and open DNA at resolutions of 8.8 Å and 3.6 Å, respectively. DNA is positioned and retained over the Pol II cleft by a network of interactions between the TATA-box-binding protein TBP and transcription factors TFIIA, TFIIB, TFIIE, and TFIIF. DNA opening occurs around the tip of the Pol II clamp and the TFIIE 'extended winged helix' domain, and can occur in the absence of TFIIH. Loading of the DNA template strand into the active centre may be facilitated by movements of obstructing protein elements triggered by allosteric binding of the TFIIE 'E-ribbon' domain. The results suggest a unified model for transcription initiation with a key event, the trapping of open promoter DNA by extended protein-protein and protein-DNA contacts.

The Compass-like locus, exclusive to the Ambulacrarians, encodes a chromatin insulator binding protein in the sea urchin embryo.

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Vincenzo Cavalieri

Full Text Available Chromatin insulators are eukaryotic genome elements that upon binding of specific proteins display barrier and/or enhancer-blocking activity. Although several insulators have been described throughout various metazoans, much less is known about proteins that mediate their functions. This article deals with the identification and functional characterization in Paracentrotus lividus of COMPASS-like (CMPl, a novel echinoderm insulator binding protein. Phylogenetic analysis shows that the CMPl factor, encoded by the alternative spliced Cmp/Cmpl transcript, is the founder of a novel ambulacrarian-specific family of Homeodomain proteins containing the Compass domain. Specific association of CMPl with the boxB cis-element of the sns5 chromatin insulator is demonstrated by using a yeast one-hybrid system, and further corroborated by ChIP-qPCR and trans-activation assays in developing sea urchin embryos. The sns5 insulator lies within the early histone gene cluster, basically between the H2A enhancer and H1 promoter. To assess the functional role of CMPl within this locus, we challenged the activity of CMPl by two distinct experimental strategies. First we expressed in the developing embryo a chimeric protein, containing the DNA-binding domain of CMPl, which efficiently compete with the endogenous CMPl for the binding to the boxB sequence. Second, to titrate the embryonic CMPl protein, we microinjected an affinity-purified CMPl antibody. In both the experimental assays we congruently observed the loss of the enhancer-blocking function of sns5, as indicated by the specific increase of the H1 expression level. Furthermore, microinjection of the CMPl antiserum in combination with a synthetic mRNA encoding a forced repressor of the H2A enhancer-bound MBF1 factor restores the normal H1 mRNA abundance. Altogether, these results strongly support the conclusion that the recruitment of CMPl on sns5 is required for buffering the H1 promoter from the H2A enhancer

When is protein binding important?

Science.gov (United States)

Heuberger, Jules; Schmidt, Stephan; Derendorf, Hartmut

2013-09-01

The present paper is an ode to a classic citation by Benet and Hoener (2002. Clin Pharm Ther 71(3):115-121). The now classic paper had a huge impact on drug development and the way the issue of protein binding is perceived and interpreted. Although the authors very clearly pointed out the limitations and underlying assumptions for their delineations, these are too often overlooked and the classic paper's message is misinterpreted by broadening to cases that were not intended. Some members of the scientific community concluded from the paper that protein binding is not important. This was clearly not intended by the authors, as they finished their paper with a paragraph entitled: "When is protein binding important?" Misinterpretation of the underlying assumptions in the classic work can result in major pitfalls in drug development. Therefore, we revisit the topic of protein binding with the intention of clarifying when clinically relevant changes should be considered during drug development. Copyright © 2013 Wiley Periodicals, Inc.

Megalin binds and mediates cellular internalization of folate binding protein

DEFF Research Database (Denmark)

Birn, Henrik; Zhai, Xiaoyue; Holm, Jan

2005-01-01

Folate is an essential vitamin involved in a number of biological processes. High affinity folate binding proteins (FBPs) exist both as glycosylphosphatidylinositol-linked, membrane associated folate binding proteins and as soluble FBPs in plasma and some secretory fluids such as milk, saliva...... to express high levels of megalin, is inhibitable by excess unlabeled FBP and by receptor associated protein, a known inhibitor of binding to megalin. Immortalized rat yolk sac cells, representing an established model for studying megalin-mediated uptake, reveal (125)I-labeled FBP uptake which is inhibited...

Protein binding of psychotropic agents

International Nuclear Information System (INIS)

Hassan, H.A.

1990-01-01

Based upon fluorescence measurements, protein binding of some psychotropic agents (chlorpromazine, promethazine, and trifluoperazine) to human IgG and HSA was studied in aqueous cacodylate buffer, PH7. The interaction parameters determined from emission quenching of the proteins. The interaction parameters determined include the equilibrium constant (K), calculated from equations derived by Borazan and coworkers, the number of binding sites (n) available to the monomer molecules on a single protein molecule. The results revealed a high level of affinity, as reflected by high values of K, and the existence of specific binding sites, since a limited number of n values are obtained. 39 tabs.; 37 figs.; 83 refs

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

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

2015-12-01

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

Predicting binding within disordered protein regions to structurally characterised peptide-binding domains.

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Waqasuddin Khan

Full Text Available Disordered regions of proteins often bind to structured domains, mediating interactions within and between proteins. However, it is difficult to identify a priori the short disordered regions involved in binding. We set out to determine if docking such peptide regions to peptide binding domains would assist in these predictions.We assembled a redundancy reduced dataset of SLiM (Short Linear Motif containing proteins from the ELM database. We selected 84 sequences which had an associated PDB structures showing the SLiM bound to a protein receptor, where the SLiM was found within a 50 residue region of the protein sequence which was predicted to be disordered. First, we investigated the Vina docking scores of overlapping tripeptides from the 50 residue SLiM containing disordered regions of the protein sequence to the corresponding PDB domain. We found only weak discrimination of docking scores between peptides involved in binding and adjacent non-binding peptides in this context (AUC 0.58.Next, we trained a bidirectional recurrent neural network (BRNN using as input the protein sequence, predicted secondary structure, Vina docking score and predicted disorder score. The results were very promising (AUC 0.72 showing that multiple sources of information can be combined to produce results which are clearly superior to any single source.We conclude that the Vina docking score alone has only modest power to define the location of a peptide within a larger protein region known to contain it. However, combining this information with other knowledge (using machine learning methods clearly improves the identification of peptide binding regions within a protein sequence. This approach combining docking with machine learning is primarily a predictor of binding to peptide-binding sites, and is not intended as a predictor of specificity of binding to particular receptors.

Molecular dynamics of TBP and DBP studied by neutron transmission

International Nuclear Information System (INIS)

Salles Filho, J.B.V.; Refinetti, M.E.; Fulfaro, R.; Vinhas, L.A.

1984-04-01

Differences between the properties of TBP and DBP, concerning the uranium extraction processes, may be related to certain characteristics of the molecular dynamics of each compound. In order to investigate the dynamical behaviour of hydrogen in these molecules, neutron transmission of TBP and DBP has been measured as a function of neutron wavelenght in the range 4.0 - 6.0 A, at room temperature. Scattering cross sections per hydrogen atom have been obtained. From the comparison with results previously obtained for n-butanol, similar dynamical behaviour of butyl radicals in these compounds could be observed. This similarity indicates that the presence of two or three butyl radicals in butylphosphate molecules does not exert influence in the hydrogen motion of methyl and methylene groups. This suggests that the different chemical behaviour between TBP and DBP is related to the dynamics of the hydrogen directly bound to the DBP phosphate group.(Author) [pt

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

Science.gov (United States)

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.

Extracellular and intracellular steroid binding proteins

International Nuclear Information System (INIS)

Wagner, R.K.

1978-01-01

Steroid hormone binding proteins can be measured, after the removal of endogenous steroids, as specific complexes with radio-labelled hormones. In this study all the requirements for a quantitative determination of steroid hormone binding proteins are defined. For different methods, agargel electrophoresis, density gradient centrifugation, equilibrium dialysis and polyacrylamide electrophoresis have been evaluated. Agar electrophoresis at low temperature was found to be the simplest and most useful procedure. With this method the dissociation rates of high affinity complexes can be assessed and absolute binding protein concentrations can be determined. The dissociation rates of the oestradiol-oestrogen receptor complex and the R-5020-progestin receptor complex are low (1-2% per h run time.) In contrast, that of complexes between androgen receptor and dihydrotestosterone (17β-hydroxy-5α-androstan-3-one (DHT), progestin receptor and progesterone, corticosteroid binding globulin (CBG) and cortisol or progesterone and sex hormone binding globulin (SHBG) and DHT were hign (16-27% per h run time). Target tissue extracts (cytosols) contain, besides soluble tissue proteins, large amounts of plasma proteins. The extent of this plasma contamination can be determined by measuring the albumin concentration in cytosols by immunodiffusion. In cytosols of 4 different human target tissues the albumin content varied from 20-30% corresponding to an even higher whole plasma concentration. Steroid binding plasma proteins, such as CBG and SHBG are constituents of this containment. (author)

Study of the extraction mechanisms by TBP saturated by uranyl nitrate; Etude des mecanismes d'extraction du TBP sature par le nitrate d'uranyle

Energy Technology Data Exchange (ETDEWEB)

Meze, F

2004-02-15

This work deals with a particular phenomenon likely to occur in the nuclear waste reprocessing process PUREX. It was shown earlier by Russian works that the extractant molecule, tributyl phosphate (TBP), saturated by uranyl nitrate keeps its extraction capacities for nitric acid and tetravalent actinides. This study is composed of three parts. Firstly, some liquid-liquid extraction experiments were conducted to verify the ability of TBP saturated by uranyl nitrate to conserve its extraction capacities for nitric acid. Then, during these experiments, the UV and infrared spectra of both phases were recorded to obtain the organic phase speciation. At last, the informations gathered during the experimental part were used to build a general species distribution model of the H{sub 2}O/HNO{sub 3}/UO{sub 2}(NO{sub 3}){sub 2}/TBP system. (author)

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

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Tam Michael WC

2010-03-01

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

Determination of tributyl phosphate (TBP) by precision densimetry in the TBP-Varsol-HNO3 system

International Nuclear Information System (INIS)

Matsuda, H.T.; Araujo, B.F. de; Araujo, J.A. de.

1981-05-01

A direct TBP determination by digital density measurements is presented. The method is based on the variation of the natural frequency of a hollow oscillator when filled with a solvent. For this purpose a digital densimeter DMA 02C, from PAAR was used. There is a simple relationship between the density of the sample and the frequency of the filled oscillator. (I.C.R.) [pt

Cation-π interactions: computational analyses of the aromatic box motif and the fluorination strategy for experimental evaluation.

Science.gov (United States)

Davis, Matthew R; Dougherty, Dennis A

2015-11-21

Cation-π interactions are common in biological systems, and many structural studies have revealed the aromatic box as a common motif. With the aim of understanding the nature of the aromatic box, several computational methods were evaluated for their ability to reproduce experimental cation-π binding energies. We find the DFT method M06 with the 6-31G(d,p) basis set performs best of several methods tested. The binding of benzene to a number of different cations (sodium, potassium, ammonium, tetramethylammonium, and guanidinium) was studied. In addition, the binding of the organic cations NH4(+) and NMe4(+) to ab initio generated aromatic boxes as well as examples of aromatic boxes from protein crystal structures were investigated. These data, along with a study of the distance dependence of the cation-π interaction, indicate that multiple aromatic residues can meaningfully contribute to cation binding, even with displacements of more than an angstrom from the optimal cation-π interaction. Progressive fluorination of benzene and indole was studied as well, and binding energies obtained were used to reaffirm the validity of the "fluorination strategy" to study cation-π interactions in vivo.

Partial characterization of GTP-binding proteins in Neurospora

International Nuclear Information System (INIS)

Hasunuma, K.; Miyamoto-Shinohara, Y.; Furukawa, K.

1987-01-01

Six fractions of GTP-binding proteins separated by gel filtration of a mycelial extract containing membrane components of Neurospora crassa were partially characterized. [ 35 S]GTP gamma S bound to GTP-binding protein was assayed by repeated treatments with a Norit solution and centrifugation. The binding of [ 35 S]GTP gamma S to GTP-binding proteins was competitively prevented in the presence of 0.1 to 1 mM GTP but not in the presence of ATP. These GTP-binding proteins fractionated by the gel column had Km values of 20, 7, 4, 4, 80 and 2 nM. All six fractions of these GTP-binding proteins showed the capacity to be ADP-ribosylated by pertussis toxin

Guardian of Genetic Messenger-RNA-Binding Proteins

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Antje Anji

2016-01-01

Full Text Available RNA in cells is always associated with RNA-binding proteins that regulate all aspects of RNA metabolism including RNA splicing, export from the nucleus, RNA localization, mRNA turn-over as well as translation. Given their diverse functions, cells express a variety of RNA-binding proteins, which play important roles in the pathologies of a number of diseases. In this review we focus on the effect of alcohol on different RNA-binding proteins and their possible contribution to alcohol-related disorders, and discuss the role of these proteins in the development of neurological diseases and cancer. We further discuss the conventional methods and newer techniques that are employed to identify RNA-binding proteins.

Internal Associations of the Acidic Region of Upstream Binding Factor Control Its Nucleolar Localization.

Science.gov (United States)

Ueshima, Shuhei; Nagata, Kyosuke; Okuwaki, Mitsuru

2017-11-15

Upstream binding factor (UBF) is a member of the high-mobility group (HMG) box protein family, characterized by multiple HMG boxes and a C-terminal acidic region (AR). UBF is an essential transcription factor for rRNA genes and mediates the formation of transcriptionally active chromatin in the nucleolus. However, it remains unknown how UBF is specifically localized to the nucleolus. Here, we examined the molecular mechanisms that localize UBF to the nucleolus. We found that the first HMG box (HMG box 1), the linker region (LR), and the AR cooperatively regulate the nucleolar localization of UBF1. We demonstrated that the AR intramolecularly associates with and attenuates the DNA binding activity of HMG boxes and confers the structured DNA preference to HMG box 1. In contrast, the LR was found to serve as a nuclear localization signal and compete with HMG boxes to bind the AR, permitting nucleolar localization of UBF1. The LR sequence binds DNA and assists the stable chromatin binding of UBF. We also showed that the phosphorylation status of the AR does not clearly affect the localization of UBF1. Our results strongly suggest that associations of the AR with HMG boxes and the LR regulate UBF nucleolar localization. Copyright © 2017 American Society for Microbiology.

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

Science.gov (United States)

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.

Polymeric competitive protein binding adsorbents for radioassay

International Nuclear Information System (INIS)

Adams, R.J.

1976-01-01

Serum protein comprising specific binding proteins such as antibodies, B 12 intrinsic factor, thyroxin binding globulin and the like may be copolymerized with globulin constituents of serum by the action of ethylchloroformate to form readily packed insoluble precipitates which, following purification as by washing, are eminently suited for employment as competitive binding protein absorbents in radioassay procedures. 10 claims, no drawings

Predator-induced defences in Daphnia pulex: Selection and evaluation of internal reference genes for gene expression studies with real-time PCR

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Gilbert Don

2010-06-01

Full Text Available Abstract Background The planktonic microcrustacean Daphnia pulex is among the best-studied animals in ecological, toxicological and evolutionary research. One aspect that has sustained interest in the study system is the ability of D. pulex to develop inducible defence structures when exposed to predators, such as the phantom midge larvae Chaoborus. The available draft genome sequence for D. pulex is accelerating research to identify genes that confer plastic phenotypes that are regularly cued by environmental stimuli. Yet for quantifying gene expression levels, no experimentally validated set of internal control genes exists for the accurate normalization of qRT-PCR data. Results In this study, we tested six candidate reference genes for normalizing transcription levels of D. pulex genes; alpha tubulin (aTub, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, TATA box binding protein (Tbp syntaxin 16 (Stx16, X-box binding protein 1 (Xbp1 and CAPON, a protein associated with the neuronal nitric oxide synthase, were selected on the basis of an earlier study and from microarray studies. One additional gene, a matrix metalloproteinase (MMP, was tested to validate its transcriptional response to Chaoborus, which was earlier observed in a microarray study. The transcription profiles of these seven genes were assessed by qRT-PCR from RNA of juvenile D. pulex that showed induced defences in comparison to untreated control animals. We tested the individual suitability of genes for expression normalization using the programs geNorm, NormFinder and BestKeeper. Intriguingly, Xbp1, Tbp, CAPON and Stx16 were selected as ideal reference genes. Analyses on the relative expression level using the software REST showed that both classical housekeeping candidate genes (aTub and GAPDH were significantly downregulated, whereas the MMP gene was shown to be significantly upregulated, as predicted. aTub is a particularly ill suited reference gene because five copies are

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

Science.gov (United States)

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.

CaMELS: In silico prediction of calmodulin binding proteins and their binding sites.

Science.gov (United States)

Abbasi, Wajid Arshad; Asif, Amina; Andleeb, Saiqa; Minhas, Fayyaz Ul Amir Afsar

2017-09-01

Due to Ca 2+ -dependent binding and the sequence diversity of Calmodulin (CaM) binding proteins, identifying CaM interactions and binding sites in the wet-lab is tedious and costly. Therefore, computational methods for this purpose are crucial to the design of such wet-lab experiments. We present an algorithm suite called CaMELS (CalModulin intEraction Learning System) for predicting proteins that interact with CaM as well as their binding sites using sequence information alone. CaMELS offers state of the art accuracy for both CaM interaction and binding site prediction and can aid biologists in studying CaM binding proteins. For CaM interaction prediction, CaMELS uses protein sequence features coupled with a large-margin classifier. CaMELS models the binding site prediction problem using multiple instance machine learning with a custom optimization algorithm which allows more effective learning over imprecisely annotated CaM-binding sites during training. CaMELS has been extensively benchmarked using a variety of data sets, mutagenic studies, proteome-wide Gene Ontology enrichment analyses and protein structures. Our experiments indicate that CaMELS outperforms simple motif-based search and other existing methods for interaction and binding site prediction. We have also found that the whole sequence of a protein, rather than just its binding site, is important for predicting its interaction with CaM. Using the machine learning model in CaMELS, we have identified important features of protein sequences for CaM interaction prediction as well as characteristic amino acid sub-sequences and their relative position for identifying CaM binding sites. Python code for training and evaluating CaMELS together with a webserver implementation is available at the URL: http://faculty.pieas.edu.pk/fayyaz/software.html#camels. © 2017 Wiley Periodicals, Inc.

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

Science.gov (United States)

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.

X-box binding protein 1 is essential for the anti-oxidant defense and cell survival in the retinal pigment epithelium.

Directory of Open Access Journals (Sweden)

Yimin Zhong

Full Text Available Damage to the retinal pigment epithelium (RPE is an early event in the pathogenesis of age-related macular degeneration (AMD. X-box binding protein 1 (XBP1 is a key transcription factor that regulates endoplasmic reticulum (ER homeostasis and cell survival. This study aimed to delineate the role of endogenous XBP1 in the RPE. Our results show that in a rat model of light-induced retinal degeneration, XBP1 activation was suppressed in the RPE/choroid complex, accompanied by decreased anti-oxidant genes and increased oxidative stress. Knockdown of XBP1 by siRNA resulted in reduced expression of SOD1, SOD2, catalase, and glutathione synthase and sensitized RPE cells to oxidative damage. Using Cre/LoxP system, we generated a mouse line that lacks XBP1 only in RPE cells. Compared to wildtype littermates, RPE-XBP1 KO mice expressed less SOD1, SOD2, and catalase in the RPE, and had increased oxidative stress. At age 3 months and older, these mice exhibited apoptosis of RPE cells, decreased number of cone photoreceptors, shortened photoreceptor outer segment, reduced ONL thickness, and deficit in retinal function. Electron microscopy showed abnormal ultrastructure, Bruch's membrane thickening, and disrupted basal membrane infolding in XBP1-deficient RPE. These results indicate that XBP1 is an important gene involved in regulation of the anti-oxidant defense in the RPE, and that impaired activation of XBP1 may contribute to RPE dysfunction and cell death during retinal degeneration and AMD.

Factor VII and protein C are phosphatidic acid-binding proteins.

Science.gov (United States)

Tavoosi, Narjes; Smith, Stephanie A; Davis-Harrison, Rebecca L; Morrissey, James H

2013-08-20

Seven proteins in the human blood clotting cascade bind, via their GLA (γ-carboxyglutamate-rich) domains, to membranes containing exposed phosphatidylserine (PS), although with membrane binding affinities that vary by 3 orders of magnitude. Here we employed nanodiscs of defined phospholipid composition to quantify the phospholipid binding specificities of these seven clotting proteins. All bound preferentially to nanobilayers in which PS headgroups contained l-serine versus d-serine. Surprisingly, however, nanobilayers containing phosphatidic acid (PA) bound substantially more of two of these proteins, factor VIIa and activated protein C, than did equivalent bilayers containing PS. Consistent with this finding, liposomes containing PA supported higher proteolytic activity by factor VIIa and activated protein C toward their natural substrates (factors X and Va, respectively) than did PS-containing liposomes. Moreover, treating activated human platelets with phospholipase D enhanced the rates of factor X activation by factor VIIa in the presence of soluble tissue factor. We hypothesize that factor VII and protein C bind preferentially to the monoester phosphate of PA because of its accessibility and higher negative charge compared with the diester phosphates of most other phospholipids. We further found that phosphatidylinositol 4-phosphate, which contains a monoester phosphate attached to its myo-inositol headgroup, also supported enhanced enzymatic activity of factor VIIa and activated protein C. We conclude that factor VII and protein C bind preferentially to monoester phosphates, which may have implications for the function of these proteases in vivo.

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

Directory of Open Access Journals (Sweden)

Chao-Hsun Yang

2013-01-01

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

TBP determination in nitric acid solutions from solvent extraction process

International Nuclear Information System (INIS)

Kuada, T.A.; Carvalho, E.I. de; Araujo, I. da C.; Cohen, V.H.

1988-07-01

Heavy organic phases have been observed on some occasions during TBP extraction process. These products, described as red oils, were considered as the main cause for process failures, specially in evaporators and concentrators. In view of safety aspects it is necessary to control organic concentration in product and waste solutions. The proposed method involves the organic removal by chloroform as a first step, followed by purification onto a silica gel column. The results are given from analysing TBP and its degradation product (DBP) by gas chromatography. (author) [pt

GTP-binding proteins in rat liver nuclear envelopes

International Nuclear Information System (INIS)

Rubins, J.B.; Benditt, J.O.; Dickey, B.F.; Riedel, N.

1990-01-01

Nuclear transport as well as reassembly of the nuclear envelope (NE) after completion of mitosis are processes that have been shown to require GTP and ATP. To study the presence and localization of GTP-binding proteins in the NE, we have combined complementary techniques of [alpha-32P]GTP binding to Western-blotted proteins and UV crosslinking of [alpha-32P]GTP with well-established procedures for NE subfractionation. GTP binding to blotted NE proteins revealed five low molecular mass GTP-binding proteins of 26, 25, 24.5, 24, and 23 kDa, and [alpha-32P]GTP photoaffinity labeling revealed major proteins with apparent molecular masses of 140, 53, 47, 33, and 31 kDa. All GTP-binding proteins appear to localize preferentially to the inner nuclear membrane, possibly to the interface between inner nuclear membrane and lamina. Despite the evolutionary conservation between the NE and the rough endoplasmic reticulum, the GTP-binding proteins identified differed between these two compartments. Most notably, the 68- and 30-kDa GTP-binding subunits of the signal recognition particle receptor, which photolabeled with [alpha-32P]GTP in the rough endoplasmic reticulum fraction, were totally excluded from the NE fraction. Conversely, a major 53-kDa photolabeled protein in the NE was absent from rough endoplasmic reticulum. Whereas Western-blotted NE proteins bound GTP specifically, all [alpha-32P]GTP photolabeled proteins could be blocked by competition with ATP, although with a competition profile that differed from that obtained with GTP. In comparative crosslinking studies with [alpha-32P]ATP, we have identified three specific ATP-binding proteins with molecular masses of 160, 78, and 74 kDa. The localization of GTP- and ATP-binding proteins within the NE appears appropriate for their involvement in nuclear transport and in the GTP-dependent fusion of nuclear membranes

Cobalamin and its binding protein in rat milk

DEFF Research Database (Denmark)

Raaberg, Lasse; Nexø, Ebba; Poulsen, Steen Seier

1989-01-01

Cobalamin and its binding protein, haptocorrin, are present in rat milk throughout the lactation period. The concentration of cobalamin is approximately 0.3-times the concentration of the unsaturated binding protein. The concentration of the unsaturated cobalamin-binding protein varies between 18...

Gonadal cell surface receptor for plasma retinol-binding protein

International Nuclear Information System (INIS)

Krishna Bhat, M.; Cama, H.R.

1979-01-01

A specific membrane receptor for plasma retinol-binding protein has been demonstrated in testicular cells. Prealbumin-2 did not show any specific binding to the membrane. The affinity of retinol-binding protein for receptor drastically decreases upon delivery of retinol and the retinol-binding protein does not enter the cell. The mechanism of delivery of retinol to the target cell by plasma retinol-binding protein has been investigated. The process involves two steps; direct binding of retinol-binding protein to the receptor and uptake of retinol by the target cell with a concomitant drastic reduction in the affinity of the retinol-binding protein to the receptor. Probably the second step of the process needs a cytosolic factor, possibly the cellular retinol-binding protein or an enzyme. The binding of retinol-binding protein to the receptor is saturable and reversible. The interaction shows a Ksub(d) value of 2.1x10 -10 . The specific binding of a retinol-binding protein with great affinity has been employed in the development of a method for radioassay of the receptor. The receptor level of the gonadal cell has been found to vary with the stage of differentiation. The receptor concentrations in 11-week-old birds and adult birds are comparable. Testosterone treatment of 11-week-old birds produced a substantial increase in the receptor concentration over control, while the protein content increased marginally, indicating that, probably, synthesis of the receptor is specifcally induced by testosterone during spermatogenesis, and the concentration of receptor is relatively higher before the formation of the acrosome. (Auth.)

Solvent extraction of thorium from nitrate medium by TBP, Cyanex272 and their mixture

International Nuclear Information System (INIS)

Mostaan Shaeri; Ahmad Rahbar Kelishami; Meisam Torab-Mostaedi

2015-01-01

The extraction behavior of thorium(IV) has been investigated with tri-butyl phosphate (TBP) and bis(2,4,4-trimethylpentyl) phosphinic acid (Cyanex272) in kerosene from nitrate medium. The effect of operating variables including time, aqueous phase acidity (pH), extractant concentration and temperature were investigated. This study also examined the synergistic enhancement of the extraction of thorium(IV) from nitrate medium by mixtures of TBP and Cyanex272 for the first time. The optimum synergistic enhancement factor of 3.86 was obtained at a Cyanex272/TBP molar ratio of 1:4. (author)

Acyl-CoA-binding protein/diazepam-binding inhibitor gene and pseudogenes

DEFF Research Database (Denmark)

Mandrup, S; Hummel, R; Ravn, S

1992-01-01

Acyl-CoA-binding protein (ACBP) is a 10 kDa protein isolated from bovine liver by virtue of its ability to bind and induce the synthesis of medium-chain acyl-CoA esters. Surprisingly, it turned out to be identical to a protein named diazepam-binding Inhibitor (DBI) claimed to be an endogenous mod...... have molecularly cloned and characterized the ACBP/DBI gene family in rat. The rat ACBP/DBI gene family comprises one expressed gene and four processed pseudogenes of which one was shown to exist in two allelic forms. The expressed gene is organized into four exons and three introns...

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

International Nuclear Information System (INIS)

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

2009-01-01

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

Global MYCN transcription factor binding analysis in neuroblastoma reveals association with distinct E-box motifs and regions of DNA hypermethylation.

LENUS (Irish Health Repository)

Murphy, Derek M

2009-01-01

BACKGROUND: Neuroblastoma, a cancer derived from precursor cells of the sympathetic nervous system, is a major cause of childhood cancer related deaths. The single most important prognostic indicator of poor clinical outcome in this disease is genomic amplification of MYCN, a member of a family of oncogenic transcription factors. METHODOLOGY: We applied MYCN chromatin immunoprecipitation to microarrays (ChIP-chip) using MYCN amplified\\/non-amplified cell lines as well as a conditional knockdown cell line to determine the distribution of MYCN binding sites within all annotated promoter regions. CONCLUSION: Assessment of E-box usage within consistently positive MYCN binding sites revealed a predominance for the CATGTG motif (p<0.0016), with significant enrichment of additional motifs CATTTG, CATCTG, CAACTG in the MYCN amplified state. For cell lines over-expressing MYCN, gene ontology analysis revealed enrichment for the binding of MYCN at promoter regions of numerous molecular functional groups including DNA helicases and mRNA transcriptional regulation. In order to evaluate MYCN binding with respect to other genomic features, we determined the methylation status of all annotated CpG islands and promoter sequences using methylated DNA immunoprecipitation (MeDIP). The integration of MYCN ChIP-chip and MeDIP data revealed a highly significant positive correlation between MYCN binding and DNA hypermethylation. This association was also detected in regions of hemizygous loss, indicating that the observed association occurs on the same homologue. In summary, these findings suggest that MYCN binding occurs more commonly at CATGTG as opposed to the classic CACGTG E-box motif, and that disease associated over expression of MYCN leads to aberrant binding to additional weaker affinity E-box motifs in neuroblastoma. The co-localization of MYCN binding and DNA hypermethylation further supports the dual role of MYCN, namely that of a classical transcription factor affecting the

Salt modulates the stability and lipid binding affinity of the adipocyte lipid-binding proteins

Science.gov (United States)

Schoeffler, Allyn J.; Ruiz, Carmen R.; Joubert, Allison M.; Yang, Xuemei; LiCata, Vince J.

2003-01-01

Adipocyte lipid-binding protein (ALBP or aP2) is an intracellular fatty acid-binding protein that is found in adipocytes and macrophages and binds a large variety of intracellular lipids with high affinity. Although intracellular lipids are frequently charged, biochemical studies of lipid-binding proteins and their interactions often focus most heavily on the hydrophobic aspects of these proteins and their interactions. In this study, we have characterized the effects of KCl on the stability and lipid binding properties of ALBP. We find that added salt dramatically stabilizes ALBP, increasing its Delta G of unfolding by 3-5 kcal/mol. At 37 degrees C salt can more than double the stability of the protein. At the same time, salt inhibits the binding of the fluorescent lipid 1-anilinonaphthalene-8-sulfonate (ANS) to the protein and induces direct displacement of the lipid from the protein. Thermodynamic linkage analysis of the salt inhibition of ANS binding shows a nearly 1:1 reciprocal linkage: i.e. one ion is released from ALBP when ANS binds, and vice versa. Kinetic experiments show that salt reduces the rate of association between ANS and ALBP while simultaneously increasing the dissociation rate of ANS from the protein. We depict and discuss the thermodynamic linkages among stability, lipid binding, and salt effects for ALBP, including the use of these linkages to calculate the affinity of ANS for the denatured state of ALBP and its dependence on salt concentration. We also discuss the potential molecular origins and potential intracellular consequences of the demonstrated salt linkages to stability and lipid binding in ALBP.

Bibliographic report on the synthesis, the chemical and physical properties and the applications of the TBP phosphate

International Nuclear Information System (INIS)

Azzouz, A.; Attou, M.

1985-02-01

This work consists of a bibliographic synthesis concerning the tri-n-butylphosphate (TBP) technology. It gathers briefly in 56 references on a table, the multiple applications of TBP such as extraction, spectroscopy and various other uses. Then, it deals with chemical instability different causes of this substance as well as its physical and chemical properties. In this way, for instance, the thermal degradation occurs well before the TBP boiling point. It comes out of this, several decomposition products such as the mono-n-butyl-phosphate (MBP), the di-n-butylphosphate (DBP) and some olefins. The acids and some impurities are known to have effects on TBP degradation. Later on, some processes of the TBP synthesis and their principles are stated. In the major cases, the POCl 3 process seems to be the best way, probably because of convenient efficiencies and moderate conditions of its making. Some purification processes according to the TBP use are also invoqued. In this case as well, Alcock et al. method is often stated in the literature. Moreover, five analysis methods corresponding to the most common situations are described

Singlet Ground State Magnetism: III Magnetic Excitons in Antiferromagnetic TbP

DEFF Research Database (Denmark)

Knorr, K.; Loidl, A.; Kjems, Jørgen

1981-01-01

The dispersion of the lowest magnetic excitations of the singlet ground state system TbP has been studied in the antiferromagnetic phase by inelastic neutron scattering. The magnetic exchange interaction and the magnetic and the rhombohedral molecular fields have been determined.......The dispersion of the lowest magnetic excitations of the singlet ground state system TbP has been studied in the antiferromagnetic phase by inelastic neutron scattering. The magnetic exchange interaction and the magnetic and the rhombohedral molecular fields have been determined....

Tritium NMR spectroscopy of ligand binding to maltose-binding protein

International Nuclear Information System (INIS)

Gehring, K.; Williams, P.G.; Pelton, J.G.; Morimoto, H.; Wemmer, D.E.

1991-01-01

Tritium-labeled α- and β-maltodextrins have been used to study their complexes with maltose-binding protein (MBP), a 40-kDa bacterial protein. Five substrates, from maltose to maltohexaose, were labeled at their reducing ends and their binding studied. Tritium NMR specctroscopy of the labeled sugars showed large upfield chamical shift changes upon binding and strong anomeric specficity. At 10 degrees C, MBP bound α-maltose with 2.7 ± 0.5-fold higher affinity than β-maltose, and, for longer maltodextrins, the ratio of affinities was even larger. The maximum chemical shift change was 2.2 ppm, suggesting that the reducing end of bound α-maltodextrin makes close contact with an aromatic residue in the MBP-binding site. Experiments with maltotriose (and longer maltodextrins) also revealed the presence of two bound β-maltotriose resonances in rapid exchange. The authors interpret these two resonances as arising from two distinct sugar-protein complexes. In one complex, the β-maltodextrin is bound by its reducing end, and, in the other complex, the β-maltodextrin is bound by the middle glucose residue(s). This interpretation also suggests how MBP is able to bind both linear and circular maltodextrins

Dysregulation of gene expression in the striatum of BACHD rats expressing full-length mutant huntingtin and associated abnormalities on molecular and protein levels.

Science.gov (United States)

Yu-Taeger, Libo; Bonin, Michael; Stricker-Shaver, Janice; Riess, Olaf; Nguyen, Hoa Huu Phuc

2017-05-01

Huntington disease (HD) is an autosomal dominantly inherited neurodegenerative disorder caused by a CAG repeat expansion in the gene coding for the huntingtin protein (HTT). Mutant HTT (mHTT) has been proposed to cause neuronal dysfunction and neuronal loss through multiple mechanisms. Transcriptional changes may be a core pathogenic feature of HD. Utilizing the Affymetrix platform we performed a genome-wide RNA expression analysis in two BACHD transgenic rat lines (TG5 and TG9) at 12 months of age, both of which carry full-length human mHTT but with different expression levels. By defining the threshold of significance at p < 0.01, we found 1608 genes and 871 genes differentially expressed in both TG5 and TG9 rats when compared to the wild type littermates, respectively. We only chose the highly up-/down-regulated genes for further analysis by setting an additional threshold of 1.5 fold change. Comparing gene expression profiles of human HD brains and BACHD rats revealed a high concordance in both functional and IPA (Ingenuity Pathway Analysis) canonical pathways relevant to HD. In addition, we investigated the causes leading to gene expression changes at molecular and protein levels in BACHD rats including the involvement of polyQ-containing transcription factors TATA box-binding protein (TBP), Sp1 and CBP as well as the chromatin structure. We demonstrate that the BACHD rat model recapitulates the gene expression changes of the human disease supporting its role as a preclinical research animal model. We also show for the first time that TFIID complex formation is reduced, while soluble TBP is increased in an HD model. This finding suggests that mHTT is a competitor instead of a recruiter of polyQ-containing transcription factors in the transcription process in HD. Copyright © 2017 Elsevier Ltd. All rights reserved.

Localization of cellular retinol-binding protein and retinol-binding protein in cells comprising the blood-brain barrier of rat and human

International Nuclear Information System (INIS)

MacDonald, P.N.; Ong, D.E.; Bok, D.

1990-01-01

Brain is not generally recognized as an organ that requires vitamin A, perhaps because no obvious histologic lesions have been observed in severely vitamin A-deficient animals. However, brain tissue does contain cellular vitamin A-binding proteins and a nuclear receptor protein for retinoic acid. In the present study, immunohistochemical techniques were used to determine the cell-specific location of cellular retinol-binding protein in human and rat brain tissue. Cellular retinol-binding protein was localized specifically within the cuboidal epithelial cells of the choroid plexus, two primary sites of the mammalian blood-brain barrier. In addition, autoradiographic procedures demonstrated binding sites for serum retinol-binding protein in the choroidal epithelium. These observations suggest that a significant movement of retinol across the blood-brain barrier may occur

Elution of Uranium and Calculation of Plate Number on the Column of Silica-TBP

International Nuclear Information System (INIS)

Endang Susiantini; Indra Suryawan

2007-01-01

Separation process of 99 Mo resulted of irradiated uranyl nitrate with an accelerator as the neutron source by the chromatographic extraction using column containing kiesel gel-TBP will be developed. Kiesel gel (silica) was used as an inert subpart, TBP as a phase stationary and simulated used natural uranyl nitrate of 200-300 g/l with the acidity of 2 N as the mobile phase. The inert support was made by means of kiesel-gel (silica) hydrophobization to change hydrophilic silica to hydrophobic silica, so that it could be impregnated by TBP. Uranium which has been attached to TBP would be eluted by dilute acid at acidity of 0.05; 0.1; 0.2 N HNO 3 ; Warm DW and cool DW. By using 0.1 N HNO 3 eluent and warm DW the uranium attached to silica-TBP could be eluted perfectly and more quickly than the three others eluent. Uranium concentration which were absorbed and eluted were analysed titrimetrically by using titan method and it was used to calculate Plate Number (N). The value of N obtained for the column which the inert support of 8 cm in height, 1 cm in diameter, 10 drops per minute by using of 0.1 N HNO 3 eluent was 300.6. (author)

CC1, a novel crenarchaeal DNA binding protein.

Science.gov (United States)

Luo, Xiao; Schwarz-Linek, Uli; Botting, Catherine H; Hensel, Reinhard; Siebers, Bettina; White, Malcolm F

2007-01-01

The genomes of the related crenarchaea Pyrobaculum aerophilum and Thermoproteus tenax lack any obvious gene encoding a single-stranded DNA binding protein (SSB). SSBs are essential for DNA replication, recombination, and repair and are found in all other genomes across the three domains of life. These two archaeal genomes also have only one identifiable gene encoding a chromatin protein (the Alba protein), while most other archaea have at least two different abundant chromatin proteins. We performed a biochemical screen for novel nucleic acid binding proteins present in cell extracts of T. tenax. An assay for proteins capable of binding to a single-stranded DNA oligonucleotide resulted in identification of three proteins. The first protein, Alba, has been shown previously to bind single-stranded DNA as well as duplex DNA. The two other proteins, which we designated CC1 (for crenarchaeal chromatin protein 1), are very closely related to one another, and homologs are restricted to the P. aerophilum and Aeropyrum pernix genomes. CC1 is a 6-kDa, monomeric, basic protein that is expressed at a high level in T. tenax. This protein binds single- and double-stranded DNAs with similar affinities. These properties are consistent with a role for CC1 as a crenarchaeal chromatin protein.

MOCVD growth of InP-related materials using TBA and TBP

International Nuclear Information System (INIS)

Czub, M.; Strupinski, W.

1995-01-01

High quality epitaxial layers of GaAs, InP, AlAs, InGaAs, InGaP, InGaAlP have been grown by low-pressure metalorganic chemical vapor deposition using TMIn, TMGa, TMAl and the less hazardous group V precursors, temperature ranges of 570-650 C and 520-650 C, respectively. The V/III ratio as low as 1.5 was used to grow epilayers of InP. The 77 K mobility of InGaAs lattice matched to InP (grown with TBA) was 72360 cm 2 /(Vs) for n = 1.5 x 10 1 5/cm 3 and the thickness of 2 μm. Comparable photoluminescence parameters of InGaAlP between layers grown with TBP and PH 3 were achieved, but for InGaAlP (TBP) photoluminescence intensity was significantly lower than for InGaAlP (PH 3 ). The promising results allow one to apply of TBA and TBP for developing of device structures. (author)

Quantitative analysis of EGR proteins binding to DNA: assessing additivity in both the binding site and the protein

Directory of Open Access Journals (Sweden)

Stormo Gary D

2005-07-01

Full Text Available Abstract Background Recognition codes for protein-DNA interactions typically assume that the interacting positions contribute additively to the binding energy. While this is known to not be precisely true, an additive model over the DNA positions can be a good approximation, at least for some proteins. Much less information is available about whether the protein positions contribute additively to the interaction. Results Using EGR zinc finger proteins, we measure the binding affinity of six different variants of the protein to each of six different variants of the consensus binding site. Both the protein and binding site variants include single and double mutations that allow us to assess how well additive models can account for the data. For each protein and DNA alone we find that additive models are good approximations, but over the combined set of data there are context effects that limit their accuracy. However, a small modification to the purely additive model, with only three additional parameters, improves the fit significantly. Conclusion The additive model holds very well for every DNA site and every protein included in this study, but clear context dependence in the interactions was detected. A simple modification to the independent model provides a better fit to the complete data.

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

Science.gov (United States)

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

Efficient identification of phosphatidylserine-binding proteins by ORF phage display

International Nuclear Information System (INIS)

Caberoy, Nora B.; Zhou, Yixiong; Alvarado, Gabriela; Fan, Xianqun; Li, Wei

2009-01-01

To efficiently elucidate the biological roles of phosphatidylserine (PS), we developed open-reading-frame (ORF) phage display to identify PS-binding proteins. The procedure of phage panning was optimized with a phage clone expressing MFG-E8, a well-known PS-binding protein. Three rounds of phage panning with ORF phage display cDNA library resulted in ∼300-fold enrichment in PS-binding activity. A total of 17 PS-binding phage clones were identified. Unlike phage display with conventional cDNA libraries, all 17 PS-binding clones were ORFs encoding 13 real proteins. Sequence analysis revealed that all identified PS-specific phage clones had dimeric basic amino acid residues. GST fusion proteins were expressed for 3 PS-binding proteins and verified for their binding activity to PS liposomes, but not phosphatidylcholine liposomes. These results elucidated previously unknown PS-binding proteins and demonstrated that ORF phage display is a versatile technology capable of efficiently identifying binding proteins for non-protein molecules like PS.

Characterization of a cocaine binding protein in human placenta

International Nuclear Information System (INIS)

Ahmed, M.S.; Zhou, D.H.; Maulik, D.; Eldefrawi, M.E.

1990-01-01

[ 3 H]-Cocaine binding sites are identified in human placental villus tissue plasma membranes. These binding sites are associated with a protein and show saturable and specific binding of [ 3 H]-cocaine with a high affinity site of 170 fmole/mg protein. The binding is lost with pretreatment with trypsin or heat. The membrane bound protein is solubilized with the detergent 3-(3-cholamidopropyl)dimethyl-ammonio-1-propane sulphonate (CHAPS) with retention of its saturable and specific binding of [ 3 H]-cocaine. The detergent-protein complex migrates on a sepharose CL-6B gel chromatography column as a protein with an apparent molecular weight of 75,900. The protein has an S 20,w value of 5.1. The binding of this protein to norcocaine, pseudococaine, nomifensine, imipramine, desipramine, amphetamine and dopamine indicates that it shares some, but not all, the properties of the brain cocaine receptor. The physiologic significance of this protein in human placenta is currently unclear

Bioactive L acidissima protein hydrolysates using Box-Behnken design.

Science.gov (United States)

Sonawane, Sachin K; Arya, Shalini S

2017-07-01

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

Mechanism of the G-protein mimetic nanobody binding to a muscarinic G-protein-coupled receptor.

Science.gov (United States)

Miao, Yinglong; McCammon, J Andrew

2018-03-20

Protein-protein binding is key in cellular signaling processes. Molecular dynamics (MD) simulations of protein-protein binding, however, are challenging due to limited timescales. In particular, binding of the medically important G-protein-coupled receptors (GPCRs) with intracellular signaling proteins has not been simulated with MD to date. Here, we report a successful simulation of the binding of a G-protein mimetic nanobody to the M 2 muscarinic GPCR using the robust Gaussian accelerated MD (GaMD) method. Through long-timescale GaMD simulations over 4,500 ns, the nanobody was observed to bind the receptor intracellular G-protein-coupling site, with a minimum rmsd of 2.48 Å in the nanobody core domain compared with the X-ray structure. Binding of the nanobody allosterically closed the orthosteric ligand-binding pocket, being consistent with the recent experimental finding. In the absence of nanobody binding, the receptor orthosteric pocket sampled open and fully open conformations. The GaMD simulations revealed two low-energy intermediate states during nanobody binding to the M 2 receptor. The flexible receptor intracellular loops contribute remarkable electrostatic, polar, and hydrophobic residue interactions in recognition and binding of the nanobody. These simulations provided important insights into the mechanism of GPCR-nanobody binding and demonstrated the applicability of GaMD in modeling dynamic protein-protein interactions.

The F-box Protein FBXO44 Mediates BRCA1 Ubiquitination and Degradation*

Science.gov (United States)

Lu, Yunzhe; Li, Jiezhi; Cheng, Dongmei; Parameswaran, Balaji; Zhang, Shaohua; Jiang, Zefei; Yew, P. Renee; Peng, Junmin; Ye, Qinong; Hu, Yanfen

2012-01-01

BRCA1 mutations account for a significant proportion of familial breast and ovarian cancers. In addition, reduced BRCA1 protein is associated with sporadic cancer cases in these tissues. At the cellular level, BRCA1 plays a critical role in multiple cellular functions such as DNA repair and cell cycle checkpoint control. Its protein level is regulated in a cell cycle-dependent manner. However, regulation of BRCA1 protein stability is not fully understood. Our earlier study showed that the amino terminus of BRCA1 harbors a degron sequence that is sufficient and necessary for conferring BRCA1 degradation. In the current study, we used mass spectrometry to identify Skp1 that regulates BRCA1 protein stability. Small interfering RNA screening that targets all human F-box proteins uncovered FBXO44 as an important protein that influences BRCA1 protein level. The Skp1-Cul1-F-box-protein44 (SCFFBXO44) complex ubiquitinates full-length BRCA1 in vitro. Furthermore, the N terminus of BRCA1 mediates the interaction between BRCA1 and FBXO44. Overexpression of SCFFBXO44 reduces BRCA1 protein level. Taken together, our work strongly suggests that SCFFBXO44 is an E3 ubiquitin ligase responsible for BRCA1 degradation. In addition, FBXO44 expression pattern in breast carcinomas suggests that SCFFBXO44-mediated BRCA1 degradation might contribute to sporadic breast tumor development. PMID:23086937

Measuring Binding Affinity of Protein-Ligand Interaction Using Spectrophotometry: Binding of Neutral Red to Riboflavin-Binding Protein

Science.gov (United States)

Chenprakhon, Pirom; Sucharitakul, Jeerus; Panijpan, Bhinyo; Chaiyen, Pimchai

2010-01-01

The dissociation constant, K[subscript d], of the binding of riboflavin-binding protein (RP) with neutral red (NR) can be determined by titrating RP to a fixed concentration of NR. Upon adding RP to the NR solution, the maximum absorption peak of NR shifts to 545 nm from 450 nm for the free NR. The change of the absorption can be used to determine…

Improved detection of calcium-binding proteins in polyacrylamide gels

International Nuclear Information System (INIS)

Anthony, F.A.; Babitch, J.A.

1984-01-01

The authors refined the method of Schibeci and Martonosi (1980) to enhance detection of calcium-binding proteins in polyacrylamide gels using 45 Ca 2+ . Their efforts have produced a method which is shorter, has 40-fold greater sensitivity over the previous method, and will detect 'EF hand'-containing calcium-binding proteins in polyacrylamide gels below the 0.5 μg level. In addition this method will detect at least one example from every described class of calcium-binding protein, including lectins and γ-carboxyglutamic acid containing calcium-binding proteins. The method should be useful for detecting calcium-binding proteins which may trigger neurotransmitter release. (Auth.)

Treatment of low level radioactive liquid waste containing appreciable concentration of TBP degraded products.

Science.gov (United States)

Valsala, T P; Sonavane, M S; Kore, S G; Sonar, N L; De, Vaishali; Raghavendra, Y; Chattopadyaya, S; Dani, U; Kulkarni, Y; Changrani, R D

2011-11-30

The acidic and alkaline low level radioactive liquid waste (LLW) generated during the concentration of high level radioactive liquid waste (HLW) prior to vitrification and ion exchange treatment of intermediate level radioactive liquid waste (ILW), respectively are decontaminated by chemical co-precipitation before discharge to the environment. LLW stream generated from the ion exchange treatment of ILW contained high concentrations of carbonates, tributyl phosphate (TBP) degraded products and problematic radio nuclides like (106)Ru and (99)Tc. Presence of TBP degraded products was interfering with the co-precipitation process. In view of this a modified chemical treatment scheme was formulated for the treatment of this waste stream. By mixing the acidic LLW and alkaline LLW, the carbonates in the alkaline LLW were destroyed and the TBP degraded products got separated as a layer at the top of the vessel. By making use of the modified co-precipitation process the effluent stream (1-2 μCi/L) became dischargeable to the environment after appropriate dilution. Based on the lab scale studies about 250 m(3) of LLW was treated in the plant. The higher activity of the TBP degraded products separated was due to short lived (90)Y isotope. The cement waste product prepared using the TBP degraded product was having good chemical durability and compressive strength. Copyright © 2011 Elsevier B.V. All rights reserved.

The optimisation study of tbp synthesis process by phosphoric acid

International Nuclear Information System (INIS)

Amedjkouh, A.; Attou, M.; Azzouz, A.; Zaoui, B.

1995-07-01

The present work deals with the optimisation study of TBP synthesis process by phosphoric acid. This way of synthesis is more advantageous than POCL3 or P2O5 as phosphatant agents. these latters are toxic and dangerous for the environnement. The optimisation study is based on a series of 16 experiences taking into account the range of variation of the following parameters : temperature, pressure, reagents mole ratio, promoter content. the yield calculation is based on the randomisation of an equation including all parameters. the resolution of this equation gave a 30% TBP molar ratio. this value is in agreement with that of experimental data

Predicting DNA-binding proteins and binding residues by complex structure prediction and application to human proteome.

Directory of Open Access Journals (Sweden)

Huiying Zhao

Full Text Available As more and more protein sequences are uncovered from increasingly inexpensive sequencing techniques, an urgent task is to find their functions. This work presents a highly reliable computational technique for predicting DNA-binding function at the level of protein-DNA complex structures, rather than low-resolution two-state prediction of DNA-binding as most existing techniques do. The method first predicts protein-DNA complex structure by utilizing the template-based structure prediction technique HHblits, followed by binding affinity prediction based on a knowledge-based energy function (Distance-scaled finite ideal-gas reference state for protein-DNA interactions. A leave-one-out cross validation of the method based on 179 DNA-binding and 3797 non-binding protein domains achieves a Matthews correlation coefficient (MCC of 0.77 with high precision (94% and high sensitivity (65%. We further found 51% sensitivity for 82 newly determined structures of DNA-binding proteins and 56% sensitivity for the human proteome. In addition, the method provides a reasonably accurate prediction of DNA-binding residues in proteins based on predicted DNA-binding complex structures. Its application to human proteome leads to more than 300 novel DNA-binding proteins; some of these predicted structures were validated by known structures of homologous proteins in APO forms. The method [SPOT-Seq (DNA] is available as an on-line server at http://sparks-lab.org.

Clinical relevance of drug binding to plasma proteins

Science.gov (United States)

Ascenzi, Paolo; Fanali, Gabriella; Fasano, Mauro; Pallottini, Valentina; Trezza, Viviana

2014-12-01

Binding to plasma proteins highly influences drug efficacy, distribution, and disposition. Serum albumin, the most abundant protein in plasma, is a monomeric multi-domain macromolecule that displays an extraordinary ligand binding capacity, providing a depot and carrier for many endogenous and exogenous compounds, such as fatty acids and most acidic drugs. α-1-Acid glycoprotein, the second main plasma protein, is a glycoprotein physiologically involved in the acute phase reaction and is the main carrier for basic and neutral drugs. High- and low-density lipoproteins play a limited role in drug binding and are natural drug delivery system only for few lipophilic drugs or lipid-based formulations. Several factors influence drug binding to plasma proteins, such as pathological conditions, concurrent administration of drugs, sex, and age. Any of these factors, in turn, influences drug efficacy and toxicity. Here, biochemical, biomedical, and biotechnological aspects of drug binding to plasma proteins are reviewed.

Loss of Hda activity stimulates replication initiation from I-box, but not R4 mutant origins in Escherichia coli.

Science.gov (United States)

Riber, Leise; Fujimitsu, Kazuyuki; Katayama, Tsutomu; Løbner-Olesen, Anders

2009-01-01

Initiation of chromosome replication in Escherichia coli is limited by the initiator protein DnaA associated with ATP. Within the replication origin, binding sites for DnaA associated with ATP or ADP (R boxes) and the DnaA(ATP) specific sites (I-boxes, tau-boxes and 6-mer sites) are found. We analysed chromosome replication of cells carrying mutations in conserved regions of oriC. Cells carrying mutations in DnaA-boxes I2, I3, R2, R3 and R5 as well as FIS and IHF binding sites resembled wild-type cells with respect to origin concentration. Initiation of replication in these mutants occurred in synchrony or with slight asynchrony only. Furthermore, lack of Hda stimulated initiation in all these mutants. The DnaA(ATP) containing complex that leads to initiation can therefore be formed in the absence of several of the origin DnaA binding sites including both DnaA(ATP) specific I-boxes. However, competition between I-box mutant and wild-type origins, revealed a positive role of I-boxes on initiation. On the other hand, mutations affecting DnaA-box R4 were found to be compromised for initiation and could not be augmented by an increase in cellular DnaA(ATP)/DnaA(ADP) ratio. Compared with the sites tested here, R4 therefore seems to contribute to initiation most critically.

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

Science.gov (United States)

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

Characterization of binding of N'-nitrosonornicotine to protein

International Nuclear Information System (INIS)

Hughes, M.F.

1986-01-01

The NADPH-dependent activation of the carcinogenic nitrosamine, N'-nitrosonornicotine (NNN) to a reactive intermediate which binds covalently to protein was assessed using male Sprague-Dawley rat liver and lung microsomes. The NADPH-dependent covalent binding of [ 14 C]NNN to liver and lung microsomes was linear with time up to 90 and 45 min, respectively and was also linear with protein concentrations up to 3.0 and 2.0 mg/ml, respectively. The apparent K/sub m/ and V/sub max/ of the NADPH-dependent binding to liver microsomes were determined from the initial velocities. Addition of the thiols glutathione, cystein, N-acetylcysteine or 2-mercapthoethanol significantly decreased the non-NADPH-dependent binding to liver microsomal protein, but did not affect the NADPH-dependent binding. Glutathione was required in order to observe any NADPH-dependent binding to lung microsomal protein. In lung microsomes, SKF-525A significantly decreased the NADPH-dependent binding by 79%. Replacement of an air atmosphere with N 2 or CO:O 2 (8:2) significantly decreased the NADPH-dependent binding of [ 14 C]NNN to liver microsomal protein by 40% or 27% respectively. Extensive covalent binding of [ 14 C]NNN to liver and muscle microsomal protein occurred in the absence of an NADPH-generating system, in the presence of 50% methanol and also to bovine serum albumin, indicating a nonenzymatic reaction. These data indicate that cytochrome P-450 is at least in part responsible for the metabolic activation of the carcinogen NNN, but also suggest additional mechanisms of activation

Substrate-Triggered Exosite Binding: Synergistic Dendrimer/Folic Acid Action for Achieving Specific, Tight-Binding to Folate Binding Protein.

Science.gov (United States)

Chen, Junjie; van Dongen, Mallory A; Merzel, Rachel L; Dougherty, Casey A; Orr, Bradford G; Kanduluru, Ananda Kumar; Low, Philip S; Marsh, E Neil G; Banaszak Holl, Mark M

2016-03-14

Polymer-ligand conjugates are designed to bind proteins for applications as drugs, imaging agents, and transport scaffolds. In this work, we demonstrate a folic acid (FA)-triggered exosite binding of a generation five poly(amidoamine) (G5 PAMAM) dendrimer scaffold to bovine folate binding protein (bFBP). The protein exosite is a secondary binding site on the protein surface, separate from the FA binding pocket, to which the dendrimer binds. Exosite binding is required to achieve the greatly enhanced binding constants and protein structural change observed in this study. The G5Ac-COG-FA1.0 conjugate bound tightly to bFBP, was not displaced by a 28-fold excess of FA, and quenched roughly 80% of the initial fluorescence. Two-step binding kinetics were measured using the intrinsic fluorescence of the FBP tryptophan residues to give a KD in the low nanomolar range for formation of the initial G5Ac-COG-FA1.0/FBP* complex, and a slow conversion to the tight complex formed between the dendrimer and the FBP exosite. The extent of quenching was sensitive to the choice of FA-dendrimer linker chemistry. Direct amide conjugation of FA to G5-PAMAM resulted in roughly 50% fluorescence quenching of the FBP. The G5Ac-COG-FA, which has a longer linker containing a 1,2,3-triazole ring, exhibited an ∼80% fluorescence quenching. The binding of the G5Ac-COG-FA1.0 conjugate was compared to poly(ethylene glycol) (PEG) conjugates of FA (PEGn-FA). PEG2k-FA had a binding strength similar to that of FA, whereas other PEG conjugates with higher molecular weight showed weaker binding. However, no PEG conjugates gave an increased degree of total fluorescence quenching.

Human plasminogen binding protein tetranectin

DEFF Research Database (Denmark)

Kastrup, J S; Rasmussen, H; Nielsen, B B

1997-01-01

The recombinant human plasminogen binding protein tetranectin (TN) and the C-type lectin CRD of this protein (TN3) have been crystallized. TN3 crystallizes in the tetragonal space group P4(2)2(1)2 with cell dimensions a = b = 64.0, c = 75.7 A and with one molecule per asymmetric unit. The crystals...... to at least 2.5 A. A full data set has been collected to 3.0 A. The asymmetric unit contains one monomer of TN. Molecular replacement solutions for TN3 and TN have been obtained using the structure of the C-type lectin CRD of rat mannose-binding protein as search model. The rhombohedral space group indicates...

Determining Membrane Protein-Lipid Binding Thermodynamics Using Native Mass Spectrometry.

Science.gov (United States)

Cong, Xiao; Liu, Yang; Liu, Wen; Liang, Xiaowen; Russell, David H; Laganowsky, Arthur

2016-04-06

Membrane proteins are embedded in the biological membrane where the chemically diverse lipid environment can modulate their structure and function. However, the thermodynamics governing the molecular recognition and interaction of lipids with membrane proteins is poorly understood. Here, we report a method using native mass spectrometry (MS), to determine thermodynamics of individual ligand binding events to proteins. Unlike conventional methods, native MS can resolve individual ligand binding events and, coupled with an apparatus to control the temperature, determine binding thermodynamic parameters, such as for protein-lipid interactions. We validated our approach using three soluble protein-ligand systems (maltose binding protein, lysozyme, and nitrogen regulatory protein) and obtained similar results to those using isothermal titration calorimetry and surface plasmon resonance. We also determined for the first time the thermodynamics of individual lipid binding to the ammonia channel (AmtB), an integral membrane protein from Escherichia coli. Remarkably, we observed distinct thermodynamic signatures for the binding of different lipids and entropy-enthalpy compensation for binding lipids of variable chain length. Additionally, using a mutant form of AmtB that abolishes a specific phosphatidylglycerol (PG) binding site, we observed distinct changes in the thermodynamic signatures for binding PG, implying these signatures can identify key residues involved in specific lipid binding and potentially differentiate between specific lipid binding sites.

Effect of diluent wash over the removal of aqueous dissolved TBP and DBP in reprocessing

International Nuclear Information System (INIS)

Manjula, R.; Dasi, Mahesh; Mohandas, Jaya; Vijaya Kumar, N.; Kumar, T.

2015-01-01

In reprocessing of nuclear spent fuels by PUREX process Tri-n-Butyl phosphate diluted with n-Dodecane (nDD) is used as solvent. This solvent undergoes degradation due to radiation yielding degradation products, mainly Di-n-butyl phosphate (HDBP). During extraction steps some amount of these organic gets dissolved in aqueous phase owing to its mutual solubility. Removal of dissolved organic from aqueous streams before evaporation is essential to prevent red oil related disasters. Diluent wash technique employing nDD as diluent is one of the commonly used method for the same. During the continuous operation of this process, the diluent will get loaded with dissolved organic and subsequently the performance of diluent will not remain same as pure diluent. While some reports are available in literature for the efficiency of removal of TBP by nDD, so far no work has been reported for the removal of DBP. The scope of the present work is to ascertain the efficiency of diluent wash technique on the removal of dissolved TBP as well as DBP. The results obtained indicate that the removal of dissolved TBP by nDD decreases with increase in percentage of TBP in nDD. In the case of DBP it is just reverse and the removal becomes more effective when the TBP percentage in the diluent increases. A/O ratio of 6:1 is found to be more suitable. As the DBP is getting extracted very effectively into nDD containing TBP, diluent wash solution should be treated as spent organic and managed accordingly for further utilization

The RNA-binding protein repertoire of Arabidopsis thaliana

KAUST Repository

Marondedze, Claudius; Thomas, Ludivine; Serano, Natalia Lorena Gorron; Lilley, Kathryn S.; Gehring, Christoph A

2016-01-01

RNA-binding proteins (RBPs) have essential roles in determining the fate of RNA from synthesis to decay and have been studied on a protein-by-protein basis, or computationally based on a number of well-characterised RNA-binding domains. Recently

Identification of the self-incompatibility locus F-box protein-containing complex in Petunia inflata.

Science.gov (United States)

Li, Shu; Sun, Penglin; Williams, Justin Stephen; Kao, Teh-hui

2014-03-01

The polymorphic S-locus regulating self-incompatibility (SI) in Petunia contains the S-RNase gene and a number of S-locus F-box (SLF) genes. While penetrating the style through the stigma, a pollen tube takes up all S-RNases, but only self S-RNase inhibits pollen tube growth. Recent evidence suggests that SLFs produced by pollen collectively interact with and detoxify non-self S-RNases, but none can interact with self S-RNase. An SLF may be the F-box protein component of an SCF complex (containing Cullin1, Skp1 and Rbx1), which mediates ubiquitination of protein substrates for degradation by the 26S proteasome. However, the precise nature of the complex is unknown. We used pollen extracts of a transgenic plant over-expressing GFP-fused S2-SLF1 (SLF1 of S 2-haplotype) for co-immunoprecipitation (Co-IP) followed by mass spectrometry (MS). We identified PiCUL1-P (a pollen-specific Cullin1), PiSSK1 (a pollen-specific Skp1-like protein) and PiRBX1 (an Rbx1). To validate the results, we raised transgenic plants over-expressing PiSSK1:FLAG:GFP and used pollen extracts for Co-IP-MS. The results confirmed the presence of PiCUL1-P and PiRBX1 in the complex and identified two different SLFs as the F-box protein component. Thus, all but Rbx1 of the complex may have evolved in SI, and all SLFs may be the F-box component of similar complexes.

Identification of FUSE-binding proteins as interacting partners of TIA proteins

International Nuclear Information System (INIS)

Rothe, Francoise; Gueydan, Cyril; Bellefroid, Eric; Huez, Georges; Kruys, Veronique

2006-01-01

TIA-1 and TIAR are closely related RNA-binding proteins involved in several mechanisms of RNA metabolism, including alternative hnRNA splicing and mRNA translation regulation. In particular, TIA-1 represses tumor necrosis factor (TNF) mRNA translation by binding to the AU-rich element (ARE) present in the mRNA 3' untranslated region. Here, we demonstrate that TIA proteins interact with FUSE-binding proteins (FBPs) and that fbp genes are co-expressed with tia genes during Xenopus embryogenesis. FBPs participate in various steps of RNA processing and degradation. In Cos cells, FBPs co-localize with TIA proteins in the nucleus and migrate into TIA-enriched cytoplasmic granules upon oxidative stress. Overexpression of FBP2-KH3 RNA-binding domain fused to EGFP induces the specific sequestration of TIA proteins in cytoplasmic foci, thereby precluding their nuclear accumulation. In cytosolic RAW 264.7 macrophage extracts, FBPs are found associated in EMSA to the TIA-1/TNF-ARE complex. Together, our results indicate that TIA and FBP proteins may thus be relevant biological involved in common events of RNA metabolism occurring both in the nucleus and the cytoplasm

Identification of actin binding protein, ABP-280, as a binding partner of human Lnk adaptor protein.

Science.gov (United States)

He, X; Li, Y; Schembri-King, J; Jakes, S; Hayashi, J

2000-08-01

Human Lnk (hLnk) is an adaptor protein with multiple functional domains that regulates T cell activation signaling. In order to identify cellular Lnk binding partners, a yeast two-hybrid screening of human spleen cDNA library was carried out using human hLnk as bait. A polypeptide sequence identical to the C-terminal segment of the actin binding protein (ABP-280) was identified as a hLnk binding protein. The expressed hLnk and the FLAG tagged C-terminal 673 amino acid residues of ABP-280 or the endogenous ABP-280 in COS-7 cells could be co-immunoprecipitated using antibodies either to hLnk, FLAG or ABP-280, respectively. Furthermore, immunofluorescence confocal microscope showed that hLnk and ABP-280 co-localized at the plasma membrane and at juxtanuclear region of COS-7 cells. In Jurkat cells, the endogenous hLnk also associates with the endogenous ABP-280 indicating that the association of these two proteins is physiological. The interacting domains of both proteins were mapped using yeast two-hybrid assays. Our results indicate that hLnk binds to the residues 2006-2454 (repeats 19-23C) of ABP-280. The domain in hLnk that associates with ABP-280 was mapped to an interdomain region of 56 amino acids between pleckstrin homology and Src homology 2 domains. These results suggest that hLnk may exert its regulatory role through its association with ABP-280.

A novel F-box protein CaF-box is involved in responses to plant hormones and abiotic stress in pepper (Capsicum annuum L.).

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Chen, Rugang; Guo, Weili; Yin, Yanxu; Gong, Zhen-Hui

2014-02-10

The F-box protein family is characterized by an F-box motif that has been shown to play an important role in regulating various developmental processes and stress responses. In this study, a novel F-box-containing gene was isolated from leaves of pepper cultivar P70 (Capsicum annuum L.) and designated CaF-box. The full-length cDNA is 2088 bp and contains an open reading frame of 1914 bp encoding a putative polypeptide of 638 amino acids with a mass of 67.8 kDa. CaF-box was expressed predominantly in stems and seeds, and the transcript was markedly upregulated in response to cold stress, abscisic acid (ABA) and salicylic acid (SA) treatment, and downregulated under osmotic and heavy metal stress. CaF-box expression was dramatically affected by salt stress, and was rapidly increased for the first hour, then sharply decreased thereafter. In order to further assess the role of CaF-box in the defense response to abiotic stress, a loss-of-function experiment in pepper plants was performed using a virus-induced gene silencing (VIGS) technique. Measurement of thiobarbituric acid reactive substances (TBARS) and electrolyte leakage revealed stronger lipid peroxidation and cell death in the CaF-box-silenced plants than in control plants, suggesting CaF-box plays an important role in regulating the defense response to abiotic stress resistance in pepper plants.

Analysis of Steam Heating of a Two-Layer TBP/N-Paraffin/Nitric Acid Mixtures

International Nuclear Information System (INIS)

Laurinat, J.E.; Hassan, N.M.; Rudisill, T.S.; Askew, N.M.

1998-01-01

This report presents an analysis of steam heating of a two-layer tri-n-butyl phosphate (TBP)/n-paraffin-nitric acid mixture.The purpose of this study is to determine if the degree of mixing provided by the steam jet or by bubbles generated by the TBP/nitric acid reaction is sufficient to prevent a runaway reaction

Structural and functional analysis of the human spliceosomal DEAD-box helicase Prp28

Energy Technology Data Exchange (ETDEWEB)

Möhlmann, Sina [Georg-August-University Göttingen, Justus-von-Liebig Weg 11, 37077 Göttingen (Germany); Mathew, Rebecca [Max-Planck-Institute for Biophysical Chemistry, Am Fassberg, 37077 Göttingen (Germany); Neumann, Piotr; Schmitt, Andreas [Georg-August-University Göttingen, Justus-von-Liebig Weg 11, 37077 Göttingen (Germany); Lührmann, Reinhard [Max-Planck-Institute for Biophysical Chemistry, Am Fassberg, 37077 Göttingen (Germany); Ficner, Ralf, E-mail: rficner@uni-goettingen.de [Georg-August-University Göttingen, Justus-von-Liebig Weg 11, 37077 Göttingen (Germany)

2014-06-01

The crystal structure of the helicase domain of the human spliceosomal DEAD-box protein Prp28 was solved by SAD. The binding of ADP and ATP by Prp28 was studied biochemically and analysed with regard to the crystal structure. The DEAD-box protein Prp28 is essential for pre-mRNA splicing as it plays a key role in the formation of an active spliceosome. Prp28 participates in the release of the U1 snRNP from the 5′-splice site during association of the U5·U4/U6 tri-snRNP, which is a crucial step in the transition from a pre-catalytic spliceosome to an activated spliceosome. Here, it is demonstrated that the purified helicase domain of human Prp28 (hPrp28ΔN) binds ADP, whereas binding of ATP and ATPase activity could not be detected. ATP binding could not be observed for purified full-length hPrp28 either, but within an assembled spliceosomal complex hPrp28 gains ATP-binding activity. In order to understand the structural basis for the ATP-binding deficiency of isolated hPrp28, the crystal structure of hPrp28ΔN was determined at 2.0 Å resolution. In the crystal the helicase domain adopts a wide-open conformation, as the two RecA-like domains are extraordinarily displaced from the productive ATPase conformation. Binding of ATP is hindered by a closed conformation of the P-loop, which occupies the space required for the γ-phosphate of ATP.

The RNA-Binding Site of Poliovirus 3C Protein Doubles as a Phosphoinositide-Binding Domain.

Science.gov (United States)

Shengjuler, Djoshkun; Chan, Yan Mei; Sun, Simou; Moustafa, Ibrahim M; Li, Zhen-Lu; Gohara, David W; Buck, Matthias; Cremer, Paul S; Boehr, David D; Cameron, Craig E

2017-12-05

Some viruses use phosphatidylinositol phosphate (PIP) to mark membranes used for genome replication or virion assembly. PIP-binding motifs of cellular proteins do not exist in viral proteins. Molecular-docking simulations revealed a putative site of PIP binding to poliovirus (PV) 3C protein that was validated using nuclear magnetic resonance spectroscopy. The PIP-binding site was located on a highly dynamic α helix, which also functions in RNA binding. Broad PIP-binding activity was observed in solution using a fluorescence polarization assay or in the context of a lipid bilayer using an on-chip, fluorescence assay. All-atom molecular dynamics simulations of the 3C protein-membrane interface revealed PIP clustering and perhaps PIP-dependent conformations. PIP clustering was mediated by interaction with residues that interact with the RNA phosphodiester backbone. We conclude that 3C binding to membranes will be determined by PIP abundance. We suggest that the duality of function observed for 3C may extend to RNA-binding proteins of other viruses. Copyright © 2017 Elsevier Ltd. All rights reserved.

The F-box protein FBXO44 mediates BRCA1 ubiquitination and degradation.

Science.gov (United States)

Lu, Yunzhe; Li, Jiezhi; Cheng, Dongmei; Parameswaran, Balaji; Zhang, Shaohua; Jiang, Zefei; Yew, P Renee; Peng, Junmin; Ye, Qinong; Hu, Yanfen

2012-11-30

BRCA1 mutations account for a significant proportion of familial breast and ovarian cancers. In addition, reduced BRCA1 protein is associated with sporadic cancer cases in these tissues. At the cellular level, BRCA1 plays a critical role in multiple cellular functions such as DNA repair and cell cycle checkpoint control. Its protein level is regulated in a cell cycle-dependent manner. However, regulation of BRCA1 protein stability is not fully understood. Our earlier study showed that the amino terminus of BRCA1 harbors a degron sequence that is sufficient and necessary for conferring BRCA1 degradation. In the current study, we used mass spectrometry to identify Skp1 that regulates BRCA1 protein stability. Small interfering RNA screening that targets all human F-box proteins uncovered FBXO44 as an important protein that influences BRCA1 protein level. The Skp1-Cul1-F-box-protein44 (SCF(FBXO44)) complex ubiquitinates full-length BRCA1 in vitro. Furthermore, the N terminus of BRCA1 mediates the interaction between BRCA1 and FBXO44. Overexpression of SCF(FBXO44) reduces BRCA1 protein level. Taken together, our work strongly suggests that SCF(FBXO44) is an E3 ubiquitin ligase responsible for BRCA1 degradation. In addition, FBXO44 expression pattern in breast carcinomas suggests that SCF(FBXO44)-mediated BRCA1 degradation might contribute to sporadic breast tumor development.

An Inductive Logic Programming Approach to Validate Hexose Binding Biochemical Knowledge.

Science.gov (United States)

Nassif, Houssam; Al-Ali, Hassan; Khuri, Sawsan; Keirouz, Walid; Page, David

2010-01-01

Hexoses are simple sugars that play a key role in many cellular pathways, and in the regulation of development and disease mechanisms. Current protein-sugar computational models are based, at least partially, on prior biochemical findings and knowledge. They incorporate different parts of these findings in predictive black-box models. We investigate the empirical support for biochemical findings by comparing Inductive Logic Programming (ILP) induced rules to actual biochemical results. We mine the Protein Data Bank for a representative data set of hexose binding sites, non-hexose binding sites and surface grooves. We build an ILP model of hexose-binding sites and evaluate our results against several baseline machine learning classifiers. Our method achieves an accuracy similar to that of other black-box classifiers while providing insight into the discriminating process. In addition, it confirms wet-lab findings and reveals a previously unreported Trp-Glu amino acids dependency.

Partitioning of actinides from high active waste solution of Purex origin counter-current extraction studies using TBP and CMPO

International Nuclear Information System (INIS)

Chitnis, R.R.; Dhami, P.S.; Gopalkrishnan, V.; Wattal, P.K.; Ramanujam, A.; Murali, M.S.; Mathur, J.N.; Bauri, A.K.; Chattopadhyay, S.

2000-10-01

A solvent extraction scheme has been formulated for the partitioning of actinides from Purex high level waste (HLW). The scheme is based on the results of earlier studies carried out with simulated waste solutions. In the present studies, the scheme was tested with high active waste (HAW) solution generated during the reprocessing of spent fuel from research reactors using laboratory scale mixer-settlers. The proposed process involved two-step extraction using tri-n-butyl phosphate (TBP) and octyl (phenyl)-N,N-diisobutylcarbamolylmethylphosphine oxide (CMPO). In the first step, uranium, neptunium and plutonium were removed from the waste using TBP as extractant. The minor actinides left in the raffinate were extracted using a mixture of CMPO and TBP in the second step. The results showed complete extraction of actinides from the waste solution. Plutonium and neptunium extracted in TBP, were stripped together using a mixture of hydrogen peroxide and ascorbic acid in 2 M nitric acid medium, leaving uranium in the organic phase. Uranium can later be stripped using dilute nitric acid. Actinides extracted in CMPO-TBP phase were stripped using a mixture of formic acid, hydrazine, hydrate and citric acid. The stripping was quantitative in both the stripping runs. An additional extraction step for the preferential recovery of uranium, neptunium and plutonium from the waste solution using TBP is a modification over the conventional Truex process. Selective stripping of neptunium and plutonium from large quantities of uranium. The extraction of uranium using TBP eliminates the possibility of third phase and undesired loading of CMPO-TBP in the following step. Use of citrate-containing strippant allows the recovery of actinides from loaded CMPO-TBP mixture without causing any reflux of the actinides during stripping. The process has been developed with due consideration to minimising the generation of secondary wastes. The proposed strippants are effective even in presence of

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

Science.gov (United States)

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.

Third phase formation revisited: the U(VI), HNO3 - TBP, n-dodecane system

International Nuclear Information System (INIS)

Chiarizia, R.; Jensen, M.P.; Borkowski, M.; Ferraro, J.R.; Thiyagarajan, P.; Littrell, K.C.

2003-01-01

In this work, the system U(VI), HNO 3 -tri-n-butylphosphate (TBP), n-dodecane has been revisited with the objective of gaining information on the coordination chemistry and structural evolution of the species formed in the organic phase before and after third phase formation. Chemical analyses, spectroscopic and EXAFS data indicate that U(VI) is extracted as the UO 2 (NO 3 ) 2 ·2TBP adduct, while the third phase species have the average composition UO 2 (NO 3 ) 2 ·2TBP·HNO 3 . Small-angle neutron scattering (SANS) measurements on TBP solutions loaded with only HNO 3 or with increasing amounts of U(VI) have revealed the presence, before phase splitting, of ellipsoidal aggregates with the major and minor axes up to about 64 and 15 A, respectively. The formation of these aggregates, very likely of the reverse micelle-type, is observed in all cases, that is, when only HNO 3 , only UO 2 (NO 3 ) 2 , or both HNO 3 and UO 2 (NO 3 ) 2 are extracted by the TBP solution. Upon third phase formation, the SANS data reveal the presence of smaller aggregates in the light organic phase, while the heavy organic phase contains pockets of diluent, each with an average of about two molecules of n-dodecane.

DNA-binding proteins essential for protein-primed bacteriophage ø29 DNA replication

Directory of Open Access Journals (Sweden)

Margarita Salas

2016-08-01

Full Text Available Bacillus subtilis phage Φ29 has a linear, double-stranded DNA 19 kb long with an inverted terminal repeat of 6 nucleotides and a protein covalently linked to the 5’ ends of the DNA. This protein, called terminal protein (TP, is the primer for the initiation of replication, a reaction catalyzed by the viral DNA polymerase at the two DNA ends. The DNA polymerase further elongates the nascent DNA chain in a processive manner, coupling strand displacement with elongation. The viral protein p5 is a single-stranded DNA binding protein (SSB that binds to the single strands generated by strand displacement during the elongation process. Viral protein p6 is a double-stranded DNA binding protein (DBP that preferentially binds to the origins of replication at the Φ29 DNA ends and is required for the initiation of replication. Both SSB and DBP are essential for Φ29 DNA amplification. This review focuses on the role of these phage DNA-binding proteins in Φ29 DNA replication both in vitro and in vivo, as well as on the implication of several B. subtilis DNA-binding proteins in different processes of the viral cycle. We will revise the enzymatic activities of the Φ29 DNA polymerase: TP-deoxynucleotidylation, processive DNA polymerization coupled to strand displacement, 3’-5’ exonucleolysis and pyrophosphorolysis. The resolution of the Φ29 DNA polymerase structure has shed light on the translocation mechanism and the determinants responsible for processivity and strand displacement. These two properties have made Φ29 DNA polymerase one of the main enzymes used in the current DNA amplification technologies. The determination of the structure of Φ29 TP revealed the existence of three domains: the priming domain, where the primer residue Ser232, as well as Phe230, involved in the determination of the initiating nucleotide, are located, the intermediate domain, involved in DNA polymerase binding, and the N-terminal domain, responsible for DNA binding

A bioinformatic survey of RNA-binding proteins in Plasmodium.

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Reddy, B P Niranjan; Shrestha, Sony; Hart, Kevin J; Liang, Xiaoying; Kemirembe, Karen; Cui, Liwang; Lindner, Scott E

2015-11-02

The malaria parasites in the genus Plasmodium have a very complicated life cycle involving an invertebrate vector and a vertebrate host. RNA-binding proteins (RBPs) are critical factors involved in every aspect of the development of these parasites. However, very few RBPs have been functionally characterized to date in the human parasite Plasmodium falciparum. Using different bioinformatic methods and tools we searched P. falciparum genome to list and annotate RBPs. A representative 3D models for each of the RBD domain identified in P. falciparum was created using I-TESSAR and SWISS-MODEL. Microarray and RNAseq data analysis pertaining PfRBPs was performed using MeV software. Finally, Cytoscape was used to create protein-protein interaction network for CITH-Dozi and Caf1-CCR4-Not complexes. We report the identification of 189 putative RBP genes belonging to 13 different families in Plasmodium, which comprise 3.5% of all annotated genes. Almost 90% (169/189) of these genes belong to six prominent RBP classes, namely RNA recognition motifs, DEAD/H-box RNA helicases, K homology, Zinc finger, Puf and Alba gene families. Interestingly, almost all of the identified RNA-binding helicases and KH genes have cognate homologs in model species, suggesting their evolutionary conservation. Exploration of the existing P. falciparum blood-stage transcriptomes revealed that most RBPs have peak mRNA expression levels early during the intraerythrocytic development cycle, which taper off in later stages. Nearly 27% of RBPs have elevated expression in gametocytes, while 47 and 24% have elevated mRNA expression in ookinete and asexual stages. Comparative interactome analyses using human and Plasmodium protein-protein interaction datasets suggest extensive conservation of the PfCITH/PfDOZI and PfCaf1-CCR4-NOT complexes. The Plasmodium parasites possess a large number of putative RBPs belonging to most of RBP families identified so far, suggesting the presence of extensive post

Aluminium fluoride and magnesium, activators of heterotrimeric GTP-binding proteins, affect high-affinity binding of the fungal toxin fusicoccin to the fusicoccin-binding protein in oat root plasma membranes.

NARCIS (Netherlands)

de Boer, A.H.; Van der Molen, G.W.; Prins, H.B.A.; Korthout, H.A.A.J.; van der Hoeven, P.C.J.

1994-01-01

The fusicoccin-binding protein was solubilised from purified oat root plasma membranes. The solubilised protein retained full binding activity, provided that protease inhibitors were included. Sodium fluoride reduced the high-affinity [H-3]fusicoccin binding to almost zero in a

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

Science.gov (United States)

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

2010-09-24

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

Molecular cloning and characterization of an F-box family gene CarF-box1 from chickpea (Cicer arietinum L.).

Science.gov (United States)

Jia, Yuying; Gu, Hanyan; Wang, Xiansheng; Chen, Quanjia; Shi, Shubing; Zhang, Jusong; Ma, Lin; Zhang, Hua; Ma, Hao

2012-03-01

F-box protein family has been found to play important roles in plant development and abiotic stress responses via the ubiquitin pathway. In this study, an F-box gene CarF-box1 (for Cicer arietinum F-box gene 1, Genbank accession no. GU247510) was isolated based on a cDNA library constructed with chickpea seedling leaves treated by polyethylene glycol. CarF-box1 encoded a putative protein with 345 amino acids and contained no intron within genomic DNA sequence. CarF-box1 is a KFB-type F-box protein, having a conserved F-box domain in the N-terminus and a Kelch repeat domain in the C-terminus. CarF-box1 was localized in the nucleus. CarF-box1 exhibited organ-specific expression and showed different expression patterns during seed development and germination processes, especially strongly expressed in the blooming flowers. In the leaves, CarF-box1 could be significantly induced by drought stress and slightly induced by IAA treatment, while in the roots, CarF-box1 could be strongly induced by drought, salinity and methyl jasmonate stresses. Our results suggest that CarF-box1 encodes an F-box protein and may be involved in various plant developmental processes and abiotic stress responses.

DPI-ELISA: a fast and versatile method to specify the binding of plant transcription factors to DNA in vitro

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Chaban Christina

2010-11-01

Full Text Available Abstract Background About 10% of all genes in eukaryote genomes are predicted to encode transcription factors. The specific binding of transcription factors to short DNA-motifs influences the expression of neighbouring genes. However, little is known about the DNA-protein interaction itself. To date there are only a few suitable methods to characterise DNA-protein-interactions, among which the EMSA is the method most frequently used in laboratories. Besides EMSA, several protocols describe the effective use of an ELISA-based transcription factor binding assay e.g. for the analysis of human NFκB binding to specific DNA sequences. Results We provide a unified protocol for this type of ELISA analysis, termed DNA-Protein-Interaction (DPI-ELISA. Qualitative analyses with His-epitope tagged plant transcription factors expressed in E. coli revealed that EMSA and DPI-ELISA result in comparable and reproducible data. The binding of AtbZIP63 to the C-box and AtWRKY11 to the W2-box could be reproduced and validated by both methods. We next examined the physical binding of the C-terminal DNA-binding domains of AtWRKY33, AtWRKY50 and AtWRKY75 to the W2-box. Although the DNA-binding domain is highly conserved among the WRKY proteins tested, the use of the DPI-ELISA discloses differences in W2-box binding properties between these proteins. In addition to these well-studied transcription factor families, we applied our protocol to AtBPC2, a member of the so far uncharacterised plant specific Basic Pentacysteine transcription factor family. We could demonstrate binding to GA/TC-dinucleotide repeat motifs by our DPI-ELISA protocol. Different buffers and reaction conditions were examined. Conclusions We successfully applied our DPI-ELISA protocol to investigate the DNA-binding specificities of three different classes of transcription factors from Arabidopsis thaliana. However, the analysis of the binding affinity of any DNA-binding protein to any given DNA

Vitiligo inducing phenols activate the unfolded protein response in melanocytes resulting in upregulation of IL6 and IL8

Science.gov (United States)

Toosi, Siavash; Orlow, Seth J.; Manga, Prashiela

2012-01-01

Vitiligo is characterized by depigmented skin patches due to loss of epidermal melanocytes. Oxidative stress may play a role in vitiligo onset, while autoimmunity contributes to disease progression. In this study we sought to identify mechanisms that link disease triggers and spreading of lesions. A hallmark of melanocytes at the periphery of vitiligo lesions is dilation of the endoplasmic reticulum (ER). We hypothesized that oxidative stress results in redox disruptions that extend to the ER, causing accumulation of misfolded peptides, which activates the unfolded protein response (UPR). We used 4-tertiary butyl phenol (4-TBP) and monobenzyl ether of hydroquinone (MBEH), known triggers of vitiligo. We show that expression of key UPR components, including the transcription factor X-box binding protein 1 (XBP1), are increased following exposure of melanocytes to phenols. XBP1 activation increases production of immune mediators interleukin-6 (IL6) and IL8. Co-treatment with XBP1 inhibitors reduced IL6 and IL8 production induced by phenols, while over-expression of XBP1 alone increased their expression. Thus, melanocytes themselves produce cytokines associated with activation of an immune response following exposure to chemical triggers of vitiligo. These results expand our understanding of the mechanisms underlying melanocyte loss in vitiligo and pathways linking environmental stressors and autoimmunity. PMID:22696056

XBP1 (X-Box-Binding Protein-1)-Dependent O-GlcNAcylation Is Neuroprotective in Ischemic Stroke in Young Mice and Its Impairment in Aged Mice Is Rescued by Thiamet-G.

Science.gov (United States)

Jiang, Meng; Yu, Shu; Yu, Zhui; Sheng, Huaxin; Li, Ying; Liu, Shuai; Warner, David S; Paschen, Wulf; Yang, Wei

2017-06-01

Impaired protein homeostasis induced by endoplasmic reticulum dysfunction is a key feature of a variety of age-related brain diseases including stroke. To restore endoplasmic reticulum function impaired by stress, the unfolded protein response is activated. A key unfolded protein response prosurvival pathway is controlled by the endoplasmic reticulum stress sensor (inositol-requiring enzyme-1), XBP1 (downstream X-box-binding protein-1), and O-GlcNAc (O-linked β-N-acetylglucosamine) modification of proteins (O-GlcNAcylation). Stroke impairs endoplasmic reticulum function, which activates unfolded protein response. The rationale of this study was to explore the potentials of the IRE1/XBP1/O-GlcNAc axis as a target for neuroprotection in ischemic stroke. Mice with Xbp1 loss and gain of function in neurons were generated. Stroke was induced by transient or permanent occlusion of the middle cerebral artery in young and aged mice. Thiamet-G was used to increase O-GlcNAcylation. Deletion of Xbp1 worsened outcome after transient and permanent middle cerebral artery occlusion. After stroke, O-GlcNAcylation was activated in neurons of the stroke penumbra in young mice, which was largely Xbp1 dependent. This activation of O-GlcNAcylation was impaired in aged mice. Pharmacological increase of O-GlcNAcylation before or after stroke improved outcome in both young and aged mice. Our study indicates a critical role for the IRE1/XBP1 unfolded protein response branch in stroke outcome. O-GlcNAcylation is a prosurvival pathway that is activated in the stroke penumbra in young mice but impaired in aged mice. Boosting prosurvival pathways to counterbalance the age-related decline in the brain's self-healing capacity could be a promising strategy to improve ischemic stroke outcome in aged brains. © 2017 American Heart Association, Inc.

Drosophila DNA-Binding Proteins in Polycomb Repression

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Maksim Erokhin

2018-01-01

Full Text Available The formation of individual gene expression patterns in different cell types is required during differentiation and development of multicellular organisms. Polycomb group (PcG proteins are key epigenetic regulators responsible for gene repression, and dysregulation of their activities leads to developmental abnormalities and diseases. PcG proteins were first identified in Drosophila, which still remains the most convenient system for studying PcG-dependent repression. In the Drosophila genome, these proteins bind to DNA regions called Polycomb response elements (PREs. A major role in the recruitment of PcG proteins to PREs is played by DNA-binding factors, several of which have been characterized in detail. However, current knowledge is insufficient for comprehensively describing the mechanism of this process. In this review, we summarize and discuss the available data on the role of DNA-binding proteins in PcG recruitment to chromatin.

Effect of cobratoxin binding on the normal mode vibration within acetylcholine binding protein.

Science.gov (United States)

Bertaccini, Edward J; Lindahl, Erik; Sixma, Titia; Trudell, James R

2008-04-01

Recent crystal structures of the acetylcholine binding protein (AChBP) have revealed surprisingly small structural alterations upon ligand binding. Here we investigate the extent to which ligand binding may affect receptor dynamics. AChBP is a homologue of the extracellular component of ligand-gated ion channels (LGICs). We have previously used an elastic network normal-mode analysis to propose a gating mechanism for the LGICs and to suggest the effects of various ligands on such motions. However, the difficulties with elastic network methods lie in their inability to account for the modest effects of a small ligand or mutation on ion channel motion. Here, we report the successful application of an elastic network normal mode technique to measure the effects of large ligand binding on receptor dynamics. The present calculations demonstrate a clear alteration in the native symmetric motions of a protein due to the presence of large protein cobratoxin ligands. In particular, normal-mode analysis revealed that cobratoxin binding to this protein significantly dampened the axially symmetric motion of the AChBP that may be associated with channel gating in the full nAChR. The results suggest that alterations in receptor dynamics could be a general feature of ligand binding.

Direct extraction of uranium and plutonium from oxide fuel using TBP-HNO3 complex for super-DIREX process

International Nuclear Information System (INIS)

Miura, S.; Kamiya, M.; Nomura, K.; Koyama, T.; Ogum, S.; Shimada, T.; Mori, Y.; Enokida, Y.

2004-01-01

Super-DIREX is a new reprocessing method which has high economical efficiency. Experimental study of this process was started on the direct extraction of U and Pu from irradiated MOX fuel by the supercritical carbon dioxide (SFCO 2 ) containing TBP-HNO 3 complex. This report describes direct extraction of U and Pu with TBP-HNO 3 complex at atmospheric pressure, as the first test for irradiated fuel, in order to investigate the applicability of SFCO 2 containing TBP-HNO 3 complex. In this test, dependency on dissolution temperature, Pu content, fuel/ TBP-HNO 3 complex ratio and effect of voloxidation were investigated. From these results, TBP-HNO 3 complex was found to be effective in the respect of the recovery of U and Pu. The number of the process step in dissolution and co-extraction is small, and amount of waste can be reduced. It is applicable to the direct extraction in Super-DIREX. (authors)

Saccharomyces cerevisiae SSB1 protein and its relationship to nucleolar RNA-binding proteins.

Science.gov (United States)

Jong, A Y; Clark, M W; Gilbert, M; Oehm, A; Campbell, J L

1987-08-01

To better define the function of Saccharomyces cerevisiae SSB1, an abundant single-stranded nucleic acid-binding protein, we determined the nucleotide sequence of the SSB1 gene and compared it with those of other proteins of known function. The amino acid sequence contains 293 amino acid residues and has an Mr of 32,853. There are several stretches of sequence characteristic of other eucaryotic single-stranded nucleic acid-binding proteins. At the amino terminus, residues 39 to 54 are highly homologous to a peptide in calf thymus UP1 and UP2 and a human heterogeneous nuclear ribonucleoprotein. Residues 125 to 162 constitute a fivefold tandem repeat of the sequence RGGFRG, the composition of which suggests a nucleic acid-binding site. Near the C terminus, residues 233 to 245 are homologous to several RNA-binding proteins. Of 18 C-terminal residues, 10 are acidic, a characteristic of the procaryotic single-stranded DNA-binding proteins and eucaryotic DNA- and RNA-binding proteins. In addition, examination of the subcellular distribution of SSB1 by immunofluorescence microscopy indicated that SSB1 is a nuclear protein, predominantly located in the nucleolus. Sequence homologies and the nucleolar localization make it likely that SSB1 functions in RNA metabolism in vivo, although an additional role in DNA metabolism cannot be excluded.

Binding free energy analysis of protein-protein docking model structures by evERdock.

Science.gov (United States)

Takemura, Kazuhiro; Matubayasi, Nobuyuki; Kitao, Akio

2018-03-14

To aid the evaluation of protein-protein complex model structures generated by protein docking prediction (decoys), we previously developed a method to calculate the binding free energies for complexes. The method combines a short (2 ns) all-atom molecular dynamics simulation with explicit solvent and solution theory in the energy representation (ER). We showed that this method successfully selected structures similar to the native complex structure (near-native decoys) as the lowest binding free energy structures. In our current work, we applied this method (evERdock) to 100 or 300 model structures of four protein-protein complexes. The crystal structures and the near-native decoys showed the lowest binding free energy of all the examined structures, indicating that evERdock can successfully evaluate decoys. Several decoys that show low interface root-mean-square distance but relatively high binding free energy were also identified. Analysis of the fraction of native contacts, hydrogen bonds, and salt bridges at the protein-protein interface indicated that these decoys were insufficiently optimized at the interface. After optimizing the interactions around the interface by including interfacial water molecules, the binding free energies of these decoys were improved. We also investigated the effect of solute entropy on binding free energy and found that consideration of the entropy term does not necessarily improve the evaluations of decoys using the normal model analysis for entropy calculation.

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

NARCIS (Netherlands)

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

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

Probing the role of intercalating protein sidechains for kink formation in DNA.

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Achim Sandmann

Full Text Available Protein binding can induce DNA kinks, which are for example important to enhance the specificity of the interaction and to facilitate the assembly of multi protein complexes. The respective proteins frequently exhibit amino acid sidechains that intercalate between the DNA base steps at the site of the kink. However, on a molecular level there is only little information available about the role of individual sidechains for kink formation. To unravel structural principles of protein-induced DNA kinking we have performed molecular dynamics (MD simulations of five complexes that varied in their architecture, function, and identity of intercalated residues. Simulations were performed for the DNA complexes of wildtype proteins (Sac7d, Sox-4, CcpA, TFAM, TBP and for mutants, in which the intercalating residues were individually or combined replaced by alanine. The work revealed that for systems with multiple intercalated residues, not all of them are necessarily required for kink formation. In some complexes (Sox-4, TBP, one of the residues proved to be essential for kink formation, whereas the second residue has only a very small effect on the magnitude of the kink. In other systems (e.g. Sac7d each of the intercalated residues proved to be individually capable of conferring a strong kink suggesting a partially redundant role of the intercalating residues. Mutation of the key residues responsible for kinking either resulted in stable complexes with reduced kink angles or caused conformational instability as evidenced by a shift of the kink to an adjacent base step. Thus, MD simulations can help to identify the role of individual inserted residues for kinking, which is not readily apparent from an inspection of the static structures. This information might be helpful for understanding protein-DNA interactions in more detail and for designing proteins with altered DNA binding properties in the future.

Hydrolytic and radiolytic degradation of TBP in TBP.30% V/V-dodecane/UO2(NO3)2.HNO3.H2O systems

International Nuclear Information System (INIS)

Barreta, L.G.

1980-01-01

The hydrolytic and radiolytic degradation of TBP is investigated in systems of TBP 30% V/V-dodecane/H 2 O . HNO 3 . UO 2 (NO 3 ) 2 by gas chromatographic determination of HDBP. No direct relation between the concentration of HDBP formed and the quantity of HNO 3 extracted by the organic phase is observed in the studies of hydrolysis of TBP. The HDBP concentration is seen to increase non-linearly with the concentration of HNO 3 extracted by the organic phase. Radiolytic studies show that for doses greater than 1 Wh/l, the concentration of HDBP formed increases with the dose absorbed by the system. Whith doses smaller than 1 Wh/l and acid concentration greater than 2 M, two distinct patterns of behavior are observed. The concentration of HDBP as a function of the radiation dose absorbed by the system presents a minimum for uranyl nitrate concentrations smaller than 0.9 M; for uranyl nitrate concentrations greater than 1.3 M the concentration of radiolytic HDBP cannot be calculated because the concentration of the hydrolytic HDBP determined is greater than the sum of the experimental concentrations of hydrolytic and radiolytic HDBP. It is known that the dose absorbed by the process solutions during the reprocessing of light water reactor fuel elements is smaller than one Wh/l. Thus, dose rates between zero and one Wh/l should be studied for this system. (Author) [pt

Factors Affecting the Binding of a Recombinant Heavy Metal-Binding Domain (CXXC motif Protein to Heavy Metals

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Kamala Boonyodying

2012-06-01

Full Text Available A number of heavy metal-binding proteins have been used to study bioremediation. CXXC motif, a metal binding domain containing Cys-X-X-Cys motif, has been identified in various organisms. These proteins are capable of binding various types of heavy metals. In this study, heavy metal binding domain (CXXC motif recombinant protein encoded from mcsA gene of S. aureus were cloned and overexpressed in Escherichia coli. The factors involved in the metal-binding activity were determined in order to analyze the potential of recombinant protein for bioremediation. A recombinant protein can be bound to Cd2+, Co2+, Cu2+ and Zn2+. The thermal stability of a recombinant protein was tested, and the results showed that the metal binding activity to Cu2+ and Zn2+ still exist after treating the protein at 85ºC for 30 min. The temperature and pH that affected the metal binding activity was tested and the results showed that recombinant protein was still bound to Cu2+ at 65ºC, whereas a pH of 3-7 did not affect the metal binding E. coli harboring a pRset with a heavy metal-binding domain CXXC motif increased the resistance of heavy metals against CuCl2 and CdCl2. This study shows that metal binding domain (CXXC motif recombinant protein can be effectively bound to various types of heavy metals and may be used as a potential tool for studying bioremediation.

Predicting the binding patterns of hub proteins: a study using yeast protein interaction networks.

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Carson M Andorf

Full Text Available Protein-protein interactions are critical to elucidating the role played by individual proteins in important biological pathways. Of particular interest are hub proteins that can interact with large numbers of partners and often play essential roles in cellular control. Depending on the number of binding sites, protein hubs can be classified at a structural level as singlish-interface hubs (SIH with one or two binding sites, or multiple-interface hubs (MIH with three or more binding sites. In terms of kinetics, hub proteins can be classified as date hubs (i.e., interact with different partners at different times or locations or party hubs (i.e., simultaneously interact with multiple partners.Our approach works in 3 phases: Phase I classifies if a protein is likely to bind with another protein. Phase II determines if a protein-binding (PB protein is a hub. Phase III classifies PB proteins as singlish-interface versus multiple-interface hubs and date versus party hubs. At each stage, we use sequence-based predictors trained using several standard machine learning techniques.Our method is able to predict whether a protein is a protein-binding protein with an accuracy of 94% and a correlation coefficient of 0.87; identify hubs from non-hubs with 100% accuracy for 30% of the data; distinguish date hubs/party hubs with 69% accuracy and area under ROC curve of 0.68; and SIH/MIH with 89% accuracy and area under ROC curve of 0.84. Because our method is based on sequence information alone, it can be used even in settings where reliable protein-protein interaction data or structures of protein-protein complexes are unavailable to obtain useful insights into the functional and evolutionary characteristics of proteins and their interactions.We provide a web server for our three-phase approach: http://hybsvm.gdcb.iastate.edu.

Exploring the binding sites and binding mechanism for hydrotrope encapsulated griseofulvin drug on γ-tubulin protein.

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Shubhadip Das

Full Text Available The protein γ-tubulin plays an important role in centrosomal clustering and this makes it an attractive therapeutic target for treating cancers. Griseofulvin, an antifungal drug, has recently been used to inhibit proliferation of various types of cancer cells. It can also affect the microtubule dynamics by targeting the γ-tubulin protein. So far, the binding pockets of γ-tubulin protein are not properly identified and the exact mechanism by which the drug binds to it is an area of intense speculation and research. The aim of the present study is to investigate the binding mechanism and binding affinity of griseofulvin on γ-tubulin protein using classical molecular dynamics simulations. Since the drug griseofulvin is sparingly soluble in water, here we also present a promising approach for formulating and achieving delivery of hydrophobic griseofulvin drug via hydrotrope sodium cumene sulfonate (SCS cluster. We observe that the binding pockets of γ-tubulin protein are mainly formed by the H8, H9 helices and S7, S8, S14 strands and the hydrophobic interactions between the drug and γ-tubulin protein drive the binding process. The release of the drug griseofulvin from the SCS cluster is confirmed by the coordination number analysis. We also find hydrotrope-induced alteration of the binding sites of γ-tubulin protein and the weakening of the drug-protein interactions.

Detection of secondary binding sites in proteins using fragment screening.

Science.gov (United States)

Ludlow, R Frederick; Verdonk, Marcel L; Saini, Harpreet K; Tickle, Ian J; Jhoti, Harren

2015-12-29

Proteins need to be tightly regulated as they control biological processes in most normal cellular functions. The precise mechanisms of regulation are rarely completely understood but can involve binding of endogenous ligands and/or partner proteins at specific locations on a protein that can modulate function. Often, these additional secondary binding sites appear separate to the primary binding site, which, for example for an enzyme, may bind a substrate. In previous work, we have uncovered several examples in which secondary binding sites were discovered on proteins using fragment screening approaches. In each case, we were able to establish that the newly identified secondary binding site was biologically relevant as it was able to modulate function by the binding of a small molecule. In this study, we investigate how often secondary binding sites are located on proteins by analyzing 24 protein targets for which we have performed a fragment screen using X-ray crystallography. Our analysis shows that, surprisingly, the majority of proteins contain secondary binding sites based on their ability to bind fragments. Furthermore, sequence analysis of these previously unknown sites indicate high conservation, which suggests that they may have a biological function, perhaps via an allosteric mechanism. Comparing the physicochemical properties of the secondary sites with known primary ligand binding sites also shows broad similarities indicating that many of the secondary sites may be druggable in nature with small molecules that could provide new opportunities to modulate potential therapeutic targets.

SCOWLP classification: Structural comparison and analysis of protein binding regions

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Anders Gerd

2008-01-01

Full Text Available Abstract Background Detailed information about protein interactions is critical for our understanding of the principles governing protein recognition mechanisms. The structures of many proteins have been experimentally determined in complex with different ligands bound either in the same or different binding regions. Thus, the structural interactome requires the development of tools to classify protein binding regions. A proper classification may provide a general view of the regions that a protein uses to bind others and also facilitate a detailed comparative analysis of the interacting information for specific protein binding regions at atomic level. Such classification might be of potential use for deciphering protein interaction networks, understanding protein function, rational engineering and design. Description Protein binding regions (PBRs might be ideally described as well-defined separated regions that share no interacting residues one another. However, PBRs are often irregular, discontinuous and can share a wide range of interacting residues among them. The criteria to define an individual binding region can be often arbitrary and may differ from other binding regions within a protein family. Therefore, the rational behind protein interface classification should aim to fulfil the requirements of the analysis to be performed. We extract detailed interaction information of protein domains, peptides and interfacial solvent from the SCOWLP database and we classify the PBRs of each domain family. For this purpose, we define a similarity index based on the overlapping of interacting residues mapped in pair-wise structural alignments. We perform our classification with agglomerative hierarchical clustering using the complete-linkage method. Our classification is calculated at different similarity cut-offs to allow flexibility in the analysis of PBRs, feature especially interesting for those protein families with conflictive binding regions

Trans-acting translational regulatory RNA binding proteins.

Science.gov (United States)

Harvey, Robert F; Smith, Tom S; Mulroney, Thomas; Queiroz, Rayner M L; Pizzinga, Mariavittoria; Dezi, Veronica; Villenueva, Eneko; Ramakrishna, Manasa; Lilley, Kathryn S; Willis, Anne E

2018-05-01

The canonical molecular machinery required for global mRNA translation and its control has been well defined, with distinct sets of proteins involved in the processes of translation initiation, elongation and termination. Additionally, noncanonical, trans-acting regulatory RNA-binding proteins (RBPs) are necessary to provide mRNA-specific translation, and these interact with 5' and 3' untranslated regions and coding regions of mRNA to regulate ribosome recruitment and transit. Recently it has also been demonstrated that trans-acting ribosomal proteins direct the translation of specific mRNAs. Importantly, it has been shown that subsets of RBPs often work in concert, forming distinct regulatory complexes upon different cellular perturbation, creating an RBP combinatorial code, which through the translation of specific subsets of mRNAs, dictate cell fate. With the development of new methodologies, a plethora of novel RNA binding proteins have recently been identified, although the function of many of these proteins within mRNA translation is unknown. In this review we will discuss these methodologies and their shortcomings when applied to the study of translation, which need to be addressed to enable a better understanding of trans-acting translational regulatory proteins. Moreover, we discuss the protein domains that are responsible for RNA binding as well as the RNA motifs to which they bind, and the role of trans-acting ribosomal proteins in directing the translation of specific mRNAs. This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes Translation > Translation Regulation Translation > Translation Mechanisms. © 2018 Medical Research Council and University of Cambridge. WIREs RNA published by Wiley Periodicals, Inc.

Nuclear factor ETF specifically stimulates transcription from promoters without a TATA box.

Science.gov (United States)

Kageyama, R; Merlino, G T; Pastan, I

1989-09-15

Transcription factor ETF stimulates the expression of the epidermal growth factor receptor (EGFR) gene which does not have a TATA box in the promoter region. Here, we show that ETF recognizes various GC-rich sequences including stretches of deoxycytidine or deoxyguanosine residues and GC boxes with similar affinities. ETF also binds to TATA boxes but with a lower affinity. ETF stimulated in vitro transcription from several promoters without TATA boxes but had little or no effect on TATA box-containing promoters even though they had strong ETF-binding sites. These inactive ETF-binding sites became functional when placed upstream of the EGFR promoter whose own ETF-binding sites were removed. Furthermore, when a TATA box was introduced into the EGFR promoter, the responsiveness to ETF was abolished. These results indicate that ETF is a specific transcription factor for promoters which do not contain TATA elements.

Characterization of cap binding proteins associated with the nucleus

International Nuclear Information System (INIS)

Patzelt, E.

1986-04-01

Eucaryotic mRNAs a carry 7-methylguanosine triphosphate residue (called cap structure) at their 5' terminus. The cap plays an important role in RNA recognition. Cap binding proteins (CBP) of HeLa cells were identified by photoaffinity labelling using the cap analogue γ-( 32 P)-(4-(benzoyl-phenyl)methylamido)-7-methylguanosine-5'-triphosphate (BP-m 7 GTP). Photoreaction of this cap analogue with HeLa cell initiation factors resulted in specific labelling of two polypeptides of Msub(r) 37000 and 26000. The latter was also labelled in crude initiation factors prepared from reticulocytes and is identical to the cap binding protein CBP I previously identified. These cap binding proteins were also affinity labelled in poliovirus infected cell extracts. Photoaffinity reaction with BP-m 7 GTP of whole HeLa cell homogenate showed three additional polypeptides with Msub(r) 120000, 89000 and 80000. These cap binding proteins were found to be associated with the nucleus and are therefore referred to as nuclear cap binding proteins, i.e. NCBP 1, NCBP 2 and NCBP 3. They were also present in splicing extracts. Photoaffinity labelling in these nuclear extracts was differentially inhibited by various cap analogues and capped mRNAs. Affinity chromatography on immobilized globin mRNA led to a partial separation of the three nuclear cap binding proteins. Chromatography on m 7 GTP-Sepharose resulted in a specific binding of NCBP 3. The different behaviour of the cap binding proteins suggests that they are functionally distinct and that they might be involved in different processes requiring cap recognition. (Author)

Separation of the components of the TBP-H2 MBP-HDBP-H3PO4 mixture

International Nuclear Information System (INIS)

Pires, M.A.F.; Abrao, A.

1981-04-01

Several schemes for the separation of dibutylphosphoric acid (HDBP), monobutylphosphoric acid (H 2 MBP) and orthophosphoric acid (H 3 PO 4 ) as hydrolytic and radiolytic degradation products from tri-n-butylphosphate (TBP) were studied. For the resolution of a HDBP, H 2 MPB and H 3 PO 4 mixture in TBP-diluent, or in TBP-diluent-heavy metal nitrate (U-VI, Th-IV or Zr-IV), techniques such as ion exchange chromatography, ion chromatography and separation onto a chromatographic alumina column were investigated. For the identification, determination and analytical resolution following up for the several systems studied, techniques such as refraction index measurement, electrical conductivity measurement, molecular spectrophotometry and gas chromatography were applied. Special emphasys was given to the separation using alumina column where the HDBP acid was retained and eluted selectively for its separation from TBP-varsol-uranyl nitrate mixtures. This analytical procedure was applied to the samples coming from the Uranium Purification Pilot Plant in operation at the Centro de Engenharia Quimica (IPEN). (Author) [pt

Solution structure of telomere binding domain of AtTRB2 derived from Arabidopsis thaliana

International Nuclear Information System (INIS)

Yun, Ji-Hye; Lee, Won Kyung; Kim, Heeyoun; Kim, Eunhee; Cheong, Chaejoon; Cho, Myeon Haeng; Lee, Weontae

2014-01-01

Highlights: • We have determined solution structure of Myb domain of AtTRB2. • The Myb domain of AtTRB2 is located in the N-terminal region. • The Myb domain of AtTRB2 binds to plant telomeric DNA without fourth helix. • Helix 2 and 3 of the Myb domain of AtTRB2 are involved in DNA recognition. • AtTRB2 is a novel protein distinguished from other known plant TBP. - Abstract: Telomere homeostasis is regulated by telomere-associated proteins, and the Myb domain is well conserved for telomere binding. AtTRB2 is a member of the SMH (Single-Myb-Histone)-like family in Arabidopsis thaliana, having an N-terminal Myb domain, which is responsible for DNA binding. The Myb domain of AtTRB2 contains three α-helices and loops for DNA binding, which is unusual given that other plant telomere-binding proteins have an additional fourth helix that is essential for DNA binding. To understand the structural role for telomeric DNA binding of AtTRB2, we determined the solution structure of the Myb domain of AtTRB2 (AtTRB2 1–64 ) using nuclear magnetic resonance (NMR) spectroscopy. In addition, the inter-molecular interaction between AtTRB2 1–64 and telomeric DNA has been characterized by the electrophoretic mobility shift assay (EMSA) and NMR titration analyses for both plant (TTTAGGG)n and human (TTAGGG)n telomere sequences. Data revealed that Trp28, Arg29, and Val47 residues located in Helix 2 and Helix 3 are crucial for DNA binding, which are well conserved among other plant telomere binding proteins. We concluded that although AtTRB2 is devoid of the additional fourth helix in the Myb-extension domain, it is able to bind to plant telomeric repeat sequences as well as human telomeric repeat sequences

Purification of degraded TBP solvent using macroreticular anion exchange resin

International Nuclear Information System (INIS)

Kartha, P.K.S.; Kutty, P.V.E.; Janaradanan, C.; Ramanujam, A.; Dhumwad, R.K.

1989-01-01

Tri-n-butyl phosphate (TBP) diluted with a suitable diluent is commonly used for solvent extraction in Purex process for the recovery of uranium and plutonium from irradiated nuclear fuels. This solvent gets degraded due to various factors, the main degradation product being dibutyl phosphoric acid (HDBP). A solvent cleanup step is generally incorporated in the process for removing the degradation products from the used solvent. A liquid-liquid cleanup system using sodium carbonate or sodium hydroxide solution is routinely used. Considering certain advantages, like the possibility of loading the resin almost to saturation capacity and the subsequent disposal of the spent resin by incineration and the feasibility of adopting it to the process, a liquid-solid system has been tried as an alternate method, employing various available macroreticular anion exchange resins in OH - form for the sorption of HDBP from TBP. After standardizing the various conditions for the satisfactory removal of HDBP from TBP using synthetic mixtures, resins were tested with process solvent in batch contacts. The parameters studied were (1) capacity of different resins for HDBP sorption (2) influence of acidity, uranium and HDBP on the sorption behaviour of the latter (3) removal of fission products from the solvent by the resin and (4) regeneration and recycling of the resin. (author). 2 figs., 13 tabs., 17 refs

Cell-cycle-specific interaction of nuclear DNA-binding proteins with a CCAAT sequence from the human thymidine kinase gene

International Nuclear Information System (INIS)

Knight, G.B.; Gudas, J.M.; Pardee, A.B.

1987-01-01

Induction of thymidine kinase parallels the onset of DNA synthesis. To investigate the transcriptional regulation of the thymidine kinase gene, the authors have examined whether specific nuclear factors interact in a cell-cycle-dependent manner with sequences upstream of this gene. Two inverted CCAAT boxes near the transcriptional initiation sites were observed to form complexes with nuclear DNA-binding proteins. The nature of the complexes changes dramatically as the cells approach DNA synthesis and correlates well with the previously reported transcriptional increase of the thymidine kinase gene

Probing protein phosphatase substrate binding

DEFF Research Database (Denmark)

Højlys-Larsen, Kim B.; Sørensen, Kasper Kildegaard; Jensen, Knud Jørgen

2012-01-01

Proteomics and high throughput analysis for systems biology can benefit significantly from solid-phase chemical tools for affinity pull-down of proteins from complex mixtures. Here we report the application of solid-phase synthesis of phosphopeptides for pull-down and analysis of the affinity...... profile of the integrin-linked kinase associated phosphatase (ILKAP), a member of the protein phosphatase 2C (PP2C) family. Phosphatases can potentially dephosphorylate these phosphopeptide substrates but, interestingly, performing the binding studies at 4 °C allowed efficient binding to phosphopeptides......, without the need for phosphopeptide mimics or phosphatase inhibitors. As no proven ILKAP substrates were available, we selected phosphopeptide substrates among known PP2Cδ substrates including the protein kinases: p38, ATM, Chk1, Chk2 and RSK2 and synthesized directly on PEGA solid supports through a BAL...

Thermodynamics of uranium and nitric acid extraction from aqueous solution of TBP/diluent

International Nuclear Information System (INIS)

Souza Freitas, R.F. de.

1982-06-01

A thermodynamically consistent procedure for predicting distribution equilibria for uranyl nitrate and nitric acid between an aqueous solution and 30 vol % tributyl phosphate (TBP) in a hydrocarbon diluent is studied. Experimental work is developed in order to obtain equilibrium data for the system uranyl nitrate, nitric acid, water and 30 vol % TBP in n-dodecane, at 25 0 C and 40 0 C. The theoretical equilibrium data, obtained with the aid of a computer, are compared with the experimental results. (Author) [pt

Further biochemical characterization of Mycobacterium leprae laminin-binding proteins

Directory of Open Access Journals (Sweden)

M.A.M. Marques

2001-04-01

Full Text Available It has been demonstrated that the alpha2 chain of laminin-2 present on the surface of Schwann cells is involved in the process of attachment of Mycobacterium leprae to these cells. Searching for M. leprae laminin-binding molecules, in a previous study we isolated and characterized the cationic proteins histone-like protein (Hlp and ribosomal proteins S4 and S5 as potential adhesins involved in M. leprae-Schwann cell interaction. Hlp was shown to bind alpha2-laminins and to greatly enhance the attachment of mycobacteria to ST88-14 Schwann cells. In the present study, we investigated the laminin-binding capacity of the ribosomal proteins S4 and S5. The genes coding for these proteins were PCR amplified and their recombinant products were shown to bind alpha2-laminins in overlay assays. However, when tested in ELISA-based assays and in adhesion assays with ST88-14 cells, in contrast to Hlp, S4 and S5 failed to bind laminin and act as adhesins. The laminin-binding property and adhesin capacity of two basic host-derived proteins were also tested, and only histones, but not cytochrome c, were able to increase bacterial attachment to ST88-14 cells. Our data suggest that the alanine/lysine-rich sequences shared by Hlp and eukaryotic H1 histones might be involved in the binding of these cationic proteins to laminin.

An overview of the prediction of protein DNA-binding sites.

Science.gov (United States)

Si, Jingna; Zhao, Rui; Wu, Rongling

2015-03-06

Interactions between proteins and DNA play an important role in many essential biological processes such as DNA replication, transcription, splicing, and repair. The identification of amino acid residues involved in DNA-binding sites is critical for understanding the mechanism of these biological activities. In the last decade, numerous computational approaches have been developed to predict protein DNA-binding sites based on protein sequence and/or structural information, which play an important role in complementing experimental strategies. At this time, approaches can be divided into three categories: sequence-based DNA-binding site prediction, structure-based DNA-binding site prediction, and homology modeling and threading. In this article, we review existing research on computational methods to predict protein DNA-binding sites, which includes data sets, various residue sequence/structural features, machine learning methods for comparison and selection, evaluation methods, performance comparison of different tools, and future directions in protein DNA-binding site prediction. In particular, we detail the meta-analysis of protein DNA-binding sites. We also propose specific implications that are likely to result in novel prediction methods, increased performance, or practical applications.

Measurement of tributyl phosphate (TBP) in groundwater at a legacy radioactive waste site and its possible role in contaminant mobilisation.

Science.gov (United States)

Rowling, Brett; Kinsela, Andrew S; Comarmond, M Josick; Hughes, Catherine E; Harrison, Jennifer J; Johansen, Mathew P; Payne, Timothy E

2017-11-01

At many legacy radioactive waste sites, organic compounds have been co-disposed, which may be a factor in mobilisation of radionuclides at these sites. Tri-butyl phosphate (TBP) is a component of waste streams from the nuclear fuel cycle, where it has been used in separating actinides during processing of nuclear fuels. Analyses of ground waters from the Little Forest Legacy Site (LFLS) in eastern Australia were undertaken using solid-phase extraction (SPE) followed by gas chromatographic mass spectrometry (GCMS). The results indicate the presence of TBP several decades after waste disposal, with TBP only being detected in the immediate vicinity of the main disposal area. TBP is generally considered to degrade in the environment relatively rapidly. Therefore, it is likely that its presence is due to relatively recent releases of TBP, possibly stemming from leakage due to container degradation. The ongoing presence and solubility of TBP has the potential to provide a mechanism for nuclide mobilisation, with implications for long term management of LFLS and similar legacy waste sites. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

Enriched uranium processing with 7-1/2% TBP

International Nuclear Information System (INIS)

Orth, D.A.; Martin, W.H.; Pickett, C.E.

1983-01-01

The 7-1/2% TBP flowsheet gives adequate recovery of uranium and neptunium or plutonium, with reduced waste volume as compared to the prior aluminum-salted 3-1/2% TBP flowsheet. Decontamination from fission products is sensitive to numerous variables, including aluminum nitrate concentration in the feed, impeller speeds, and prior treatment of the fuel solution in head end operations. The impeller speed in the 1A bank also influences uranium losses as well as the fission product decontamination. The magnitudes of these effects suggest that stage efficiency is poor with this flowsheet in this mixer settler unit. The existing continuous solvent washers give evidence of low washing efficiency that limits permissible feed activity and that may be related to low contact time between the solvent and the carbonate wash solution. The most general conclusion is that satisfactory operation can be obtained with all projected domestic and foreign fuels under consideration for processing, by suitable adjustment of operating conditions. Also, possible flowsheet and equipment changes are known that could improve operations with these fuels further. 7 references

Resolution of the mixture TBP-H2 MPB-HDB-PH3PO4

International Nuclear Information System (INIS)

Pires, M.A.F.; Abrao, A.

1980-01-01

Tributylphosphate (TBP) is presently the most used extractant for uranium, thorium and zirconium technologies, both for the purification of these elements from their concentrates and for the recovery of heavy metals in the reprocessing of irradiated fuels. Nevertheless, in the presence of phosphoric acid, some degradation products are formed, such as: di-n-butylphosphoric acid (HDBP), monobutylphosphoric ancd (H 2 MBP) and ortophosphoric acid. The accumulation of these degradation products in the organic phase of the liquid-liquid extraction processes is highly undesirable. In order to avoid this problem, the separation of the mixture HDBP, H 2 MBP and H 3 PO 4 in TBP-dilutent or TBP-diluent-uranyl, thorium of zirconyl nitrate was then studied. Several separation techniques were investigated: adsorption using a cationic resin saturated with U(VI), chromatogtraphy in macroporous polymers, chromatography in alumina columns. Refractive index and condutimetric measurements were carried out to follow up the separation processes. The best results were obtained in an alumina column. (C.L.B.) [pt

The development of 126Sn separation procedure by means of TBP resin

International Nuclear Information System (INIS)

Andris, Boris; Bena, Jozef

2016-01-01

Separation possibilities of 126 Sn with a new extraction-chromatographic material TBP Resin were studied. Suitable conditions for tin separation were determined in hydrochloric acid medium. 126 Sn was concentrated on TBP resin from 6 mol L -1 HCl and was eluted with 0.1 mol L -1 HCl. A purification step to remove 137 Cs with AMP-PAN column was necessary to obtain sufficiently purified samples which were directly measured with gamma spectrometry for 126 Sn activity. Separation of 126 Sn from a raw sludge sample was done according to proposed procedure, 126 Sn was detected and its activity was determined. (author)

Dominant Alcohol-Protein Interaction via Hydration-Enabled Enthalpy-Driven Binding Mechanism

Science.gov (United States)

Chong, Yuan; Kleinhammes, Alfred; Tang, Pei; Xu, Yan; Wu, Yue

2015-01-01

Water plays an important role in weak associations of small drug molecules with proteins. Intense focus has been on binding-induced structural changes in the water network surrounding protein binding sites, especially their contributions to binding thermodynamics. However, water is also tightly coupled to protein conformations and dynamics, and so far little is known about the influence of water-protein interactions on ligand binding. Alcohols are a type of low-affinity drugs, and it remains unclear how water affects alcohol-protein interactions. Here, we present alcohol adsorption isotherms under controlled protein hydration using in-situ NMR detection. As functions of hydration level, Gibbs free energy, enthalpy, and entropy of binding were determined from the temperature dependence of isotherms. Two types of alcohol binding were found. The dominant type is low-affinity nonspecific binding, which is strongly dependent on temperature and the level of hydration. At low hydration levels, this nonspecific binding only occurs above a threshold of alcohol vapor pressure. An increased hydration level reduces this threshold, with it finally disappearing at a hydration level of h~0.2 (g water/g protein), gradually shifting alcohol binding from an entropy-driven to an enthalpy-driven process. Water at charged and polar groups on the protein surface was found to be particularly important in enabling this binding. Although further increase in hydration has smaller effects on the changes of binding enthalpy and entropy, it results in significant negative change in Gibbs free energy due to unmatched enthalpy-entropy compensation. These results show the crucial role of water-protein interplay in alcohol binding. PMID:25856773

Synergistic inhibition of the intrinsic factor X activation by protein S and C4b-binding protein

NARCIS (Netherlands)

Koppelman, S.J.

1995-01-01

The complement protein C4b-binding protein plays an important role in the regulation of the protein C anticoagulant pathway. C4b-binding protein can bind to protein S, thereby inhibiting the cofactor activity of protein S for activated protein C. In this report, we describe a new role for

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

Science.gov (United States)

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

2013-10-09

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

Specificity of DNA-binding by the FAX-1 and NHR-67 nuclear receptors of Caenorhabditis elegans is partially mediated via a subclass-specific P-box residue

Directory of Open Access Journals (Sweden)

Smith Eric L

2008-01-01

Full Text Available Abstract Background The nuclear receptors of the NR2E class play important roles in pattern formation and nervous system development. Based on a phylogenetic analysis of DNA-binding domains, we define two conserved groups of orthologous NR2E genes: the NR2E1 subclass, which includes C. elegans nhr-67, Drosophila tailless and dissatisfaction, and vertebrate Tlx (NR2E2, NR2E4, NR2E1, and the NR2E3 subclass, which includes C. elegans fax-1 and vertebrate PNR (NR2E5, NR2E3. PNR and Tll nuclear receptors have been shown to bind the hexamer half-site AAGTCA, instead of the hexamer AGGTCA recognized by most other nuclear receptors, suggesting unique DNA-binding properties for NR2E class members. Results We show that NR2E3 subclass member FAX-1, unlike NHR-67 and other NR2E1 subclass members, binds to hexamer half-sites with relaxed specificity: it will bind hexamers with the sequence ANGTCA, although it prefers a purine to a pyrimidine at the second position. We use site-directed mutagenesis to demonstrate that the difference between FAX-1 and NHR-67 binding preference is partially mediated by a conserved subclass-specific asparagine or aspartate residue at position 19 of the DNA-binding domain. This amino acid position is part of the "P box" that plays a critical role in defining binding site specificity and has been shown to make hydrogen-bond contacts to the second position of the hexamer in co-crystal structures for other nuclear receptors. The relaxed specificity allows FAX-1 to bind a much larger repertoire of half-sites than NHR-67. While NR2E1 class proteins bind both monomeric and dimeric sites, the NR2E3 class proteins bind only dimeric sites. The presence of a single strong site adjacent to a very weak site allows dimeric FAX-1 binding, further increasing the number of dimeric binding sites to which FAX-1 may bind in vivo. Conclusion These findings identify subclass-specific DNA-binding specificities and dimerization properties for the NR2E1

New insights in third phase formation in the U(VI)-HNO3, TBP-alkane system

International Nuclear Information System (INIS)

Jensen, M. P.; Chiarizia, R.; Ferraro, J. R.; Borkowski, M.; Nash, K. L.; Thiyagarajan, P.; Littrell, K. C.

2001-01-01

In this work, the system U(VI)-HNO 3 -tributylphosphate (TBP)-n-dodecane has been revisited with the objective of gaining coordination chemistry and structural information on the species that are formed in the organic phase before and after third phase formation. Chemical analyses, spectroscopic and EXAFS data indicate that U(VI) is extracted as the UO 2 (NO 3 ) 2 · 2TBP adduct, while the third phase species has the composition UO 2 (NO 3 ) 2 · 2TBP · HNO 3 . Small-angle neutron scattering (SANS) data reveal the presence in the organic phase, both before and after phase splitting, of ellipsoidal aggregates whose formation seems to depend more on the extraction of HNO 3 than that of U(VI)

Promotion of minTBP-1-PRGDN on the attachment, proliferation and collagen I synthesis of human keratocyte on titanium

Directory of Open Access Journals (Sweden)

Xin-Yu Li

2014-02-01

Full Text Available AIM:To investigate the influence of minTBP-1-PRGDN on the attachment, proliferation and collagen I synthesis of human keratocyte on titanium (Ti surface.METHODS:The chimeric peptide RKLPDAPRGDN (minTBP-1-PRGDN was synthesized by connecting RKLPDA (minTBP-1 to the N-terminal of PRGDN , the influence of minTBP-1-PRGDN on the attachment, proliferation and collagen I synthesis of human keratocyte on Ti surface were tested using PRGDN and minTBP-1as controls. The keratocytes attached to the surface of Ti were either stained with FITC-labeled phalloidin and viewed with fluorescence microscope or quantified with alamar Blue method. The proliferation of keratocytes on Ti were quantified with 3-(4,5-dim- ethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide up-taking methods. The secretion of type I collagen were determined using an ELISA kit.RESULTS:The results showed that minTBP-1-PRGDN at a concentration of 100ng/mL was the most potent peptide to enhance the attachment of human keratocytes to the surface of Ti (1.40±0.03 folds, P=0.003, to promote the proliferation (1.26±0.05 folds, P=0.014 and the synthesis of type I collagen (1.530±0.128, P=0.008. MinTBP-1 at the same concentration could only promote the attachment (1.13±0.04 folds, P=0.020 and proliferation(1.15±0.06 folds, P=0.021, while PRGDN had no significant influence (P＞0.05.CONCLUSION:Our data shows that the novel chimeric peptide minTBP-1-PRGDN could promote the attachment, proliferation and type I collagen synthesis of human keratocytes on the surface of Ti.

Computational analysis of protein-protein interfaces involving an alpha helix: insights for terphenyl-like molecules binding.

Science.gov (United States)

Isvoran, Adriana; Craciun, Dana; Martiny, Virginie; Sperandio, Olivier; Miteva, Maria A

2013-06-14

Protein-Protein Interactions (PPIs) are key for many cellular processes. The characterization of PPI interfaces and the prediction of putative ligand binding sites and hot spot residues are essential to design efficient small-molecule modulators of PPI. Terphenyl and its derivatives are small organic molecules known to mimic one face of protein-binding alpha-helical peptides. In this work we focus on several PPIs mediated by alpha-helical peptides. We performed computational sequence- and structure-based analyses in order to evaluate several key physicochemical and surface properties of proteins known to interact with alpha-helical peptides and/or terphenyl and its derivatives. Sequence-based analysis revealed low sequence identity between some of the analyzed proteins binding alpha-helical peptides. Structure-based analysis was performed to calculate the volume, the fractal dimension roughness and the hydrophobicity of the binding regions. Besides the overall hydrophobic character of the binding pockets, some specificities were detected. We showed that the hydrophobicity is not uniformly distributed in different alpha-helix binding pockets that can help to identify key hydrophobic hot spots. The presence of hydrophobic cavities at the protein surface with a more complex shape than the entire protein surface seems to be an important property related to the ability of proteins to bind alpha-helical peptides and low molecular weight mimetics. Characterization of similarities and specificities of PPI binding sites can be helpful for further development of small molecules targeting alpha-helix binding proteins.

Ion Binding Energies Determining Functional Transport of ClC Proteins

Science.gov (United States)

Yu, Tao; Guo, Xu; Zou, Xian-Wu; Sang, Jian-Ping

2014-06-01

The ClC-type proteins, a large family of chloride transport proteins ubiquitously expressed in biological organisms, have been extensively studied for decades. Biological function of ClC proteins can be reflected by analyzing the binding situation of Cl- ions. We investigate ion binding properties of ClC-ec1 protein with the atomic molecular dynamics simulation approach. The calculated electrostatic binding energy results indicate that Cl- at the central binding site Scen has more binding stability than the internal binding site Sint. Quantitative comparison between the latest experimental heat release data isothermal titration calorimetry (ITC) and our calculated results demonstrates that chloride ions prefer to bind at Scen than Sint in the wild-type ClC-ec1 structure and prefer to bind at Sext and Scen than Sint in mutant E148A/E148Q structures. Even though the chloride ions make less contribution to heat release when binding to Sint and are relatively unstable in the Cl- pathway, they are still part contributors for the Cl- functional transport. This work provides a guide rule to estimate the importance of Cl- at the binding sites and how chloride ions have influences on the function of ClC proteins.

Rapid identification of DNA-binding proteins by mass spectrometry

DEFF Research Database (Denmark)

Nordhoff, E.; Korgsdam, A.-M.; Jørgensen, H.F.

1999-01-01

We report a protocol for the rapid identification of DNA-binding proteins. Immobilized DNA probes harboring a specific sequence motif are incubated with cell or nuclear extract. Proteins are analyzed directly off the solid support by matrix-assisted laser desorption/ionization time-of-flight mass...... was validated by the identification of known prokaryotic and eukaryotic DNA-binding proteins, and its use provided evidence that poly(ADP-ribose) polymerase exhibits DNA sequence-specific binding to DNA....

Protein Binding Capacity of Different Forages Tannin

Science.gov (United States)

Yusiati, L. M.; Kurniawati, A.; Hanim, C.; Anas, M. A.

2018-02-01

Eight forages of tannin sources(Leucaena leucocephala, Arachis hypogaea, Mimosa pudica, Morus alba L, Swietenia mahagoni, Manihot esculenta, Gliricidia sepium, and Bauhinia purpurea)were evaluated their tannin content and protein binding capacity. The protein binding capacity of tannin were determined using precipitation of bovine serum albumin (BSA). Swietenia mahagonihas higest total tannin level and condensed tannin (CT) compared with other forages (P<0.01). The Leucaena leucocephala has highest hydrolysable tannin (HT) level (P<0.01). The total and condensed tannin content of Swietenia mahagoni were 11.928±0.04 mg/100 mg and 9.241±0.02mg/100mg dry matter (DM) of leaves. The hydrolysable tannin content of Leucaena leucocephala was 5.338±0.03 mg/100 mg DM of leaves. Binding capacity was highest in Swietenia mahagoni and Leucaena leucocephala compared to the other forages (P<0.01). The optimum binding of BSA to tannin in Leucaena leucocephala and Swietenia mahagoniwere1.181±0.44 and 1.217±0.60mg/mg dry matter of leaves. The present study reports that Swietenia mahagoni has highest of tannin content and Leucaena leucocephala and Swietenia mahagoni capacity of protein binding.

Study of Soil Decontamination Method Using Supercritical Carbon Dioxide and TBP

International Nuclear Information System (INIS)

Park, Jihye; Park, Kwangheon; Jung, Wonyoung

2014-01-01

The result of this study means that we have a possible new method for cheap and less wasteful nuclear waste decontamination. When severe accidents such as the incident at the Fukushima nuclear site occur, the soil near the power plant is contaminated with fission products or the activation metal structure of the power plant. The soil pollution form depends on the environment and soil characteristics of the contaminated areas. Thus, a- single-decontamination method is not effective for site cleanup. In addition, some soil decontamination methods are expensive and large amounts of secondary waste are generated. Therefore, we need new soil decontamination methods. In this study, instead of using a conventional solvent method that generates secondary waste, supercritical carbon dioxide was used to remove metal ions from the soil. Supercritical carbon dioxide is known for good permeation characteristics. We expect that we will reduce the cost of soil pollution management. Supercritical carbon dioxide can decontaminate soil easily, as it has the ability to penetrate even narrow gaps with very good moisture permeability. We used TBP, which is a known for extractant of actinium metal. TBP is usually used for uranium and strontium extraction. Using TBP-HNO 3 complex and supercritical carbon dioxide, we did extraction experiments for several heavy metals in contaminated soil

Study of Soil Decontamination Method Using Supercritical Carbon Dioxide and TBP

Energy Technology Data Exchange (ETDEWEB)

Park, Jihye; Park, Kwangheon; Jung, Wonyoung [Kyunghee Univ., Yongin (Korea, Republic of)

2014-05-15

The result of this study means that we have a possible new method for cheap and less wasteful nuclear waste decontamination. When severe accidents such as the incident at the Fukushima nuclear site occur, the soil near the power plant is contaminated with fission products or the activation metal structure of the power plant. The soil pollution form depends on the environment and soil characteristics of the contaminated areas. Thus, a- single-decontamination method is not effective for site cleanup. In addition, some soil decontamination methods are expensive and large amounts of secondary waste are generated. Therefore, we need new soil decontamination methods. In this study, instead of using a conventional solvent method that generates secondary waste, supercritical carbon dioxide was used to remove metal ions from the soil. Supercritical carbon dioxide is known for good permeation characteristics. We expect that we will reduce the cost of soil pollution management. Supercritical carbon dioxide can decontaminate soil easily, as it has the ability to penetrate even narrow gaps with very good moisture permeability. We used TBP, which is a known for extractant of actinium metal. TBP is usually used for uranium and strontium extraction. Using TBP-HNO{sub 3} complex and supercritical carbon dioxide, we did extraction experiments for several heavy metals in contaminated soil.

DNABP: Identification of DNA-Binding Proteins Based on Feature Selection Using a Random Forest and Predicting Binding Residues.

Science.gov (United States)

Ma, Xin; Guo, Jing; Sun, Xiao

2016-01-01

DNA-binding proteins are fundamentally important in cellular processes. Several computational-based methods have been developed to improve the prediction of DNA-binding proteins in previous years. However, insufficient work has been done on the prediction of DNA-binding proteins from protein sequence information. In this paper, a novel predictor, DNABP (DNA-binding proteins), was designed to predict DNA-binding proteins using the random forest (RF) classifier with a hybrid feature. The hybrid feature contains two types of novel sequence features, which reflect information about the conservation of physicochemical properties of the amino acids, and the binding propensity of DNA-binding residues and non-binding propensities of non-binding residues. The comparisons with each feature demonstrated that these two novel features contributed most to the improvement in predictive ability. Furthermore, to improve the prediction performance of the DNABP model, feature selection using the minimum redundancy maximum relevance (mRMR) method combined with incremental feature selection (IFS) was carried out during the model construction. The results showed that the DNABP model could achieve 86.90% accuracy, 83.76% sensitivity, 90.03% specificity and a Matthews correlation coefficient of 0.727. High prediction accuracy and performance comparisons with previous research suggested that DNABP could be a useful approach to identify DNA-binding proteins from sequence information. The DNABP web server system is freely available at http://www.cbi.seu.edu.cn/DNABP/.

IGF binding proteins.

Science.gov (United States)

Bach, Leon A

2017-12-18

Insulin-like growth factor binding proteins (IGFBPs) 1-6 bind IGFs but not insulin with high affinity. They were initially identified as serum carriers and passive inhibitors of IGF actions. However, subsequent studies showed that, although IGFBPs inhibit IGF actions in many circumstances, they may also potentiate these actions. IGFBPs are widely expressed in most tissues, and they are flexible endocrine and autocrine/paracrine regulators of IGF activity, which is essential for this important physiological system. More recently, individual IGFBPs have been shown to have IGF-independent actions. Mechanisms underlying these actions include (i) interaction with non-IGF proteins in compartments including the extracellular space and matrix, the cell surface and intracellularly; (ii) interaction with and modulation of other growth factor pathways including EGF, TGF- and VEGF; and (iii) direct or indirect transcriptional effects following nuclear entry of IGFBPs. Through these IGF-dependent and IGF-independent actions, IGFBPs modulate essential cellular processes including proliferation, survival, migration, senescence, autophagy and angiogenesis. They have been implicated in a range of disorders including malignant, metabolic, neurological and immune diseases. A more complete understanding of their cellular roles may lead to the development of novel IGFBP-based therapeutic opportunities.

Ligand Binding Domain Protein in Tetracycline-Inducible Expression

African Journals Online (AJOL)

Purpose: To investigate tetracycline-inducible expression system for producing clinically usable, highquality liver X receptor ligand-binding domain recombinant protein. Methods: In this study, we have expressed and purified the recombinant liver X receptor β-ligand binding domain proteins in E. coli using a tetracycline ...

B1-induced caspase-independent apoptosis in MCF-7 cells is mediated by down-regulation of Bcl-2 via p53 binding to P2 promoter TATA box

International Nuclear Information System (INIS)

Liang Xin; Xu Ke; Xu Yufang; Liu Jianwen; Qian Xuhong

2011-01-01

The Bcl-2 family contains a panel of proteins which are conserved regulators of apoptosis in mammalian cells, like the anti-apoptotic protein Bcl-2. According to its significant role in altering susceptibility to apoptosis, the deciphering of the mechanism of Bcl-2 expression modulation may be crucial for identifying therapeutics strategies for cancer. Treatment with naphthalimide-based DNA intercalators, including M2-A and R16, generally leads to a decrease in Bcl-2 intracellular amounts. Whereas the interest for these chemotherapeutics is accompanied by advances in the fundamental understanding of their anticancer properties, the molecular mechanism underlying changes in Bcl-2 expression remains poorly understood. We report here that p53 contributes to Bcl-2 down-regulation induced by B1, a novel naphthalimide-based DNA intercalating agent. Indeed, the decrease in Bcl-2 protein levels observed during B1-induced apoptosis was correlated to the decrease in mRNA levels, as a result of the inhibition of Bcl-2 transcription and promoter activity. In this context, we evaluated p53 contribution in the Bcl-2 transcriptional down-regulation. We found a significant increase of p53 binding to P 2 promoter TATA box in MCF7 cells by chromatin immunoprecipitation. These data suggest that B1-induced caspase-independent apoptosis in MCF-7 cells is associated with the activation of p53 and the down-regulation of Bcl-2. Our study strengthens the links between p53 and Bcl-2 at a transcriptional level, upon naphthalimide-based DNA intercalator treatment. - Research highlights: → B1 induced apoptosis in MCF-7 cells, following a transcriptional decrease in Bcl-2. → B1 treatment triggered p53 activation and leads to a p53-dependent down-regulation of Bcl-2. → B1 induced significant increase of p53 binding to Bcl-2 P 2 promoter TATA box.

An Overview of the Prediction of Protein DNA-Binding Sites

Directory of Open Access Journals (Sweden)

Jingna Si

2015-03-01

Full Text Available Interactions between proteins and DNA play an important role in many essential biological processes such as DNA replication, transcription, splicing, and repair. The identification of amino acid residues involved in DNA-binding sites is critical for understanding the mechanism of these biological activities. In the last decade, numerous computational approaches have been developed to predict protein DNA-binding sites based on protein sequence and/or structural information, which play an important role in complementing experimental strategies. At this time, approaches can be divided into three categories: sequence-based DNA-binding site prediction, structure-based DNA-binding site prediction, and homology modeling and threading. In this article, we review existing research on computational methods to predict protein DNA-binding sites, which includes data sets, various residue sequence/structural features, machine learning methods for comparison and selection, evaluation methods, performance comparison of different tools, and future directions in protein DNA-binding site prediction. In particular, we detail the meta-analysis of protein DNA-binding sites. We also propose specific implications that are likely to result in novel prediction methods, increased performance, or practical applications.

STRUCTURAL FEATURES OF PLANT CHITINASES AND CHITIN-BINDING PROTEINS

NARCIS (Netherlands)

BEINTEMA, JJ

1994-01-01

Structural features of plant chitinases and chitin-binding proteins are discussed. Many of these proteins consist of multiple domains,of which the chitin-binding hevein domain is a predominant one. X-ray and NMR structures of representatives of the major classes of these proteins are available now,

A tool for calculating binding-site residues on proteins from PDB structures

Directory of Open Access Journals (Sweden)

Hu Jing

2009-08-01

Full Text Available Abstract Background In the research on protein functional sites, researchers often need to identify binding-site residues on a protein. A commonly used strategy is to find a complex structure from the Protein Data Bank (PDB that consists of the protein of interest and its interacting partner(s and calculate binding-site residues based on the complex structure. However, since a protein may participate in multiple interactions, the binding-site residues calculated based on one complex structure usually do not reveal all binding sites on a protein. Thus, this requires researchers to find all PDB complexes that contain the protein of interest and combine the binding-site information gleaned from them. This process is very time-consuming. Especially, combing binding-site information obtained from different PDB structures requires tedious work to align protein sequences. The process becomes overwhelmingly difficult when researchers have a large set of proteins to analyze, which is usually the case in practice. Results In this study, we have developed a tool for calculating binding-site residues on proteins, TCBRP http://yanbioinformatics.cs.usu.edu:8080/ppbindingsubmit. For an input protein, TCBRP can quickly find all binding-site residues on the protein by automatically combining the information obtained from all PDB structures that consist of the protein of interest. Additionally, TCBRP presents the binding-site residues in different categories according to the interaction type. TCBRP also allows researchers to set the definition of binding-site residues. Conclusion The developed tool is very useful for the research on protein binding site analysis and prediction.

Human Mediator Enhances Activator-Facilitated Recruitment of RNA Polymerase II and Promoter Recognition by TATA-Binding Protein (TBP) Independently of TBP-Associated Factors

OpenAIRE

Wu, Shwu-Yuan; Zhou, Tianyuan; Chiang, Cheng-Ming

2003-01-01

Mediator is a general cofactor implicated in the functions of many transcriptional activators. Although Mediator with different protein compositions has been isolated, it remains unclear how Mediator facilitates activator-dependent transcription, independent of its general stimulation of basal transcription. To define the mechanisms of Mediator function, we isolated two forms of human Mediator complexes (Mediator-P.5 and Mediator-P.85) and demonstrated that Mediator-P.5 clearly functions by e...

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

Science.gov (United States)

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

2017-06-01

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

Plant RNA binding proteins for control of RNA virus infection

Directory of Open Access Journals (Sweden)

Sung Un eHuh

2013-12-01

Full Text Available Plant RNA viruses have effective strategies to infect host plants through either direct or indirect interactions with various host proteins, thus suppressing the host immune system. When plant RNA viruses enter host cells exposed RNAs of viruses are recognized by the host immune system through processes such as siRNA-dependent silencing. Interestingly, some host RNA binding proteins have been involved in the inhibition of RNA virus replication, movement, and translation through RNA-specific binding. Host plants intensively use RNA binding proteins for defense against viral infections in nature. In this mini review, we will summarize the function of some host RNA binding proteins which act in a sequence-specific binding manner to the infecting virus RNA. It is important to understand how plants effectively suppresses RNA virus infections via RNA binding proteins, and this defense system can be potentially developed as a synthetic virus defense strategy for use in crop engineering.

Fragile X mental retardation protein: A paradigm for translational control by RNA-binding proteins.

Science.gov (United States)

Chen, Eileen; Joseph, Simpson

2015-07-01

Translational control is a common mechanism used to regulate gene expression and occur in bacteria to mammals. Typically in translational control, an RNA-binding protein binds to a unique sequence in the mRNA to regulate protein synthesis by the ribosomes. Alternatively, a protein may bind to or modify a translation factor to globally regulate protein synthesis by the cell. Here, we review translational control by the fragile X mental retardation protein (FMRP), the absence of which causes the neurological disease, fragile X syndrome (FXS). Copyright © 2015 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.

Human HMG box transcription factor HBP1: a role in hCD2 LCR function.

Science.gov (United States)

Zhuma, T; Tyrrell, R; Sekkali, B; Skavdis, G; Saveliev, A; Tolaini, M; Roderick, K; Norton, T; Smerdon, S; Sedgwick, S; Festenstein, R; Kioussis, D

1999-01-01

The locus control region (LCR) of the human CD2 gene (hCD2) confers T cell-specific, copy-dependent and position-independent gene expression in transgenic mice. This LCR consists of a strong T cell-specific enhancer and an element without enhancer activity (designated HSS3), which is required for prevention of position effect variegation (PEV) in transgenic mice. Here, we identified the HMG box containing protein-1 (HBP1) as a factor binding to HSS3 of the hCD2 LCR. Within the LCR, HBP1 binds to a novel TTCATTCATTCA sequence that is higher in affinity than other recently reported HBP1-binding sites. Mice transgenic for a hCD2 LCR construct carrying a deletion of the HBP1-binding sequences show a propensity for PEV if the transgene integrates in a heterochromatic region of the chromosome such as the centromere or telomere. We propose that HBP1 plays an important role in chromatin opening and remodelling activities by binding to and bending the DNA, thus allowing DNA-protein and/or protein-protein interactions, which increase the probability of establishing an active locus. PMID:10562551

Detection and properties of A-factor-binding protein from Streptomyces griseus

International Nuclear Information System (INIS)

Miyake, K.; Horinouchi, S.; Yoshida, M.; Chiba, N.; Mori, K.; Nogawa, N.; Morikawa, N.; Beppu, T.

1989-01-01

The optically active form of tritium-labeled A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone), a pleiotropic autoregulator responsible for streptomycin production, streptomycin resistance, and sporulation in Streptomyces griseus, was chemically synthesized. By using the radioactive A-factor, a binding protein for A-factor was detected in the cytoplasmic fraction of this organism. The binding protein had an apparent molecular weight of approximately 26,000, as determined by gel filtration. Scatchard analysis suggested that A-factor bound the protein in the molar ratio of 1:1 with a binding constant, Kd, of 0.7 nM. The number of the binding protein was roughly estimated to be 37 per genome. The inducing material virginiae butanolide C (VB-C), which has a structure very similar to that of A-factor and is essential for virginiamycin production in Streptomyces virginiae, did not inhibit binding. In addition, no protein capable of specifically binding 3 H-labeled VB-C was found in S. griseus. Together with the observation that VB-C had almost no biological activity on the restoration of streptomycin production or sporulation in an A-factor-deficient mutant of S. griseus, these results indicated that the binding protein had a strict ligand specificity. Examination for an A-factor-binding protein in Streptomyces coelicolor A3(2) and Streptomyces lividans showed the absence of any specifically binding protein

A mosquito hemolymph odorant-binding protein family member specifically binds juvenile hormone

Energy Technology Data Exchange (ETDEWEB)

Kim, Il Hwan; Pham, Van; Jablonka, Willy; Goodman, Walter G.; Ribeiro, José M. C.; Andersen, John F.

2017-07-27

Juvenile hormone (JH) is a key regulator of insect development and reproduction. In adult mosquitoes, it is essential for maturation of the ovary and normal male reproductive behavior, but how JH distribution and activity is regulated after secretion is unclear. Here, we report a new type of specific JH-binding protein, given the name mosquito juvenile hormone-binding protein (mJHBP), which circulates in the hemolymph of pupal and adult Aedes aegypti males and females. mJHBP is a member of the odorant-binding protein (OBP) family, and orthologs are present in the genomes of Aedes, Culex, and Anopheles mosquito species. Using isothermal titration calorimetry, we show that mJHBP specifically binds JH II and JH III but not eicosanoids or JH derivatives. mJHBP was crystallized in the presence of JH III and found to have a double OBP domain structure reminiscent of salivary “long” D7 proteins of mosquitoes. We observed that a single JH III molecule is contained in the N-terminal domain binding pocket that is closed in an apparent conformational change by a C-terminal domain-derived α-helix. The electron density for the ligand indicated a high occupancy of the natural 10R enantiomer of JH III. Of note, mJHBP is structurally unrelated to hemolymph JHBP from lepidopteran insects. A low level of expression of mJHBP in Ae. aegypti larvae suggests that it is primarily active during the adult stage where it could potentially influence the effects of JH on egg development, mating behavior, feeding, or other processes.

A mosquito hemolymph odorant-binding protein family member specifically binds juvenile hormone.

Science.gov (United States)

Kim, Il Hwan; Pham, Van; Jablonka, Willy; Goodman, Walter G; Ribeiro, José M C; Andersen, John F

2017-09-15

Juvenile hormone (JH) is a key regulator of insect development and reproduction. In adult mosquitoes, it is essential for maturation of the ovary and normal male reproductive behavior, but how JH distribution and activity is regulated after secretion is unclear. Here, we report a new type of specific JH-binding protein, given the name mosquito juvenile hormone-binding protein (mJHBP), which circulates in the hemolymph of pupal and adult Aedes aegypti males and females. mJHBP is a member of the odorant-binding protein (OBP) family, and orthologs are present in the genomes of Aedes , Culex , and Anopheles mosquito species. Using isothermal titration calorimetry, we show that mJHBP specifically binds JH II and JH III but not eicosanoids or JH derivatives. mJHBP was crystallized in the presence of JH III and found to have a double OBP domain structure reminiscent of salivary "long" D7 proteins of mosquitoes. We observed that a single JH III molecule is contained in the N-terminal domain binding pocket that is closed in an apparent conformational change by a C-terminal domain-derived α-helix. The electron density for the ligand indicated a high occupancy of the natural 10 R enantiomer of JH III. Of note, mJHBP is structurally unrelated to hemolymph JHBP from lepidopteran insects. A low level of expression of mJHBP in Ae. aegypti larvae suggests that it is primarily active during the adult stage where it could potentially influence the effects of JH on egg development, mating behavior, feeding, or other processes.

Conformational Dynamics of the Receptor Protein Galactose/Glucose Binding Protein

Science.gov (United States)

Messina, Troy; Talaga, David

2006-03-01

We have performed time-correlated single photon counting (TCSPC) anisotropy and Stokes Shift measurements on bulk solutions of galactose/glucose binding protein. Site-directed mutagenesis was used to provide a single cysteine amino acid near the sugar-binding center of the protein (glutamine 26 to cysteine -- Q26C). The cysteine was covalently labeled with the environmentally-sensitive fluorophore acrylodan, and a long-lived ruthenium complex was covalently attached to the N-terminus to provide a fluorescent reference. The TCSPC data were analyzed using global convolute-and-compare fitting routines over the entire glucose titration and temperature range to provide minimal reduced chi-squared values and the highest time resolution possible. Using a standard ligand-binding model, the resulting distributions show that the closed (ligand-bound) conformation exists even at zero glucose concentration. At 20^oC, the relative abundance of this conformation is as high as 40%. The temperature dependence of this conformational study will be discussed and related to the ligand-binding free energy surface.

Functional Advantages of Conserved Intrinsic Disorder in RNA-Binding Proteins.

Science.gov (United States)

Varadi, Mihaly; Zsolyomi, Fruzsina; Guharoy, Mainak; Tompa, Peter

2015-01-01

Proteins form large macromolecular assemblies with RNA that govern essential molecular processes. RNA-binding proteins have often been associated with conformational flexibility, yet the extent and functional implications of their intrinsic disorder have never been fully assessed. Here, through large-scale analysis of comprehensive protein sequence and structure datasets we demonstrate the prevalence of intrinsic structural disorder in RNA-binding proteins and domains. We addressed their functionality through a quantitative description of the evolutionary conservation of disordered segments involved in binding, and investigated the structural implications of flexibility in terms of conformational stability and interface formation. We conclude that the functional role of intrinsically disordered protein segments in RNA-binding is two-fold: first, these regions establish extended, conserved electrostatic interfaces with RNAs via induced fit. Second, conformational flexibility enables them to target different RNA partners, providing multi-functionality, while also ensuring specificity. These findings emphasize the functional importance of intrinsically disordered regions in RNA-binding proteins.

Functional Advantages of Conserved Intrinsic Disorder in RNA-Binding Proteins.

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Mihaly Varadi

Full Text Available Proteins form large macromolecular assemblies with RNA that govern essential molecular processes. RNA-binding proteins have often been associated with conformational flexibility, yet the extent and functional implications of their intrinsic disorder have never been fully assessed. Here, through large-scale analysis of comprehensive protein sequence and structure datasets we demonstrate the prevalence of intrinsic structural disorder in RNA-binding proteins and domains. We addressed their functionality through a quantitative description of the evolutionary conservation of disordered segments involved in binding, and investigated the structural implications of flexibility in terms of conformational stability and interface formation. We conclude that the functional role of intrinsically disordered protein segments in RNA-binding is two-fold: first, these regions establish extended, conserved electrostatic interfaces with RNAs via induced fit. Second, conformational flexibility enables them to target different RNA partners, providing multi-functionality, while also ensuring specificity. These findings emphasize the functional importance of intrinsically disordered regions in RNA-binding proteins.

In vitro binding of germanium to proteins of rice shoots

International Nuclear Information System (INIS)

Matsumoto, Hideaki; Takahashi, Eiichi

1976-01-01

The possibility of in vitro binding between proteins of rice shoots and germanium (Ge) was investigated. The proteins in mixtures of aqueous extracts of rice shoots and radioactive germanium ( 68 GeO 2 ) were fractionated. The binding of radioactivity to the proteins was observed even after 5 successive fractionation steps from the original mixtures. At the final fractionation step using polyacrylamide gel electrophoresis, a constant proportionality between protein concentration and associated radioactivity was found in most samples although not all. These results indicate that the binding of 68 Ge to proteins is not due to the simple adsorption by proteins. (auth.)

The bZIP protein from Tamarix hispida, ThbZIP1, is ACGT elements binding factor that enhances abiotic stress signaling in transgenic Arabidopsis.

Science.gov (United States)

Ji, Xiaoyu; Liu, Guifeng; Liu, Yujia; Zheng, Lei; Nie, Xianguang; Wang, Yucheng

2013-10-04

Tamarix spp. are woody halophyte, which are very tolerant to abiotic stresses such as salinity and drought, but little is known about their specific stress response systems. Basic leucine zipper proteins (bZIPs) play important roles in the ability of plants to withstand adverse environmental conditions. However, their exact roles in abiotic stress tolerance are still not fully known. In the current study, we functionally characterized a bZIP gene (ThbZIP1) from Tamarix hispida in response to abiotic stresses. We addressed the regulatory network of ThbZIP1 in three levels, i.e. its upstream regulators, the cis-acting elements recognized by ThbZIP1, and its downstream target genes. Two MYCs were found to bind to E-box, in the promoter of ThbZIP1 to activate its expression. Expression of ThbZIP1 is induced by ABA, salt, drought, methyl viologen and cold. ThbZIP1 can specifically bind to ACGT elements, with the highest binding affinity to the C-box, followed by the G-box and lastly the A-box. Compared with wild-type (Col-0) Arabidopsis, transgenic plants expressing ThbZIP1 had an increased tolerance to drought and salt, but had an increased sensitivity to ABA during seed germination and root growth; meanwhile, ROS level, cell death and water loss rate in transgenic plants were significantly reduced. Microarray analyses showed that many ROS scavenging genes were up-regulated by ThbZIP1 under salt stress conditions. Based on these data, we suggest that ThbZIP1 confers abiotic stress tolerance through activating stress tolerance genes to modulate ROS scavenging ability and other physiological changes involved in stress tolerance, and plays an important role in the ABA-mediated stress response of T. hispida.

Peptoid–Peptide Hybrid Ligands Targeting the Polo Box Domain of Polo-Like Kinase 1k | Center for Cancer Research

Science.gov (United States)

The cover picture shows the binding of a PLHSpT derivative, 6q, to the polo-like kinase 1 (Plk1) polo-box domain (PBD), thereby uncovering a new hydrophobic channel (magnified upper right), which is absent in the unliganded protein (magnified lower left). The authors explain how, as a consequence of the additional interaction with the channel, the peptide binds to the Plk1 PBD

Probing binding hot spots at protein-RNA recognition sites.

Science.gov (United States)

Barik, Amita; Nithin, Chandran; Karampudi, Naga Bhushana Rao; Mukherjee, Sunandan; Bahadur, Ranjit Prasad

2016-01-29

We use evolutionary conservation derived from structure alignment of polypeptide sequences along with structural and physicochemical attributes of protein-RNA interfaces to probe the binding hot spots at protein-RNA recognition sites. We find that the degree of conservation varies across the RNA binding proteins; some evolve rapidly compared to others. Additionally, irrespective of the structural class of the complexes, residues at the RNA binding sites are evolutionary better conserved than those at the solvent exposed surfaces. For recognitions involving duplex RNA, residues interacting with the major groove are better conserved than those interacting with the minor groove. We identify multi-interface residues participating simultaneously in protein-protein and protein-RNA interfaces in complexes where more than one polypeptide is involved in RNA recognition, and show that they are better conserved compared to any other RNA binding residues. We find that the residues at water preservation site are better conserved than those at hydrated or at dehydrated sites. Finally, we develop a Random Forests model using structural and physicochemical attributes for predicting binding hot spots. The model accurately predicts 80% of the instances of experimental ΔΔG values in a particular class, and provides a stepping-stone towards the engineering of protein-RNA recognition sites with desired affinity. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Relationship between hot spot residues and ligand binding hot spots in protein-protein interfaces.

Science.gov (United States)

Zerbe, Brandon S; Hall, David R; Vajda, Sandor; Whitty, Adrian; Kozakov, Dima

2012-08-27

In the context of protein-protein interactions, the term "hot spot" refers to a residue or cluster of residues that makes a major contribution to the binding free energy, as determined by alanine scanning mutagenesis. In contrast, in pharmaceutical research, a hot spot is a site on a target protein that has high propensity for ligand binding and hence is potentially important for drug discovery. Here we examine the relationship between these two hot spot concepts by comparing alanine scanning data for a set of 15 proteins with results from mapping the protein surfaces for sites that can bind fragment-sized small molecules. We find the two types of hot spots are largely complementary; the residues protruding into hot spot regions identified by computational mapping or experimental fragment screening are almost always themselves hot spot residues as defined by alanine scanning experiments. Conversely, a residue that is found by alanine scanning to contribute little to binding rarely interacts with hot spot regions on the partner protein identified by fragment mapping. In spite of the strong correlation between the two hot spot concepts, they fundamentally differ, however. In particular, while identification of a hot spot by alanine scanning establishes the potential to generate substantial interaction energy with a binding partner, there are additional topological requirements to be a hot spot for small molecule binding. Hence, only a minority of hot spots identified by alanine scanning represent sites that are potentially useful for small inhibitor binding, and it is this subset that is identified by experimental or computational fragment screening.

Characterization of a Chitin-Binding Protein from Bacillus thuringiensis HD-1.

Directory of Open Access Journals (Sweden)

Naresh Arora

Full Text Available Strains of Bacillus thuringiensis produce insecticidal proteins. These strains have been isolated from diverse ecological niches, such as soil, phylloplane, insect cadavers and grain dust. To effectively propagate, these strains produce a range of molecules that facilitate its multiplication in a competing environment. In this report, we have examined synthesis of a chitin-binding protein and evaluated its effect on fungi encountered in environment and its interaction with insecticidal proteins synthesized by B. thuringiensis. The gene encoding chitin-binding protein has been cloned and expressed. The purified protein has been demonstrated to interact with Cry insecticidal protein, Cry1Ac by Circular Dichrosim spectroscopy (CD and in vitro pull down assays. The chitin-binding protein potentiates insecticidal activity of bacillar insecticidal protein, Cry1Ac. Further, chitin-binding protein was fungistatic against several soil fungi. The chitin binding protein is expressed in spore mother cell and deposited along with insecticidal protein, Cry1Ac. It interacts with Cry1Ac to potentiate its insecticidal activity and facilitate propagation of Bacillus strain in environment by inhibiting growth of certain fungi.

Yersinia enterocolitica serum resistance proteins YadA and ail bind the complement regulator C4b-binding protein.

Directory of Open Access Journals (Sweden)

Vesa Kirjavainen

Full Text Available Many pathogens are equipped with factors providing resistance against the bactericidal action of complement. Yersinia enterocolitica, a Gram-negative enteric pathogen with invasive properties, efficiently resists the deleterious action of human complement. The major Y. enterocolitica serum resistance determinants include outer membrane proteins YadA and Ail. Lipopolysaccharide (LPS O-antigen (O-ag and outer core (OC do not contribute directly to complement resistance. The aim of this study was to analyze a possible mechanism whereby Y. enterocolitica could inhibit the antibody-mediated classical pathway of complement activation. We show that Y. enterocolitica serotypes O:3, O:8, and O:9 bind C4b-binding protein (C4bp, an inhibitor of both the classical and lectin pathways of complement. To identify the C4bp receptors on Y. enterocolitica serotype O:3 surface, a set of mutants expressing YadA, Ail, O-ag, and OC in different combinations was tested for the ability to bind C4bp. The studies showed that both YadA and Ail acted as C4bp receptors. Ail-mediated C4bp binding, however, was blocked by the O-ag and OC, and could be observed only with mutants lacking these LPS structures. C4bp bound to Y. enterocolitica was functionally active and participated in the factor I-mediated degradation of C4b. These findings show that Y. enterocolitica uses two proteins, YadA and Ail, to bind C4bp. Binding of C4bp could help Y. enterocolitica to evade complement-mediated clearance in the human host.

Echinococcus granulosus fatty acid binding proteins subcellular localization.

Science.gov (United States)

Alvite, Gabriela; Esteves, Adriana

2016-05-01

Two fatty acid binding proteins, EgFABP1 and EgFABP2, were isolated from the parasitic platyhelminth Echinococcus granulosus. These proteins bind fatty acids and have particular relevance in flatworms since de novo fatty acids synthesis is absent. Therefore platyhelminthes depend on the capture and intracellular distribution of host's lipids and fatty acid binding proteins could participate in lipid distribution. To elucidate EgFABP's roles, we investigated their intracellular distribution in the larval stage by a proteomic approach. Our results demonstrated the presence of EgFABP1 isoforms in cytosolic, nuclear, mitochondrial and microsomal fractions, suggesting that these molecules could be involved in several cellular processes. Copyright © 2016 Elsevier Inc. All rights reserved.

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

International Nuclear Information System (INIS)

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

2016-01-01

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

Quantifying drug-protein binding in vivo

International Nuclear Information System (INIS)

Buchholz, B; Bench, G; Keating III, G; Palmblad, M; Vogel, J; Grant, P G; Hillegonds, D

2004-01-01

Accelerator mass spectrometry (AMS) provides precise quantitation of isotope labeled compounds that are bound to biological macromolecules such as DNA or proteins. The sensitivity is high enough to allow for sub-pharmacological (''micro-'') dosing to determine macromolecular targets without inducing toxicities or altering the system under study, whether it is healthy or diseased. We demonstrated an application of AMS in quantifying the physiologic effects of one dosed chemical compound upon the binding level of another compound in vivo at sub-toxic doses [4].We are using tissues left from this study to develop protocols for quantifying specific binding to isolated and identified proteins. We also developed a new technique to quantify nanogram to milligram amounts of isolated protein at precisions that are comparable to those for quantifying the bound compound by AMS

Identification and characterization of riboflavin-binding proteins in human circulation

International Nuclear Information System (INIS)

Innis-Whitehouse, W.S.A.

1988-01-01

Riboflavin binding by plasma proteins from healthy human subjects was examined by equilibrium dialysis and binding was observed to vary over a greater than 10-fold range. Upon fractionation of plasma by gel filtration, the major riboflavin-binding components eluted with albumin and gamma-globulins. Albumin was purified and found to bind riboflavin only very weakly, although FMN and photo-chemical degradation products were more tightly bound. Most of the binding occurred in the gamma-globulin fraction and was attributed to immunoglobulins because the binding proteins and immunoglobulins copurified using various methods, were removed by treatment of plasma with protein A-agarose, and were coincident upon immuno-electrophoresis followed by autoradiography to detect [2- 14 C]-riboflavin. Binding differences among plasma samples were reflected in the binding recovered with the immunoglobulin fractions; however, there was not a direct relationship between the amount of immunoglobulin and the amount of [2- 14 C]riboflavin bound. Hence, it appeared that the binding was due to a subfraction of immunoglobulins

NifI inhibits nitrogenase by competing with Fe protein for binding to the MoFe protein

International Nuclear Information System (INIS)

Dodsworth, Jeremy A.; Leigh, John A.

2007-01-01

Reduction of substrate by nitrogenase requires direct electron transfer from the Fe protein to the MoFe protein. Inhibition of nitrogenase activity in Methanococcus maripaludis occurs when the regulatory protein NifI 1,2 binds the MoFe protein. This inhibition is relieved by 2-oxoglutarate. Here we present evidence that NifI 1,2 binding prevents association of the two nitrogenase components. Increasing amounts of Fe protein competed with NifI 1,2 , decreasing its inhibitory effect. NifI 1,2 prevented the co-purification of MoFe protein with a mutant form of the Fe protein that forms a stable complex with the MoFe protein, and NifI 1,2 was unable to bind to an AlF 4 - -stabilized Fe protein:MoFe protein complex. NifI 1,2 inhibited ATP- and MoFe protein-dependent oxidation of the Fe protein, and 2OG relieved this inhibition. These results support a model where NifI 1,2 competes with the Fe protein for binding to MoFe protein and prevents electron transfer

Selective regulation of YB-1 mRNA translation by the mTOR signaling pathway is not mediated by 4E-binding protein.

Science.gov (United States)

Lyabin, D N; Ovchinnikov, L P

2016-03-02

The Y-box binding protein 1 (YB-1) is a key regulator of gene expression at the level of both translation and transcription. The mode of its action on cellular events depends on its subcellular distribution and the amount in the cell. So far, the regulatory mechanisms of YB-1 synthesis have not been adequately studied. Our previous finding was that selective inhibition of YB-1 mRNA translation was caused by suppression of activity of the mTOR signaling pathway. It was suggested that this event may be mediated by phosphorylation of the 4E-binding protein (4E-BP). Here, we report that 4E-BP alone can only slightly inhibit YB-1 synthesis both in the cell and in vitro, although it essentially decreases binding of the 4F-group translation initiation factors to mRNA. With inhibited mTOR kinase, the level of mRNA binding to the eIF4F-group factors was decreased, while that to 4E-BP1 was increased, as was observed for both mTOR kinase-sensitive mRNAs and those showing low sensitivity. This suggests that selective inhibition of translation of YB-1 mRNA, and probably some other mRNAs as well, by mTOR kinase inhibitors is not mediated by the action of the 4E-binding protein upon functions of the 4F-group translation initiation factors.

CLIPZ: a database and analysis environment for experimentally determined binding sites of RNA-binding proteins.

Science.gov (United States)

Khorshid, Mohsen; Rodak, Christoph; Zavolan, Mihaela

2011-01-01

The stability, localization and translation rate of mRNAs are regulated by a multitude of RNA-binding proteins (RBPs) that find their targets directly or with the help of guide RNAs. Among the experimental methods for mapping RBP binding sites, cross-linking and immunoprecipitation (CLIP) coupled with deep sequencing provides transcriptome-wide coverage as well as high resolution. However, partly due to their vast volume, the data that were so far generated in CLIP experiments have not been put in a form that enables fast and interactive exploration of binding sites. To address this need, we have developed the CLIPZ database and analysis environment. Binding site data for RBPs such as Argonaute 1-4, Insulin-like growth factor II mRNA-binding protein 1-3, TNRC6 proteins A-C, Pumilio 2, Quaking and Polypyrimidine tract binding protein can be visualized at the level of the genome and of individual transcripts. Individual users can upload their own sequence data sets while being able to limit the access to these data to specific users, and analyses of the public and private data sets can be performed interactively. CLIPZ, available at http://www.clipz.unibas.ch, aims to provide an open access repository of information for post-transcriptional regulatory elements.

Discover binding pathways using the sliding binding-box docking approach: application to binding pathways of oseltamivir to avian influenza H5N1 neuraminidase

Science.gov (United States)

Tran, Diem-Trang T.; Le, Ly T.; Truong, Thanh N.

2013-08-01

Drug binding and unbinding are transient processes which are hardly observed by experiment and difficult to analyze by computational techniques. In this paper, we employed a cost-effective method called "pathway docking" in which molecular docking was used to screen ligand-receptor binding free energy surface to reveal possible paths of ligand approaching protein binding pocket. A case study was applied on oseltamivir, the key drug against influenza a virus. The equilibrium pathways identified by this method are found to be similar to those identified in prior studies using highly expensive computational approaches.

Discrete persistent-chain model for protein binding on DNA.

Science.gov (United States)

Lam, Pui-Man; Zhen, Yi

2011-04-01

We describe and solve a discrete persistent-chain model of protein binding on DNA, involving an extra σ(i) at a site i of the DNA. This variable takes the value 1 or 0, depending on whether or not the site is occupied by a protein. In addition, if the site is occupied by a protein, there is an extra energy cost ɛ. For a small force, we obtain analytic expressions for the force-extension curve and the fraction of bound protein on the DNA. For higher forces, the model can be solved numerically to obtain force-extension curves and the average fraction of bound proteins as a function of applied force. Our model can be used to analyze experimental force-extension curves of protein binding on DNA, and hence deduce the number of bound proteins in the case of nonspecific binding. ©2011 American Physical Society

Identification of DNA-binding sites for the activator involved in late transcription of the temperate lactococcal phage TP901-1

DEFF Research Database (Denmark)

Pedersen, Margit; Kilstrup, Mogens; Hammer, Karin

2006-01-01

Alt, encoded by the lactococcal phage TP901-1, is needed for late transcription. We identify Alt as a DNA-binding protein, and footprint analysis shows that Alt binds to a region containing four imperfect direct repeats (ALT boxes) located -76 to -32 relative to the P-late transcriptional start...... site. The importance of the ALT boxes was confirmed by deletion of one or two ALT boxes and by introducing mutations in ALT boxes 1 and 4. Alt is proposed to act as a tetramer or higher multimer activating transcription of TP901-1 late genes by binding to the four ALT boxes, and bending of the DNA may...... be important for transcriptional activation of P-late. Furthermore, our results suggest that DNA replication may be required for late transcription in TP901-1. Additionally, we identify gp28 of the related lactococcal phage Tuc2009 as an activator and show that the activators required for late transcription...

[Determination of plasma protein binding rate of arctiin and arctigenin with ultrafiltration].

Science.gov (United States)

Han, Xue-Ying; Wang, Wei; Tan, Ri-Qiu; Dou, De-Qiang

2013-02-01

To determine the plasma protein binding rate of arctiin and arctigenin. The ultrafiltration combined with HPLC was employed to determine the plasma protein binding rate of arctiin and arctigenin as well as rat plasma and healthy human plasma proteins. The plasma protein binding rate of arctiin with rat plasma at the concentrations of 64. 29, 32.14, 16.07 mg x L(-1) were (71.2 +/- 2.0)%, (73.4 +/- 0.61)%, (78.2 +/- 1.9)%, respectively; while the plasma protein binding rate of arctiin with healthy human plasma at the above concentrations were (64.8 +/- 3.1)%, (64.5 +/- 2.5)%, (77.5 +/- 1.7)%, respectively. The plasma protein binding rate of arctigenin with rat plasma at the concentrations of 77.42, 38.71, 19.36 mg x L(-1) were (96.7 +/- 0.41)%, (96.8 +/- 1.6)%, (97.3 +/- 0.46)%, respectively; while the plasma protein binding rate of arctigenin with normal human plasma at the above concentrations were (94.7 +/- 3.1)%, (96.8 +/- 1.6)%, (97.9 +/- 1.3)%, respectively. The binding rate of arctiin with rat plasma protein was moderate, which is slightly higher than the binding rate of arctiin with healthy human plasma protein. The plasma protein binding rates of arctigenin with both rat plasma and healthy human plasma are very high.

Predicting binding affinities of protein ligands from three-dimensional models: application to peptide binding to class I major histocompatibility proteins

DEFF Research Database (Denmark)

Rognan, D; Lauemoller, S L; Holm, A

1999-01-01

A simple and fast free energy scoring function (Fresno) has been developed to predict the binding free energy of peptides to class I major histocompatibility (MHC) proteins. It differs from existing scoring functions mainly by the explicit treatment of ligand desolvation and of unfavorable protein...... coordinates of the MHC-bound peptide have first been determined with an accuracy of about 1-1.5 A. Furthermore, it may be easily recalibrated for any protein-ligand complex.......) and of a series of 16 peptides to H-2K(k). Predictions were more accurate for HLA-A2-binding peptides as the training set had been built from experimentally determined structures. The average error in predicting the binding free energy of the test peptides was 3.1 kJ/mol. For the homology model-derived equation...

Zirconium distribution in the system HNO3-H2O-TBP-diluent

International Nuclear Information System (INIS)

Shu, J.; Araujo, B.F. de.

1984-01-01

The extraction behaviour of zirconium in TBP/diluent-HNO 3 -H 2 O systems is studied in order to increase the uranium decontamination factor by adjusting the extraction conditions so that zirconium extraction is kept at a minimum. Equilibrium diagram, TBP concentration, aqueous: organic phases ratio, salting-out effects and uranium loading in the organic phase were the main factors studied. All the experiments have been carried out with zirconium in the 10 -2 - 10 -3 M concentration range. The extractant degradation products influence upon zirconium behaviour was also verified. With the data obtained it was possible to introduce some modifications in the standard Purex flowsheet with the increase of the decontamination of uranium product from zirconium. (Author) [pt

The RNA-binding protein repertoire of Arabidopsis thaliana

KAUST Repository

Marondedze, Claudius

2016-07-11

RNA-binding proteins (RBPs) have essential roles in determining the fate of RNA from synthesis to decay and have been studied on a protein-by-protein basis, or computationally based on a number of well-characterised RNA-binding domains. Recently, high-throughput methods enabled the capture of mammalian RNA-binding proteomes. To gain insight into the role of Arabidopsis thaliana RBPs at the systems level, we have employed interactome capture techniques using cells from different ecotypes grown in cultures and leaves. In vivo UV-crosslinking of RNA to RBPs, oligo(dT) capture and mass spectrometry yielded 1,145 different proteins including 550 RBPs that either belong to the functional category ‘RNA-binding’, have known RNA-binding domains or have orthologs identified in mammals, C. elegans, or S. cerevisiae in addition to 595 novel candidate RBPs. We noted specific subsets of RBPs in cultured cells and leaves and a comparison of Arabidopsis, mammalian, C. elegans, and S. cerevisiae RBPs reveals a common set of proteins with a role in intermediate metabolism, as well as distinct differences suggesting that RBPs are also species and tissue specific. This study provides a foundation for studies that will advance our understanding of the biological significance of RBPs in plant developmental and stimulus specific responses.

Molecular characterization and expression pattern of X box-binding protein-1 (XBP1) in common carp (Cyprinus carpio L.): Indications for a role of XBP1 in antibacterial and antiviral immunity.

Science.gov (United States)

Li, Ting; Li, Hua; Peng, Shaoqing; Zhang, Fumiao; An, Liguo; Yang, Guiwen

2017-08-01

X box-binding protein-1 (XBP1) is a transcription factor that is essential for the unfolded protein response (UPR) and the differentiation of plasma cells, and some findings have also uncovered its function in innate immunity. XBP1 typically has two different transcripts, un-spliced (XBP1u) and spliced forms (XBP1s), but XBP1s is an active transcription factor in the regulation of target genes. To date, there is no evidence about the identification and function of XBP1 in common carp. Moreover, no data are currently available regarding the role of fish XBP1 in innate immunity. Thus, to determine whether XBP1 is involved in innate immune response in common carp, we cloned CcXBP1s and examined the expression of XBP1s and a XBP1s stimulated gene (IL-6) after Aeromonas hydrophila (A. hydrophila) and polyinosinic-polycytidylic acid (polyI:C) challenges. The results imply that CcXBP1s, as an active transcription factor, might play regulation roles in the antibacterial and antiviral innate immune responses of common carp. This allows us to gain new insights into the immunological function of XBP1 in fish innate immunity and the evolution of this important class of genes across vertebrates. Copyright © 2017 Elsevier Ltd. All rights reserved.

Predicting nucleic acid binding interfaces from structural models of proteins.

Science.gov (United States)

Dror, Iris; Shazman, Shula; Mukherjee, Srayanta; Zhang, Yang; Glaser, Fabian; Mandel-Gutfreund, Yael

2012-02-01

The function of DNA- and RNA-binding proteins can be inferred from the characterization and accurate prediction of their binding interfaces. However, the main pitfall of various structure-based methods for predicting nucleic acid binding function is that they are all limited to a relatively small number of proteins for which high-resolution three-dimensional structures are available. In this study, we developed a pipeline for extracting functional electrostatic patches from surfaces of protein structural models, obtained using the I-TASSER protein structure predictor. The largest positive patches are extracted from the protein surface using the patchfinder algorithm. We show that functional electrostatic patches extracted from an ensemble of structural models highly overlap the patches extracted from high-resolution structures. Furthermore, by testing our pipeline on a set of 55 known nucleic acid binding proteins for which I-TASSER produces high-quality models, we show that the method accurately identifies the nucleic acids binding interface on structural models of proteins. Employing a combined patch approach we show that patches extracted from an ensemble of models better predicts the real nucleic acid binding interfaces compared with patches extracted from independent models. Overall, these results suggest that combining information from a collection of low-resolution structural models could be a valuable approach for functional annotation. We suggest that our method will be further applicable for predicting other functional surfaces of proteins with unknown structure. Copyright © 2011 Wiley Periodicals, Inc.

Neurochemical aftermath of amateur boxing.

Science.gov (United States)

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

2006-09-01

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

Analysis of electric moments of RNA-binding proteins: implications for mechanism and prediction

Directory of Open Access Journals (Sweden)

Sarai Akinori

2011-02-01

Full Text Available Abstract Background Protein-RNA interactions play important role in many biological processes such as gene regulation, replication, protein synthesis and virus assembly. Although many structures of various types of protein-RNA complexes have been determined, the mechanism of protein-RNA recognition remains elusive. We have earlier shown that the simplest electrostatic properties viz. charge, dipole and quadrupole moments, calculated from backbone atomic coordinates of proteins are biased relative to other proteins, and these quantities can be used to identify DNA-binding proteins. Closely related, RNA-binding proteins are investigated in this study. In particular, discrimination between various types of RNA-binding proteins, evolutionary conservation of these bulk electrostatic features and effect of conformational changes by complex formation are investigated. Basic binding mechanism of a putative RNA-binding protein (HI1333 from Haemophilus influenza is suggested as a potential application of this study. Results We found that similar to DNA-binding proteins (DBPs, RNA-binding proteins (RBPs also show significantly higher values of electric moments. However, higher moments in RBPs are found to strongly depend on their functional class: proteins binding to ribosomal RNA (rRNA constitute the only class with all three of the properties (charge, dipole and quadrupole moments being higher than control proteins. Neural networks were trained using leave-one-out cross-validation to predict RBPs from control data as well as pair-wise classification capacity between proteins binding to various RNA types. RBPs and control proteins reached up to 78% accuracy measured by the area under the ROC curve. Proteins binding to rRNA are found to be best distinguished (AUC = 79%. Changes in dipole and quadrupole moments between unbound and bound structures were small and these properties are found to be robust under complex formation. Conclusions Bulk electric

A Venom Gland Extracellular Chitin-Binding-Like Protein from Pupal Endoparasitoid Wasps, Pteromalus Puparum, Selectively Binds Chitin

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Yu Zhu

2015-11-01

Full Text Available Chitin-binding proteins (CBPs are present in many species and they act in a variety of biological processes. We analyzed a Pteromalus puparum venom apparatus proteome and transcriptome and identified a partial gene encoding a possible CBP. Here, we report cloning a full-length cDNA of a sequence encoding a chitin-binding-like protein (PpCBP from P. puparum, a pupal endoparasitoid of Pieris rapae. The cDNA encoded a 96-amino-acid protein, including a secretory signal peptide and a chitin-binding peritrophin-A domain. Phylogenetic analysis of chitin binding domains (CBDs of cuticle proteins and peritrophic matrix proteins in selected insects revealed that the CBD of PpCBP clustered with the CBD of Nasonia vitripennis. The PpCBP is specifically expressed in the venom apparatus of P. puparum, mostly in the venom gland. PpCBP expression was highest at day one after adult eclosion and much lower for the following five days. We produced a recombinant PpCBP and binding assays showed the recombinant protein selectively binds chitin but not cellulose in vitro. We infer that PpCBP serves a structural role in the venom reservoir, or may be injected into the host to help wound healing of the host exoskeleton.

Evaluation of Th1/Th2-Related Immune Response against Recombinant Proteins of Brucella abortus Infection in Mice.

Science.gov (United States)

Im, Young Bin; Park, Woo Bin; Jung, Myunghwan; Kim, Suk; Yoo, Han Sang

2016-06-28

Brucellosis is a zoonotic disease caused by Brucella, a genus of gram-negative bacteria. Cytokines have key roles in the activation of innate and acquired immunities. Despite several research attempts to reveal the immune responses, the mechanism of Brucella infection remains unclear. Therefore, immune responses were analyzed in mice immunized with nine recombinant proteins. Cytokine production profiles were analyzed in the RAW 264.7 cells and naive splenocytes after stimulation with three recombinant proteins, metal-dependent hydrolase (r0628), bacterioferritin (rBfr), and thiamine transporter substrate-binding protein (rTbpA). Immune responses were analyzed by ELISA and ELISpot assay after immunization with proteins in mice. The production levels of NO, TNF-α, and IL-6 were time-dependently increased after having been stimulated with proteins in the RAW 264.7 cells. In naive splenocytes, the production of IFN-γ and IL-2 was increased after stimulation with the proteins. It was concluded that two recombinant proteins, r0628 and rTbpA, showed strong immunogenicity that was induced with Th1-related cytokines IFN-γ, IL-2, and TNF-α more than Th2-related cytokines IL-6, IL-4, and IL-5 in vitro. Conversely, a humoral immune response was activated by increasing the number of antigen-secreting cells specifically. Furthermore, these could be candidate diagnosis antigens for better understanding of brucellosis.

Informing the Human Plasma Protein Binding of ...

Science.gov (United States)

The free fraction of a xenobiotic in plasma (Fub) is an important determinant of chemical adsorption, distribution, metabolism, elimination, and toxicity, yet experimental plasma protein binding data is scarce for environmentally relevant chemicals. The presented work explores the merit of utilizing available pharmaceutical data to predict Fub for environmentally relevant chemicals via machine learning techniques. Quantitative structure-activity relationship (QSAR) models were constructed with k nearest neighbors (kNN), support vector machines (SVM), and random forest (RF) machine learning algorithms from a training set of 1045 pharmaceuticals. The models were then evaluated with independent test sets of pharmaceuticals (200 compounds) and environmentally relevant ToxCast chemicals (406 total, in two groups of 238 and 168 compounds). The selection of a minimal feature set of 10-15 2D molecular descriptors allowed for both informative feature interpretation and practical applicability domain assessment via a bounded box of descriptor ranges and principal component analysis. The diverse pharmaceutical and environmental chemical sets exhibit similarities in terms of chemical space (99-82% overlap), as well as comparable bias and variance in constructed learning curves. All the models exhibit significant predictability with mean absolute errors (MAE) in the range of 0.10-0.18 Fub. The models performed best for highly bound chemicals (MAE 0.07-0.12), neutrals (MAE 0

Identification of Arsenic Direct-Binding Proteins in Acute Promyelocytic Leukaemia Cells

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Tao Zhang

2015-11-01

Full Text Available The identification of arsenic direct-binding proteins is essential for determining the mechanism by which arsenic trioxide achieves its chemotherapeutic effects. At least two cysteines close together in the amino acid sequence are crucial to the binding of arsenic and essential to the identification of arsenic-binding proteins. In the present study, arsenic binding proteins were pulled down with streptavidin and identified using a liquid chromatograph-mass spectrometer (LC-MS/MS. More than 40 arsenic-binding proteins were separated, and redox-related proteins, glutathione S-transferase P1 (GSTP1, heat shock 70 kDa protein 9 (HSPA9 and pyruvate kinase M2 (PKM2, were further studied using binding assays in vitro. Notably, PKM2 has a high affinity for arsenic. In contrast to PKM2, GSTP1and HSPA9 did not combine with arsenic directly in vitro. These observations suggest that arsenic-mediated acute promyelocytic leukaemia (APL suppressive effects involve PKM2. In summary, we identified several arsenic binding proteins in APL cells and investigated the therapeutic mechanisms of arsenic trioxide for APL. Further investigation into specific signal pathways by which PKM2 mediates APL developments may lead to a better understanding of arsenic effects on APL.

DMPD: LPS-binding proteins and receptors. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 9665271 LPS-binding proteins and receptors. Fenton MJ, Golenbock DT. J Leukoc Biol.... 1998 Jul;64(1):25-32. (.png) (.svg) (.html) (.csml) Show LPS-binding proteins and receptors. PubmedID 9665271 Title LPS-binding prot...eins and receptors. Authors Fenton MJ, Golenbock DT. Publication J Leukoc Biol. 199

Thermodynamics of ligand binding to acyl-coenzyme A binding protein studied by titration calorimetry

DEFF Research Database (Denmark)

Færgeman, Nils J.; Sigurskjold, B W; Kragelund, B B

1996-01-01

Ligand binding to recombinant bovine acyl-CoA binding protein (ACBP) was examined using isothermal microcalorimetry. Microcalorimetric measurements confirm that the binding affinity of acyl-CoA esters for ACBP is strongly dependent on the length of the acyl chain with a clear preference for acyl-...

Phosphorylation inhibits DNA-binding of alternatively spliced aryl hydrocarbon receptor nuclear translocator

International Nuclear Information System (INIS)

Kewley, Robyn J.; Whitelaw, Murray L.

2005-01-01

The basic helix-loop-helix/PER-ARNT-SIM homology (bHLH/PAS) transcription factor ARNT (aryl hydrocarbon receptor nuclear translocator) is a key component of various pathways which induce the transcription of cytochrome P450 and hypoxia response genes. ARNT can be alternatively spliced to express Alt ARNT, containing an additional 15 amino acids immediately N-terminal to the DNA-binding basic region. Here, we show that ARNT and Alt ARNT proteins are differentially phosphorylated by protein kinase CKII in vitro. Phosphorylation had an inhibitory effect on DNA-binding to an E-box probe by Alt ARNT, but not ARNT, homodimers. This inhibitory phosphorylation occurs through Ser77. Moreover, a point mutant, Alt ARNT S77A, shows increased activity on an E-box reporter gene, consistent with Ser77 being a regulatory site in vivo. In contrast, DNA binding by an Alt ARNT/dioxin receptor heterodimer to the xenobiotic response element is not inhibited by phosphorylation with CKII, nor does Alt ARNT S77A behave differently from wild type Alt ARNT in the context of a dioxin receptor heterodimer

A role for the weak DnaA binding sites in bacterial replication origins

DEFF Research Database (Denmark)

Charbon, Godefroid; Løbner-Olesen, Anders

2011-01-01

DnaA initiates the chromosomal DNA replication in nearly all bacteria, and replication origins are characterized by binding sites for the DnaA protein (DnaA-boxes) along with an ‘AT-rich’ region. However, great variation in number, spatial organization and specificity of DnaA-boxes is observed...... between species. In the study by Taylor et al. (2011), new and unexpectedly weak DnaA-boxes were identified within the Caulobacter crescentus origin of replication (Cori). The position of weak and stronger DnaA-boxes follows a pattern seen in Escherichia coli oriC. This raises the possibility...... that bacterial origins might be more alike than previously thought....

Genome-wide identification, sequence characterization, and protein-protein interaction properties of DDB1 (damaged DNA binding protein-1)-binding WD40-repeat family members in Solanum lycopersicum.

Science.gov (United States)

Zhu, Yunye; Huang, Shengxiong; Miao, Min; Tang, Xiaofeng; Yue, Junyang; Wang, Wenjie; Liu, Yongsheng

2015-06-01

One hundred DDB1 (damaged DNA binding protein-1)-binding WD40-repeat domain (DWD) family genes were identified in the S. lycopersicum genome. The DWD genes encode proteins presumably functioning as the substrate recognition subunits of the cullin4-ring ubiquitin E3 ligase complex. These findings provide candidate genes and a research platform for further gene functionality and molecular breeding study. A subclass of DDB1 (damaged DNA binding protein-1)-binding WD40-repeat domain (DWD) family proteins has been demonstrated to function as the substrate recognition subunits of the cullin4-ring ubiquitin E3 ligase complex. However, little information is available about the cognate subfamily genes in tomato (S. lycopersicum). In this study, based on the recently released tomato genome sequences, 100 tomato genes encoding DWD proteins that potentially interact with DDB1 were identified and characterized, including analyses of the detailed annotations, chromosome locations and compositions of conserved amino acid domains. In addition, a phylogenetic tree, which comprises of three main groups, of the subfamily genes was constructed. The physical interaction between tomato DDB1 and 14 representative DWD proteins was determined by yeast two-hybrid and co-immunoprecipitation assays. The subcellular localization of these 14 representative DWD proteins was determined. Six of them were localized in both nucleus and cytoplasm, seven proteins exclusively in cytoplasm, and one protein either in nucleus and cytoplasm, or exclusively in cytoplasm. Comparative genomic analysis demonstrated that the expansion of these subfamily members in tomato predominantly resulted from two whole-genome triplication events in the evolution history.

Study on the effect of free acidity and entrained TBP in UNPS on the quality of ADU powder

International Nuclear Information System (INIS)

Prudhvi Raju, P.V.S.N.; Mandal, D.

2015-01-01

Graphical abstract: From the experimental study it was found that mean particle size of precipitated Ammonium Di-Uranate (ADU) depends on the free acidity content and entrained TBP in Uranyl Nitrate Pure Solution (UNPS). It was found that as the free acidity as well as the entrained TBP content in UNPS increases, mean particle size of precipitated ADU decreases. Based on the experimental results two correlations were developed as shown in below figures. - Highlights: • The mean particle size of precipitated ADU depends on the free acidity in UNPS. • The mean particle size of precipitated ADU also depends on the entrained TBP in UNPS. • As free acidity in UNPS increases, mean particle size of precipitated ADU decreases. • As entrained TBP in UNPS increases, particle size of precipitated ADU decreases. • Based on the experimental results two correlations were developed to find d p of ADU. - Abstract: The mean particle size and size distribution of Ammonium Di-Uranate (ADU) particles, precipitated during the precipitation reaction of Uranyl Nitrate Pure Solution (UNPS) with ammonia play an important role on the sintered density of UO 2 pellets. The quality of precipitated ADU depends on number of process parameters viz., pH of UNPS, concentration of uranium in UNPS, flow rate of ammonium hydroxide, temperature etc. However, the effects of the presence of free acid and entrained Tri-Butyl-Phosphate (TBP) in UNPS on the quality of ADU powder were not studied till date. Experiments were conducted to study the effect of free acidity and the presence of entrained TBP on the quality of precipitated ADU particles. It was found that as the concentration of free acid as well as the concentration of entrained TBP in UNPS increases, the particle size of precipitated ADU decreases. Based on the experimental results two correlations were developed to determine the mean particle size of ADU; one is based on the free acid content of UNPS and the other is based on the

Study on the effect of free acidity and entrained TBP in UNPS on the quality of ADU powder

Energy Technology Data Exchange (ETDEWEB)

Prudhvi Raju, P.V.S.N. [Nuclear Fuel Complex, Hyderabad 500 062 (India); Mandal, D., E-mail: dmandal@barc.gov.in [Chemical Engineering Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085 (India)

2015-09-15

Graphical abstract: From the experimental study it was found that mean particle size of precipitated Ammonium Di-Uranate (ADU) depends on the free acidity content and entrained TBP in Uranyl Nitrate Pure Solution (UNPS). It was found that as the free acidity as well as the entrained TBP content in UNPS increases, mean particle size of precipitated ADU decreases. Based on the experimental results two correlations were developed as shown in below figures. - Highlights: • The mean particle size of precipitated ADU depends on the free acidity in UNPS. • The mean particle size of precipitated ADU also depends on the entrained TBP in UNPS. • As free acidity in UNPS increases, mean particle size of precipitated ADU decreases. • As entrained TBP in UNPS increases, particle size of precipitated ADU decreases. • Based on the experimental results two correlations were developed to find d{sub p} of ADU. - Abstract: The mean particle size and size distribution of Ammonium Di-Uranate (ADU) particles, precipitated during the precipitation reaction of Uranyl Nitrate Pure Solution (UNPS) with ammonia play an important role on the sintered density of UO{sub 2} pellets. The quality of precipitated ADU depends on number of process parameters viz., pH of UNPS, concentration of uranium in UNPS, flow rate of ammonium hydroxide, temperature etc. However, the effects of the presence of free acid and entrained Tri-Butyl-Phosphate (TBP) in UNPS on the quality of ADU powder were not studied till date. Experiments were conducted to study the effect of free acidity and the presence of entrained TBP on the quality of precipitated ADU particles. It was found that as the concentration of free acid as well as the concentration of entrained TBP in UNPS increases, the particle size of precipitated ADU decreases. Based on the experimental results two correlations were developed to determine the mean particle size of ADU; one is based on the free acid content of UNPS and the other is based on

Solution structure of telomere binding domain of AtTRB2 derived from Arabidopsis thaliana

Energy Technology Data Exchange (ETDEWEB)

Yun, Ji-Hye [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Lee, Won Kyung [Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Kim, Heeyoun [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Kim, Eunhee; Cheong, Chaejoon [Magnetic Resonance Team, Korea Basic Science Institute (KBSI), Ochang, Chungbuk 363-883 (Korea, Republic of); Cho, Myeon Haeng [Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Lee, Weontae, E-mail: wlee@spin.yonsei.ac.kr [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of)

2014-09-26

Highlights: • We have determined solution structure of Myb domain of AtTRB2. • The Myb domain of AtTRB2 is located in the N-terminal region. • The Myb domain of AtTRB2 binds to plant telomeric DNA without fourth helix. • Helix 2 and 3 of the Myb domain of AtTRB2 are involved in DNA recognition. • AtTRB2 is a novel protein distinguished from other known plant TBP. - Abstract: Telomere homeostasis is regulated by telomere-associated proteins, and the Myb domain is well conserved for telomere binding. AtTRB2 is a member of the SMH (Single-Myb-Histone)-like family in Arabidopsis thaliana, having an N-terminal Myb domain, which is responsible for DNA binding. The Myb domain of AtTRB2 contains three α-helices and loops for DNA binding, which is unusual given that other plant telomere-binding proteins have an additional fourth helix that is essential for DNA binding. To understand the structural role for telomeric DNA binding of AtTRB2, we determined the solution structure of the Myb domain of AtTRB2 (AtTRB2{sub 1–64}) using nuclear magnetic resonance (NMR) spectroscopy. In addition, the inter-molecular interaction between AtTRB2{sub 1–64} and telomeric DNA has been characterized by the electrophoretic mobility shift assay (EMSA) and NMR titration analyses for both plant (TTTAGGG)n and human (TTAGGG)n telomere sequences. Data revealed that Trp28, Arg29, and Val47 residues located in Helix 2 and Helix 3 are crucial for DNA binding, which are well conserved among other plant telomere binding proteins. We concluded that although AtTRB2 is devoid of the additional fourth helix in the Myb-extension domain, it is able to bind to plant telomeric repeat sequences as well as human telomeric repeat sequences.

Serotonin binding in vitro by releasable proteins from human blood platelets

International Nuclear Information System (INIS)

Heemstra, V.L.

1983-11-01

Among the substances released from human blood platelets are serotonin and various proteins. It was hypothesized that one of these proteins binds serotonin and that serotonin might be important to the protein's function or that the protein might be important to serotonin's function. Two platelet-specific proteins, platelet factor 4 (PF4) and β-thromboglobulin (βTG) were found to bind serotonin in vitro. Endogenous PF4 was isolated by serotonin-affinity chromatography and was identified by radioimmunoassay. Purified [ 125 I] -PF4 and native PF4 bound to and eluted from a serotonin-affinity column similarly. Ultrafiltration of the homologous protein, βTG, with [ 14 C]-serotonin demonstrated binding of about 8 moles serotonin per mole tetrameric βTG with a dissociation constant of about 4 X 10(sup-8) M. Equilibrium dialysis of PF4 with radiolabelled serotonin was attempted, but no binding constant values were obtained because serotonin apparently bound to the dialysis membrane. Since EDTA was one of the two agents that eluted PF4 from the serotonin-affinity gel, calcium binding by PF4 was investigated by equilibrium dialysis. Evidence was obtained for positively cooperative binding of calcium ions by PF4. It is concluded that PF4 and βTG bind serotonin in vitro, that they may also bind in vivo when platelets undergo release, and that the functions of serotonin, PF4 and βTG may be mediated in part by serotonin-protein associations

Differential plasma protein binding to metal oxide nanoparticles

International Nuclear Information System (INIS)

Deng, Zhou J; Mortimer, Gysell; Minchin, Rodney F; Schiller, Tara; Musumeci, Anthony; Martin, Darren

2009-01-01

Nanoparticles rapidly interact with the proteins present in biological fluids, such as blood. The proteins that are adsorbed onto the surface potentially dictate the biokinetics of the nanomaterials and their fate in vivo. Using nanoparticles with different sizes and surface characteristics, studies have reported the effects of physicochemical properties on the composition of adsorbed plasma proteins. However, to date, few studies have been conducted focusing on the nanoparticles that are commonly exposed to the general public, such as the metal oxides. Using previously established ultracentrifugation approaches, two-dimensional gel electrophoresis and mass spectrometry, the current study investigated the binding of human plasma proteins to commercially available titanium dioxide, silicon dioxide and zinc oxide nanoparticles. We found that, despite these particles having similar surface charges in buffer, they bound different plasma proteins. For TiO 2 , the shape of the nanoparticles was also an important determinant of protein binding. Agglomeration in water was observed for all of the nanoparticles and both TiO 2 and ZnO further agglomerated in biological media. This led to an increase in the amount and number of different proteins bound to these nanoparticles. Proteins with important biological functions were identified, including immunoglobulins, lipoproteins, acute-phase proteins and proteins involved in complement pathways and coagulation. These results provide important insights into which human plasma proteins bind to particular metal oxide nanoparticles. Because protein absorption to nanoparticles may determine their interaction with cells and tissues in vivo, understanding how and why plasma proteins are adsorbed to these particles may be important for understanding their biological responses.

Identification of stable reference genes for quantitative PCR in cells derived from chicken lymphoid organs.

Science.gov (United States)

Borowska, D; Rothwell, L; Bailey, R A; Watson, K; Kaiser, P

2016-02-01

Quantitative polymerase chain reaction (qPCR) is a powerful technique for quantification of gene expression, especially genes involved in immune responses. Although qPCR is a very efficient and sensitive tool, variations in the enzymatic efficiency, quality of RNA and the presence of inhibitors can lead to errors. Therefore, qPCR needs to be normalised to obtain reliable results and allow comparison. The most common approach is to use reference genes as internal controls in qPCR analyses. In this study, expression of seven genes, including β-actin (ACTB), β-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-glucuronidase (GUSB), TATA box binding protein (TBP), α-tubulin (TUBAT) and 28S ribosomal RNA (r28S), was determined in cells isolated from chicken lymphoid tissues and stimulated with three different mitogens. The stability of the genes was measured using geNorm, NormFinder and BestKeeper software. The results from both geNorm and NormFinder were that the three most stably expressed genes in this panel were TBP, GAPDH and r28S. BestKeeper did not generate clear answers because of the highly heterogeneous sample set. Based on these data we will include TBP in future qPCR normalisation. The study shows the importance of appropriate reference gene normalisation in other tissues before qPCR analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

Comparative kinetic and equilibrium investigations on the extraction of U(IV) in nitric acid/Tbp or TLA-Kerosene systems

Energy Technology Data Exchange (ETDEWEB)

Daoud, J A; Khalifa, S M; Abdel Rahman, N; Aly, H F [Hot laboratories center, Atomic energy authority post code 13758, (Egypt)

1995-10-01

The extraction equilibrium of U(IV) by tributyl-phosphate (Tbp) or trilaurylamine (TLA) in kerosene from nitric acid media was investigated. The effect of the different parameters affecting the extraction was separately studied and compared. The kinetics of extraction of U(IV) by Tbp or TLA was also investigated using a stirred lewis cell. The data showed that the rate of extraction of U(IV) by Tbp is controlled by chemical reactions at the interface. In the TLA system, the extraction of U(IV) is controlled by chemical reactions at the interface and bulk phase. Rate equations show that the extraction of U(IV) is first order dependent on Tbp concentration while it is of fractional negative order with respect to TLA. The effect of temperature on the rate of extraction was also studied and the thermodynamic functions were evaluated for the two systems. 5 figs.

The expression of selenium-binding protein 1 is decreased in uterine leiomyoma

Directory of Open Access Journals (Sweden)

Quddus M Ruhul

2010-12-01

Full Text Available Abstract Background Selenium has been shown to inhibit cancer development and growth through the mediation of selenium-binding proteins. Decreased expression of selenium-binding protein 1 has been reported in cancers of the prostate, stomach, colon, and lungs. No information, however, is available concerning the roles of selenium-binding protein 1 in uterine leiomyoma. Methods Using Western Blot analysis and immunohistochemistry, we examined the expression of selenium-binding protein 1 in uterine leiomyoma and normal myometrium in 20 patients who had undergone hysterectomy for uterine leiomyoma. Results and Discussion The patient age ranged from 34 to 58 years with a mean of 44.3 years. Proliferative endometrium was seen in 8 patients, secretory endometrium in 7 patients, and atrophic endometrium in 5 patients. Two patients showed solitary leiomyoma, and eighteen patients revealed 2 to 5 tumors. Tumor size ranged from 1 to 15.5 cm with a mean of 4.3 cm. Both Western Blot analysis and immunohistochemistry showed a significant lower level of selenium-binding protein 1 in leiomyoma than in normal myometrium. Larger tumors had a tendency to show a lower level of selenium-binding protein 1 than smaller ones, but the difference did not reach a statistical significance. The expression of selenium-binding protein 1 was the same among patients with proliferative, secretory, and atrophic endometrium in either leiomyoma or normal myometrium. Also, we did not find a difference of selenium-binding protein 1 level between patients younger than 45 years and older patients in either leiomyoma or normal myometrium. Conclusions Decreased expression of selenium-binding protein 1 in uterine leiomyoma may indicate a role of the protein in tumorigenesis. Our findings may provide a basis for future studies concerning the molecular mechanisms of selenium-binding protein 1 in tumorigenesis as well as the possible use of selenium in prevention and treatment of uterine

A conserved NAD+ binding pocket that regulates protein-protein interactions during aging.

Science.gov (United States)

Li, Jun; Bonkowski, Michael S; Moniot, Sébastien; Zhang, Dapeng; Hubbard, Basil P; Ling, Alvin J Y; Rajman, Luis A; Qin, Bo; Lou, Zhenkun; Gorbunova, Vera; Aravind, L; Steegborn, Clemens; Sinclair, David A

2017-03-24

DNA repair is essential for life, yet its efficiency declines with age for reasons that are unclear. Numerous proteins possess Nudix homology domains (NHDs) that have no known function. We show that NHDs are NAD + (oxidized form of nicotinamide adenine dinucleotide) binding domains that regulate protein-protein interactions. The binding of NAD + to the NHD domain of DBC1 (deleted in breast cancer 1) prevents it from inhibiting PARP1 [poly(adenosine diphosphate-ribose) polymerase], a critical DNA repair protein. As mice age and NAD + concentrations decline, DBC1 is increasingly bound to PARP1, causing DNA damage to accumulate, a process rapidly reversed by restoring the abundance of NAD + Thus, NAD + directly regulates protein-protein interactions, the modulation of which may protect against cancer, radiation, and aging. Copyright © 2017, American Association for the Advancement of Science.

Saccharomyces cerevisiae SSB1 protein and its relationship to nucleolar RNA-binding proteins.

OpenAIRE

Jong, A Y; Clark, M W; Gilbert, M; Oehm, A; Campbell, J L

1987-01-01

To better define the function of Saccharomyces cerevisiae SSB1, an abundant single-stranded nucleic acid-binding protein, we determined the nucleotide sequence of the SSB1 gene and compared it with those of other proteins of known function. The amino acid sequence contains 293 amino acid residues and has an Mr of 32,853. There are several stretches of sequence characteristic of other eucaryotic single-stranded nucleic acid-binding proteins. At the amino terminus, residues 39 to 54 are highly ...

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

Science.gov (United States)

Gong, Jian; Lv, Liang; Huo, Jirong

2014-12-01

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

A novel signal transduction protein: Combination of solute binding and tandem PAS-like sensor domains in one polypeptide chain: Periplasmic Ligand Binding Protein Dret_0059

Energy Technology Data Exchange (ETDEWEB)

Wu, R. [Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Biosciences Division, Argonne National Laboratory, Argonne Illinois 60439; Wilton, R. [Biosciences Division, Argonne National Laboratory, Argonne Illinois 60439; Cuff, M. E. [Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Biosciences Division, Argonne National Laboratory, Argonne Illinois 60439; Structural Biology Center, Argonne National Laboratory, Argonne Illinois 60439; Endres, M. [Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Babnigg, G. [Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Biosciences Division, Argonne National Laboratory, Argonne Illinois 60439; Edirisinghe, J. N. [Mathematics and Computer Science Division, Argonne National Laboratory, Argonne Illinois 60439; Computation Institute, University of Chicago, Chicago Illinois 60637; Henry, C. S. [Mathematics and Computer Science Division, Argonne National Laboratory, Argonne Illinois 60439; Computation Institute, University of Chicago, Chicago Illinois 60637; Joachimiak, A. [Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Biosciences Division, Argonne National Laboratory, Argonne Illinois 60439; Structural Biology Center, Argonne National Laboratory, Argonne Illinois 60439; Department of Biochemistry and Molecular Biology, University of Chicago, Chicago Illinois 60637; Schiffer, M. [Biosciences Division, Argonne National Laboratory, Argonne Illinois 60439; Pokkuluri, P. R. [Biosciences Division, Argonne National Laboratory, Argonne Illinois 60439

2017-03-06

We report the structural and biochemical characterization of a novel periplasmic ligand-binding protein, Dret_0059, from Desulfohalobium retbaense DSM 5692, an organism isolated from the Salt Lake Retba in Senegal. The structure of the protein consists of a unique combination of a periplasmic solute binding protein (SBP) domain at the N-terminal and a tandem PAS-like sensor domain at the C-terminal region. SBP domains are found ubiquitously and their best known function is in solute transport across membranes. PAS-like sensor domains are commonly found in signal transduction proteins. These domains are widely observed as parts of many protein architectures and complexes but have not been observed previously within the same polypeptide chain. In the structure of Dret_0059, a ketoleucine moiety is bound to the SBP, whereas a cytosine molecule is bound in the distal PAS-like domain of the tandem PAS-like domain. Differential scanning flourimetry support the binding of ligands observed in the crystal structure. There is significant interaction between the SBP and tandem PAS-like domains, and it is possible that the binding of one ligand could have an effect on the binding of the other. We uncovered three other proteins with this structural architecture in the non-redundant sequence data base, and predict that they too bind the same substrates. The genomic context of this protein did not offer any clues for its function. We did not find any biological process in which the two observed ligands are coupled. The protein Dret_0059 could be involved in either signal transduction or solute transport.

Binding Direction-Based Two-Dimensional Flattened Contact Area Computing Algorithm for Protein-Protein Interactions.

Science.gov (United States)

Kang, Beom Sik; Pugalendhi, GaneshKumar; Kim, Ku-Jin

2017-10-13

Interactions between protein molecules are essential for the assembly, function, and regulation of proteins. The contact region between two protein molecules in a protein complex is usually complementary in shape for both molecules and the area of the contact region can be used to estimate the binding strength between two molecules. Although the area is a value calculated from the three-dimensional surface, it cannot represent the three-dimensional shape of the surface. Therefore, we propose an original concept of two-dimensional contact area which provides further information such as the ruggedness of the contact region. We present a novel algorithm for calculating the binding direction between two molecules in a protein complex, and then suggest a method to compute the two-dimensional flattened area of the contact region between two molecules based on the binding direction.

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

Science.gov (United States)

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

2016-02-01

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

Coupling ligand recognition to protein folding in an engineered variant of rabbit ileal lipid binding protein.

Science.gov (United States)

Kouvatsos, Nikolaos; Meldrum, Jill K; Searle, Mark S; Thomas, Neil R

2006-11-28

We have engineered a variant of the beta-clam shell protein ILBP which lacks the alpha-helical motif that caps the central binding cavity; the mutant protein is sufficiently destabilised that it is unfolded under physiological conditions, however, it unexpectedly binds its natural bile acid substrates with high affinity forming a native-like beta-sheet rich structure and demonstrating strong thermodynamic coupling between ligand binding and protein folding.

The interrelationship between ligand binding and self-association of the folate binding protein

DEFF Research Database (Denmark)

Holm, Jan; Schou, Christian; Babol, Linnea N.

2011-01-01

The folate binding protein (FBP) regulates homeostasis and intracellular trafficking of folic acid, a vitamin of decisive importance in cell division and growth. We analyzed whether interrelationship between ligand binding and self-association of FBP plays a significant role in the physiology of ...

Cellulose binding domain proteins

Science.gov (United States)

Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc; Doi, Roy

1998-01-01

A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

Functional Advantages of Conserved Intrinsic Disorder in RNA-Binding Proteins

OpenAIRE

Varadi, Mihaly; Zsolyomi, Fruzsina; Guharoy, Mainak; Tompa, Peter

2015-01-01

Proteins form large macromolecular assemblies with RNA that govern essential molecular processes. RNA-binding proteins have often been associated with conformational flexibility, yet the extent and functional implications of their intrinsic disorder have never been fully assessed. Here, through large-scale analysis of comprehensive protein sequence and structure datasets we demonstrate the prevalence of intrinsic structural disorder in RNA-binding proteins and domains. We addressed their func...

Does protein binding modulate the effect of angiotensin II receptor antagonists?

Directory of Open Access Journals (Sweden)

Marc P Maillard

2001-03-01

Full Text Available IntroductionAngiotensin II AT 1-receptor antagonists are highly bound to plasma proteins (≥ 99%. With some antagonists, such as DuP-532, the protein binding was such that no efficacy of the drug could be demonstrated clinically. Whether protein binding interferes with the efficacy of other antagonists is not known. We have therefore investigated in vitro how plasma proteins may affect the antagonistic effect of different AT1-receptor antagonists.MethodsA radio-receptor binding assay was used to analyse the interaction between proteins and the ability of various angiotensin II (Ang II antagonists to block AT1-receptors. In addition, the Biacore technology, a new technique which enables the real-time monitoring of binding events between two molecules, was used to evaluate the dissociation rate constants of five AT1-receptor antagonists from human serum albumin.ResultsThe in vitro AT 1-antagonistic effects of different Ang II receptor antagonists were differentially affected by the presence of human plasma, with rightward shifts of the IC50 ranging from one to several orders of magnitude. The importance of the shift correlates with the dissociation rate constants of these drugs from albumin. Our experiments also show that the way that AT1-receptor antagonists bind to proteins differs from one compound to another. These results suggest that the interaction with plasma proteins appears to modulate the efficacy of some Ang II antagonists.ConclusionAlthough the high binding level of Ang II receptor antagonist to plasma proteins appears to be a feature common to this class of compounds, the kinetics and characteristics of this binding is of great importance. With some antagonists, protein binding interferes markedly with their efficacy to block AT1-receptors.

TBP production plant effluent treatment process

International Nuclear Information System (INIS)

Sriniwas, C.; Sugilal, G.; Wattal, P.K.

2004-06-01

TBP production facility at Heavy Water Plant, Talcher generates about 2000 litres of effluent per 200 kg batch. The effluent is basically an aqueous solution containing dissolved and dispersed organics such as dibutyl phosphate, butanol etc. The effluent has high salinity, chemical oxygen demand (30-80 g/L) and pungent odour. It requires treatment before discharge. A chemical precipitation process using ferric chloride was developed for quantitative separation of organics from the aqueous part of the effluent. This process facilitates the discharge of the aqueous effluent. Results of the laboratory and bench scale experiments on actual effluent samples are presented in this report. (author)

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

Directory of Open Access Journals (Sweden)

Qinxue Li

2018-04-01

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

Monomeric Yeast Frataxin is an Iron-Binding Protein

International Nuclear Information System (INIS)

Cook, J.; Bencze, K.; Jankovic, A.; Crater, A.; Busch, C.; Bradley, P.; Stemmler, A.; Spaller, M.; Stemmler, T.

2006-01-01

Friedreich's ataxia, an autosomal cardio- and neurodegenerative disorder that affects 1 in 50 000 humans, is caused by decreased levels of the protein frataxin. Although frataxin is nuclear-encoded, it is targeted to the mitochondrial matrix and necessary for proper regulation of cellular iron homeostasis. Frataxin is required for the cellular production of both heme and iron-sulfur (Fe-S) clusters. Monomeric frataxin binds with high affinity to ferrochelatase, the enzyme involved in iron insertion into porphyrin during heme production. Monomeric frataxin also binds to Isu, the scaffold protein required for assembly of Fe-S cluster intermediates. These processes (heme and Fe-S cluster assembly) share requirements for iron, suggesting that monomeric frataxin might function as the common iron donor. To provide a molecular basis to better understand frataxin's function, we have characterized the binding properties and metal-site structure of ferrous iron bound to monomeric yeast frataxin. Yeast frataxin is stable as an iron-loaded monomer, and the protein can bind two ferrous iron atoms with micromolar binding affinity. Frataxin amino acids affected by the presence of iron are localized within conserved acidic patches located on the surfaces of both helix-1 and strand-1. Under anaerobic conditions, bound metal is stable in the high-spin ferrous state. The metal-ligand coordination geometry of both metal-binding sites is consistent with a six-coordinate iron-(oxygen/nitrogen) based ligand geometry, surely constructed in part from carboxylate and possibly imidazole side chains coming from residues within these conserved acidic patches on the protein. On the basis of our results, we have developed a model for how we believe yeast frataxin interacts with iron

SECRET domain of variola virus CrmB protein can be a member of poxviral type II chemokine-binding proteins family.

Science.gov (United States)

Antonets, Denis V; Nepomnyashchikh, Tatyana S; Shchelkunov, Sergei N

2010-10-27

Variola virus (VARV) the causative agent of smallpox, eradicated in 1980, have wide spectrum of immunomodulatory proteins to evade host immunity. Recently additional biological activity was discovered for VARV CrmB protein, known to bind and inhibit tumour necrosis factor (TNF) through its N-terminal domain homologous to cellular TNF receptors. Besides binding TNF, this protein was also shown to bind with high affinity several chemokines which recruit B- and T-lymphocytes and dendritic cells to sites of viral entry and replication. Ability to bind chemokines was shown to be associated with unique C-terminal domain of CrmB protein. This domain named SECRET (Smallpox virus-Encoded Chemokine Receptor) is unrelated to the host proteins and lacks significant homology with other known viral chemokine-binding proteins or any other known protein. De novo modelling of VARV-CrmB SECRET domain spatial structure revealed its apparent structural homology with cowpox virus CC-chemokine binding protein (vCCI) and vaccinia virus A41 protein, despite low sequence identity between these three proteins. Potential ligand-binding surface of modelled VARV-CrmB SECRET domain was also predicted to bear prominent electronegative charge which is characteristic to known orthopoxviral chemokine-binding proteins. Our results suggest that SECRET should be included into the family of poxviral type II chemokine-binding proteins and that it might have been evolved from the vCCI-like predecessor protein.

Evaluating the binding efficiency of pheromone binding protein with its natural ligand using molecular docking and fluorescence analysis

Science.gov (United States)

Ilayaraja, Renganathan; Rajkumar, Ramalingam; Rajesh, Durairaj; Muralidharan, Arumugam Ramachandran; Padmanabhan, Parasuraman; Archunan, Govindaraju

2014-06-01

Chemosignals play a crucial role in social and sexual communication among inter- and intra-species. Chemical cues are bound with protein that is present in the pheromones irrespective of sex are commonly called as pheromone binding protein (PBP). In rats, the pheromone compounds are bound with low molecular lipocalin protein α2u-globulin (α2u). We reported farnesol is a natural endogenous ligand (compound) present in rat preputial gland as a bound volatile compound. In the present study, an attempt has been made through computational method to evaluating the binding efficiency of α2u with the natural ligand (farnesol) and standard fluorescent molecule (2-naphthol). The docking analysis revealed that the binding energy of farnesol and 2-naphthol was almost equal and likely to share some binding pocket of protein. Further, to extrapolate the results generated through computational approach, the α2u protein was purified and subjected to fluorescence titration and binding assay. The results showed that the farnesol is replaced by 2-naphthol with high hydrophobicity of TYR120 in binding sites of α2u providing an acceptable dissociation constant indicating the binding efficiency of α2u. The obtained results are in corroboration with the data made through computational approach.

Chromate Binding and Removal by the Molybdate-Binding Protein ModA.

Science.gov (United States)

Karpus, Jason; Bosscher, Michael; Ajiboye, Ifedayo; Zhang, Liang; He, Chuan

2017-04-04

Effective and cheap methods and techniques for the safe removal of hexavalent chromate from the environment are in increasingly high demand. High concentrations of hexavalent chromate have been shown to have numerous harmful effects on human biology. We show that the E. coli molybdate-binding protein ModA is a genetically encoded tool capable of removing chromate from aqueous solutions. Although previously reported to not bind chromate, we show that ModA binds chromate tightly and is capable of removing chromate to levels well below current US federal standards. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Investigations of foam formation and its stabilization in the extraction systems: TBP in kerosene-nitric acid solutions

International Nuclear Information System (INIS)

Zielinski, A.

1980-01-01

The paper is devoted to studies of foam formation and its stabilization in TBP - kerosene - nitric acid solutions extracting systems. It was experimentally found, that TBP acts as a stabilizator of thin, liquid foam films as well as an emulgator in forming dispersions. The stabilizing effect of fine emulsions w/o on formed foams column was observed. Relevant references on the subject are also reviewed. (author)

Actin, actin-binding proteins, and actin-related proteins in the nucleus.

Science.gov (United States)

Kristó, Ildikó; Bajusz, Izabella; Bajusz, Csaba; Borkúti, Péter; Vilmos, Péter

2016-04-01

Extensive research in the past decade has significantly broadened our view about the role actin plays in the life of the cell and added novel aspects to actin research. One of these new aspects is the discovery of the existence of nuclear actin which became evident only recently. Nuclear activities including transcriptional activation in the case of all three RNA polymerases, editing and nuclear export of mRNAs, and chromatin remodeling all depend on actin. It also became clear that there is a fine-tuned equilibrium between cytoplasmic and nuclear actin pools and that this balance is ensured by an export-import system dedicated to actin. After over half a century of research on conventional actin and its organizing partners in the cytoplasm, it was also an unexpected finding that the nucleus contains more than 30 actin-binding proteins and new classes of actin-related proteins which are not able to form filaments but had evolved nuclear-specific functions. The actin-binding and actin-related proteins in the nucleus have been linked to RNA transcription and processing, nuclear transport, and chromatin remodeling. In this paper, we attempt to provide an overview of the wide range of information that is now available about actin, actin-binding, and actin-related proteins in the nucleus.

Characterization of fatty acid binding by the P2 myelin protein

International Nuclear Information System (INIS)

Gudaitis, P.G.; Weise, M.J.

1987-01-01

In recent years, significant sequence homology has been found between the P2 protein of peripheral myelin and intracellular retinoid- and fatty acid-binding proteins. They have found that salt extracts of bovine intradural nerve roots contain the P2 basic protein in association with free fatty acid. Preliminary results from quantitative analyses showed a ratio of 0.4-1.1 fatty acid (mainly oleate and palmitate) per P2 molecule. P2/ligand interactions were partially characterized using ( 3 H)-oleate in gel permeation assays and binding studies using lipidex to separated bound and free fatty acid. Methyloleate was found to displace ( 3 H)-oleate from P2, indicating that ligand binding interactions are predominantly hydrophobic in nature. On the other hand, myristic acid and retinol did not inhibit the binding of oleate to the protein, results consistent with a decided affinity for long chain fatty acids but not for the retinoids. The binding between P2 and oleic acid showed an apparent Kd in the micromolar range, a value comparable to those found for other fatty acid-binding proteins. From these results they conclude that P2 shares not only structural homology with certain fatty acid binding proteins but also an ability to bind long chain fatty acids. Although the significance of these similarities is not yet clear, they may, by analogy, expect P2 to have a role in PNS lipid metabolism

A sequence-based dynamic ensemble learning system for protein ligand-binding site prediction

KAUST Repository

Chen, Peng

2015-12-03

Background: Proteins have the fundamental ability to selectively bind to other molecules and perform specific functions through such interactions, such as protein-ligand binding. Accurate prediction of protein residues that physically bind to ligands is important for drug design and protein docking studies. Most of the successful protein-ligand binding predictions were based on known structures. However, structural information is not largely available in practice due to the huge gap between the number of known protein sequences and that of experimentally solved structures

A sequence-based dynamic ensemble learning system for protein ligand-binding site prediction

KAUST Repository

Chen, Peng; Hu, ShanShan; Zhang, Jun; Gao, Xin; Li, Jinyan; Xia, Junfeng; Wang, Bing

2015-01-01

Background: Proteins have the fundamental ability to selectively bind to other molecules and perform specific functions through such interactions, such as protein-ligand binding. Accurate prediction of protein residues that physically bind to ligands is important for drug design and protein docking studies. Most of the successful protein-ligand binding predictions were based on known structures. However, structural information is not largely available in practice due to the huge gap between the number of known protein sequences and that of experimentally solved structures