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Sample records for binding motif pbm

  1. A comparative analysis of transcription factor binding models learned from PBM, HT-SELEX and ChIP data.

    Science.gov (United States)

    Orenstein, Yaron; Shamir, Ron

    2014-04-01

    Understanding gene regulation is a key challenge in today's biology. The new technologies of protein-binding microarrays (PBMs) and high-throughput SELEX (HT-SELEX) allow measurement of the binding intensities of one transcription factor (TF) to numerous synthetic double-stranded DNA sequences in a single experiment. Recently, Jolma et al. reported the results of 547 HT-SELEX experiments covering human and mouse TFs. Because 162 of these TFs were also covered by PBM technology, for the first time, a large-scale comparison between implementations of these two in vitro technologies is possible. Here we assessed the similarities and differences between binding models, represented as position weight matrices, inferred from PBM and HT-SELEX, and also measured how well these models predict in vivo binding. Our results show that HT-SELEX- and PBM-derived models agree for most TFs. For some TFs, the HT-SELEX-derived models are longer versions of the PBM-derived models, whereas for other TFs, the HT-SELEX models match the secondary PBM-derived models. Remarkably, PBM-based 8-mer ranking is more accurate than that of HT-SELEX, but models derived from HT-SELEX predict in vivo binding better. In addition, we reveal several biases in HT-SELEX data including nucleotide frequency bias, enrichment of C-rich k-mers and oligos and underrepresentation of palindromes.

  2. A Comparison Study for DNA Motif Modeling on Protein Binding Microarray.

    Science.gov (United States)

    Wong, Ka-Chun; Li, Yue; Peng, Chengbin; Wong, Hau-San

    2016-01-01

    Transcription factor binding sites (TFBSs) are relatively short (5-15 bp) and degenerate. Identifying them is a computationally challenging task. In particular, protein binding microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner; for instance, a typical PBM experiment can measure binding signal intensities of a protein to all possible DNA k-mers (k = 8∼10). Since proteins can often bind to DNA with different binding intensities, one of the major challenges is to build TFBS (also known as DNA motif) models which can fully capture the quantitative binding affinity data. To learn DNA motif models from the non-convex objective function landscape, several optimization methods are compared and applied to the PBM motif model building problem. In particular, representative methods from different optimization paradigms have been chosen for modeling performance comparison on hundreds of PBM datasets. The results suggest that the multimodal optimization methods are very effective for capturing the binding preference information from PBM data. In particular, we observe a general performance improvement if choosing di-nucleotide modeling over mono-nucleotide modeling. In addition, the models learned by the best-performing method are applied to two independent applications: PBM probe rotation testing and ChIP-Seq peak sequence prediction, demonstrating its biological applicability.

  3. A Comparison Study for DNA Motif Modeling on Protein Binding Microarray

    KAUST Repository

    Wong, Ka-Chun

    2015-06-11

    Transcription Factor Binding Sites (TFBSs) are relatively short (5-15 bp) and degenerate. Identifying them is a computationally challenging task. In particular, Protein Binding Microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner; for instance, a typical PBM experiment can measure binding signal intensities of a protein to all possible DNA k-mers (k=810). Since proteins can often bind to DNA with different binding intensities, one of the major challenges is to build motif models which can fully capture the quantitative binding affinity data. To learn DNA motif models from the non-convex objective function landscape, several optimization methods are compared and applied to the PBM motif model building problem. In particular, representative methods from different optimization paradigms have been chosen for modeling performance comparison on hundreds of PBM datasets. The results suggest that the multimodal optimization methods are very effective for capturing the binding preference information from PBM data. In particular, we observe a general performance improvement using di-nucleotide modeling over mono-nucleotide modeling. In addition, the models learned by the best-performing method are applied to two independent applications: PBM probe rotation testing and ChIP-Seq peak sequence prediction, demonstrating its biological applicability.

  4. The Human Papillomavirus E6 PDZ Binding Motif: From Life Cycle to Malignancy

    Directory of Open Access Journals (Sweden)

    Ketaki Ganti

    2015-07-01

    Full Text Available Cancer-causing HPV E6 oncoproteins are characterized by the presence of a PDZ binding motif (PBM at their extreme carboxy terminus. It was long thought that this region of E6 had a sole function to confer interaction with a defined set of cellular substrates. However, more recent studies have shown that the E6 PBM has a complex pattern of regulation, whereby phosphorylation within the PBM can regulate interaction with two classes of cellular proteins: those containing PDZ domains and the members of the 14-3-3 family of proteins. In this review, we explore the roles that the PBM and its ligands play in the virus life cycle, and subsequently how these can inadvertently contribute towards the development of malignancy. We also explore how subtle alterations in cellular signal transduction pathways might result in aberrant E6 phosphorylation, which in turn might contribute towards disease progression.

  5. Assessment of algorithms for inferring positional weight matrix motifs of transcription factor binding sites using protein binding microarray data.

    Directory of Open Access Journals (Sweden)

    Yaron Orenstein

    Full Text Available The new technology of protein binding microarrays (PBMs allows simultaneous measurement of the binding intensities of a transcription factor to tens of thousands of synthetic double-stranded DNA probes, covering all possible 10-mers. A key computational challenge is inferring the binding motif from these data. We present a systematic comparison of four methods developed specifically for reconstructing a binding site motif represented as a positional weight matrix from PBM data. The reconstructed motifs were evaluated in terms of three criteria: concordance with reference motifs from the literature and ability to predict in vivo and in vitro bindings. The evaluation encompassed over 200 transcription factors and some 300 assays. The results show a tradeoff between how the methods perform according to the different criteria, and a dichotomy of method types. Algorithms that construct motifs with low information content predict PBM probe ranking more faithfully, while methods that produce highly informative motifs match reference motifs better. Interestingly, in predicting high-affinity binding, all methods give far poorer results for in vivo assays compared to in vitro assays.

  6. PDlim2 selectively interacts with the PDZ binding motif of highly pathogenic avian H5N1 influenza A virus NS1.

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    Jia Yu

    Full Text Available The multi-functional NS1 protein of influenza A virus is a viral virulence determining factor. The last four residues at the C-terminus of NS1 constitute a type I PDZ domain binding motif (PBM. Avian influenza viruses currently in circulation carry an NS1 PBM with consensus sequence ESEV, whereas human influenza viruses bear an NS1 PBM with consensus sequence RSKV or RSEV. The PBM sequence of the influenza A virus NS1 is reported to contribute to high viral pathogenicity in animal studies. Here, we report the identification of PDlim2 as a novel binding target of the highly pathogenic avian influenza virus H5N1 strain with an NS1 PBM of ESEV (A/Chicken/Henan/12/2004/H5N1, HN12-NS1 by yeast two-hybrid screening. The interaction was confirmed by in vitro GST pull-down assays, as well as by in vivo mammalian two-hybrid assays and bimolecular fluorescence complementation assays. The binding was also confirmed to be mediated by the interaction of the PDlim2 PDZ domain with the NS1 PBM motif. Interestingly, our assays showed that PDlim2 bound specifically with HN12-NS1, but exhibited no binding to NS1 from a human influenza H1N1 virus bearing an RSEV PBM (A/Puerto Rico/8/34/H1N1, PR8-NS1. A crystal structure of the PDlim2 PDZ domain fused with the C-terminal hexapeptide from HN12-NS1, together with GST pull-down assays on PDlim2 mutants, reveals that residues Arg16 and Lys31 of PDlim2 are critical for the binding between PDlim2 and HN12-NS1. The identification of a selective binding target of HN12-NS1 (ESEV, but not PR8-NS1 (RSEV, enables us to propose a structural mechanism for the interaction between NS1 PBM and PDlim2 or other PDZ-containing proteins.

  7. An RNA motif that binds ATP

    Science.gov (United States)

    Sassanfar, M.; Szostak, J. W.

    1993-01-01

    RNAs that contain specific high-affinity binding sites for small molecule ligands immobilized on a solid support are present at a frequency of roughly one in 10(10)-10(11) in pools of random sequence RNA molecules. Here we describe a new in vitro selection procedure designed to ensure the isolation of RNAs that bind the ligand of interest in solution as well as on a solid support. We have used this method to isolate a remarkably small RNA motif that binds ATP, a substrate in numerous biological reactions and the universal biological high-energy intermediate. The selected ATP-binding RNAs contain a consensus sequence, embedded in a common secondary structure. The binding properties of ATP analogues and modified RNAs show that the binding interaction is characterized by a large number of close contacts between the ATP and RNA, and by a change in the conformation of the RNA.

  8. GABPα Binding to Overlapping ETS and CRE DNA Motifs Is Enhanced by CREB1: Custom DNA Microarrays.

    Science.gov (United States)

    He, Ximiao; Syed, Khund Sayeed; Tillo, Desiree; Mann, Ishminder; Weirauch, Matthew T; Vinson, Charles

    2015-07-16

    To achieve proper spatiotemporal control of gene expression, transcription factors cooperatively assemble onto specific DNA sequences. The ETS domain protein monomer of GABPα and the B-ZIP domain protein dimer of CREB1 cooperatively bind DNA only when the ETS ((C)/GCGGAA GT: ) and CRE ( GT: GACGTCAC) motifs overlap precisely, producing the ETS↔CRE motif ((C)/GCGGAA GT: GACGTCAC). We designed a Protein Binding Microarray (PBM) with 60-bp DNAs containing four identical sectors, each with 177,440 features that explore the cooperative interactions between GABPα and CREB1 upon binding the ETS↔CRE motif. The DNA sequences include all 15-mers of the form (C)/GCGGA--CG-, the ETS↔CRE motif, and all single nucleotide polymorphisms (SNPs), and occurrences in the human and mouse genomes. CREB1 enhanced GABPα binding to the canonical ETS↔CRE motif CCGGAAGT two-fold, and up to 23-fold for several SNPs at the beginning and end of the ETS motif, which is suggestive of two separate and distinct allosteric mechanisms of cooperative binding. We show that the ETS-CRE array data can be used to identify regions likely cooperatively bound by GABPα and CREB1 in vivo, and demonstrate their ability to identify human genetic variants that might inhibit cooperative binding. Copyright © 2015 He et al.

  9. DNA motif elucidation using belief propagation

    OpenAIRE

    Wong, Ka-Chun; Chan, Tak-Ming; Peng, Chengbin; Li, Yue; Zhang, Zhaolei

    2013-01-01

    Protein-binding microarray (PBM) is a high-throughout platform that can measure the DNA-binding preference of a protein in a comprehensive and unbiased manner. A typical PBM experiment can measure binding signal intensities of a protein to all the possible DNA k-mers (k = 8 ∼10); such comprehensive binding affinity data usually need to be reduced and represented as motif models before they can be further analyzed and applied. Since proteins can often bind to DNA in multiple modes, one of the ...

  10. Space-related pharma-motifs for fast search of protein binding motifs and polypharmacological targets.

    Science.gov (United States)

    Chiu, Yi-Yuan; Lin, Chun-Yu; Lin, Chih-Ta; Hsu, Kai-Cheng; Chang, Li-Zen; Yang, Jinn-Moon

    2012-01-01

    To discover a compound inhibiting multiple proteins (i.e. polypharmacological targets) is a new paradigm for the complex diseases (e.g. cancers and diabetes). In general, the polypharmacological proteins often share similar local binding environments and motifs. As the exponential growth of the number of protein structures, to find the similar structural binding motifs (pharma-motifs) is an emergency task for drug discovery (e.g. side effects and new uses for old drugs) and protein functions. We have developed a Space-Related Pharmamotifs (called SRPmotif) method to recognize the binding motifs by searching against protein structure database. SRPmotif is able to recognize conserved binding environments containing spatially discontinuous pharma-motifs which are often short conserved peptides with specific physico-chemical properties for protein functions. Among 356 pharma-motifs, 56.5% interacting residues are highly conserved. Experimental results indicate that 81.1% and 92.7% polypharmacological targets of each protein-ligand complex are annotated with same biological process (BP) and molecular function (MF) terms, respectively, based on Gene Ontology (GO). Our experimental results show that the identified pharma-motifs often consist of key residues in functional (active) sites and play the key roles for protein functions. The SRPmotif is available at http://gemdock.life.nctu.edu.tw/SRP/. SRPmotif is able to identify similar pharma-interfaces and pharma-motifs sharing similar binding environments for polypharmacological targets by rapidly searching against the protein structure database. Pharma-motifs describe the conservations of binding environments for drug discovery and protein functions. Additionally, these pharma-motifs provide the clues for discovering new sequence-based motifs to predict protein functions from protein sequence databases. We believe that SRPmotif is useful for elucidating protein functions and drug discovery.

  11. The MHC motif viewer: a visualization tool for MHC binding motifs

    DEFF Research Database (Denmark)

    Rapin, Nicolas; Hoof, Ilka; Lund, Ole

    2010-01-01

    of peptides, and knowledge of their binding specificities is important for understanding differences in the immune response between individuals. Algorithms predicting which peptides bind a given MHC molecule have recently been developed with high prediction accuracy. The utility of these algorithms...... is hampered by the lack of tools for browsing and comparing specificity of these molecules. We have developed a Web server, MHC Motif Viewer, which allows the display of the binding motif for MHC class I proteins for human, chimpanzee, rhesus monkey, mouse, and swine, as well as HLA-DR protein sequences...

  12. Binding properties of SUMO-interacting motifs (SIMs) in yeast.

    Science.gov (United States)

    Jardin, Christophe; Horn, Anselm H C; Sticht, Heinrich

    2015-03-01

    Small ubiquitin-like modifier (SUMO) conjugation and interaction play an essential role in many cellular processes. A large number of yeast proteins is known to interact non-covalently with SUMO via short SUMO-interacting motifs (SIMs), but the structural details of this interaction are yet poorly characterized. In the present work, sequence analysis of a large dataset of 148 yeast SIMs revealed the existence of a hydrophobic core binding motif and a preference for acidic residues either within or adjacent to the core motif. Thus the sequence properties of yeast SIMs are highly similar to those described for human. Molecular dynamics simulations were performed to investigate the binding preferences for four representative SIM peptides differing in the number and distribution of acidic residues. Furthermore, the relative stability of two previously observed alternative binding orientations (parallel, antiparallel) was assessed. For all SIMs investigated, the antiparallel binding mode remained stable in the simulations and the SIMs were tightly bound via their hydrophobic core residues supplemented by polar interactions of the acidic residues. In contrary, the stability of the parallel binding mode is more dependent on the sequence features of the SIM motif like the number and position of acidic residues or the presence of additional adjacent interaction motifs. This information should be helpful to enhance the prediction of SIMs and their binding properties in different organisms to facilitate the reconstruction of the SUMO interactome.

  13. Variable structure motifs for transcription factor binding sites.

    Science.gov (United States)

    Reid, John E; Evans, Kenneth J; Dyer, Nigel; Wernisch, Lorenz; Ott, Sascha

    2010-01-14

    Classically, models of DNA-transcription factor binding sites (TFBSs) have been based on relatively few known instances and have treated them as sites of fixed length using position weight matrices (PWMs). Various extensions to this model have been proposed, most of which take account of dependencies between the bases in the binding sites. However, some transcription factors are known to exhibit some flexibility and bind to DNA in more than one possible physical configuration. In some cases this variation is known to affect the function of binding sites. With the increasing volume of ChIP-seq data available it is now possible to investigate models that incorporate this flexibility. Previous work on variable length models has been constrained by: a focus on specific zinc finger proteins in yeast using restrictive models; a reliance on hand-crafted models for just one transcription factor at a time; and a lack of evaluation on realistically sized data sets. We re-analysed binding sites from the TRANSFAC database and found motivating examples where our new variable length model provides a better fit. We analysed several ChIP-seq data sets with a novel motif search algorithm and compared the results to one of the best standard PWM finders and a recently developed alternative method for finding motifs of variable structure. All the methods performed comparably in held-out cross validation tests. Known motifs of variable structure were recovered for p53, Stat5a and Stat5b. In addition our method recovered a novel generalised version of an existing PWM for Sp1 that allows for variable length binding. This motif improved classification performance. We have presented a new gapped PWM model for variable length DNA binding sites that is not too restrictive nor over-parameterised. Our comparison with existing tools shows that on average it does not have better predictive accuracy than existing methods. However, it does provide more interpretable models of motifs of variable

  14. Variable structure motifs for transcription factor binding sites

    Directory of Open Access Journals (Sweden)

    Wernisch Lorenz

    2010-01-01

    Full Text Available Abstract Background Classically, models of DNA-transcription factor binding sites (TFBSs have been based on relatively few known instances and have treated them as sites of fixed length using position weight matrices (PWMs. Various extensions to this model have been proposed, most of which take account of dependencies between the bases in the binding sites. However, some transcription factors are known to exhibit some flexibility and bind to DNA in more than one possible physical configuration. In some cases this variation is known to affect the function of binding sites. With the increasing volume of ChIP-seq data available it is now possible to investigate models that incorporate this flexibility. Previous work on variable length models has been constrained by: a focus on specific zinc finger proteins in yeast using restrictive models; a reliance on hand-crafted models for just one transcription factor at a time; and a lack of evaluation on realistically sized data sets. Results We re-analysed binding sites from the TRANSFAC database and found motivating examples where our new variable length model provides a better fit. We analysed several ChIP-seq data sets with a novel motif search algorithm and compared the results to one of the best standard PWM finders and a recently developed alternative method for finding motifs of variable structure. All the methods performed comparably in held-out cross validation tests. Known motifs of variable structure were recovered for p53, Stat5a and Stat5b. In addition our method recovered a novel generalised version of an existing PWM for Sp1 that allows for variable length binding. This motif improved classification performance. Conclusions We have presented a new gapped PWM model for variable length DNA binding sites that is not too restrictive nor over-parameterised. Our comparison with existing tools shows that on average it does not have better predictive accuracy than existing methods. However, it does

  15. Photobiomodulation (PBM) Applications in Ophthalmology

    Science.gov (United States)

    Dotson, Robert

    2011-03-01

    In a very real sense, we are all creatures of light. This fact is just now beginning to impact medicine, as quantum theory begins to spread outside the confines of physics and into the life sciences. No longer can living organisms simply be viewed as retorts for biochemical reactions. They also demonstrate an energy component that will prove to be the unifying force of life in all its varied forms. With the advent of this shift in the life sciences, light is becoming an increasingly important diagnostic and therapeutic tool within medicine. Ophthalmologists have long been concerned with light and its application and, consequently, have an interest in the coming scientific revolution, photomedicine. A brief history of the use of low energy light for healing, a review of known mechanisms by which photons interact with living cells, and a review of some of the established cellular effects will be presented. Finally, brief clinical studies will be presented illustrating the benefits of PBM - specifically regarding: corneal healing, glaucoma, and dry age-related macular degeneration. The purpose of this talk is to introduce the emerging field of PBM to the physics community at large.

  16. High resolution genome wide binding event finding and motif discovery reveals transcription factor spatial binding constraints.

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    Yuchun Guo

    Full Text Available An essential component of genome function is the syntax of genomic regulatory elements that determine how diverse transcription factors interact to orchestrate a program of regulatory control. A precise characterization of in vivo spacing constraints between key transcription factors would reveal key aspects of this genomic regulatory language. To discover novel transcription factor spatial binding constraints in vivo, we developed a new integrative computational method, genome wide event finding and motif discovery (GEM. GEM resolves ChIP data into explanatory motifs and binding events at high spatial resolution by linking binding event discovery and motif discovery with positional priors in the context of a generative probabilistic model of ChIP data and genome sequence. GEM analysis of 63 transcription factors in 214 ENCODE human ChIP-Seq experiments recovers more known factor motifs than other contemporary methods, and discovers six new motifs for factors with unknown binding specificity. GEM's adaptive learning of binding-event read distributions allows it to further improve upon previous methods for processing ChIP-Seq and ChIP-exo data to yield unsurpassed spatial resolution and discovery of closely spaced binding events of the same factor. In a systematic analysis of in vivo sequence-specific transcription factor binding using GEM, we have found hundreds of spatial binding constraints between factors. GEM found 37 examples of factor binding constraints in mouse ES cells, including strong distance-specific constraints between Klf4 and other key regulatory factors. In human ENCODE data, GEM found 390 examples of spatially constrained pair-wise binding, including such novel pairs as c-Fos:c-Jun/USF1, CTCF/Egr1, and HNF4A/FOXA1. The discovery of new factor-factor spatial constraints in ChIP data is significant because it proposes testable models for regulatory factor interactions that will help elucidate genome function and the

  17. High resolution genome wide binding event finding and motif discovery reveals transcription factor spatial binding constraints.

    Science.gov (United States)

    Guo, Yuchun; Mahony, Shaun; Gifford, David K

    2012-01-01

    An essential component of genome function is the syntax of genomic regulatory elements that determine how diverse transcription factors interact to orchestrate a program of regulatory control. A precise characterization of in vivo spacing constraints between key transcription factors would reveal key aspects of this genomic regulatory language. To discover novel transcription factor spatial binding constraints in vivo, we developed a new integrative computational method, genome wide event finding and motif discovery (GEM). GEM resolves ChIP data into explanatory motifs and binding events at high spatial resolution by linking binding event discovery and motif discovery with positional priors in the context of a generative probabilistic model of ChIP data and genome sequence. GEM analysis of 63 transcription factors in 214 ENCODE human ChIP-Seq experiments recovers more known factor motifs than other contemporary methods, and discovers six new motifs for factors with unknown binding specificity. GEM's adaptive learning of binding-event read distributions allows it to further improve upon previous methods for processing ChIP-Seq and ChIP-exo data to yield unsurpassed spatial resolution and discovery of closely spaced binding events of the same factor. In a systematic analysis of in vivo sequence-specific transcription factor binding using GEM, we have found hundreds of spatial binding constraints between factors. GEM found 37 examples of factor binding constraints in mouse ES cells, including strong distance-specific constraints between Klf4 and other key regulatory factors. In human ENCODE data, GEM found 390 examples of spatially constrained pair-wise binding, including such novel pairs as c-Fos:c-Jun/USF1, CTCF/Egr1, and HNF4A/FOXA1. The discovery of new factor-factor spatial constraints in ChIP data is significant because it proposes testable models for regulatory factor interactions that will help elucidate genome function and the implementation of combinatorial

  18. DNA motif elucidation using belief propagation.

    Science.gov (United States)

    Wong, Ka-Chun; Chan, Tak-Ming; Peng, Chengbin; Li, Yue; Zhang, Zhaolei

    2013-09-01

    Protein-binding microarray (PBM) is a high-throughout platform that can measure the DNA-binding preference of a protein in a comprehensive and unbiased manner. A typical PBM experiment can measure binding signal intensities of a protein to all the possible DNA k-mers (k=8∼10); such comprehensive binding affinity data usually need to be reduced and represented as motif models before they can be further analyzed and applied. Since proteins can often bind to DNA in multiple modes, one of the major challenges is to decompose the comprehensive affinity data into multimodal motif representations. Here, we describe a new algorithm that uses Hidden Markov Models (HMMs) and can derive precise and multimodal motifs using belief propagations. We describe an HMM-based approach using belief propagations (kmerHMM), which accepts and preprocesses PBM probe raw data into median-binding intensities of individual k-mers. The k-mers are ranked and aligned for training an HMM as the underlying motif representation. Multiple motifs are then extracted from the HMM using belief propagations. Comparisons of kmerHMM with other leading methods on several data sets demonstrated its effectiveness and uniqueness. Especially, it achieved the best performance on more than half of the data sets. In addition, the multiple binding modes derived by kmerHMM are biologically meaningful and will be useful in interpreting other genome-wide data such as those generated from ChIP-seq. The executables and source codes are available at the authors' websites: e.g. http://www.cs.toronto.edu/∼wkc/kmerHMM.

  19. DNA motif elucidation using belief propagation

    KAUST Repository

    Wong, Ka-Chun

    2013-06-29

    Protein-binding microarray (PBM) is a high-throughout platform that can measure the DNA-binding preference of a protein in a comprehensive and unbiased manner. A typical PBM experiment can measure binding signal intensities of a protein to all the possible DNA k-mers (k = 8 ?10); such comprehensive binding affinity data usually need to be reduced and represented as motif models before they can be further analyzed and applied. Since proteins can often bind to DNA in multiple modes, one of the major challenges is to decompose the comprehensive affinity data into multimodal motif representations. Here, we describe a new algorithm that uses Hidden Markov Models (HMMs) and can derive precise and multimodal motifs using belief propagations. We describe an HMM-based approach using belief propagations (kmerHMM), which accepts and preprocesses PBM probe raw data into median-binding intensities of individual k-mers. The k-mers are ranked and aligned for training an HMM as the underlying motif representation. Multiple motifs are then extracted from the HMM using belief propagations. Comparisons of kmerHMM with other leading methods on several data sets demonstrated its effectiveness and uniqueness. Especially, it achieved the best performance on more than half of the data sets. In addition, the multiple binding modes derived by kmerHMM are biologically meaningful and will be useful in interpreting other genome-wide data such as those generated from ChIP-seq. The executables and source codes are available at the authors\\' websites: e.g. http://www.cs.toronto.edu/?wkc/kmerHMM. 2013 The Author(s).

  20. Evolutionarily conserved bias of amino-acid usage refines the definition of PDZ-binding motif.

    Science.gov (United States)

    Chimura, Takahiko; Launey, Thomas; Ito, Masao

    2011-06-08

    The interactions between PDZ (PSD-95, Dlg, ZO-1) domains and PDZ-binding motifs play central roles in signal transductions within cells. Proteins with PDZ domains bind to PDZ-binding motifs almost exclusively when the motifs are located at the carboxyl (C-) terminal ends of their binding partners. However, it remains little explored whether PDZ-binding motifs show any preferential location at the C-terminal ends of proteins, at genome-level. Here, we examined the distribution of the type-I (x-x-S/T-x-I/L/V) or type-II (x-x-V-x-I/V) PDZ-binding motifs in proteins encoded in the genomes of five different species (human, mouse, zebrafish, fruit fly and nematode). We first established that these PDZ-binding motifs are indeed preferentially present at their C-terminal ends. Moreover, we found specific amino acid (AA) bias for the 'x' positions in the motifs at the C-terminal ends. In general, hydrophilic AAs were favored. Our genomics-based findings confirm and largely extend the results of previous interaction-based studies, allowing us to propose refined consensus sequences for all of the examined PDZ-binding motifs. An ontological analysis revealed that the refined motifs are functionally relevant since a large fraction of the proteins bearing the motif appear to be involved in signal transduction. Furthermore, co-precipitation experiments confirmed two new protein interactions predicted by our genomics-based approach. Finally, we show that influenza virus pathogenicity can be correlated with PDZ-binding motif, with high-virulence viral proteins bearing a refined PDZ-binding motif. Our refined definition of PDZ-binding motifs should provide important clues for identifying functional PDZ-binding motifs and proteins involved in signal transduction.

  1. Evolutionarily conserved bias of amino-acid usage refines the definition of PDZ-binding motif

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    Launey Thomas

    2011-06-01

    Full Text Available Abstract Background The interactions between PDZ (PSD-95, Dlg, ZO-1 domains and PDZ-binding motifs play central roles in signal transductions within cells. Proteins with PDZ domains bind to PDZ-binding motifs almost exclusively when the motifs are located at the carboxyl (C- terminal ends of their binding partners. However, it remains little explored whether PDZ-binding motifs show any preferential location at the C-terminal ends of proteins, at genome-level. Results Here, we examined the distribution of the type-I (x-x-S/T-x-I/L/V or type-II (x-x-V-x-I/V PDZ-binding motifs in proteins encoded in the genomes of five different species (human, mouse, zebrafish, fruit fly and nematode. We first established that these PDZ-binding motifs are indeed preferentially present at their C-terminal ends. Moreover, we found specific amino acid (AA bias for the 'x' positions in the motifs at the C-terminal ends. In general, hydrophilic AAs were favored. Our genomics-based findings confirm and largely extend the results of previous interaction-based studies, allowing us to propose refined consensus sequences for all of the examined PDZ-binding motifs. An ontological analysis revealed that the refined motifs are functionally relevant since a large fraction of the proteins bearing the motif appear to be involved in signal transduction. Furthermore, co-precipitation experiments confirmed two new protein interactions predicted by our genomics-based approach. Finally, we show that influenza virus pathogenicity can be correlated with PDZ-binding motif, with high-virulence viral proteins bearing a refined PDZ-binding motif. Conclusions Our refined definition of PDZ-binding motifs should provide important clues for identifying functional PDZ-binding motifs and proteins involved in signal transduction.

  2. The GTP binding motif: variations on a theme.

    Science.gov (United States)

    Kjeldgaard, M; Nyborg, J; Clark, B F

    1996-10-01

    GTP binding proteins (G-proteins) have wide-ranging functions in biology, being involved in cell proliferation, signal transduction, protein synthesis, and protein targeting. Common to their functioning is that they are active in the GTP-bound form and inactive in the GDP-bound form. The protein synthesis elongation factor EF-Tu was the first G-protein whose nucleotide binding domain was solved structurally by X-ray crystallography to yield a structural definition of the GDP-bound form, but a still increasing number of new structures of G-proteins are appearing in the literature, in both GDP and GTP bound forms. A common structural core for nucleotide binding is present in all these structures, and this core has long been known to include common consensus sequence elements involved in binding of the nucleotide. Nevertheless, subtle changes in the common sequences reflect functional differences. Therefore, it becomes increasingly important to focus on how these differences are reflected in the structures, and how these structural differences are related to function. The aim of this review is to describe to what extent this structural motif for GDP/GTP binding is common to other known structures of this class of proteins. We first describe the common structural core of the G-proteins. Next, examples are based on information available on the Ras protein superfamily, the targeting protein ARF, elongation factors EF-Tu and EF-G, and the heterotrimeric G-proteins. Finally, we discuss the important structures of complexes between GTP binding proteins and their substrates that have appeared in the literature recently.

  3. Comprehensive human transcription factor binding site map for combinatory binding motifs discovery.

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    Arnoldo J Müller-Molina

    Full Text Available To know the map between transcription factors (TFs and their binding sites is essential to reverse engineer the regulation process. Only about 10%-20% of the transcription factor binding motifs (TFBMs have been reported. This lack of data hinders understanding gene regulation. To address this drawback, we propose a computational method that exploits never used TF properties to discover the missing TFBMs and their sites in all human gene promoters. The method starts by predicting a dictionary of regulatory "DNA words." From this dictionary, it distills 4098 novel predictions. To disclose the crosstalk between motifs, an additional algorithm extracts TF combinatorial binding patterns creating a collection of TF regulatory syntactic rules. Using these rules, we narrowed down a list of 504 novel motifs that appear frequently in syntax patterns. We tested the predictions against 509 known motifs confirming that our system can reliably predict ab initio motifs with an accuracy of 81%-far higher than previous approaches. We found that on average, 90% of the discovered combinatorial binding patterns target at least 10 genes, suggesting that to control in an independent manner smaller gene sets, supplementary regulatory mechanisms are required. Additionally, we discovered that the new TFBMs and their combinatorial patterns convey biological meaning, targeting TFs and genes related to developmental functions. Thus, among all the possible available targets in the genome, the TFs tend to regulate other TFs and genes involved in developmental functions. We provide a comprehensive resource for regulation analysis that includes a dictionary of "DNA words," newly predicted motifs and their corresponding combinatorial patterns. Combinatorial patterns are a useful filter to discover TFBMs that play a major role in orchestrating other factors and thus, are likely to lock/unlock cellular functional clusters.

  4. Motif decomposition of the phosphotyrosine proteome reveals a new N-terminal binding motif for SHIP2

    DEFF Research Database (Denmark)

    Miller, Martin Lee; Hanke, S.; Hinsby, A. M.

    2008-01-01

    and validated as a binding motif for the SH2 domain-containing inositol phosphatase SHIP2. Our decomposition of the in vivo Tyr(P) proteome furthermore suggests that two-thirds of the Tyr(P) sites mediate interaction, whereas the remaining third govern processes such as enzyme activation and nucleic acid...

  5. Genome-wide conserved consensus transcription factor binding motifs are hyper-methylated

    Directory of Open Access Journals (Sweden)

    Down Thomas A

    2010-09-01

    Full Text Available Abstract Background DNA methylation can regulate gene expression by modulating the interaction between DNA and proteins or protein complexes. Conserved consensus motifs exist across the human genome ("predicted transcription factor binding sites": "predicted TFBS" but the large majority of these are proven by chromatin immunoprecipitation and high throughput sequencing (ChIP-seq not to be biological transcription factor binding sites ("empirical TFBS". We hypothesize that DNA methylation at conserved consensus motifs prevents promiscuous or disorderly transcription factor binding. Results Using genome-wide methylation maps of the human heart and sperm, we found that all conserved consensus motifs as well as the subset of those that reside outside CpG islands have an aggregate profile of hyper-methylation. In contrast, empirical TFBS with conserved consensus motifs have a profile of hypo-methylation. 40% of empirical TFBS with conserved consensus motifs resided in CpG islands whereas only 7% of all conserved consensus motifs were in CpG islands. Finally we further identified a minority subset of TF whose profiles are either hypo-methylated or neutral at their respective conserved consensus motifs implicating that these TF may be responsible for establishing or maintaining an un-methylated DNA state, or whose binding is not regulated by DNA methylation. Conclusions Our analysis supports the hypothesis that at least for a subset of TF, empirical binding to conserved consensus motifs genome-wide may be controlled by DNA methylation.

  6. Miz-1 activates gene expression via a novel consensus DNA binding motif.

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    Bonnie L Barrilleaux

    Full Text Available The transcription factor Miz-1 can either activate or repress gene expression in concert with binding partners including the Myc oncoprotein. The genomic binding of Miz-1 includes both core promoters and more distal sites, but the preferred DNA binding motif of Miz-1 has been unclear. We used a high-throughput in vitro technique, Bind-n-Seq, to identify two Miz-1 consensus DNA binding motif sequences--ATCGGTAATC and ATCGAT (Mizm1 and Mizm2--bound by full-length Miz-1 and its zinc finger domain, respectively. We validated these sequences directly as high affinity Miz-1 binding motifs. Competition assays using mutant probes indicated that the binding affinity of Miz-1 for Mizm1 and Mizm2 is highly sequence-specific. Miz-1 strongly activates gene expression through the motifs in a Myc-independent manner. MEME-ChIP analysis of Miz-1 ChIP-seq data in two different cell types reveals a long motif with a central core sequence highly similar to the Mizm1 motif identified by Bind-n-Seq, validating the in vivo relevance of the findings. Miz-1 ChIP-seq peaks containing the long motif are predominantly located outside of proximal promoter regions, in contrast to peaks without the motif, which are highly concentrated within 1.5 kb of the nearest transcription start site. Overall, our results indicate that Miz-1 may be directed in vivo to the novel motif sequences we have identified, where it can recruit its specific binding partners to control gene expression and ultimately regulate cell fate.

  7. Mitoxantrone and Analogues Bind and Stabilize i-Motif Forming DNA Sequences

    Science.gov (United States)

    Wright, Elisé P.; Day, Henry A.; Ibrahim, Ali M.; Kumar, Jeethendra; Boswell, Leo J. E.; Huguin, Camille; Stevenson, Clare E. M.; Pors, Klaus; Waller, Zoë A. E.

    2016-12-01

    There are hundreds of ligands which can interact with G-quadruplex DNA, yet very few which target i-motif. To appreciate an understanding between the dynamics between these structures and how they can be affected by intervention with small molecule ligands, more i-motif binding compounds are required. Herein we describe how the drug mitoxantrone can bind, induce folding of and stabilise i-motif forming DNA sequences, even at physiological pH. Additionally, mitoxantrone was found to bind i-motif forming sequences preferentially over double helical DNA. We also describe the stabilisation properties of analogues of mitoxantrone. This offers a new family of ligands with potential for use in experiments into the structure and function of i-motif forming DNA sequences.

  8. The Verrucomicrobia LexA-binding Motif: Insights into the Evolutionary Dynamics of the SOS Response

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    Ivan Erill

    2016-07-01

    Full Text Available The SOS response is the primary bacterial mechanism to address DNA damage, coordinating multiple cellular processes that include DNA repair, cell division and translesion synthesis. In contrast to other regulatory systems, the composition of the SOS genetic network and the binding motif of its transcriptional repressor, LexA, have been shown to vary greatly across bacterial clades, making it an ideal system to study the co-evolution of transcription factors and their regulons. Leveraging comparative genomics approaches and prior knowledge on the core SOS regulon, here we define the binding motif of the Verrucomicrobia, a recently described phylum of emerging interest due to its association with eukaryotic hosts. Site directed mutagenesis of the Verrucomicrobium spinosum recA promoter confirms that LexA binds a 14 bp palindromic motif with consensus sequence TGTTC-N4-GAACA. Computational analyses suggest that recognition of this novel motif is determined primarily by changes in base-contacting residues of the third alpha helix of the LexA helix-turn-helix DNA binding motif. In conjunction with comparative genomics analysis of the LexA regulon in the Verrucomicrobia phylum, electrophoretic shift assays reveal that LexA binds to operators in the promoter region of DNA repair genes and a mutagenesis cassette in this organism, and identify previously unreported components of the SOS response. The identification of tandem LexA-binding sites generating instances of other LexA-binding motifs in the lexA gene promoter of Verrucomicrobia species leads us to postulate a novel mechanism for LexA-binding motif evolution. This model, based on gene duplication, successfully addresses outstanding questions in the intricate co-evolution of the LexA protein, its binding motif and the regulatory network it controls.

  9. The Verrucomicrobia LexA-Binding Motif: Insights into the Evolutionary Dynamics of the SOS Response.

    Science.gov (United States)

    Erill, Ivan; Campoy, Susana; Kılıç, Sefa; Barbé, Jordi

    2016-01-01

    The SOS response is the primary bacterial mechanism to address DNA damage, coordinating multiple cellular processes that include DNA repair, cell division, and translesion synthesis. In contrast to other regulatory systems, the composition of the SOS genetic network and the binding motif of its transcriptional repressor, LexA, have been shown to vary greatly across bacterial clades, making it an ideal system to study the co-evolution of transcription factors and their regulons. Leveraging comparative genomics approaches and prior knowledge on the core SOS regulon, here we define the binding motif of the Verrucomicrobia, a recently described phylum of emerging interest due to its association with eukaryotic hosts. Site directed mutagenesis of the Verrucomicrobium spinosum recA promoter confirms that LexA binds a 14 bp palindromic motif with consensus sequence TGTTC-N4-GAACA. Computational analyses suggest that recognition of this novel motif is determined primarily by changes in base-contacting residues of the third alpha helix of the LexA helix-turn-helix DNA binding motif. In conjunction with comparative genomics analysis of the LexA regulon in the Verrucomicrobia phylum, electrophoretic shift assays reveal that LexA binds to operators in the promoter region of DNA repair genes and a mutagenesis cassette in this organism, and identify previously unreported components of the SOS response. The identification of tandem LexA-binding sites generating instances of other LexA-binding motifs in the lexA gene promoter of Verrucomicrobia species leads us to postulate a novel mechanism for LexA-binding motif evolution. This model, based on gene duplication, successfully addresses outstanding questions in the intricate co-evolution of the LexA protein, its binding motif and the regulatory network it controls.

  10. Discovering structural motifs using a structural alphabet: Application to magnesium-binding sites

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    Lim Carmay

    2007-03-01

    Full Text Available Abstract Background For many metalloproteins, sequence motifs characteristic of metal-binding sites have not been found or are so short that they would not be expected to be metal-specific. Striking examples of such metalloproteins are those containing Mg2+, one of the most versatile metal cofactors in cellular biochemistry. Even when Mg2+-proteins share insufficient sequence homology to identify Mg2+-specific sequence motifs, they may still share similarity in the Mg2+-binding site structure. However, no structural motifs characteristic of Mg2+-binding sites have been reported. Thus, our aims are (i to develop a general method for discovering structural patterns/motifs characteristic of ligand-binding sites, given the 3D protein structures, and (ii to apply it to Mg2+-proteins sharing 2+-structural motifs are identified as recurring structural patterns. Results The structural alphabet-based motif discovery method has revealed the structural preference of Mg2+-binding sites for certain local/secondary structures: compared to all residues in the Mg2+-proteins, both first and second-shell Mg2+-ligands prefer loops to helices. Even when the Mg2+-proteins share no significant sequence homology, some of them share a similar Mg2+-binding site structure: 4 Mg2+-structural motifs, comprising 21% of the binding sites, were found. In particular, one of the Mg2+-structural motifs found maps to a specific functional group, namely, hydrolases. Furthermore, 2 of the motifs were not found in non metalloproteins or in Ca2+-binding proteins. The structural motifs discovered thus capture some essential biochemical and/or evolutionary properties, and hence may be useful for discovering proteins where Mg2+ plays an important biological role. Conclusion The structural motif discovery method presented herein is general and can be applied to any set of proteins with known 3D structures. This new method is timely considering the increasing number of structures for

  11. A Conserved Motif Provides Binding Specificity to the PP2A-B56 Phosphatase

    DEFF Research Database (Denmark)

    Hertz, Emil Peter Thrane; Kruse, Thomas; Davey, Norman E

    2016-01-01

    -exposed pocket on PP2A regulatory B56 subunits binds to a consensus sequence on interacting proteins, which we term the LxxIxE motif. The composition of the motif modulates the affinity for B56, which in turn determines the phosphorylation status of associated substrates. Phosphorylation of amino acid residues...... a molecular description of PP2A binding specificity with broad implications for understanding signaling in eukaryotes....

  12. Structural basis for the binding of tryptophan-based motifs by δ-COP.

    Science.gov (United States)

    Suckling, Richard J; Poon, Pak Phi; Travis, Sophie M; Majoul, Irina V; Hughson, Frederick M; Evans, Philip R; Duden, Rainer; Owen, David J

    2015-11-17

    Coatomer consists of two subcomplexes: the membrane-targeting, ADP ribosylation factor 1 (Arf1):GTP-binding βγδζ-COP F-subcomplex, which is related to the adaptor protein (AP) clathrin adaptors, and the cargo-binding αβ'ε-COP B-subcomplex. We present the structure of the C-terminal μ-homology domain of the yeast δ-COP subunit in complex with the WxW motif from its binding partner, the endoplasmic reticulum-localized Dsl1 tether. The motif binds at a site distinct from that used by the homologous AP μ subunits to bind YxxΦ cargo motifs with its two tryptophan residues sitting in compatible pockets. We also show that the Saccharomyces cerevisiae Arf GTPase-activating protein (GAP) homolog Gcs1p uses a related WxxF motif at its extreme C terminus to bind to δ-COP at the same site in the same way. Mutations designed on the basis of the structure in conjunction with isothermal titration calorimetry confirm the mode of binding and show that mammalian δ-COP binds related tryptophan-based motifs such as that from ArfGAP1 in a similar manner. We conclude that δ-COP subunits bind Wxn(1-6)[WF] motifs within unstructured regions of proteins that influence the lifecycle of COPI-coated vesicles; this conclusion is supported by the observation that, in the context of a sensitizing domain deletion in Dsl1p, mutating the tryptophan-based motif-binding site in yeast causes defects in both growth and carboxypeptidase Y trafficking/processing.

  13. Composite Structural Motifs of Binding Sites for Delineating Biological Functions of Proteins

    Science.gov (United States)

    Kinjo, Akira R.; Nakamura, Haruki

    2012-01-01

    Most biological processes are described as a series of interactions between proteins and other molecules, and interactions are in turn described in terms of atomic structures. To annotate protein functions as sets of interaction states at atomic resolution, and thereby to better understand the relation between protein interactions and biological functions, we conducted exhaustive all-against-all atomic structure comparisons of all known binding sites for ligands including small molecules, proteins and nucleic acids, and identified recurring elementary motifs. By integrating the elementary motifs associated with each subunit, we defined composite motifs that represent context-dependent combinations of elementary motifs. It is demonstrated that function similarity can be better inferred from composite motif similarity compared to the similarity of protein sequences or of individual binding sites. By integrating the composite motifs associated with each protein function, we define meta-composite motifs each of which is regarded as a time-independent diagrammatic representation of a biological process. It is shown that meta-composite motifs provide richer annotations of biological processes than sequence clusters. The present results serve as a basis for bridging atomic structures to higher-order biological phenomena by classification and integration of binding site structures. PMID:22347478

  14. A Novel Protein Interaction between Nucleotide Binding Domain of Hsp70 and p53 Motif

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    Asita Elengoe

    2015-01-01

    Full Text Available Currently, protein interaction of Homo sapiens nucleotide binding domain (NBD of heat shock 70 kDa protein (PDB: 1HJO with p53 motif remains to be elucidated. The NBD-p53 motif complex enhances the p53 stabilization, thereby increasing the tumor suppression activity in cancer treatment. Therefore, we identified the interaction between NBD and p53 using STRING version 9.1 program. Then, we modeled the three-dimensional structure of p53 motif through homology modeling and determined the binding affinity and stability of NBD-p53 motif complex structure via molecular docking and dynamics (MD simulation. Human DNA binding domain of p53 motif (SCMGGMNR retrieved from UniProt (UniProtKB: P04637 was docked with the NBD protein, using the Autodock version 4.2 program. The binding energy and intermolecular energy for the NBD-p53 motif complex were −0.44 Kcal/mol and −9.90 Kcal/mol, respectively. Moreover, RMSD, RMSF, hydrogen bonds, salt bridge, and secondary structure analyses revealed that the NBD protein had a strong bond with p53 motif and the protein-ligand complex was stable. Thus, the current data would be highly encouraging for designing Hsp70 structure based drug in cancer therapy.

  15. Mycobacterial PE_PGRS Proteins Contain Calcium-Binding Motifs with Parallel β-roll Folds

    Institute of Scientific and Technical Information of China (English)

    Nandita; Bachhawat; Balvinder; Singh

    2007-01-01

    The PE_PGRS family of proteins unique to mycobacteria is demonstrated to con- rain multiple calcium-binding and glycine-rich sequence motifs GGXGXD/NXUX. This sequence repeat constitutes a calcium-binding parallel/3-roll or parallel β-helix structure and is found in RTX toxins secreted by many Gram-negative bacteria. It is predicted that the highly homologous PE_PGRS proteins containing multiple copies of the nona-peptide motif could fold into similar calcium-binding structures. The implication of the predicted calcium-binding property of PE_PGRS proteins in the Ught of macrophage-pathogen interaction and pathogenesis is presented.

  16. Spodoptera frugiperda FKBP-46 is a consensus p53 motif binding protein.

    Science.gov (United States)

    Mohareer, Krishnaveni; Sahdev, Sudhir; Hasnain, Seyed E

    2013-04-01

    p53 protein, the central molecule of the apoptosis pathway, is mutated in 50% of the human cancers. Of late, p53 homologues have been identified from different invertebrates including Drosophila melanogaster, Caenorhabditis elegans, Squid, and Clams. We report the identification of a p53-like protein in Spodoptera frugiperda (Sf9) insect cells, which is activated during oxidative stress, caused by exposure to UV-B or H(2) O(2) , and binds to p53 consensus DNA binding motifs as well as other p53 cognate motifs. Sf9 p53 motif-binding protein is similar to murine and Drosophila p53 in terms of molecular size, which is around 50-60 kDa, as evident from UV cross-linking, and displays DNA binding characteristics similar to both insect and vertebrate p53 as seen from electrophoretic mobility shift assays. The N-terminal sequencing of the purified Sf9 p53 motif-binding protein reveals extensive homology to the pro-apoptotic FK-506 binding protein (FKBP-46), earlier identified in Sf9 cells as a factor which interacts with murine casein kinase. FKBP, an evolutionarily conserved protein of mammalian origin functions as a pro-apoptotic factor. Identification of FKBP-46 as a novel p53 motif-binding protein in insect cells adds a new facet to our understanding of the mechanisms of apoptosis under oxidative stress in the absence of a typical p53 homologue.

  17. Discovery and validation of information theory-based transcription factor and cofactor binding site motifs.

    Science.gov (United States)

    Lu, Ruipeng; Mucaki, Eliseos J; Rogan, Peter K

    2016-11-28

    Data from ChIP-seq experiments can derive the genome-wide binding specificities of transcription factors (TFs) and other regulatory proteins. We analyzed 765 ENCODE ChIP-seq peak datasets of 207 human TFs with a novel motif discovery pipeline based on recursive, thresholded entropy minimization. This approach, while obviating the need to compensate for skewed nucleotide composition, distinguishes true binding motifs from noise, quantifies the strengths of individual binding sites based on computed affinity and detects adjacent cofactor binding sites that coordinate with the targets of primary, immunoprecipitated TFs. We obtained contiguous and bipartite information theory-based position weight matrices (iPWMs) for 93 sequence-specific TFs, discovered 23 cofactor motifs for 127 TFs and revealed six high-confidence novel motifs. The reliability and accuracy of these iPWMs were determined via four independent validation methods, including the detection of experimentally proven binding sites, explanation of effects of characterized SNPs, comparison with previously published motifs and statistical analyses. We also predict previously unreported TF coregulatory interactions (e.g. TF complexes). These iPWMs constitute a powerful tool for predicting the effects of sequence variants in known binding sites, performing mutation analysis on regulatory SNPs and predicting previously unrecognized binding sites and target genes.

  18. A minimum of three motifs is essential for optimal binding of pseudomurein cell wall-binding domain of Methanothermobacter thermautotrophicus.

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    Ganesh Ram R Visweswaran

    Full Text Available We have biochemically and functionally characterized the pseudomurein cell wall-binding (PMB domain that is present at the C-terminus of the Surface (S-layer protein MTH719 from Methanothermobacter thermautotrophicus. Chemical denaturation of the protein with guanidinium hydrochloride occurred at 3.8 M. A PMB-GFP fusion protein not only binds to intact pseudomurein of methanogenic archaea, but also to spheroplasts of lysozyme-treated bacterial cells. This binding is pH dependent. At least two of the three motifs that are present in the domain are necessary for binding. Limited proteolysis revealed a possible cleavage site in the spacing sequence between motifs 1 and 2 of the PMB domain, indicating that the motif region itself is protected from proteases.

  19. 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.

  20. The Q Motif Is Involved in DNA Binding but Not ATP Binding in ChlR1 Helicase.

    Directory of Open Access Journals (Sweden)

    Hao Ding

    Full Text Available Helicases are molecular motors that couple the energy of ATP hydrolysis to the unwinding of structured DNA or RNA and chromatin remodeling. The conversion of energy derived from ATP hydrolysis into unwinding and remodeling is coordinated by seven sequence motifs (I, Ia, II, III, IV, V, and VI. The Q motif, consisting of nine amino acids (GFXXPXPIQ with an invariant glutamine (Q residue, has been identified in some, but not all helicases. Compared to the seven well-recognized conserved helicase motifs, the role of the Q motif is less acknowledged. Mutations in the human ChlR1 (DDX11 gene are associated with a unique genetic disorder known as Warsaw Breakage Syndrome, which is characterized by cellular defects in genome maintenance. To examine the roles of the Q motif in ChlR1 helicase, we performed site directed mutagenesis of glutamine to alanine at residue 23 in the Q motif of ChlR1. ChlR1 recombinant protein was overexpressed and purified from HEK293T cells. ChlR1-Q23A mutant abolished the helicase activity of ChlR1 and displayed reduced DNA binding ability. The mutant showed impaired ATPase activity but normal ATP binding. A thermal shift assay revealed that ChlR1-Q23A has a melting point value similar to ChlR1-WT. Partial proteolysis mapping demonstrated that ChlR1-WT and Q23A have a similar globular structure, although some subtle conformational differences in these two proteins are evident. Finally, we found ChlR1 exists and functions as a monomer in solution, which is different from FANCJ, in which the Q motif is involved in protein dimerization. Taken together, our results suggest that the Q motif is involved in DNA binding but not ATP binding in ChlR1 helicase.

  1. Functional diversification of paralogous transcription factors via divergence in DNA binding site motif and in expression.

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    Larry N Singh

    Full Text Available BACKGROUND: Gene duplication is a major driver of evolutionary innovation as it allows for an organism to elaborate its existing biological functions via specialization or diversification of initially redundant gene paralogs. Gene function can diversify in several ways. Transcription factor gene paralogs in particular, can diversify either by changes in their tissue-specific expression pattern or by changes in the DNA binding site motif recognized by their protein product, which in turn alters their gene targets. The relationship between these two modes of functional diversification of transcription factor paralogs has not been previously investigated, and is essential for understanding adaptive evolution of transcription factor gene families. FINDINGS: Based on a large set of human paralogous transcription factor pairs, we show that when the DNA binding site motifs of transcription factor paralogs are similar, the expressions of the genes that encode the paralogs have diverged, so in general, at most one of the paralogs is highly expressed in a tissue. Moreover, paralogs with diverged DNA binding site motifs tend to be diverged in their function. Conversely, two paralogs that are highly expressed in a tissue tend to have dissimilar DNA binding site motifs. We have also found that in general, within a paralogous family, tissue-specific decrease in gene expression is more frequent than what is expected by chance. CONCLUSIONS: While previous investigations of paralogous gene diversification have only considered coding sequence divergence, by explicitly quantifying divergence in DNA binding site motif, our work presents a new paradigm for investigating functional diversification. Consistent with evolutionary expectation, our quantitative analysis suggests that paralogous transcription factors have survived extinction in part, either through diversification of their DNA binding site motifs or through alterations in their tissue-specific expression

  2. Comparative Analysis of Regulatory Motif Discovery Tools for Transcription Factor Binding Sites

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    In the post-genomic era, identification of specific regulatory motifs or transcription factor binding sites (TFBSs) in non-coding DNA sequences, which is essential to elucidate transcriptional regulatory networks, has emerged as an obstacle that frustrates many researchers. Consequently, numerous motif discovery tools and correlated databases have been applied to solving this problem. However, these existing methods, based on different computational algorithms, show diverse motif prediction efficiency in non-coding DNA sequences. Therefore, understanding the similarities and differences of computational algorithms and enriching the motif discovery literatures are important for users to choose the most appropriate one among the online available tools. Moreover, there still lacks credible criterion to assess motif discovery tools and instructions for researchers to choose the best according to their own projects. Thus integration of the related resources might be a good approach to improve accuracy of the application. Recent studies integrate regulatory motif discovery tools with experimental methods to offer a complementary approach for researchers, and also provide a much-needed model for current researches on transcriptional regulatory networks. Here we present a comparative analysis of regulatory motif discovery tools for TFBSs.

  3. The extended AT-hook is a novel RNA binding motif.

    Science.gov (United States)

    Filarsky, Michael; Zillner, Karina; Araya, Ingrid; Villar-Garea, Ana; Merkl, Rainer; Längst, Gernot; Németh, Attila

    2015-01-01

    The AT-hook has been defined as a DNA binding peptide motif that contains a glycine-arginine-proline (G-R-P) tripeptide core flanked by basic amino acids. Recent reports documented variations in the sequence of AT-hooks and revealed RNA binding activity of some canonical AT-hooks, suggesting a higher structural and functional variability of this protein domain than previously anticipated. Here we describe the discovery and characterization of the extended AT-hook peptide motif (eAT-hook), in which basic amino acids appear symmetrical mainly at a distance of 12-15 amino acids from the G-R-P core. We identified 80 human and 60 mouse eAT-hook proteins and biochemically characterized the eAT-hooks of Tip5/BAZ2A, PTOV1 and GPBP1. Microscale thermophoresis and electrophoretic mobility shift assays reveal the nucleic acid binding features of this peptide motif, and show that eAT-hooks bind RNA with one order of magnitude higher affinity than DNA. In addition, cellular localization studies suggest a role for the N-terminal eAT-hook of PTOV1 in nucleocytoplasmic shuttling. In summary, our findings classify the eAT-hook as a novel nucleic acid binding motif, which potentially mediates various RNA-dependent cellular processes.

  4. MICU1 motifs define mitochondrial calcium uniporter binding and activity.

    Science.gov (United States)

    Hoffman, Nicholas E; Chandramoorthy, Harish C; Shamugapriya, Santhanam; Zhang, Xueqian; Rajan, Sudarsan; Mallilankaraman, Karthik; Gandhirajan, Rajesh Kumar; Vagnozzi, Ronald J; Ferrer, Lucas M; Sreekrishnanilayam, Krishnalatha; Natarajaseenivasan, Kalimuthusamy; Vallem, Sandhya; Force, Thomas; Choi, Eric T; Cheung, Joseph Y; Madesh, Muniswamy

    2013-12-26

    Resting mitochondrial matrix Ca(2+) is maintained through a mitochondrial calcium uptake 1 (MICU1)-established threshold inhibition of mitochondrial calcium uniporter (MCU) activity. It is not known how MICU1 interacts with MCU to establish this Ca(2+) threshold for mitochondrial Ca(2+) uptake and MCU activity. Here, we show that MICU1 localizes to the mitochondrial matrix side of the inner mitochondrial membrane and MICU1/MCU binding is determined by a MICU1 N-terminal polybasic domain and two interacting coiled-coil domains of MCU. Further investigation reveals that MICU1 forms homo-oligomers, and this oligomerization is independent of the polybasic region. However, the polybasic region confers MICU1 oligomeric binding to MCU and controls mitochondrial Ca(2+) current (IMCU). Moreover, MICU1 EF hands regulate MCU channel activity, but do not determine MCU binding. Loss of MICU1 promotes MCU activation leading to oxidative burden and a halt to cell migration. These studies establish a molecular mechanism for MICU1 control of MCU-mediated mitochondrial Ca(2+) accumulation, and dysregulation of this mechanism probably enhances vascular dysfunction.

  5. 24 CFR 990.275 - Project-based management (PBM).

    Science.gov (United States)

    2010-04-01

    ... 24 Housing and Urban Development 4 2010-04-01 2010-04-01 false Project-based management (PBM). 990... URBAN DEVELOPMENT THE PUBLIC HOUSING OPERATING FUND PROGRAM Asset Management § 990.275 Project-based... of rental housing at the project level. Under PBM, these property management services are arranged...

  6. Exploiting the peptidoglycan-binding motif, LysM, for medical and industrial applications

    NARCIS (Netherlands)

    Visweswaran, Ganesh Ram R.; Leenhouts, Kees; van Roosmalen, Maarten; Kok, Jan; Buist, Girbe

    The lysin motif (LysM) was first identified by Garvey et al. in 1986 and, in subsequent studies, has been shown to bind noncovalently to peptidoglycan and chitin by interacting with N-acetylglucosamine moieties. The LysM sequence is present singly or repeatedly in a large number of proteins of

  7. Exploiting the peptidoglycan-binding motif, LysM, for medical and industrial applications

    NARCIS (Netherlands)

    Visweswaran, Ganesh Ram R.; Leenhouts, Kees; van Roosmalen, Maarten; Kok, Jan; Buist, Girbe

    2014-01-01

    The lysin motif (LysM) was first identified by Garvey et al. in 1986 and, in subsequent studies, has been shown to bind noncovalently to peptidoglycan and chitin by interacting with N-acetylglucosamine moieties. The LysM sequence is present singly or repeatedly in a large number of proteins of proka

  8. Nuclear localization and pro-apoptotic signaling of YAP2 require intact PDZ-binding motif.

    Science.gov (United States)

    Oka, Tsutomu; Sudol, Marius

    2009-05-01

    The Hippo signaling pathway regulates the intrinsic size of organs by controlling two opposing processes, proliferation and apoptosis. The nuclear effector of this pathway is Yes kinase-associated protein (YAP) which is a WW domain-containing transcriptional co-activator. In addition to WW domains, YAP2 has a Post-synaptic density, Discs large, Zonula occludens-1 (PDZ)-binding motif that is located at its COOH terminus. To determine whether the localization of YAP2 in cells is PDZ-binding motif dependent, we generated a delta C mutant of YAP2 lacking the five most COOH terminal amino acids, -FLTWL, which constitute a well-conserved PDZ-binding motif. We report here that the PDZ-binding motif is necessary for YAP2 localization in the nucleus, for the stabilization of p73, and for promoting apoptosis of HEK293 cells maintained at low concentration of serum. We suggest that an unknown PDZ domain-containing protein (or proteins) functions as a shuttle, facilitating YAP2 translocation from the cytoplasm to the nucleus. Since the Hippo pathway acts as a tumor suppressor pathway, the PDZ complex of YAP represents a potential target of cancer therapy.

  9. Recombinant spider silk with cell binding motifs for specific adherence of cells.

    Science.gov (United States)

    Widhe, Mona; Johansson, Ulrika; Hillerdahl, Carl-Olof; Hedhammar, My

    2013-11-01

    Silk matrices have previously been shown to possess general properties governing cell viability. However, many cell types also require specific adhesion sites for successful in vitro culture. Herein, we have shown that cell binding motifs can be genetically fused to a partial spider silk protein, 4RepCT, without affecting its ability to self-assemble into stable matrices directly in a physiological-like buffer. The incorporated motifs were exposed in the formed matrices, and available for binding of integrins. Four different human primary cell types; fibroblasts, keratinocytes, endothelial cells and Schwann cells, were applied to the matrices and investigated under serum-free culture conditions. Silk matrices with cell binding motifs, especially RGD, were shown to promote early adherence of cells, which formed stress fibers and distinct focal adhesion points. Schwann cells acquired most spread-out morphology on silk matrices with IKVAV, where significantly more viable cells were found, also when compared to wells coated with laminin. This strategy is thus suitable for development of matrices that allow screening of various cell binding motifs and their effect on different cell types. © 2013 Elsevier Ltd. All rights reserved.

  10. Generation of high-performance binding proteins for peptide motifs by affinity clamping

    OpenAIRE

    Koide, Shohei; Huang, Jin

    2013-01-01

    We describe concepts and methodologies for generating “Affinity Clamps”, a new class of recombinant binding proteins that achieve high affinity and high specificity toward short peptide motifs of biological importance, which is a major challenge in protein engineering. The Affinity Clamping concept exploits the potential of nonhomologous recombination of protein domains in generating large changes in protein function and the inherent binding affinity and specificity of the so-called modular i...

  11. Coregulator control of androgen receptor action by a novel nuclear receptor-binding motif.

    Science.gov (United States)

    Jehle, Katja; Cato, Laura; Neeb, Antje; Muhle-Goll, Claudia; Jung, Nicole; Smith, Emmanuel W; Buzon, Victor; Carbó, Laia R; Estébanez-Perpiñá, Eva; Schmitz, Katja; Fruk, Ljiljana; Luy, Burkhard; Chen, Yu; Cox, Marc B; Bräse, Stefan; Brown, Myles; Cato, Andrew C B

    2014-03-28

    The androgen receptor (AR) is a ligand-activated transcription factor that is essential for prostate cancer development. It is activated by androgens through its ligand-binding domain (LBD), which consists predominantly of 11 α-helices. Upon ligand binding, the last helix is reorganized to an agonist conformation termed activator function-2 (AF-2) for coactivator binding. Several coactivators bind to the AF-2 pocket through conserved LXXLL or FXXLF sequences to enhance the activity of the receptor. Recently, a small compound-binding surface adjacent to AF-2 has been identified as an allosteric modulator of the AF-2 activity and is termed binding function-3 (BF-3). However, the role of BF-3 in vivo is currently unknown, and little is understood about what proteins can bind to it. Here we demonstrate that a duplicated GARRPR motif at the N terminus of the cochaperone Bag-1L functions through the BF-3 pocket. These findings are supported by the fact that a selective BF-3 inhibitor or mutations within the BF-3 pocket abolish the interaction between the GARRPR motif(s) and the BF-3. Conversely, amino acid exchanges in the two GARRPR motifs of Bag-1L can impair the interaction between Bag-1L and AR without altering the ability of Bag-1L to bind to chromatin. Furthermore, the mutant Bag-1L increases androgen-dependent activation of a subset of AR targets in a genome-wide transcriptome analysis, demonstrating a repressive function of the GARRPR/BF-3 interaction. We have therefore identified GARRPR as a novel BF-3 regulatory sequence important for fine-tuning the activity of the AR.

  12. Molecular diversity of LysM carbohydrate-binding motifs in fungi.

    Science.gov (United States)

    Akcapinar, Gunseli Bayram; Kappel, Lisa; Sezerman, Osman Ugur; Seidl-Seiboth, Verena

    2015-05-01

    LysM motifs are carbohydrate-binding modules found in prokaryotes and eukaryotes. They bind to N-acetylglucosamine-containing carbohydrates, such as chitin, chitio-oligosaccharides and peptidoglycan. In this review, we summarize the features of the protein architecture of LysM-containing proteins in fungi and discuss their so far known biochemical properties, transcriptional profiles and biological functions. Further, based on data from evolutionary analyses and consensus pattern profiling of fungal LysM motifs, we show that they can be classified into a fungal-specific group and a fungal/bacterial group. This facilitates the classification and selection of further LysM proteins for detailed analyses and will contribute to widening our understanding of the functional spectrum of this protein family in fungi. Fungal LysM motifs are predominantly found in subgroup C chitinases and in LysM effector proteins, which are secreted proteins with LysM motifs but no catalytic domains. In enzymes, LysM motifs mediate the attachment to insoluble carbon sources. In plants, receptors containing LysM motifs are responsible for the perception of chitin-oligosaccharides and are involved in beneficial symbiotic interactions between plants and bacteria or fungi, as well as plant defence responses. In plant pathogenic fungi, LysM effector proteins have already been shown to have important functions in the dampening of host defence responses as well as protective functions of fungal hyphae against chitinases. However, the large number and diversity of proteins with LysM motifs that are being unravelled in fungal genome sequencing projects suggest that the functional repertoire of LysM effector proteins in fungi is only partially discovered so far.

  13. Evolving Transcription Factor Binding Site Models From Protein Binding Microarray Data

    KAUST Repository

    Wong, Ka-Chun

    2016-02-02

    Protein binding microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner. In this paper, we describe the PBM motif model building problem. We apply several evolutionary computation methods and compare their performance with the interior point method, demonstrating their performance advantages. In addition, given the PBM domain knowledge, we propose and describe a novel method called kmerGA which makes domain-specific assumptions to exploit PBM data properties to build more accurate models than the other models built. The effectiveness and robustness of kmerGA is supported by comprehensive performance benchmarking on more than 200 datasets, time complexity analysis, convergence analysis, parameter analysis, and case studies. To demonstrate its utility further, kmerGA is applied to two real world applications: 1) PBM rotation testing and 2) ChIP-Seq peak sequence prediction. The results support the biological relevance of the models learned by kmerGA, and thus its real world applicability.

  14. Metal-binding and redox properties of substituted linear and cyclic ATCUN motifs.

    Science.gov (United States)

    Neupane, Kosh P; Aldous, Amanda R; Kritzer, Joshua A

    2014-10-01

    The amino-terminal copper and nickel binding (ATCUN) motif is a short peptide sequence found in human serum albumin and other proteins. Synthetic ATCUN-metal complexes have been used to oxidatively cleave proteins and DNA, cross-link proteins, and damage cancer cells. The ATCUN motif consists of a tripeptide that coordinates Cu(II) and Ni(II) ions in a square planar geometry, anchored by chelation sites at the N-terminal amine, histidine imidazole and two backbone amides. Many studies have shown that the histidine is required for tight binding and square planar geometry. Previously, we showed that macrocyclization of the ATCUN motif can lead to high-affinity binding with altered metal ion selectivity and enhanced Cu(II)/Cu(III) redox cycling (Inorg. Chem. 2013, 52, 2729-2735). In this work, we synthesize and characterize several linear and cyclic ATCUN variants to explore how substitutions at the histidine alter the metal-binding and catalytic properties. UV-visible spectroscopy, EPR spectroscopy and mass spectrometry indicate that cyclization can promote the formation of ATCUN-like complexes even in the absence of imidazole. We also report several novel ATCUN-like complexes and quantify their redox properties. These findings further demonstrate the effects of conformational constraints on short, metal-binding peptides, and also provide novel redox-active metallopeptides suitable for testing as catalysts for stereoselective or regioselective oxidation reactions.

  15. A Common Structural Motif in the Binding of Virulence Factors to Bacterial Secretion Chaperones

    Energy Technology Data Exchange (ETDEWEB)

    Lilic,M.; Vujanac, M.; Stebbins, C.

    2006-01-01

    Salmonella invasion protein A (SipA) is translocated into host cells by a type III secretion system (T3SS) and comprises two regions: one domain binds its cognate type III secretion chaperone, InvB, in the bacterium to facilitate translocation, while a second domain functions in the host cell, contributing to bacterial uptake by polymerizing actin. We present here the crystal structures of the SipA chaperone binding domain (CBD) alone and in complex with InvB. The SipA CBD is found to consist of a nonglobular polypeptide as well as a large globular domain, both of which are necessary for binding to InvB. We also identify a structural motif that may direct virulence factors to their cognate chaperones in a diverse range of pathogenic bacteria. Disruption of this structural motif leads to a destabilization of several chaperone-substrate complexes from different species, as well as an impairment of secretion in Salmonella.

  16. Discovery of widespread GTP-binding motifs in genomic DNA and RNA.

    Science.gov (United States)

    Curtis, Edward A; Liu, David R

    2013-04-18

    Biological RNAs that bind small molecules have been implicated in a variety of regulatory and catalytic processes. Inspired by these examples, we used in vitro selection to search a pool of genome-encoded RNA fragments for naturally occurring GTP aptamers. Several aptamer classes were identified, including one (the "G motif") with a G-quadruplex structure. Further analysis revealed that most RNA and DNA G-quadruplexes bind GTP. The G motif is abundant in eukaryotes, and the human genome contains ~75,000 examples with dissociation constants comparable to the GTP concentration of a eukaryotic cell (~300 μM). G-quadruplexes play roles in diverse cellular processes, and our findings raise the possibility that GTP may play a role in the function of these elements. Consistent with this possibility, the sequence requirements of several classes of regulatory G-quadruplexes parallel those of GTP binding.

  17. Transcription factor binding site positioning in yeast: proximal promoter motifs characterize TATA-less promoters.

    Science.gov (United States)

    Erb, Ionas; van Nimwegen, Erik

    2011-01-01

    The availability of sequence specificities for a substantial fraction of yeast's transcription factors and comparative genomic algorithms for binding site prediction has made it possible to comprehensively annotate transcription factor binding sites genome-wide. Here we use such a genome-wide annotation for comprehensively studying promoter architecture in yeast, focusing on the distribution of transcription factor binding sites relative to transcription start sites, and the architecture of TATA and TATA-less promoters. For most transcription factors, binding sites are positioned further upstream and vary over a wider range in TATA promoters than in TATA-less promoters. In contrast, a group of 6 'proximal promoter motifs' (GAT1/GLN3/DAL80, FKH1/2, PBF1/2, RPN4, NDT80, and ROX1) occur preferentially in TATA-less promoters and show a strong preference for binding close to the transcription start site in these promoters. We provide evidence that suggests that pre-initiation complexes are recruited at TATA sites in TATA promoters and at the sites of the other proximal promoter motifs in TATA-less promoters. TATA-less promoters can generally be classified by the proximal promoter motif they contain, with different classes of TATA-less promoters showing different patterns of transcription factor binding site positioning and nucleosome coverage. These observations suggest that different modes of regulation of transcription initiation may be operating in the different promoter classes. In addition we show that, across all promoter classes, there is a close match between nucleosome free regions and regions of highest transcription factor binding site density. This close agreement between transcription factor binding site density and nucleosome depletion suggests a direct and general competition between transcription factors and nucleosomes for binding to promoters.

  18. Transcription factor binding site positioning in yeast: proximal promoter motifs characterize TATA-less promoters.

    Directory of Open Access Journals (Sweden)

    Ionas Erb

    Full Text Available The availability of sequence specificities for a substantial fraction of yeast's transcription factors and comparative genomic algorithms for binding site prediction has made it possible to comprehensively annotate transcription factor binding sites genome-wide. Here we use such a genome-wide annotation for comprehensively studying promoter architecture in yeast, focusing on the distribution of transcription factor binding sites relative to transcription start sites, and the architecture of TATA and TATA-less promoters. For most transcription factors, binding sites are positioned further upstream and vary over a wider range in TATA promoters than in TATA-less promoters. In contrast, a group of 6 'proximal promoter motifs' (GAT1/GLN3/DAL80, FKH1/2, PBF1/2, RPN4, NDT80, and ROX1 occur preferentially in TATA-less promoters and show a strong preference for binding close to the transcription start site in these promoters. We provide evidence that suggests that pre-initiation complexes are recruited at TATA sites in TATA promoters and at the sites of the other proximal promoter motifs in TATA-less promoters. TATA-less promoters can generally be classified by the proximal promoter motif they contain, with different classes of TATA-less promoters showing different patterns of transcription factor binding site positioning and nucleosome coverage. These observations suggest that different modes of regulation of transcription initiation may be operating in the different promoter classes. In addition we show that, across all promoter classes, there is a close match between nucleosome free regions and regions of highest transcription factor binding site density. This close agreement between transcription factor binding site density and nucleosome depletion suggests a direct and general competition between transcription factors and nucleosomes for binding to promoters.

  19. Novel DNA motif binding activity observed in vivo with an estrogen receptor α mutant mouse.

    Science.gov (United States)

    Hewitt, Sylvia C; Li, Leping; Grimm, Sara A; Winuthayanon, Wipawee; Hamilton, Katherine J; Pockette, Brianna; Rubel, Cory A; Pedersen, Lars C; Fargo, David; Lanz, Rainer B; DeMayo, Francesco J; Schütz, Günther; Korach, Kenneth S

    2014-06-01

    Estrogen receptor α (ERα) interacts with DNA directly or indirectly via other transcription factors, referred to as "tethering." Evidence for tethering is based on in vitro studies and a widely used "KIKO" mouse model containing mutations that prevent direct estrogen response element DNA- binding. KIKO mice are infertile, due in part to the inability of estradiol (E2) to induce uterine epithelial proliferation. To elucidate the molecular events that prevent KIKO uterine growth, regulation of the pro-proliferative E2 target gene Klf4 and of Klf15, a progesterone (P4) target gene that opposes the pro-proliferative activity of KLF4, was evaluated. Klf4 induction was impaired in KIKO uteri; however, Klf15 was induced by E2 rather than by P4. Whole uterine chromatin immunoprecipitation-sequencing revealed enrichment of KIKO ERα binding to hormone response elements (HREs) motifs. KIKO binding to HRE motifs was verified using reporter gene and DNA-binding assays. Because the KIKO ERα has HRE DNA-binding activity, we evaluated the "EAAE" ERα, which has more severe DNA-binding domain mutations, and demonstrated a lack of estrogen response element or HRE reporter gene induction or DNA-binding. The EAAE mouse has an ERα null-like phenotype, with impaired uterine growth and transcriptional activity. Our findings demonstrate that the KIKO mouse model, which has been used by numerous investigators, cannot be used to establish biological functions for ERα tethering, because KIKO ERα effectively stimulates transcription using HRE motifs. The EAAE-ERα DNA-binding domain mutant mouse demonstrates that ERα DNA-binding is crucial for biological and transcriptional processes in reproductive tissues and that ERα tethering may not contribute to estrogen responsiveness in vivo.

  20. Structural basis for the binding of tryptophan-based motifs by δ-COP

    OpenAIRE

    Suckling, Richard J.; Poon, Pak P.; Travis, Sophie M.; Majoul, Irina V.; Hughson, Frederick M.; Evans, Philip R.; Duden, Rainer; Owen, David J.

    2015-01-01

    This is the author accepted manuscript. The final version is available from PNAS via http://dx.doi.org/10.1073/pnas.1506186112 Coatomer consists of two subcomplexes: the membrane-targeting, Arf1:GTP binding βγδζ-COP F-subcomplex, which is related to the AP clathrin adaptors, and the cargo binding αβ’ε-COP B-subcomplex. We present the structure of the C-terminal μ-homology domain of the yeast δ-COP subunit in complex with the WxW motif from its binding partner, the ER-localised Dsl1 tether....

  1. Identification of putative DnaN-binding motifs in plasmid replication initiation proteins.

    Science.gov (United States)

    Dalrymple, Brian P; Kongsuwan, Kritaya; Wijffels, Gene

    2007-01-01

    Recently the plasmid RK2 replication initiation protein, TrfA, has been shown to bind to the beta subunit of DNA Polymerase III (DnaN) via a short pentapeptide with the consensus QL[S/D]LF. A second consensus peptide, the hexapeptide QLxLxL, has also been demonstrated to mediate binding to DnaN. Here we describe the results of a comprehensive survey of replication initiation proteins encoded by bacterial plasmids to identify putative DnaN-binding sites. Both pentapeptide and hexapeptide motifs have been identified in a number of families of replication initiation proteins. The distribution of sites is sporadic and closely related families of proteins may differ in the presence, location, or type of putative DnaN-binding motif. Neither motif has been identified in replication initiation proteins encoded by plasmids that replicate via rolling circles or strand displacement. The results suggest that the recruitment of DnaN to the origin of replication of a replisome by plasmid replication initiation proteins is not generally required for plasmid replication, but that in some cases it may be beneficial for efficiency of replication initiation.

  2. UniPROBE, update 2015: new tools and content for the online database of protein-binding microarray data on protein–DNA interactions

    Science.gov (United States)

    Hume, Maxwell A.; Barrera, Luis A.; Gisselbrecht, Stephen S.; Bulyk, Martha L.

    2015-01-01

    The Universal PBM Resource for Oligonucleotide Binding Evaluation (UniPROBE) serves as a convenient source of information on published data generated using universal protein-binding microarray (PBM) technology, which provides in vitro data about the relative DNA-binding preferences of transcription factors for all possible sequence variants of a length k (‘k-mers’). The database displays important information about the proteins and displays their DNA-binding specificity data in terms of k-mers, position weight matrices and graphical sequence logos. This update to the database documents the growth of UniPROBE since the last update 4 years ago, and introduces a variety of new features and tools, including a new streamlined pipeline that facilitates data deposition by universal PBM data generators in the research community, a tool that generates putative nonbinding (i.e. negative control) DNA sequences for one or more proteins and novel motifs obtained by analyzing the PBM data using the BEEML-PBM algorithm for motif inference. The UniPROBE database is available at http://uniprobe.org. PMID:25378322

  3. ATtRACT-a database of RNA-binding proteins and associated motifs.

    Science.gov (United States)

    Giudice, Girolamo; Sánchez-Cabo, Fátima; Torroja, Carlos; Lara-Pezzi, Enrique

    2016-01-01

    RNA-binding proteins (RBPs) play a crucial role in key cellular processes, including RNA transport, splicing, polyadenylation and stability. Understanding the interaction between RBPs and RNA is key to improve our knowledge of RNA processing, localization and regulation in a global manner. Despite advances in recent years, a unified non-redundant resource that includes information on experimentally validated motifs, RBPs and integrated tools to exploit this information is lacking. Here, we developed a database named ATtRACT (available athttp://attract.cnic.es) that compiles information on 370 RBPs and 1583 RBP consensus binding motifs, 192 of which are not present in any other database. To populate ATtRACT we (i) extracted and hand-curated experimentally validated data from CISBP-RNA, SpliceAid-F, RBPDB databases, (ii) integrated and updated the unavailable ASD database and (iii) extracted information from Protein-RNA complexes present in Protein Data Bank database through computational analyses. ATtRACT provides also efficient algorithms to search a specific motif and scan one or more RNA sequences at a time. It also allows discoveringde novomotifs enriched in a set of related sequences and compare them with the motifs included in the database.Database URL:http:// attract. cnic. es.

  4. Comparative analyses of the thermodynamic RNA binding signatures of different types of RNA recognition motifs

    Science.gov (United States)

    Cléry, Antoine; Allain, Frédéric H-T

    2017-01-01

    Abstract RNA recognition motifs (RRMs) are structurally versatile domains important in regulation of alternative splicing. Structural mechanisms of sequence-specific recognition of single-stranded RNAs (ssRNAs) by RRMs are well understood. The thermodynamic strategies are however unclear. Therefore, we utilized microcalorimetry and semi-empirical analyses to comparatively analyze the cognate ssRNA binding thermodynamics of four different RRM domains, each with a different RNA binding mode. The different binding modes are: canonical binding to the β-sheet surface; canonical binding with involvement of N- and C-termini; binding to conserved loops; and binding to an α-helix. Our results identify enthalpy as the sole and general force driving association at physiological temperatures. Also, networks of weak interactions are a general feature regulating stability of the different RRM–ssRNA complexes. In agreement, non-polyelectrolyte effects contributed between ∼75 and 90% of the overall free energy of binding in the considered complexes. The various RNA binding modes also displayed enormous heat capacity differences, that upon dissection revealed large differential changes in hydration, conformations and dynamics upon binding RNA. Altogether, different modes employed by RRMs to bind cognate ssRNAs utilize various thermodynamics strategies during the association process. PMID:28334819

  5. Genome-Wide Motif Statistics are Shaped by DNA Binding Proteins over Evolutionary Time Scales

    Science.gov (United States)

    Qian, Long; Kussell, Edo

    2016-10-01

    The composition of a genome with respect to all possible short DNA motifs impacts the ability of DNA binding proteins to locate and bind their target sites. Since nonfunctional DNA binding can be detrimental to cellular functions and ultimately to organismal fitness, organisms could benefit from reducing the number of nonfunctional DNA binding sites genome wide. Using in vitro measurements of binding affinities for a large collection of DNA binding proteins, in multiple species, we detect a significant global avoidance of weak binding sites in genomes. We demonstrate that the underlying evolutionary process leaves a distinct genomic hallmark in that similar words have correlated frequencies, a signal that we detect in all species across domains of life. We consider the possibility that natural selection against weak binding sites contributes to this process, and using an evolutionary model we show that the strength of selection needed to maintain global word compositions is on the order of point mutation rates. Likewise, we show that evolutionary mechanisms based on interference of protein-DNA binding with replication and mutational repair processes could yield similar results and operate with similar rates. On the basis of these modeling and bioinformatic results, we conclude that genome-wide word compositions have been molded by DNA binding proteins acting through tiny evolutionary steps over time scales spanning millions of generations.

  6. LysM, a widely distributed protein motif for binding to (peptido)glycans.

    Science.gov (United States)

    Buist, Girbe; Steen, Anton; Kok, Jan; Kuipers, Oscar P

    2008-05-01

    Bacteria retain certain proteins at their cell envelopes by attaching them in a non-covalent manner to peptidoglycan, using specific protein domains, such as the prominent LysM (Lysin Motif) domain. More than 4000 (Pfam PF01476) proteins of both prokaryotes and eukaryotes have been found to contain one or more Lysin Motifs. Notably, this collection contains not only truly secreted proteins, but also (outer-)membrane proteins, lipoproteins or proteins bound to the cell wall in a (non-)covalent manner. The motif typically ranges in length from 44 to 65 amino acid residues and binds to various types of peptidoglycan and chitin, most likely recognizing the N-acetylglucosamine moiety. Most bacterial LysM-containing proteins are peptidoglycan hydrolases with various cleavage specificities. Binding of certain LysM proteins to cells of Gram-positive bacteria has been shown to occur at specific sites, as binding elsewhere is hindered by the presence of other cell wall components such as lipoteichoic acids. Interestingly, LysM domains of certain plant kinases enable the plant to recognize its symbiotic bacteria or sense and induce resistance against fungi. This interaction is triggered by chitin-like compounds that are secreted by the symbiotic bacteria or released from fungi, demonstrating an important sensing function of LysMs.

  7. DNA familial binding profiles made easy: comparison of various motif alignment and clustering strategies.

    Directory of Open Access Journals (Sweden)

    Shaun Mahony

    2007-03-01

    Full Text Available Transcription factor (TF proteins recognize a small number of DNA sequences with high specificity and control the expression of neighbouring genes. The evolution of TF binding preference has been the subject of a number of recent studies, in which generalized binding profiles have been introduced and used to improve the prediction of new target sites. Generalized profiles are generated by aligning and merging the individual profiles of related TFs. However, the distance metrics and alignment algorithms used to compare the binding profiles have not yet been fully explored or optimized. As a result, binding profiles depend on TF structural information and sometimes may ignore important distinctions between subfamilies. Prediction of the identity or the structural class of a protein that binds to a given DNA pattern will enhance the analysis of microarray and ChIP-chip data where frequently multiple putative targets of usually unknown TFs are predicted. Various comparison metrics and alignment algorithms are evaluated (a total of 105 combinations. We find that local alignments are generally better than global alignments at detecting eukaryotic DNA motif similarities, especially when combined with the sum of squared distances or Pearson's correlation coefficient comparison metrics. In addition, multiple-alignment strategies for binding profiles and tree-building methods are tested for their efficiency in constructing generalized binding models. A new method for automatic determination of the optimal number of clusters is developed and applied in the construction of a new set of familial binding profiles which improves upon TF classification accuracy. A software tool, STAMP, is developed to host all tested methods and make them publicly available. This work provides a high quality reference set of familial binding profiles and the first comprehensive platform for analysis of DNA profiles. Detecting similarities between DNA motifs is a key step in the

  8. A Review of Protein-DNA Binding Motif using Association Rule Mining

    Directory of Open Access Journals (Sweden)

    Virendra Kumar Tripathi

    2013-03-01

    Full Text Available The survival of gene regulation and life mechanisms is pre-request of finding unknown pattern of transcription factor binding sites. The discovery motif of gene regulation in bioinformatics is challenging jobs for getting relation between transcription factors and transcription factor binding sites. The increasing size and length of string pattern of motif is issued a problem related to modeling and optimization of gene selection process. In this paper we give a survey of protein-DNA binding using association rule mining. Association rule mining well known data mining technique for pattern analysis. The capability of negative and positive pattern generation help full for discovering of new pattern in DNA binding bioinformatics data. The other data mining approach such as clustering and classification also applied the process of gene selection grouping for known and unknown pattern. But faced a problem of valid string of DNA data, the rule mining principle find a better relation between transcription factors and transcription factor binding sites.

  9. Automatic generation of 3D motifs for classification of protein binding sites

    Directory of Open Access Journals (Sweden)

    Herzyk Pawel

    2007-08-01

    Full Text Available Abstract Background Since many of the new protein structures delivered by high-throughput processes do not have any known function, there is a need for structure-based prediction of protein function. Protein 3D structures can be clustered according to their fold or secondary structures to produce classes of some functional significance. A recent alternative has been to detect specific 3D motifs which are often associated to active sites. Unfortunately, there are very few known 3D motifs, which are usually the result of a manual process, compared to the number of sequential motifs already known. In this paper, we report a method to automatically generate 3D motifs of protein structure binding sites based on consensus atom positions and evaluate it on a set of adenine based ligands. Results Our new approach was validated by generating automatically 3D patterns for the main adenine based ligands, i.e. AMP, ADP and ATP. Out of the 18 detected patterns, only one, the ADP4 pattern, is not associated with well defined structural patterns. Moreover, most of the patterns could be classified as binding site 3D motifs. Literature research revealed that the ADP4 pattern actually corresponds to structural features which show complex evolutionary links between ligases and transferases. Therefore, all of the generated patterns prove to be meaningful. Each pattern was used to query all PDB proteins which bind either purine based or guanine based ligands, in order to evaluate the classification and annotation properties of the pattern. Overall, our 3D patterns matched 31% of proteins with adenine based ligands and 95.5% of them were classified correctly. Conclusion A new metric has been introduced allowing the classification of proteins according to the similarity of atomic environment of binding sites, and a methodology has been developed to automatically produce 3D patterns from that classification. A study of proteins binding adenine based ligands showed that

  10. A sialoreceptor binding motif in the Mycoplasma synoviae adhesin VlhA.

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    Meghan May

    Full Text Available Mycoplasma synoviae depends on its adhesin VlhA to mediate cytadherence to sialylated host cell receptors. Allelic variants of VlhA arise through recombination between an assemblage of promoterless vlhA pseudogenes and a single transcription promoter site, creating lineages of M. synoviae that each express a different vlhA allele. The predicted full-length VlhA sequences adjacent to the promoter of nine lineages of M. synoviae varying in avidity of cytadherence were aligned with that of the reference strain MS53 and with a 60-a.a. hemagglutinating VlhA C-terminal fragment from a Tunisian lineage of strain WVU1853(T. Seven different sequence variants of an imperfectly conserved, single-copy, 12-a.a. candidate cytadherence motif were evident amid the flanking variable residues of the 11 total sequences examined. The motif was predicted to adopt a short hairpin structure in a low-complexity region near the C-terminus of VlhA. Biotinylated synthetic oligopeptides representing four selected variants of the 12-a.a. motif, with the whole synthesized 60-a.a. fragment as a positive control, differed (P<0.01 in the extent they bound to chicken erythrocyte membranes. All bound to a greater extent (P<0.01 than scrambled or irrelevant VlhA domain negative control peptides did. Experimentally introduced branched-chain amino acid (BCAA substitutions Val3Ile and Leu7Ile did not significantly alter binding, whereas fold-destabilizing substitutions Thr4Gly and Ala9Gly tended to reduce it (P<0.05. Binding was also reduced to background levels (P<0.01 when the peptides were exposed to desialylated membranes, or were pre-saturated with free sialic acid before exposure to untreated membranes. From this evidence we conclude that the motif P-X-(BCAA-X-F-X-(BCAA-X-A-K-X-G binds sialic acid and likely mediates VlhA-dependent M. synoviae attachment to host cells. This conserved mechanism retains the potential for fine-scale rheostasis in binding avidity, which could be a

  11. Viral protein inhibits RISC activity by argonaute binding through conserved WG/GW motifs.

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    Ana Giner

    Full Text Available RNA silencing is an evolutionarily conserved sequence-specific gene-inactivation system that also functions as an antiviral mechanism in higher plants and insects. To overcome antiviral RNA silencing, viruses express silencing-suppressor proteins. These viral proteins can target one or more key points in the silencing machinery. Here we show that in Sweet potato mild mottle virus (SPMMV, type member of the Ipomovirus genus, family Potyviridae, the role of silencing suppressor is played by the P1 protein (the largest serine protease among all known potyvirids despite the presence in its genome of an HC-Pro protein, which, in potyviruses, acts as the suppressor. Using in vivo studies we have demonstrated that SPMMV P1 inhibits si/miRNA-programmed RISC activity. Inhibition of RISC activity occurs by binding P1 to mature high molecular weight RISC, as we have shown by immunoprecipitation. Our results revealed that P1 targets Argonaute1 (AGO1, the catalytic unit of RISC, and that suppressor/binding activities are localized at the N-terminal half of P1. In this region three WG/GW motifs were found resembling the AGO-binding linear peptide motif conserved in metazoans and plants. Site-directed mutagenesis proved that these three motifs are absolutely required for both binding and suppression of AGO1 function. In contrast to other viral silencing suppressors analyzed so far P1 inhibits both existing and de novo formed AGO1 containing RISC complexes. Thus P1 represents a novel RNA silencing suppressor mechanism. The discovery of the molecular bases of P1 mediated silencing suppression may help to get better insight into the function and assembly of the poorly explored multiprotein containing RISC.

  12. A novel fibronectin binding motif in MSCRAMMs targets F3 modules.

    Directory of Open Access Journals (Sweden)

    Sabitha Prabhakaran

    Full Text Available BACKGROUND: BBK32 is a surface expressed lipoprotein and fibronectin (Fn-binding microbial surface component recognizing adhesive matrix molecule (MSCRAMM of Borrelia burgdorferi, the causative agent of Lyme disease. Previous studies from our group showed that BBK32 is a virulence factor in experimental Lyme disease and located the Fn-binding region to residues 21-205 of the lipoprotein. METHODOLOGY/PRINCIPAL FINDINGS: Studies aimed at identifying interacting sites between BBK32 and Fn revealed an interaction between the MSCRAMM and the Fn F3 modules. Further analysis of this interaction showed that BBK32 can cause the aggregation of human plasma Fn in a similar concentration-dependent manner to that of anastellin, the superfibronectin (sFn inducing agent. The resulting Fn aggregates are conformationally distinct from plasma Fn as indicated by a change in available thermolysin cleavage sites. Recombinant BBK32 and anastellin affect the structure of Fn matrices formed by cultured fibroblasts and inhibit endothelial cell proliferation similarly. Within BBK32, we have located the sFn-forming activity to a region between residues 160 and 175 which contains two sequence motifs that are also found in anastellin. Synthetic peptides mimicking these motifs induce Fn aggregation, whereas a peptide with a scrambled sequence motif was inactive, suggesting that these motifs represent the sFn-inducing sequence. CONCLUSIONS/SIGNIFICANCE: We conclude that BBK32 induces the formation of Fn aggregates that are indistinguishable from those formed by anastellin. The results of this study provide evidence for how bacteria can target host proteins to manipulate host cell activities.

  13. Characterization of high affinity binding motifs for the discoidin domain receptor DDR2 in collagen.

    Science.gov (United States)

    Konitsiotis, Antonios D; Raynal, Nicolas; Bihan, Dominique; Hohenester, Erhard; Farndale, Richard W; Leitinger, Birgit

    2008-03-14

    The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases that are activated by native triple-helical collagen. Here we have located three specific DDR2 binding sites by screening the entire triple-helical domain of collagen II, using the Collagen II Toolkit, a set of overlapping triple-helical peptides. The peptide sequence that bound DDR2 with highest affinity interestingly contained the sequence for the high affinity binding site for von Willebrand factor in collagen III. Focusing on this sequence, we used a set of truncated and alanine-substituted peptides to characterize the sequence GVMGFO (O is hydroxyproline) as the minimal collagen sequence required for DDR2 binding. Based on a recent NMR analysis of the DDR2 collagen binding domain, we generated a model of the DDR2-collagen interaction that explains why a triple-helical conformation is required for binding. Triple-helical peptides comprising the DDR2 binding motif not only inhibited DDR2 binding to collagen II but also activated DDR2 transmembrane signaling. Thus, DDR2 activation may be effected by single triple-helices rather than fibrillar collagen.

  14. Trans-Regulation of RNA-Binding Protein Motifs by MicroRNA

    Directory of Open Access Journals (Sweden)

    Scott eTenenbaum

    2014-04-01

    Full Text Available The wide array of vital functions that RNA performs is dependent on its ability to dynamically fold into different structures in response to intracellular and extracellular changes. RNA-binding proteins regulate much of this activity by targeting specific RNA structures or motifs. One of these structures, the 3-way RNA junction, is characteristically found in ribosomal RNA and results from the RNA folding in cis, to produce three separate helices that meet around a central unpaired region. Here we demonstrate that 3-way junctions can also form in trans as a result of the binding of microRNAs in an unconventional manner with mRNA by splinting two non-contiguous regions together. This may be used to reinforce the base of a stem-loop motif being targeted by an RNA-binding protein. Trans interactions between non-coding RNA and mRNA may be used to control the post-transcriptional regulatory code and suggests a possible role for some of the recently described transcripts of unknown function expressed from the human genome.

  15. Proteomic screening method for phosphopeptide motif binding proteins using peptide libraries.

    Science.gov (United States)

    Christofk, Heather R; Wu, Ning; Cantley, Lewis C; Asara, John M

    2011-09-02

    Phosphopeptide binding domains mediate the directed and localized assembly of protein complexes essential to intracellular kinase signaling. To identify phosphopeptide binding proteins, we developed a proteomic screening method using immobilized partially degenerate phosphopeptide mixtures combined with SILAC and microcapillary LC-MS/MS. The method was used to identify proteins that specifically bound to phosphorylated peptide library affinity matrices, including pTyr, and the motifs pSer/pThr-Pro, pSer/pThr-X-X-X-pSer/pThr, pSer/pThr-Glu/Asp, or pSer/pThr-pSer/pThr in degenerate sequence contexts. Heavy and light SILAC lysates were applied to columns containing these phosphorylated and nonphosphorylated (control) peptide libraries respectively, and bound proteins were eluted, combined, digested, and analyzed by LC-MS/MS using a hybrid quadrupole-TOF mass spectrometer. Heavy/light peptide ion ratios were calculated, and peptides that yielded ratios greater than ∼3:1 were considered as being from potential phosphopeptide binding proteins since this ratio represents the lowest ratio from a known positive control. Many of those identified were known phosphopeptide-binding proteins, including the SH2 domain containing p85 subunit of PI3K bound to pTyr, 14-3-3 bound to pSer/pThr-Asp/Glu, polo-box domain containing PLK1 and Pin1 bound to pSer/pThr-Pro, and pyruvate kinase M2 binding to pTyr. Approximately half of the hits identified by the peptide library screens were novel. Protein domain enrichment analysis revealed that most pTyr hits contain SH2 domains, as expected, and to a lesser extent SH3, C1, STAT, Tyr phosphatase, Pkinase, C2, and PH domains; however, pSer/pThr motifs did not reveal enriched domains across hits.

  16. Binding Mode of Acetylated Histones to Bromodomains: Variations on a Common Motif.

    Science.gov (United States)

    Marchand, Jean-Rémy; Caflisch, Amedeo

    2015-08-01

    Bromodomains, epigenetic readers that recognize acetylated lysine residues in histone tails, are potential drug targets in cancer and inflammation. Herein we review the crystal structures of human bromodomains in complex with histone tails and analyze the main interaction motifs. The histone backbone is extended and occupies, in one of the two possible orientations, the bromodomain surface groove lined by the ZA and BC loops. The acetyl-lysine side chain is buried in the cavity between the four helices of the bromodomain, and its oxygen atom accepts hydrogen bonds from a structural water molecule and a conserved asparagine residue in the BC loop. In stark contrast to this common binding motif, a large variety of ancillary interactions emerge from our analysis. In 10 of 26 structures, a basic side chain (up to five residues up- or downstream in sequence with respect to the acetyl-lysine) interacts with the carbonyl groups of the C-terminal turn of helix αB. Furthermore, the complexes reveal many heterogeneous backbone hydrogen bonds (direct or water-bridged). These interactions contribute unselectively to the binding of acetylated histone tails to bromodomains, which provides further evidence that specific recognition is modulated by combinations of multiple histone modifications and multiple modules of the proteins involved in transcription. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. A calmodulin binding protein from Arabidopsis is induced by ethylene and contains a DNA-binding motif

    Science.gov (United States)

    Reddy, A. S.; Reddy, V. S.; Golovkin, M.

    2000-01-01

    Calmodulin (CaM), a key calcium sensor in all eukaryotes, regulates diverse cellular processes by interacting with other proteins. To isolate CaM binding proteins involved in ethylene signal transduction, we screened an expression library prepared from ethylene-treated Arabidopsis seedlings with 35S-labeled CaM. A cDNA clone, EICBP (Ethylene-Induced CaM Binding Protein), encoding a protein that interacts with activated CaM was isolated in this screening. The CaM binding domain in EICBP was mapped to the C-terminus of the protein. These results indicate that calcium, through CaM, could regulate the activity of EICBP. The EICBP is expressed in different tissues and its expression in seedlings is induced by ethylene. The EICBP contains, in addition to a CaM binding domain, several features that are typical of transcription factors. These include a DNA-binding domain at the N terminus, an acidic region at the C terminus, and nuclear localization signals. In database searches a partial cDNA (CG-1) encoding a DNA-binding motif from parsley and an ethylene up-regulated partial cDNA from tomato (ER66) showed significant similarity to EICBP. In addition, five hypothetical proteins in the Arabidopsis genome also showed a very high sequence similarity with EICBP, indicating that there are several EICBP-related proteins in Arabidopsis. The structural features of EICBP are conserved in all EICBP-related proteins in Arabidopsis, suggesting that they may constitute a new family of DNA binding proteins and are likely to be involved in modulating gene expression in the presence of ethylene.

  18. Crystal structure of bacterial cell-surface alginate-binding protein with an M75 peptidase motif

    Energy Technology Data Exchange (ETDEWEB)

    Maruyama, Yukie; Ochiai, Akihito [Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Mikami, Bunzo [Laboratory of Applied Structural Biology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Hashimoto, Wataru [Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Murata, Kousaku, E-mail: kmurata@kais.kyoto-u.ac.jp [Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan)

    2011-02-18

    Research highlights: {yields} Bacterial alginate-binding Algp7 is similar to component EfeO of Fe{sup 2+} transporter. {yields} We determined the crystal structure of Algp7 with a metal-binding motif. {yields} Algp7 consists of two helical bundles formed through duplication of a single bundle. {yields} A deep cleft involved in alginate binding locates around the metal-binding site. {yields} Algp7 may function as a Fe{sup 2+}-chelated alginate-binding protein. -- Abstract: A gram-negative Sphingomonas sp. A1 directly incorporates alginate polysaccharide into the cytoplasm via the cell-surface pit and ABC transporter. A cell-surface alginate-binding protein, Algp7, functions as a concentrator of the polysaccharide in the pit. Based on the primary structure and genetic organization in the bacterial genome, Algp7 was found to be homologous to an M75 peptidase motif-containing EfeO, a component of a ferrous ion transporter. Despite the presence of an M75 peptidase motif with high similarity, the Algp7 protein purified from recombinant Escherichia coli cells was inert on insulin B chain and N-benzoyl-Phe-Val-Arg-p-nitroanilide, both of which are substrates for a typical M75 peptidase, imelysin, from Pseudomonas aeruginosa. The X-ray crystallographic structure of Algp7 was determined at 2.10 A resolution by single-wavelength anomalous diffraction. Although a metal-binding motif, HxxE, conserved in zinc ion-dependent M75 peptidases is also found in Algp7, the crystal structure of Algp7 contains no metal even at the motif. The protein consists of two structurally similar up-and-down helical bundles as the basic scaffold. A deep cleft between the bundles is sufficiently large to accommodate macromolecules such as alginate polysaccharide. This is the first structural report on a bacterial cell-surface alginate-binding protein with an M75 peptidase motif.

  19. A phosphoserine/threonine-binding pocket in AGC kinases and PDK1 mediates activation by hydrophobic motif phosphorylation

    DEFF Research Database (Denmark)

    Frödin, Morten; Antal, Torben L; Dümmler, Bettina A;

    2002-01-01

    The growth factor-activated AGC protein kinases RSK, S6K, PKB, MSK and SGK are activated by serine/threonine phosphorylation in the activation loop and in the hydrophobic motif, C-terminal to the kinase domain. In some of these kinases, phosphorylation of the hydrophobic motif creates a specific...... docking site that recruits and activates PDK1, which then phosphorylates the activation loop. Here, we discover a pocket in the kinase domain of PDK1 that recognizes the phosphoserine/phosphothreonine in the hydrophobic motif by identifying two oppositely positioned arginine and lysine residues that bind...... the phosphate. Moreover, we demonstrate that RSK2, S6K1, PKBalpha, MSK1 and SGK1 contain a similar phosphate-binding pocket, which they use for intramolecular interaction with their own phosphorylated hydrophobic motif. Molecular modelling and experimental data provide evidence for a common activation mechanism...

  20. A method for identifying a proposed carbohydrate-binding motif of proteins.

    Science.gov (United States)

    Baumann, M A; Anderson, B E

    1991-11-01

    An examination of the binding sites of four carbohydrate binding proteins (Escherichia coli lactose repressor, E. coli arabinose-binding protein, yeast hexokinase A and Concanavalin A) revealed certain similarities of amino acid sequences and residues forming hydrogen bonds and hydrophobic interactions with the bound carbohydrate. These were: (i) Asx-Asx, hydrogen bonding to the pyranose ring oxygen and anomeric-OH group; (ii) Arg-X-X-X-(Ser/Thr), or the reverse sequence, with the Arg hydrogen bonding to the pyranose ring oxygen; (iii) Lys-(Ser/Thr)-X-X-Asp, or the reverse sequence and with interchange of the Lys-(Ser/Thr) positions, with hydrogen bonding of either or both the Lys and Asp residues to the -OH groups at carbons 2, 3, 4 or 6; (iv) a diaromatic sequence with possible hydrophobic interactions to the faces of the pyranose ring structure. An algorithm was devised to search the amino acid sequences of a large number of proteins, those known to bind carbohydrates as well as those without known carbohydrate-binding activities, for the four amino acid sequence criteria. The algorithm incorporated a weighted distance value (WDV) to assess the approximate distance between any two criteria, with the WDV being based on the predicted secondary structure of the protein amino acid sequence. When the algorithm using criteria 1 and 2 plus the WDV was applied to the sequences of 125 proteins, the method indicated the presence of the potential carbohydrate-binding site motif for 42% of proteins with known carbohydrate binding, only 8% of proteins were predicted as false positives, and the accuracy of the method was calculated to be 61.6%.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. Identification of HI-like loop in CELO adenovirus fiber for incorporation of receptor binding motifs.

    Science.gov (United States)

    Logunov, Denis Y; Zubkova, Olga V; Karyagina-Zhulina, Anna S; Shuvalova, Eugenia A; Karpov, Andrei P; Shmarov, Maxim M; Tutykhina, Irina L; Alyapkina, Yulia S; Grezina, Natalia M; Zinovieva, Natalia A; Ernst, Lev K; Gintsburg, Alexsandr L; Naroditsky, Boris S

    2007-09-01

    Vectors based on the chicken embryo lethal orphan (CELO) avian adenovirus (Ad) have two attractive properties for gene transfer applications: resistance to preformed immune responses to human Ads and the ability to grow in chicken embryos, allowing low-cost production of recombinant viruses. However, a major limitation of this technology is that CELO vectors demonstrate decreased efficiency of gene transfer into cells expressing low levels of the coxsackie-Ad receptor (CAR). In order to improve the efficacy of gene transfer into CAR-deficient cells, we modified viral tropism via genetic alteration of the CELO fiber 1 protein. The alphav integrin-binding motif (RGD) was incorporated at two different sites of the fiber 1 knob domain, within an HI-like loop that we identified and at the C terminus. Recombinant fiber-modified CELO viruses were constructed containing secreted alkaline phosphatase (SEAP) and enhanced green fluorescent protein genes as reporter genes. Our data show that insertion of the RGD motif within the HI-like loop of the fiber resulted in significant enhancement of gene transfer into CAR-negative and CAR-deficient cells. In contrast, CELO vectors containing the RGD motif at the fiber 1 C terminus showed reduced transduction of all cell lines. CELO viruses modified with RGD at the HI-like loop transduced the SEAP reporter gene into rabbit mammary gland cells in vivo with an efficiency significantly greater than that of unmodified CELO vector and similar to that of Ad type 5 vector. These results illustrate the potential for efficient CELO-mediated gene transfer into a broad range of cell types through modification of the identified HI-like loop of the fiber 1 protein.

  2. Model-based Comparative Prediction of Transcription-Factor Binding Motifs in Anabolic Responses in Bone

    Institute of Scientific and Technical Information of China (English)

    Andy; B.; Chen; Kazunori; Hamamura; Guohua; Wang; Weirong; Xing; Subburaman; Mohan; Hiroki; Yokota; Yunlong; Liu

    2007-01-01

    Understanding the regulatory mechanism that controls the alteration of global gene expression patterns continues to be a challenging task in computational biology. We previously developed an ant algorithm, a biologically-inspired computational technique for microarray data, and predicted putative transcription-factor binding motifs (TFBMs) through mimicking interactive behaviors of natural ants. Here we extended the algorithm into a set of web-based software, Ant Modeler, and applied it to investigate the transcriptional mechanism underlying bone formation. Mechanical loading and administration of bone morphogenic proteins (BMPs) are two known treatments to strengthen bone. We addressed a question: Is there any TFBM that stimulates both "anabolic responses of mechanical loading" and "BMP-mediated osteogenic signaling"? Although there is no significant overlap among genes in the two responses, a comparative model-based analysis suggests that the two independent osteogenic processes employ common TFBMs, such as a stress responsive element and a motif for peroxisome proliferator-activated recep- tor (PPAR). The post-modeling in vitro analysis using mouse osteoblast cells sup- ported involvements of the predicted TFBMs such as PPAR, Ikaros 3, and LMO2 in response to mechanical loading. Taken together, the results would be useful to derive a set of testable hypotheses and examine the role of specific regulators in complex transcriptional control of bone formation.

  3. Improved bioactivity of antimicrobial peptides by addition of amino-terminal copper and nickel (ATCUN) binding motifs.

    Science.gov (United States)

    Libardo, M Daben; Cervantes, Jorge L; Salazar, Juan C; Angeles-Boza, Alfredo M

    2014-08-01

    Antimicrobial peptides (AMPs) are promising candidates to help circumvent antibiotic resistance, which is an increasing clinical problem. Amino-terminal copper and nickel (ATCUN) binding motifs are known to actively form reactive oxygen species (ROS) upon metal binding. The combination of these two peptidic constructs could lead to a novel class of dual-acting antimicrobial agents. To test this hypothesis, a set of ATCUN binding motifs were screened for their ability to induce ROS formation, and the most potent were then used to modify AMPs with different modes of action. ATCUN binding motif-containing derivatives of anoplin (GLLKRIKTLL-NH2), pro-apoptotic peptide (PAP; KLAKLAKKLAKLAK-NH2), and sh-buforin (RAGLQFPVGRVHRLLRK-NH2) were synthesized and found to be more active than the parent AMPs against a panel of clinically relevant bacteria. The lower minimum inhibitory concentration (MIC) values for the ATCUN-anoplin peptides are attributed to the higher pore-forming activity along with their ability to cause ROS-induced membrane damage. The addition of the ATCUN motifs to PAP also increases its ability to disrupt membranes. DNA damage is the major contributor to the activity of the ATCUN-sh-buforin peptides. Our findings indicate that the addition of ATCUN motifs to AMPs is a simple strategy that leads to AMPs with higher antibacterial activity and possibly to more potent, usable antibacterial agents.

  4. Novel Strategy for Discrimination of Transcription Factor Binding Motifs Employing Mathematical Neural Network

    Science.gov (United States)

    Sugimoto, Asuka; Sumi, Takuya; Kang, Jiyoung; Tateno, Masaru

    2017-07-01

    Recognition in biological macromolecular systems, such as DNA-protein recognition, is one of the most crucial problems to solve toward understanding the fundamental mechanisms of various biological processes. Since specific base sequences of genome DNA are discriminated by proteins, such as transcription factors (TFs), finding TF binding motifs (TFBMs) in whole genome DNA sequences is currently a central issue in interdisciplinary biophysical and information sciences. In the present study, a novel strategy to create a discriminant function for discrimination of TFBMs by constituting mathematical neural networks (NNs) is proposed, together with a method to determine the boundary of signals (TFBMs) and noise in the NN-score (output) space. This analysis also leads to the mathematical limitation of discrimination in the recognition of features representing TFBMs, in an information geometrical manifold. Thus, the present strategy enables the identification of the whole space of TFBMs, right up to the noise boundary.

  5. Identification of Ubiquinol Binding Motifs at the Qo-Site of the Cytochrome bc1 Complex

    DEFF Research Database (Denmark)

    Barragan, Angela M.; Crofts, Antony R.; Schulten, Klaus;

    2015-01-01

    for the function of the bc1 complex is the initial redox process that involves a bifurcated electron transfer in which the two electrons from a quinol substrate are passed to different electron acceptors in the bc1 complex. The electron transfer is coupled to proton transfer. The overall mechanism of quinol......Enzymes of the bc1 complex family power the biosphere through their central role in respiration and photosynthesis. These enzymes couple the oxidation of quinol molecules by cytochrome c to the transfer of protons across the membrane, to generate a proton-motive force that drives ATP synthesis. Key...... oxidation by the bc1 complex is well enough characterized to allow exploration at the atomistic level, but details are still highly controversial. The controversy stems from the uncertain binding motifs of quinol at the so-called Qo active site of the bc1 complex. Here we employ a combination of classical...

  6. A poxvirus protein with a RING finger motif binds zinc and localizes in virus factories.

    Science.gov (United States)

    Upton, C; Schiff, L; Rice, S A; Dowdeswell, T; Yang, X; McFadden, G

    1994-07-01

    Shope fibroma virus (SFV) is a Leporipoxvirus closely related to the highly virulent myxoma virus. The DNA sequence of the BamHI N fragment of the SFV DNA genome was determined, and the single complete open reading frame (N1R) was characterized. The protein encoded by the N1R gene was found to contain a C3HC4 RING finger motif at the C terminus. This C3HC4 motif is the hallmark of a growing family of proteins, many of which are involved in regulation of gene expression, DNA repair, or DNA recombination. Complete homologs of the SFV N1R gene were also detected in variola virus, myxoma virus, and vaccinia virus strain IHD-W. In contrast, the gene is completely absent from vaccinia virus strain Copenhagen, and in vaccinia virus strain WR, the open reading frame is truncated prior to the zinc binding domain because of an 11-bp deletion, thus producing a frameshift and premature stop codon. Recombinant N1R protein from SFV was expressed in Escherichia coli and shown to bind zinc in a specific manner. Using fluorescence microscopy to visualize a peptide epitope tag (derived from ICP27 of herpes simplex virus) fused to the N terminus of the poxvirus proteins, we observed that the N1R protein of SFV and its homologs in myxoma virus and vaccinia virus IHD-W were localized primarily to the virus factories in the cytoplasm of infected cells and, to a lesser degree, the host cell nucleus. The truncated protein of vaccinia virus strain WR failed to localize in this manner but instead was observed throughout the cytoplasm.

  7. In vitro evolution of a peptide with a hematite binding motif that may constitute a natural metal-oxide binding archetype.

    Science.gov (United States)

    Lower, Brian H; Lins, Roberto D; Oestreicher, Zachery; Straatsma, Tjerk P; Hochella, Michael F; Shi, Liang; Lower, Steven K

    2008-05-15

    Phage-display technology was used to evolve peptides that selectively bind to the metal-oxide hematite (Fe2O3) from a library of approximately 3 billion different polypeptides. The sequences of these peptides contained the highly conserved amino acid motif, Ser/Thr-hydrophobic/aromatic-Ser/Thr-Pro-Ser/Thr. To better understand the nature of the peptide-metal oxide binding demonstrated by these experiments, molecular dynamics simulations were carried out for Ser-Pro-Ser at a hematite surface. These simulations show that hydrogen bonding occurs between the two serine amino acids and the hydroxylated hematite surface and that the presence of proline between the hydroxide residues restricts the peptide flexibility, thereby inducing a structural-binding motif. A search of published sequence data revealed that the binding motif (Ser/Thr-Pro-Ser/Thr) is adjacent to the terminal heme-binding domain of both OmcA and MtrC, which are outer membrane cytochromes from the metal-reducing bacterium Shewanella oneidensis MR-1. The entire five amino acid consensus sequence (Ser/Thr-hydrophobic/ aromatic-Ser/Thr-Pro-Ser/Thr) was also found as multiple copies in the primary sequences of metal-oxide binding proteins Sil1 and Sil2 from Thalassiosira pseudonana. We suggest that this motif constitutes a natural metal-oxide binding archetype that could be exploited in enzyme-based biofuel cell design and approaches to synthesize tailored metal-oxide nanostructures.

  8. Structural motif screening reveals a novel, conserved carbohydrate-binding surface in the pathogenesis-related protein PR-5d

    Directory of Open Access Journals (Sweden)

    Moffatt Barbara A

    2010-08-01

    Full Text Available Abstract Background Aromatic amino acids play a critical role in protein-glycan interactions. Clusters of surface aromatic residues and their features may therefore be useful in distinguishing glycan-binding sites as well as predicting novel glycan-binding proteins. In this work, a structural bioinformatics approach was used to screen the Protein Data Bank (PDB for coplanar aromatic motifs similar to those found in known glycan-binding proteins. Results The proteins identified in the screen were significantly associated with carbohydrate-related functions according to gene ontology (GO enrichment analysis, and predicted motifs were found frequently within novel folds and glycan-binding sites not included in the training set. In addition to numerous binding sites predicted in structural genomics proteins of unknown function, one novel prediction was a surface motif (W34/W36/W192 in the tobacco pathogenesis-related protein, PR-5d. Phylogenetic analysis revealed that the surface motif is exclusive to a subfamily of PR-5 proteins from the Solanaceae family of plants, and is absent completely in more distant homologs. To confirm PR-5d's insoluble-polysaccharide binding activity, a cellulose-pulldown assay of tobacco proteins was performed and PR-5d was identified in the cellulose-binding fraction by mass spectrometry. Conclusions Based on the combined results, we propose that the putative binding site in PR-5d may be an evolutionary adaptation of Solanaceae plants including potato, tomato, and tobacco, towards defense against cellulose-containing pathogens such as species of the deadly oomycete genus, Phytophthora. More generally, the results demonstrate that coplanar aromatic clusters on protein surfaces are a structural signature of glycan-binding proteins, and can be used to computationally predict novel glycan-binding proteins from 3 D structure.

  9. HLA DR4w4-binding motifs illustrate the biochemical basis of degeneracy and specificity in peptide-DR interactions.

    Science.gov (United States)

    Sette, A; Sidney, J; Oseroff, C; del Guercio, M F; Southwood, S; Arrhenius, T; Powell, M F; Colón, S M; Gaeta, F C; Grey, H M

    1993-09-15

    In the present study, we describe the definition of a DRB1*0401 (DR4w4)-specific motif. The strategy used entailed a three-step process: 1) screening a large set of unrelated peptide ligands to identify good MHC binders; 2) truncation analysis of several DR4w4 binding peptides of high affinity to identify the crucial core-binding regions; 3) the use of single amino acid substitutions of the DR4w4-binding peptide hemagglutinin (HA) 307-319 to elucidate the specific residues crucial for binding. The DR4w4 motif is characterized by the presence of a hydrophobic or aromatic (F, W, Y, L, I, V, M) anchor residue in position 1, and a second hydroxyl (S, T) or aliphatic (L, I, V, or M) anchor residue in position 6. Furthermore, positive charges (R, K) are not allowed in positions 4, 7, and 9, and negative charges (D, E) are not allowed in position 9. This motif was present in 92% of good (IC50 45 microM) binders, indicating that the presence of the motif is necessary, but not sufficient for good DR4w4 binding capacity. The results of the present study are discussed in relation to previous work defining binding motifs and rules for other DR alleles, illustrating how different DR alleles bind variations on a similar structural theme. Finally, using two different peptide ligands [tetanus toxoid 830-843 and HA 307-319] as model systems, it is demonstrated how the fine allelic specificity of the DR binders can be predictably modulated by introducing subtle changes in the primary amino acid sequence.

  10. Computational identification of transcription factor binding sites by functional analysis of sets of genes sharing overrep-resented upstream motifs

    Directory of Open Access Journals (Sweden)

    Silengo Lorenzo

    2004-05-01

    Full Text Available Abstract Background Transcriptional regulation is a key mechanism in the functioning of the cell, and is mostly effected through transcription factors binding to specific recognition motifs located upstream of the coding region of the regulated gene. The computational identification of such motifs is made easier by the fact that they often appear several times in the upstream region of the regulated genes, so that the number of occurrences of relevant motifs is often significantly larger than expected by pure chance. Results To exploit this fact, we construct sets of genes characterized by the statistical overrepresentation of a certain motif in their upstream regions. Then we study the functional characterization of these sets by analyzing their annotation to Gene Ontology terms. For the sets showing a statistically significant specific functional characterization, we conjecture that the upstream motif characterizing the set is a binding site for a transcription factor involved in the regulation of the genes in the set. Conclusions The method we propose is able to identify many known binding sites in S. cerevisiae and new candidate targets of regulation by known transcritpion factors. Its application to less well studied organisms is likely to be valuable in the exploration of their regulatory interaction network.

  11. Hyaluronan binding motifs of USP17 and SDS3 exhibit anti-tumor activity.

    Directory of Open Access Journals (Sweden)

    Suresh Ramakrishna

    Full Text Available BACKGROUND: We previously reported that the USP17 deubiquitinating enzyme having hyaluronan binding motifs (HABMs interacts with human SDS3 (suppressor of defective silencing 3 and specifically deubiquitinates Lys-63 branched polyubiquitination of SDS3 resulting in negative regulation of histone deacetylase (HDAC activity in cancer cells. Furthermore, USP17 and SDS3 mutually interact with each other to block cell proliferation in HeLa cells but the mechanism for this inhibition in cell proliferation is not known. We wished to investigate whether the HABMs of USP17 were responsible for tumor suppression activity. METHODOLOGY/PRINCIPAL FINDINGS: Similarly to USP17, we have identified that SDS3 also has three consecutive HABMs and shows direct binding with hyaluronan (HA using cetylpyridinium chloride (CPC assay. Additionally, HA oligosaccharides (6-18 sugar units competitively block binding of endogenous HA polymer to HA binding proteins. Thus, administration of HA oligosaccharides antagonizes the interaction between HA and USP17 or SDS3. Interestingly, HABMs deleted USP17 showed lesser interaction with SDS3 but retain its deubiquitinating activity towards SDS3. The deletion of HABMs of USP17 could not alter its functional regulation on SDS3-associated HDAC activity. Furthermore, to explore whether HABMs in USP17 and SDS3 are responsible for the inhibition of cell proliferation, we investigated the effect of USP17 and SDS3-lacking HABMs on cell proliferation by soft agar, apoptosis, cell migration and cell proliferation assays. CONCLUSIONS: Our results have demonstrated that these HABMs in USP17 and its substrate SDS3 are mainly involved in the inhibition of anchorage-independent tumor growth.

  12. Enhanced Binding Affinity for an i-Motif DNA Substrate Exhibited by a Protein Containing Nucleobase Amino Acids.

    Science.gov (United States)

    Bai, Xiaoguang; Talukder, Poulami; Daskalova, Sasha M; Roy, Basab; Chen, Shengxi; Li, Zhongxian; Dedkova, Larisa M; Hecht, Sidney M

    2017-03-17

    Several variants of a nucleic acid binding motif (RRM1) of putative transcription factor hnRNP LL containing nucleobase amino acids at specific positions have been prepared and used to study binding affinity for the BCL2 i-motif DNA. Molecular modeling suggested a number of amino acids in RRM1 likely to be involved in interaction with the i-motif DNA, and His24 and Arg26 were chosen for modification based on their potential ability to interact with G14 of the i-motif DNA. Four nucleobase amino acids were introduced into RRM1 at one or both of positions 24 and 26. The introduction of cytosine nucleobase 2 into position 24 of RRM1 increased the affinity of the modified protein for the i-motif DNA, consistent with the possible Watson-Crick interaction of 2 and G14. In comparison, the introduction of uracil nucleobase 3 had a minimal effect on DNA affinity. Two structurally simplified nucleobase analogues (1 and 4) lacking both the N-1 and the 2-oxo substituents were also introduced in lieu of His24. Again, the RRM1 analogue containing 1 exhibited enhanced affinity for the i-motif DNA, while the protein analogue containing 4 bound less tightly to the DNA substrate. Finally, the modified protein containing 1 in lieu of Arg26 also bound to the i-motif DNA more strongly than the wild-type protein, but a protein containing 1 both at positions 24 and 26 bound to the DNA less strongly than wild type. The results support the idea of using nucleobase amino acids as protein constituents for controlling and enhancing DNA-protein interaction. Finally, modification of the i-motif DNA at G14 diminished RRM1-DNA interaction, as well as the ability of nucleobase amino acid 1 to stabilize RRM1-DNA interaction.

  13. Exploiting the peptidoglycan-binding motif, LysM, for medical and industrial applications.

    Science.gov (United States)

    Visweswaran, Ganesh Ram R; Leenhouts, Kees; van Roosmalen, Maarten; Kok, Jan; Buist, Girbe

    2014-05-01

    The lysin motif (LysM) was first identified by Garvey et al. in 1986 and, in subsequent studies, has been shown to bind noncovalently to peptidoglycan and chitin by interacting with N-acetylglucosamine moieties. The LysM sequence is present singly or repeatedly in a large number of proteins of prokaryotes and eukaryotes. Since the mid-1990s, domains containing one or more of these LysM sequences originating from different LysM-containing proteins have been examined for purely scientific reasons as well as for their possible use in various medical and industrial applications. These studies range from detecting localized binding of LysM-containing proteins onto bacteria to actual bacterial cell surface analysis. On a more applied level, the possibilities of employing the LysM domains for cell immobilization, for the display of peptides, proteins, or enzymes on (bacterial) surfaces as well as their utility in the development of novel vaccines have been scrutinized. To serve these purposes, the chimeric proteins containing one or more of the LysM sequences have been produced and isolated from various prokaryotic and eukaryotic expression hosts. This review gives a succinct overview of the characteristics of the LysM domain and of current developments in its application potential.

  14. The First Residue of the PWWP Motif Modulates HATH Domain Binding, Stability, and Protein-Protein Interaction.

    Science.gov (United States)

    Hung, Yi-Lin; Lee, Hsia-Ju; Jiang, Ingjye; Lin, Shang-Chi; Lo, Wei-Cheng; Lin, Yi-Jan; Sue, Shih-Che

    2015-07-01

    Hepatoma-derived growth factor (hHDGF) and HDGF-related proteins (HRPs) contain conserved N-terminal HATH domains with a characteristic structural motif, namely the PWWP motif. The HATH domain has attracted attention because of its ability to bind with heparin/heparan sulfate, DNA, and methylated histone peptide. Depending on the sequence of the PWWP motif, HRP HATHs are classified into P-type (Pro-His-Trp-Pro) and A-type (Ala-His-Trp-Pro) forms. A-type HATH is highly unstable and tends to precipitate in solution. We replaced the Pro residue in P-type HATHHDGF with Ala and evaluated the influence on structure, dynamics, and ligand binding. Nuclear magnetic resonance (NMR) hydrogen/deuterium exchange and circular dichroism (CD) measurements revealed reduced stability. Analysis of NMR backbone (15)N relaxations (R1, R2, and nuclear Overhauser effect) revealed additional backbone dynamics in the interface between the β-barrel and the C-terminal helix bundle. The β1-β2 loop, where the AHWP sequence is located, has great structural flexibility, which aids HATH-HATH interaction through the loop. A-type HATH, therefore, shows a stronger tendency to aggregate when binding with heparin and DNA oligomers. This study defines the role of the first residue of the PWWP motif in modulating HATH domain stability and oligomer formation in binding.

  15. Mammalian cell-based optimization of the biarsenical-binding tetracysteine motif for improved fluorescence and affinity

    NARCIS (Netherlands)

    Martin, Brent R; Giepmans, Ben N G; Adams, Stephen R; Tsien, Roger Y

    2005-01-01

    Membrane-permeant biarsenical dyes such as FlAsH and ReAsH fluoresce upon binding to genetically encoded tetracysteine motifs expressed in living cells, yet spontaneous nonspecific background staining can prevent detection of weakly expressed or dilute proteins. If the affinity of the tetracysteine

  16. The CXXC motifs in the metal binding domains are required for ATP7B to mediate resistance to cisplatin.

    Science.gov (United States)

    Safaei, Roohangiz; Adams, Preston L; Maktabi, Mohammad H; Mathews, Ryan A; Howell, Stephen B

    2012-05-01

    The copper (Cu) exporter ATP7B mediates resistance to cisplatin (cDDP) but details of the mechanism are unknown. We explored the role of the CXXC motifs in the metal binding domains (MBDs) of ATP7B by investigating binding of cDDP to the sixth metal binding domain (MBD6) or a variant in which the CXXC motif was converted to SXXS. Platinum measurement showed that cDDP bound to wild type MBD6 but not to the SXXS variant. Wild type ATP7B rendered ovarian 2008 cells resistant to cDDP. In 2008 and in HEK293T cells, wild type ATP7B trafficked from TGN to peripheral locations in response to Cu or cDDP. A variant in which the CXXC motifs in all 6 MBDs were converted to SXXS localized correctly to the TGN but failed to traffic when exposed to either Cu or cDDP. Deletion of either the first 5 MBDs or all 6 MBDs resulted in failure to localize to the TGN. Neither the SXXS variant nor the deletion variant was able to mediate resistance to cDDP. We conclude that cDDP binds to the CXXC motifs of ATP7B and that this interaction is essential to the trafficking of ATP7B and to its ability to mediate resistance to cDDP. Copyright © 2012 Elsevier Inc. All rights reserved.

  17. The CXXC motifs in the metal binding domains are required for ATP7B to mediate resistance to cisplatin☆

    Science.gov (United States)

    Safaei, Roohangiz; Adams, Preston L.; Maktabi, Mohammad H.; Mathews, Ryan A.; Howell, Stephen B.

    2013-01-01

    The copper (Cu) exporter ATP7B mediates resistance to cisplatin (cDDP) but details of the mechanism are unknown. We explored the role of the CXXC motifs in the metal binding domains (MBDs) of ATP7B by investigating binding of cDDP to the sixth metal binding domain (MBD6) or a variant in which the CXXC motif was converted to SXXS. Platinum measurement showed that cDDP bound to wild type MBD6 but not to the SXXS variant. Wild type ATP7B rendered ovarian 2008 cells resistant to cDDP. In 2008 and in HEK293T cells, wild type ATP7B trafficked from TGN to peripheral locations in response to Cu or cDDP. A variant in which the CXXC motifs in all 6 MBDs were converted to SXXS localized correctly to the TGN but failed to traffic when exposed to either Cu or cDDP. Deletion of either the first 5 MBDs or all 6 MBDs resulted in failure to localize to the TGN. Neither the SXXS variant nor the deletion variant was able to mediate resistance to cDDP. We conclude that cDDP binds to the CXXC motifs of ATP7B and that this interaction is essential to the trafficking of ATP7B and to its ability to mediate resistance to cDDP. PMID:22459168

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

    Science.gov (United States)

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

    2013-01-01

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

  19. RNA-protein binding motifs mining with a new hybrid deep learning based cross-domain knowledge integration approach.

    Science.gov (United States)

    Pan, Xiaoyong; Shen, Hong-Bin

    2017-02-28

    RNAs play key roles in cells through the interactions with proteins known as the RNA-binding proteins (RBP) and their binding motifs enable crucial understanding of the post-transcriptional regulation of RNAs. How the RBPs correctly recognize the target RNAs and why they bind specific positions is still far from clear. Machine learning-based algorithms are widely acknowledged to be capable of speeding up this process. Although many automatic tools have been developed to predict the RNA-protein binding sites from the rapidly growing multi-resource data, e.g. sequence, structure, their domain specific features and formats have posed significant computational challenges. One of current difficulties is that the cross-source shared common knowledge is at a higher abstraction level beyond the observed data, resulting in a low efficiency of direct integration of observed data across domains. The other difficulty is how to interpret the prediction results. Existing approaches tend to terminate after outputting the potential discrete binding sites on the sequences, but how to assemble them into the meaningful binding motifs is a topic worth of further investigation. In viewing of these challenges, we propose a deep learning-based framework (iDeep) by using a novel hybrid convolutional neural network and deep belief network to predict the RBP interaction sites and motifs on RNAs. This new protocol is featured by transforming the original observed data into a high-level abstraction feature space using multiple layers of learning blocks, where the shared representations across different domains are integrated. To validate our iDeep method, we performed experiments on 31 large-scale CLIP-seq datasets, and our results show that by integrating multiple sources of data, the average AUC can be improved by 8% compared to the best single-source-based predictor; and through cross-domain knowledge integration at an abstraction level, it outperforms the state-of-the-art predictors by 6

  20. Human transcriptional coactivator with PDZ-binding motif (TAZ) is downregulated during decidualization.

    Science.gov (United States)

    Strakova, Zuzana; Reed, Jennifer; Ihnatovych, Ivanna

    2010-06-01

    Transcriptional coactivator with PDZ-binding motif (TAZ) is known to bind to a variety of transcription factors to control cell differentiation and organ development. However, its role in uterine physiology has not yet been described. To study its regulation during the unique process of differentiation of fibroblasts into decidual cells (decidualization), we utilized the human uterine fibroblast (HuF) in vitro cell model. Immunocytochemistry data demonstrated that the majority of the TAZ protein is localized in the nucleus. Treatment of HuF cells with the embryonic stimulus cytokine interleukin 1 beta in the presence of steroid hormones (estradiol-17 beta and medroxyprogesterone acetate) for 13 days did not cause any apparent TAZ mRNA changes but resulted in a significant TAZ protein decline (approximately 62%) in total cell lysates. Analysis of cytosolic and nuclear extracts revealed that the decline of total TAZ was caused primarily by a drop of TAZ protein levels in the nucleus. TAZ was localized on the peroxisome proliferator-activated receptor response element site (located at position -1200 bp relative to the transcription start site) of the genomic region of decidualization marker insulin-like growth factor-binding protein 1 (IGFBP1) in HuF cells as detected by chromatin immunoprecipitation. TAZ is also present in human endometrium tissue as confirmed by immunohistochemistry. During the secretory phase of the menstrual cycle, specific TAZ staining particularly diminishes in the stroma, suggesting its participation during the decidualization process, as well as implantation. During early baboon pregnancy, TAZ protein expression remains minimal in the endometrium close to the implantation site. In summary, the presented evidence shows for the first time to date TAZ protein in the human uterine tract, its downregulation during in vitro decidualization, and its localization on the IGFBP1 promoter region, all of which indicate its presence in the uterine

  1. Hinderin, a five-domains protein including coiled-coil motifs that binds to SMC3

    Directory of Open Access Journals (Sweden)

    Ghiselli Giancarlo

    2005-01-01

    Full Text Available Abstract Background The structural maintenance of chromosome proteins SMC1 and SMC3 play an important role in the maintenance of chromosomal integrity by preventing the premature separation of the sister chromatids at the onset of anaphase. The two proteins are constitutive components of the multimeric complex cohesin and form dimers by interacting at their central globular regions. Results In order to identify proteins that by binding to SMC3 may interfere with the protein dimerization process, a human cDNA library was screened by the yeast two-hybrid system by using the hinge region of SMC3 as bait. This has lead to the identification of Hinderin, a novel five domains protein including two coiled-coil motifs and sharing a strikingly structural similarity to the SMC family of proteins. Hinderin is ubiquitously expressed in human tissues. Orthologue forms of the protein are present in other vertebrates but not in lower organisms. A mapping of the interaction sites revealed that the N- and C-terminal globular domains mediate the binding of Hinderin to SMC3. Hinderin/SMC3 complexes could be recovered by immunoprecipitation from cell lysates using an anti-SMC3 antibody, thus demonstrating that the two proteins interact in vivo. On the contrary, Hinderin did not interact with SMC1. In vivo the rate of SMC1/SMC3 interaction was decreased by the ectopic expression of Hinderin. Conclusions Hinderin is a novel binding partner of SMC3. Based on its ability to modulate SMC1/SMC3 interaction we postulate that Hinderin affects the availability of SMC3 to engage in the formation of multimeric protein complexes.

  2. DNA consensus sequence motif for binding response regulator PhoP, a virulence regulator of Mycobacterium tuberculosis.

    Science.gov (United States)

    He, Xiaoyuan; Wang, Shuishu

    2014-12-30

    Tuberculosis has reemerged as a serious threat to human health because of the increasing prevalence of drug-resistant strains and synergetic infection with HIV, prompting an urgent need for new and more efficient treatments. The PhoP-PhoR two-component system of Mycobacterium tuberculosis plays an important role in the virulence of the pathogen and thus represents a potential drug target. To study the mechanism of gene transcription regulation by response regulator PhoP, we identified a high-affinity DNA sequence for PhoP binding using systematic evolution of ligands by exponential enrichment. The sequence contains a direct repeat of two 7 bp motifs separated by a 4 bp spacer, TCACAGC(N4)TCACAGC. The specificity of the direct-repeat sequence for PhoP binding was confirmed by isothermal titration calorimetry and electrophoretic mobility shift assays. PhoP binds to the direct repeat as a dimer in a highly cooperative manner. We found many genes previously identified to be regulated by PhoP that contain the direct-repeat motif in their promoter sequences. Synthetic DNA fragments at the putative promoter-binding sites bind PhoP with variable affinity, which is related to the number of mismatches in the 7 bp motifs, the positions of the mismatches, and the spacer and flanking sequences. Phosphorylation of PhoP increases the affinity but does not change the specificity of DNA binding. Overall, our results confirm the direct-repeat sequence as the consensus motif for PhoP binding and thus pave the way for identification of PhoP directly regulated genes in different mycobacterial genomes.

  3. An unusual helix turn helix motif in the catalytic core of HIV-1 integrase binds viral DNA and LEDGF.

    Directory of Open Access Journals (Sweden)

    Hayate Merad

    Full Text Available BACKGROUND: Integrase (IN of the type 1 human immunodeficiency virus (HIV-1 catalyzes the integration of viral DNA into host cellular DNA. We identified a bi-helix motif (residues 149-186 in the crystal structure of the catalytic core (CC of the IN-Phe185Lys variant that consists of the alpha(4 and alpha(5 helices connected by a 3 to 5-residue turn. The motif is embedded in a large array of interactions that stabilize the monomer and the dimer. PRINCIPAL FINDINGS: We describe the conformational and binding properties of the corresponding synthetic peptide. This displays features of the protein motif structure thanks to the mutual intramolecular interactions of the alpha(4 and alpha(5 helices that maintain the fold. The main properties are the binding to: 1- the processing-attachment site at the LTR (long terminal repeat ends of virus DNA with a K(d (dissociation constant in the sub-micromolar range; 2- the whole IN enzyme; and 3- the IN binding domain (IBD but not the IBD-Asp366Asn variant of LEDGF (lens epidermal derived growth factor lacking the essential Asp366 residue. In our motif, in contrast to the conventional HTH (helix-turn-helix, it is the N terminal helix (alpha(4 which has the role of DNA recognition helix, while the C terminal helix (alpha(5 would rather contribute to the motif stabilization by interactions with the alpha(4 helix. CONCLUSION: The motif, termed HTHi (i, for inverted emerges as a central piece of the IN structure and function. It could therefore represent an attractive target in the search for inhibitors working at the DNA-IN, IN-IN and IN-LEDGF interfaces.

  4. Prediction of DNA binding motifs from 3D models of transcription factors; identifying TLX3 regulated genes.

    Science.gov (United States)

    Pujato, Mario; Kieken, Fabien; Skiles, Amanda A; Tapinos, Nikos; Fiser, Andras

    2014-12-16

    Proper cell functioning depends on the precise spatio-temporal expression of its genetic material. Gene expression is controlled to a great extent by sequence-specific transcription factors (TFs). Our current knowledge on where and how TFs bind and associate to regulate gene expression is incomplete. A structure-based computational algorithm (TF2DNA) is developed to identify binding specificities of TFs. The method constructs homology models of TFs bound to DNA and assesses the relative binding affinity for all possible DNA sequences using a knowledge-based potential, after optimization in a molecular mechanics force field. TF2DNA predictions were benchmarked against experimentally determined binding motifs. Success rates range from 45% to 81% and primarily depend on the sequence identity of aligned target sequences and template structures, TF2DNA was used to predict 1321 motifs for 1825 putative human TF proteins, facilitating the reconstruction of most of the human gene regulatory network. As an illustration, the predicted DNA binding site for the poorly characterized T-cell leukemia homeobox 3 (TLX3) TF was confirmed with gel shift assay experiments. TLX3 motif searches in human promoter regions identified a group of genes enriched in functions relating to hematopoiesis, tissue morphology, endocrine system and connective tissue development and function. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. DNase I-hypersensitive sites and transcription factor-binding motifs within the mouse E beta meiotic recombination hot spot.

    Science.gov (United States)

    Shenkar, R; Shen, M H; Arnheim, N

    1991-04-01

    The second intron of the E beta gene in the mouse major histocompatibility complex is the site of a meiotic recombination hot spot. We detected two DNase I-hypersensitive sites in this intron in meiotic cells isolated from mouse testes. One site appears to be constitutive and is found in other tissues regardless of whether or not they express the E beta gene. Near this hypersensitive site are potential binding motifs for H2TF1/KBF1, NF kappa B, and octamer transcription factors. Gel retardation studies with mouse lymphoma cell nuclear extracts confirmed that each of these motifs is capable of binding protein. The binding of transcription factors may contribute to the enhancement of recombination potential by altering chromatin structure and increasing the accessibility of the DNA to the recombination machinery.

  6. The PDZ-binding motif of Yes-associated protein is required for its co-activation of TEAD-mediated CTGF transcription and oncogenic cell transforming activity

    Energy Technology Data Exchange (ETDEWEB)

    Shimomura, Tadanori; Miyamura, Norio; Hata, Shoji; Miura, Ryota; Hirayama, Jun, E-mail: hirayama.dbio@mri.tmd.ac.jp; Nishina, Hiroshi, E-mail: nishina.dbio@mri.tmd.ac.jp

    2014-01-17

    Highlights: •Loss of the PDZ-binding motif inhibits constitutively active YAP (5SA)-induced oncogenic cell transformation. •The PDZ-binding motif of YAP promotes its nuclear localization in cultured cells and mouse liver. •Loss of the PDZ-binding motif inhibits YAP (5SA)-induced CTGF transcription in cultured cells and mouse liver. -- Abstract: YAP is a transcriptional co-activator that acts downstream of the Hippo signaling pathway and regulates multiple cellular processes, including proliferation. Hippo pathway-dependent phosphorylation of YAP negatively regulates its function. Conversely, attenuation of Hippo-mediated phosphorylation of YAP increases its ability to stimulate proliferation and eventually induces oncogenic transformation. The C-terminus of YAP contains a highly conserved PDZ-binding motif that regulates YAP’s functions in multiple ways. However, to date, the importance of the PDZ-binding motif to the oncogenic cell transforming activity of YAP has not been determined. In this study, we disrupted the PDZ-binding motif in the YAP (5SA) protein, in which the sites normally targeted by Hippo pathway-dependent phosphorylation are mutated. We found that loss of the PDZ-binding motif significantly inhibited the oncogenic transformation of cultured cells induced by YAP (5SA). In addition, the increased nuclear localization of YAP (5SA) and its enhanced activation of TEAD-dependent transcription of the cell proliferation gene CTGF were strongly reduced when the PDZ-binding motif was deleted. Similarly, in mouse liver, deletion of the PDZ-binding motif suppressed nuclear localization of YAP (5SA) and YAP (5SA)-induced CTGF expression. Taken together, our results indicate that the PDZ-binding motif of YAP is critical for YAP-mediated oncogenesis, and that this effect is mediated by YAP’s co-activation of TEAD-mediated CTGF transcription.

  7. A new calmodulin-binding motif for inositol 1,4,5-trisphosphate 3-kinase regulation.

    Science.gov (United States)

    Franco-Echevarría, Elsa; Baños-Sanz, Jose I; Monterroso, Begoña; Round, Adam; Sanz-Aparicio, Julia; González, Beatriz

    2014-11-01

    IP3-3K [Ins(1,4,5)P3 3-kinase] is a key enzyme that catalyses the synthesis of Ins(1,3,4,5)P4, using Ins(1,4,5)P3 and ATP as substrates. Both inositides, substrate and product, present crucial roles in the cell. Ins(1,4,5)P3 is a key point in Ca2+ metabolism that promotes Ca2+ release from intracellular stores and together with Ins(1,3,4,5)P4 regulates Ca2+ homoeostasis. In addition, Ins(1,3,4,5)P4 is involved in immune cell development. It has been proved that Ca2+/CaM (calmodulin) regulates the activity of IP3-3K, via direct interaction between both enzymes. Although we have extensive structural knowledge of the kinase domains of the three IP3-3K isoforms, no structural information is available about the interaction between IP3-3K and Ca2+/CaM. In the present paper we describe the crystal structure of the complex between human Ca2+/CaM and the CaM-binding region of human IP3-3K isoform A (residues 158-183) and propose a model for a complex including the kinase domain. The structure obtained allowed us to identify all of the key residues involved in the interaction, which have been evaluated by site-directed mutagenesis, pull-down and fluorescence anisotropy experiments. The results allowed the identification of a new CaM-binding motif, expanding our knowledge about how CaM interacts with its partners.

  8. Conserved retinoblastoma protein-binding motif in human cytomegalovirus UL97 kinase minimally impacts viral replication but affects susceptibility to maribavir

    Directory of Open Access Journals (Sweden)

    Chou Sunwen

    2009-01-01

    Full Text Available Abstract The UL97 kinase has been shown to phosphorylate and inactivate the retinoblastoma protein (Rb and has three consensus Rb-binding motifs that might contribute to this activity. Recombinant viruses containing mutations in the Rb-binding motifs generally replicated well in human foreskin fibroblasts with only a slight delay in replication kinetics. Their susceptibility to the specific UL97 kinase inhibitor, maribavir, was also examined. Mutation of the amino terminal motif, which is involved in the inactivation of Rb, also renders the virus hypersensitive to the drug and suggests that the motif may play a role in its mechanism of action.

  9. The PDZ-binding motif of Yes-associated protein is required for its co-activation of TEAD-mediated CTGF transcription and oncogenic cell transforming activity.

    Science.gov (United States)

    Shimomura, Tadanori; Miyamura, Norio; Hata, Shoji; Miura, Ryota; Hirayama, Jun; Nishina, Hiroshi

    2014-01-17

    YAP is a transcriptional co-activator that acts downstream of the Hippo signaling pathway and regulates multiple cellular processes, including proliferation. Hippo pathway-dependent phosphorylation of YAP negatively regulates its function. Conversely, attenuation of Hippo-mediated phosphorylation of YAP increases its ability to stimulate proliferation and eventually induces oncogenic transformation. The C-terminus of YAP contains a highly conserved PDZ-binding motif that regulates YAP's functions in multiple ways. However, to date, the importance of the PDZ-binding motif to the oncogenic cell transforming activity of YAP has not been determined. In this study, we disrupted the PDZ-binding motif in the YAP (5SA) protein, in which the sites normally targeted by Hippo pathway-dependent phosphorylation are mutated. We found that loss of the PDZ-binding motif significantly inhibited the oncogenic transformation of cultured cells induced by YAP (5SA). In addition, the increased nuclear localization of YAP (5SA) and its enhanced activation of TEAD-dependent transcription of the cell proliferation gene CTGF were strongly reduced when the PDZ-binding motif was deleted. Similarly, in mouse liver, deletion of the PDZ-binding motif suppressed nuclear localization of YAP (5SA) and YAP (5SA)-induced CTGF expression. Taken together, our results indicate that the PDZ-binding motif of YAP is critical for YAP-mediated oncogenesis, and that this effect is mediated by YAP's co-activation of TEAD-mediated CTGF transcription.

  10. Characterizing the binding motifs of 11 common human HLA‐DP and HLA‐DQ molecules using NNAlign

    DEFF Research Database (Denmark)

    Andreatta, Massimo; Nielsen, Morten

    2012-01-01

    in the pattern of amino acid preferences at core positions, with conserved hydrophobic/aromatic anchors at P1 and P6, and an additional hydrophobic anchor at P9 in some variants. These results confirm the existence of a previously hypothesized supertype encompassing the most common DP alleles. Conversely......, the binding motifs for DQ molecules appear more divergent, displaying unconventional anchor positions and in some cases rather unspecific amino acid preferences....

  11. Mutation of the conserved calcium-binding motif in Neisseria gonorrhoeae PilC1 impacts adhesion but not piliation.

    Science.gov (United States)

    Cheng, Yuan; Johnson, Michael D L; Burillo-Kirch, Christine; Mocny, Jeffrey C; Anderson, James E; Garrett, Christopher K; Redinbo, Matthew R; Thomas, Christopher E

    2013-11-01

    Neisseria gonorrhoeae PilC1 is a member of the PilC family of type IV pilus-associated adhesins found in Neisseria species and other type IV pilus-producing genera. Previously, a calcium-binding domain was described in the C-terminal domains of PilY1 of Pseudomonas aeruginosa and in PilC1 and PilC2 of Kingella kingae. Genetic analysis of N. gonorrhoeae revealed a similar calcium-binding motif in PilC1. To evaluate the potential significance of this calcium-binding region in N. gonorrhoeae, we produced recombinant full-length PilC1 and a PilC1 C-terminal domain fragment. We show that, while alterations of the calcium-binding motif disrupted the ability of PilC1 to bind calcium, they did not grossly affect the secondary structure of the protein. Furthermore, we demonstrate that both full-length wild-type PilC1 and full-length calcium-binding-deficient PilC1 inhibited gonococcal adherence to cultured human cervical epithelial cells, unlike the truncated PilC1 C-terminal domain. Similar to PilC1 in K. kingae, but in contrast to the calcium-binding mutant of P. aeruginosa PilY1, an equivalent mutation in N. gonorrhoeae PilC1 produced normal amounts of pili. However, the N. gonorrhoeae PilC1 calcium-binding mutant still had partial defects in gonococcal adhesion to ME180 cells and genetic transformation, which are both essential virulence factors in this human pathogen. Thus, we conclude that calcium binding to PilC1 plays a critical role in pilus function in N. gonorrhoeae.

  12. Enhancement of binding activity of soluble human CD40 to CD40 ligand through incorporation of an isoleucine zipper motif

    Institute of Scientific and Technical Information of China (English)

    Xian-hui HE; Li-hui XU; Yi LIU

    2006-01-01

    Aim:To investigate the effect of incorporation of all isoleucine zipper(IZ)motif into CD40 on binding activity of CD40 for the CD40 ligand (CD40L).Methods:Prokaryotic expression vectors for 2 soluble CD40 derivatives,shCD40His and shCD40IZ containing an IZ dowain,were constructed and expressed in Escherichia coli.The recombinant proteins were purified to homogeneity after refolding from inclusion bodies.Their molecular weights in solution of shCD40His and shCD40IZ were compared by size-exclusion chromatography,and their binding activity for CD40L on Jurkat T cells was determined by flow cytometry.Results:shCD40His and shCD40IZ were generated.Both of them possessed significant binding activity for the cognate ligand CD40L expressed on the cell surface.shCD40IZ had much higher binding activity to its ligand(CD40L)than did shCD40His.Furthermore,size-exclusion chromatography demonstrated that shCD40IZ existed in high molecular mass forms that were most likely to be trimers in solution.Conclusion:Incorporation of an IZ motif into CD40 enhances its binding activity for CD40L through trimerization of the CD40 derivative.

  13. The Arabidopsis SUPERMAN protein is able to specifically bind DNA through its single Cys2-His2 zinc finger motif.

    Science.gov (United States)

    Dathan, Nina; Zaccaro, Laura; Esposito, Sabrina; Isernia, Carla; Omichinski, James G; Riccio, Andrea; Pedone, Carlo; Di Blasio, Benedetto; Fattorusso, Roberto; Pedone, Paolo V

    2002-11-15

    The Arabidopsis SUPERMAN (SUP) gene has been shown to be important in maintaining the boundary between stamens and carpels, and is presumed to act by regulating cell proliferation. In this work, we show that the SUP protein, which contains a single Cys2-His2 zinc finger domain including the QALGGH sequence, highly conserved in the plant zinc finger proteins, binds DNA. Using a series of deletion mutants, it was determined that the minimal domain required for specific DNA binding (residues 15-78) includes the single zinc finger and two basic regions located on either side of this motif. Furthermore, amino acid substitutions in the zinc finger or in the basic regions, including a mutation that knocks out the function of the SUP protein in vivo (glycine 63 to aspartate), have been found to abolish the activity of the SUP DNA-binding domain. These results strongly suggest that the SUP protein functions in vivo by acting as a DNA-binding protein, likely involved in transcriptional regulation. The association of both an N-terminal and a C-terminal basic region with a single Cys2-His2 zinc finger represents a novel DNA-binding motif suggesting that the mechanism of DNA recognition adopted by the SUP protein is different from that described so far in other zinc finger proteins.

  14. Asp residues of βDELSEED-motif are required for peptide binding in the Escherichia coli ATP synthase.

    Science.gov (United States)

    Ahmad, Zulfiqar; Tayou, Junior; Laughlin, Thomas F

    2015-04-01

    This study demonstrates the requirement of Asp-380 and Asp-386 in the βDELSEED-motif of Escherichia coli ATP synthase for peptide binding and inhibition. We studied the inhibition profiles of wild-type and mutant E. coli ATP synthase in presence of c-terminal amide bound melittin and melittin related peptide. Melittin and melittin related peptide inhibited wild-type ATPase almost completely while only partial inhibition was observed in single mutations with replacement of Asp to Ala, Gln, or Arg. Additionally, very little or no inhibition occurred among double mutants βD380A/βD386A, βD380Q/βD386Q, or βD380R/βD386R signifying that removal of one Asp residue allows limited peptide binding. Partial or substantial loss of oxidative phosphorylation among double mutants demonstrates the functional requirement of βD380 and βD386 Asp residues. Moreover, abrogation of wild-type E. coli cell growth and normal growth of mutant cells in presence of peptides provides strong evidence for the requirement of βDELSEED-motif Asp residues for peptide binding. It is concluded that while presence of one Asp residue may allow partial peptide binding, both Asp residues, βD380 and βD386, are essential for proper peptide binding and inhibition of ATP synthase. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Differential expression of the RNA-binding motif protein 3 in human astrocytoma

    Institute of Scientific and Technical Information of China (English)

    ZHANG Hai-tao; ZHANG Zhi-wen; XUE Jing-hui; KONG Hai-bo; LIU Ai-jun; LI Shou-chun; LIU Yu-xiao

    2013-01-01

    Background The RNA-binding motif protein 3 (RBM3),which is transcriptionally induced by low temperature and hypoxia,has recently been found to be upregulated in human tumors.However,its expression status in human astrocytoma is not well defined.This article focuses on the differential expression of RBM3 in human astrocytomas of different grades and normal brain tissues.Methods RBM3 was detected in astrocytomas and normal brain tissues by quantitative real-time PCR,immunohistochemistry,and Western blotting.Analysis of variance was performed on the data from quantitative real-time PCR.The Fisher's exact test was used to analyze the immunohistochemistry results.A P-value of less than 0.05 indicates a statistically significant difference.Results On one hand,the mRNA expression levels of three X-chromosome-related RBM genes (RBMX,RBM3,and RBM10) were detected by quantitative real-time PCR.The results showed that there were no significant differences in RBMX and RBM10 mRNA expression levels in human astrocytomas of different grades and normal brain tissues.However,RBM3 mRNA expression levels were elevated in high-grade (World Health Organization (WHO) Grade Ⅲ-Ⅳ) astrocytomas versus low-grade (WHO Grade Ⅰ-Ⅱ) astrocytomas (5.06±0.66 vs.1.60±0.58; P <0.05) or normal controls (5.06±0.66 vs.1.03±0.22; P <0.05) as determined by quantitative real-time PCR analysis.On the other hand,immunohistochemistry showed an increased RBM3 labeling index in astrocytomas of different grades and normal brain tissues (positive staining rate:astrocytoma Grade Ⅳ,92.9%; astrocytoma Grade Ⅲ,81.8%; astrocytoma Grade Ⅰ-Ⅱ,50%;normal brain tissues,37.5%; high-grade astrocytoma versus normal brain tissues,P <0.05; high-grade astrocytoma versus low-grade astrocytoma,P <0.05).The higher protein levels of RBM3 were also validated in high-grade astrocytomas and low-grade astrocytomas compared with normal brain tissues by Western blotting.Conclusions These

  16. An Unexpected Duo: Rubredoxin Binds Nine TPR Motifs to Form LapB, an Essential Regulator of Lipopolysaccharide Synthesis.

    Science.gov (United States)

    Prince, Chelsy; Jia, Zongchao

    2015-08-01

    Lipopolysaccharide (LPS) synthesis and export are essential pathways for bacterial growth, proliferation, and virulence. The essential protein LapB from Escherichia coli has recently been identified as a regulator of LPS synthesis. We have determined the crystal structure of LapB (without the N-terminal transmembrane helix) at 2 Å resolution using zinc single-wavelength anomalous diffraction phasing derived from a single bound zinc atom. This structure demonstrates the presence of nine tetratricopeptide repeats (TPR) motifs, including two TPR folds that were not predicted from sequence, and a rubredoxin-type metal binding domain. The rubredoxin domain is bound intimately to the TPR motifs, which has not been previously observed or predicted. Mutations in the rubredoxin/TPR interface inhibit in vivo cell growth, and in vitro studies indicate that these modifications cause local displacement of rubredoxin from its binding site without changing the secondary structure of LapB. LapB is the first reported structure to contain both a rubredoxin domain and TPR motifs.

  17. The N-terminal zinc finger of the erythroid transcription factor GATA-1 binds GATC motifs in DNA.

    Science.gov (United States)

    Newton, A; Mackay, J; Crossley, M

    2001-09-21

    The mammalian transcription factor GATA-1 is required for normal erythroid and megakaryocytic development. GATA-1 contains two zinc fingers, the C-terminal finger, which is known to bind (A/T)GATA(A/G) motifs in DNA and the N-finger, which is important for interacting with co-regulatory proteins such as Friend of GATA (FOG). We now show that, like the C-finger, the N-finger of GATA-1 is also capable of binding DNA but recognizes distinct sequences with the core GATC. We demonstrate that the GATA-1 N-finger can bind these sequences in vitro and that in cellular assays, GATA-1 can activate promoters containing GATC motifs. Experiments with mutant GATA-1 proteins confirm the importance of the N-finger, as the C-finger is not required for transactivation from GATC sites. Recently four naturally occurring mutations in GATA-1 have been shown to be associated with familial blood disorders. These mutations all map to the N-finger domain. We have investigated the effect of these mutations on the recognition of GATC sites by the N-finger and show that one mutation R216Q abolishes DNA binding, whereas the others have only minor effects.

  18. Thermodynamic contribution of backbone conformational entropy in the binding between SH3 domain and proline-rich motif.

    Science.gov (United States)

    Zeng, Danyun; Shen, Qingliang; Cho, Jae-Hyun

    2017-02-26

    Biological functions of intrinsically disordered proteins (IDPs), and proteins containing intrinsically disordered regions (IDRs) are often mediated by short linear motifs, like proline-rich motifs (PRMs). Upon binding to their target proteins, IDPs undergo a disorder-to-order transition which is accompanied by a large conformational entropy penalty. Hence, the molecular mechanisms underlying control of conformational entropy are critical for understanding the binding affinity and selectivity of IDPs-mediated protein-protein interactions (PPIs). Here, we investigated the backbone conformational entropy change accompanied by binding of the N-terminal SH3 domain (nSH3) of CrkII and PRM derived from guanine nucleotide exchange factor 1 (C3G). In particular, we focused on the estimation of conformational entropy change of disordered PRM upon binding to the nSH3 domain. Quantitative characterization of conformational dynamics of disordered peptides like PRMs is limited. Hence, we combined various methods, including NMR model-free analysis, δ2D, DynaMine, and structure-based calculation of entropy loss. This study demonstrates that the contribution of backbone conformational entropy change is significant in the PPIs mediated by IDPs/IDRs.

  19. Synergic approach to XAFS analysis for the identification of most probable binding motifs for mononuclear zinc sites in metalloproteins.

    Science.gov (United States)

    Giachini, Lisa; Veronesi, Giulia; Francia, Francesco; Venturoli, Giovanni; Boscherini, Federico

    2010-01-01

    In the present work a data analysis approach, based on XAFS data, is proposed for the identification of most probable binding motifs of unknown mononuclear zinc sites in metalloproteins. This approach combines multiple-scattering EXAFS analysis performed within the rigid-body refinement scheme, non-muffin-tin ab initio XANES simulations, average structural information on amino acids and metal binding clusters provided by the Protein Data Bank, and Debye-Waller factor calculations based on density functional theory. The efficiency of the method is tested by using three reference zinc proteins for which the local structure around the metal is already known from protein crystallography. To show the applicability of the present analysis to structures not deposited in the Protein Data Bank, the XAFS spectra of six mononuclear zinc binding sites present in diverse membrane proteins, for which we have previously proposed the coordinating amino acids by applying a similar approach, is also reported. By comparing the Zn K-edge XAFS features exhibited by these proteins with those pertaining to the reference structures, key spectral characteristics, related to specific binding motifs, are observed. These case studies exemplify the combined data analysis proposed and further support its validity.

  20. MEGARA Optics: stain removal in PBM2Y prisms

    Science.gov (United States)

    Aguirre-Aguirre, D.; Izazaga-Pérez, R.; Villalobos-Mendoza, B.; Carrasco, E.; Gil de Paz, A.; Gallego, J.; Iglesias, J.

    2017-01-01

    MEGARA is the new integral-field and multi-object optical spectrograph for the GTC. For medium and high resolution, the dispersive elements are volume phase holographic gratings, sandwiched between two flat windows and two prisms of high optical precision. The prisms are made of Ohara PBM2Y optical glass. After the prisms polishing process, some stains appeared on the surfaces. For this, in this work is shown the comparative study of five different products (muriatic acid, paint remover, sodium hydroxide, aqua regia and rare earth liquid polish) used for trying to eliminate the stains of the HR MEGARA prisms. It was found that by polishing with the hands the affected area, and using a towel like a kind of pad, and polish during five minutes using rare earth, the stains disappear completely affecting only a 5% the rms of the surface quality. Not so the use of the other products that did not show any apparent result.

  1. DeFCoM: analysis and modeling of transcription factor binding sites using a motif-centric genomic footprinter.

    Science.gov (United States)

    Quach, Bryan; Furey, Terrence S

    2017-04-01

    Identifying the locations of transcription factor binding sites is critical for understanding how gene transcription is regulated across different cell types and conditions. Chromatin accessibility experiments such as DNaseI sequencing (DNase-seq) and Assay for Transposase Accessible Chromatin sequencing (ATAC-seq) produce genome-wide data that include distinct 'footprint' patterns at binding sites. Nearly all existing computational methods to detect footprints from these data assume that footprint signals are highly homogeneous across footprint sites. Additionally, a comprehensive and systematic comparison of footprinting methods for specifically identifying which motif sites for a specific factor are bound has not been performed. Using DNase-seq data from the ENCODE project, we show that a large degree of previously uncharacterized site-to-site variability exists in footprint signal across motif sites for a transcription factor. To model this heterogeneity in the data, we introduce a novel, supervised learning footprinter called Detecting Footprints Containing Motifs (DeFCoM). We compare DeFCoM to nine existing methods using evaluation sets from four human cell-lines and eighteen transcription factors and show that DeFCoM outperforms current methods in determining bound and unbound motif sites. We also analyze the impact of several biological and technical factors on the quality of footprint predictions to highlight important considerations when conducting footprint analyses and assessing the performance of footprint prediction methods. Finally, we show that DeFCoM can detect footprints using ATAC-seq data with similar accuracy as when using DNase-seq data. Python code available at https://bitbucket.org/bryancquach/defcom. bquach@email.unc.edu or tsfurey@email.unc.edu. Supplementary data are available at Bioinformatics online.

  2. Structure-activity relationship of the peptide binding-motif mediating the BRCA2:RAD51 protein-protein interaction.

    Science.gov (United States)

    Scott, Duncan E; Marsh, May; Blundell, Tom L; Abell, Chris; Hyvönen, Marko

    2016-04-01

    RAD51 is a recombinase involved in the homologous recombination of double-strand breaks in DNA. RAD51 forms oligomers by binding to another molecule of RAD51 via an 'FxxA' motif, and the same recognition sequence is similarly utilised to bind BRCA2. We have tabulated the effects of mutation of this sequence, across a variety of experimental methods and from relevant mutations observed in the clinic. We use mutants of a tetrapeptide sequence to probe the binding interaction, using both isothermal titration calorimetry and X-ray crystallography. Where possible, comparison between our tetrapeptide mutational study and the previously reported mutations is made, discrepancies are discussed and the importance of secondary structure in interpreting alanine scanning and mutational data of this nature is considered.

  3. LysM, a widely distributed protein motif for binding to (peptido)glycans

    NARCIS (Netherlands)

    Buist, Girbe; Steen, Anton; Kok, Jan; Kuipers, Oscar P.

    Bacteria retain certain proteins at their cell envelopes by attaching them in a non-covalent manner to peptidoglycan, using specific protein domains, such as the prominent LysM (Lysin Motif) domain. More than 4000 (Pfam PF01476) proteins of both prokaryotes and eukaryotes have been found to contain

  4. The neuronal nitric oxide synthase PDZ motif binds to -G(D,E)XV* carboxyterminal sequences

    NARCIS (Netherlands)

    Schepens, J.; Cuppen, E.; Wieringa, B.; Hendriks, W.

    1997-01-01

    PDZ motifs are small protein-protein interaction modules that are thought to play a role in the clustering of submembranous signalling molecules. The specificity and functional consequences of their associative actions is still largely unknown. Using two-hybrid methodology we here demonstrate that t

  5. Affinity of the heparin binding motif of Noggin1 to heparan sulfate and its visualization in the embryonic tissues.

    Science.gov (United States)

    Nesterenko, Alexey M; Orlov, Eugeny E; Ermakova, Galina V; Ivanov, Igor A; Semenyuk, Pavel I; Orlov, Victor N; Martynova, Natalia Y; Zaraisky, Andrey G

    Heparin binding motifs were found in many secreted proteins and it was suggested that they are responsible for retardation of the protein diffusion within the intercellular space due to the binding to heparan sulfate proteoglycanes (HSPG). Here we used synthetic FITC labeled heparin binding motif (HBM peptide) of the Xenopus laevis secreted BMP inhibitor Noggin1 to study its diffusion along the surface of the heparin beads by FRAP method. As a result, we have found out that diffusivity of HBM-labeled FITC was indeed much lesser than those predicted by theoretical calculations even for whole protein of the Noggin size. We also compared by isothermal titration calorimetry the binding affinity of HBM and the control oligolysine peptide to several natural polyanions including heparan sulfate (HS), heparin, the bacterial dextran sulfate and salmon sperm DNA, and demonstrated that HBM significantly exceeds oligolysine peptide in the affinity to HS, heparin and DNA. By contrast, oligolysine peptide bound with higher affinity to dextran sulfate. We speculate that such a difference may ensure specificity of the morphogen binding to HSPG and could be explained by steric constrains imposed by different distribution of the negative charges along a given polymeric molecule. Finally, by using EGFP-HBM recombinant protein we have visualized the natural pattern of the Noggin1 binding sites within the X. laevis gastrula and demonstrated that these sites forms a dorsal-ventral concentration gradient, with a maximum in the dorsal blastopore lip. In sum, our data provide a quantitative basis for modeling the process of Noggin1 diffusion in embryonic tissues, considering its interaction with HSPG.

  6. Berberine inhibits cyclin D1 expression via suppressed binding of AP-1 transcription factors to CCND1 AP-1 motif

    Institute of Scientific and Technical Information of China (English)

    Ye LUO; Yu HAO; Tai-ping SHI; Wei-wei DENG; Na LI

    2008-01-01

    Aim: To verify the suppressive effect of berberine on the proliferation of the human pulmonary giant cell carcinoma cell line PG and to demonstrate the mecha-nisms behind the antitumoral effects of berberine. Methods: The proliferative effects of PG cells were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetry. The cell cycle was examined by flow cytometry. The expression level of cyclin D1 was detected by RT-PCR. The activities of the activating protein-1 (AP-1) and NF-κB signaling pathways related to cyclin D1 were examined by luciferase assay. The cytoplasmic level of c-Jun was detected by Western blot analysis. An electrophoretic mobility shift assay was used to examiae the binding of transcription factors to the cyclin D1 gene (CCNDl) AP-1 motif. Results: The results showed that the proliferation of PG cells treated with different concentrations (10, 20, and 40 μg/mL) of berberine for 24 and 48 h was suppressed significantly compared to the control group. After treatment with berberine, the proportion of PG cells at the G0/G1 phase increased, while cells at the S and G2/M phases decreased. Berberine could inhibit the expression of cyclin D1 in PG cells. Berberine inhibited the activity of the AP-1 signaling pathway, but had no significant effect on the NF-κB signaling pathway. Berberine suppressed the expression of c-Jun and decreased the binding of tran-scription factors to the CCND1 AP-1 motif. Conclusion: Berberine suppresses the activity of the AP-1 signaling pathway and decreases the binding of transcrip-tion factors to the CCND1 AP-1 motif. This is one of the important mechanisms behind the antitumoral effects of berberine as a regulator of cyclin D1.

  7. TGF-β Signaling Cooperates with AT Motif-Binding Factor-1 for Repression of the α-Fetoprotein Promoter

    Directory of Open Access Journals (Sweden)

    Nobuo Sakata

    2014-01-01

    Full Text Available α-Fetoprotein (AFP is known to be highly produced in fetal liver despite its barely detectable level in normal adult liver. On the other hand, hepatocellular carcinoma often shows high expression of AFP. Thus, AFP seems to be an oncogenic marker. In our present study, we investigated how TGF-β signaling cooperates with AT motif-binding factor-1 (ATBF1 to inhibit AFP transcription. Indeed, the expression of AFP mRNA in HuH-7 cells was negatively regulated by TGF-β signaling. To further understand how TGF-β suppresses the transcription of the AFP gene, we analyzed the activity of the AFP promoter in the presence of TGF-β. We found that the TGF-β signaling and ATBF1 suppressed AFP transcription through two ATBF1 binding elements (AT-motifs. Using a heterologous reporter system, both AT-motifs were required for transcriptional repression upon TGF-β stimulation. Furthermore, Smads were found to interact with ATBF1 at both its N-terminal and C-terminal regions. Since the N-terminal (ATBF1N and C-terminal regions of ATBF1 (ATBF1C lack the ability of DNA binding, both truncated mutants rescued the cooperative inhibitory action by the TGF-β signaling and ATBF1 in a dose-dependent manner. Taken together, these findings indicate that TGF-β signaling can act in concert with ATBF1 to suppress the activity of the AFP promoter through direct interaction of ATBF1 with Smads.

  8. Solution structure of the ETS domain from murine Ets-1: a winged helix-turn-helix DNA binding motif.

    OpenAIRE

    Donaldson, L W; Petersen, J.M.; Graves, B J; McIntosh, L. P.

    1996-01-01

    Ets-1 is the prototypic member of the ets family of transcription factors. This family is characterized by the conserved ETS domain that mediates specific DNA binding. Using NMR methods, we have determined the structure of a fragment of murine Ets-1 composed of the 85 residue ETS domain and a 25 amino acid extension that ends at its native C-terminus. The ETS domain folds into a helix-turn-helix motif on a four-stranded anti-parallel beta-sheet scaffold. This structure places Ets-1 in the win...

  9. Translation initiation factor eIF4G1 preferentially binds yeast transcript leaders containing conserved oligo-uridine motifs.

    Science.gov (United States)

    Zinshteyn, Boris; Rojas-Duran, Maria F; Gilbert, Wendy V

    2017-09-01

    Translational control of gene expression plays essential roles in cellular stress responses and organismal development by enabling rapid, selective, and localized control of protein production. Translational regulation depends on context-dependent differences in the protein output of mRNAs, but the key mRNA features that distinguish efficiently translated mRNAs are largely unknown. Here, we comprehensively determined the RNA-binding preferences of the eukaryotic initiation factor 4G (eIF4G) to assess whether this core translation initiation factor has intrinsic sequence preferences that may contribute to preferential translation of specific mRNAs. We identified a simple RNA sequence motif-oligo-uridine-that mediates high-affinity binding to eIF4G in vitro. Oligo(U) motifs occur naturally in the transcript leader (TL) of hundreds of yeast genes, and mRNAs with unstructured oligo(U) motifs were enriched in immunoprecipitations against eIF4G. Ribosome profiling following depletion of eIF4G in vivo showed preferentially reduced translation of mRNAs with long TLs, including those that contain oligo(U). Finally, TL oligo(U) elements are enriched in genes with regulatory roles and are conserved between yeast species, consistent with an important cellular function. Taken together, our results demonstrate RNA sequence preferences for a general initiation factor, which cells potentially exploit for translational control of specific mRNAs. © 2017 Zinshteyn et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  10. A sequence upstream of canonical PDZ-binding motif within CFTR COOH-terminus enhances NHERF1 interaction.

    Science.gov (United States)

    Sharma, Neeraj; LaRusch, Jessica; Sosnay, Patrick R; Gottschalk, Laura B; Lopez, Andrea P; Pellicore, Matthew J; Evans, Taylor; Davis, Emily; Atalar, Melis; Na, Chan-Hyun; Rosson, Gedge D; Belchis, Deborah; Milewski, Michal; Pandey, Akhilesh; Cutting, Garry R

    2016-12-01

    The development of cystic fibrosis transmembrane conductance regulator (CFTR) targeted therapy for cystic fibrosis has generated interest in maximizing membrane residence of mutant forms of CFTR by manipulating interactions with scaffold proteins, such as sodium/hydrogen exchange regulatory factor-1 (NHERF1). In this study, we explored whether COOH-terminal sequences in CFTR beyond the PDZ-binding motif influence its interaction with NHERF1. NHERF1 displayed minimal self-association in blot overlays (NHERF1, Kd = 1,382 ± 61.1 nM) at concentrations well above physiological levels, estimated at 240 nM from RNA-sequencing and 260 nM by liquid chromatography tandem mass spectrometry in sweat gland, a key site of CFTR function in vivo. However, NHERF1 oligomerized at considerably lower concentrations (10 nM) in the presence of the last 111 amino acids of CFTR (20 nM) in blot overlays and cross-linking assays and in coimmunoprecipitations using differently tagged versions of NHERF1. Deletion and alanine mutagenesis revealed that a six-amino acid sequence (1417)EENKVR(1422) and the terminal (1478)TRL(1480) (PDZ-binding motif) in the COOH-terminus were essential for the enhanced oligomerization of NHERF1. Full-length CFTR stably expressed in Madin-Darby canine kidney epithelial cells fostered NHERF1 oligomerization that was substantially reduced (∼5-fold) on alanine substitution of EEN, KVR, or EENKVR residues or deletion of the TRL motif. Confocal fluorescent microscopy revealed that the EENKVR and TRL sequences contribute to preferential localization of CFTR to the apical membrane. Together, these results indicate that COOH-terminal sequences mediate enhanced NHERF1 interaction and facilitate the localization of CFTR, a property that could be manipulated to stabilize mutant forms of CFTR at the apical surface to maximize the effect of CFTR-targeted therapeutics.

  11. Novel hinge-binding motifs for Janus kinase 3 inhibitors: a comprehensive structure-activity relationship study on tofacitinib bioisosteres.

    Science.gov (United States)

    Gehringer, Matthias; Forster, Michael; Pfaffenrot, Ellen; Bauer, Silke M; Laufer, Stefan A

    2014-11-01

    The Janus kinases (JAKs) are a family of cytosolic tyrosine kinases crucially involved in cytokine signaling. JAKs have been demonstrated to be valid targets in the treatment of inflammatory and myeloproliferative disorders, and two inhibitors, tofacitinib and ruxolitinib, recently received their marketing authorization. Despite this success, selectivity within the JAK family remains a major issue. Both approved compounds share a common 7H-pyrrolo[2,3-d]pyrimidine hinge binding motif, and little is known about modifications tolerated at this heterocyclic core. In the current study, a library of tofacitinib bioisosteres was prepared and tested against JAK3. The compounds possessed the tofacitinib piperidinyl side chain, whereas the hinge binding motif was replaced by a variety of heterocycles mimicking its pharmacophore. In view of the promising expectations obtained from molecular modeling, most of the compounds proved to be poorly active. However, strategies for restoring activity within this series of novel chemotypes were discovered and crucial structure-activity relationships were deduced. The compounds presented may serve as starting point for developing novel JAK inhibitors and as a valuable training set for in silico models.

  12. Copper-binding tripeptide motif increases potency of the antimicrobial peptide Anoplin via Reactive Oxygen Species generation.

    Science.gov (United States)

    Libardo, M Daben J; Nagella, Sai; Lugo, Andrea; Pierce, Scott; Angeles-Boza, Alfredo M

    2015-01-02

    Antimicrobial peptides (AMPs) are broad spectrum antimicrobial agents that act through diverse mechanisms, this characteristic makes them suitable starting points for development of novel classes of antibiotics. We have previously reported the increase in activity of AMPs upon addition of the Amino Terminal Copper and Nickel (ATCUN) Binding Unit. Herein we synthesized the membrane active peptide, Anoplin and two ATCUN-Anoplin derivatives and show that the increase in activity is indeed due to the ROS formation by the Cu(II)-ATCUN complex. We found that the ATCUN-Anoplin peptides were up to four times more potent compared to Anoplin alone against standard test bacteria. We studied membrane disruption, and cellular localization and found that addition of the ATCUN motif did not lead to a difference in these properties. When helical content was calculated, we observed that ATCUN-Anoplin had a lower helical composition. We found that ATCUN-Anoplin are able to oxidatively damage lipids in the bacterial membrane and that their activity trails the rate at which ROS is formed by the Cu(II)-ATCUN complexes alone. This study shows that addition of a metal binding tripeptide motif is a simple strategy to increase potency of AMPs by conferring a secondary action.

  13. Surface binding sites in amylase have distinct roles in recognition of starch structure motifs and degradation

    DEFF Research Database (Denmark)

    Cockburn, Darrell; Nielsen, Morten M.; Christiansen, Camilla

    2015-01-01

    Carbohydrate converting enzymes often possess extra substrate binding regions that enhance their activity. These can be found either on separate domains termed carbohydrate binding modules or as so-called surface binding sites (SBSs) situated on the catalytic domain. SBSs are common in starch...... to soluble polysaccharides and oligosaccharides with α-1,6 linkages, suggesting that branch points are key structural elements in recognition by SBS2. Mutation at both SBS1 and SBS2 eliminated binding to all starch granule types tested. Taken together, the findings indicate that the two SBSs act in concert...

  14. Non-canonical binding interactions of the RNA recognition motif (RRM) domains of P34 protein modulate binding within the 5S ribonucleoprotein particle (5S RNP).

    Science.gov (United States)

    Kamina, Anyango D; Williams, Noreen

    2017-01-01

    RNA binding proteins are involved in many aspects of RNA metabolism. In Trypanosoma brucei, our laboratory has identified two trypanosome-specific RNA binding proteins P34 and P37 that are involved in the maturation of the 60S subunit during ribosome biogenesis. These proteins are part of the T. brucei 5S ribonucleoprotein particle (5S RNP) and P34 binds to 5S ribosomal RNA (rRNA) and ribosomal protein L5 through its N-terminus and its RNA recognition motif (RRM) domains. We generated truncated P34 proteins to determine these domains' interactions with 5S rRNA and L5. Our analyses demonstrate that RRM1 of P34 mediates the majority of binding with 5S rRNA and the N-terminus together with RRM1 contribute the most to binding with L5. We determined that the consensus ribonucleoprotein (RNP) 1 and 2 sequences, characteristic of canonical RRM domains, are not fully conserved in the RRM domains of P34. However, the aromatic amino acids previously described to mediate base stacking interactions with their RNA target are conserved in both of the RRM domains of P34. Surprisingly, mutation of these aromatic residues did not disrupt but instead enhanced 5S rRNA binding. However, we identified four arginine residues located in RRM1 of P34 that strongly impact L5 binding. These mutational analyses of P34 suggest that the binding site for 5S rRNA and L5 are near each other and specific residues within P34 regulate the formation of the 5S RNP. These studies show the unique way that the domains of P34 mediate binding with the T. brucei 5S RNP.

  15. Pseudouridine synthases: four families of enzymes containing a putative uridine-binding motif also conserved in dUTPases and dCTP deaminases.

    Science.gov (United States)

    Koonin, E V

    1996-06-15

    Using a combination of several methods for protein sequence comparison and motif analysis, it is shown that the four recently described pseudouridine syntheses with different specificities belong to four distinct families. Three of these families share two conserved motifs that are likely to be directly involved in catalysis. One of these motifs is detected also in two other families of enzymes that specifically bind uridine, namely deoxycitidine triphosphate deaminases and deoxyuridine triphosphatases. It is proposed that this motif is an essential part of the uridine-binding site. Two of the pseudouridine syntheses, one of which modifies the anticodon arm of tRNAs and the other is predicted to modify a portion of the large ribosomal subunit RNA belonging to the peptidyltransferase center, are encoded in all extensively sequenced genomes, including the 'minimal' genome of Mycoplasma genitalium. These particular RNA modifications and the respective enzymes are likely to be essential for the functioning of any cell.

  16. MPBind: a Meta-motif-based statistical framework and pipeline to Predict Binding potential of SELEX-derived aptamers.

    Science.gov (United States)

    Jiang, Peng; Meyer, Susanne; Hou, Zhonggang; Propson, Nicholas E; Soh, H Tom; Thomson, James A; Stewart, Ron

    2014-09-15

    Aptamers are 'synthetic antibodies' that can bind to target molecules with high affinity and specificity. Aptamers are chemically synthesized and their discovery can be performed completely in vitro, rather than relying on in vivo biological processes, making them well-suited for high-throughput discovery. However, a large fraction of the most enriched aptamers in Systematic Evolution of Ligands by EXponential enrichment (SELEX) rounds display poor binding activity. Here, we present MPBind, a Meta-motif-based statistical framework and pipeline to Predict the BIND: ing potential of SELEX-derived aptamers. Using human embryonic stem cell SELEX-Seq data, MPBind achieved high prediction accuracy for binding potential. Further analysis showed that MPBind is robust to both polymerase chain reaction amplification bias and incomplete sequencing of aptamer pools. These two biases usually confound aptamer analysis. MPBind software and documents are available at http://www.morgridge.net/MPBind.html. The human embryonic stem cells whole-cell SELEX-Seq data are available at http://www.morgridge.net/Aptamer/. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  17. Examination of the transcription factor NtcA-binding motif by in vitro selection of DNA sequences from a random library.

    Science.gov (United States)

    Jiang, F; Wisén, S; Widersten, M; Bergman, B; Mannervik, B

    2000-08-25

    A recursive in vitro selection among random DNA sequences was used for analysis of the cyanobacterial transcription factor NtcA-binding motifs. An eight-base palindromic sequence, TGTA-(N(8))-TACA, was found to be the optimal NtcA-binding sequence. The more divergent the binding sequences, compared to this consensus sequence, the lower the NtcA affinity. The second and third bases in each four-nucleotide half of the consensus sequence were crucial for NtcA binding, and they were in general highly conserved. The most frequently occurring sequence in the middle weakly conserved region was similar to that of the NtcA-binding motif of the Anabaena sp. strain PCC 7120 glnA gene, previously known to have high affinity for NtcA. This indicates that the middle sequences were selected for high NtcA affinity. Analysis of natural NtcA-binding motifs showed that these could be classified into two groups based on differences in recognition consensus sequences. It is suggested that NtcA naturally recognizes different DNA-binding motifs, or has differential affinities to these sequences under different physiological conditions.

  18. Surface binding sites in amylase have distinct roles in recognition of starch structure motifs and degradation.

    Science.gov (United States)

    Cockburn, Darrell; Nielsen, Morten M; Christiansen, Camilla; Andersen, Joakim M; Rannes, Julie B; Blennow, Andreas; Svensson, Birte

    2015-04-01

    Carbohydrate converting enzymes often possess extra substrate binding regions that enhance their activity. These can be found either on separate domains termed carbohydrate binding modules or as so-called surface binding sites (SBSs) situated on the catalytic domain. SBSs are common in starch degrading enzymes and critically important for their function. The affinity towards a variety of starch granules as well as soluble poly- and oligosaccharides of barley α-amylase 1 (AMY1) wild-type and mutants of two SBSs (SBS1 and SBS2) was investigated using Langmuir binding analysis, confocal laser scanning microscopy, affinity gel electrophoresis and surface plasmon resonance to unravel functional roles of the SBSs. SBS1 was critical for binding to different starch types as Kd increased by 7-62-fold or was not measurable upon mutation. By contrast SBS2 was particularly important for binding to soluble polysaccharides and oligosaccharides with α-1,6 linkages, suggesting that branch points are key structural elements in recognition by SBS2. Mutation at both SBS1 and SBS2 eliminated binding to all starch granule types tested. Taken together, the findings indicate that the two SBSs act in concert to localize AMY1 to the starch granule surface and that SBS2 works synergistically with the active site in the degradation of amylopectin.

  19. SH3 domains of Grb2 adaptor bind to PXpsiPXR motifs within the Sos1 nucleotide exchange factor in a discriminate manner.

    Science.gov (United States)

    McDonald, Caleb B; Seldeen, Kenneth L; Deegan, Brian J; Farooq, Amjad

    2009-05-19

    Ubiquitously encountered in a wide variety of cellular processes, the Grb2-Sos1 interaction is mediated through the combinatorial binding of nSH3 and cSH3 domains of Grb2 to various sites containing PXpsiPXR motifs within Sos1. Here, using isothermal titration calorimetry, we demonstrate that while the nSH3 domain binds with affinities in the physiological range to all four sites containing PXpsiPXR motifs, designated S1, S2, S3, and S4, the cSH3 domain can only do so at the S1 site. Further scrutiny of these sites yields rationale for the recognition of various PXpsiPXR motifs by the SH3 domains in a discriminate manner. Unlike the PXpsiPXR motifs at S2, S3, and S4 sites, the PXpsiPXR motif at the S1 site is flanked at its C-terminus with two additional arginine residues that are absolutely required for high-affinity binding of the cSH3 domain. In striking contrast, these two additional arginine residues augment the binding of the nSH3 domain to the S1 site, but their role is not critical for the recognition of S2, S3, and S4 sites. Site-directed mutagenesis suggests that the two additional arginine residues flanking the PXpsiPXR motif at the S1 site contribute to free energy of binding via the formation of salt bridges with specific acidic residues in SH3 domains. Molecular modeling is employed to project these novel findings into the 3D structures of SH3 domains in complex with a peptide containing the PXpsiPXR motif and flanking arginine residues at the S1 site. Taken together, this study furthers our understanding of the assembly of a key signaling complex central to cellular machinery.

  20. Identification of DNA binding motifs of the Mycobacterium tuberculosis PhoP/PhoR two-component signal transduction system.

    Science.gov (United States)

    Cimino, Mena; Thomas, Christophe; Namouchi, Amine; Dubrac, Sarah; Gicquel, Brigitte; Gopaul, Deshmukh N

    2012-01-01

    The Mycobacterium tuberculosis PhoP/PhoR two-component signal transduction system controls the expression of about 2% of the genome and plays a major role in pathogenicity. However, its regulon has not been well characterized. The binding site of PhoP transcription regulator was identified in the upstream regions of msl3, pks2, lipF and fadD21 genes, by using gene fusions, electrophoretic mobility shift assays and DNase I footprinting experiments. A consensus sequence for PhoP binding was deduced. It consists of two direct repeats, DR1/DR2, associated with a third repeat, DR3, important in some cases for PhoP binding to DR1/DR2 but located at a variable distance from these direct repeats. DR1/DR2 and DR3 consensus sequences were used to screen the whole-genome sequence for other putative binding sites potentially corresponding to genes directly regulated by PhoP. The identified 87 genes, encoding transcription regulators, and proteins involved in secondary metabolites biosynthesis, transport and catabolism are proposed to belong to the PhoP regulon. A consensus sequence derived from the analysis of PhoP binding to four gene promoter regions is proposed. We show for the first time the involvement of a third direct repeat motif in this binding reaction. The consensus sequence was instrumented to study the global regulation mediated by PhoP in M. tuberculosis. This analysis leads to the identification of several genes that are potentially regulated by this key player.

  1. Selective Targeting of G-Quadruplex Structures by a Benzothiazole-Based Binding Motif.

    Science.gov (United States)

    Buchholz, Ina; Karg, Beatrice; Dickerhoff, Jonathan; Sievers-Engler, Adrian; Lämmerhofer, Michael; Weisz, Klaus

    2017-03-09

    A benzothiazole derivative was identified as potent ligand for DNA G-quadruplex structures. Fluorescence titrations revealed selective binding to quadruplexes of different topologies including parallel, antiparallel and (3+1) hybrid structures. The parallel c-MYC sequence was found to constitute the preferred target with dissociation constants in the micromolar range. Binding of the benzothiazole-based ligand to c-MYC was structurally and thermodynamically characterized in detail by employing a comprehensive set of spectroscopic and calorimetric techniques. Job plot analyses and mass spectral data indicate non-cooperative ligand binding to form 1:1 and 2:1 complex stoichiometries. Whereas stacking interactions are suggested by optical methods, NMR chemical shift perturbations also indicate significant rearrangements of both 5'- and 3'-flanking sequences upon ligand binding. Additional isothermal calorimetry studies yield a thermodynamic profile of the ligand-quadruplex association and reveal enthalpic contributions to be the major driving force for binding. The structural and thermodynamic information obtained in the present work provides the basis for the rational development of benzothiazole derivatives as promising quadruplex binding agents.

  2. One motif to bind them: A small-XXX-small motif affects transmembrane domain 1 oligomerization, function, localization, and cross-talk between two yeast GPCRs.

    Science.gov (United States)

    Lock, Antonia; Forfar, Rachel; Weston, Cathryn; Bowsher, Leo; Upton, Graham J G; Reynolds, Christopher A; Ladds, Graham; Dixon, Ann M

    2014-12-01

    G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors in mammals and facilitate a range of physiological responses triggered by a variety of ligands. GPCRs were thought to function as monomers, however it is now accepted that GPCR homo- and hetero-oligomers also exist and influence receptor properties. The Schizosaccharomyces pombe GPCR Mam2 is a pheromone-sensing receptor involved in mating and has previously been shown to form oligomers in vivo. The first transmembrane domain (TMD) of Mam2 contains a small-XXX-small motif, overrepresented in membrane proteins and well-known for promoting helix-helix interactions. An ortholog of Mam2 in Saccharomyces cerevisiae, Ste2, contains an analogous small-XXX-small motif which has been shown to contribute to receptor homo-oligomerization, localization and function. Here we have used experimental and computational techniques to characterize the role of the small-XXX-small motif in function and assembly of Mam2 for the first time. We find that disruption of the motif via mutagenesis leads to reduction of Mam2 TMD1 homo-oligomerization and pheromone-responsive cellular signaling of the full-length protein. It also impairs correct targeting to the plasma membrane. Mutation of the analogous motif in Ste2 yielded similar results, suggesting a conserved mechanism for assembly. Using co-expression of the two fungal receptors in conjunction with computational models, we demonstrate a functional change in G protein specificity and propose that this is brought about through hetero-dimeric interactions of Mam2 with Ste2 via the complementary small-XXX-small motifs. This highlights the potential of these motifs to affect a range of properties that can be investigated in other GPCRs.

  3. Human CTP:phosphoethanolamine cytidylyltransferase: enzymatic properties and unequal catalytic roles of CTP-binding motifs in two cytidylyltransferase domains.

    Science.gov (United States)

    Tian, Siqi; Ohtsuka, Jun; Wang, Shipeng; Nagata, Koji; Tanokura, Masaru; Ohta, Akinori; Horiuchi, Hiroyuki; Fukuda, Ryouichi

    2014-06-20

    CTP:phosphoethanolamine cytidylyltransferase (ECT) is a key enzyme in the CDP-ethanolamine branch of the Kennedy pathway, which is the primary pathway of phosphatidylethanolamine (PE) synthesis in mammalian cells. Here, the enzymatic properties of recombinant human ECT (hECT) were characterized. The catalytic reaction of hECT obeyed Michaelis-Menten kinetics with respect to both CTP and phosphoethanolamine. hECT is composed of two tandem cytidylyltransferase (CT) domains as ECTs of other organisms. The histidines, especially the first histidine, in the CTP-binding motif HxGH in the N-terminal CT domain were critical for its catalytic activity in vitro, while those in the C-terminal CT domain were not. Overexpression of the wild-type hECT and hECT mutants containing amino acid substitutions in the HxGH motif in the C-terminal CT domain suppressed the growth defect of the Saccharomyces cerevisiae mutant of ECT1 encoding ECT in the absence of a PE supply via the decarboxylation of phosphatidylserine, but overexpression of hECT mutants of the N-terminal CT domain did not. These results suggest that the N-terminal CT domain of hECT contributes to its catalytic reaction, but C-terminal CT domain does not. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Analysis of the key elements of FFAT-like motifs identifies new proteins that potentially bind VAP on the ER, including two AKAPs and FAPP2.

    Directory of Open Access Journals (Sweden)

    Veronika Mikitova

    Full Text Available BACKGROUND: Two phenylalanines (FF in an acidic tract (FFAT-motifs were originally described as having seven elements: an acidic flanking region followed by 6 residues (EFFDA-E. Such motifs are found in several lipid transfer protein (LTP families, and they interact with a protein on the cytosolic face of the ER called vesicle-associated membrane protein-associated protein (VAP. Mutation of which causes ER stress and motor neuron disease, making it important to determine which proteins bind VAP. Among other proteins that bind VAP, some contain FFAT-like motifs that are missing one or more of the seven elements. Defining how much variation is tolerated in FFAT-like motifs is a preliminary step prior to the identification of the full range of VAP interactors. RESULTS: We used a quantifiable in vivo system that measured ER targeting in a reporter yeast strain that over-expressed VAP to study the effect of substituting different elements of FFAT-like motifs in turn. By defining FFAT-like motifs more widely than before, we found them in novel proteins the functions of which had not previously been directly linked to the ER, including: two PKA anchoring proteins, AKAP220 and AKAP110; a family of plant LTPs; and the glycolipid LTP phosphatidylinositol-four-phosphate adaptor-protein-2 (FAPP-2. CONCLUSION: All of the seven essential elements of a FFAT motif tolerate variation, and weak targeting to the ER via VAP is still detected if two elements are substituted. In addition to the strong FFAT motifs already known, there are additional proteins with weaker FFAT-like motifs, which might be functionally important VAP interactors.

  5. In silico analysis of molecular mechanisms of Galanthus nivalis agglutinin-related lectin-induced cancer cell death from carbohydrate-binding motif evolution hypothesis.

    Science.gov (United States)

    Yu, Qi-Jia; Li, Zi-Yue; Yao, Shun; Ming, Miao; Wang, Shu-Ya; Liu, Bo; Bao, Jin-Ku

    2011-10-01

    Galanthus nivalis agglutinin-related lectins, a superfamily of strictly mannose-binding-specific lectins widespread amongst monotyledonous plants, have drawn a rising attention for their remarkable anti-proliferative and apoptosis-inducing activities toward various types of cancer cells; however, the precise molecular mechanisms by which they induce tumor cell apoptosis are still only rudimentarily understood. Herein, we found that the three conserved motifs "QXDXNXVXY," the mannose-specific binding sites, could mutate at one or more amino acid sites, which might be a driving force for the sequential evolution and thus ultimately leading to the complete disappearance of the three conserved motifs. In addition, we found that the motif evolution could result in the diversification of sugar-binding types that G. nivalis agglutinin-related lectins could bind from specific mannose receptors to more types of sugar-containing receptors in cancer cells. Subsequently, we indicated that some sugar-containing receptors such as TNFR1, EGFR, Hsp90, and Hsp70 could block downstream anti-apoptotic or survival signaling pathways, which, in turn, resulted in tumor cell apoptosis. Taken together, our hypothesis that carbohydrate-binding motif evolution may impact the G. nivalis agglutinin-related lectin-induced survival or anti-apoptotic pathways would provide a new perspective for further elucidating the intricate relationships between the carbohydrate-binding specificities and complex molecular mechanisms by which G. nivalis agglutinin-related lectins induce cancer cell death.

  6. Binding site structure of one LRP-RAP complex: implications for a common ligand-receptor binding motif

    DEFF Research Database (Denmark)

    Jensen, Gitte A; Andersen, Olav M; Bonvin, Alexandre M J J

    2006-01-01

    domains of RAP and alpha2-macroglobulin, which promotes the catabolism of the Abeta-peptide implicated in Alzheimer's disease. To understand the receptor-ligand cross-talk, the NMR structure of CR56 has been solved and ligand binding experiments with RAP domain 1 (RAPd1) have been performed. From chemical...

  7. Regulation of T cell receptor activation by dynamic membrane binding of the CD3epsilon cytoplasmic tyrosine-based motif.

    Science.gov (United States)

    Xu, Chenqi; Gagnon, Etienne; Call, Matthew E; Schnell, Jason R; Schwieters, Charles D; Carman, Christopher V; Chou, James J; Wucherpfennig, Kai W

    2008-11-14

    Many immune system receptors signal through cytoplasmic tyrosine-based motifs (ITAMs), but how receptor ligation results in ITAM phosphorylation remains unknown. Live-cell imaging studies showed a close interaction of the CD3epsilon cytoplasmic domain of the T cell receptor (TCR) with the plasma membrane through fluorescence resonance energy transfer between a C-terminal fluorescent protein and a membrane fluorophore. Electrostatic interactions between basic CD3epsilon residues and acidic phospholipids enriched in the inner leaflet of the plasma membrane were required for binding. The nuclear magnetic resonance structure of the lipid-bound state of this cytoplasmic domain revealed deep insertion of the two key tyrosines into the hydrophobic core of the lipid bilayer. Receptor ligation thus needs to result in unbinding of the CD3epsilon ITAM from the membrane to render these tyrosines accessible to Src kinases. Sequestration of key tyrosines into the lipid bilayer represents a previously unrecognized mechanism for control of receptor activation.

  8. Zinc binding to RNA recognition motif of TDP-43 induces the formation of amyloid-like aggregates.

    Science.gov (United States)

    Garnier, Cyrille; Devred, François; Byrne, Deborah; Puppo, Rémy; Roman, Andrei Yu; Malesinski, Soazig; Golovin, Andrey V; Lebrun, Régine; Ninkina, Natalia N; Tsvetkov, Philipp O

    2017-07-28

    Aggregation of TDP-43 (transactive response DNA binding protein 43 kDa) is a hallmark of certain forms of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Moreover, intracellular TDP-43-positive inclusions are often found in other neurodegenerative diseases. Recently it was shown that zinc ions can provoke the aggregation of endogenous TDP-43 in cells, allowing to assume a direct interaction of TDP-43 with zinc ions. In this work, we investigated zinc binding to the 102-269 TDP-43 fragment, which comprise the two RNA recognition motifs. Using isothermal titration calorimetry, mass spectrometry, and differential scanning fluorimetry, we showed that zinc binds to this TDP-43 domain with a dissociation constant in the micromolar range and modifies its tertiary structure leading to a decrease of its thermostability. Moreover, the study by dynamic light scattering and negative stain electron microscopy demonstrated that zinc ions induce auto-association process of this TDP-43 fragment into rope-like structures. These structures are thioflavin-T-positive allowing to hypothesize the direct implication of zinc ions in pathological aggregation of TDP-43.

  9. New structural and functional contexts of the Dx[DN]xDG linear motif: insights into evolution of calcium-binding proteins.

    Science.gov (United States)

    Rigden, Daniel J; Woodhead, Duncan D; Wong, Prudence W H; Galperin, Michael Y

    2011-01-01

    Binding of calcium ions (Ca²⁺) to proteins can have profound effects on their structure and function. Common roles of calcium binding include structure stabilization and regulation of activity. It is known that diverse families--EF-hands being one of at least twelve--use a Dx[DN]xDG linear motif to bind calcium in near-identical fashion. Here, four novel structural contexts for the motif are described. Existing experimental data for one of them, a thermophilic archaeal subtilisin, demonstrate for the first time a role for Dx[DN]xDG-bound calcium in protein folding. An integrin-like embedding of the motif in the blade of a β-propeller fold--here named the calcium blade--is discovered in structures of bacterial and fungal proteins. Furthermore, sensitive database searches suggest a common origin for the calcium blade in β-propeller structures of different sizes and a pan-kingdom distribution of these proteins. Factors favouring the multiple convergent evolution of the motif appear to include its general Asp-richness, the regular spacing of the Asp residues and the fact that change of Asp into Gly and vice versa can occur though a single nucleotide change. Among the known structural contexts for the Dx[DN]xDG motif, only the calcium blade and the EF-hand are currently found intracellularly in large numbers, perhaps because the higher extracellular concentration of Ca²⁺ allows for easier fixing of newly evolved motifs that have acquired useful functions. The analysis presented here will inform ongoing efforts toward prediction of similar calcium-binding motifs from sequence information alone.

  10. Specific binding of the replication protein of plasmid pPS10 to direct and inverted repeats is mediated by an HTH motif.

    Science.gov (United States)

    García de Viedma, D; Serrano-López, A; Díaz-Orejas, R

    1995-01-01

    The initiator protein of the plasmid pPS10, RepA, has a putative helix-turn-helix (HTH) motif at its C-terminal end. RepA dimers bind to an inverted repeat at the repA promoter (repAP) to autoregulate RepA synthesis. [D. García de Viedma, et al. (1996) EMBO J. in press]. RepA monomers bind to four direct repeats at the origin of replication (oriV) to initiate pPS10 replication This report shows that randomly generated mutations in RepA, associated with defficiencies in autoregulation, map either at the putative HTH motif or in its vicinity. These mutant proteins do not promote pPS10 replication and are severely affected in binding to both the repAP and oriV regions in vitro. Revertants of a mutant that map in the vicinity of the HTH motif have been obtained and correspond to a second amino acid substitution far upstream of the motif. However, reversion of mutants that map in the helices of the motif occurs less frequently, at least by an order of magnitude. All these data indicate that the helices of the HTH motif play an essential role in specific RepA-DNA interactions, although additional regions also seem to be involved in DNA binding activity. Some mutations have slightly different effects in replication and autoregulation, suggesting that the role of the HTH motif in the interaction of RepA dimers or monomers with their respective DNA targets (IR or DR) is not the same. Images PMID:8559664

  11. Condensin II Regulates Interphase Chromatin Organization Through the Mrg-Binding Motif of Cap-H2.

    Science.gov (United States)

    Wallace, Heather A; Klebba, Joseph E; Kusch, Thomas; Rogers, Gregory C; Bosco, Giovanni

    2015-03-09

    The spatial organization of the genome within the eukaryotic nucleus is a dynamic process that plays a central role in cellular processes such as gene expression, DNA replication, and chromosome segregation. Condensins are conserved multi-subunit protein complexes that contribute to chromosome organization by regulating chromosome compaction and homolog pairing. Previous work in our laboratory has shown that the Cap-H2 subunit of condensin II physically and genetically interacts with the Drosophila homolog of human MORF4-related gene on chromosome 15 (MRG15). Like Cap-H2, Mrg15 is required for interphase chromosome compaction and homolog pairing. However, the mechanism by which Mrg15 and Cap-H2 cooperate to maintain interphase chromatin organization remains unclear. Here, we show that Cap-H2 localizes to interband regions on polytene chromosomes and co-localizes with Mrg15 at regions of active transcription across the genome. We show that co-localization of Cap-H2 on polytene chromosomes is partially dependent on Mrg15. We have identified a binding motif within Cap-H2 that is essential for its interaction with Mrg15, and have found that mutation of this motif results in loss of localization of Cap-H2 on polytene chromosomes and results in partial suppression of Cap-H2-mediated compaction and homolog unpairing. Our data are consistent with a model in which Mrg15 acts as a loading factor to facilitate Cap-H2 binding to chromatin and mediate changes in chromatin organization. Copyright © 2015 Wallace et al.

  12. A Plasmodium falciparum copper-binding membrane protein with copper transport motifs

    Directory of Open Access Journals (Sweden)

    Choveaux David L

    2012-11-01

    Full Text Available Abstract Background Copper is an essential catalytic co-factor for metabolically important cellular enzymes, such as cytochrome-c oxidase. Eukaryotic cells acquire copper through a copper transport protein and distribute intracellular copper using molecular chaperones. The copper chelator, neocuproine, inhibits Plasmodium falciparum ring-to-trophozoite transition in vitro, indicating a copper requirement for malaria parasite development. How the malaria parasite acquires or secretes copper still remains to be fully elucidated. Methods PlasmoDB was searched for sequences corresponding to candidate P. falciparum copper-requiring proteins. The amino terminal domain of a putative P. falciparum copper transport protein was cloned and expressed as a maltose binding fusion protein. The copper binding ability of this protein was examined. Copper transport protein-specific anti-peptide antibodies were generated in chickens and used to establish native protein localization in P. falciparum parasites by immunofluorescence microscopy. Results Six P. falciparum copper-requiring protein orthologs and a candidate P. falciparum copper transport protein (PF14_0369, containing characteristic copper transport protein features, were identified in PlasmoDB. The recombinant amino terminal domain of the transport protein bound reduced copper in vitro and within Escherichia coli cells during recombinant expression. Immunolocalization studies tracked the copper binding protein translocating from the erythrocyte plasma membrane in early ring stage to a parasite membrane as the parasites developed to schizonts. The protein appears to be a PEXEL-negative membrane protein. Conclusion Plasmodium falciparum parasites express a native protein with copper transporter characteristics that binds copper in vitro. Localization of the protein to the erythrocyte and parasite plasma membranes could provide a mechanism for the delivery of novel anti-malarial compounds.

  13. LIN28 binds messenger RNAs at GGAGA motifs and regulates splicing factor abundance.

    Science.gov (United States)

    Wilbert, Melissa L; Huelga, Stephanie C; Kapeli, Katannya; Stark, Thomas J; Liang, Tiffany Y; Chen, Stella X; Yan, Bernice Y; Nathanson, Jason L; Hutt, Kasey R; Lovci, Michael T; Kazan, Hilal; Vu, Anthony Q; Massirer, Katlin B; Morris, Quaid; Hoon, Shawn; Yeo, Gene W

    2012-10-26

    LIN28 is a conserved RNA-binding protein implicated in pluripotency, reprogramming, and oncogenesis. It was previously shown to act primarily by blocking let-7 microRNA (miRNA) biogenesis, but here we elucidate distinct roles of LIN28 regulation via its direct messenger RNA (mRNA) targets. Through crosslinking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq) in human embryonic stem cells and somatic cells expressing exogenous LIN28, we have defined discrete LIN28-binding sites in a quarter of human transcripts. These sites revealed that LIN28 binds to GGAGA sequences enriched within loop structures in mRNAs, reminiscent of its interaction with let-7 miRNA precursors. Among LIN28 mRNA targets, we found evidence for LIN28 autoregulation and also direct but differing effects on the protein abundance of splicing regulators in somatic and pluripotent stem cells. Splicing-sensitive microarrays demonstrated that exogenous LIN28 expression causes widespread downstream alternative splicing changes. These findings identify important regulatory functions of LIN28 via direct mRNA interactions. Copyright © 2012 Elsevier Inc. All rights reserved.

  14. Function of the PEX19-binding site of human adrenoleukodystrophy protein as targeting motif in man and yeast. PMP targeting is evolutionarily conserved.

    Science.gov (United States)

    Halbach, André; Lorenzen, Stephan; Landgraf, Christiane; Volkmer-Engert, Rudolf; Erdmann, Ralf; Rottensteiner, Hanspeter

    2005-06-01

    We predicted in human peroxisomal membrane proteins (PMPs) the binding sites for PEX19, a key player in the topogenesis of PMPs, by virtue of an algorithm developed for yeast PMPs. The best scoring PEX19-binding site was found in the adrenoleukodystrophy protein (ALDP). The identified site was indeed bound by human PEX19 and was also recognized by the orthologous yeast PEX19 protein. Likewise, both human and yeast PEX19 bound with comparable affinities to the PEX19-binding site of the yeast PMP Pex13p. Interestingly, the identified PEX19-binding site of ALDP coincided with its previously determined targeting motif. We corroborated the requirement of the ALDP PEX19-binding site for peroxisomal targeting in human fibroblasts and showed that the minimal ALDP fragment targets correctly also in yeast, again in a PEX19-binding site-dependent manner. Furthermore, the human PEX19-binding site of ALDP proved interchangeable with that of yeast Pex13p in an in vivo targeting assay. Finally, we showed in vitro that most of the predicted binding sequences of human PMPs represent true binding sites for human PEX19, indicating that human PMPs harbor common PEX19-binding sites that do resemble those of yeast. Our data clearly revealed a role for PEX19-binding sites as PMP-targeting motifs across species, thereby demonstrating the evolutionary conservation of PMP signal sequences from yeast to man.

  15. Structure prediction of LDLR-HNP1 complex based on docking enhanced by LDLR binding 3D motif.

    Science.gov (United States)

    Esmaielbeiki, Reyhaneh; Naughton, Declan P; Nebel, Jean-Christophe

    2012-04-01

    Human antimicrobial peptides (AMPs), including defensins, have come under intense scrutiny owing to their key multiple roles as antimicrobial agents. Not only do they display direct action on microbes, but also recently they have been shown to interact with the immune system to increase antimicrobial activity. Unfortunately, since mechanisms involved in the binding of AMPs to mammalian cells are largely unknown, their potential as novel anti-infective agents cannot be exploited yet. Following the reported interaction of Human Neutrophil Peptide 1 dimer (HNP1) with a low density lipoprotein receptor (LDLR), a computational study was conducted to discover their putative mode of interaction. State-of-the-art docking software produced a set of LDLR-HNP1 complex 3D models. Creation of a 3D motif capturing atomic interactions of the LDLR binding interface allowed selection of the most plausible configurations. Eventually, only two models were in agreement with the literature. Binding energy estimations revealed that only one of them is particularly stable, but also interaction with LDLR weakens significantly bonds within the HNP1 dimer. This may be significant since it suggests a mechanism for internalisation of HNP1 in mammalian cells. In addition to a novel approach for complex structure prediction, this study proposes a 3D model of the LDLR-HNP1 complex which highlights the key residues which are involved in the interactions. The putative identification of the receptor binding mechanism should inform the future design of synthetic HNPs to afford maximum internalisation, which could lead to novel anti-infective drugs.

  16. A "coiled-coil" motif is important for oligomerization and DNA binding properties of human cytomegalovirus protein UL77.

    Directory of Open Access Journals (Sweden)

    Christina Sylvia Meissner

    Full Text Available Human cytomegalovirus (HCMV UL77 gene encodes the essential protein UL77, its function is characterized in the present study. Immunoprecipitation identified monomeric and oligomeric pUL77 in HCMV infected cells. Immunostaining of purified virions and subviral fractions showed that pUL77 is a structural protein associated with capsids. In silico analysis revealed the presence of a coiled-coil motif (CCM at the N-terminus of pUL77. Chemical cross-linking of either wild-type pUL77 or CCM deletion mutant (pUL77ΔCCM implicated that CCM is critical for oligomerization of pUL77. Furthermore, co-immunoprecipitations of infected and transfected cells demonstrated that pUL77 interacts with the capsid-associated DNA packaging motor components, pUL56 and pUL104, as well as the major capsid protein. The ability of pUL77 to bind dsDNA was shown by an in vitro assay. Binding to certain DNA was further confirmed by an assay using biotinylated 36-, 250-, 500-, 1000-meric dsDNA and 966-meric HCMV-specific dsDNA designed for this study. The binding efficiency (BE was determined by image processing program defining values above 1.0 as positive. While the BE of the pUL56 binding to the 36-mer bio-pac1 containing a packaging signal was 10.0 ± 0.63, the one for pUL77 was only 0.2±0.03. In contrast to this observation the BE of pUL77 binding to bio-500 bp or bio-1000 bp was 2.2 ± 0.41 and 4.9 ± 0.71, respectively. By using pUL77ΔCCM it was demonstrated that this protein could not bind to dsDNA. These data indicated that pUL77 (i could form homodimers, (ii CCM of pUL77 is crucial for oligomerization and (iii could bind to dsDNA in a sequence independent manner.

  17. Polycystin-2 activity is controlled by transcriptional coactivator with PDZ binding motif and PALS1-associated tight junction protein.

    Science.gov (United States)

    Duning, Kerstin; Rosenbusch, Deike; Schlüter, Marc A; Tian, Yuemin; Kunzelmann, Karl; Meyer, Nina; Schulze, Ulf; Markoff, Arseni; Pavenstädt, Hermann; Weide, Thomas

    2010-10-29

    Autosomal dominant polycystic kidney disease (ADPKD) is the most frequent monogenic cause of kidney failure, characterized by the development of renal cysts. ADPKD is caused by mutations of the polycystin-1 (PC1) or polycystin-2 (PC2) genes. PC2 encodes a Ca(2+)-permeable cation channel, and its dysfunction has been implicated in cyst development. The transcriptional coactivator with PDZ binding motif (TAZ) is required for the integrity of renal cilia. Its absence results in the development of renal cysts in a knock-out mouse model. TAZ directly interacts with PC2, and it has been suggested that another yet unidentified PDZ domain protein may be involved in the TAZ/PC2 interaction. Here we describe a novel interaction of TAZ with the multi-PDZ-containing PALS1-associated tight junction protein (PATJ). TAZ interacts with both the N-terminal PDZ domains 1-3 and the C-terminal PDZ domains 8-10 of PATJ, suggesting two distinct TAZ binding domains. We also show that the C terminus of PC2 strongly interacts with PDZ domains 8-10 and to a weaker extent with PDZ domains 1-3 of PATJ. Finally, we demonstrate that both TAZ and PATJ impair PC2 channel activity when co-expressed with PC2 in oocytes of Xenopus laevis. These results implicate TAZ and PATJ as novel regulatory elements of the PC2 channel and might thus be involved in ADPKD pathology.

  18. Basal lamina strengthens cell membrane integrity via the laminin G domain-binding motif of α-dystroglycan

    Science.gov (United States)

    Han, Renzhi; Kanagawa, Motoi; Yoshida-Moriguchi, Takako; Rader, Erik P.; Ng, Rainer A.; Michele, Daniel E.; Muirhead, David E.; Kunz, Stefan; Moore, Steven A.; Iannaccone, Susan T.; Miyake, Katsuya; McNeil, Paul L.; Mayer, Ulrike; Oldstone, Michael B. A.; Faulkner, John A.; Campbell, Kevin P.

    2009-01-01

    Skeletal muscle basal lamina is linked to the sarcolemma through transmembrane receptors, including integrins and dystroglycan. The function of dystroglycan relies critically on posttranslational glycosylation, a common target shared by a genetically heterogeneous group of muscular dystrophies characterized by α-dystroglycan hypoglycosylation. Here we show that both dystroglycan and integrin α7 contribute to force-production of muscles, but that only disruption of dystroglycan causes detachment of the basal lamina from the sarcolemma and renders muscle prone to contraction-induced injury. These phenotypes of dystroglycan-null muscles are recapitulated by Largemyd muscles, which have an intact dystrophin–glycoprotein complex and lack only the laminin globular domain-binding motif on α-dystroglycan. Compromised sarcolemmal integrity is directly shown in Largemyd muscles and similarly in normal muscles when arenaviruses compete with matrix proteins for binding α-dystroglycan. These data provide direct mechanistic insight into how the dystroglycan-linked basal lamina contributes to the maintenance of sarcolemmal integrity and protects muscles from damage. PMID:19633189

  19. Identification of the Raptor-binding motif on Arabidopsis S6 kinase and its use as a TOR signaling suppressor

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    Son, Ora; Kim, Sunghan; Hur, Yoon-Sun; Cheon, Choong-Ill, E-mail: ccheon@sookmyung.ac.kr

    2016-03-25

    TOR (target of rapamycin) kinase signaling plays central role as a regulator of growth and proliferation in all eukaryotic cells and its key signaling components and effectors are also conserved in plants. Unlike the mammalian and yeast counterparts, however, we found through yeast two-hybrid analysis that multiple regions of the Arabidopsis Raptor (regulatory associated protein of TOR) are required for binding to its substrate. We also identified that a 44-amino acid region at the N-terminal end of Arabidopsis ribosomal S6 kinase 1 (AtS6K1) specifically interacted with AtRaptor1, indicating that this region may contain a functional equivalent of the TOS (TOR-Signaling) motif present in the mammalian TOR substrates. Transient over-expression of this 44-amino acid fragment in Arabidopsis protoplasts resulted in significant decrease in rDNA transcription, demonstrating a feasibility of developing a new plant-specific TOR signaling inhibitor based upon perturbation of the Raptor-substrate interaction. - Highlights: • Multiple regions on the Arabidopsis Raptor protein were found to be involved in substrate binding. • N-terminal end of the Arabidopsis ribosomal S6 kinase 1 (AtS6K1) was responsible for interacting with AtRaptor1. • The Raptor-interacting fragment of AtS6K1 could be utilized as an effective inhibitor of plant TOR signaling.

  20. Identification of a positively evolving putative binding region with increased variability in posttranslational motifs in zonadhesin MAM domain 2.

    Science.gov (United States)

    Herlyn, Holger; Zischler, Hans

    2005-10-01

    Positive selection has been shown to be pervasive in sex-related proteins of many metazoan taxa. However, we are only beginning to understand molecular evolutionary processes on the lineage to humans. To elucidate the evolution of proteins involved in human reproduction, we studied the sequence evolution of MAM domains of the sperm-ligand zonadhesin in respect to single amino acid sites, solvent accessibility, and posttranslational modification. GenBank-data were supplemented by new cDNA-sequences of a representative non-human primate panel. Solvent accessibility predictions identified a probably exposed fragment of 30 amino acids belonging to MAM domain 2 (i.e., MAM domain 3 in mouse). The fragment is characterized by significantly increased rate of positively selected amino acid sites and exhibits high variability in predicted posttranslational modification, and, thus, might represent a binding region in the mature protein. At the same time, there is a significant coincidence of positively selected amino acid sites and non-conserved posttranslational motifs. We conclude that the binding specificity of zonadhesin MAM domains, especially of the presumed epitope, is achieved by positive selection at the level of single amino acid sites and posttranslational modifications, respectively.

  1. An Essential Role for the Glut1 PDZ-Binding Motif in Growth Factor Regulation of Glut1 Degradation and Trafficking

    OpenAIRE

    Wieman, Heather L.; Horn, Sarah R.; Jacobs, Sarah R.; Altman, Brian J.; Kornbluth, Sally; Rathmell, Jeffrey C.

    2009-01-01

    Cell surface localization of the glucose transporter, Glut1, is a cytokine-controlled process essential to support the metabolism and survival of hematopoietic cells. Molecular mechanisms that regulate Glut1 trafficking, however, are not certain. Here we show a C-terminal PDZ-binding motif in Glut1 is critical to promote maximal cytokine-stimulated Glut1 cell surface localization and prevent Glut1 lysosomal degradation in the absence of growth factor. Disruption of this PDZ-binding sequence t...

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

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    Derek M Murphy

    Full Text Available 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

  3. 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

  4. Guidelines for choosing molecular "alligator clip" binding motifs in electron transport devices

    Science.gov (United States)

    Reuter, Matthew G.; Seideman, Tamar; Ratner, Mark A.

    2011-04-01

    We employ a one-electron, tight-binding model of an electrode-molecule-electrode junction to explore the fundamental relationship between adsorption geometry and electron transport, producing exact results (within this model). By varying the chemisorption location (e.g., atop a surface atom or in a hollow site between surface atoms) and the molecule-electrode coupling, we find that the largest currents are realized when the molecule (i) is highly coordinated by the surface and (ii) has favorable overlap with electrode states near the Fermi level. We also show the importance of electrode-induced molecular level shifting for certain adsorption geometries, which can cause molecular levels far from the Fermi level to conduct better than those near the Fermi level. Since all of these factors are greatly influenced by the chemical moiety used to link the molecule to an electrode, these results present a set of guidelines to help choose "alligator clips" for molecular electronic devices.

  5. Uncommon structural motifs dominate the antigen binding site in human autoantibodies reactive with basement membrane collagen.

    Science.gov (United States)

    Foster, Mary H; Buckley, Elizabeth S; Chen, Benny J; Hwang, Kwan-Ki; Clark, Amy G

    2016-08-01

    Autoantibodies mediate organ destruction in multiple autoimmune diseases, yet their origins in patients remain poorly understood. To probe the genetic origins and structure of disease-associated autoantibodies, we engrafted immunodeficient mice with human CD34+ hematopoietic stem cells and immunized with the non-collagenous-1 (NC1) domain of the alpha3 chain of type IV collagen. This antigen is expressed in lungs and kidneys and is targeted by autoantibodies in anti-glomerular basement membrane (GBM) nephritis and Goodpasture syndrome (GPS), prototypic human organ-specific autoimmune diseases. Using Epstein Barr virus transformation and cell fusion, six human anti-alpha3(IV)NC1 collagen monoclonal autoantibodies (mAb) were recovered, including subsets reactive with human kidney and with epitopes recognized by patients' IgG. Sequence analysis reveals a long to exceptionally long heavy chain complementarity determining region3 (HCDR3), the major site of antigen binding, in all six mAb. Mean HCDR3 length is 25.5 amino acids (range 20-36), generated from inherently long DH and JH genes and extended regions of non-templated N-nucleotides. Long HCDR3 are suited to forming noncontiguous antigen contacts and to binding recessed, immunologically silent epitopes hidden from conventional antibodies, as seen with self-antigen crossreactive broadly neutralizing anti-HIV Ig (bnAb). The anti-alpha3(IV)NC1 collagen mAb also show preferential use of unmutated variable region genes that are enriched among human chronic lymphocytic leukemia antibodies that share features with natural polyreactive Ig. Our findings suggest unexpected relationships between pathogenic anti-collagen Ig, bnAb, and autoreactive Ig associated with malignancy, all of which arise from B cells expressing unconventional structural elements that may require transient escape from tolerance for successful expansion. Published by Elsevier Ltd.

  6. Uncommon Structural Motifs Dominate the Antigen Binding Site in Human Autoantibodies Reactive with Basement Membrane Collagen

    Science.gov (United States)

    Foster, Mary H.; Buckley, Elizabeth S.; Chen, Benny J.; Hwang, Kwan-Ki; Clark, Amy G.

    2016-01-01

    Autoantibodies mediate organ destruction in multiple autoimmune diseases, yet their origins in patients remain poorly understood. To probe the genetic origins and structure of disease-associated autoantibodies, we engrafted immunodeficient mice with human CD34+ hematopoietic stem cells and immunized with the non-collagenous-1 (NC1) domain of the alpha3 chain of type IV collagen. This antigen is expressed in lungs and kidneys and is targeted by autoantibodies in anti-glomerular basement membrane (GBM) nephritis and Goodpasture syndrome (GPS), prototypic human organ-specific autoimmune diseases. Using Epstein Barr virus transformation and cell fusion, six human anti-alpha3(IV)NC1 collagen monoclonal autoantibodies (mAb) were recovered, including subsets reactive with human kidney and with epitopes recognized by patients’ IgG. Sequence analysis reveals a long to exceptionally long heavy chain complementarity determining region3 (HCDR3), the major site of antigen binding, in all six mAb. Mean HCDR3 length is 25.5 amino acids (range 20–36), generated from inherently long DH and JH genes and extended regions of non-templated N-nucleotides. Long HCDR3 are suited to forming noncontiguous antigen contacts and to binding recessed, immunologically silent epitopes hidden from conventional antibodies, as seen with self-antigen crossreactive broadly neutralizing anti-HIV Ig (bnAb). The anti-alpha3(IV)NC1 collagen mAb also show preferential use of unmutated variable region genes that are enriched among human chronic lymphocytic leukemia antibodies that share features with natural polyreactive Ig. Our findings suggest unexpected relationships between pathogenic anti-collagen Ig, bnAb, and autoreactive Ig associated with malignancy, all of which arise from B cells expressing unconventional structural elements that may require transient escape from tolerance for successful expansion. PMID:27450516

  7. Flexibility of EF-hand motifs: structural and thermodynamic studies of Calcium Binding Protein-1 from Entamoeba histolytica with Pb2+, Ba2+, and Sr2+

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    Kumar Shivesh

    2012-08-01

    Full Text Available Abstract Background EF-hand proteins can be activated by the binding of various heavy metals other than calcium, and such complexes can disturb the calcium-signaling pathway and cause toxicity and disease causing state. So far, no comprehensive study has been done to understand different heavy metals binding to calcium signaling proteins. Results In this work, the flexibility of the EF-hand motifs are examined by crystallographic and thermodynamic studies of binding of Pb2+, Ba2+ and Sr2+ to Calcium Binding Protein-1 from Entamoeba histolytica (EhCaBP1. The structures of the EhCaBP1- heavy metal complexes are found to be overall similar, nevertheless specific differences in metal coordination, and small differences in the coordination distances between the metal and the ligands in the metal binding loop. The largest such distances occur for the Ba2+- EhCaBP1 complex, where two bariums are bound with partial occupancy at the EF2 motif. Thermodynamic studies confirm that EhCaBP1 has five binding sites for Ba2+ compared to four binding sites for the other metals. These structures and thermodynamic studies reveal that the EF-hand motifs can accommodate several heavy atoms with similar binding affinities. The binding of Ca2+ to the 1st, 2nd and 4th sites and the binding of Ba2+ to the 1st, 2nd, 4th and 5th sites are both enthalpically and entropically driven, whereas the binding of Sr2+ to the 1st, 2nd and 4th sites are simply enthalpy driven, interestingly in agreement with ITC data, Sr2+ do not coordinate with water in this structure. For all the metals, binding to the 3rd site is only entropy driven. Conclusion Energetically, Ca2+ is preferred in three sites, while in one site Ba2+ has better binding energy. The Sr2+-coordination in the EF hand motifs is similar to that of the native Ca2+ bound structure, except for the lack of water coordination. Sr2+ coordination seems to be a pre-formed in nature since all seven coordinating atoms are from the

  8. Identification of a novel calcium binding motif based on the detection of sequence insertions in the animal peroxidase domain of bacterial proteins.

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    Saray Santamaría-Hernando

    Full Text Available Proteins of the animal heme peroxidase (ANP superfamily differ greatly in size since they have either one or two catalytic domains that match profile PS50292. The orf PP_2561 of Pseudomonas putida KT2440 that we have called PepA encodes a two-domain ANP. The alignment of these domains with those of PepA homologues revealed a variable number of insertions with the consensus G-x-D-G-x-x-[GN]-[TN]-x-D-D. This motif has also been detected in the structure of pseudopilin (pdb 3G20, where it was found to be involved in Ca(2+ coordination although a sequence analysis did not reveal the presence of any known calcium binding motifs in this protein. Isothermal titration calorimetry revealed that a peptide containing this consensus motif bound specifically calcium ions with affinities ranging between 33-79 µM depending on the pH. Microcalorimetric titrations of the purified N-terminal ANP-like domain of PepA revealed Ca(2+ binding with a K(D of 12 µM and stoichiometry of 1.25 calcium ions per protein monomer. This domain exhibited peroxidase activity after its reconstitution with heme. These data led to the definition of a novel calcium binding motif that we have termed PERCAL and which was abundantly present in animal peroxidase-like domains of bacterial proteins. Bacterial heme peroxidases thus possess two different types of calcium binding motifs, namely PERCAL and the related hemolysin type calcium binding motif, with the latter being located outside the catalytic domains and in their C-terminal end. A phylogenetic tree of ANP-like catalytic domains of bacterial proteins with PERCAL motifs, including single domain peroxidases, was divided into two major clusters, representing domains with and without PERCAL motif containing insertions. We have verified that the recently reported classification of bacterial heme peroxidases in two families (cd09819 and cd09821 is unrelated to these insertions. Sequences matching PERCAL were detected in all kingdoms of

  9. ChIP-exo signal associated with DNA-binding motifs provides insight into the genomic binding of the glucocorticoid receptor and cooperating transcription factors.

    Science.gov (United States)

    Starick, Stephan R; Ibn-Salem, Jonas; Jurk, Marcel; Hernandez, Céline; Love, Michael I; Chung, Ho-Ryun; Vingron, Martin; Thomas-Chollier, Morgane; Meijsing, Sebastiaan H

    2015-06-01

    The classical DNA recognition sequence of the glucocorticoid receptor (GR) appears to be present at only a fraction of bound genomic regions. To identify sequences responsible for recruitment of this transcription factor (TF) to individual loci, we turned to the high-resolution ChIP-exo approach. We exploited this signal by determining footprint profiles of TF binding at single-base-pair resolution using ExoProfiler, a computational pipeline based on DNA binding motifs. When applied to our GR and the few available public ChIP-exo data sets, we find that ChIP-exo footprints are protein- and recognition sequence-specific signatures of genomic TF association. Furthermore, we show that ChIP-exo captures information about TFs other than the one directly targeted by the antibody in the ChIP procedure. Consequently, the shape of the ChIP-exo footprint can be used to discriminate between direct and indirect (tethering to other DNA-bound proteins) DNA association of GR. Together, our findings indicate that the absence of classical recognition sequences can be explained by direct GR binding to a broader spectrum of sequences than previously known, either as a homodimer or as a heterodimer binding together with a member of the ETS or TEAD families of TFs, or alternatively by indirect recruitment via FOX or STAT proteins. ChIP-exo footprints also bring structural insights and locate DNA:protein cross-link points that are compatible with crystal structures of the studied TFs. Overall, our generically applicable footprint-based approach uncovers new structural and functional insights into the diverse ways of genomic cooperation and association of TFs. © 2015 Starick et al.; Published by Cold Spring Harbor Laboratory Press.

  10. BEESEM: estimation of binding energy models using HT-SELEX data.

    Science.gov (United States)

    Ruan, Shuxiang; Swamidass, S Joshua; Stormo, Gary D

    2017-08-01

    Characterizing the binding specificities of transcription factors (TFs) is crucial to the study of gene expression regulation. Recently developed high-throughput experimental methods, including protein binding microarrays (PBM) and high-throughput SELEX (HT-SELEX), have enabled rapid measurements of the specificities for hundreds of TFs. However, few studies have developed efficient algorithms for estimating binding motifs based on HT-SELEX data. Also the simple method of constructing a position weight matrix (PWM) by comparing the frequency of the preferred sequence with single-nucleotide variants has the risk of generating motifs with higher information content than the true binding specificity. We developed an algorithm called BEESEM that builds on a comprehensive biophysical model of protein-DNA interactions, which is trained using the expectation maximization method. BEESEM is capable of selecting the optimal motif length and calculating the confidence intervals of estimated parameters. By comparing BEESEM with the published motifs estimated using the same HT-SELEX data, we demonstrate that BEESEM provides significant improvements. We also evaluate several motif discovery algorithms on independent PBM and ChIP-seq data. BEESEM provides significantly better fits to in vitro data, but its performance is similar to some other methods on in vivo data under the criterion of the area under the receiver operating characteristic curve (AUROC). This highlights the limitations of the purely rank-based AUROC criterion. Using quantitative binding data to assess models, however, demonstrates that BEESEM improves on prior models. Freely available on the web at http://stormo.wustl.edu/resources.html . stormo@wustl.edu. Supplementary data are available at Bioinformatics online.

  11. Adhesive and migratory effects of phosphophoryn are modulated by flanking peptides of the integrin binding motif.

    Directory of Open Access Journals (Sweden)

    Shigeki Suzuki

    Full Text Available Phosphophoryn (PP is generated from the proteolytic cleavage of dentin sialophosphoprotein (DSPP. Gene duplications in the ancestor dentin matrix protein-1 (DMP-1 genomic sequence created the DSPP gene in toothed animals. PP and DMP-1 are phosphorylated extracellular matrix proteins that belong to the family of small integrin-binding ligand N-linked glycoproteins (SIBLINGs. Many SIBLING members have been shown to evoke various cell responses through the integrin-binding Arg-Gly-Asp (RGD domain; however, the RGD-dependent function of PP is not yet fully understood. We demonstrated that recombinant PP did not exhibit any obvious cell adhesion ability, whereas the simultaneously purified recombinant DMP-1 did. A cell adhesion inhibitory analysis was performed by pre-incubating human osteosarcoma MG63 cells with various PP peptides before seeding onto vitronectin. The results obtained revealed that the incorporation of more than one amino acid on both sides of the PP-RGD domain was unable to inhibit the adhesion of MG63 cells onto vitronectin. Furthermore, the inhibitory activity of a peptide containing the PP-RGD domain with an open carboxyl-terminal side (H-463SDESDTNSESANESGSRGDA482-OH was more potent than that of a peptide containing the RGD domain with an open amino-terminal side (H-478SRGDASYTSDESSDDDNDSDSH499-OH. This phenomenon was supported by the potent cell adhesion and migration abilities of the recombinant truncated PP, which terminated with Ala482. Furthermore, various point mutations in Ala482 and/or Ser483 converted recombinant PP into cell-adhesive proteins. Therefore, we concluded that the Ala482-Ser483 flanking sequence, which was detected in primates and mice, was the key peptide bond that allowed the PP-RGD domain to be sequestered. The differential abilities of PP and DMP-1 to act on integrin imply that DSPP was duplicated from DMP-1 to serve as a crucial extracellular protein for tooth development rather than as an integrin

  12. An RNA Aptamer Targets the PDZ-Binding Motif of the HPV16 E6 Oncoprotein

    Energy Technology Data Exchange (ETDEWEB)

    Belyaeva, Tamara A.; Nicol, Clare; Cesur, Özlem [School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT (United Kingdom); Travé, Gilles [UMR 7242 CNRS-Université de Strasbourg, Ecole Supérieure de Biotechnologie, Boulevard Sébastien Brant, Illkirch 67412 (France); Blair, George Eric; Stonehouse, Nicola J., E-mail: n.j.stonehouse@leeds.ac.uk [School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT (United Kingdom)

    2014-07-24

    Human papillomavirus 16 (HPV16) is a high-risk DNA tumour virus which is the primary causative agent of cervical cancer. Cell transformation arises from deregulated expression of the E6 and E7 oncogenes. E6 has been shown to bind a number of cellular proteins, including p53 and proteins containing a PDZ domain. This study reports the first RNA aptamers to E6. These have been employed as molecular tools to further investigate E6-p53 and E6-PDZ interactions. This study is focussed on two aptamers (termed F2 and F4) which induced apoptosis in cells derived from an HPV16-transformed cervical carcinoma. The molecules were able to inhibit the interaction between E6 and PDZ1 from Magi1, with F2 being the most effective inhibitor. Neither of the aptamers inhibited E6-p53 interaction or p53 degradation. This study shows the specificity of this approach and highlights the potential benefits of the E6 aptamers as potential therapeutic or diagnostic agents in the future.

  13. An RNA Aptamer Targets the PDZ-Binding Motif of the HPV16 E6 Oncoprotein

    Directory of Open Access Journals (Sweden)

    Tamara A. Belyaeva

    2014-07-01

    Full Text Available Human papillomavirus 16 (HPV16 is a high-risk DNA tumour virus which is the primary causative agent of cervical cancer. Cell transformation arises from deregulated expression of the E6 and E7 oncogenes. E6 has been shown to bind a number of cellular proteins, including p53 and proteins containing a PDZ domain. This study reports the first RNA aptamers to E6. These have been employed as molecular tools to further investigate E6-p53 and E6-PDZ interactions. This study is focussed on two aptamers (termed F2 and F4 which induced apoptosis in cells derived from an HPV16-transformed cervical carcinoma. The molecules were able to inhibit the interaction between E6 and PDZ1 from Magi1, with F2 being the most effective inhibitor. Neither of the aptamers inhibited E6-p53 interaction or p53 degradation. This study shows the specificity of this approach and highlights the potential benefits of the E6 aptamers as potential therapeutic or diagnostic agents in the future.

  14. A CAF40-binding motif facilitates recruitment of the CCR4-NOT complex to mRNAs targeted by Drosophila Roquin

    Science.gov (United States)

    Sgromo, Annamaria; Raisch, Tobias; Bawankar, Praveen; Bhandari, Dipankar; Chen, Ying; Kuzuoğlu-Öztürk, Duygu; Weichenrieder, Oliver; Izaurralde, Elisa

    2017-01-01

    Human (Hs) Roquin1 and Roquin2 are RNA-binding proteins that promote mRNA target degradation through the recruitment of the CCR4-NOT deadenylase complex and are implicated in the prevention of autoimmunity. Roquin1 recruits CCR4-NOT via a C-terminal region that is not conserved in Roquin2 or in invertebrate Roquin. Here we show that Roquin2 and Drosophila melanogaster (Dm) Roquin also interact with the CCR4-NOT complex through their C-terminal regions. The C-terminal region of Dm Roquin contains multiple motifs that mediate CCR4-NOT binding. One motif binds to the CAF40 subunit of the CCR4-NOT complex. The crystal structure of the Dm Roquin CAF40-binding motif (CBM) bound to CAF40 reveals that the CBM adopts an α-helical conformation upon binding to a conserved surface of CAF40. Thus, despite the lack of sequence conservation, the C-terminal regions of Roquin proteins act as an effector domain that represses the expression of mRNA targets via recruitment of the CCR4-NOT complex. PMID:28165457

  15. An activating mutation reveals a second binding mode of the integrin α2 I domain to the GFOGER motif in collagens.

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    Federico Carafoli

    Full Text Available The GFOGER motif in collagens (O denotes hydroxyproline represents a high-affinity binding site for all collagen-binding integrins. Other GxOGER motifs require integrin activation for maximal binding. The E318W mutant of the integrin α2β1 I domain displays a relaxed collagen specificity, typical of an active state. E318W binds more strongly than the wild-type α2 I domain to GMOGER, and forms a 2:1 complex with a homotrimeric, collagen-like, GFOGER peptide. Crystal structure analysis of this complex reveals two E318W I domains, A and B, bound to a single triple helix. The E318W I domains are virtually identical to the collagen-bound wild-type I domain, suggesting that the E318W mutation activates the I domain by destabilising the unligated conformation. E318W I domain A interacts with two collagen chains similarly to wild-type I domain (high-affinity mode. E318W I domain B makes favourable interactions with only one collagen chain (low-affinity mode. This observation suggests that single GxOGER motifs in the heterotrimeric collagens V and IX may support binding of activated integrins.

  16. A tandem sequence motif acts as a distance-dependent enhancer in a set of genes involved in translation by binding the proteins NonO and SFPQ

    Directory of Open Access Journals (Sweden)

    Roepcke Stefan

    2011-12-01

    Full Text Available Abstract Background Bioinformatic analyses of expression control sequences in promoters of co-expressed or functionally related genes enable the discovery of common regulatory sequence motifs that might be involved in co-ordinated gene expression. By studying promoter sequences of the human ribosomal protein genes we recently identified a novel highly specific Localized Tandem Sequence Motif (LTSM. In this work we sought to identify additional genes and LTSM-binding proteins to elucidate potential regulatory mechanisms. Results Genome-wide analyses allowed finding a considerable number of additional LTSM-positive genes, the products of which are involved in translation, among them, translation initiation and elongation factors, and 5S rRNA. Electromobility shift assays then showed specific signals demonstrating the binding of protein complexes to LTSM in ribosomal protein gene promoters. Pull-down assays with LTSM-containing oligonucleotides and subsequent mass spectrometric analysis identified the related multifunctional nucleotide binding proteins NonO and SFPQ in the binding complex. Functional characterization then revealed that LTSM enhances the transcriptional activity of the promoters in dependency of the distance from the transcription start site. Conclusions Our data demonstrate the power of bioinformatic analyses for the identification of biologically relevant sequence motifs. LTSM and the here found LTSM-binding proteins NonO and SFPQ were discovered through a synergistic combination of bioinformatic and biochemical methods and are regulators of the expression of a set of genes of the translational apparatus in a distance-dependent manner.

  17. The histidine of the c-type cytochrome CXXCH haem-binding motif is essential for haem attachment by the Escherichia coli cytochrome c maturation (Ccm) apparatus.

    Science.gov (United States)

    Allen, James W A; Leach, Nicholas; Ferguson, Stuart J

    2005-07-15

    c-type cytochromes are characterized by covalent attachment of haem to the protein by two thioether bonds formed between the haem vinyl groups and the cysteine sulphurs in a CXXCH peptide motif. In Escherichia coli and many other Gram-negative bacteria, this post-translational haem attachment is catalysed by the Ccm (cytochrome c maturation) system. The features of the apocytochrome substrate required and recognized by the Ccm apparatus are uncertain. In the present study, we report investigations of maturation of cytochrome b562 variants containing CXXCR, CXXCK or CXXCM haem-binding motifs. None of them showed any evidence for correct maturation by the Ccm system. However, we have determined, for each variant, that the proteins (i) were expressed in large amounts, (ii) could bind haem in vivo and/or in vitro and (iii) were not degraded in the cell. Together with previous observations, these results strongly suggest that the apocytochrome substrate feature recognized by the Ccm system is simply the two cysteine residues and the histidine of the CXXCH haem-binding motif. Using the same experimental approach, we have also investigated a cytochrome b562 variant containing the special CWSCK motif that binds the active-site haem of E. coli nitrite reductase NrfA. Whereas a CWSCH analogue was matured by the Ccm apparatus in large amounts, the CWSCK form was not detectably matured either by the Ccm system or by the dedicated Nrf biogenesis proteins, implying that the substrate recognition features for haem attachment in NrfA may be more extensive than the CWSCK motif.

  18. A Polybasic Plasma Membrane Binding Motif in the I-II Linker Stabilizes Voltage-gated CaV1.2 Calcium Channel Function.

    Science.gov (United States)

    Kaur, Gurjot; Pinggera, Alexandra; Ortner, Nadine J; Lieb, Andreas; Sinnegger-Brauns, Martina J; Yarov-Yarovoy, Vladimir; Obermair, Gerald J; Flucher, Bernhard E; Striessnig, Jörg

    2015-08-21

    L-type voltage-gated Ca(2+) channels (LTCCs) regulate many physiological functions like muscle contraction, hormone secretion, gene expression, and neuronal excitability. Their activity is strictly controlled by various molecular mechanisms. The pore-forming α1-subunit comprises four repeated domains (I-IV), each connected via an intracellular linker. Here we identified a polybasic plasma membrane binding motif, consisting of four arginines, within the I-II linker of all LTCCs. The primary structure of this motif is similar to polybasic clusters known to interact with polyphosphoinositides identified in other ion channels. We used de novo molecular modeling to predict the conformation of this polybasic motif, immunofluorescence microscopy and live cell imaging to investigate the interaction with the plasma membrane, and electrophysiology to study its role for Cav1.2 channel function. According to our models, this polybasic motif of the I-II linker forms a straight α-helix, with the positive charges facing the lipid phosphates of the inner leaflet of the plasma membrane. Membrane binding of the I-II linker could be reversed after phospholipase C activation, causing polyphosphoinositide breakdown, and was accelerated by elevated intracellular Ca(2+) levels. This indicates the involvement of negatively charged phospholipids in the plasma membrane targeting of the linker. Neutralization of four arginine residues eliminated plasma membrane binding. Patch clamp recordings revealed facilitated opening of Cav1.2 channels containing these mutations, weaker inhibition by phospholipase C activation, and reduced expression of channels (as quantified by ON-gating charge) at the plasma membrane. Our data provide new evidence for a membrane binding motif within the I-II linker of LTCC α1-subunits essential for stabilizing normal Ca(2+) channel function. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. [Expression of transcriptional coactivator with PDZ-binding motif (TAZ) in colon cancer tissues and its clinical significance].

    Science.gov (United States)

    Zeng, Changqing; Huang, Liangxiang; Zheng, Yu; Huang, Haixiao; Chen, Linhao; Chi, Liangjie

    2015-11-01

    To investigate the expression of transcriptional coactivator with PDZ-binding motif(TAZ) in colon cancer tissues and its association with clinicopathological parameters and prognosis of patients. The expression of TAZ protein was detected in 56 resected colon cancer tissues and matched tumor-adjacent tissues using immunohistochemistry. The positive expression rate of TAZ was compared between patients with different clinicopathological features. The association between TAZ expression and prognosis was analyzed. Expression of TAZ protein located in the nucleolus. The positive expression rate of TAZ in colon cancer tissues was significantly higher than that in matched tumor-adjacent tissues(73.2% vs. 12.5%, P=0.000). Clinicopathological evaluation suggested that the expression of TAZ protein was associated with tumor size(P=0.009), depth of infiltration(P=0.026), lymph node metastasis (P=0.007) and TNM staging(P=0.004). Colon cancer patients with negative expression of TAZ showed a better 5-year survival as compared with those with positive expression of TAZ (66.7% vs. 22.9%, P=0.0017). Multivariate Cox regression analysis revealed that positive TAZ expression was an independent factor for predicting poor prognosis in colon cancer (HR:3.532, 95% CI: 1.3-9.9, P=0.016). The expression of TAZ protein is up-regulated in colon cancer tissues and its high expression is associated with poor prognosis of colon cancer patients.

  20. Human HDAC7 Harbors a Class IIa Histone Deacetylase-specific Zinc Binding Motif and Cryptic Deacetylase Activity

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    Schuetz, Anja; Min, Jinrong; Allali-Hassani, Abdellah; Schapira, Matthieu; Shuen, Michael; Loppnau, Peter; Mazitschek, Ralph; Kwiatkowski, Nick P.; Lewis, Timothy A.; Maglathin, Rebecca L.; McLean, Thomas H.; Bochkarev, Alexey; Plotnikov, Alexander N.; Vedadi, Masoud; Arrowsmith, Cheryl H. (MIT); (Toronto)

    2010-10-18

    Histone deacetylases (HDACs) are protein deacetylases that play a role in repression of gene transcription and are emerging targets in cancer therapy. Here, we characterize the structure and enzymatic activity of the catalytic domain of human HDAC7 (cdHDAC7). Although HDAC7 normally exists as part of a multiprotein complex, we show that cdHDAC7 has a low level of deacetylase activity which can be inhibited by known HDAC inhibitors. The crystal structures of human cdHDAC7 and its complexes with two hydroxamate inhibitors are the first structures of the catalytic domain of class IIa HDACs and demonstrate significant differences with previously reported class I and class IIb-like HDAC structures. We show that cdHDAC7 has an additional class IIa HDAC-specific zinc binding motif adjacent to the active site which is likely to participate in substrate recognition and protein-protein interaction and may provide a site for modulation of activity. Furthermore, a different active site topology results in modified catalytic properties and in an enlarged active site pocket. Our studies provide mechanistic insights into class IIa HDACs and facilitate the design of specific modulators.

  1. Novel Catalytically-Inactive PII Metalloproteinases from a Viperid Snake Venom with Substitutions in the Canonical Zinc-Binding Motif

    Science.gov (United States)

    Camacho, Erika; Sanz, Libia; Escalante, Teresa; Pérez, Alicia; Villalta, Fabián; Lomonte, Bruno; Neves-Ferreira, Ana Gisele C.; Feoli, Andrés; Calvete, Juan J.; Gutiérrez, José María; Rucavado, Alexandra

    2016-01-01

    Snake venom metalloproteinases (SVMPs) play key biological roles in prey immobilization and digestion. The majority of these activities depend on the hydrolysis of relevant protein substrates in the tissues. Hereby, we describe several isoforms and a cDNA clone sequence, corresponding to PII SVMP homologues from the venom of the Central American pit viper Bothriechis lateralis, which have modifications in the residues of the canonical sequence of the zinc-binding motif HEXXHXXGXXH. As a consequence, the proteolytic activity of the isolated proteins was undetectable when tested on azocasein and gelatin. These PII isoforms comprise metalloproteinase and disintegrin domains in the mature protein, thus belonging to the subclass PIIb of SVMPs. PII SVMP homologues were devoid of hemorrhagic and in vitro coagulant activities, effects attributed to the enzymatic activity of SVMPs, but induced a mild edema. One of the isoforms presents the characteristic RGD sequence in the disintegrin domain and inhibits ADP- and collagen-induced platelet aggregation. Catalytically-inactive SVMP homologues may have been hitherto missed in the characterization of snake venoms. The presence of such enzymatically-inactive homologues in snake venoms and their possible toxic and adaptive roles deserve further investigation. PMID:27754342

  2. Identification of the Raptor-binding motif on Arabidopsis S6 kinase and its use as a TOR signaling suppressor.

    Science.gov (United States)

    Son, Ora; Kim, Sunghan; Hur, Yoon-Sun; Cheon, Choong-Ill

    2016-03-25

    TOR (target of rapamycin) kinase signaling plays central role as a regulator of growth and proliferation in all eukaryotic cells and its key signaling components and effectors are also conserved in plants. Unlike the mammalian and yeast counterparts, however, we found through yeast two-hybrid analysis that multiple regions of the Arabidopsis Raptor (regulatory associated protein of TOR) are required for binding to its substrate. We also identified that a 44-amino acid region at the N-terminal end of Arabidopsis ribosomal S6 kinase 1 (AtS6K1) specifically interacted with AtRaptor1, indicating that this region may contain a functional equivalent of the TOS (TOR-Signaling) motif present in the mammalian TOR substrates. Transient over-expression of this 44-amino acid fragment in Arabidopsis protoplasts resulted in significant decrease in rDNA transcription, demonstrating a feasibility of developing a new plant-specific TOR signaling inhibitor based upon perturbation of the Raptor-substrate interaction.

  3. Regulation of T cell Receptor Activation by Dynamic Membrane Binding of the CD3ε Cytoplasmic Tyrosine-Based Motif

    Science.gov (United States)

    Xu, Chenqi; Gagnon, Etienne; Call, Matthew E.; Schnell, Jason R.; Schwieters, Charles D.; Carman, Christopher V.; Chou, James J.; Wucherpfennig, Kai W.

    2008-01-01

    Summary Many immune system receptors signal through cytoplasmic tyrosine-based motifs (ITAMs), but how receptor ligation results in ITAM phosphorylation remains unknown. Live cell imaging studies showed a close interaction of the CD3ε cytoplasmic domain of the T cell receptor (TCR) with the plasma membrane through fluorescence resonance energy transfer (FRET) between a C-terminal fluorescent protein and a membrane fluorophore. Electrostatic interactions between basic CD3ε residues and acidic phospholipids enriched in the inner leaflet of the plasma membrane were required for binding. The nuclear magnetic resonance (NMR) structure of the lipid-bound state of this cytoplasmic domain revealed deep insertion of the two key tyrosines into the hydrophobic core of the lipid bilayer. Receptor ligation thus needs to result in unbinding of the CD3ε ITAM from the membrane to render these tyrosines accessible to Src kinases. Sequestration of key tyrosines into the lipid bilayer represents a previously unrecognized mechanism for control of receptor activation. PMID:19013279

  4. MicroRNA genes preferentially expressed in dendritic cells contain sites for conserved transcription factor binding motifs in their promoters

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    Huynen Martijn A

    2011-06-01

    Full Text Available Abstract Background MicroRNAs (miRNAs play a fundamental role in the regulation of gene expression by translational repression or target mRNA degradation. Regulatory elements in miRNA promoters are less well studied, but may reveal a link between their expression and a specific cell type. Results To explore this link in myeloid cells, miRNA expression profiles were generated from monocytes and dendritic cells (DCs. Differences in miRNA expression among monocytes, DCs and their stimulated progeny were observed. Furthermore, putative promoter regions of miRNAs that are significantly up-regulated in DCs were screened for Transcription Factor Binding Sites (TFBSs based on TFBS motif matching score, the degree to which those TFBSs are over-represented in the promoters of the up-regulated miRNAs, and the extent of conservation of the TFBSs in mammals. Conclusions Analysis of evolutionarily conserved TFBSs in DC promoters revealed preferential clustering of sites within 500 bp upstream of the precursor miRNAs and that many mRNAs of cognate TFs of the conserved TFBSs were indeed expressed in the DCs. Taken together, our data provide evidence that selected miRNAs expressed in DCs have evolutionarily conserved TFBSs relevant to DC biology in their promoters.

  5. The CcpA regulon of Streptococcus suis reveals novel insights into the regulation of the streptococcal central carbon metabolism by binding of CcpA to two distinct binding motifs.

    Science.gov (United States)

    Willenborg, Jörg; de Greeff, Astrid; Jarek, Michael; Valentin-Weigand, Peter; Goethe, Ralph

    2014-04-01

    Streptococcus suis (S. suis) is a neglected zoonotic streptococcus causing fatal diseases in humans and in pigs. The transcriptional regulator CcpA (catabolite control protein A) is involved in the metabolic adaptation to different carbohydrate sources and virulence of S. suis and other pathogenic streptococci. In this study, we determined the DNA binding characteristics of CcpA and identified the CcpA regulon during growth of S. suis. Electrophoretic mobility shift analyses showed promiscuous DNA binding of CcpA to cognate cre sites in vitro. In contrast, sequencing of immunoprecipitated chromatin revealed two specific consensus motifs, a pseudo-palindromic cre motif (WWGAAARCGYTTTCWW) and a novel cre2 motif (TTTTYHWDHHWWTTTY), within the regulatory elements of the genes directly controlled by CcpA. Via these elements CcpA regulates expression of genes involved in carbohydrate uptake and conversion, and in addition in important metabolic pathways of the central carbon metabolism, like glycolysis, mixed-acid fermentation, and the fragmentary TCA cycle. Furthermore, our analyses provide evidence that CcpA regulates the genes of the central carbon metabolism by binding either the pseudo-palindromic cre motif or the cre2 motif in a HPr(Ser)∼P independent conformation.

  6. Validation of chemical compound library screening for transcriptional co-activator with PDZ-binding motif inhibitors using GFP-fused transcriptional co-activator with PDZ-binding motif.

    Science.gov (United States)

    Nagashima, Shunta; Maruyama, Junichi; Kawano, Shodai; Iwasa, Hiroaki; Nakagawa, Kentaro; Ishigami-Yuasa, Mari; Kagechika, Hiroyuki; Nishina, Hiroshi; Hata, Yutaka

    2016-06-01

    Transcriptional co-activator with PDZ-binding motif (TAZ) plays versatile roles in cell proliferation and differentiation. It is phosphorylated by large tumor suppressor kinases, the core kinases of the tumor-suppressive Hippo pathway. Phosphorylation induces the cytoplasmic accumulation of TAZ and its degradation. In human cancers, the deregulation of the Hippo pathway and gene amplification enhance TAZ activity. TAZ interacts with TEA domain family members (TEAD), and upregulates genes implicated in epithelial-mesenchymal transition. It also confers stemness to cancer cells. Thus, TAZ activation provides cancer cells with malignant properties and worsens the clinical prognosis. Therefore, TAZ attracts attention as a therapeutic target in cancer therapy. We applied 18 606 small chemical compounds to human osteosarcoma U2OS cells expressing GFP-fused TAZ (GFP-TAZ), monitored the subcellular localization of GFP-TAZ, and selected 33 compounds that shifted GFP-TAZ to the cytoplasm. Unexpectedly, only a limited number of compounds suppressed TAZ-mediated enhancement of TEAD-responsive reporter activity. Moreover, the compounds that weakened TEAD reporter activity did not necessarily decrease the unphosphorylated TAZ. In this study, we focused on three compounds that decreased both TEAD reporter activity and unphosphorylated TAZ, and treated several human cancer cells with these compounds. One compound did not show a remarkable effect, whereas the other two compounds compromised the cell viability in certain cancer cells. In conclusion, the GFP-TAZ-based assay can be used as the first screening for compounds that inhibit TAZ and show anticancer properties. To develop anticancer drugs, we need additional assays to select the compounds.

  7. Complementarity of Binding Motifs is a General Property of HLA-A and HLA-B Molecules and Does Not Seem to Effect HLA Haplotype Composition.

    Science.gov (United States)

    Rao, Xiangyu; De Boer, Rob J; van Baarle, Debbie; Maiers, Martin; Kesmir, Can

    2013-01-01

    Different human leukocyte antigen (HLA) haplotypes (i.e., the specific combinations of HLA-A, -B, -DR alleles inherited together from one parent) are observed in different frequencies in human populations. Some haplotypes, like HLA-A1-B8, are very frequent, reaching up to 10% in the Caucasian population, while others are very rare. Numerous studies have identified associations between HLA haplotypes and diseases, and differences in haplotype frequencies can in part be explained by these associations: the stronger the association with a severe (autoimmune) disease, the lower the expected HLA haplotype frequency. The peptide repertoires of the HLA molecules composing a haplotype can also influence the frequency of a haplotype. For example, it would seem advantageous to have HLA molecules with non-overlapping binding specificities within a haplotype, as individuals expressing such an haplotype would present a diverse set of peptides from viruses and pathogenic bacteria on the cell surface. To test this hypothesis, we collect the proteome data from a set of common viruses, and estimate the total ligand repertoire of HLA class I haplotypes (HLA-A-B) using in silico predictions. We compare the size of these repertoires to the HLA haplotype frequencies reported in the National Marrow Donor Program (NMDP). We find that in most HLA-A and HLA-B pairs have fairly distinct binding motifs, and that the observed haplotypes do not contain HLA-A and -B molecules with more distinct binding motifs than random HLA-A and HLA-B pairs. In addition, the population frequency of a haplotype is not correlated to the distinctness of its HLA-A and HLA-B peptide binding motifs. These results suggest that there is a not a strong selection pressure on the haplotype level favoring haplotypes having HLA molecules with distinct binding motifs, which would result the largest possible presented peptide repertoires in the context of infectious diseases.

  8. Complementarity of binding motifs is a general property of HLA-A and HLA-B molecules and does not seem to effect HLA haplotype composition

    Directory of Open Access Journals (Sweden)

    Xiangyu eRao

    2013-11-01

    Full Text Available Different HLA haplotypes (i.e., the specific combinations of HLA-A, -B, -DR alleles inherited together from one parent are observed in different frequencies in human populations. Some haplotypes, like HLA-A1-B8, are very frequent, reaching up to 10% in the Caucasian population, while others are very rare. Numerous studies have identified associations between HLA haplotypes and diseases, and differences in haplotype frequencies can in part be explained by these associations: the stronger the association with a severe (autoimmune disease, the lower the expected HLA haplotype frequency. The peptide repertoires of the HLA molecules composing a haplotype can also influence the frequency of a haplotype. For example, it would seem advantageous to have HLA molecules with non-overlapping binding specificities within a haplotype, as individuals expressing such an haplotype would present a diverse set of peptides from viruses and pathogenic bacteria on the cell surface. To test this hypothesis, we collect the proteome data from a set of common viruses, and estimate the total ligand repertoire of HLA class I haplotypes (HLA-A-B using in silico predictions. We compare the size of these repertoires to the HLA haplotype frequencies reported in the National Marrow Donor Program (NMDP. We find that in most HLA-A and HLA-B pairs have fairly distinct binding motifs, and that the observed haplotypes do not contain HLA-A and –B molecules with more distinct binding motifs than random HLA-A and HLA-B pairs. In addition, the population frequency of a haplotype is not correlated to the distinctness of its HLA-A and HLA-B peptide binding motifs. These results suggest that there is a not a strong selection pressure on the haplotype level favouring haplotypes having HLA molecules with distinct binding motifs, which would result the largest possible presented peptide repertoires in the context of infectious diseases.

  9. Role of NH{sub 2}-terminal hydrophobic motif in the subcellular localization of ATP-binding cassette protein subfamily D: Common features in eukaryotic organisms

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Asaka; Asahina, Kota; Okamoto, Takumi; Kawaguchi, Kosuke [Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Kostsin, Dzmitry G. [Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Institute of Biophysics and Cell Engineering, National Academy of Sciences of Belarus, Academicheskaya Str. 27, Minsk 220072 (Belarus); Kashiwayama, Yoshinori [Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Takanashi, Kojiro; Yazaki, Kazufumi [Laboratory of Plant Gene Expression, Research Institute for Sustainable Humanosphere, Kyoko University, Uji, Kyoto 611-0011 (Japan); Imanaka, Tsuneo, E-mail: imanaka@pha.u-toyama.ac.jp [Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Morita, Masashi [Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan)

    2014-10-24

    Highlights: • ABCD proteins classifies based on with or without NH{sub 2}-terminal hydrophobic segment. • The ABCD proteins with the segment are targeted peroxisomes. • The ABCD proteins without the segment are targeted to the endoplasmic reticulum. • The role of the segment in organelle targeting is conserved in eukaryotic organisms. - Abstract: In mammals, four ATP-binding cassette (ABC) proteins belonging to subfamily D have been identified. ABCD1–3 possesses the NH{sub 2}-terminal hydrophobic region and are targeted to peroxisomes, while ABCD4 lacking the region is targeted to the endoplasmic reticulum (ER). Based on hydropathy plot analysis, we found that several eukaryotes have ABCD protein homologs lacking the NH{sub 2}-terminal hydrophobic segment (H0 motif). To investigate whether the role of the NH{sub 2}-terminal H0 motif in subcellular localization is conserved across species, we expressed ABCD proteins from several species (metazoan, plant and fungi) in fusion with GFP in CHO cells and examined their subcellular localization. ABCD proteins possessing the NH{sub 2}-terminal H0 motif were localized to peroxisomes, while ABCD proteins lacking this region lost this capacity. In addition, the deletion of the NH{sub 2}-terminal H0 motif of ABCD protein resulted in their localization to the ER. These results suggest that the role of the NH{sub 2}-terminal H0 motif in organelle targeting is widely conserved in living organisms.

  10. Two 14-3-3 binding motifs are required for stable association of Forkhead transcription factor FOXO4 with 14-3-3 proteins and inhibition of DNA binding.

    Science.gov (United States)

    Obsil, Tomas; Ghirlando, Rodolfo; Anderson, D Eric; Hickman, Alison Burgess; Dyda, Fred

    2003-12-30

    The 14-3-3 proteins, a family of dimeric regulatory proteins, are involved in many biologically important processes. The common feature of 14-3-3 proteins is their ability to bind to other proteins in a phosphorylation-dependent manner. Through these binding interactions, 14-3-3 proteins work as molecular scaffolds, modulating the biological functions of their partners. 14-3-3 proteins recognize short motifs containing a phosphorylated serine or threonine residue. In this study, we have quantitatively characterized the in vitro interactions among 14-3-3, the Forkhead transcription factor FOXO4, and its target DNA, the insulin response element. Phosphorylation of FOXO4 (residues 11-213) by protein kinase B at Thr-28 and Ser-193 creates two 14-3-3 binding motifs. Analytical gel filtration and sedimentation equilibrium experiments indicate that doubly phosphorylated FOXO4 and 14-3-3zeta form a complex with 1:2 molar stoichiometry and a K(D) of less than 30 nM. In contrast, singly phosphorylated FOXO4 mutants bind 14-3-3zeta with significantly lower affinity while retaining the ability to bind DNA. An active role for 14-3-3 in the disassembly of the FOXO4/DNA complex is demonstrated by the fact that, in the presence of 14-3-3, two phosphorylated 14-3-3 binding motifs are needed for the complete inhibition of FOXO4 binding to its target DNA.

  11. Binding of ABI4 to a CACCG motif mediates the ABA-induced expression of the ZmSSI gene in maize (Zea mays L.) endosperm.

    Science.gov (United States)

    Hu, Yu-Feng; Li, Yang-Ping; Zhang, Junjie; Liu, Hanmei; Tian, Mengliang; Huang, Yubi

    2012-10-01

    Starch synthase I (SSI) contributes the majority of the starch synthase activity in developing maize endosperm. In this work, the effects of various plant hormones and sugars on the expression of the starch synthase I gene (ZmSSI) in developing maize endosperms were examined. The accumulation of ZmSSI mRNA was induced using abscisic acid (ABA) but not with glucose, sucrose, or gibberellin treatment. To investigate the molecular mechanism underlying this effect, the ZmSSI promoter region (-1537 to +51) was isolated and analysed. A transient expression assay in maize endosperm tissue showed that the full-length ZmSSI promoter is activated by ABA. The results of deletion and mutation assays demonstrated that a CACCG motif in the ZmSSI promoter is responsible for the ABA inducibility. The results of binding shift assays indicated that this CACCG motif interacts with the maize ABI4 protein in vitro. The overexpression of ABI4 in endosperm tissue enhanced the activity of a promoter containing the CACCG motif in the absence of ABA treatment. Expression pattern analysis indicated that the transcription pattern of ABI4 in the developing maize endosperm was induced by ABA treatment but was only slightly affected by glucose or sucrose treatment. Taken together, these data indicate that ABI4 binds to the CACCG motif in the ZmSSI promoter and mediates its ABA inducibility.

  12. Crystal Structures of the Scaffolding Protein LGN Reveal the General Mechanism by Which GoLoco Binding Motifs Inhibit the Release of GDP from Gαi *

    Science.gov (United States)

    Jia, Min; Li, Jianchao; Zhu, Jinwei; Wen, Wenyu; Zhang, Mingjie; Wang, Wenning

    2012-01-01

    GoLoco (GL) motif-containing proteins regulate G protein signaling by binding to Gα subunit and acting as guanine nucleotide dissociation inhibitors. GLs of LGN are also known to bind the GDP form of Gαi/o during asymmetric cell division. Here, we show that the C-terminal GL domain of LGN binds four molecules of Gαi·GDP. The crystal structures of Gαi·GDP in complex with LGN GL3 and GL4, respectively, reveal distinct GL/Gαi interaction features when compared with the only high resolution structure known with GL/Gαi interaction between RGS14 and Gαi1. Only a few residues C-terminal to the conserved GL sequence are required for LGN GLs to bind to Gαi·GDP. A highly conserved “double Arg finger” sequence (RΨ(D/E)(D/E)QR) is responsible for LGN GL to bind to GDP bound to Gαi. Together with the sequence alignment, we suggest that the LGN GL/Gαi interaction represents a general binding mode between GL motifs and Gαi. We also show that LGN GLs are potent guanine nucleotide dissociation inhibitors. PMID:22952234

  13. KIR polymorphisms modulate peptide-dependent binding to an MHC class I ligand with a Bw6 motif.

    Directory of Open Access Journals (Sweden)

    Arnaud D Colantonio

    2011-03-01

    Full Text Available Molecular interactions between killer immunoglobulin-like receptors (KIRs and their MHC class I ligands play a central role in the regulation of natural killer (NK cell responses to viral pathogens and tumors. Here we identify Mamu-A1*00201 (Mamu-A*02, a common MHC class I molecule in the rhesus macaque with a canonical Bw6 motif, as a ligand for Mamu-KIR3DL05. Mamu-A1*00201 tetramers folded with certain SIV peptides, but not others, directly stained primary NK cells and Jurkat cells expressing multiple allotypes of Mamu-KIR3DL05. Differences in binding avidity were associated with polymorphisms in the D0 and D1 domains of Mamu-KIR3DL05, whereas differences in peptide-selectivity mapped to the D1 domain. The reciprocal exchange of the third predicted MHC class I-contact loop of the D1 domain switched the specificity of two Mamu-KIR3DL05 allotypes for different Mamu-A1*00201-peptide complexes. Consistent with the function of an inhibitory KIR, incubation of lymphocytes from Mamu-KIR3DL05(+ macaques with target cells expressing Mamu-A1*00201 suppressed the degranulation of tetramer-positive NK cells. These observations reveal a previously unappreciated role for D1 polymorphisms in determining the selectivity of KIRs for MHC class I-bound peptides, and identify the first functional KIR-MHC class I interaction in the rhesus macaque. The modulation of KIR-MHC class I interactions by viral peptides has important implications to pathogenesis, since it suggests that the immunodeficiency viruses, and potentially other types of viruses and tumors, may acquire changes in epitopes that increase the affinity of certain MHC class I ligands for inhibitory KIRs to prevent the activation of specific NK cell subsets.

  14. The biological function of some human transcription factor binding motifs varies with position relative to the transcription start site.

    Science.gov (United States)

    Tharakaraman, Kannan; Bodenreider, Olivier; Landsman, David; Spouge, John L; Mariño-Ramírez, Leonardo

    2008-05-01

    A number of previous studies have predicted transcription factor binding sites (TFBSs) by exploiting the position of genomic landmarks like the transcriptional start site (TSS). The studies' methods are generally too computationally intensive for genome-scale investigation, so the full potential of 'positional regulomics' to discover TFBSs and determine their function remains unknown. Because databases often annotate the genomic landmarks in DNA sequences, the methodical exploitation of positional regulomics has become increasingly urgent. Accordingly, we examined a set of 7914 human putative promoter regions (PPRs) with a known TSS. Our methods identified 1226 eight-letter DNA words with significant positional preferences with respect to the TSS, of which only 608 of the 1226 words matched known TFBSs. Many groups of genes whose PPRs contained a common word displayed similar expression profiles and related biological functions, however. Most interestingly, our results included 78 words, each of which clustered significantly in two or three different positions relative to the TSS. Often, the gene groups corresponding to different positional clusters of the same word corresponded to diverse functions, e.g. activation or repression in different tissues. Thus, different clusters of the same word likely reflect the phenomenon of 'positional regulation', i.e. a word's regulatory function can vary with its position relative to a genomic landmark, a conclusion inaccessible to methods based purely on sequence. Further integrative analysis of words co-occurring in PPRs also yielded 24 different groups of genes, likely identifying cis-regulatory modules de novo. Whereas comparative genomics requires precise sequence alignments, positional regulomics exploits genomic landmarks to provide a 'poor man's alignment'. By exploiting the phenomenon of positional regulation, it uses position to differentiate the biological functions of subsets of TFBSs sharing a common sequence motif.

  15. The clathrin-binding motif and the J-domain of Drosophila Auxilin are essential for facilitating Notch ligand endocytosis

    Science.gov (United States)

    Kandachar, Vasundhara; Bai, Ting; Chang, Henry C

    2008-01-01

    Background Ligand endocytosis plays a critical role in regulating the activity of the Notch pathway. The Drosophila homolog of auxilin (dAux), a J-domain-containing protein best known for its role in the disassembly of clathrin coats from clathrin-coated vesicles, has recently been implicated in Notch signaling, although its exact mechanism remains poorly understood. Results To understand the role of auxilin in Notch ligand endocytosis, we have analyzed several point mutations affecting specific domains of dAux. In agreement with previous work, analysis using these stronger dAux alleles shows that dAux is required for several Notch-dependent processes, and its function during Notch signaling is required in the signaling cells. In support of the genetic evidences, the level of Delta appears elevated in dAux deficient cells, suggesting that the endocytosis of Notch ligand is disrupted. Deletion analysis shows that the clathrin-binding motif and the J-domain, when over-expressed, are sufficient for rescuing dAux phenotypes, implying that the recruitment of Hsc70 to clathrin is a critical role for dAux. However, surface labeling experiment shows that, in dAux mutant cells, Delta accumulates at the cell surface. In dAux mutant cells, clathrin appears to form large aggregates, although Delta is not enriched in these aberrant clathrin-positive structures. Conclusion Our data suggest that dAux mutations inhibit Notch ligand internalization at an early step during clathrin-mediated endocytosis, before the disassembly of clathrin-coated vesicles. Further, the inhibition of ligand endocytosis in dAux mutant cells possibly occurs due to depletion of cytosolic pools of clathrin via the formation of clathrin aggregates. Together, our observations argue that ligand endocytosis is critical for Notch signaling and auxilin participates in Notch signaling by facilitating ligand internalization. PMID:18466624

  16. The clathrin-binding motif and the J-domain of Drosophila Auxilin are essential for facilitating Notch ligand endocytosis

    Directory of Open Access Journals (Sweden)

    Chang Henry C

    2008-05-01

    Full Text Available Abstract Background Ligand endocytosis plays a critical role in regulating the activity of the Notch pathway. The Drosophila homolog of auxilin (dAux, a J-domain-containing protein best known for its role in the disassembly of clathrin coats from clathrin-coated vesicles, has recently been implicated in Notch signaling, although its exact mechanism remains poorly understood. Results To understand the role of auxilin in Notch ligand endocytosis, we have analyzed several point mutations affecting specific domains of dAux. In agreement with previous work, analysis using these stronger dAux alleles shows that dAux is required for several Notch-dependent processes, and its function during Notch signaling is required in the signaling cells. In support of the genetic evidences, the level of Delta appears elevated in dAux deficient cells, suggesting that the endocytosis of Notch ligand is disrupted. Deletion analysis shows that the clathrin-binding motif and the J-domain, when over-expressed, are sufficient for rescuing dAux phenotypes, implying that the recruitment of Hsc70 to clathrin is a critical role for dAux. However, surface labeling experiment shows that, in dAux mutant cells, Delta accumulates at the cell surface. In dAux mutant cells, clathrin appears to form large aggregates, although Delta is not enriched in these aberrant clathrin-positive structures. Conclusion Our data suggest that dAux mutations inhibit Notch ligand internalization at an early step during clathrin-mediated endocytosis, before the disassembly of clathrin-coated vesicles. Further, the inhibition of ligand endocytosis in dAux mutant cells possibly occurs due to depletion of cytosolic pools of clathrin via the formation of clathrin aggregates. Together, our observations argue that ligand endocytosis is critical for Notch signaling and auxilin participates in Notch signaling by facilitating ligand internalization.

  17. The histone chaperone sNASP binds a conserved peptide motif within the globular core of histone H3 through its TPR repeats.

    Science.gov (United States)

    Bowman, Andrew; Lercher, Lukas; Singh, Hari R; Zinne, Daria; Timinszky, Gyula; Carlomagno, Teresa; Ladurner, Andreas G

    2016-04-20

    Eukaryotic chromatin is a complex yet dynamic structure, which is regulated in part by the assembly and disassembly of nucleosomes. Key to this process is a group of proteins termed histone chaperones that guide the thermodynamic assembly of nucleosomes by interacting with soluble histones. Here we investigate the interaction between the histone chaperone sNASP and its histone H3 substrate. We find that sNASP binds with nanomolar affinity to a conserved heptapeptide motif in the globular domain of H3, close to the C-terminus. Through functional analysis of sNASP homologues we identified point mutations in surface residues within the TPR domain of sNASP that disrupt H3 peptide interaction, but do not completely disrupt binding to full length H3 in cells, suggesting that sNASP interacts with H3 through additional contacts. Furthermore, chemical shift perturbations from(1)H-(15)N HSQC experiments show that H3 peptide binding maps to the helical groove formed by the stacked TPR motifs of sNASP. Our findings reveal a new mode of interaction between a TPR repeat domain and an evolutionarily conserved peptide motif found in canonical H3 and in all histone H3 variants, including CenpA and have implications for the mechanism of histone chaperoning within the cell.

  18. The human Ago2 MC region does not contain an eIF4E-like mRNA cap binding motif

    Directory of Open Access Journals (Sweden)

    Grishin Nick V

    2009-01-01

    Full Text Available Abstract Background Argonaute (Ago proteins interact with small regulatory RNAs to mediate gene regulatory pathways. A recent report by Kiriakidou et al. 1 describes an MC sequence region identified in Ago2 that displays similarity to the cap-binding motif in translation initiation factor 4E (eIF4E. In a cap-bound eIF4E structure, two important aromatic residues of the motif stack on either side of a 7-methylguanosine 5'-triphosphate (m7Gppp base. The corresponding Ago2 aromatic residues (F450 and F505 were hypothesized to perform the same cap-binding function. However, the detected similarity between the MC sequence and the eIF4E cap-binding motif was questionable. Results A number of sequence-based and structure-based bioinformatics methods reveal the reported similarity between the Ago2 MC sequence region and the eIF4E cap-binding motif to be spurious. Alternatively, the MC sequence region is confidently assigned to the N-terminus of the Ago piwi module, within the mid domain of experimentally determined prokaryotic Ago structures. Confident mapping of the Ago2 MC sequence region to the piwi mid domain results in a homology-based structure model that positions the identified aromatic residues over 20 Å apart, with one of the aromatic side chains (F450 contributing instead to the hydrophobic core of the domain. Conclusion Correct functional prediction based on weak sequence similarity requires substantial evolutionary and structural support. The evolutionary context of the Ago mid domain suggested by multiple sequence alignment is limited to a conserved hydrophobicity profile required for the fold and a motif following the MC region that binds guide RNA. Mapping of the MC sequence to the mid domain structure reveals Ago2 aromatics that are incompatible with eIF4E-like mRNA cap-binding, yet display some limited local structure similarities that cause the chance sequence match to eIF4E. Reviewers This article was reviewed by Arcady Mushegian

  19. Identification of second arginine-glycine-aspartic acid motif of ovine vitronectin as the complement C9 binding site and its implication in bacterial infection.

    Science.gov (United States)

    T, Prasada Rao; T, Lakshmi Prasanth; R, Parvathy; S, Murugavel; Devi, Karuna; Joshi, Paritosh

    2017-02-02

    Vitronectin (Vn), a multifunctional protein of blood and extracellular matrix interacts with complement C9. This interaction may modulate innate immunity. Details of Vn-C9 interaction are limited. An assessment of Vn-C9 interaction was made employing goat homologous system. Vn binding to C9 was observed in three different assays. Using recombinant fragments, the C9 binding was mapped to the N-terminus of Vn. Site directed mutagenesis was performed to alter the second RGD sequence (RGD-2) of Vn. Change of R to G or D to A in RGD-2 caused significant decrease in Vn binding to C9 whereas change of R to G in the first RGD motif (RGD-1) had no effect on Vn binding to C9. These results imply that the RGD-2 of goat Vn is involved in C9 binding. In competitive binding assay, the presence of soluble RGD peptide inhibited Vn binding to C9 whereas heparin had no effect. Vn binding to C9 in terms of bacterial pathogenesis was also evaluated. Serum dependent inhibition of E. coli growth was significantly reverted when Vn or its N-fragment were included in the assay. The C-fragment, which did not support C9 binding, also partly nullified serum dependent inhibition of bacterial growth probably through other serum component(s).

  20. The seven amino acids (547-553) of rat glucocorticoid receptor required for steroid and hsp90 binding contain a functionally independent LXXLL motif that is critical for steroid binding.

    Science.gov (United States)

    Giannoukos, G; Silverstein, A M; Pratt, W B; Simons, S S

    1999-12-17

    Hsp90 association with glucocorticoid receptors (GRs) is required for steroid binding. We recently reported that seven amino acids (547-553) overlapping the amino-terminal end of the rat GR ligand-binding domain are necessary for hsp90 binding, and consequently steroid binding. The role of a LXXLL motif at the COOH terminus of this sequence has now been analyzed by determining the properties of Leu to Ser mutations in full-length GR and glutathione S-transferase chimeras. Surprisingly, these mutations decreased steroid binding capacity without altering receptor levels, steroid binding affinity, or hsp90 binding. Single mutations in the context of the full-length receptor did not affect the transcriptional activity but the double mutant (L550S/L553S) was virtually inactive. This biological inactivity was found to be due to an increased rate of steroid dissociation from the activated mutant complex. These results, coupled with those from trypsin digestion studies, suggest a model in which the GR ligand-binding domain is viewed as having a "hinged pocket," with the hinge being in the region of the trypsin digestion site at Arg(651). The pocket would normally be kept shut via the intramolecular interactions of the LXXLL motif at amino acids 550-554 acting as a hydrophobic clasp.

  1. Accessory Interaction Motifs in the Atg19 Cargo Receptor Enable Strong Binding to the Clustered Ubiquitin-related Atg8 Protein.

    Science.gov (United States)

    Abert, Christine; Kontaxis, Georg; Martens, Sascha

    2016-09-02

    Selective autophagy contributes to cellular homeostasis by delivering harmful material into the lysosomal system for degradation via vesicular intermediates referred to as autophagosomes. The cytoplasm-to-vacuole targeting pathway is a variant of selective autophagy in Saccharomyces cerevisiae during which hydrolases such as prApe1 are transported into the vacuole. In general, selectivity is achieved by autophagic cargo receptors that link the cargo to autophagosomal membranes because of their ability to simultaneously interact with the cargo and Atg8 proteins that coat the membrane. The Atg19 receptor contains multiple Atg8 interaction sites in its C terminus in addition to a canonical Atg8-interacting LC3-interacting region (LIR, with LC3 being a homolog of Atg8) motif, but their mode of interaction with Atg8 is unclear. Here we show, using a combination of NMR, microscopy-based interaction assays, and prApe1 processing experiments, that two additional sites interact with Atg8 in a LIR-like and thus mutually exclusive manner. We term these motifs accessory LIR motifs because their affinities are lower than that of the canonical LIR motif. Thus, one Atg19 molecule has the ability to interact with multiple Atg8 proteins simultaneously, resulting in a high-avidity interaction that may confer specific binding to the Atg8-coated autophagosomal membrane on which Atg8 is concentrated.

  2. Exploratory Study on the RNA-Binding Structural Motifs by Library Screening Targeting pre-miRNA-29 a.

    Science.gov (United States)

    Fukuzumi, Takeo; Murata, Asako; Aikawa, Haruo; Harada, Yasue; Nakatani, Kazuhiko

    2015-11-16

    The metabolic stream of microRNA (miRNA) production, the so-called maturation process of miRNAs, became one of important metabolic paths for drug-targeting to modulate the expression of genes related to a number of diseases. We carried out discovery studies on small molecules binding to the precursor of miR-29a (pre-miR-29a) from a chemical library containing 41,119 compounds (AQ library) by the fluorescent indicator displacement (FID) assay using the xanthone derivative X2SdiMe as a fluorescent indicator. The FID assay provided 1075 compounds, which showed an increase of fluorescence. These compounds were subsequently submitted to a binding analysis in a surface plasmon resonance (SPR) assay on a pre-miR-29a immobilized surface. 21 hit compounds were identified with a good reproducibility in the binding. These compounds have not been reported to bind to RNA until now and can be classified into two groups on the basis of the kinetics in the binding. To gain more information on the motif structures that could be necessary for the binding to pre-miR-29a, 19 substructures were selected from the hit compounds. The substructure library (SS library) which consisted of 362 compounds was prepared from the AQ library. An SPR assay of the SS library on pre-miR-29a-immobilized surface suggested that five substructures could potentially be important structural motifs to bind to pre-miR-29a. These studies demonstrate that the combination of FID-based screening of chemical library and subsequent SPR assay would be one way for obtaining practical solutions for the discovery of molecules which bind to the target pre-miRNAs.

  3. Identification of a phosphorylation-dependent nuclear localization motif in interferon regulatory factor 2 binding protein 2.

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    Allen C T Teng

    Full Text Available BACKGROUND: Interferon regulatory factor 2 binding protein 2 (IRF2BP2 is a muscle-enriched transcription factor required to activate vascular endothelial growth factor-A (VEGFA expression in muscle. IRF2BP2 is found in the nucleus of cardiac and skeletal muscle cells. During the process of skeletal muscle differentiation, some IRF2BP2 becomes relocated to the cytoplasm, although the functional significance of this relocation and the mechanisms that control nucleocytoplasmic localization of IRF2BP2 are not yet known. METHODOLOGY/PRINCIPAL FINDINGS: Here, by fusing IRF2BP2 to green fluorescent protein and testing a series of deletion and site-directed mutagenesis constructs, we mapped the nuclear localization signal (NLS to an evolutionarily conserved sequence (354ARKRKPSP(361 in IRF2BP2. This sequence corresponds to a classical nuclear localization motif bearing positively charged arginine and lysine residues. Substitution of arginine and lysine with negatively charged aspartic acid residues blocked nuclear localization. However, these residues were not sufficient because nuclear targeting of IRF2BP2 also required phosphorylation of serine 360 (S360. Many large-scale phosphopeptide proteomic studies had reported previously that serine 360 of IRF2BP2 is phosphorylated in numerous human cell types. Alanine substitution at this site abolished IRF2BP2 nuclear localization in C(2C(12 myoblasts and CV1 cells. In contrast, substituting serine 360 with aspartic acid forced nuclear retention and prevented cytoplasmic redistribution in differentiated C(2C(12 muscle cells. As for the effects of these mutations on VEGFA promoter activity, the S360A mutation interfered with VEGFA activation, as expected. Surprisingly, the S360D mutation also interfered with VEGFA activation, suggesting that this mutation, while enforcing nuclear entry, may disrupt an essential activation function of IRF2BP2. CONCLUSIONS/SIGNIFICANCE: Nuclear localization of IRF2BP2 depends on

  4. Structural and functional studies of a phosphatidic acid-binding antifungal plant defensin MtDef4: Identification of an RGFRRR motif governing fungal cell entry

    Energy Technology Data Exchange (ETDEWEB)

    Sagaram, Uma S.; El-Mounadi, Kaoutar; Buchko, Garry W.; Berg, Howard R.; Kaur, Jagdeep; Pandurangi, Raghoottama; Smith, Thomas J.; Shah, Dilip

    2013-12-04

    A highly conserved plant defensin MtDef4 potently inhibits the growth of a filamentous fungus Fusarium graminearum. MtDef4 is internalized by cells of F. graminearum. To determine its mechanism of fungal cell entry and antifungal action, NMR solution structure of MtDef4 has been determined. The analysis of its structure has revealed a positively charged patch on the surface of the protein consisting of arginine residues in its γ-core signature, a major determinant of the antifungal activity of MtDef4. Here, we report functional analysis of the RGFRRR motif of the γ-core signature of MtDef4. The replacement of RGFRRR to AAAARR or to RGFRAA not only abolishes fungal cell entry but also results in loss of the antifungal activity of MtDef4. MtDef4 binds strongly to phosphatidic acid (PA), a precursor for the biosynthesis of membrane phospholipids and a signaling lipid known to recruit cytosolic proteins to membranes. Mutations of RGFRRR which abolish fungal cell entry of MtDef4 also impair its binding to PA. Our results suggest that RGFRRR motif is a translocation signal for entry of MtDef4 into fungal cells and that this positively charged motif likely mediates interaction of this defensin with PA as part of its antifungal action.

  5. Vaccinia protein F12 has structural similarity to kinesin light chain and contains a motor binding motif required for virion export.

    Directory of Open Access Journals (Sweden)

    Gareth W Morgan

    2010-02-01

    Full Text Available Vaccinia virus (VACV uses microtubules for export of virions to the cell surface and this process requires the viral protein F12. Here we show that F12 has structural similarity to kinesin light chain (KLC, a subunit of the kinesin-1 motor that binds cargo. F12 and KLC share similar size, pI, hydropathy and cargo-binding tetratricopeptide repeats (TPRs. Moreover, molecular modeling of F12 TPRs upon the crystal structure of KLC2 TPRs showed a striking conservation of structure. We also identified multiple TPRs in VACV proteins E2 and A36. Data presented demonstrate that F12 is critical for recruitment of kinesin-1 to virions and that a conserved tryptophan and aspartic acid (WD motif, which is conserved in the kinesin-1-binding sequence (KBS of the neuronal protein calsyntenin/alcadein and several other cellular kinesin-1 binding proteins, is essential for kinesin-1 recruitment and virion transport. In contrast, mutation of WD motifs in protein A36 revealed they were not required for kinesin-1 recruitment or IEV transport. This report of a viral KLC-like protein containing a KBS that is conserved in several cellular proteins advances our understanding of how VACV recruits the kinesin motor to virions, and exemplifies how viruses use molecular mimicry of cellular components to their advantage.

  6. Gene cloning and function analysis of ABP9 protein which specifically binds to ABRE2 motif of maize Cat1 gene

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A cDNA library was constructed using mRNA extracted from 17 days post-pollination (dpp) maize embryos and was screened by employing a yeast one-hybrid system for proteins specifically interacting with ABRE2 motif of maize Cat1 gene. Three truncated overlapping positive clones designated ABP9 were obtained and the full-length cDNA was isolated by 5′ RACE. Searching the database revealed that ABP9 protein belongs to a bZIP-type transcription factor family. ABP9 protein specifically binds to ABRE2 motif and activates the expression of downstream reporter gene in yeast cells. Our results strongly suggest that the ABP9 protein functions as a transcription activator.

  7. Molecular sensing of bacteria in plants. The highly conserved RNA-binding motif RNP-1 of bacterial cold shock proteins is recognized as an elicitor signal in tobacco.

    Science.gov (United States)

    Felix, Georg; Boller, Thomas

    2003-02-21

    To detect microbial infection multicellular organisms have evolved sensing systems for pathogen-associated molecular patterns (PAMPs). Here, we identify bacterial cold shock protein (CSP) as a new such PAMP that acts as a highly active elicitor of defense responses in tobacco. Tobacco cells perceive a conserved domain of CSP and synthetic peptides representing 15 amino acids of this domain-induced responses at subnanomolar concentrations. Central to the elicitor-active domain is the RNP-1 motif KGFGFITP, a motif conserved also in many RNA- and DNA-binding proteins of eukaryotes. Csp15-Nsyl, a peptide representing the domain with highest homology to csp15 in a protein of Nicotiana sylvestris exhibited only weak activity in tobacco cells. Crystallographic and genetic data from the literature show that the RNP-1 domain of bacterial CSPs resides on a protruding loop and exposes a series of aromatic and basic side chains to the surface that are essential for the nucleotide-binding activity of CSPs. Similarly, these side chains were also essential for elicitor activity and replacement of single residues in csp15 with Ala strongly reduced or abolished activity. Most strikingly, csp15-Ala10, a peptide with the RNP-1 motif modified to KGAGFITP, lacked elicitor activity but acted as a competitive antagonist for CSP-related elicitors. Bacteria commonly have a small family of CSP-like proteins including both cold-inducible and noninducible members, and Csp-related elicitor activity was detected in extracts from all bacteria tested. Thus, the CSP domain containing the RNP-1 motif provides a structure characteristic for bacteria in general, and tobacco plants have evolved a highly sensitive chemoperception system to detect this bacterial PAMP.

  8. Consensus PP1 binding motifs regulate transcriptional corepression and alternative RNA splicing activities of the steroid receptor coregulators, p54nrb and PSF.

    Science.gov (United States)

    Liu, Liangliang; Xie, Ning; Rennie, Paul; Challis, John R G; Gleave, Martin; Lye, Stephen J; Dong, Xuesen

    2011-07-01

    Originally identified as essential pre-mRNA splicing factors, non-POU-domain-containing, octamer binding protein (p54nrb) and PTB-associated RNA splicing factor (PSF) are also steroid receptor corepressors. The mechanisms by which p54nrb and PSF regulate gene transcription remain unclear. Both p54nrb and PSF contain protein phosphatase 1 (PP1) consensus binding RVxF motifs, suggesting that PP1 may regulate phosphorylation status of p54nrb and PSF and thus their function in gene transcription. In this report, we demonstrated that PP1 forms a protein complex with both p54nrb and PSF. PP1 interacts directly with the RVxF motif only in p54nrb, but not in PSF. Association with PP1 results in dephosphorylation of both p54nrb and PSF in vivo and the loss of their transcriptional corepressor activities. Using the CD44 minigene as a reporter, we showed that PP1 regulates p54nrb and PSF alternative splicing activities that determine exon skipping vs. inclusion in the final mature RNA for translation. In addition, changes in transcriptional corepression and RNA splicing activities of p54nrb and PSF are correlated with alterations in protein interactions of p54nrb and PSF with transcriptional corepressors such as Sin3A and histone deacetylase 1, and RNA splicing factors such as U1A and U2AF. Furthermore, we demonstrated a novel function of the RVxF motif within PSF that enhances its corepression and RNA splicing activities independent of PP1. We conclude that the RVxF motifs play an important role in controlling the multifunctional properties of p54nrb and PSF in the regulation of gene transcription.

  9. Crystal structure of the N-terminal region of human Ash2L shows a winged-helix motif involved in DNA binding

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Yong; Wan, Bingbing; Wang, Kevin C.; Cao, Fang; Yang, Yuting; Protacio, Angeline; Dou, Yali; Chang, Howard Y.; Lei, Ming (Michigan-Med); (HHMI)

    2011-09-06

    Ash2L is a core component of the MLL family histone methyltransferases and has an important role in regulating the methylation of histone H3 on lysine 4. Here, we report the crystal structure of the N-terminal domain of Ash2L and reveal a new function of Ash2L. The structure shows that Ash2L contains an atypical PHD finger that does not have histone tail-binding activity. Unexpectedly, the structure shows a previously unrecognized winged-helix motif that directly binds to DNA. The DNA-binding-deficient mutants of Ash2L reduced Ash2L localization to the HOX locus. Strikingly, a single mutation in Ash2L{sub WH} (K131A) breaks the chromatin domain boundary, suggesting that Ash2L also has a role in chromosome demarcation.

  10. Gas-phase complexes of Ni2+ and Ca2+ with deprotonated histidylhistidine (HisHis): A model case for polyhistidyl-metal binding motifs

    Science.gov (United States)

    Peckelsen, Katrin; Martens, Jonathan; Berden, Giel; Oomens, Jos; Dunbar, Robert C.; Meijer, Anthony J. H. M.; Schäfer, Mathias

    2017-02-01

    In the complex formed between the calcium cation (Ca2+) and a deprotonated HisHis dipeptide, the complex adopts a charge solvation (CS) structure. Ca2+, a weak binding main group metal cation, interacts with the oxygens of the peptide carbonyl moiety and the deprotonated C-terminus. In contrast, the much stronger binding Ni2+ cation deprotonates the peptide nitrogen and induces an iminolate (Im) ligand structure in the [Ni(HisHis-H)]+ complex ion. The combination of infrared multiple-photon dissociation (IRMPD) spectroscopy and quantum chemistry evidence these two representative binding motifs. The iminolate coordination pattern identified and characterized in the [Ni(HisHis-H)]+ complex serves as a model case for nickel complexes of poly-histidyl-domains and is thereby also of interest to better understand the fundamentals of immobilized metal ion affinity chromatography as well as of Ni co-factor chemistry in enzymology.

  11. Nicotine induced CpG methylation of Pax6 binding motif in StAR promoter reduces the gene expression and cortisol production

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Tingting [Department of Pharmacology, Basic Medical School of Wuhan University, Wuhan 430071 (China); Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland (United States); Chen, Man; Liu, Lian [Department of Pharmacology, Basic Medical School of Wuhan University, Wuhan 430071 (China); Cheng, Huaiyan [Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland (United States); Yan, You-E [Department of Pharmacology, Basic Medical School of Wuhan University, Wuhan 430071 (China); Feng, Ying-Hong, E-mail: yhfeng@usuhs.edu [Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland (United States); Wang, Hui, E-mail: wanghui19@whu.edu.cn [Department of Pharmacology, Basic Medical School of Wuhan University, Wuhan 430071 (China); Research Center of Food and Drug Evaluation, Wuhan University, Wuhan 430071 (China)

    2011-12-15

    Steroidogenic acute regulatory protein (StAR) mediates the rate-limiting step in the synthesis of steroid hormones, essential to fetal development. We have reported that the StAR expression in fetal adrenal is inhibited in a rat model of nicotine-induced intrauterine growth retardation (IUGR). Here using primary human fetal adrenal cortex (pHFAC) cells and a human fetal adrenal cell line NCI-H295A, we show that nicotine inhibits StAR expression and cortisol production in a dose- and time-dependent manner, and prolongs the inhibitory effect on cells proliferating over 5 passages after termination of nicotine treatment. Methylation detection within the StAR promoter region uncovers a single site CpG methylation at nt -377 that is sensitive to nicotine treatment. Nicotine-induced alterations in frequency of this point methylation correlates well with the levels of StAR expression, suggesting an important role of the single site in regulating StAR expression. Further studies using bioinformatics analysis and siRNA approach reveal that the single CpG site is part of the Pax6 binding motif (CGCCTGA) in the StAR promoter. The luciferase activity assays validate that Pax6 increases StAR gene expression by binding to the glucagon G3-like motif (CGCCTGA) and methylation of this site blocks Pax6 binding and thus suppresses StAR expression. These data identify a nicotine-sensitive CpG site at the Pax6 binding motif in the StAR promoter that may play a central role in regulating StAR expression. The results suggest an epigenetic mechanism that may explain how nicotine contributes to onset of adult diseases or disorders such as metabolic syndrome via fetal programming. -- Highlights: Black-Right-Pointing-Pointer Nicotine-induced StAR inhibition in two human adrenal cell models. Black-Right-Pointing-Pointer Nicotine-induced single CpG site methylation in StAR promoter. Black-Right-Pointing-Pointer Persistent StAR inhibition and single CpG methylation after nicotine termination

  12. Cloning of an SNF2/SWI2-related protein that binds specifically to the SPH motifs of the SV40 enhancer and to the HIV-1 promoter.

    Science.gov (United States)

    Sheridan, P L; Schorpp, M; Voz, M L; Jones, K A

    1995-03-03

    We have isolated a human cDNA clone encoding HIP116, a protein that binds to the SPH repeats of the SV40 enhancer and to the TATA/inhibitor region of the human immunodeficiency virus (HIV)-1 promoter. The predicted HIP116 protein is related to the yeast SNF2/SWI2 transcription factor and to other members of this extended family and contains seven domains similar to those found in the vaccinia NTP1 ATPase. Interestingly, HIP116 also contains a C3HC4 zinc-binding motif (RING finger) interspersed between the ATPase motifs in an arrangement similar to that found in the yeast RAD5 and RAD16 proteins. The HIP116 amino terminus is unique among the members of this family, and houses a specific DNA-binding domain. Antiserum raised against HIP116 recognizes a 116-kDa nuclear protein in Western blots and specifically supershifts SV40 and HIV-1 protein-DNA complexes in gel shift experiments. The binding site for HIP116 on the SV40 enhancer directly overlaps the site for TEF-1, and like TEF-1, binding of HIP116 to the SV40 enhancer is destroyed by mutations that inhibit SPH enhancer activity in vivo. Purified fractions of HIP116 display strong ATPase activity that is preferentially stimulated by SPH DNA and can be inhibited specifically by antibodies to HIP116. These findings suggest that HIP116 might affect transcription, directly or indirectly, by acting as a DNA binding site-specific ATPase.

  13. The Role of Glutamic or Aspartic Acid in Position Four of the Epitope Binding Motif and Thyrotropin Receptor-Extracellular Domain Epitope Selection in Graves' Disease

    Science.gov (United States)

    Inaba, Hidefumi; Martin, William; Ardito, Matt; De Groot, Anne Searls; De Groot, Leslie J.

    2010-01-01

    Context: Development of Graves' disease (GD) is related to HLA-DRB1*0301 (DR3),and more specifically to arginine at position 74 of the DRB1 molecule. The extracellular domain (ECD) of human TSH receptor (hTSH-R) contains the target antigen. Objective and Design: We analyzed the relation between hTSH-R-ECD peptides and DR molecules to determine whether aspartic acid (D) or glutamic acid (E) at position four in the binding motif influenced selection of functional epitopes. Results: Peptide epitopes from TSH-R-ECD with D or E in position four (D/E+) had higher affinity for binding to DR3 than peptides without D/E (D/E−) (IC50 29.3 vs. 61.4, P = 0.0024). HLA-DR7, negatively correlated with GD, and DRB1*0302 (HLA-DR18), not associated with GD, had different profiles of epitope binding. Toxic GD patients who are DR3+ had higher responses to D/E+ peptides than D/E− peptides (stimulation index 1.42 vs. 1.22, P = 0.028). All DR3+ GD patients (toxic + euthyroid) had higher responses, with borderline significance (Sl; 1.32 vs. 1.18, P = 0.051). Splenocytes of DR3 transgenic mice immunized to TSH-R-ECD responded to D/E+ peptides more than D/E− peptides (stimulation index 1.95 vs. 1.69, P = 0.036). Seven of nine hTSH-R-ECD peptide epitopes reported to be reactive with GD patients' peripheral blood mononuclear cells contain binding motifs with D/E at position four. Conclusions: TSH-R-ECD epitopes with D/E in position four of the binding motif bind more strongly to DRB1*0301 than epitopes that are D/E− and are more stimulatory to GD patients' peripheral blood mononuclear cells and to splenocytes from mice immunized to hTSH-R. These epitopes appear important in immunogenicity to TSH-R due to their favored binding to HLA-DR3, thus increasing presentation to T cells. PMID:20392871

  14. ATP regulation of type-1 inositol 1,4,5-trisphosphate receptor activity does not require walker A-type ATP-binding motifs.

    Science.gov (United States)

    Betzenhauser, Matthew J; Wagner, Larry E; Park, Hyung Seo; Yule, David I

    2009-06-12

    ATP is known to increase the activity of the type-1 inositol 1,4,5-trisphosphate receptor (InsP3R1). This effect is attributed to the binding of ATP to glycine rich Walker A-type motifs present in the regulatory domain of the receptor. Only two such motifs are present in neuronal S2+ splice variant of InsP3R1 and are designated the ATPA and ATPB sites. The ATPA site is unique to InsP3R1, and the ATPB site is conserved among all three InsP3R isoforms. Despite the fact that both the ATPA and ATPB sites are known to bind ATP, the relative contribution of these two sites to the enhancing effects of ATP on InsP3R1 function is not known. We report here a mutational analysis of the ATPA and ATPB sites and conclude neither of these sites is required for ATP modulation of InsP3R1. ATP augmented InsP3-induced Ca2+ release from permeabilized cells expressing wild type and ATP-binding site-deficient InsP3R1. Similarly, ATP increased the single channel open probability of the mutated InsP3R1 to the same extent as wild type. ATP likely exerts its effects on InsP3R1 channel function via a novel and as yet unidentified mechanism.

  15. Evidence for a Structural Motif in Toxins and Interleukin-2 That May Be Responsible for Binding to Endothelial Cells and Initiating Vascular Leak Syndrome

    Science.gov (United States)

    Baluna, Roxana; Rizo, Josep; Gordon, Brian E.; Ghetie, Victor; Vitetta, Ellen S.

    1999-03-01

    The dose-limiting toxicity of interleukin-2 (IL-2) and immunotoxin (IT) therapy in humans is vascular leak syndrome (VLS). VLS has a complex etiology involving damage to vascular endothelial cells (ECs), extravasation of fluids and proteins, interstitial edema, and organ failure. IL-2 and ITs prepared with the catalytic A chain of the plant toxin, ricin (RTA), and other toxins, damage human ECs in vitro and in vivo. Damage to ECs may initiate VLS; if this damage could be avoided without losing the efficacy of ITs or IL-2, larger doses could be administered. In this paper, we provide evidence that a three amino acid sequence motif, (x)D(y), in toxins and IL-2 damages ECs. Thus, when peptides from RTA or IL-2 containing this sequence motif are coupled to mouse IgG, they bind to and damage ECs both in vitro and, in the case of RTA, in vivo. In contrast, the same peptides with a deleted or mutated sequence do not. Furthermore, the peptide from RTA attached to mouse IgG can block the binding of intact RTA to ECs in vitro and vice versa. In addition, RTA, a fragment of Pseudomonas exotoxin A (PE38-lys), and fibronectin also block the binding of the mouse IgG-RTA peptide to ECs, suggesting that an (x)D(y) motif is exposed on all three molecules. Our results suggest that deletions or mutations in this sequence or the use of nondamaging blocking peptides may increase the therapeutic index of both IL-2, as well as ITs prepared with a variety of plant or bacterial toxins.

  16. IGD motifs, which are required for migration stimulatory activity of fibronectin type I modules, do not mediate binding in matrix assembly.

    Directory of Open Access Journals (Sweden)

    Lisa M Maurer

    Full Text Available Picomolar concentrations of proteins comprising only the N-terminal 70-kDa region (70K of fibronectin (FN stimulate cell migration into collagen gels. The Ile-Gly-Asp (IGD motifs in four of the nine FN type 1 (FNI modules in 70K are important for such migratory stimulating activity. The 70K region mediates binding of nanomolar concentrations of intact FN to cell-surface sites where FN is assembled. Using baculovirus, we expressed wildtype 70K and 70K with Ile-to-Ala mutations in (3FNI and (5FNI; (7FNI and (9FNI; or (3FNI, (5FNI, (7FNI, and (9FNI. Wildtype 70K and 70K with Ile-to-Ala mutations were equally active in binding to assembly sites of FN-null fibroblasts. This finding indicates that IGD motifs do not mediate the interaction between 70K and the cell-surface that is important for FN assembly. Further, FN fragment N-(3FNIII, which does not stimulate migration, binds to assembly sites on FN-null fibroblast. The Ile-to-Ala mutations had effects on the structure of FNI modules as evidenced by decreases in abilities of 70K with Ile-to-Ala mutations to bind to monoclonal antibody 5C3, which recognizes an epitope in (9FNI, or to bind to FUD, a polypeptide based on the F1 adhesin of Streptococcus pyogenes that interacts with 70K by the β-zipper mechanism. These results suggest that the picomolar interactions of 70K with cells that stimulate cell migration require different conformations of FNI modules than the nanomolar interactions required for assembly.

  17. Specific recognition of the collagen triple helix by chaperone HSP47. II. The HSP47-binding structural motif in collagens and related proteins.

    Science.gov (United States)

    Koide, Takaki; Nishikawa, Yoshimi; Asada, Shinichi; Yamazaki, Chisato M; Takahara, Yoshifumi; Homma, Daisuke L; Otaka, Akira; Ohtani, Katsuki; Wakamiya, Nobutaka; Nagata, Kazuhiro; Kitagawa, Kouki

    2006-04-21

    The endoplasmic reticulum-resident chaperone heat-shock protein 47 (HSP47) plays an essential role in procollagen biosynthesis. The function of HSP47 relies on its specific interaction with correctly folded triple-helical regions comprised of Gly-Xaa-Yaa repeats, and Arg residues at Yaa positions have been shown to be important for this interaction. The amino acid at the Yaa position (Yaa(-3)) in the N-terminal-adjoining triplet containing the critical Arg (defined as Arg(0)) was also suggested to be directly recognized by HSP47 (Koide, T., Asada, S., Takahara, Y., Nishikawa, Y., Nagata, K., and Kitagawa, K. (2006) J. Biol. Chem. 281, 3432-3438). Based on this finding, we examined the relationship between the structure of Yaa(-3) and HSP47 binding using synthetic collagenous peptides. The results obtained indicated that the structure of Yaa(-3) determined the binding affinity for HSP47. Maximal binding was observed when Yaa(-3) was Thr. Moreover, the required relative spatial arrangement of these key residues in the triple helix was analyzed by taking advantage of heterotrimeric collagen-model peptides, each of which contains one Thr(-3) and one Arg(0). The results revealed that HSP47 recognizes the Yaa(-3) and Arg(0) residues only when they are on the same peptide strand. Taken together, the data obtained led us to define the HSP47-binding structural epitope in the collagen triple helix and also define the HSP47-binding motif in the primary structure. A motif search against human protein database predicted candidate clients for this molecular chaperone. The search result indicated that not all collagen family proteins require the chaperoning by HSP47.

  18. A tailor-made specific anion-binding motif in the side chain transforms a tetrapeptide into an efficient vector for gene delivery.

    Science.gov (United States)

    Li, Mao; Schlesiger, Stefanie; Knauer, Shirley K; Schmuck, Carsten

    2015-03-02

    Arginine-rich cell-penetrating peptides are widely utilized as vectors for gene delivery. However, their transfection efficacy still needs to be optimized. To accomplish this, guanidinocarbonylpyrrole groups, which are tailor-made anion binding sites, were introduced into the side chains of tetralysine to obtain the peptide analogue 1. In contrast to the common strategy of adding a lipophilic tail to peptide vectors, this novel method most likely enhances transfection efficacy through more specific interactions between the binding motifs and DNA or the cell membrane. Tetrapeptide analogue 1 is thus the smallest peptidic transfection vector that has been reported to date. The transfection efficacy of 1, which on average has less than two positive charges under physiological conditions, is even better than that of polyethylenimine (PEI). Furthermore, 1 exhibits only negligible cytotoxicity, which makes it an interesting candidate for further development.

  19. Nucleoporin NUP153 phenylalanine-glycine motifs engage a common binding pocket within the HIV-1 capsid protein to mediate lentiviral infectivity.

    Directory of Open Access Journals (Sweden)

    Kenneth A Matreyek

    Full Text Available Lentiviruses can infect non-dividing cells, and various cellular transport proteins provide crucial functions for lentiviral nuclear entry and integration. We previously showed that the viral capsid (CA protein mediated the dependency on cellular nucleoporin (NUP 153 during HIV-1 infection, and now demonstrate a direct interaction between the CA N-terminal domain and the phenylalanine-glycine (FG-repeat enriched NUP153 C-terminal domain (NUP153(C. NUP153(C fused to the effector domains of the rhesus Trim5α restriction factor (Trim-NUP153(C potently restricted HIV-1, providing an intracellular readout for the NUP153(C-CA interaction during retroviral infection. Primate lentiviruses and equine infectious anemia virus (EIAV bound NUP153(C under these conditions, results that correlated with direct binding between purified proteins in vitro. These binding phenotypes moreover correlated with the requirement for endogenous NUP153 protein during virus infection. Mutagenesis experiments concordantly identified NUP153(C and CA residues important for binding and lentiviral infectivity. Different FG motifs within NUP153(C mediated binding to HIV-1 versus EIAV capsids. HIV-1 CA binding mapped to residues that line the common alpha helix 3/4 hydrophobic pocket that also mediates binding to the small molecule PF-3450074 (PF74 inhibitor and cleavage and polyadenylation specific factor 6 (CPSF6 protein, with Asn57 (Asp58 in EIAV playing a particularly important role. PF74 and CPSF6 accordingly each competed with NUP153(C for binding to the HIV-1 CA pocket, and significantly higher concentrations of PF74 were needed to inhibit HIV-1 infection in the face of Trim-NUP153(C expression or NUP153 knockdown. Correlation between CA mutant viral cell cycle and NUP153 dependencies moreover indicates that the NUP153(C-CA interaction underlies the ability of HIV-1 to infect non-dividing cells. Our results highlight similar mechanisms of binding for disparate host factors

  20. Sites for phosphates and iron-sulfur thiolates in the first membranes: 3 to 6 residue anion-binding motifs (nests).

    Science.gov (United States)

    Milner-White, E James; Russell, Michael J

    2005-02-01

    Nests are common three to six amino acid residue motifs in proteins where successive main chain NH groups bind anionic atoms or groups. On average 8% of residues in proteins belong to nests. Nests form a key part of a number of phosphate binding sites, notably the P-loop, which is the commonest of the binding sites for the phosphates of ATP and GTP. They also occur regularly in sites that bind [Fe2S2](RS)4 [Fe3S4](RS)3and [Fe4S4](RS)4 iron-sulfur centers, which are also anionic groups. Both phosphates and iron-sulfur complexes would have occurred in the precipitates within hydrothermal vents of moderate temperature as key components of the earliest metabolism and it is likely existing organisms emerging in this milieu would have benefited from evolving molecules binding such anions. The nest conformation is favored by high proportions of glycine residues and there is evidence for glycine being the commonest amino acid during the stage of evolution when proteins were evolving so it is likely nests would have been common features in peptides occupying the membranes at the dawn of life.

  1. Dendrimeric template of Plasmodium falciparum histidine rich protein II repeat motifs bearing Asp→Asn mutation exhibits heme binding and β-hematin formation.

    Directory of Open Access Journals (Sweden)

    Pinky Kumari

    Full Text Available Plasmodium falciparum (Pf employs a crucial PfHRPII catalyzed reaction that converts toxic heme into hemozoin. Understanding heme polymerization mechanism is the first step for rational design of new drugs, targeting this pathway. Heme binding and hemozoin formation have been ascribed to PfHRPII aspartate carboxylate-heme metal ionic interactions. To investigate, if this ionic interaction is indeed pivotal, we examined the comparative heme binding and β-hematin forming abilities of a wild type dendrimeric peptide BNT1 {harboring the native sequence motif of PfHRPII (AHHAHHAADA} versus a mutant dendrimeric peptide BNTM {in which ionic Aspartate residues have been replaced by the neutral Asparaginyl residues (AHHAHHAANA}. UV and IR data reported here reveal that at pH 5, both BNT1 and BNTM exhibit comparable heme binding as well as β-hematin forming abilities, thus questioning the role of PfHRPII aspartate carboxylate-heme metal ionic interactions in heme binding and β-hematin formation. Based on our data and information in the literature we suggest the possible role of weak dispersive interactions like N-H···π and lone-pair···π in heme binding and hemozoin formation.

  2. Associations of homologous RNA-binding motif gene on the X chromosome (RBMX) and its like sequence on chromosome 9(RBMXL9) with non-obstructive azoospermia

    Institute of Scientific and Technical Information of China (English)

    Akira Tsujimura; Masao Ota; Akihiko Okuyama; Kazutoshi Fujita; Kazuhiko Komori; Phanu Tanjapatkul; Yasushi Miyagawa; Shingo Takada; Kiyomi Matsumiya; Masaharu Sada; Yoshihiko Katsuyama

    2006-01-01

    Aim: To investigate the associations of autosomal and X-chromosome homologs of the RNA-binding-motif (RNA-binding-motif on the Y chromosome, RBMY) gene with non-obstructive azoospermia (NOA), as genetic factors for NOA may map to chromosomes other than the Y chromosome. Methods: Genomic DNA was extracted using a salting-out procedure after treatment of peripheral blood leukocytes with proteinase K from Japanese patients with NOA (n = 67) and normal fertile volunteers (n = 105). The DNA were analyzed for RBMX by expressed sequence tag (EST) deletion and for the like sequence on chromosome 9 (RBMXL9) by microsatellite polymorphism. Results: We examined six ESTs in and around RBMX and found a deletion of SHGC31764 in one patient with NOA and a deletion of DXS7491 in one other patient with NOA. No deletions were detected in control subjects. The association study with nine microsatellite markers near RBMXL9 revealed that D9S319 was less prevalent in patients than in control subjects, whereas D9S1853 was detected more frequently in patients than that in control subjects. Conclusion: We provide evidence that deletions in or around RBMX may be involved in NOA. In addition, analyses of markers in the vicinity of RBMXL9 on chromosome 9 suggest the possibility that variants of this gene may be associated with NOA.Although further studies are necessary, this is the first report of the association between RBMX and RBMXL9 with NOA.

  3. Cutting edge: the nucleotide receptor P2X7 contains multiple protein- and lipid-interaction motifs including a potential binding site for bacterial lipopolysaccharide.

    Science.gov (United States)

    Denlinger, L C; Fisette, P L; Sommer, J A; Watters, J J; Prabhu, U; Dubyak, G R; Proctor, R A; Bertics, P J

    2001-08-15

    The nucleotide receptor P2X7 has been shown to modulate LPS-induced macrophage production of numerous inflammatory mediators. Although the C-terminal portion of P2X7 is thought to be essential for multiple receptor functions, little is known regarding the structural motifs that lie within this region. We show here that the P2X7 C-terminal domain contains several apparent protein-protein and protein-lipid interaction motifs with potential importance to macrophage signaling and LPS action. Surprisingly, P2X7 also contains a conserved LPS-binding domain. In this report, we demonstrate that peptides derived from this P2X7 sequence bind LPS in vitro. Moreover, these peptides neutralize the ability of LPS to activate the extracellular signal-regulated kinases (ERK1, ERK2) and to promote the degradation of the inhibitor of kappaB-alpha isoform (IkappaB-alpha) in RAW 264.7 macrophages. Collectively, these data suggest that the C-terminal domain of P2X7 may directly coordinate several signal transduction events related to macrophage function and LPS action.

  4. Allosteric regulation of helicase core activities of the DEAD-box helicase YxiN by RNA binding to its RNA recognition motif.

    Science.gov (United States)

    Samatanga, Brighton; Andreou, Alexandra Z; Klostermeier, Dagmar

    2017-01-23

    DEAD-box proteins share a structurally similar core of two RecA-like domains (RecA_N and RecA_C) that contain the conserved motifs for ATP-dependent RNA unwinding. In many DEAD-box proteins the helicase core is flanked by ancillary domains. To understand the regulation of the DEAD-box helicase YxiN by its C-terminal RNA recognition motif (RRM), we investigated the effect of RNA binding to the RRM on its position relative to the core, and on core activities. RRM/RNA complex formation substantially shifts the RRM from a position close to the RecA_C to the proximity of RecA_N, independent of RNA contacts with the core. RNA binding to the RRM is communicated to the core, and stimulates ATP hydrolysis and RNA unwinding. The conformational space of the core depends on the identity of the RRM-bound RNA. Allosteric regulation of core activities by RNA-induced movement of ancillary domains may constitute a general regulatory mechanism of DEAD-box protein activity.

  5. DETECTION OF BRUGIA MALAYI INFECTED MOSQUITOES WITH SPECIES SPECIFIC DNA PROBE pBm 15, IN RIAU, INDONESIA

    Directory of Open Access Journals (Sweden)

    L. Kurniawan

    2012-09-01

    Full Text Available A species specific DNA probe (pBm15 was used in a field area where 2 filarial infections coexist: B.malayi in man and B.pahangi in cats. In our laboratory in Jakarta, this DNA probe proved to be sensitive enough to detect 500 ng DNA. One to two infective larvae of B.malayi could be detected with ease. This DNA probe did not react with infective larvae of wuchereria bancrofti, B.pahangi, and Dirofilaria spp. Non specific binding caused by undefined mosquito components was overcome with proteinase K and chitinase treatment. This additional step, made it possible for whole body mosquitoes to be squashed directly onto nitrocellulose paper. A comparative study of experimental infections of laboratory bred mosquitoes infected with B.malayi, showed no difference in infection rate between the group examined by dissection or by DNA probing. Mosquitoes which are vectors in Riau were collected and fed on microfilaremic patients of Riau. The set of mosquitoes were tested in parallel with mosquitoes infected with B.pahangi from cats. All fed mosquitoes were tested after 10-12 days. Only mosquitoes infected with B.malayi reacted in the assay. This study shows a success in applying the DNA probe technique in Jakarta. Further application in the field should be encouraged, with some modification of the DNA probing technique, for cheaper and easier implementation.

  6. Crystallization and preliminary crystallographic analysis of calcium-binding protein-2 from Entamoeba histolytica and its complexes with strontium and the IQ1 motif of myosin V

    Energy Technology Data Exchange (ETDEWEB)

    Gourinath, S., E-mail: sgourinath@mail.jnu.ac.in; Padhan, Narendra; Alam, Neelima; Bhattacharya, Alok [School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067 (India)

    2005-04-01

    Calcium-binding protein-2 (EhCaBP2) crystals were grown using MPD as a precipitant. EhCaBP2 also crystallized in complex with strontium (replacing calcium) at similar conditions. Preliminary data for EhCaBP2 crystals in complex with an IQ motif are also reported. Calcium plays a pivotal role in the pathogenesis of amoebiasis, a major disease caused by Entamoeba histolytica. Two domains with four canonical EF-hand-containing calcium-binding proteins (CaBPs) have been identified from E. histolytica. Even though they have very high sequence similarity, these bind to different target proteins in a Ca{sup 2+}-dependent manner, leading to different functional pathways. Calcium-binding protein-2 (EhCaBP2) crystals were grown using MPD as a precipitant. The crystals belong to space group P2{sub 1}, with unit-cell parameters a = 111.74, b = 68.83, c = 113.25 Å, β = 116.7°. EhCaBP2 also crystallized in complex with strontium (replacing calcium) at similar conditions. The crystals belong to space group P2{sub 1}, with unit-cell parameters a = 69.18, b = 112.03, c = 93.42 Å, β = 92.8°. Preliminary data for EhCaBP2 crystals in complex with an IQ motif are also reported. This complex was crystallized with MPD and ethanol as precipitating agents. These crystals belong to space group P2{sub 1}, with unit-cell parameters a = 60.5, b = 69.86, c = 86.5 Å, β = 97.9°.

  7. MHC motif viewer

    DEFF Research Database (Denmark)

    Rapin, Nicolas Philippe Jean-Pierre; Hoof, Ilka; Lund, Ole

    2008-01-01

    . Algorithms that predict which peptides MHC molecules bind have recently been developed and cover many different alleles, but the utility of these algorithms is hampered by the lack of tools for browsing and comparing the specificity of these molecules. We have, therefore, developed a web server, MHC motif...... viewer, that allows the display of the likely binding motif for all human class I proteins of the loci HLA A, B, C, and E and for MHC class I molecules from chimpanzee (Pan troglodytes), rhesus monkey (Macaca mulatta), and mouse (Mus musculus). Furthermore, it covers all HLA-DR protein sequences...

  8. Characterization of the fibronectin-attachment protein of Mycobacterium avium reveals a fibronectin-binding motif conserved among mycobacteria.

    Science.gov (United States)

    Schorey, J S; Holsti, M A; Ratliff, T L; Allen, P M; Brown, E J

    1996-07-01

    Mycobacterium avium is an intracellular pathogen and a major opportunistic infectious agent observed in patients with acquired immune deficiency syndrome (AIDS). Evidence suggests that the initial portal of infection by M. avium is often the gastrointestinal tract. However, the mechanism by which the M. avium crosses the epithelial barrier is unclear. A possible mechanism is suggested by the ability of M. avium to bind fibronectin, an extracellular matrix protein that is a virulence factor for several extracellular pathogenic bacteria which bind to mucosal surfaces. To further characterize fibronectin binding by M. avium, we have cloned the M. avium fibronectin-attachment protein (FAP). The M. avium FAP (FAP-A) has an unusually large number of Pro and Ala residues (40% overall) and is 50% identical to FAP of both Mycobacterium leprae and Mycobacterium tuberculosis. Using recombinant FAP-A and FAP-A peptides, we show that two non-continuous regions in FAP-A bind fibronectin. Peptides from these regions and homologous sequences from M. leprae FAP inhibit fibronectin binding by both M. avium and Mycobacterium bovis Bacillus Calmette-Guerin (BCG). These regions have no homology to eukaryotic fibronectin-binding proteins and are only distantly related to fibronectin-binding peptides of Gram-positive bacteria. Nevertheless, these fibronectin-binding regions are highly conserved among the mycobacterial FAPs, suggesting an essential function for this interaction in mycobacteria infection of their metazoan hosts.

  9. ø29 DNA polymerase residue Lys383, invariant at motif B of DNA-dependent polymerases, is involved in dNTP binding.

    Science.gov (United States)

    Saturno, J; Lázaro, J M; Esteban, F J; Blanco, L; Salas, M

    1997-06-13

    Bacteriophage ø29 DNA polymerase shares with other DNA-dependent DNA polymerases several regions of amino acid homology along the primary structure. Among them, motif B, characterized by the consensus +x3Kx(6-7)YG (+ being a positively charged amino acid), appears to be specifically conserved in those polymerases that use DNA but not RNA as template. In particular, the lysine residue of this motif is invariant in all members of DNA-dependent polymerases. In this paper we report a mutational analysis of this invariant residue of motif B with the construction and characterization of two mutant proteins in the corresponding residue (Lys383) of ø29 DNA polymerase. Mutant proteins (K383R and K383P) were overexpressed, purified and analyzed under steady-state conditions. In agreement with the modular organization proposed for ø29 DNA polymerase, the exonuclease activity was not affected in either mutant protein. Conversely, mutant K383P showed no detectable capacity to incorporate dNTP substrates using either DNA or TP as primer, although its affinity for DNA was not affected. The conservative substitution of Lys383 by arginine (K383R) resulted in a considerable impairment to use dNTPs, in both processive and non-processive DNA synthesis; the Km for dNTPs being 200-fold higher than that of the wild-type enzyme. Mutant K383R recovered the wild-type polymerase/exonuclease ratio when Mn2+ was used instead of Mg2+ as metal activator, indicating a distorted binding of the [dNTP-metal] chelate at the mutant enzyme active site. The positive charge at residue Lys383 was also critical in the catalysis of deoxynucleotidylation of the terminal protein by ø29 DNA polymerase. The results obtained suggest a direct role for the lysine residue in motif B in forming an evolutionarily conserved DNA templated dNTP binding pocket. Additionally, K383R mutant protein was also affected in the progression from protein-primed initiation to DNA elongation, a switch between two modes of

  10. Coach simplified structure modeling and optimization study based on the PBM method

    Science.gov (United States)

    Zhang, Miaoli; Ren, Jindong; Yin, Ying; Du, Jian

    2016-09-01

    For the coach industry, rapid modeling and efficient optimization methods are desirable for structure modeling and optimization based on simplified structures, especially for use early in the concept phase and with capabilities of accurately expressing the mechanical properties of structure and with flexible section forms. However, the present dimension-based methods cannot easily meet these requirements. To achieve these goals, the property-based modeling (PBM) beam modeling method is studied based on the PBM theory and in conjunction with the characteristics of coach structure of taking beam as the main component. For a beam component of concrete length, its mechanical characteristics are primarily affected by the section properties. Four section parameters are adopted to describe the mechanical properties of a beam, including the section area, the principal moments of inertia about the two principal axles, and the torsion constant of the section. Based on the equivalent stiffness strategy, expressions for the above section parameters are derived, and the PBM beam element is implemented in HyperMesh software. A case is realized using this method, in which the structure of a passenger coach is simplified. The model precision is validated by comparing the basic performance of the total structure with that of the original structure, including the bending and torsion stiffness and the first-order bending and torsional modal frequencies. Sensitivity analysis is conducted to choose design variables. The optimal Latin hypercube experiment design is adopted to sample the test points, and polynomial response surfaces are used to fit these points. To improve the bending and torsion stiffness and the first-order torsional frequency and taking the allowable maximum stresses of the braking and left turning conditions as constraints, the multi-objective optimization of the structure is conducted using the NSGA-II genetic algorithm on the ISIGHT platform. The result of the

  11. FACT Disrupts Nucleosome Structure by Binding H2A-H2B with Conserved Peptide Motifs.

    Science.gov (United States)

    Kemble, David J; McCullough, Laura L; Whitby, Frank G; Formosa, Tim; Hill, Christopher P

    2015-10-15

    FACT, a heterodimer of Spt16 and Pob3, is an essential histone chaperone. We show that the H2A-H2B binding activity that is central to FACT function resides in short acidic regions near the C termini of each subunit. Mutations throughout these regions affect binding and cause correlated phenotypes that range from mild to lethal, with the largest individual contributions unexpectedly coming from an aromatic residue and a nearby carboxylate residue within each domain. Spt16 and Pob3 bind overlapping sites on H2A-H2B, and Spt16-Pob3 heterodimers simultaneously bind two H2A-H2B dimers, the same stoichiometry as the components of a nucleosome. An Spt16:H2A-H2B crystal structure explains the biochemical and genetic data, provides a model for Pob3 binding, and implies a mechanism for FACT reorganization that we confirm biochemically. Moreover, unexpected similarity to binding of ANP32E and Swr1 with H2A.Z-H2B reveals that diverse H2A-H2B chaperones use common mechanisms of histone binding and regulating nucleosome functions.

  12. Coaggregation of RNA-Binding Proteins in a Model of TDP-43 Proteinopathy with Selective RGG Motif Methylation and a Role for RRM1 Ubiquitination

    Science.gov (United States)

    Dammer, Eric B.; Fallini, Claudia; Gozal, Yair M.; Duong, Duc M.; Rossoll, Wilfried; Xu, Ping; Lah, James J.; Levey, Allan I.; Peng, Junmin; Bassell, Gary J.; Seyfried, Nicholas T.

    2012-01-01

    TAR DNA-binding protein 43 (TDP-43) is a major component within ubiquitin-positive inclusions of a number of neurodegenerative diseases that increasingly are considered as TDP-43 proteinopathies. Identities of other inclusion proteins associated with TDP-43 aggregation remain poorly defined. In this study, we identify and quantitate 35 co-aggregating proteins in the detergent-resistant fraction of HEK-293 cells in which TDP-43 or a particularly aggregate prone variant, TDP-S6, were enriched following overexpression, using stable isotope-labeled (SILAC) internal standards and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). We also searched for differential post-translational modification (PTM) sites of ubiquitination. Four sites of ubiquitin conjugation to TDP-43 or TDP-S6 were confirmed by dialkylated GST-TDP-43 external reference peptides, occurring on or near RNA binding motif (RRM) 1. RRM-containing proteins co-enriched in cytoplasmic granular structures in HEK-293 cells and primary motor neurons with insoluble TDP-S6, including cytoplasmic stress granule associated proteins G3BP, PABPC1, and eIF4A1. Proteomic evidence for TDP-43 co-aggregation with paraspeckle markers RBM14, PSF and NonO was also validated by western blot and by immunocytochemistry in HEK-293 cells. An increase in peptides from methylated arginine-glycine-glycine (RGG) RNA-binding motifs of FUS/TLS and hnRNPs was found in the detergent-insoluble fraction of TDP-overexpressing cells. Finally, TDP-43 and TDP-S6 detergent-insoluble species were reduced by mutagenesis of the identified ubiquitination sites, even following oxidative or proteolytic stress. Together, these findings define some of the aggregation partners of TDP-43, and suggest that TDP-43 ubiquitination influences TDP-43 oligomerization. PMID:22761693

  13. Hitchcock's Motifs

    NARCIS (Netherlands)

    Walker, Michael

    2005-01-01

    Among the abundant Alfred Hitchcock literature, Hitchcock's Motifs has found a fresh angle. Starting from recurring objects, settings, character-types and events, Michael Walker tracks some forty motifs, themes and clusters across the whole of Hitchcock's oeuvre, including not only all his 52 extant

  14. Conformational consequences of cooperative binding of a coiled-coil peptide motif to poly(N-(2-hydroxypropyl) methacrylamide) HPMA copolymers.

    Science.gov (United States)

    Griffiths, Peter C; Paul, Alison; Apostolovic, Bojana; Klok, Harm-Anton; de Luca, Edoardo; King, Stephen M; Heenan, Richard K

    2011-07-30

    Small-angle neutron scattering and pulsed-gradient spin-echo NMR have been used to examine the solution conformation of a series of water soluble poly(N-(2-hydroxypropyl) methacrylamide) P(HPMA) co-polymer drug delivery vehicles incorporating a coiled-coil peptide motif as a novel pH sensitive non-covalent linker. The conformation of the HPMA homopolymer is well-described by a Gaussian coil model and changing pH from pH 7 to pH 5 has little effect on the solution conformation, as quantified via the radius of gyration. Copolymerisation with 5-10mol% of the K3 peptide bearing methacrylate monomer (K3-MA), gave a series of copolymers that exhibited an increase in radius of gyration at both pH's, despite being typically 30% lower in molecular weight, indicating that the K3-MA causes a perturbation (expansion) of the copolymer conformation. Subsequent addition of an equimolar amount of the complementary peptide E3 makes little further difference to the conformation, indicative of the intimate binding (coiled-coil motif) between the two peptides. Again, the effects of pH are small. Only the addition of a large aromatic structure such as methotrexate causes a further perturbation of the structure - the hydrophobic interaction between the MTX units causes a significant collapse of the polymer coil. These findings further elaborate the understanding of those factors that determine the solution conformation of novel polymer therapeutics.

  15. The cyclic cystine ladder of theta-defensins as a stable, bifunctional scaffold: A proof-of-concept study using the integrin-binding RGD motif.

    Science.gov (United States)

    Conibear, Anne C; Bochen, Alexander; Rosengren, K Johan; Stupar, Petar; Wang, Conan; Kessler, Horst; Craik, David J

    2014-02-10

    Peptides have the specificity and size required to target the protein-protein interactions involved in many diseases. Some cyclic peptides have been utilised as scaffolds for peptide drugs because of their stability; however, other cyclic peptide scaffolds remain to be explored. θ-Defensins are cyclic peptides from mammals; they are characterised by a cyclic cystine ladder motif and have low haemolytic and cytotoxic activity. Here we demonstrate the potential of the cyclic cystine ladder as a scaffold for peptide drug design by introducing the integrin-binding Arg-Gly-Asp (RGD) motif into the θ-defensin RTD-1. The most active analogue had an IC50 of 18 nM for the αv β3 integrin as well as high serum stability, thus demonstrating that a desired bioactivity can be imparted to the cyclic cystine ladder. This study highlights how θ-defensins can provide a stable and conformationally restrained scaffold for bioactive epitopes in a β-strand or turn conformation. Furthermore, the symmetry of the cyclic cystine ladder presents the opportunity to design peptides with dual bioactive epitopes to increase activity and specificity.

  16. La-related protein 4 binds poly(A), interacts with the poly(A)-binding protein MLLE domain via a variant PAM2w motif, and can promote mRNA stability.

    Science.gov (United States)

    Yang, Ruiqing; Gaidamakov, Sergei A; Xie, Jingwei; Lee, Joowon; Martino, Luigi; Kozlov, Guennadi; Crawford, Amanda K; Russo, Amy N; Conte, Maria R; Gehring, Kalle; Maraia, Richard J

    2011-02-01

    The conserved RNA binding protein La recognizes UUU-3'OH on its small nuclear RNA ligands and stabilizes them against 3'-end-mediated decay. We report that newly described La-related protein 4 (LARP4) is a factor that can bind poly(A) RNA and interact with poly(A) binding protein (PABP). Yeast two-hybrid analysis and reciprocal immunoprecipitations (IPs) from HeLa cells revealed that LARP4 interacts with RACK1, a 40S ribosome- and mRNA-associated protein. LARP4 cosediments with 40S ribosome subunits and polyribosomes, and its knockdown decreases translation. Mutagenesis of the RNA binding or PABP interaction motifs decrease LARP4 association with polysomes. Several translation and mRNA metabolism-related proteins use a PAM2 sequence containing a critical invariant phenylalanine to make direct contact with the MLLE domain of PABP, and their competition for the MLLE is thought to regulate mRNA homeostasis. Unlike all ∼150 previously analyzed PAM2 sequences, LARP4 contains a variant PAM2 (PAM2w) with tryptophan in place of the phenylalanine. Binding and nuclear magnetic resonance (NMR) studies have shown that a peptide representing LARP4 PAM2w interacts with the MLLE of PABP within the affinity range measured for other PAM2 motif peptides. A cocrystal of PABC bound to LARP4 PAM2w shows tryptophan in the pocket in PABC-MLLE otherwise occupied by phenylalanine. We present evidence that LARP4 expression stimulates luciferase reporter activity by promoting mRNA stability, as shown by mRNA decay analysis of luciferase and cellular mRNAs. We propose that LARP4 activity is integrated with other PAM2 protein activities by PABP as part of mRNA homeostasis.

  17. A motif unique to the human DEAD-box protein DDX3 is important for nucleic acid binding, ATP hydrolysis, RNA/DNA unwinding and HIV-1 replication.

    Directory of Open Access Journals (Sweden)

    Anna Garbelli

    Full Text Available DEAD-box proteins are enzymes endowed with nucleic acid-dependent ATPase, RNA translocase and unwinding activities. The human DEAD-box protein DDX3 has been shown to play important roles in tumor proliferation and viral infections. In particular, DDX3 has been identified as an essential cofactor for HIV-1 replication. Here we characterized a set of DDX3 mutants biochemically with respect to nucleic acid binding, ATPase and helicase activity. In particular, we addressed the functional role of a unique insertion between motifs I and Ia of DDX3 and provide evidence for its implication in nucleic acid binding and HIV-1 replication. We show that human DDX3 lacking this domain binds HIV-1 RNA with lower affinity. Furthermore, a specific peptide ligand for this insertion selected by phage display interferes with HIV-1 replication after transduction into HelaP4 cells. Besides broadening our understanding of the structure-function relationships of this important protein, our results identify a specific domain of DDX3 which may be suited as target for antiviral drugs designed to inhibit cellular cofactors for HIV-1 replication.

  18. A motif unique to the human DEAD-box protein DDX3 is important for nucleic acid binding, ATP hydrolysis, RNA/DNA unwinding and HIV-1 replication.

    Science.gov (United States)

    Garbelli, Anna; Beermann, Sandra; Di Cicco, Giulia; Dietrich, Ursula; Maga, Giovanni

    2011-05-12

    DEAD-box proteins are enzymes endowed with nucleic acid-dependent ATPase, RNA translocase and unwinding activities. The human DEAD-box protein DDX3 has been shown to play important roles in tumor proliferation and viral infections. In particular, DDX3 has been identified as an essential cofactor for HIV-1 replication. Here we characterized a set of DDX3 mutants biochemically with respect to nucleic acid binding, ATPase and helicase activity. In particular, we addressed the functional role of a unique insertion between motifs I and Ia of DDX3 and provide evidence for its implication in nucleic acid binding and HIV-1 replication. We show that human DDX3 lacking this domain binds HIV-1 RNA with lower affinity. Furthermore, a specific peptide ligand for this insertion selected by phage display interferes with HIV-1 replication after transduction into HelaP4 cells. Besides broadening our understanding of the structure-function relationships of this important protein, our results identify a specific domain of DDX3 which may be suited as target for antiviral drugs designed to inhibit cellular cofactors for HIV-1 replication.

  19. An inverted repeat motif stabilizes binding of E2F and enhances transcription of the dihydrofolate reductase gene

    DEFF Research Database (Denmark)

    Wade, M; Blake, M C; Jambou, R C

    1995-01-01

    An overlapping inverted repeat sequence that binds the eukaryotic transcription factor E2F is 100% conserved near the major transcription start sites in the promoters of three mammalian genes encoding dihydrofolate reductase, and is also found in the promoters of several other important cellular ...

  20. A Synthetic Peptide with the Putative Iron Binding Motif of Amyloid Precursor Protein (APP) Does Not Catalytically Oxidize Iron

    NARCIS (Netherlands)

    Honarmand Ebrahimi, K.; Hagedoorn, P.L.; Hagen, W.R.

    2012-01-01

    The β-amyloid precursor protein (APP), which is a key player in Alzheimer's disease, was recently reported to possess an Fe(II) binding site within its E2 domain which exhibits ferroxidase activity [Duce et al. 2010, Cell 142: 857]. The putative ligands of this site were compared to those in the fer

  1. The HIV-1 Envelope Transmembrane Domain Binds TLR2 through a Distinct Dimerization Motif and Inhibits TLR2-Mediated Responses

    Science.gov (United States)

    Rotem, Etai; Schwarzter, Roland; Gramatica, Andrea; Futerman, Anthony H.; Shai, Yechiel

    2014-01-01

    HIV-1 uses a number of means to manipulate the immune system, to avoid recognition and to highjack signaling pathways. HIV-1 infected cells show limited Toll-Like Receptor (TLR) responsiveness via as yet unknown mechanisms. Using biochemical and biophysical approaches, we demonstrate that the trans-membrane domain (TMD) of the HIV-1 envelope (ENV) directly interacts with TLR2 TMD within the membrane milieu. This interaction attenuates TNFα, IL-6 and MCP-1 secretion in macrophages, induced by natural ligands of TLR2 both in in vitro and in vivo models. This was associated with decreased levels of ERK phosphorylation. Furthermore, mutagenesis demonstrated the importance of a conserved GxxxG motif in driving this interaction within the membrane milieu. The administration of the ENV TMD in vivo to lipotechoic acid (LTA)/Galactosamine-mediated septic mice resulted in a significant decrease in mortality and in tissue damage, due to the weakening of systemic macrophage activation. Our findings suggest that the TMD of ENV is involved in modulation of the innate immune response during HIV infection. Furthermore, due to the high functional homology of viral ENV proteins this function may be a general character of viral-induced immune modulation. PMID:25121610

  2. The HIV-1 envelope transmembrane domain binds TLR2 through a distinct dimerization motif and inhibits TLR2-mediated responses.

    Science.gov (United States)

    Reuven, Eliran Moshe; Ali, Mohammad; Rotem, Etai; Schwarzer, Roland; Schwarzter, Roland; Gramatica, Andrea; Futerman, Anthony H; Shai, Yechiel

    2014-08-01

    HIV-1 uses a number of means to manipulate the immune system, to avoid recognition and to highjack signaling pathways. HIV-1 infected cells show limited Toll-Like Receptor (TLR) responsiveness via as yet unknown mechanisms. Using biochemical and biophysical approaches, we demonstrate that the trans-membrane domain (TMD) of the HIV-1 envelope (ENV) directly interacts with TLR2 TMD within the membrane milieu. This interaction attenuates TNFα, IL-6 and MCP-1 secretion in macrophages, induced by natural ligands of TLR2 both in in vitro and in vivo models. This was associated with decreased levels of ERK phosphorylation. Furthermore, mutagenesis demonstrated the importance of a conserved GxxxG motif in driving this interaction within the membrane milieu. The administration of the ENV TMD in vivo to lipotechoic acid (LTA)/Galactosamine-mediated septic mice resulted in a significant decrease in mortality and in tissue damage, due to the weakening of systemic macrophage activation. Our findings suggest that the TMD of ENV is involved in modulation of the innate immune response during HIV infection. Furthermore, due to the high functional homology of viral ENV proteins this function may be a general character of viral-induced immune modulation.

  3. The HIV-1 envelope transmembrane domain binds TLR2 through a distinct dimerization motif and inhibits TLR2-mediated responses.

    Directory of Open Access Journals (Sweden)

    Eliran Moshe Reuven

    2014-08-01

    Full Text Available HIV-1 uses a number of means to manipulate the immune system, to avoid recognition and to highjack signaling pathways. HIV-1 infected cells show limited Toll-Like Receptor (TLR responsiveness via as yet unknown mechanisms. Using biochemical and biophysical approaches, we demonstrate that the trans-membrane domain (TMD of the HIV-1 envelope (ENV directly interacts with TLR2 TMD within the membrane milieu. This interaction attenuates TNFα, IL-6 and MCP-1 secretion in macrophages, induced by natural ligands of TLR2 both in in vitro and in vivo models. This was associated with decreased levels of ERK phosphorylation. Furthermore, mutagenesis demonstrated the importance of a conserved GxxxG motif in driving this interaction within the membrane milieu. The administration of the ENV TMD in vivo to lipotechoic acid (LTA/Galactosamine-mediated septic mice resulted in a significant decrease in mortality and in tissue damage, due to the weakening of systemic macrophage activation. Our findings suggest that the TMD of ENV is involved in modulation of the innate immune response during HIV infection. Furthermore, due to the high functional homology of viral ENV proteins this function may be a general character of viral-induced immune modulation.

  4. Characterization of sialyloligosaccharide binding by recombinant soluble and native cell-associated CD22. Evidence for a minimal structural recognition motif and the potential importance of multisite binding.

    Science.gov (United States)

    Powell, L D; Jain, R K; Matta, K L; Sabesan, S; Varki, A

    1995-03-31

    CD22, a B cell-specific receptor of the immunoglobulin superfamily, has been demonstrated to bind to oligosaccharides containing alpha 2-6-linked sialic acid (Sia) residues. Previously, we demonstrated that the minimal structure recognized by this lectin is the trisaccharide Sia alpha 2-6Gal beta 1-4GlcNAc, as found on N-linked, O-linked, or glycolipid structures (Powell, L., and Varki, A. (1994) J. Biol. Chem. 269, 10628-10636). Here we utilize a soluble immunoglobulin fusion construct (CD22Rg) to determine directly by equilibrium dialysis the stoichiometry (2:1) and dissociation constant (32 microM) for Neu5Ac alpha 2-6Gal beta 1-4Glc binding. Inhibition assays performed with over 30 different natural and synthetic sialylated and/or sulfated compounds are utilized to define in greater detail specific structural features involved in oligosaccharide-protein binding. Specifically, the critical features required for binding include the exocyclic hydroxylated side chain of the Sia residue and the alpha 2-6 linkage position to the underlying Gal unit. Surprisingly, alterations of the 2-, 3-, and 4-positions of the latter residue have limited effect on the binding. The nature of the residue to which the Gal is attached may affect binding. Bi(alpha 2-6)-sialylated biantennary oligosaccharides are capable of simultaneously interacting with both lectin sites present on the dimeric CD22Rg fusion construct, giving a marked improvement in binding over monosialylated compounds. Furthermore, data are presented indicating that full-length native CD22, expressed on the surface of Chinese hamster ovary cells, is structurally and functionally a multimeric protein, demonstrating a higher apparent affinity for multiply sialylated compounds over monosialylated compounds. These observations provide a mechanism for strong CD22-dependent cell adhesion despite the relatively low Kd for protein-sugar binding.

  5. The κB transcriptional enhancer motif and signal sequences of V(DJ recombination are targets for the zinc finger protein HIVEP3/KRC: a site selection amplification binding study

    Directory of Open Access Journals (Sweden)

    Wu Lai-Chu

    2002-08-01

    Full Text Available Abstract Background The ZAS family is composed of proteins that regulate transcription via specific gene regulatory elements. The amino-DNA binding domain (ZAS-N and the carboxyl-DNA binding domain (ZAS-C of a representative family member, named κB DNA binding and recognition component (KRC, were expressed as fusion proteins and their target DNA sequences were elucidated by site selection amplification binding assays, followed by cloning and DNA sequencing. The fusion proteins-selected DNA sequences were analyzed by the MEME and MAST computer programs to obtain consensus motifs and DNA elements bound by the ZAS domains. Results Both fusion proteins selected sequences that were similar to the κB motif or the canonical elements of the V(DJ recombination signal sequences (RSS from a pool of degenerate oligonucleotides. Specifically, the ZAS-N domain selected sequences similar to the canonical RSS nonamer, while ZAS-C domain selected sequences similar to the canonical RSS heptamer. In addition, both KRC fusion proteins selected oligonucleoties with sequences identical to heptamer and nonamer sequences within endogenous RSS. Conclusions The RSS are cis-acting DNA motifs which are essential for V(DJ recombination of antigen receptor genes. Due to its specific binding affinity for RSS and κB-like transcription enhancer motifs, we hypothesize that KRC may be involved in the regulation of V(DJ recombination.

  6. Histatins: salivary peptides with copper(II)- and zinc(II)-binding motifs: perspectives for biomedical applications.

    Science.gov (United States)

    Melino, Sonia; Santone, Celeste; Di Nardo, Paolo; Sarkar, Bibudhendra

    2014-02-01

    Natural antimicrobial peptides represent a primordial mechanism of immunity in both vertebrate and nonvertebrate organisms. Among them, histatins belong to a family of human salivary metal-binding peptides displaying potent antibacterial, antifungal and wound-healing activities. These properties, along with the ability of histatins to inhibit collagenases and cysteine proteases, have attracted much attention for their potential use in the treatment of several oral diseases. This review critically assesses the studies carried out to date in order to provide a comprehensive and systematic vision of the information accumulated so far. In particular, the relationship between metal-binding and peptide activity is extensively analysed. The review provides important clues for developing possible therapeutic applications of histatins and their synthetic peptide analogues by creating a set of necessary resource materials to support investigators and industries interested in exploiting their unique properties.

  7. Elucidation of a C-Rich Signature Motif in Target mRNAs of RNA-Binding Protein TIAR▿ †

    OpenAIRE

    Kim, Henry S.; Kuwano, Yuki; Zhan, Ming; Pullmann, Rudolf; Mazan-Mamczarz, Krystyna; Li, Huai; Kedersha, Nancy; Anderson, Paul; Wilce, Matthew C J; Gorospe, Myriam; Wilce, Jacqueline A.

    2007-01-01

    The RNA-binding protein TIAR (related to TIA-1 [T-cell-restricted intracellular antigen 1]) was shown to associate with subsets of mRNAs bearing U-rich sequences in their 3′ untranslated regions. TIAR can function as a translational repressor, particularly in response to cytotoxic agents. Using unstressed colon cancer cells, collections of mRNAs associated with TIAR were isolated by immunoprecipitation (IP) of (TIAR-RNA) ribonucleoprotein (RNP) complexes, identified by microarray analysis, an...

  8. T-cell memory responses elicited by yellow fever vaccine are targeted to overlapping epitopes containing multiple HLA-I and -II binding motifs.

    Directory of Open Access Journals (Sweden)

    Andréa Barbosa de Melo

    Full Text Available The yellow fever vaccines (YF-17D-204 and 17DD are considered to be among the safest vaccines and the presence of neutralizing antibodies is correlated with protection, although other immune effector mechanisms are known to be involved. T-cell responses are known to play an important role modulating antibody production and the killing of infected cells. However, little is known about the repertoire of T-cell responses elicited by the YF-17DD vaccine in humans. In this report, a library of 653 partially overlapping 15-mer peptides covering the envelope (Env and nonstructural (NS proteins 1 to 5 of the vaccine was utilized to perform a comprehensive analysis of the virus-specific CD4(+ and CD8(+ T-cell responses. The T-cell responses were screened ex-vivo by IFN-γ ELISPOT assays using blood samples from 220 YF-17DD vaccinees collected two months to four years after immunization. Each peptide was tested in 75 to 208 separate individuals of the cohort. The screening identified sixteen immunodominant antigens that elicited activation of circulating memory T-cells in 10% to 33% of the individuals. Biochemical in-vitro binding assays and immunogenetic and immunogenicity studies indicated that each of the sixteen immunogenic 15-mer peptides contained two or more partially overlapping epitopes that could bind with high affinity to molecules of different HLAs. The prevalence of the immunogenicity of a peptide in the cohort was correlated with the diversity of HLA-II alleles that they could bind. These findings suggest that overlapping of HLA binding motifs within a peptide enhances its T-cell immunogenicity and the prevalence of the response in the population. In summary, the results suggests that in addition to factors of the innate immunity, "promiscuous" T-cell antigens might contribute to the high efficacy of the yellow fever vaccines.

  9. Investigating actinomycin D binding to G-quadruplex, i-motif and double-stranded DNA in 27-nt segment of c-MYC gene promoter.

    Science.gov (United States)

    Niknezhad, Zhila; Hassani, Leila; Norouzi, Davood

    2016-01-01

    c-MYC DNA is an attractive target for drug design, especially for cancer chemotherapy. Around 90% of c-MYC transcription is controlled by NHE III1, whose 27-nt purine-rich strand has the ability to form G-quadruplex structure. In this investigation, interaction of ActD with 27-nt G-rich strand (G/c-MYC) and its equimolar mixture with the complementary sequence, (GC/c-MYC) as well as related C-rich oligonucleotide (C/c-MYC) was evaluated. Molecular dynamic simulations showed that phenoxazine and lactone rings of ActD come close to the outer G-tetrad nucleotides indicating that ActD binds through end-stacking to the quadruplex DNA. RMSD and RMSF revealed that fluctuation of the quadruplex DNA increases upon interaction with the drug. The results of spectrophotometry and spectrofluorometry indicated that ActD most probably binds to the c-MYC quadruplex and duplex DNA via end-stacking and intercalation, respectively and polarity of ActD environment decreases due to the interaction. It was also found that binding of ActD to the GC-rich DNA is stronger than the two other forms of DNA. Circular dichroism results showed that the type of the three forms of DNA structures doesn't change, but their compactness alters due to their interaction with ActD. Finally, it can be concluded that ActD binds differently to double stranded DNA, quadruplex DNA and i-motif. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Interleukin-1β induced nuclear factor-κB binds to a disintegrin-like and metalloproteinase with thrombospondin type 1 motif 9 promoter in human chondrosarcoma cells.

    Science.gov (United States)

    Altuntas, Aynur; Halacli, Sevil Oskay; Cakmak, Ozlem; Erden, Gonul; Akyol, Sumeyya; Ugurcu, Veli; Hirohata, Satoshi; Demircan, Kadir

    2015-07-01

    Nuclear factor-κB (NF-κB) is involved in the regulation of inflammation‑associated genes. NF-κB forms dimers which bind with sequences referred to as NF-κB sites (9-11 bp). A disintegrin-like and metalloproteinase with thrombospondin type 1 motif 9 (ADAMTS9) is a type of proteoglycanase, which proteolytically cleaves versican and aggrecan. ADAMTS9 is a cytokine-inducible gene that contains binding sites for NF-κB within its promoter region. Interleukin-1β (IL-1β) affects cartilage metabolism and is involved in the NF-κB pathway. It is therefore hypothesized that NF-κB binding with ADAMTS9 promoters may activate IL-1β, thereby promoting chondrocytic cell growth. In the present study, the OUMS-27 chondrocytic human chondrosarcoma cell line was treated with IL-1β with or without inhibitors of NF-κB signaling pathways. Chromatin immunoprecipitation (ChIP) and electromobility shift assays (EMSA) were conducted order to analyze the binding of NF-κB with the ADAMTS9 promoter region. NF-κB-p65 subunit phosphorylation was promoted in IL-1β-treated cells, which were not treated with inhibitors of NF-κB signaling pathways. By contrast, NF-κB-p65 subunit phosphorylation was inhibited in cells that had been treated with BAY-117085, an NF-κB pathway inhibitor. ChIP and EMSA assays demonstrated that, following treatment with IL-1β, NF-κB‑p65 bound to elements located at -1177 and -1335 in the ADAMTS9 promoter region, in contrast to the untreated samples. The results of the present study suggested that NF-κB may be involved in IL-1β-induced activation of ADAMTS9 in human chondrocytes.

  11. An SH3 binding motif within the nucleocapsid protein of porcine reproductive and respiratory syndrome virus interacts with the host cellular signaling proteins STAMI, TXK, Fyn, Hck, and cortactin.

    Science.gov (United States)

    Kenney, Scott P; Meng, Xiang-Jin

    2015-06-02

    Porcine reproductive and respiratory syndrome virus (PRRSV) causes an economically important global swine disease, and has a complicated virus-host immunomodulation that often leads to a weak Th2 immune response and viral persistence. In this study, we identified a Src homology 3 (SH3) binding motif, PxxPxxP, that is conserved within the N protein of PRRSV strains. Subsequently, we identified five host cellular proteins [signal transducing adaptor molecule (STAM)I, TXK tyrosine kinase (TXK), protein tyrosine kinase fyn (Fyn), hematopoietic cell kinase (Hck), and cortactin] that interact with this SH3 motif. We demonstrated that binding of SH3 proteins with PRRSV N protein depends on at least one intact PxxP motif as disruption of P53 within the motif significantly reduced interaction of each of the 5 proteins. The first PxxP motif appears to be more important for STAMI-N protein interactions whereas the second PxxP motif was more important for Hck interaction. Both STAMI and Hck interactions with PRRSV N protein required an unhindered C-terminal domain as the interaction was only observed with STAMI and Hck proteins with N-terminal but not C-terminal fluorescent tags. We showed that the P56 residue within the SH3 motif is critical for virus lifecycle as mutation resulted in a loss of virus infectivity, however the P50 and P53 mutations did not abolish virus infectivity suggesting that these highly conserved proline residues within the SH3 motif may provide a selective growth advantage through interactions with the host rather than a vital functional element. These results have important implications in understanding PRRSV-host interactions.

  12. The NS1 polypeptide of the murine parvovirus minute virus of mice binds to DNA sequences containing the motif [ACCA]2-3.

    Science.gov (United States)

    Cotmore, S F; Christensen, J; Nüesch, J P; Tattersall, P

    1995-03-01

    A DNA fragment containing the minute virus of mice 3' replication origin was specifically coprecipitated in immune complexes containing the virally coded NS1, but not the NS2, polypeptide. Antibodies directed against the amino- or carboxy-terminal regions of NS1 precipitated the NS1-origin complexes, but antibodies directed against NS1 amino acids 284 to 459 blocked complex formation. Using affinity-purified histidine-tagged NS1 preparations, we have shown that the specific protein-DNA interaction is of moderate affinity, being stable in 0.1 M salt but rapidly lost at higher salt concentrations. In contrast, generalized (or nonspecific) DNA binding by NS1 could be demonstrated only in low salt. Addition of ATP or gamma S-ATP enhanced specific DNA binding by wild-type NS1 severalfold, but binding was lost under conditions which favored ATP hydrolysis. NS1 molecules with mutations in a critical lysine residue (amino acid 405) in the consensus ATP-binding site bound to the origin, but this binding could not be enhanced by ATP addition. DNase I protection assays carried out with wild-type NS1 in the presence of gamma S-ATP gave footprints which extended over 43 nucleotides on both DNA strands, from the middle of the origin bubble sequence to a position some 14 bp beyond the nick site. The DNA-binding site for NS1 was mapped to a 22-bp fragment from the middle of the 3' replication origin which contains the sequence ACCAACCA. This conforms to a reiterated motif (ACCA)2-3, which occurs, in more or less degenerate form, at many sites throughout the minute virus of mice genome (J. W. Bodner, Virus Genes 2:167-182, 1989). Insertion of a single copy of the sequence (ACCA)3 was shown to be sufficient to confer NS1 binding on an otherwise unrecognized plasmid fragment. The functions of NS1 in the viral life cycle are reevaluated in the light of this result.

  13. A synthetic peptide with the putative iron binding motif of amyloid precursor protein (APP) does not catalytically oxidize iron.

    Science.gov (United States)

    Ebrahimi, Kourosh Honarmand; Hagedoorn, Peter-Leon; Hagen, Wilfred R

    2012-01-01

    The β-amyloid precursor protein (APP), which is a key player in Alzheimer's disease, was recently reported to possess an Fe(II) binding site within its E2 domain which exhibits ferroxidase activity [Duce et al. 2010, Cell 142: 857]. The putative ligands of this site were compared to those in the ferroxidase site of ferritin. The activity was indirectly measured using transferrin, which scavenges the Fe(III) product of the reaction. A 22-residue synthetic peptide, named FD1, with the putative ferroxidase site of APP, and the E2 domain of APP were each reported to exhibit 40% of the ferroxidase activity of APP and of ceruloplasmin. It was also claimed that the ferroxidase activity of APP is inhibited by Zn(II) just as in ferritin. We measured the ferroxidase activity indirectly (i) by the incorporation of the Fe(III) product of the ferroxidase reaction into transferrin and directly (ii) by monitoring consumption of the substrate molecular oxygen. The results with the FD1 peptide were compared to the established ferroxidase activities of human H-chain ferritin and of ceruloplasmin. For FD1 we observed no activity above the background of non-enzymatic Fe(II) oxidation by molecular oxygen. Zn(II) binds to transferrin and diminishes its Fe(III) incorporation capacity and rate but it does not specifically bind to a putative ferroxidase site of FD1. Based on these results, and on comparison of the putative ligands of the ferroxidase site of APP with those of ferritin, we conclude that the previously reported results for ferroxidase activity of FD1 and - by implication - of APP should be re-evaluated.

  14. A synthetic peptide with the putative iron binding motif of amyloid precursor protein (APP does not catalytically oxidize iron.

    Directory of Open Access Journals (Sweden)

    Kourosh Honarmand Ebrahimi

    Full Text Available The β-amyloid precursor protein (APP, which is a key player in Alzheimer's disease, was recently reported to possess an Fe(II binding site within its E2 domain which exhibits ferroxidase activity [Duce et al. 2010, Cell 142: 857]. The putative ligands of this site were compared to those in the ferroxidase site of ferritin. The activity was indirectly measured using transferrin, which scavenges the Fe(III product of the reaction. A 22-residue synthetic peptide, named FD1, with the putative ferroxidase site of APP, and the E2 domain of APP were each reported to exhibit 40% of the ferroxidase activity of APP and of ceruloplasmin. It was also claimed that the ferroxidase activity of APP is inhibited by Zn(II just as in ferritin. We measured the ferroxidase activity indirectly (i by the incorporation of the Fe(III product of the ferroxidase reaction into transferrin and directly (ii by monitoring consumption of the substrate molecular oxygen. The results with the FD1 peptide were compared to the established ferroxidase activities of human H-chain ferritin and of ceruloplasmin. For FD1 we observed no activity above the background of non-enzymatic Fe(II oxidation by molecular oxygen. Zn(II binds to transferrin and diminishes its Fe(III incorporation capacity and rate but it does not specifically bind to a putative ferroxidase site of FD1. Based on these results, and on comparison of the putative ligands of the ferroxidase site of APP with those of ferritin, we conclude that the previously reported results for ferroxidase activity of FD1 and - by implication - of APP should be re-evaluated.

  15. Long Non-Coding RNA HOTAIR Promotes Cell Migration and Invasion via Down-Regulation of RNA Binding Motif Protein 38 in Hepatocellular Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Chaofeng Ding

    2014-03-01

    Full Text Available Long non-coding RNA HOTAIR exerts regulatory functions in various biological processes in cancer cells, such as proliferation, apoptosis, mobility, and invasion. We previously found that HOX transcript antisense RNA (HOTAIR is a negative prognostic factor and exhibits oncogenic activity in hepatocellular carcinoma (HCC. In this study, we aimed to investigate the role and molecular mechanism of HOTAIR in promoting HCC cell migration and invasion. Firstly, we profiled its gene expression pattern by microarray analysis of HOTAIR loss in Bel-7402 HCC cell line. The results showed that 129 genes were significantly down-regulated, while 167 genes were significantly up-regulated (fold change >2, p < 0.05. Bioinformatics analysis indicated that RNA binding proteins were involved in this biological process. HOTAIR suppression using RNAi strategy with HepG2 and Bel-7402 cells increased the mRNA and protein expression levels of RNA binding motif protein 38 (RBM38. Moreover, the expression levels of RBM38 in HCC specimens were significantly lower than paired adjacent noncancerous tissues. In addition, knockdown of HOTAIR resulted in a decrease of cell migration and invasion, which could be specifically rescued by down-regulation of RBM38. Taken together, HOTAIR could promote migration and invasion of HCC cells by inhibiting RBM38, which indicated critical roles of HOTAIR and RBM38 in HCC progression.

  16. Rice MEL2, the RNA recognition motif (RRM) protein, binds in vitro to meiosis-expressed genes containing U-rich RNA consensus sequences in the 3'-UTR.

    Science.gov (United States)

    Miyazaki, Saori; Sato, Yutaka; Asano, Tomoya; Nagamura, Yoshiaki; Nonomura, Ken-Ichi

    2015-10-01

    Post-transcriptional gene regulation by RNA recognition motif (RRM) proteins through binding to cis-elements in the 3'-untranslated region (3'-UTR) is widely used in eukaryotes to complete various biological processes. Rice MEIOSIS ARRESTED AT LEPTOTENE2 (MEL2) is the RRM protein that functions in the transition to meiosis in proper timing. The MEL2 RRM preferentially associated with the U-rich RNA consensus, UUAGUU[U/A][U/G][A/U/G]U, dependently on sequences and proportionally to MEL2 protein amounts in vitro. The consensus sequences were located in the putative looped structures of the RNA ligand. A genome-wide survey revealed a tendency of MEL2-binding consensus appearing in 3'-UTR of rice genes. Of 249 genes that conserved the consensus in their 3'-UTR, 13 genes spatiotemporally co-expressed with MEL2 in meiotic flowers, and included several genes whose function was supposed in meiosis; such as Replication protein A and OsMADS3. The proteome analysis revealed that the amounts of small ubiquitin-related modifier-like protein and eukaryotic translation initiation factor3-like protein were dramatically altered in mel2 mutant anthers. Taken together with transcriptome and gene ontology results, we propose that the rice MEL2 is involved in the translational regulation of key meiotic genes on 3'-UTRs to achieve the faithful transition of germ cells to meiosis.

  17. Binding of human prothymosin alpha to the leucine-motif/activation domains of HTLV-I Rex and HIV-1 Rev.

    Science.gov (United States)

    Kubota, S; Adachi, Y; Copeland, T D; Oroszlan, S

    1995-10-01

    Rex of human T-cell leukemia virus type I (HTLV-I) and Rev of human immunodeficiency virus 1 (HIV-1) are post-transcriptional regulators of viral gene expression. By means of affinity chromatography, we purified an 18-kDa cellular protein that bound to the conserved leucine-motif/activation domain of HTLV-I Rex or HIV-1 Rev. The protein that was purified through a Rev-affinity column was found to bind to Rex immunoprecipitated with anti-Rex IgG from an HTLV-I-producing cell line. We analyzed the purified approximately 18-kDa protein biochemically and identified it as prothymosin alpha. The binding activity of prothymosin alpha to Rev or Rex was completely abolished when the epsilon-amino groups of its lysine residues were chemically modified by N-succinimidyl-3-(4-hydroxy-3,5-diodo- phenyl)propionate. The functional relationship between the nuclear protein prothymosin alpha and Rex-Rev is discussed.

  18. The cholesterol-dependent cytolysin signature motif: a critical element in the allosteric pathway that couples membrane binding to pore assembly.

    Directory of Open Access Journals (Sweden)

    Kelley J Dowd

    Full Text Available The cholesterol-dependent cytolysins (CDCs constitute a family of pore-forming toxins that contribute to the pathogenesis of a large number of Gram-positive bacterial pathogens.The most highly conserved region in the primary structure of the CDCs is the signature undecapeptide sequence (ECTGLAWEWWR. The CDC pore forming mechanism is highly sensitive to changes in its structure, yet its contribution to the molecular mechanism of the CDCs has remained enigmatic. Using a combination of fluorescence spectroscopic methods we provide evidence that shows the undecapeptide motif of the archetype CDC, perfringolysin O (PFO, is a key structural element in the allosteric coupling of the cholesterol-mediated membrane binding in domain 4 (D4 to distal structural changes in domain 3 (D3 that are required for the formation of the oligomeric pore complex. Loss of the undecapeptide function prevents all measurable D3 structural transitions, the intermolecular interaction of membrane bound monomers and the assembly of the oligomeric pore complex. We further show that this pathway does not exist in intermedilysin (ILY, a CDC that exhibits a divergent undecapeptide and that has evolved to use human CD59 rather than cholesterol as its receptor. These studies show for the first time that the undecapeptide of the cholesterol-binding CDCs forms a critical element of the allosteric pathway that controls the assembly of the pore complex.

  19. The serine protease motif of Pic mediates a dose-dependent mucolytic activity after binding to sugar constituents of the mucin substrate.

    Science.gov (United States)

    Gutiérrez-Jiménez, Javier; Arciniega, Ivonne; Navarro-García, Fernando

    2008-08-01

    The pic gene is harbored on the chromosomes of three important pathogens: enteroaggregative Escherichia coli (EAEC), uropathogenic E. coli (UPEC), and Shigella flexneri. Since Pic is secreted into the intestinal lumen during EAEC infection, we sought to identify intestinal-mucosal substrates for Pic. Pic did not damage epithelial cells, cleave fodrin, or degrade host defense proteins embedded in the mucus layer (sIgA, lactoferrin and lysozyme). However, by using a solid-phase assay to evaluate the mucinolytic activity of EAEC Pic, we documented a specific, dose-dependent mucinolytic activity. A serine protease inhibitor and an enzymatically inactive variant of Pic were used to show that the Pic serine protease motif is required for mucinolytic activity. Pic binds mucin, and this binding was blocked in competition assays using monosaccharide constituents of the oligosaccharide side chains of mucin. Moreover, Pic mucinolytic activity decreased when sialic acid was removed from mucin. Thus, Pic is a mucinase with lectin-like activity that can be related to its reported hemagglutinin activity. Our results suggest that EAEC may secrete Pic into the intestinal lumen as a strategy for penetrating the gel-like mucus layer during EAEC colonization.

  20. Automated discovery of tissue-targeting enhancers and transcription factors from binding motif and gene function data.

    Directory of Open Access Journals (Sweden)

    Geetu Tuteja

    2014-01-01

    Full Text Available Identifying enhancers regulating gene expression remains an important and challenging task. While recent sequencing-based methods provide epigenomic characteristics that correlate well with enhancer activity, it remains onerous to comprehensively identify all enhancers across development. Here we introduce a computational framework to identify tissue-specific enhancers evolving under purifying selection. First, we incorporate high-confidence binding site predictions with target gene functional enrichment analysis to identify transcription factors (TFs likely functioning in a particular context. We then search the genome for clusters of binding sites for these TFs, overcoming previous constraints associated with biased manual curation of TFs or enhancers. Applying our method to the placenta, we find 33 known and implicate 17 novel TFs in placental function, and discover 2,216 putative placenta enhancers. Using luciferase reporter assays, 31/36 (86% tested candidates drive activity in placental cells. Our predictions agree well with recent epigenomic data in human and mouse, yet over half our loci, including 7/8 (87% tested regions, are novel. Finally, we establish that our method is generalizable by applying it to 5 additional tissues: heart, pancreas, blood vessel, bone marrow, and liver.

  1. Fox-2 Splicing Factor Binds to a Conserved Intron Motif to PromoteInclusion of Protein 4.1R Alternative Exon 16

    Energy Technology Data Exchange (ETDEWEB)

    Ponthier, Julie L.; Schluepen, Christina; Chen, Weiguo; Lersch,Robert A.; Gee, Sherry L.; Hou, Victor C.; Lo, Annie J.; Short, Sarah A.; Chasis, Joel A.; Winkelmann, John C.; Conboy, John G.

    2006-03-01

    Activation of protein 4.1R exon 16 (E16) inclusion during erythropoiesis represents a physiologically important splicing switch that increases 4.1R affinity for spectrin and actin. Previous studies showed that negative regulation of E16 splicing is mediated by the binding of hnRNP A/B proteins to silencer elements in the exon and that downregulation of hnRNP A/B proteins in erythroblasts leads to activation of E16 inclusion. This paper demonstrates that positive regulation of E16 splicing can be mediated by Fox-2 or Fox-1, two closely related splicing factors that possess identical RNA recognition motifs. SELEX experiments with human Fox-1 revealed highly selective binding to the hexamer UGCAUG. Both Fox-1 and Fox-2 were able to bind the conserved UGCAUG elements in the proximal intron downstream of E16, and both could activate E16 splicing in HeLa cell co-transfection assays in a UGCAUG-dependent manner. Conversely, knockdown of Fox-2 expression, achieved with two different siRNA sequences resulted in decreased E16 splicing. Moreover, immunoblot experiments demonstrate mouse erythroblasts express Fox-2, but not Fox-1. These findings suggest that Fox-2 is a physiological activator of E16 splicing in differentiating erythroid cells in vivo. Recent experiments show that UGCAUG is present in the proximal intron sequence of many tissue-specific alternative exons, and we propose that the Fox family of splicing enhancers plays an important role in alternative splicing switches during differentiation in metazoan organisms.

  2. Activation of nonspecific cytotoxic cells (NCC) with synthetic oligodeoxynucleotides and bacterial genomic DNA: binding, specificity and identification of unique immunostimulatory motifs.

    Science.gov (United States)

    Oumouna, M; Jaso-Friedmann, L; Evans, D L

    2002-04-01

    We have analyzed the effects of synthetic oligodeoxynucleotides (sODNs) and bacterial DNA (bDNA) on the in vitro activation of NCC. Teleost NCC recognition of DNA appeared to differ from that which occurs in higher vertebrates. NCC contain at least two different receptor specificities for DNA. Both oligodeoxyguanosine 20-mers (dG20) and 5'-TGCTGCTTGTGCTTGTGCTT-3' (4GC-2T) bound specifically to NCC. The existence of different receptor specificities was indicated by reciprocal cold target inhibition experiments. dG20 competed with 4GC-2T binding but sODNs composed of GpC or CpG nests did not compete with recognition by NCC of the dG20. ODN binding by NCC primarily depended on the presence of GpC or CpG nests with a preference for -G- serving as the anchor nucleotide. Secondarily, and similar to models of ODN activation in mammals, palindrome sequences of pu-pu-CpG-py-py activated NCC cytotoxicity. Additional analysis of the requirements for ODN activation indicated that guanosine could not substitute for adenosine as a purine spacer and that CpG motifs containing flanking thymidine (i.e.-GTCpGTT-) augmented the activity of the sODN containing this flanking base. Other evidence for the participation of both G and C in the recognition of specific nucleotides by NCC was that poly-dC20, dA20 or dT20 had no activating properties. Methylation of all cytosine nucleotides within an ODN abrogated activation. A canonical ODN motif of 5'-C/AT/AGCTT-3' can now be suggested for teleosts. Additional studies were done to examine the effects of in vitro treatment of NCC with bDNA. bDNA from three different disease isolates of Streptococcus iniae activated NCC cytotoxicity. Treatment of the bDNA with DNase abrogated the enhancement of cytotoxicity. Also, treatment of NCC with eukaryotic DNA had no effects on cytotoxicity. These studies suggested that NCC recognize bacterial nonmethylated DNA. The consequences of these interactions may be increased innate and acquired anti

  3. The p53 cofactor Strap exhibits an unexpected TPR motif and oligonucleotide-binding (OB)-fold structure.

    Science.gov (United States)

    Adams, Cassandra J; Pike, Ashley C W; Maniam, Sandra; Sharpe, Timothy D; Coutts, Amanda S; Knapp, Stefan; La Thangue, Nicholas B; Bullock, Alex N

    2012-03-01

    Activation of p53 target genes for tumor suppression depends on the stress-specific regulation of transcriptional coactivator complexes. Strap (stress-responsive activator of p300) is activated upon DNA damage by ataxia telangiectasia mutated (ATM) and Chk2 kinases and is a key regulator of the p53 response. In addition to antagonizing Mdm2, Strap facilitates the recruitment of p53 coactivators, including JMY and p300. Strap is a predicted TPR-repeat protein, but shows only limited sequence identity with any protein of known structure. To address this and to elucidate the molecular mechanism of Strap activity we determined the crystal structure of the full-length protein at 2.05 Å resolution. The structure of Strap reveals an atypical six tetratricopeptide repeat (TPR) protein that also contains an unexpected oligonucleotide/oligosaccharide-binding (OB)-fold domain. This previously unseen domain organization provides an extended superhelical scaffold allowing for protein-protein as well as protein-DNA interaction. We show that both of the TPR and OB-fold domains localize to the chromatin of p53 target genes and exhibit intrinsic regulatory activity necessary for the Strap-dependent p53 response.

  4. Identification of a binding motif in the S5 helix that confers cholesterol sensitivity to the TRPV1 ion channel.

    Science.gov (United States)

    Picazo-Juárez, Giovanni; Romero-Suárez, Silvina; Nieto-Posadas, Andrés; Llorente, Itzel; Jara-Oseguera, Andrés; Briggs, Margaret; McIntosh, Thomas J; Simon, Sidney A; Ladrón-de-Guevara, Ernesto; Islas, León D; Rosenbaum, Tamara

    2011-07-15

    The TRPV1 ion channel serves as an integrator of noxious stimuli with its activation linked to pain and neurogenic inflammation. Cholesterol, a major component of cell membranes, modifies the function of several types of ion channels. Here, using measurements of capsaicin-activated currents in excised patches from TRPV1-expressing HEK cells, we show that enrichment with cholesterol, but not its diastereoisomer epicholesterol, markedly decreased wild-type rat TRPV1 currents. Substitutions in the S5 helix, rTRPV1-R579D, and rTRPV1-F582Q, decreased this cholesterol response and rTRPV1-L585I was insensitive to cholesterol addition. Two human TRPV1 variants, with different amino acids at position 585, had different responses to cholesterol with hTRPV1-Ile(585) being insensitive to this molecule. However, hTRPV1-I585L was inhibited by cholesterol addition similar to rTRPV1 with the same S5 sequence. In the absence of capsaicin, cholesterol enrichment also inhibited TRPV1 currents induced by elevated temperature and voltage. These data suggest that there is a cholesterol-binding site in TRPV1 and that the functions of TRPV1 depend on the genetic variant and membrane cholesterol content.

  5. In silico cloning and characterization of the TGA (TGACG MOTIF-BINDING FACTOR) transcription factors subfamily in Carica papaya.

    Science.gov (United States)

    Idrovo Espín, Fabio Marcelo; Peraza-Echeverria, Santy; Fuentes, Gabriela; Santamaría, Jorge M

    2012-05-01

    The TGA transcription factors belong to the subfamily of bZIP group D that play a major role in disease resistance and development. Most of the TGA identified in Arabidopsis interact with the master regulator of SAR, NPR1 that controls the expression of PR genes. As a first approach to determine the possible involvement of these transcription factors in papaya defense, we characterized Arabidopsis TGA orthologs from the genome of Carica papaya cv. SunUp. Six orthologs CpTGA1 to CpTGA6, were identified. The predicted CpTGA proteins were highly similar to AtTGA sequences and probably share the same DNA binding properties and transcriptional regulation features. The protein sequences alignment evidenced the presence of conserved domains, characteristic of this group of transcription factors. The phylogeny showed that CpTGA evolved into three different subclades associated with defense and floral development. This is the first report of basal expression patterns assessed by RT-PCR, from the whole subfamily of CpTGA members in different tissues from papaya cv. Maradol mature plants. Overall, CpTGA1, CpTGA3 CpTGA6 and CpTGA4 showed a basal expression in all tissues tested; CpTGA2 expressed strongly in all tissues except in petioles while CpTGA5 expressed only in petals and to a lower extent in petioles. Although more detailed studies in anthers and other floral structures are required, we suggest that CpTGA5 might be tissue-specific, and it might be involved in papaya floral development. On the other hand, we report here for the first time, the expression of the whole family of CpTGA in response to salicylic acid (SA). The expression of CpTGA3, CpTGA4 and CpTGA6 increased in response to SA, what would suggest its involvement in the SAR response in papaya.

  6. N-terminal GNBP homology domain of Gram-negative binding protein 3 functions as a beta-1,3-glucan binding motif in Tenebrio molitor.

    Science.gov (United States)

    Lee, Hanna; Kwon, Hyun-Mi; Park, Ji-Won; Kurokawa, Kenji; Lee, Bok Luel

    2009-08-31

    The Toll signalling pathway in invertebrates is responsible for defense against Gram-positive bacteria and fungi, leading to the expression of antimicrobial peptides via NF-kappaB-like transcription factors. Gram-negative binding protein 3 (GNBP3) detects beta-1,3-glucan, a fungal cell wall component, and activates a three step serine protease cascade for activation of the Toll signalling pathway. Here, we showed that the recombinant N-terminal domain of Tenebrio molitor GNBP3 bound to beta-1,3-glucan, but did not activate down-stream serine protease cascade in vitro. Reversely, the N-terminal domain blocked GNBP3-mediated serine protease cascade activation in vitro and also inhibited beta-1,3-glucan-mediated antimicrobial peptide induction in Tenebrio molitor larvae. These results suggest that the N-terminal GNBP homology domain of GNBP3 functions as a beta-1,3-glucan binding domain and the C-terminal domain of GNBP3 may be required for the recruitment of immediate down-stream serine protease zymogen during Toll signalling pathway activation.

  7. (-)-Epicatechin gallate (ECG) stimulates osteoblast differentiation via Runt-related transcription factor 2 (RUNX2) and transcriptional coactivator with PDZ-binding motif (TAZ)-mediated transcriptional activation.

    Science.gov (United States)

    Byun, Mi Ran; Sung, Mi Kyung; Kim, A Rum; Lee, Cham Han; Jang, Eun Jung; Jeong, Mi Gyeong; Noh, Minsoo; Hwang, Eun Sook; Hong, Jeong-Ho

    2014-04-01

    Osteoporosis is a degenerative bone disease characterized by low bone mass and is caused by an imbalance between osteoblastic bone formation and osteoclastic bone resorption. It is known that the bioactive compounds present in green tea increase osteogenic activity and decrease the risk of fracture by improving bone mineral density. However, the detailed mechanism underlying these beneficial effects has yet to be elucidated. In this study, we investigated the osteogenic effect of (-)-epicatechin gallate (ECG), a major bioactive compound found in green tea. We found that ECG effectively stimulates osteoblast differentiation, indicated by the increased expression of osteoblastic marker genes. Up-regulation of osteoblast marker genes is mediated by increased expression and interaction of the transcriptional coactivator with PDZ-binding motif (TAZ) and Runt-related transcription factor 2 (RUNX2). ECG facilitates nuclear localization of TAZ through PP1A. PP1A is essential for osteoblast differentiation because inhibition of PP1A activity was shown to suppress ECG-mediated osteogenic differentiation. Taken together, the results showed that ECG stimulates osteoblast differentiation through the activation of TAZ and RUNX2, revealing a novel mechanism for green tea-stimulated osteoblast differentiation.

  8. Cysteine S-Glutathionylation Promotes Stability and Activation of the Hippo Downstream Effector Transcriptional Co-activator with PDZ-binding Motif (TAZ)*

    Science.gov (United States)

    Gandhirajan, Rajesh Kumar; Jain, Manaswita; Walla, Benedikt; Johnsen, Marc; Bartram, Malte P.; Huynh Anh, Minh; Rinschen, Markus M.; Benzing, Thomas; Schermer, Bernhard

    2016-01-01

    Transcriptional co-activator with PDZ-binding motif (TAZ) and Yes-associated protein (YAP) are critical transcriptional co-activators downstream of the Hippo pathway involved in the regulation of organ size, tissue regeneration, proliferation, and apoptosis. Recent studies suggested common and distinct functions of TAZ and YAP and their diverse impact under several pathological conditions. Here we report differential regulation of TAZ and YAP in response to oxidative stress. H2O2 exposure leads to increased stability and activation of TAZ but not of YAP. H2O2 induces reversible S-glutathionylation at conserved cysteine residues within TAZ. We further demonstrate that TAZ S-glutathionylation is critical for reactive oxygen species (ROS)-mediated, TAZ-dependent TEA domain transcription factor (TEAD) trans-activation. Lysophosphatidic acid, a physiological activator of YAP and TAZ, induces ROS elevation and, subsequently, TAZ S-glutathionylation, which promotes TAZ-mediated target gene expression. TAZ expression is essential for renal homeostasis in mice, and we identify basal TAZ S-glutathionylation in murine kidney lysates, which is elevated during ischemia/reperfusion injury in vivo. This induced nuclear localization of TAZ and increased expression of connective tissue growth factor. These results describe a novel mechanism by which ROS sustains total cellular levels of TAZ. This preferential regulation suggests TAZ to be a redox sensor of the Hippo pathway. PMID:27048650

  9. DNA polymerases BI and D from the hyperthermophilic archaeon Pyrococcus furiosus both bind to proliferating cell nuclear antigen with their C-terminal PIP-box motifs.

    Science.gov (United States)

    Tori, Kazuo; Kimizu, Megumi; Ishino, Sonoko; Ishino, Yoshizumi

    2007-08-01

    Proliferating cell nuclear antigen (PCNA) is the sliding clamp that is essential for the high processivity of DNA synthesis during DNA replication. Pyrococcus furiosus, a hyperthermophilic archaeon, has at least two DNA polymerases, polymerase BI (PolBI) and PolD. Both of the two DNA polymerases interact with the archaeal P. furiosus PCNA (PfuPCNA) and perform processive DNA synthesis in vitro. This phenomenon, in addition to the fact that both enzymes display 3'-5' exonuclease activity, suggests that both DNA polymerases work in replication fork progression. We demonstrated here that both PolBI and PolD functionally interact with PfuPCNA at their C-terminal PIP boxes. The mutant PolBI and PolD enzymes lacking the PIP-box sequence do not respond to the PfuPCNA at all in an in vitro primer extension reaction. This is the first experimental evidence that the PIP-box motif, located at the C termini of the archaeal DNA polymerases, is actually critical for PCNA binding to form a processive DNA-synthesizing complex.

  10. Worldwide genetic features of HIV-1 Env α4β7 binding motif: the Local Dissemination Impact of the LDI tripeptide

    Science.gov (United States)

    Hait, Sabrina H.; Soares, Esmeralda A.; Sprinz, Eduardo; Arthos, James; Machado, Elizabeth S.; Soares, Marcelo A.

    2015-01-01

    Background HIV-1 gp120 binds to integrin α4β7, a homing receptor of lymphocytes to gut-associated lymphoid tissues. This interaction is mediated by the LDI/V tripeptide encoded in the V2-loop. This tripeptide mimics similar motifs in MAdCAM and VCAM, the natural ligands of α4β7. In this study we explored the association of V2-loop LDI/V mimotopes with transmission routes and patterns of disease progression in HIV-infected adult and pediatric patients. HIV-1 env sequences available in the Los Alamos HIV Sequence Database were included in the analyses. Methodology HIV-1 V2-loop sequences generated from infected adults and infants from South and Southeast Brazil, and also retrieved from the Los Alamos Database, were assessed for α4β7 binding tripeptide composition. Chi-Square/Fisher’s Exact test and Mann Whitney U-test were used for tripeptide comparisons. Shannon entropy was assessed for conservancy of the α4β7 tripeptide mimotope. Results We observed no association between the tripeptide composition or conservation and virus transmission route or disease progression. However, LDI was linked to successful epidemic dissemination of HIV-1 subtype C in South America, and further to other expanding non-B subtypes in Europe and Asia. In Africa, subtypes showing increased LDV prevalence evidenced an ongoing process of selection towards LDI expansion, an observation also extended to subtype B in the Americas and Western Europe. Conclusions The V2-loop LDI mimotope was conserved in HIV-1C from South America and other expanding subtypes across the globe, which suggests that LDI may promote successful dissemination of HIV at local geographic levels by means of increased transmission fitness. PMID:26569174

  11. N-termini of fungal CSL transcription factors are disordered, enriched in regulatory motifs and inhibit DNA binding in fission yeast.

    Directory of Open Access Journals (Sweden)

    Martin Převorovský

    Full Text Available BACKGROUND: CSL (CBF1/RBP-Jκ/Suppressor of Hairless/LAG-1 transcription factors are the effector components of the Notch receptor signalling pathway, which is critical for metazoan development. The metazoan CSL proteins (class M can also function in a Notch-independent manner. Recently, two novel classes of CSL proteins, designated F1 and F2, have been identified in fungi. The role of the fungal CSL proteins is unclear, because the Notch pathway is not present in fungi. In fission yeast, the Cbf11 and Cbf12 CSL paralogs play antagonistic roles in cell adhesion and the coordination of cell and nuclear division. Unusually long N-terminal extensions are typical for fungal and invertebrate CSL family members. In this study, we investigate the functional significance of these extended N-termini of CSL proteins. METHODOLOGY/PRINCIPAL FINDINGS: We identify 15 novel CSL family members from 7 fungal species and conduct bioinformatic analyses of a combined dataset containing 34 fungal and 11 metazoan CSL protein sequences. We show that the long, non-conserved N-terminal tails of fungal CSL proteins are likely disordered and enriched in phosphorylation sites and PEST motifs. In a case study of Cbf12 (class F2, we provide experimental evidence that the protein is proteolytically processed and that the N-terminus inhibits the Cbf12-dependent DNA binding activity in an electrophoretic mobility shift assay. CONCLUSIONS/SIGNIFICANCE: This study provides insight into the characteristics of the long N-terminal tails of fungal CSL proteins that may be crucial for controlling DNA-binding and CSL function. We propose that the regulation of DNA binding by Cbf12 via its N-terminal region represents an important means by which fission yeast strikes a balance between the class F1 and class F2 paralog activities. This mode of regulation might be shared with other CSL-positive fungi, some of which are relevant to human disease and biotechnology.

  12. Comprehensive discovery of DNA motifs in 349 human cells and tissues reveals new features of motifs.

    Science.gov (United States)

    Zheng, Yiyu; Li, Xiaoman; Hu, Haiyan

    2015-01-01

    Comprehensive motif discovery under experimental conditions is critical for the global understanding of gene regulation. To generate a nearly complete list of human DNA motifs under given conditions, we employed a novel approach to de novo discover significant co-occurring DNA motifs in 349 human DNase I hypersensitive site datasets. We predicted 845 to 1325 motifs in each dataset, for a total of 2684 non-redundant motifs. These 2684 motifs contained 54.02 to 75.95% of the known motifs in seven large collections including TRANSFAC. In each dataset, we also discovered 43 663 to 2 013 288 motif modules, groups of motifs with their binding sites co-occurring in a significant number of short DNA regions. Compared with known interacting transcription factors in eight resources, the predicted motif modules on average included 84.23% of known interacting motifs. We further showed new features of the predicted motifs, such as motifs enriched in proximal regions rarely overlapped with motifs enriched in distal regions, motifs enriched in 5' distal regions were often enriched in 3' distal regions, etc. Finally, we observed that the 2684 predicted motifs classified the cell or tissue types of the datasets with an accuracy of 81.29%. The resources generated in this study are available at http://server.cs.ucf.edu/predrem/.

  13. RNA-binding motif protein 5 inhibits the proliferation of cigarette smoke-transformed BEAS-2B cells through cell cycle arrest and apoptosis.

    Science.gov (United States)

    Lv, Xue-Jiao; Du, Yan-Wei; Hao, Yu-Qiu; Su, Zhen-Zhong; Zhang, Lin; Zhao, Li-Jing; Zhang, Jie

    2016-04-01

    Cigarette smoking has been shown to be the most significant risk factor for lung cancer. Recent studies have also indicated that RNA-binding motif protein 5 (RBM5) can modulate apoptosis and suppress tumor growth. The present study focused on the role of RBM5 in the regulation of cigarette smoke extract (CSE)-induced transformation of bronchial epithelial cells into the cancerous phenotype and its mechanism of action. Herein, we exposed normal BEAS-2B cells for 8 days to varying concentrations of CSE or dimethylsulfoxide (DMSO), followed by a recovery period of 2 weeks. Next, the RBM5 protein was overexpressed in these transformed BEAS-2B cells though lentiviral infection. Later, the morphological changes, cell proliferation, cell cycle, apoptosis, invasion and migration were assessed. In addition, we analyzed the role of RBM5 in xenograft growth. The expression of RBM5 along with the genes related to cell cycle regulation, apoptosis and invasion were also examined. Finally, our results revealed that BEAS-2B cells exposed to 100 µg/ml CSE acquired phenotypic changes and formed tumors in nude mice, indicative of their cancerous transformation and had reduced RBM5 expression. Subsequent overexpression of RBM5 in these cells significantly inhibited their proliferation, induced G1/S arrest, triggered apoptosis and inhibited their invasion and migration, including xenograft growth. Thus, we established an in vitro model of CSE-induced cancerous transformation and concluded that RBM5 overexpression inhibited the growth of these transformed cells through cell cycle arrest and induction of apoptosis. Therefore, our study suggests the importance of RBM5 in the pathogenesis of smoking-related cancer.

  14. Aberrant Assembly of RNA Recognition Motif 1 Links to Pathogenic Conversion of TAR DNA-binding Protein of 43 kDa (TDP-43)*

    Science.gov (United States)

    Shodai, Akemi; Morimura, Toshifumi; Ido, Akemi; Uchida, Tsukasa; Ayaki, Takashi; Takahashi, Rina; Kitazawa, Soichiro; Suzuki, Sakura; Shirouzu, Mikako; Kigawa, Takanori; Muto, Yutaka; Yokoyama, Shigeyuki; Takahashi, Ryosuke; Kitahara, Ryo; Ito, Hidefumi; Fujiwara, Noriko; Urushitani, Makoto

    2013-01-01

    Aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) is a pathological signature of amyotrophic lateral sclerosis (ALS). Although accumulating evidence suggests the involvement of RNA recognition motifs (RRMs) in TDP-43 proteinopathy, it remains unclear how native TDP-43 is converted to pathogenic forms. To elucidate the role of homeostasis of RRM1 structure in ALS pathogenesis, conformations of RRM1 under high pressure were monitored by NMR. We first found that RRM1 was prone to aggregation and had three regions showing stable chemical shifts during misfolding. Moreover, mass spectrometric analysis of aggregated RRM1 revealed that one of the regions was located on protease-resistant β-strands containing two cysteines (Cys-173 and Cys-175), indicating that this region served as a core assembly interface in RRM1 aggregation. Although a fraction of RRM1 aggregates comprised disulfide-bonded oligomers, the substitution of cysteine(s) to serine(s) (C/S) resulted in unexpected acceleration of amyloid fibrils of RRM1 and disulfide-independent aggregate formation of full-length TDP-43. Notably, TDP-43 aggregates with RRM1-C/S required the C terminus, and replicated cytopathologies of ALS, including mislocalization, impaired RNA splicing, ubiquitination, phosphorylation, and motor neuron toxicity. Furthermore, RRM1-C/S accentuated inclusions of familial ALS-linked TDP-43 mutants in the C terminus. The relevance of RRM1-C/S-induced TDP-43 aggregates in ALS pathogenesis was verified by immunolabeling of inclusions of ALS patients and cultured cells overexpressing the RRM1-C/S TDP-43 with antibody targeting misfolding-relevant regions. Our results indicate that cysteines in RRM1 crucially govern the conformation of TDP-43, and aberrant self-assembly of RRM1 at amyloidogenic regions contributes to pathogenic conversion of TDP-43 in ALS. PMID:23558684

  15. 2,6-Diiminopiperidin-1-ol: an overlooked motif relevant to uranyl and transition metal binding on poly(amidoxime) adsorbents

    Energy Technology Data Exchange (ETDEWEB)

    Kennedy, Zachary C.; Cardenas, Allan J.; Corbey, Jordan F.; Warner, Marvin G.

    2016-06-06

    Glutardiamidoxime, a structural motif on sorbents used in uranium extraction from seawater, was discovered to cyclize in situ at room temperature to 2,6-diimino-piperidin-1-ol in the presence of uranyl nitrate. The new diimino motif was also generated when exposed to competing transition metals Cu(II) and Ni(II). Multinuclear μ-O bridged U(VI), Cu(II), and Ni(II) complexes featuring bound diimino ligands were isolated. A Cu(II) complex with the historically relevant cyclic imide dioxime motif is also reported for structural comparison to the reported diimino complexes.

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

    Directory of Open Access Journals (Sweden)

    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.

  17. Parametric bootstrapping for biological sequence motifs.

    Science.gov (United States)

    O'Neill, Patrick K; Erill, Ivan

    2016-10-06

    Biological sequence motifs drive the specific interactions of proteins and nucleic acids. Accordingly, the effective computational discovery and analysis of such motifs is a central theme in bioinformatics. Many practical questions about the properties of motifs can be recast as random sampling problems. In this light, the task is to determine for a given motif whether a certain feature of interest is statistically unusual among relevantly similar alternatives. Despite the generality of this framework, its use has been frustrated by the difficulties of defining an appropriate reference class of motifs for comparison and of sampling from it effectively. We define two distributions over the space of all motifs of given dimension. The first is the maximum entropy distribution subject to mean information content, and the second is the truncated uniform distribution over all motifs having information content within a given interval. We derive exact sampling algorithms for each. As a proof of concept, we employ these sampling methods to analyze a broad collection of prokaryotic and eukaryotic transcription factor binding site motifs. In addition to positional information content, we consider the informational Gini coefficient of the motif, a measure of the degree to which information is evenly distributed throughout a motif's positions. We find that both prokaryotic and eukaryotic motifs tend to exhibit higher informational Gini coefficients (IGC) than would be expected by chance under either reference distribution. As a second application, we apply maximum entropy sampling to the motif p-value problem and use it to give elementary derivations of two new estimators. Despite the historical centrality of biological sequence motif analysis, this study constitutes to our knowledge the first use of principled null hypotheses for sequence motifs given information content. Through their use, we are able to characterize for the first time differerences in global motif statistics

  18. Growth and antioxidant response of Brassica rapa var. rapa L. (turnip) irrigated with different compositions of paper and board mill (PBM) effluent.

    Science.gov (United States)

    Iqbal, Shahid; Younas, Umer; Chan, Kim Wei; Saeed, Zohaib; Shaheen, Muhammad Ashraf; Akhtar, Naeem; Majeed, Abdul

    2013-05-01

    Current study presents the effect of irrigation with different compositions (0%, 20%, 40%, 60%, 80% and 100%) of PBM effluent on growth and antioxidant potential of Brassica rapa var. rapa L. plants. Seeds were exposed to different PBM effluent compositions, which resulted in significant decrease in their germination potential with elevated delay index. Significant changes in growth parameters (plant height, number of leaves and leaf area) were recorded for turnip plants at regular intervals (25, 50 and 75 d) as function of PBM effluent proportion. Response of biochemical and antioxidant constituents in different parts of turnip, against stress induced by PBM effluent, was assessed by estimating the contents of chlorophyll (a+b), carotenoids, protein, phenolics, flavonoids, ascorbic acid and malondialdehyde. Antioxidant activity was evaluated by measuring DPPH radical scavenging potential. The results of this study suggest that the impact of PBM effluent irrigation is dependent on concentration of effluent in irrigation mixture and is very clear on plant growth and antioxidant attributes. Maximum benefits were secured at 40% PBM effluent to irrigate turnip plants till maturity while higher concentrations were found useful for shorter period (25-50 d).

  19. Reference: TCA1MOTIF [PLACE

    Lifescience Database Archive (English)

    Full Text Available TCA1MOTIF Goldsbrough AP, Albrecht H, Stratford R Salicylic acid-inducible binding ...of a tobacco nuclear protein to a 10 bp sequence which is highly conserved amongst stress-inducible genes. Plant J 3:563-571 (1993) PubMed: 8220463; ...

  20. rMotifGen: random motif generator for DNA and protein sequences

    Directory of Open Access Journals (Sweden)

    Hardin C Timothy

    2007-08-01

    Full Text Available Abstract Background Detection of short, subtle conserved motif regions within a set of related DNA or amino acid sequences can lead to discoveries about important regulatory domains such as transcription factor and DNA binding sites as well as conserved protein domains. In order to help assess motif detection algorithms on motifs with varying properties and levels of conservation, we have developed a computational tool, rMotifGen, with the sole purpose of generating a number of random DNA or protein sequences containing short sequence motifs. Each motif consensus can be user-defined, randomly generated, or created from a position-specific scoring matrix (PSSM. Insertions and mutations within these motifs are created according to user-defined parameters and substitution matrices. The resulting sequences can be helpful in mutational simulations and in testing the limits of motif detection algorithms. Results Two implementations of rMotifGen have been created, one providing a graphical user interface (GUI for random motif construction, and the other serving as a command line interface. The second implementation has the added advantages of platform independence and being able to be called in a batch mode. rMotifGen was used to construct sample sets of sequences containing DNA motifs and amino acid motifs that were then tested against the Gibbs sampler and MEME packages. Conclusion rMotifGen provides an efficient and convenient method for creating random DNA or amino acid sequences with a variable number of motifs, where the instance of each motif can be incorporated using a position-specific scoring matrix (PSSM or by creating an instance mutated from its corresponding consensus using an evolutionary model based on substitution matrices. rMotifGen is freely available at: http://bioinformatics.louisville.edu/brg/rMotifGen/.

  1. A peptide motif from the second fibronectin module of the neural cell adhesion molecule, NCAM, NLIKQDDGGSPIRHY, is a binding site for the FGF receptor

    DEFF Research Database (Denmark)

    Jacobsen, Jacob Hedemand; Kiselyov, Vladislav; Bock, Elisabeth

    2008-01-01

    The mechanism of fibroblast growth factor receptor (FGFR) activation by the neural cell adhesion molecule (NCAM) is not well understood. A motif in the second NCAM fibronectin type III (FN3) module, termed FGL, has by means of nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) a...

  2. The DNA-binding domain of BenM reveals the structural basis for the recognition of a T-N11-A sequence motif by LysR-type transcriptional regulators.

    Science.gov (United States)

    Alanazi, Amer M; Neidle, Ellen L; Momany, Cory

    2013-10-01

    LysR-type transcriptional regulators (LTTRs) play critical roles in metabolism and constitute the largest family of bacterial regulators. To understand protein-DNA interactions, atomic structures of the DNA-binding domain and linker-helix regions of a prototypical LTTR, BenM, were determined by X-ray crystallography. BenM structures with and without bound DNA reveal a set of highly conserved amino acids that interact directly with DNA bases. At the N-terminal end of the recognition helix (α3) of a winged-helix-turn-helix DNA-binding motif, several residues create hydrophobic pockets (Pro30, Pro31 and Ser33). These pockets interact with the methyl groups of two thymines in the DNA-recognition motif and its complementary strand, T-N11-A. This motif usually includes some dyad symmetry, as exemplified by a sequence that binds two subunits of a BenM tetramer (ATAC-N7-GTAT). Gln29 forms hydrogen bonds to adenine in the first position of the recognition half-site (ATAC). Another hydrophobic pocket defined by Ala28, Pro30 and Pro31 interacts with the methyl group of thymine, complementary to the base at the third position of the half-site. Arg34 interacts with the complementary base of the 3' position. Arg53, in the wing, provides AT-tract recognition in the minor groove. For DNA recognition, LTTRs use highly conserved interactions between amino acids and nucleotide bases as well as numerous less-conserved secondary interactions.

  3. The calmodulin-binding, short linear motif, NSCaTE is conserved in L-type channel ancestors of vertebrate Cav1.2 and Cav1.3 channels.

    Directory of Open Access Journals (Sweden)

    Valentina Taiakina

    Full Text Available NSCaTE is a short linear motif of (xWxxx(I or Lxxxx, composed of residues with a high helix-forming propensity within a mostly disordered N-terminus that is conserved in L-type calcium channels from protostome invertebrates to humans. NSCaTE is an optional, lower affinity and calcium-sensitive binding site for calmodulin (CaM which competes for CaM binding with a more ancient, C-terminal IQ domain on L-type channels. CaM bound to N- and C- terminal tails serve as dual detectors to changing intracellular Ca(2+ concentrations, promoting calcium-dependent inactivation of L-type calcium channels. NSCaTE is absent in some arthropod species, and is also lacking in vertebrate L-type isoforms, Cav1.1 and Cav1.4 channels. The pervasiveness of a methionine just downstream from NSCaTE suggests that L-type channels could generate alternative N-termini lacking NSCaTE through the choice of translational start sites. Long N-terminus with an NSCaTE motif in L-type calcium channel homolog LCav1 from pond snail Lymnaea stagnalis has a faster calcium-dependent inactivation than a shortened N-termini lacking NSCaTE. NSCaTE effects are present in low concentrations of internal buffer (0.5 mM EGTA, but disappears in high buffer conditions (10 mM EGTA. Snail and mammalian NSCaTE have an alpha-helical propensity upon binding Ca(2+-CaM and can saturate both CaM N-terminal and C-terminal domains in the absence of a competing IQ motif. NSCaTE evolved in ancestors of the first animals with internal organs for promoting a more rapid, calcium-sensitive inactivation of L-type channels.

  4. Pengaruh Pembelajaran Berbasis Masalah (PBM terhadap Prestasi Belajar Fisika Dasar II Ditinjau dari Kerja Ilmiah dan Sikap Ilmiah Mahasiswa

    Directory of Open Access Journals (Sweden)

    Eddy Supramono

    2014-04-01

    Full Text Available Kuliah teori Fisika Dasar cenderung berupa informasi, dosen cenderung menuntaskan materi yang ada pada silabi, akibatnya adanya pemahaman yang keliru pada mahasiswa tentang pemecahan masalah dalam fisika. Penelitian ini bertujuan menguji perbedaan prestasi belajar mahasiswa pada penerapan model PBM ditinjau dari kerja ilmiah dan sikap ilmiah. Penelitian ini menggunakan jenis penelitian Quasy Experimentation (semi eksperimen dengan desain faktorial yang menggunakan dua faktor dan masing-masing faktor menggunakan dua katagori. Populasi penelitian yang digunakan adalah seluruh mahasiswa semester 2, angkatan 2011-2012 sebanyak 6 kelas. Sampel penelitian yangdiambil sebanyak 2 kelas yang setiap kelasnya terdiri 36 mahasiswa secara purposive sampling. Instrumen tes prestasi berupa soal pilihan ganda sebanyak 25 butir soal yang telah diuji validitasnya, menggunakan 87 mahasiswa angkatan 2010 dan 2011 sebagai responden. Reliabilitas instrumen dicari dengan menggunakan KR 20. Uji hipotesis menggunakan statistik Anava Dua Jalur. Hasil penelitianmenunjukkan adanya perbedaan prestasi kerja ilmiah tinggi kelas eksperimen dengan kontrol tetapi tidak signifikan. Ada perbedaan prestasi belajar mahasiswa kerja ilmiah rendah yang signifikan antara kelas eksperimen dengan konvensional. Tidak terdapat perbedaan prestasi belajar mahasiswa sikap ilmiah tinggi antara kelas eksperimen dengan konvensional dan juga tidak terdapat perbedaanprestasi belajar mahasiswa sikap ilmiah rendah antara kelas eksperimen dengan konvensional pada materi Optika-Geometrik.Kata kunci: PBM, prestasi, kerja ilmiah, sikap ilmiah

  5. Structure-Function Analysis of PPP1R3D, a Protein Phosphatase 1 Targeting Subunit, Reveals a Binding Motif for 14-3-3 Proteins which Regulates its Glycogenic Properties.

    Science.gov (United States)

    Rubio-Villena, Carla; Sanz, Pascual; Garcia-Gimeno, Maria Adelaida

    2015-01-01

    Protein phosphatase 1 (PP1) is one of the major protein phosphatases in eukaryotic cells. It plays a key role in regulating glycogen synthesis, by dephosphorylating crucial enzymes involved in glycogen homeostasis such as glycogen synthase (GS) and glycogen phosphorylase (GP). To play this role, PP1 binds to specific glycogen targeting subunits that, on one hand recognize the substrates to be dephosphorylated and on the other hand recruit PP1 to glycogen particles. In this work we have analyzed the functionality of the different protein binding domains of one of these glycogen targeting subunits, namely PPP1R3D (R6) and studied how binding properties of different domains affect its glycogenic properties. We have found that the PP1 binding domain of R6 comprises a conserved RVXF motif (R102VRF) located at the N-terminus of the protein. We have also identified a region located at the C-terminus of R6 (W267DNND) that is involved in binding to the PP1 glycogenic substrates. Our results indicate that although binding to PP1 and glycogenic substrates are independent processes, impairment of any of them results in lack of glycogenic activity of R6. In addition, we have characterized a novel site of regulation in R6 that is involved in binding to 14-3-3 proteins (RARS74LP). We present evidence indicating that when binding of R6 to 14-3-3 proteins is prevented, R6 displays hyper-glycogenic activity although is rapidly degraded by the lysosomal pathway. These results define binding to 14-3-3 proteins as an additional pathway in the control of the glycogenic properties of R6.

  6. Integrated microfluidic approach for quantitative high-throughput measurements of transcription factor binding affinities.

    Science.gov (United States)

    Glick, Yair; Orenstein, Yaron; Chen, Dana; Avrahami, Dorit; Zor, Tsaffrir; Shamir, Ron; Gerber, Doron

    2016-04-07

    Protein binding to DNA is a fundamental process in gene regulation. Methodologies such as ChIP-Seq and mapping of DNase I hypersensitive sites provide global information on this regulation in vivo In vitro methodologies provide valuable complementary information on protein-DNA specificities. However, current methods still do not measure absolute binding affinities. There is a real need for large-scale quantitative protein-DNA affinity measurements. We developed QPID, a microfluidic application for measuring protein-DNA affinities. A single run is equivalent to 4096 gel-shift experiments. Using QPID, we characterized the different affinities of ATF1, c-Jun, c-Fos and AP-1 to the CRE consensus motif and CRE half-site in two different genomic sequences on a single device. We discovered that binding of ATF1, but not of AP-1, to the CRE half-site is highly affected by its genomic context. This effect was highly correlated with ATF1 ChIP-seq and PBM experiments. Next, we characterized the affinities of ATF1 and ATF3 to 128 genomic CRE and CRE half-site sequences. Our affinity measurements explained that in vivo binding differences between ATF1 and ATF3 to CRE and CRE half-sites are partially mediated by differences in the minor groove width. We believe that QPID would become a central tool for quantitative characterization of biophysical aspects affecting protein-DNA binding.

  7. Prediction of a key role of motifs binding E2F and NR2F in down-regulation of numerous genes during the development of the mouse hippocampus

    Directory of Open Access Journals (Sweden)

    Kaminska Bozena

    2006-08-01

    Full Text Available Abstract Background We previously demonstrated that gene expression profiles during neuronal differentiation in vitro and hippocampal development in vivo were very similar, due to a conservation of the important second singular value decomposition (SVD mode (Mode 2 of expression. The conservation of Mode 2 suggests that it reflects a regulatory mechanism conserved between the two systems. In either dataset, the expression vectors of all the genes form two large clusters that differ in the sign of the contribution of Mode 2, which for the majority of them reflects the difference between down- or up-regulation. Results In the current work, we used a novel approach of analyzing cis-regulation of gene expression in a subspace of a single SVD mode of temporal expression profiles. In the putative upstream regulatory sequences identified by mouse-human homology for all the genes represented in either dataset, we searched for simple features (motifs and pairs of motifs associated with either sign of the loading of Mode 2. Using a cross-system training-test set approach, we identified E2F binding sites as predictors of down-regulation of gene expression during hippocampal development. NR2F binding sites, for the transcription factors Nr2f/COUP and Hnf4, and also NR2F_SP1 pairs of binding sites, were predictors of down-regulation of expression both during hippocampal development and neuronal differentiation. Analysis of another dataset, from gene profiling of myoblast differentiation in vitro, shows that the conservation of Mode 2 extends to the differentiation of mesenchymal cells. This permitted the identification of two more pairs of motifs, one of which included the CDE/CHR tandem element, as features associated with down-regulation both in the differentiating myoblasts and in the developing hippocampus. Of the features we identified, the E2F and CDE/CHR motifs may be associated with the cycling progenitor cell status, while NR2F may be related to the

  8. MSDmotif: exploring protein sites and motifs

    Directory of Open Access Journals (Sweden)

    Henrick Kim

    2008-07-01

    Full Text Available Abstract Background Protein structures have conserved features – motifs, which have a sufficient influence on the protein function. These motifs can be found in sequence as well as in 3D space. Understanding of these fragments is essential for 3D structure prediction, modelling and drug-design. The Protein Data Bank (PDB is the source of this information however present search tools have limited 3D options to integrate protein sequence with its 3D structure. Results We describe here a web application for querying the PDB for ligands, binding sites, small 3D structural and sequence motifs and the underlying database. Novel algorithms for chemical fragments, 3D motifs, ϕ/ψ sequences, super-secondary structure motifs and for small 3D structural motif associations searches are incorporated. The interface provides functionality for visualization, search criteria creation, sequence and 3D multiple alignment options. MSDmotif is an integrated system where a results page is also a search form. A set of motif statistics is available for analysis. This set includes molecule and motif binding statistics, distribution of motif sequences, occurrence of an amino-acid within a motif, correlation of amino-acids side-chain charges within a motif and Ramachandran plots for each residue. The binding statistics are presented in association with properties that include a ligand fragment library. Access is also provided through the distributed Annotation System (DAS protocol. An additional entry point facilitates XML requests with XML responses. Conclusion MSDmotif is unique by combining chemical, sequence and 3D data in a single search engine with a range of search and visualisation options. It provides multiple views of data found in the PDB archive for exploring protein structures.

  9. DNA vaccine encoding HPV-16 E7 with mutation in L-Y-C-Y-E pRb-binding motif induces potent anti-tumor responses in mice.

    Science.gov (United States)

    Bahrami, Armina Alagheband; Ghaemi, Amir; Tabarraei, Alijan; Sajadian, Azadeh; Gorji, Ali; Soleimanjahi, Hoorieh

    2014-09-01

    Cervical cancer is the second most common cancer among women worldwide and remains a clinical problem despite improvements in early detection and therapy. The human papillomavirus (HPV) type 16 (HPV16) E7 oncoprotein expressed in cervical carcinoma cells are considered as attractive tumor-specific antigen targets for immunotherapy. Since the transformation potential of the oncogenes, vaccination based of these oncogenes is not safe. In present study, DNA vaccine expressing the modified variant with mutation in pRb-binding motif of the HPV-16 E7 oncoprotein was generated. A novel modified E7 gene with mutation in LYCYE motif was designed and constructed and the immunogenicity and antitumor effect of therapeutic DNA vaccines encoding the mutant and wild type of E7 gene were investigated. The L-Y-C-Y-E pRb-binding motif of E7 proteins has been involved in the immortalization and transformation of the host cell. The results showed that the mutant and wild type HPV-16 E7 vectors expressed the desired protein. Furthermore, the immunological mechanism behind mutant E7 DNA vaccine can be attributed at least partially to increased cytotoxic T lymphocyte, accompanied by the up-regulation of Th1-cytokine IFN-γ and TNF-β and down-regulation of Th3-cytokine TGF-β. Immunized mice with mutant plasmid demonstrated significantly stronger cell immune responses and higher levels of tumor protection than wild-type E7 DNA vaccine. The results exhibit that modified E7 DNA vaccine may be a promising candidate for development of therapeutic vaccine against HPV-16 cancers.

  10. Binding of Substrates to the Central Pore of the Vps4 ATPase Is Autoinhibited by the Microtubule Interacting and Trafficking (MIT) Domain and Activated by MIT Interacting Motifs (MIMs)*

    Science.gov (United States)

    Han, Han; Monroe, Nicole; Votteler, Jörg; Shakya, Binita; Sundquist, Wesley I.; Hill, Christopher P.

    2015-01-01

    The endosomal sorting complexes required for transport (ESCRT) pathway drives reverse topology membrane fission events within multiple cellular pathways, including cytokinesis, multivesicular body biogenesis, repair of the plasma membrane, nuclear membrane vesicle formation, and HIV budding. The AAA ATPase Vps4 is recruited to membrane necks shortly before fission, where it catalyzes disassembly of the ESCRT-III lattice. The N-terminal Vps4 microtubule-interacting and trafficking (MIT) domains initially bind the C-terminal MIT-interacting motifs (MIMs) of ESCRT-III subunits, but it is unclear how the enzyme then remodels these substrates in response to ATP hydrolysis. Here, we report quantitative binding studies that demonstrate that residues from helix 5 of the Vps2p subunit of ESCRT-III bind to the central pore of an asymmetric Vps4p hexamer in a manner that is dependent upon the presence of flexible nucleotide analogs that can mimic multiple states in the ATP hydrolysis cycle. We also find that substrate engagement is autoinhibited by the Vps4p MIT domain and that this inhibition is relieved by binding of either Type 1 or Type 2 MIM elements, which bind the Vps4p MIT domain through different interfaces. These observations support the model that Vps4 substrates are initially recruited by an MIM-MIT interaction that activates the Vps4 central pore to engage substrates and generate force, thereby triggering ESCRT-III disassembly. PMID:25833946

  11. Binding of Substrates to the Central Pore of the Vps4 ATPase Is Autoinhibited by the Microtubule Interacting and Trafficking (MIT) Domain and Activated by MIT Interacting Motifs (MIMs).

    Science.gov (United States)

    Han, Han; Monroe, Nicole; Votteler, Jörg; Shakya, Binita; Sundquist, Wesley I; Hill, Christopher P

    2015-05-22

    The endosomal sorting complexes required for transport (ESCRT) pathway drives reverse topology membrane fission events within multiple cellular pathways, including cytokinesis, multivesicular body biogenesis, repair of the plasma membrane, nuclear membrane vesicle formation, and HIV budding. The AAA ATPase Vps4 is recruited to membrane necks shortly before fission, where it catalyzes disassembly of the ESCRT-III lattice. The N-terminal Vps4 microtubule-interacting and trafficking (MIT) domains initially bind the C-terminal MIT-interacting motifs (MIMs) of ESCRT-III subunits, but it is unclear how the enzyme then remodels these substrates in response to ATP hydrolysis. Here, we report quantitative binding studies that demonstrate that residues from helix 5 of the Vps2p subunit of ESCRT-III bind to the central pore of an asymmetric Vps4p hexamer in a manner that is dependent upon the presence of flexible nucleotide analogs that can mimic multiple states in the ATP hydrolysis cycle. We also find that substrate engagement is autoinhibited by the Vps4p MIT domain and that this inhibition is relieved by binding of either Type 1 or Type 2 MIM elements, which bind the Vps4p MIT domain through different interfaces. These observations support the model that Vps4 substrates are initially recruited by an MIM-MIT interaction that activates the Vps4 central pore to engage substrates and generate force, thereby triggering ESCRT-III disassembly. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. A discriminative approach for unsupervised clustering of DNA sequence motifs.

    Directory of Open Access Journals (Sweden)

    Philip Stegmaier

    Full Text Available Algorithmic comparison of DNA sequence motifs is a problem in bioinformatics that has received increased attention during the last years. Its main applications concern characterization of potentially novel motifs and clustering of a motif collection in order to remove redundancy. Despite growing interest in motif clustering, the question which motif clusters to aim at has so far not been systematically addressed. Here we analyzed motif similarities in a comprehensive set of vertebrate transcription factor classes. For this we developed enhanced similarity scores by inclusion of the information coverage (IC criterion, which evaluates the fraction of information an alignment covers in aligned motifs. A network-based method enabled us to identify motif clusters with high correspondence to DNA-binding domain phylogenies and prior experimental findings. Based on this analysis we derived a set of motif families representing distinct binding specificities. These motif families were used to train a classifier which was further integrated into a novel algorithm for unsupervised motif clustering. Application of the new algorithm demonstrated its superiority to previously published methods and its ability to reproduce entrained motif families. As a result, our work proposes a probabilistic approach to decide whether two motifs represent common or distinct binding specificities.

  13. Assessment of composite motif discovery methods

    Directory of Open Access Journals (Sweden)

    Johansen Jostein

    2008-02-01

    Full Text Available Abstract Background Computational discovery of regulatory elements is an important area of bioinformatics research and more than a hundred motif discovery methods have been published. Traditionally, most of these methods have addressed the problem of single motif discovery – discovering binding motifs for individual transcription factors. In higher organisms, however, transcription factors usually act in combination with nearby bound factors to induce specific regulatory behaviours. Hence, recent focus has shifted from single motifs to the discovery of sets of motifs bound by multiple cooperating transcription factors, so called composite motifs or cis-regulatory modules. Given the large number and diversity of methods available, independent assessment of methods becomes important. Although there have been several benchmark studies of single motif discovery, no similar studies have previously been conducted concerning composite motif discovery. Results We have developed a benchmarking framework for composite motif discovery and used it to evaluate the performance of eight published module discovery tools. Benchmark datasets were constructed based on real genomic sequences containing experimentally verified regulatory modules, and the module discovery programs were asked to predict both the locations of these modules and to specify the single motifs involved. To aid the programs in their search, we provided position weight matrices corresponding to the binding motifs of the transcription factors involved. In addition, selections of decoy matrices were mixed with the genuine matrices on one dataset to test the response of programs to varying levels of noise. Conclusion Although some of the methods tested tended to score somewhat better than others overall, there were still large variations between individual datasets and no single method performed consistently better than the rest in all situations. The variation in performance on individual

  14. Main: TCA1MOTIF [PLACE

    Lifescience Database Archive (English)

    Full Text Available TCA1MOTIF S000159 17-May-1998 (last modified) kehi TCA-1 (tobacco nuclear protein 1...) binding site; Related to salicylic acid-inducible expression of many genes; Found in barley beta-1,3-gluca...nase and over 30 different plant genes which are known to be induced by one or more forms of stress; A similar sequence (TCA... et al., 1997); SA; salicylic acid; stress; TCA-1; barley (Hordeum vulgare); tobacco (Nicotiana tabacum); TCATCTTCTT ...

  15. The lysin motif receptor-like kinase (LysM-RLK) CERK1 is a major chitin-binding protein in Arabidopsis thaliana and subject to chitin-induced phosphorylation.

    Science.gov (United States)

    Petutschnig, Elena K; Jones, Alexandra M E; Serazetdinova, Liliya; Lipka, Ulrike; Lipka, Volker

    2010-09-10

    Plants detect potential pathogens by sensing microbe-associated molecular patterns via pattern recognition receptors. In the dicot model plant Arabidopsis, the lysin motif (LysM)-containing chitin elicitor receptor kinase 1 (CERK1) has been shown to be essential for perception of the fungal cell wall component chitin and for resistance to fungal pathogens. Recent in vitro studies with CERK1 protein expressed heterologously in yeast suggested direct chitin binding activity. Here we show in an affinity purification approach that CERK1 is a major chitin-binding protein of Arabidopsis cells, along with several known and putative chitinases. The ectodomain of CERK1 harbors three distinct LysM domains with potential ligand binding capacity. We demonstrate that the CERK1 ectodomain binds chitin and partially deacetylated chitosan directly without any requirement for interacting proteins and that all three LysM domains are necessary for chitin binding. Ligand-induced phosphorylation events are a general feature of animal and plant signal transduction pathways. Our studies show that chitin, chitin oligomers, and chitosan rapidly induce in vivo phosphorylation of CERK1 at multiple residues in the juxtamembrane and kinase domain. Functional analyses with a kinase dead variant provide evidence that kinase activity of CERK1 is required for its chitin-dependent in vivo phosphorylation, as well as for early defense responses and downstream signaling. Collectively, our data suggest that in Arabidopsis, CERK1 is a major chitin, chitosan, and chito-oligomer binding component and that chitin signaling depends on CERK1 post-translational modification and kinase activity.

  16. The structure of Prp40 FF1 domain and its interaction with the crn-TPR1 motif of Clf1 gives a new insight into the binding mode of FF domains.

    Science.gov (United States)

    Gasch, Alexander; Wiesner, Silke; Martin-Malpartida, Pau; Ramirez-Espain, Ximena; Ruiz, Lidia; Macias, Maria J

    2006-01-06

    The yeast splicing factor Prp40 (pre-mRNA processing protein 40) consists of a pair of WW domains followed by several FF domains. The region comprising the FF domains has been shown to associate with the 5' end of U1 small nuclear RNA and to interact directly with two proteins, the Clf1 (Crooked neck-like factor 1) and the phosphorylated repeats of the C-terminal domain of RNA polymerase II (CTD-RNAPII). In this work we reported the solution structure of the first FF domain of Prp40 and the identification of a novel ligand-binding site in FF domains. By using chemical shift assays, we found a binding site for the N-terminal crooked neck tetratricopeptide repeat of Clf1 that is distinct and structurally separate from the previously identified CTD-RNAPII binding pocket of the FBP11 (formin-binding protein 11) FF1 domain. No interaction, however, was observed between the Prp40 FF1 domain and three different peptides derived from the CTD-RNAPII protein. Indeed, the equivalent CTD-RNAPII-binding site in the Prp40 FF1 domain is predominantly negatively charged and thus unfavorable for an interaction with phosphorylated peptide sequences. Sequence alignments and phylogenetic tree reconstructions using the FF domains of three functionally related proteins, Prp40, FBP11, and CA150, revealed that Prp40 and FBP11 are not orthologous proteins and supported the different ligand specificities shown by their respective FF1 domains. Our results also revealed that not all FF domains in Prp40 are functionally equivalent. We proposed that at least two different interaction surfaces exist in FF domains that have evolved to recognize distinct binding motifs.

  17. The btp [2,6-bis(1,2,3-triazol-4-yl)pyridine] binding motif: a new versatile terdentate ligand for supramolecular and coordination chemistry.

    Science.gov (United States)

    Byrne, Joseph P; Kitchen, Jonathan A; Gunnlaugsson, Thorfinnur

    2014-08-07

    Ligands containing the btp [2,6-bis(1,2,3-triazol-4-yl)pyridine] motif have appeared with increasing regularity over the last decade. This class of ligands, formed in a one pot ‘click’ reaction, has been studied for various purposes, such as for generating d and f metal coordination complexes and supramolecular self-assemblies, and in the formation of dendritic and polymeric networks, etc. This review article introduces btp as a novel and highly versatile terdentate building block with huge potential in inorganic supramolecular chemistry. We will focus on the coordination chemistry of btp ligands with a wide range of metals, and how it compares with other classical pyridyl and polypyridyl based ligands, and then present a selection of applications including use in catalysis, enzyme inhibition, photochemistry, molecular logic and materials, e.g. polymers, dendrimers and gels. The photovoltaic potential of triazolium derivatives of btp and its interactions with anions will also be discussed.

  18. Binding of S-methyl-5'-thioadenosine and S-adenosyl-L-methionine to protein MJ0100 triggers an open-to-closed conformational change in its CBS motif pair.

    Science.gov (United States)

    Lucas, María; Encinar, José Antonio; Arribas, Egoitz Astigarraga; Oyenarte, Iker; García, Inmaculada Gómez; Kortazar, Danel; Fernández, José A; Mato, Jose M; Martínez-Chantar, María Luz; Martínez-Cruz, Luis Alfonso

    2010-02-26

    Cystathionine beta-synthase (CBS) domains are small motifs that are present in proteins with completely different functions. Several genetic diseases in humans have been associated with mutations in their sequence, which has made them promising targets for rational drug design. The protein MJ0100 from Methanocaldococcus jannaschii includes a DUF39 domain of so far unknown function and a CBS domain pair (Bateman domain) at its C-terminus. This work presents the crystallographic analysis of four different states of the CBS motif pair of MJ0100 in complex with different numbers of S-adenosyl-L-methionine (SAM) and S-methyl-5'-thioadenosine (MTA) ligands, providing evidence that ligand-induced conformational reorganization of Bateman domain dimers could be an important regulatory mechanism. These observations are in contrast to what is known from most of the other Bateman domain structures but are supported by recent studies on the magnesium transporter MgtE. Our structures represent the first example of a CBS domain protein complexed with SAM and/or MTA and might provide a structural basis for understanding the molecular mechanisms regulated by SAM upon binding to the C-terminal domain of human CBS, whose structure remains unknown. Copyright (c) 2009. Elsevier Ltd. All rights reserved.

  19. Unsupervised statistical discovery of spaced motifs in prokaryotic genomes.

    Science.gov (United States)

    Tong, Hao; Schliekelman, Paul; Mrázek, Jan

    2017-01-05

    DNA sequences contain repetitive motifs which have various functions in the physiology of the organism. A number of methods have been developed for discovery of such sequence motifs with a primary focus on detection of regulatory motifs and particularly transcription factor binding sites. Most motif-finding methods apply probabilistic models to detect motifs characterized by unusually high number of copies of the motif in the analyzed sequences. We present a novel method for detection of pairs of motifs separated by spacers of variable nucleotide sequence but conserved length. Unlike existing methods for motif discovery, the motifs themselves are not required to occur at unusually high frequency but only to exhibit a significant preference to occur at a specific distance from each other. In the present implementation of the method, motifs are represented by pentamers and all pairs of pentamers are evaluated for statistically significant preference for a specific distance. An important step of the algorithm eliminates motif pairs where the spacers separating the two motifs exhibit a high degree of sequence similarity; such motif pairs likely arise from duplications of the whole segment including the motifs and the spacer rather than due to selective constraints indicative of a functional importance of the motif pair. The method was used to scan 569 complete prokaryotic genomes for novel sequence motifs. Some motifs detected were previously known but other motifs found in the search appear to be novel. Selected motif pairs were subjected to further investigation and in some cases their possible biological functions were proposed. We present a new motif-finding technique that is applicable to scanning complete genomes for sequence motifs. The results from analysis of 569 genomes suggest that the method detects previously known motifs that are expected to be found as well as new motifs that are unlikely to be discovered by traditional motif-finding methods. We conclude

  20. Toxin inhibition in C. crescentus VapBC1 is mediated by a flexible pseudo-palindromic protein motif and modulated by DNA binding

    DEFF Research Database (Denmark)

    Bendtsen, Kirstine L; Xu, Kehan; Luckmann, Majbritt

    2017-01-01

    for binding and inactivation of the VapC1 toxin dimer. Sequence analysis of 4127 orthologous VapB sequences reveals that such palindromic protein sequences are widespread and unique to bacterial and archaeal VapB antitoxins suggesting a general principle governing regulation of VapBC TA systems. Finally......Expression of bacterial type II toxin-antitoxin (TA) systems is regulated at the transcriptional level through direct binding of the antitoxin to pseudo-palindromic sequences on operator DNA. In this context, the toxin functions as a co-repressor by stimulating DNA binding through direct...... architectural rearrangement of conserved TA interactions in which C-terminal extended structures of the antitoxin VapB1 swap positions to interlock the complex in the DNA-bound state. We further show that a pseudo-palindromic protein sequence in the antitoxin is responsible for this interaction and required...

  1. Bacteria binding by DMBT1/SAG/gp-340 is confined to the VEVLXXXXW motif in its scavenger receptor cysteine-rich domains

    DEFF Research Database (Denmark)

    Bikker, Floris J; Ligtenberg, Antoon J M; End, Caroline

    2004-01-01

    for group B. The protein DMBT1 (deleted in malignant brain tumors 1), which is identical to salivary agglutinin and lung gp-340, belongs to the group B SRCR proteins and is considered to be involved in tumor suppression and host defense by pathogen binding. In a previous study we used nonoverlapping......The scavenger receptor cysteine-rich (SRCR) proteins form an archaic group of metazoan proteins characterized by the presence of SRCR domains. These proteins are classified in group A and B based on the number of conserved cysteine residues in their SRCR domains, i.e. six for group A and eight...... synthetic peptides covering the SRCR consensus sequence to identify a 16-amino acid bacteria-binding protein loop (peptide SRCRP2; QGRVEVLYRGSWGTVC) within the SRCR domains. In this study, using overlapping peptides, we pinpointed the minimal bacteria-binding site on SRCRP2, and thus DMBT1, to an 11-amino...

  2. Seq2Logo: a method for construction and visualization of amino acid binding motifs and sequence profiles including sequence weighting, pseudo counts and two-sided representation of amino acid enrichment and depletion

    DEFF Research Database (Denmark)

    Thomsen, Martin Christen Frølund; Nielsen, Morten

    2012-01-01

    Seq2Logo is a web-based sequence logo generator. Sequence logos are a graphical representation of the information content stored in a multiple sequence alignment (MSA) and provide a compact and highly intuitive representation of the position-specific amino acid composition of binding motifs, active...... sites, etc. in biological sequences. Accurate generation of sequence logos is often compromised by sequence redundancy and low number of observations. Moreover, most methods available for sequence logo generation focus on displaying the position-specific enrichment of amino acids, discarding the equally...... valuable information related to amino acid depletion. Seq2logo aims at resolving these issues allowing the user to include sequence weighting to correct for data redundancy, pseudo counts to correct for low number of observations and different logotype representations each capturing different aspects...

  3. Mapping of Potent and Specific Binding Motifs, GLOGEN and GVOGEA, for Integrin α1β1 Using Collagen Toolkits II and III*

    OpenAIRE

    Hamaia, Samir W.; Pugh, Nicholas; Raynal, Nicolas; Némoz, Benjamin; Stone, Rachael; Gullberg, Donald; Bihan, Dominique; Farndale, Richard W.

    2012-01-01

    Integrins are well characterized cell surface receptors for extracellular matrix proteins. Mapping integrin-binding sites within the fibrillar collagens identified GFOGER as a high affinity site recognized by α2β1, but with lower affinity for α1β1. Here, to identify specific ligands for α1β1, we examined binding of the recombinant human α1 I domain, the rat pheochromocytoma cell line (PC12), and the rat glioma Rugli cell line to our collagen Toolkit II and III peptides using solid-phase and r...

  4. PARP-1 expression in the mouse is controlled by an autoregulatory loop: PARP-1 binding to an upstream S/MAR element and to a novel recognition motif in its promoter suppresses transcription.

    Science.gov (United States)

    Vidaković, Melita; Gluch, Angela; Qiao, Junhua; Oumard, Andrè; Frisch, Matthias; Poznanović, Goran; Bode, Juergen

    2009-05-15

    This work identifies central components of a feedback mechanism for the expression of mouse poly(ADP-ribose) polymerase-1 (PARP-1). Using the stress-induced duplex destabilization algorithm, multiple base-unpairing regions (BURs) could be localized in the 5' region of the mouse PARP-1 gene (muPARP-1). Some of these could be identified as scaffold/matrix-attachment regions (S/MARs), suggesting an S/MAR-mediated transcriptional regulation. PARP-1 binding to the most proximal element, S/MAR 1, and to three consensus motifs, AGGCC, in its own promoter (basepairs -956 to +100), could be traced by electrophoretic mobility-shift assay. The AGGCC-complementary GGCCT motif was detected by cis-diammine-dichloro platinum cross-linking and functionally characterized by the effects of site-directed mutagenesis on its performance in wild type (PARP-1(+/+)) and PARP-1 knockout cells (PARP-1(-/-)). Mutation of the central AGGCC tract at basepairs -554 to -550 prevented PARP-1/promoter interactions, whereby muPARP-1 expression became up-regulated. Transfection of a series of reporter gene constructs with or without S/MAR 1 (basepairs -1523 to -1007) and the more distant S/MAR 2 (basepairs -8373 to -6880), into PARP-1(+/+) as well as PARP-1(-/-) cells, revealed an additional, major level of muPARP-1 promoter down-regulation, triggered by PARP-1 binding to S/MAR 1. We conclude that S/MAR 1 represents an upstream control element that acts in conjunction with the muPARP-1 promoter. These interactions are part of a negative autoregulatory loop.

  5. MEME SUITE: tools for motif discovery and searching.

    Science.gov (United States)

    Bailey, Timothy L; Boden, Mikael; Buske, Fabian A; Frith, Martin; Grant, Charles E; Clementi, Luca; Ren, Jingyuan; Li, Wilfred W; Noble, William S

    2009-07-01

    The MEME Suite web server provides a unified portal for online discovery and analysis of sequence motifs representing features such as DNA binding sites and protein interaction domains. The popular MEME motif discovery algorithm is now complemented by the GLAM2 algorithm which allows discovery of motifs containing gaps. Three sequence scanning algorithms--MAST, FIMO and GLAM2SCAN--allow scanning numerous DNA and protein sequence databases for motifs discovered by MEME and GLAM2. Transcription factor motifs (including those discovered using MEME) can be compared with motifs in many popular motif databases using the motif database scanning algorithm TOMTOM. Transcription factor motifs can be further analyzed for putative function by association with Gene Ontology (GO) terms using the motif-GO term association tool GOMO. MEME output now contains sequence LOGOS for each discovered motif, as well as buttons to allow motifs to be conveniently submitted to the sequence and motif database scanning algorithms (MAST, FIMO and TOMTOM), or to GOMO, for further analysis. GLAM2 output similarly contains buttons for further analysis using GLAM2SCAN and for rerunning GLAM2 with different parameters. All of the motif-based tools are now implemented as web services via Opal. Source code, binaries and a web server are freely available for noncommercial use at http://meme.nbcr.net.

  6. Structural Biology of The sequestration & Transport of Heavy Metal Toxins: NMR Structure Determination of Proteins Containing the CYS-X-Y-Metal Binding Motif

    Energy Technology Data Exchange (ETDEWEB)

    Stanley J. Opella

    2004-03-10

    The support from the Department of Energy enabled us to initiate research on several proteins from the bacterial mercury detoxification system; in particular, we were able to determine the structures of MerP and related metal binding sequences. We have also worked on the membrane transport proteins MerF and MerT.

  7. Regulation and function of the CD3¿ DxxxLL motif: a binding site for adaptor protein-1 and adaptor protein-2 in vitro

    DEFF Research Database (Denmark)

    Dietrich, J; Kastrup, J; Nielsen, B L

    1997-01-01

    Several receptors are downregulated by internalization after ligand binding. Regulation of T cell receptor (TCR) expression is an important step in T cell activation, desensitization, and tolerance induction. One way T cells regulate TCR expression is by phosphorylation/dephosphorylation of the TCR...

  8. Computational analysis of a novel mutation in ETFDH gene highlights its long-range effects on the FAD-binding motif

    Directory of Open Access Journals (Sweden)

    Chang Jan-Gowth

    2011-10-01

    Full Text Available Abstract Background Multiple acyl-coenzyme A dehydrogenase deficiency (MADD is an autosomal recessive disease caused by the defects in the mitochondrial electron transfer system and the metabolism of fatty acids. Recently, mutations in electron transfer flavoprotein dehydrogenase (ETFDH gene, encoding electron transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO have been reported to be the major causes of riboflavin-responsive MADD. To date, no studies have been performed to explore the functional impact of these mutations or their mechanism of disrupting enzyme activity. Results High resolution melting (HRM analysis and sequencing of the entire ETFDH gene revealed a novel mutation (p.Phe128Ser and the hotspot mutation (p.Ala84Thr from a patient with MADD. According to the predicted 3D structure of ETF:QO, the two mutations are located within the flavin adenine dinucleotide (FAD binding domain; however, the two residues do not have direct interactions with the FAD ligand. Using molecular dynamics (MD simulations and normal mode analysis (NMA, we found that the p.Ala84Thr and p.Phe128Ser mutations are most likely to alter the protein structure near the FAD binding site as well as disrupt the stability of the FAD binding required for the activation of ETF:QO. Intriguingly, NMA revealed that several reported disease-causing mutations in the ETF:QO protein show highly correlated motions with the FAD-binding site. Conclusions Based on the present findings, we conclude that the changes made to the amino acids in ETF:QO are likely to influence the FAD-binding stability.

  9. Mapping of potent and specific binding motifs, GLOGEN and GVOGEA, for integrin α1β1 using collagen toolkits II and III.

    Science.gov (United States)

    Hamaia, Samir W; Pugh, Nicholas; Raynal, Nicolas; Némoz, Benjamin; Stone, Rachael; Gullberg, Donald; Bihan, Dominique; Farndale, Richard W

    2012-07-27

    Integrins are well characterized cell surface receptors for extracellular matrix proteins. Mapping integrin-binding sites within the fibrillar collagens identified GFOGER as a high affinity site recognized by α2β1, but with lower affinity for α1β1. Here, to identify specific ligands for α1β1, we examined binding of the recombinant human α1 I domain, the rat pheochromocytoma cell line (PC12), and the rat glioma Rugli cell line to our collagen Toolkit II and III peptides using solid-phase and real-time label-free adhesion assays. We observed Mg(2+)-dependent binding of the α1 I domain to the peptides in the following rank order: III-7 (GLOGEN), II-28 (GFOGER), II-7 and II-8 (GLOGER), II-18 (GAOGER), III-4 (GROGER). PC12 cells showed a similar profile. Using antibody blockade, we confirmed that binding of PC12 cells to peptide III-7 was mediated by integrin α1β1. We also identified a new α1β1-binding activity within peptide II-27. The sequence GVOGEA bound weakly to PC12 cells and strongly to activated Rugli cells or to an activated α1 I domain, but not to the α2 I domain or to C2C12 cells expressing α2β1 or α11β1. Thus, GVOGEA is specific for α1β1. Although recognized by both α2β1 and α11β1, GLOGEN is a better ligand for α1β1 compared with GFOGER. Finally, using biosensor assays, we show that although GLOGEN is able to compete for the α1 I domain from collagen IV (IC(50) ∼3 μm), GFOGER is much less potent (IC(50) ∼90 μm), as shown previously. These data confirm the selectivity of GFOGER for α2β1 and establish GLOGEN as a high affinity site for α1β1.

  10. AISMOTIF-An Artificial Immune System for DNA Motif Discovery

    CERN Document Server

    Seeja, K R

    2011-01-01

    Discovery of transcription factor binding sites is a much explored and still exploring area of research in functional genomics. Many computational tools have been developed for finding motifs and each of them has their own advantages as well as disadvantages. Most of these algorithms need prior knowledge about the data to construct background models. However there is not a single technique that can be considered as best for finding regulatory motifs. This paper proposes an artificial immune system based algorithm for finding the transcription factor binding sites or motifs and two new weighted scores for motif evaluation. The algorithm is enumerative, but sufficient pruning of the pattern search space has been incorporated using immune system concepts. The performance of AISMOTIF has been evaluated by comparing it with eight state of art composite motif discovery algorithms and found that AISMOTIF predicts known motifs as well as new motifs from the benchmark dataset without any prior knowledge about the data...

  11. Human Cannabinoid Receptor 2 Ligand-Interaction Motif: Transmembrane Helix 2 Cysteine, C2.59(89), as Determinant of Classical Cannabinoid Agonist Activity and Binding Pose.

    Science.gov (United States)

    Zhou, Han; Peng, Yan; Halikhedkar, Aneetha; Fan, Pusheng; Janero, David R; Thakur, Ganesh A; Mercier, Richard W; Sun, Xin; Ma, Xiaoyu; Makriyannis, Alexandros

    2017-06-21

    Cannabinoid receptor 2 (CB2R)-dependent signaling is implicated in neuronal physiology and immune surveillance by brain microglia. Selective CB2R agonists hold therapeutic promise for inflammatory and other neurological disorders. Information on human CB2R (hCB2R) ligand-binding and functional domains is needed to inform the rational design and optimization of candidate druglike hCB2R agonists. Prior demonstration that hCB2R transmembrane helix 2 (TMH2) cysteine C2.59(89) reacts with small-molecule methanethiosulfonates showed that this cysteine residue is accessible to sulfhydryl derivatization reagents. We now report the design and application of two novel, pharmacologically active, high-affinity molecular probes, AM4073 and AM4099, as chemical reporters to interrogate directly the interaction of classical cannabinoid agonists with hCB2R cysteine residues. AM4073 has one electrophilic isothiocyanate (NCS) functionality at the C9 position of its cyclohexenyl C-ring, whereas AM4099 has NCS groups at that position and at the terminus of its aromatic A-ring C3 side chain. Pretreatment of wild-type hCB2R with either probe reduced subsequent [(3)H]CP55,940 specific binding by ∼60%. Conservative serine substitution of any hCB2R TMH cysteine residue except C2.59(89) did not affect the reduction of [(3)H]CP55,940 specific binding by either probe, suggesting that AM4073 and AM4099 interact irreversibly with this TMH2 cysteine. In contrast, AM841, an exceptionally potent hCB2R megagonist and direct AM4073/4099 congener bearing a single electrophilic NCS group at the terminus of its C3 side chain, had been demonstrated to bind covalently to TMH6 cysteine C6.47(257) and not C2.59(89). Molecular modeling indicates that the AM4073-hCB2R* interaction at C2.59(89) orients this classical cannabinoid away from TMH6 and toward the TMH2-TMH3 interface in the receptor's hydrophobic binding pocket, whereas the AM841-hCB2R* interaction at C6.47(257) favors agonist orientation toward

  12. Schistosoma mansoni venom allergen-like protein 4 (SmVAL4) is a novel lipid-binding SCP/TAPS protein that lacks the prototypical CAP motifs

    Energy Technology Data Exchange (ETDEWEB)

    Kelleher, Alan [Baylor College of Medicine, Houston, TX 77030 (United States); Darwiche, Rabih [University of Fribourg, Chemin du Musée 10, CH 1700 Fribourg (Switzerland); Rezende, Wanderson C. [Baylor College of Medicine, Houston, TX 77030 (United States); Farias, Leonardo P.; Leite, Luciana C. C. [Instituto Butantan, São Paulo, SP (Brazil); Schneiter, Roger [University of Fribourg, Chemin du Musée 10, CH 1700 Fribourg (Switzerland); Asojo, Oluwatoyin A., E-mail: asojo@bcm.edu [Baylor College of Medicine, Houston, TX 77030 (United States)

    2014-08-01

    The first structure of an S. mansoni venom allergen-like protein is presented. Schistosomiasis is a parasitic disease that affects over 200 million people. Vaccine candidates have been identified, including Schistosoma mansoni venom allergen-like proteins (SmVALs) from the SCP/TAPS (sperm-coating protein/Tpx/antigen 5/pathogenesis related-1/Sc7) superfamily. The first SmVAL structure, SmVAL4, was refined to a resolution limit of 2.16 Å. SmVAL4 has a unique structure that could not be predicted from homologous structures, with longer loops and an unusual C-terminal extension. SmVAL4 has the characteristic α/β-sandwich and central SCP/TAPS cavity. Furthermore, SmVAL4 has only one of the signature CAP cavity tetrad amino-acid residues and is missing the histidines that coordinate divalent cations such as Zn{sup 2+} in other SCP/TAPS proteins. SmVAL4 has a cavity between α-helices 1 and 4 that was observed to bind lipids in tablysin-15, suggesting the ability to bind lipids. Subsequently, SmVAL4 was shown to bind cholesterol in vitro. Additionally, SmVAL4 was shown to complement the in vivo sterol-export phenotype of yeast mutants lacking their endogenous CAP proteins. Expression of SmVAL4 in yeast cells lacking endogenous CAP function restores the block in sterol export. These studies suggest an evolutionarily conserved lipid-binding function shared by CAP proteins such as SmVAL4 and yeast CAP proteins such as Pry1.

  13. Toxin inhibition in C. crescentus VapBC1 is mediated by a flexible pseudo-palindromic protein motif and modulated by DNA binding.

    Science.gov (United States)

    Bendtsen, Kirstine L; Xu, Kehan; Luckmann, Majbritt; Winther, Kristoffer S; Shah, Shiraz A; Pedersen, Christian N S; Brodersen, Ditlev E

    2017-03-17

    Expression of bacterial type II toxin-antitoxin (TA) systems is regulated at the transcriptional level through direct binding of the antitoxin to pseudo-palindromic sequences on operator DNA. In this context, the toxin functions as a co-repressor by stimulating DNA binding through direct interaction with the antitoxin. Here, we determine crystal structures of the complete 90 kDa heterooctameric VapBC1 complex from Caulobacter crescentus CB15 both in isolation and bound to its cognate DNA operator sequence at 1.6 and 2.7 Å resolution, respectively. DNA binding is associated with a dramatic architectural rearrangement of conserved TA interactions in which C-terminal extended structures of the antitoxin VapB1 swap positions to interlock the complex in the DNA-bound state. We further show that a pseudo-palindromic protein sequence in the antitoxin is responsible for this interaction and required for binding and inactivation of the VapC1 toxin dimer. Sequence analysis of 4127 orthologous VapB sequences reveals that such palindromic protein sequences are widespread and unique to bacterial and archaeal VapB antitoxins suggesting a general principle governing regulation of VapBC TA systems. Finally, a structure of C-terminally truncated VapB1 bound to VapC1 reveals discrete states of the TA interaction that suggest a structural basis for toxin activation in vivo. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. WW domains of the yes-kinase-associated-protein (YAP transcriptional regulator behave as independent units with different binding preferences for PPxY motif-containing ligands.

    Directory of Open Access Journals (Sweden)

    Manuel Iglesias-Bexiga

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

  15. WW domains of the yes-kinase-associated-protein (YAP) transcriptional regulator behave as independent units with different binding preferences for PPxY motif-containing ligands.

    Science.gov (United States)

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

    2015-01-01

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

  16. Crystal Structures of Two Novel Dye-Decolorizing Peroxidases Reveal a Beta-Bar Fold With a Conserved Heme-Binding Motif

    Energy Technology Data Exchange (ETDEWEB)

    Zubieta, C.; Krishna, S.S.; Kapoor, M.; Kozbial, P.; McMullan, D.; Axelrod, H.L.; Miller, M.D.; Abdubek, P.; Ambing, E.; Astakhova, T.; Carlton, D.; Chiu, H.J.; Clayton, T.; Deller, M.C.; Duan, L.; Elsliger, M.A.; Feuerhelm, J.; Grzechnik, S.K.; Hale, J.; Hampton, E.; Han, G.W.; /JCSG /SLAC, SSRL /Burnham Inst. Med. Res. /UC, San Diego /Scripps Res. Inst. /Novartis Res. Found.

    2007-10-31

    BtDyP from Bacteroides thetaiotaomicron (strain VPI-5482) and TyrA from Shewanella oneidensis are dye-decolorizing peroxidases (DyPs), members of a new family of heme-dependent peroxidases recently identified in fungi and bacteria. Here, we report the crystal structures of BtDyP and TyrA at 1.6 and 2.7 Angstroms, respectively. BtDyP assembles into a hexamer, while TyrA assembles into a dimer; the dimerization interface is conserved between the two proteins. Each monomer exhibits a two-domain, {alpha}+{beta} ferredoxin-like fold. A site for heme binding was identified computationally, and modeling of a heme into the proposed active site allowed for identification of residues likely to be functionally important. Structural and sequence comparisons with other DyPs demonstrate a conservation of putative heme-binding residues, including an absolutely conserved histidine. Isothermal titration calorimetry experiments confirm heme binding, but with a stoichiometry of 0.3:1 (heme:protein).

  17. Structure of L-Xylulose-5-Phosphate 3-Epimerase (UlaE) from the Anaerobic L-Ascorbate Utilization Pathway of Escherichia coli: Identification of a Novel Phosphate Binding Motif within a TIM Barrel Fold

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Rong; Pineda, Marco; Ajamian, Eunice; Cui, Qizhi; Matte, Allan; Cygler, Miroslaw (McGill); (NRC-Canada)

    2009-01-15

    Three catabolic enzymes, UlaD, UlaE, and UlaF, are involved in a pathway leading to fermentation of L-ascorbate under anaerobic conditions. UlaD catalyzes a {beta}-keto acid decarboxylation reaction to produce L-xylulose-5-phosphate, which undergoes successive epimerization reactions with UlaE (L-xylulose-5-phosphate 3-epimerase) and UlaF (L-ribulose-5-phosphate 4-epimerase), yielding D-xylulose-5-phosphate, an intermediate in the pentose phosphate pathway. We describe here crystallographic studies of UlaE from Escherichia coli O157:H7 that complete the structural characterization of this pathway. UlaE has a triosephosphate isomerase (TIM) barrel fold and forms dimers. The active site is located at the C-terminal ends of the parallel {beta}-strands. The enzyme binds Zn{sup 2+}, which is coordinated by Glu155, Asp185, His211, and Glu251. We identified a phosphate-binding site formed by residues from the {beta}1/{alpha}1 loop and {alpha}3' helix in the N-terminal region. This site differs from the well-characterized phosphate-binding motif found in several TIM barrel superfamilies that is located at strands {beta}7 and {beta}8. The intrinsic flexibility of the active site region is reflected by two different conformations of loops forming part of the substrate-binding site. Based on computational docking of the L-xylulose 5-phosphate substrate to UlaE and structural similarities of the active site of this enzyme to the active sites of other epimerases, a metal-dependent epimerization mechanism for UlaE is proposed, and Glu155 and Glu251 are implicated as catalytic residues. Mutation and activity measurements for structurally equivalent residues in related epimerases supported this mechanistic proposal.

  18. Identification of a gene for an ancient cytokine, interleukin 15-like, in mammals; interleukins 2 and 15 co-evolved with this third family member, all sharing binding motifs for IL-15Rα.

    Science.gov (United States)

    Dijkstra, Johannes M; Takizawa, Fumio; Fischer, Uwe; Friedrich, Maik; Soto-Lampe, Veronica; Lefèvre, Christophe; Lenk, Matthias; Karger, Axel; Matsui, Taei; Hashimoto, Keiichiro

    2014-02-01

    Interleukins 2 and 15 (IL-2 and IL-15) are highly differentiated but related cytokines with overlapping, yet also distinct functions, and established benefits for medical drug use. The present study identified a gene for an ancient third IL-2/15 family member in reptiles and mammals, interleukin 15-like (IL-15L), which hitherto was only reported in fish. IL-15L genes with intact open reading frames (ORFs) and evidence of transcription, and a recent past of purifying selection, were found for cattle, horse, sheep, pig and rabbit. In human and mouse the IL-15L ORF is incapacitated. Although deduced IL-15L proteins share only ~21 % overall amino acid identity with IL-15, they share many of the IL-15 residues important for binding to receptor chain IL-15Rα, and recombinant bovine IL-15L was shown to interact with IL-15Rα indeed. Comparison of sequence motifs indicates that capacity for binding IL-15Rα is an ancestral characteristic of the IL-2/15/15L family, in accordance with a recent study which showed that in fish both IL-2 and IL-15 can bind IL-15Rα. Evidence reveals that the species lineage leading to mammals started out with three similar cytokines IL-2, IL-15 and IL-15L, and that later in evolution (1) IL-2 and IL-2Rα receptor chain acquired a new and specific binding mode and (2) IL-15L was lost in several but not all groups of mammals. The present study forms an important step forward in understanding this potent family of cytokines, and may help to improve future strategies for their application in veterinarian and human medicine.

  19. The RNA-binding motif 45 (RBM45) protein accumulates in inclusion bodies in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) patients.

    Science.gov (United States)

    Collins, Mahlon; Riascos, David; Kovalik, Tina; An, Jiyan; Krupa, Kelly; Krupa, Kristin; Hood, Brian L; Conrads, Thomas P; Renton, Alan E; Traynor, Bryan J; Bowser, Robert

    2012-11-01

    RNA-binding protein pathology now represents one of the best characterized pathologic features of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration patients with TDP-43 or FUS pathology (FTLD-TDP and FTLD-FUS). Using liquid chromatography tandem mass spectrometry, we identified altered levels of the RNA-binding motif 45 (RBM45) protein in the cerebrospinal fluid (CSF) of ALS patients. This protein contains sequence similarities to TAR DNA-binding protein 43 (TDP-43) and fused-in-sarcoma (FUS) that are contained in cytoplasmic inclusions of ALS and FTLD-TDP or FTLD-FUS patients. To further characterize RBM45, we first verified the presence of RBM45 in CSF and spinal cord tissue extracts of ALS patients by immunoblot. We next used immunohistochemistry to examine the subcellular distribution of RBM45 and observed in a punctate staining pattern within nuclei of neurons and glia in the brain and spinal cord. We also detected RBM45 cytoplasmic inclusions in 91 % of ALS, 100 % of FTLD-TDP and 75 % of Alzheimer's disease (AD) cases. The most extensive RBM45 pathology was observed in patients that harbor the C9ORF72 hexanucleotide repeat expansion. These RBM45 inclusions were observed in spinal cord motor neurons, glia and neurons of the dentate gyrus. By confocal microscopy, RBM45 co-localizes with ubiquitin and TDP-43 in inclusion bodies. In neurons containing RBM45 cytoplasmic inclusions we often detected the protein in a punctate pattern within the nucleus that lacked either TDP-43 or ubiquitin. We identified RBM45 using a proteomic screen of CSF from ALS and control subjects for candidate biomarkers, and link this RNA-binding protein to inclusion pathology in ALS, FTLD-TDP and AD.

  20. Functional characterization of variations on regulatory motifs.

    Directory of Open Access Journals (Sweden)

    Michal Lapidot

    2008-03-01

    Full Text Available Transcription factors (TFs regulate gene expression through specific interactions with short promoter elements. The same regulatory protein may recognize a variety of related sequences. Moreover, once they are detected it is hard to predict whether highly similar sequence motifs will be recognized by the same TF and regulate similar gene expression patterns, or serve as binding sites for distinct regulatory factors. We developed computational measures to assess the functional implications of variations on regulatory motifs and to compare the functions of related sites. We have developed computational means for estimating the functional outcome of substituting a single position within a binding site and applied them to a collection of putative regulatory motifs. We predict the effects of nucleotide variations within motifs on gene expression patterns. In cases where such predictions could be compared to suitable published experimental evidence, we found very good agreement. We further accumulated statistics from multiple substitutions across various binding sites in an attempt to deduce general properties that characterize nucleotide substitutions that are more likely to alter expression. We found that substitutions involving Adenine are more likely to retain the expression pattern and that substitutions involving Guanine are more likely to alter expression compared to the rest of the substitutions. Our results should facilitate the prediction of the expression outcomes of binding site variations. One typical important implication is expected to be the ability to predict the phenotypic effect of variation in regulatory motifs in promoters.

  1. Nonconsensus Protein Binding to Repetitive DNA Sequence Elements Significantly Affects Eukaryotic Genomes.

    Science.gov (United States)

    Afek, Ariel; Cohen, Hila; Barber-Zucker, Shiran; Gordân, Raluca; Lukatsky, David B

    2015-08-01

    Recent genome-wide experiments in different eukaryotic genomes provide an unprecedented view of transcription factor (TF) binding locations and of nucleosome occupancy. These experiments revealed that a large fraction of TF binding events occur in regions where only a small number of specific TF binding sites (TFBSs) have been detected. Furthermore, in vitro protein-DNA binding measurements performed for hundreds of TFs indicate that TFs are bound with wide range of affinities to different DNA sequences that lack known consensus motifs. These observations have thus challenged the classical picture of specific protein-DNA binding and strongly suggest the existence of additional recognition mechanisms that affect protein-DNA binding preferences. We have previously demonstrated that repetitive DNA sequence elements characterized by certain symmetries statistically affect protein-DNA binding preferences. We call this binding mechanism nonconsensus protein-DNA binding in order to emphasize the point that specific consensus TFBSs do not contribute to this effect. In this paper, using the simple statistical mechanics model developed previously, we calculate the nonconsensus protein-DNA binding free energy for the entire C. elegans and D. melanogaster genomes. Using the available chromatin immunoprecipitation followed by sequencing (ChIP-seq) results on TF-DNA binding preferences for ~100 TFs, we show that DNA sequences characterized by low predicted free energy of nonconsensus binding have statistically higher experimental TF occupancy and lower nucleosome occupancy than sequences characterized by high free energy of nonconsensus binding. This is in agreement with our previous analysis performed for the yeast genome. We suggest therefore that nonconsensus protein-DNA binding assists the formation of nucleosome-free regions, as TFs outcompete nucleosomes at genomic locations with enhanced nonconsensus binding. In addition, here we perform a new, large-scale analysis using

  2. Nonconsensus Protein Binding to Repetitive DNA Sequence Elements Significantly Affects Eukaryotic Genomes.

    Directory of Open Access Journals (Sweden)

    Ariel Afek

    2015-08-01

    Full Text Available Recent genome-wide experiments in different eukaryotic genomes provide an unprecedented view of transcription factor (TF binding locations and of nucleosome occupancy. These experiments revealed that a large fraction of TF binding events occur in regions where only a small number of specific TF binding sites (TFBSs have been detected. Furthermore, in vitro protein-DNA binding measurements performed for hundreds of TFs indicate that TFs are bound with wide range of affinities to different DNA sequences that lack known consensus motifs. These observations have thus challenged the classical picture of specific protein-DNA binding and strongly suggest the existence of additional recognition mechanisms that affect protein-DNA binding preferences. We have previously demonstrated that repetitive DNA sequence elements characterized by certain symmetries statistically affect protein-DNA binding preferences. We call this binding mechanism nonconsensus protein-DNA binding in order to emphasize the point that specific consensus TFBSs do not contribute to this effect. In this paper, using the simple statistical mechanics model developed previously, we calculate the nonconsensus protein-DNA binding free energy for the entire C. elegans and D. melanogaster genomes. Using the available chromatin immunoprecipitation followed by sequencing (ChIP-seq results on TF-DNA binding preferences for ~100 TFs, we show that DNA sequences characterized by low predicted free energy of nonconsensus binding have statistically higher experimental TF occupancy and lower nucleosome occupancy than sequences characterized by high free energy of nonconsensus binding. This is in agreement with our previous analysis performed for the yeast genome. We suggest therefore that nonconsensus protein-DNA binding assists the formation of nucleosome-free regions, as TFs outcompete nucleosomes at genomic locations with enhanced nonconsensus binding. In addition, here we perform a new, large

  3. Diverted Total Synthesis of Promysalin Analogs Demonstrates That an Iron-Binding Motif Is Responsible for Its Narrow-Spectrum Antibacterial Activity.

    Science.gov (United States)

    Steele, Andrew D; Keohane, Colleen E; Knouse, Kyle W; Rossiter, Sean E; Williams, Sierra J; Wuest, William M

    2016-05-11

    Promysalin is a species-specific Pseudomonad metabolite with unique bioactivity. To better understand the mode of action of this natural product, we synthesized 16 analogs utilizing diverted total synthesis (DTS). Our analog studies revealed that the bioactivity of promysalin is sensitive to changes within its hydrogen bond network whereby alteration has drastic biological consequences. The DTS library not only yielded three analogs that retained potency but also provided insights that resulted in the identification of a previously unknown ability of promysalin to bind iron. These findings coupled with previous observations hint at a complex multifaceted role of the natural product within the rhizosphere.

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

    OpenAIRE

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

    2015-01-01

    YAP is a WW domain-containing effector of the Hippo tumor suppressor pathway, and the object of heightened interest as a potent oncogene and stemness factor. YAP has two major isoforms that differ in the number of WW domains they harbor. Elucidating the degree of co-operation between these WW domains is important for a full understanding of the molecular function of YAP. We present here a detailed biophysical study of the structural stability and binding properties of the two YAP WW domains a...

  5. Binding of trans-acting protein AngCP to the CCAAT-containing motifs in Aspergillus niger glaA promoter

    Institute of Scientific and Technical Information of China (English)

    ZHU Xingguo; QIU Runxiang; LIU Li; TANG Guomin

    2004-01-01

    CCAAT-binding proteins AngCP1 and AngCP2 of Aspergillus niger binding to DC(- 489- - 414 bp)and PC(- 390~ - 345 bp)of A.Niger glaA gene were respectively purified by 20 % ~ 40 % saturated ammonium sulfate,gel filtration,Heparin SepharoseC1-6B chromatography and DNA sequence-specific affinity chromatography.Gel filtration and SDS-PAGE revealed that both AngCP1 and AngCP2 were of 120 kD,comprised of two suhunits of 34 kD and 50 kD.Western blot showed that the 34 kD subunits of both AngCP1 and AngCP2 cross-reacted specifically with the anti-AngHAPC antiserum.Further electrophoretic mobility shift assay identified that AngCP1 and AngCP2 were the same protein,designated AngCP.Southwestern blot showed that the affinity of the 34 kD subunit to DC was stronger than that of the 50 kD subunit to PC.These results suggested that interaction between AngCP,DC and PC plays an important role in the regulation of transcription of Aspergillus niger glaA gene.

  6. A stage—specific protein factor binding to a CACCC motif in both human β—globin gene promoter and 5‘—HS2 region

    Institute of Scientific and Technical Information of China (English)

    SUNTONG; YADICHEN; 等

    1994-01-01

    The DNaseI hypersensitive site 2 (HS2) of human β-globin locus control region(LCR) is required for the high level expression of human β-globin genes.In the present study,a stage-specific protein factor (LPF-β) was identified in the nuclear extract prepared from mouse fetal liver at d 18 of gestation,which could bind to the HS2 region of human β-globin LCR.We also found that the shift band of LPF-β factor could be competed by human β-globin promoter.However,it couldn't be competed by human ε-globin promoter or by human Aγ-globin promoter.Furthermore,our data demonstrated that the binding-sequence of LPF-β factor is 5'CACACCCTA 3',which is located at the HS2 region of β-LCR(from-10845 to-10853 bp)and human β-globin promoter(from-92 to -84 bp).We speculated that these regions containing the CACCC box in both the human β-globin promoter and HS2 might function as stage selector elements in the regulation of human β-globin switching and the LPF-β factor might be a stage-specific protein factor involved in the regulation of human β-globin gene expression.

  7. An autoinhibited conformation of LGN reveals a distinct interaction mode between GoLoco motifs and TPR motifs.

    Science.gov (United States)

    Pan, Zhu; Zhu, Jinwei; Shang, Yuan; Wei, Zhiyi; Jia, Min; Xia, Caihao; Wen, Wenyu; Wang, Wenning; Zhang, Mingjie

    2013-06-01

    LGN plays essential roles in asymmetric cell divisions via its N-terminal TPR-motif-mediated binding to mInsc and NuMA. This scaffolding activity requires the release of the autoinhibited conformation of LGN by binding of Gα(i) to its C-terminal GoLoco (GL) motifs. The interaction between the GL and TPR motifs of LGN represents a distinct GL/target binding mode with an unknown mechanism. Here, we show that two consecutive GL motifs of LGN form a minimal TPR-motif-binding unit. GL12 and GL34 bind to TPR0-3 and TPR4-7, respectively. The crystal structure of a truncated LGN reveals that GL34 forms a pair of parallel α helices and binds to the concave surface of TPR4-7, thereby preventing LGN from binding to other targets. Importantly, the GLs bind to TPR motifs with a mode distinct from that observed in the GL/Gα(i)·GDP complexes. Our results also indicate that multiple and orphan GL motif proteins likely respond to G proteins with distinct mechanisms.

  8. Mycoplasma hyopneumoniae Surface proteins Mhp385 and Mhp384 bind host cilia and glycosaminoglycans and are endoproteolytically processed by proteases that recognize different cleavage motifs.

    Science.gov (United States)

    Deutscher, Ania T; Tacchi, Jessica L; Minion, F Chris; Padula, Matthew P; Crossett, Ben; Bogema, Daniel R; Jenkins, Cheryl; Kuit, Tracey A; Walker, Mark J; Djordjevic, Steven P

    2012-03-02

    P97 and P102 paralogues occur as endoproteolytic cleavage fragments on the surface of Mycoplasma hyopneumoniae that bind glycosaminoglycans, plasminogen, and fibronectin and perform essential roles in colonization of ciliated epithelia. We show that the P102 paralogue Mhp384 is efficiently cleaved at an S/T-X-F↓X-D/E-like site, creating P60(384) and P50(384). The P97 paralogue Mhp385 is inefficiently cleaved, with tryptic peptides from a 115 kDa protein (P115(385)) and 88 kDa (P88(385)) and 27 kDa (P27(385)) cleavage fragments identified by LC-MS/MS. This is the first time a preprotein belonging to the P97 and P102 paralogue families has been identified by mass spectrometry. The semitryptic peptide (752)IQFELEPISLNV(763) denotes the C-terminus of P88(385) and defines the novel cleavage site (761)L-N-V↓A-V-S(766) in Mhp385. P115(385), P88(385), P27(385), P60(384), and P50(384) were shown to reside extracellularly, though it is unknown how the fragments remain attached to the cell surface. Heparin- and cilium-binding sites were identified within P60(384), P50(384), and P88(385). No primary function was attributed to P27(385); however, this molecule contains four tandem R1 repeats with similarity to porcine collagen type VI (α3 chain). P97 and P102 paralogue families are adhesins targeted by several proteases with different cleavage efficiencies, and this process generates combinatorial complexity on the surface of M. hyopneumoniae.

  9. A poly(A) binding protein-specific sequence motif: MRTENGKSKGFGFVC binding to mRNA poly(A) and polynucleotides and its role on mRNA translation.

    Science.gov (United States)

    Rubin, H N; Halim, M N; Leavis, P C

    1994-06-01

    A consensus sequence (GKSKGFGFV) was recognized in all the sequenced poly(A) binding proteins. We synthesized a 15-amino acid peptide (corresponding to 354-368 in the yeast poly(A) binding protein) which includes the consensus sequence to test its binding affinity to different nucleotides, polynucleotides and mRNA with or without a poly(A) tail. Biochemical and biophysical studies revealed that the 15-amino acid peptide has a strong binding affinity to poly(A) alone or poly(A) attached at the 3' end of mRNA. Circular dichroism spectroscopy demonstrated that the secondary structure of the 15-mer is consistent with that expected based on the structure of the native RNP domain. Furthermore, among the various mononucleotides performed in the present studies, ATP was preferentially found to bind to the 15-mer. To further examine the biological significance of the binding of the 15-mer to the poly(A) tail of mRNA, in vitro translation of the mRNA poly(A)+ in the presence of the 15-mer drastically increased globin synthesis by almost 2-fold, while translation of the deadenylated mRNA in the presence of the 15-mer almost did not alter the rate of incorporation of radiolabeled leucine into globin.

  10. Detecting DNA regulatory motifs by incorporating positional trendsin information content

    Energy Technology Data Exchange (ETDEWEB)

    Kechris, Katherina J.; van Zwet, Erik; Bickel, Peter J.; Eisen,Michael B.

    2004-05-04

    On the basis of the observation that conserved positions in transcription factor binding sites are often clustered together, we propose a simple extension to the model-based motif discovery methods. We assign position-specific prior distributions to the frequency parameters of the model, penalizing deviations from a specified conservation profile. Examples with both simulated and real data show that this extension helps discover motifs as the data become noisier or when there is a competing false motif.

  11. The methyltransferase NSD3 has chromatin-binding motifs, PHD5-C5HCH, that are distinct from other NSD (nuclear receptor SET domain) family members in their histone H3 recognition.

    Science.gov (United States)

    He, Chao; Li, Fudong; Zhang, Jiahai; Wu, Jihui; Shi, Yunyu

    2013-02-15

    The NSD (nuclear receptor SET domain-containing) family members, consisting of NSD1, NSD2 (MMSET/WHSC1), and NSD3 (WHSC1L1), are SET domain-containing methyltransferases and aberrant expression of each member has been implicated in multiple diseases. They have specific mono- and dimethylase activities for H3K36, whereas play nonredundant roles during development. Aside from the well characterized catalytic SET domain, NSD proteins have multiple potential chromatin-binding motifs that are clinically relevant, including the fifth plant homeodomain (PHD5) and the adjacent Cys-His-rich domain (C5HCH) located at the C terminus. Herein, we report the crystal structures of the PHD5-C5HCH module of NSD3, in the free state and in complex with H3(1-7) (H3 residues 1-7), H3(1-15) (H3 residues 1-15), and H3(1-15)K9me3 (H3 residues 1-15 with trimethylation on K9) peptides. These structures reveal that the PHD5 and C5HCH domains fold into a novel integrated PHD-PHD-like structural module with H3 peptide bound only on the surface of PHD5 and provide the molecular basis for the recognition of unmodified H3K4 and trimethylated H3K9 by NSD3 PHD5. Structural studies and binding assays show that differences exist in histone binding specificity of the PHD5 domain between three members of the NSD family. For NSD2, the PHD5-C5HCH:H3 N terminus interaction is largely conserved, although with a stronger preference for unmethylated H3K9 (H3K9me0) than trimethylated H3K9 (H3K9me3), and NSD1 PHD5-C5HCH does not bind to H3 peptides. Our results shed light on how NSD proteins that mediate H3K36 methylation are localized to specific genomic sites and provide implications for the mechanism of functional diversity of NSD proteins.

  12. Using SCOPE to identify potential regulatory motifs in coregulated genes.

    Science.gov (United States)

    Martyanov, Viktor; Gross, Robert H

    2011-05-31

    SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and motif position preference. Each component algorithm is optimized to find a different kind of motif. By taking the best of these three approaches, SCOPE performs better than any single algorithm, even in the presence of noisy data. In this article, we utilize a web version of SCOPE to examine genes that are involved in telomere maintenance. SCOPE has been incorporated into at least two other motif finding programs and has been used in other studies. The three algorithms that comprise SCOPE are BEAM, which finds non-degenerate motifs (ACCGGT), PRISM, which finds degenerate motifs (ASCGWT), and SPACER, which finds longer bipartite motifs (ACCnnnnnnnnGGT). These three algorithms have been optimized to find their corresponding type of motif. Together, they allow SCOPE to perform extremely well. Once a gene set has been analyzed and candidate motifs identified, SCOPE can look for other genes that contain the motif which, when added to the original set, will improve the motif score. This can occur through over-representation or motif position preference. Working with partial gene sets that have biologically verified transcription factor binding sites, SCOPE was able to identify most of the rest of the genes also regulated by the given transcription factor. Output from SCOPE shows candidate motifs, their significance, and other information both as a table and as a graphical motif map. FAQs and video tutorials are available at the SCOPE web site which also includes a "Sample Search" button that allows the user to perform a trial run. Scope has a very friendly user interface that enables novice users to access the algorithm's full power without having to become an expert in the bioinformatics of motif finding. As input, SCOPE can take a list of genes, or FASTA sequences. These can be entered in browser text fields, or read from

  13. Large-scale discovery of promoter motifs in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Thomas A Down

    2007-01-01

    Full Text Available A key step in understanding gene regulation is to identify the repertoire of transcription factor binding motifs (TFBMs that form the building blocks of promoters and other regulatory elements. Identifying these experimentally is very laborious, and the number of TFBMs discovered remains relatively small, especially when compared with the hundreds of transcription factor genes predicted in metazoan genomes. We have used a recently developed statistical motif discovery approach, NestedMICA, to detect candidate TFBMs from a large set of Drosophila melanogaster promoter regions. Of the 120 motifs inferred in our initial analysis, 25 were statistically significant matches to previously reported motifs, while 87 appeared to be novel. Analysis of sequence conservation and motif positioning suggested that the great majority of these discovered motifs are predictive of functional elements in the genome. Many motifs showed associations with specific patterns of gene expression in the D. melanogaster embryo, and we were able to obtain confident annotation of expression patterns for 25 of our motifs, including eight of the novel motifs. The motifs are available through Tiffin, a new database of DNA sequence motifs. We have discovered many new motifs that are overrepresented in D. melanogaster promoter regions, and offer several independent lines of evidence that these are novel TFBMs. Our motif dictionary provides a solid foundation for further investigation of regulatory elements in Drosophila, and demonstrates techniques that should be applicable in other species. We suggest that further improvements in computational motif discovery should narrow the gap between the set of known motifs and the total number of transcription factors in metazoan genomes.

  14. CLIMP: Clustering Motifs via Maximal Cliques with Parallel Computing Design.

    Science.gov (United States)

    Zhang, Shaoqiang; Chen, Yong

    2016-01-01

    A set of conserved binding sites recognized by a transcription factor is called a motif, which can be found by many applications of comparative genomics for identifying over-represented segments. Moreover, when numerous putative motifs are predicted from a collection of genome-wide data, their similarity data can be represented as a large graph, where these motifs are connected to one another. However, an efficient clustering algorithm is desired for clustering the motifs that belong to the same groups and separating the motifs that belong to different groups, or even deleting an amount of spurious ones. In this work, a new motif clustering algorithm, CLIMP, is proposed by using maximal cliques and sped up by parallelizing its program. When a synthetic motif dataset from the database JASPAR, a set of putative motifs from a phylogenetic foot-printing dataset, and a set of putative motifs from a ChIP dataset are used to compare the performances of CLIMP and two other high-performance algorithms, the results demonstrate that CLIMP mostly outperforms the two algorithms on the three datasets for motif clustering, so that it can be a useful complement of the clustering procedures in some genome-wide motif prediction pipelines. CLIMP is available at http://sqzhang.cn/climp.html.

  15. MEME-ChIP: motif analysis of large DNA datasets.

    Science.gov (United States)

    Machanick, Philip; Bailey, Timothy L

    2011-06-15

    Advances in high-throughput sequencing have resulted in rapid growth in large, high-quality datasets including those arising from transcription factor (TF) ChIP-seq experiments. While there are many existing tools for discovering TF binding site motifs in such datasets, most web-based tools cannot directly process such large datasets. The MEME-ChIP web service is designed to analyze ChIP-seq 'peak regions'--short genomic regions surrounding declared ChIP-seq 'peaks'. Given a set of genomic regions, it performs (i) ab initio motif discovery, (ii) motif enrichment analysis, (iii) motif visualization, (iv) binding affinity analysis and (v) motif identification. It runs two complementary motif discovery algorithms on the input data--MEME and DREME--and uses the motifs they discover in subsequent visualization, binding affinity and identification steps. MEME-ChIP also performs motif enrichment analysis using the AME algorithm, which can detect very low levels of enrichment of binding sites for TFs with known DNA-binding motifs. Importantly, unlike with the MEME web service, there is no restriction on the size or number of uploaded sequences, allowing very large ChIP-seq datasets to be analyzed. The analyses performed by MEME-ChIP provide the user with a varied view of the binding and regulatory activity of the ChIP-ed TF, as well as the possible involvement of other DNA-binding TFs. MEME-ChIP is available as part of the MEME Suite at http://meme.nbcr.net.

  16. The Motif Tracking Algorithm

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The search for patterns or motifs in data represents a problem area of key interest to finance and economic researchers. In this paper, we introduce the motif tracking algorithm (MTA), a novel immune inspired (IS) pattern identification tool that is able to identify unknown motifs of a non specified length which repeat within time series data. The power of the algorithm comes from the fact that it uses a small number of parameters with minimal assumptions regarding the data being examined or the underlying motifs. Our interest lies in applying the algorithm to financial time series data to identify unknown patterns that exist. The algorithm is tested using three separate data sets. Particular suitability to financial data is shown by applying it to oil price data. In all cases, the algorithm identifies the presence of a motif population in a fast and efficient manner due to the utilization of an intuitive symbolic representation.The resulting population of motifs is shown to have considerable potential value for other applications such as forecasting and algorithm seeding.

  17. The Motif Tracking Algorithm

    CERN Document Server

    Wilson, William; Aickelin, Uwe; 10.1007/s11633.008.0032.0

    2010-01-01

    The search for patterns or motifs in data represents a problem area of key interest to finance and economic researchers. In this paper we introduce the Motif Tracking Algorithm, a novel immune inspired pattern identification tool that is able to identify unknown motifs of a non specified length which repeat within time series data. The power of the algorithm comes from the fact that it uses a small number of parameters with minimal assumptions regarding the data being examined or the underlying motifs. Our interest lies in applying the algorithm to financial time series data to identify unknown patterns that exist. The algorithm is tested using three separate data sets. Particular suitability to financial data is shown by applying it to oil price data. In all cases the algorithm identifies the presence of a motif population in a fast and efficient manner due to the utilisation of an intuitive symbolic representation. The resulting population of motifs is shown to have considerable potential value for other ap...

  18. New type of starch-binding domain: the direct repeat motif in the C-terminal region of Bacillus sp. no. 195 alpha-amylase contributes to starch binding and raw starch degrading.

    Science.gov (United States)

    Sumitani, J; Tottori, T; Kawaguchi, T; Arai, M

    2000-09-01

    The alpha-amylase from Bacillus sp. no. 195 (BAA) consists of two domains: one is the catalytic domain similar to alpha-amylases from animals and Streptomyces in the N-terminal region; the other is the functionally unknown domain composed of an approx. 90-residue direct repeat in the C-terminal region. The gene coding for BAA was expressed in Streptomyces lividans TK24. Three active forms of the gene products were found. The pH and thermal profiles of BAAs, and their catalytic activities for p-nitrophenyl maltopentaoside and soluble starch, showed almost the same behaviours. The largest, 69 kDa, form (BAA-alpha) was of the same molecular mass as that of the mature protein estimated from the nucleotide sequence, and had raw-starch-binding and -degrading abilities. The second largest, 60 kDa, form (BAA-beta), whose molecular mass was the same as that of the natural enzyme from Bacillus sp. no. 195, was generated by proteolytic processing between the two repeat sequences in the C-terminal region, and had lower activities for raw starch binding and degrading than those of BAA-alpha. The smallest, 50 kDa, form (BAA-gamma) contained only the N-terminal catalytic domain as a result of removal of the C-terminal repeat sequence, which led to loss of binding and degradation of insoluble starches. Thus the starch adsorption capacity and raw-starch-degrading activity of BAAs depends on the existence of the repeat sequence in the C-terminal region. BAA-alpha was specifically adsorbed on starch or dextran (alpha-1,4 or alpha-1,6 glucan), and specifically desorbed with maltose or beta-cyclodextrin. These observations indicated that the repeat sequence of the enzyme was functional in the starch-binding domain (SBD). We propose the designation of the homologues to the SBD of glucoamylase from Aspergillus niger as family I SBDs, the homologues to that of glucoamylase from Rhizopus oryzae as family II, and the homologues of this repeat sequence of BAA as family III.

  19. Comparison of the local binding motifs in the imidazolium-based ionic liquids [EMIM][BF4] and [EMMIM][BF4] through cryogenic ion vibrational predissociation spectroscopy: Unraveling the roles of anharmonicity and intermolecular interactions

    Science.gov (United States)

    Fournier, Joseph A.; Wolke, Conrad T.; Johnson, Christopher J.; McCoy, Anne B.; Johnson, Mark A.

    2015-02-01

    We clarify the role of the critical imidazolium C(2)H position (the central C between N atoms in the heterocycle) in the assembly motif of the [EMIM][BF4] ionic liquid by analyzing the vibrational spectra of the bare EMIM+ ion as well as that of the cationic [EMIM]2[BF4]+ (EMIM+ = 1-ethyl-3-methylimidazolium, C6H11N2+) cluster. Vibrational spectra of the cold, mass-selected ions are obtained using cryogenic ion vibrational predissociation of weakly bound D2 molecules formed in a 10 K ion trap. The C(2)H behavior is isolated by following the evolution of key vibrational features when the C(2) hydrogen, the proposed binding location of the anion to the imidazolium ring, is replaced by either deuterium or a methyl group (i.e., in the EMMIM+ analogue). Strong features in the ring CH stretching region of the bare ion are traced to Fermi resonances with overtones of lower frequency modes. Upon incorporation into the EMIM+ ṡ ṡ ṡ BF4- ṡ ṡ ṡ EMIM+ ternary complex, the C(2)H oscillator strength is dramatically increased, accounting for the much more complicated patterns derived from the EMIM+ ring CH stretches in the light isotopomer, which are strongly suppressed in the deuterated analogue. Further changes in the spectra that occur when the C(2)H is replaced by a methyl group are consistent with BF4- attachment directly to the imidazolium ring in an arrangement that maximizes the electrostatic interaction between the molecular ions.

  20. Down regulation of RNA binding motif, single-stranded interacting protein 3, along with up regulation of nuclear HIF1A correlates with poor prognosis in patients with gastric cancer

    Science.gov (United States)

    Zhao, Yingjie; Wang, Yuqi; Sun, Ruochuan; Yan, Qiang; Zhang, Shangxin; Lu, Mingdian; Zhang, Zhen; Lu, Daru; Li, Yongxiang

    2017-01-01

    Frequent loss of multiple regions in short arm of chromosome 3 is found in various tumors including gastric cancer (GC). RNA binding motif, single-stranded interacting protein 3 (RBMS3) is a tumor suppressor gene located in this region and mediates cancer angiogenesis. However, the role of RBMS3 in GC remains unclear. To evaluate whether RBMS3, together with HIF1A, another key regulator of angiogenesis, predicts GC prognosis, the levels of RBMS3 and HIF1A were first examined by quantitative PCR (qPCR) and western blot from 27 fresh frozen GC and paired normal gastric tissues and then tested by immunohistochemistry (IHC) from 191 GC and 46 normal controls. Moreover, uni- and multivariate analysis were employed to assess the correlations between their levels and microvessel density (MVD) and clinical prognosis. To further identify RBMS3 function in vitro, cell proliferation assay, clonogenic assay, flow cytometry analysis and endothelial cell tube formation assay were employed. We found that RBMS3 level was decreased, whereas HIF1A was elevated in GC. Furthermore, we demonstrated that RBMS3 was an independent prognostic factor and the levels of RBMS3 and HIF1A were associated with GC angiogenesis and histopathological differentiation: patients with lower RBMS3 level and higher nuclear HIF1A expression had poorer prognosis. Besides, gain- and loss-of-function study revealed RBMS3 regulation on G1/S progression, cell proliferation and the tube formation of human umbilical vein endothelial cells (HUVECs) in vitro. These findings implicated that RBMS3 and nuclear HIF1A could act as prognostic biomarkers and therapeutic targets for GC. PMID:27902480

  1. Comparison of the local binding motifs in the imidazolium-based ionic liquids [EMIM][BF{sub 4}] and [EMMIM][BF{sub 4}] through cryogenic ion vibrational predissociation spectroscopy: Unraveling the roles of anharmonicity and intermolecular interactions

    Energy Technology Data Exchange (ETDEWEB)

    Fournier, Joseph A.; Wolke, Conrad T.; Johnson, Christopher J.; Johnson, Mark A., E-mail: mark.johnson@yale.edu, E-mail: mccoy@chemistry.ohio-state.edu [Sterling Chemistry Laboratory, Yale University, New Haven, Connecticut 06520 (United States); McCoy, Anne B., E-mail: mark.johnson@yale.edu, E-mail: mccoy@chemistry.ohio-state.edu [Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210 (United States)

    2015-02-14

    We clarify the role of the critical imidazolium C{sub (2)}H position (the central C between N atoms in the heterocycle) in the assembly motif of the [EMIM][BF{sub 4}] ionic liquid by analyzing the vibrational spectra of the bare EMIM{sup +} ion as well as that of the cationic [EMIM]{sub 2}[BF{sub 4}]{sup +} (EMIM{sup +} = 1-ethyl-3-methylimidazolium, C{sub 6}H{sub 11}N{sub 2}{sup +}) cluster. Vibrational spectra of the cold, mass-selected ions are obtained using cryogenic ion vibrational predissociation of weakly bound D{sub 2} molecules formed in a 10 K ion trap. The C{sub (2)}H behavior is isolated by following the evolution of key vibrational features when the C{sub (2)} hydrogen, the proposed binding location of the anion to the imidazolium ring, is replaced by either deuterium or a methyl group (i.e., in the EMMIM{sup +} analogue). Strong features in the ring CH stretching region of the bare ion are traced to Fermi resonances with overtones of lower frequency modes. Upon incorporation into the EMIM{sup +} ⋅ ⋅ ⋅ BF{sub 4}{sup −} ⋅ ⋅ ⋅ EMIM{sup +} ternary complex, the C{sub (2)}H oscillator strength is dramatically increased, accounting for the much more complicated patterns derived from the EMIM{sup +} ring CH stretches in the light isotopomer, which are strongly suppressed in the deuterated analogue. Further changes in the spectra that occur when the C{sub (2)}H is replaced by a methyl group are consistent with BF{sub 4}{sup −} attachment directly to the imidazolium ring in an arrangement that maximizes the electrostatic interaction between the molecular ions.

  2. An Affinity Propagation-Based DNA Motif Discovery Algorithm

    Directory of Open Access Journals (Sweden)

    Chunxiao Sun

    2015-01-01

    Full Text Available The planted (l,d motif search (PMS is one of the fundamental problems in bioinformatics, which plays an important role in locating transcription factor binding sites (TFBSs in DNA sequences. Nowadays, identifying weak motifs and reducing the effect of local optimum are still important but challenging tasks for motif discovery. To solve the tasks, we propose a new algorithm, APMotif, which first applies the Affinity Propagation (AP clustering in DNA sequences to produce informative and good candidate motifs and then employs Expectation Maximization (EM refinement to obtain the optimal motifs from the candidate motifs. Experimental results both on simulated data sets and real biological data sets show that APMotif usually outperforms four other widely used algorithms in terms of high prediction accuracy.

  3. An Affinity Propagation-Based DNA Motif Discovery Algorithm.

    Science.gov (United States)

    Sun, Chunxiao; Huo, Hongwei; Yu, Qiang; Guo, Haitao; Sun, Zhigang

    2015-01-01

    The planted (l, d) motif search (PMS) is one of the fundamental problems in bioinformatics, which plays an important role in locating transcription factor binding sites (TFBSs) in DNA sequences. Nowadays, identifying weak motifs and reducing the effect of local optimum are still important but challenging tasks for motif discovery. To solve the tasks, we propose a new algorithm, APMotif, which first applies the Affinity Propagation (AP) clustering in DNA sequences to produce informative and good candidate motifs and then employs Expectation Maximization (EM) refinement to obtain the optimal motifs from the candidate motifs. Experimental results both on simulated data sets and real biological data sets show that APMotif usually outperforms four other widely used algorithms in terms of high prediction accuracy.

  4. Probabilistic models for semisupervised discriminative motif discovery in DNA sequences.

    Science.gov (United States)

    Kim, Jong Kyoung; Choi, Seungjin

    2011-01-01

    Methods for discriminative motif discovery in DNA sequences identify transcription factor binding sites (TFBSs), searching only for patterns that differentiate two sets (positive and negative sets) of sequences. On one hand, discriminative methods increase the sensitivity and specificity of motif discovery, compared to generative models. On the other hand, generative models can easily exploit unlabeled sequences to better detect functional motifs when labeled training samples are limited. In this paper, we develop a hybrid generative/discriminative model which enables us to make use of unlabeled sequences in the framework of discriminative motif discovery, leading to semisupervised discriminative motif discovery. Numerical experiments on yeast ChIP-chip data for discovering DNA motifs demonstrate that the best performance is obtained between the purely-generative and the purely-discriminative and the semisupervised learning improves the performance when labeled sequences are limited.

  5. Solution structure of the two RNA recognition motifs of hnRNP A1 using segmental isotope labeling: how the relative orientation between RRMs influences the nucleic acid binding topology

    Energy Technology Data Exchange (ETDEWEB)

    Barraud, Pierre; Allain, Frederic H.-T., E-mail: allain@mol.biol.ethz.ch [ETH Zurich, Institute of Molecular Biology and Biophysics (Switzerland)

    2013-01-15

    Human hnRNP A1 is a multi-functional protein involved in many aspects of nucleic-acid processing such as alternative splicing, micro-RNA biogenesis, nucleo-cytoplasmic mRNA transport and telomere biogenesis and maintenance. The N-terminal region of hnRNP A1, also named unwinding protein 1 (UP1), is composed of two closely related RNA recognition motifs (RRM), and is followed by a C-terminal glycine rich region. Although crystal structures of UP1 revealed inter-domain interactions between RRM1 and RRM2 in both the free and bound form of UP1, these interactions have never been established in solution. Moreover, the relative orientation of hnRNP A1 RRMs is different in the free and bound crystal structures of UP1, raising the question of the biological significance of this domain movement. In the present study, we have used NMR spectroscopy in combination with segmental isotope labeling techniques to carefully analyze the inter-RRM contacts present in solution and subsequently determine the structure of UP1 in solution. Our data unambiguously demonstrate that hnRNP A1 RRMs interact in solution, and surprisingly, the relative orientation of the two RRMs observed in solution is different from the one found in the crystal structure of free UP1 and rather resembles the one observed in the nucleic-acid bound form of the protein. This strongly supports the idea that the two RRMs of hnRNP A1 have a single defined relative orientation which is the conformation previously observed in the bound form and now observed in solution using NMR. It is likely that the conformation in the crystal structure of the free form is a less stable form induced by crystal contacts. Importantly, the relative orientation of the RRMs in proteins containing multiple-RRMs strongly influences the RNA binding topologies that are practically accessible to these proteins. Indeed, RRM domains are asymmetric binding platforms contacting single-stranded nucleic acids in a single defined orientation

  6. EXTREME: an online EM algorithm for motif discovery

    Science.gov (United States)

    Quang, Daniel; Xie, Xiaohui

    2014-01-01

    Motivation: Identifying regulatory elements is a fundamental problem in the field of gene transcription. Motif discovery—the task of identifying the sequence preference of transcription factor proteins, which bind to these elements—is an important step in this challenge. MEME is a popular motif discovery algorithm. Unfortunately, MEME’s running time scales poorly with the size of the dataset. Experiments such as ChIP-Seq and DNase-Seq are providing a rich amount of information on the binding preference of transcription factors. MEME cannot discover motifs in data from these experiments in a practical amount of time without a compromising strategy such as discarding a majority of the sequences. Results: We present EXTREME, a motif discovery algorithm designed to find DNA-binding motifs in ChIP-Seq and DNase-Seq data. Unlike MEME, which uses the expectation-maximization algorithm for motif discovery, EXTREME uses the online expectation-maximization algorithm to discover motifs. EXTREME can discover motifs in large datasets in a practical amount of time without discarding any sequences. Using EXTREME on ChIP-Seq and DNase-Seq data, we discover many motifs, including some novel and infrequent motifs that can only be discovered by using the entire dataset. Conservation analysis of one of these novel infrequent motifs confirms that it is evolutionarily conserved and possibly functional. Availability and implementation: All source code is available at the Github repository http://github.com/uci-cbcl/EXTREME. Contact: xhx@ics.uci.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:24532725

  7. The limits of de novo DNA motif discovery.

    Directory of Open Access Journals (Sweden)

    David Simcha

    Full Text Available A major challenge in molecular biology is reverse-engineering the cis-regulatory logic that plays a major role in the control of gene expression. This program includes searching through DNA sequences to identify "motifs" that serve as the binding sites for transcription factors or, more generally, are predictive of gene expression across cellular conditions. Several approaches have been proposed for de novo motif discovery-searching sequences without prior knowledge of binding sites or nucleotide patterns. However, unbiased validation is not straightforward. We consider two approaches to unbiased validation of discovered motifs: testing the statistical significance of a motif using a DNA "background" sequence model to represent the null hypothesis and measuring performance in predicting membership in gene clusters. We demonstrate that the background models typically used are "too null," resulting in overly optimistic assessments of significance, and argue that performance in predicting TF binding or expression patterns from DNA motifs should be assessed by held-out data, as in predictive learning. Applying this criterion to common motif discovery methods resulted in universally poor performance, although there is a marked improvement when motifs are statistically significant against real background sequences. Moreover, on synthetic data where "ground truth" is known, discriminative performance of all algorithms is far below the theoretical upper bound, with pronounced "over-fitting" in training. A key conclusion from this work is that the failure of de novo discovery approaches to accurately identify motifs is basically due to statistical intractability resulting from the fixed size of co-regulated gene clusters, and thus such failures do not necessarily provide evidence that unfound motifs are not active biologically. Consequently, the use of prior knowledge to enhance motif discovery is not just advantageous but necessary. An implementation of

  8. Visibility graph motifs

    CERN Document Server

    Iacovacci, Jacopo

    2015-01-01

    Visibility algorithms transform time series into graphs and encode dynamical information in their topology, paving the way for graph-theoretical time series analysis as well as building a bridge between nonlinear dynamics and network science. In this work we introduce and study the concept of visibility graph motifs, smaller substructures that appear with characteristic frequencies. We develop a theory to compute in an exact way the motif profiles associated to general classes of deterministic and stochastic dynamics. We find that this simple property is indeed a highly informative and computationally efficient feature capable to distinguish among different dynamics and robust against noise contamination. We finally confirm that it can be used in practice to perform unsupervised learning, by extracting motif profiles from experimental heart-rate series and being able, accordingly, to disentangle meditative from other relaxation states. Applications of this general theory include the automatic classification a...

  9. 1-t-motifs

    CERN Document Server

    Taelman, Lenny

    2009-01-01

    We show that the module of rational points on an abelian t-module E is canonically isomorphic with the module Ext^1(M_E, K[t]) of extensions of the trivial t-motif K[t] by the t-motif M_E associated with E. This generalizes prior results of Anderson and Thakur and of Papanikolas and Ramachandran. In case E is uniformizable then we show that this extension module is canonically isomorphic with the corresponding extension module of Pink-Hodge structures. This situation is formally very similar to Deligne's theory of 1-motifs and we have tried to build up the theory in a way that makes this analogy as clear as possible.

  10. Transcription factor motif quality assessment requires systematic comparative analysis [version 2; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Caleb Kipkurui Kibet

    2016-03-01

    Full Text Available Transcription factor (TF binding site prediction remains a challenge in gene regulatory research due to degeneracy and potential variability in binding sites in the genome. Dozens of algorithms designed to learn binding models (motifs have generated many motifs available in research papers with a subset making it to databases like JASPAR, UniPROBE and Transfac. The presence of many versions of motifs from the various databases for a single TF and the lack of a standardized assessment technique makes it difficult for biologists to make an appropriate choice of binding model and for algorithm developers to benchmark, test and improve on their models. In this study, we review and evaluate the approaches in use, highlight differences and demonstrate the difficulty of defining a standardized motif assessment approach. We review scoring functions, motif length, test data and the type of performance metrics used in prior studies as some of the factors that influence the outcome of a motif assessment. We show that the scoring functions and statistics used in motif assessment influence ranking of motifs in a TF-specific manner. We also show that TF binding specificity can vary by source of genomic binding data. We also demonstrate that information content of a motif is not in isolation a measure of motif quality but is influenced by TF binding behaviour. We conclude that there is a need for an easy-to-use tool that presents all available evidence for a comparative analysis.

  11. Verification of a novel NADH-binding motif: combinatorial mutagenesis of three amino acids in the cofactor-binding pocket of Corynebacterium 2,5-diketo-D-gluconic acid reductase.

    Science.gov (United States)

    Banta, Scott; Anderson, Stephen

    2002-12-01

    A screening method has been developed to support randomized mutagenesis of amino acids in the cofactor-binding pocket of the NADPH-dependent 2,5-diketo-D-gluconic acid (2,5-DKG) reductase. Such an approach could enable the isolation of an enzyme that can better catalyze the reduction of 2,5-DKG to 2-keto-L-gulonic acid (2-KLG) using NADH as a cofactor. 2-KLG is a valuable precursor to ascorbic acid, or vitamin C, and an enzyme with increased activity with NADH may be able to improve two potential vitamin C production processes. Previously we have identified three amino acid residues that can be mutated to improve activity with NADH as a cofactor. As a pilot study to show feasibility, a library was made with these three amino acids randomized, and 300 random colonies were screened for increased NADH activity. The activities of seven mutants with apparent improvements were verified using activity-stained native gels, and sequencing showed that the amino acids obtained were similar to some of those already discovered using rational design. The four most active mutants were purified and kinetically characterized. All of the new mutations resulted in apparent kcat values that were equal to or higher than that of the best mutant obtained through rational design. At saturating levels of cofactor, the best mutant obtained was almost twice as active with NADH as a cofactor as the wild-type enzyme is with NADPH. This screen is a valuable tool for improving 2,5-DKG reductase, and it could easily be modified for improving other aspects of this protein or similar enzymes.

  12. A novel pro-Arg motif recognized by WW domains.

    Science.gov (United States)

    Bedford, M T; Sarbassova, D; Xu, J; Leder, P; Yaffe, M B

    2000-04-07

    WW domains mediate protein-protein interactions through binding to short proline-rich sequences. Two distinct sequence motifs, PPXY and PPLP, are recognized by different classes of WW domains, and another class binds to phospho-Ser-Pro sequences. We now describe a novel Pro-Arg sequence motif recognized by a different class of WW domains using data from oriented peptide library screening, expression cloning, and in vitro binding experiments. The prototype member of this group is the WW domain of formin-binding protein 30 (FBP30), a p53-regulated molecule whose WW domains bind to Pro-Arg-rich cellular proteins. This new Pro-Arg sequence motif re-classifies the organization of WW domains based on ligand specificity, and the Pro-Arg class now includes the WW domains of FBP21 and FE65. A structural model is presented which rationalizes the distinct motifs selected by the WW domains of YAP, Pin1, and FBP30. The Pro-Arg motif identified for WW domains often overlaps with SH3 domain motifs within protein sequences, suggesting that the same extended proline-rich sequence could form discrete SH3 or WW domain complexes to transduce distinct cellular signals.

  13. [Personal motif in art].

    Science.gov (United States)

    Gerevich, József

    2015-01-01

    One of the basic questions of the art psychology is whether a personal motif is to be found behind works of art and if so, how openly or indirectly it appears in the work itself. Analysis of examples and documents from the fine arts and literature allow us to conclude that the personal motif that can be identified by the viewer through symbols, at times easily at others with more difficulty, gives an emotional plus to the artistic product. The personal motif may be found in traumatic experiences, in communication to the model or with other emotionally important persons (mourning, disappointment, revenge, hatred, rivalry, revolt etc.), in self-searching, or self-analysis. The emotions are expressed in artistic activity either directly or indirectly. The intention nourished by the artist's identity (Kunstwollen) may stand in the way of spontaneous self-expression, channelling it into hidden paths. Under the influence of certain circumstances, the artist may arouse in the viewer, consciously or unconsciously, an illusionary, misleading image of himself. An examination of the personal motif is one of the important research areas of art therapy.

  14. Tumor suppressor, AT motif binding factor 1 (ATBF1), translocates to the nucleus with runt domain transcription factor 3 (RUNX3) in response to TGF-{beta} signal transduction

    Energy Technology Data Exchange (ETDEWEB)

    Mabuchi, Motoshi [Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya (Japan); Koseiin Medical Welfare Center, Nagoya (Japan); Kataoka, Hiromi, E-mail: hkataoka@med.nagoya-cu.ac.jp [Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya (Japan); Miura, Yutaka; Kim, Tae-Sun [Department of Molecular Neurobiology, Nagoya City University Graduate School of Medical Sciences, Nagoya (Japan); Kawaguchi, Makoto [Department of Pathology, Niigata Rosai Hospital, Japan Labor Health and Welfare Organization, Niigata (Japan); Ebi, Masahide; Tanaka, Mamoru; Mori, Yoshinori; Kubota, Eiji; Mizushima, Takashi; Shimura, Takaya; Mizoshita, Tsutomu; Tanida, Satoshi; Kamiya, Takeshi [Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya (Japan); Asai, Kiyofumi [Department of Molecular Neurobiology, Nagoya City University Graduate School of Medical Sciences, Nagoya (Japan); Joh, Takashi [Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya (Japan)

    2010-07-23

    Research highlights: {yields} Significant correlation between ATBF1 and RUNX3 nuclear localization in gastric cancer. {yields} Co-IP reveals a physical association between ATBF1 and RUNX3. {yields} ATBF1 and RUNX3 up-regulates p21 promoter activity synergistically. {yields} TGF-{beta}1 induces endogenous ATBF1 and RUNX3 nuclear translocation. -- Abstract: Background and aims: AT motif binding factor 1 (ATBF1), a homeotic transcription factor, was identified as a tumor suppressor, and loss of heterozygosity at ATBF1 locus occurs frequently in gastric cancers. We previously showed that ATBF1 expression inversely correlated with the malignant character of gastric cancer and that ATBF1 enhanced the promoter activity of p21{sup Waf1/Cip1}. We also found that ATBF1 moves between cytoplasm and nucleus, but the precise mechanism of translocation is unknown. In this study, we investigated the mechanism of ATBF1 translocation to the nucleus with the runt domain transcription factor 3 (RUNX3) in cooperation with TGF-{beta} signal transduction. Materials and methods: To analyze the expression of ATBF1 and RUNX3 in gastric cancer cells, we performed immunohistochemistry on 98 resected gastric cancer tissue samples and scored the nuclear staining intensity as grade 0 to grade 5. Co-immunoprecipitation (co-IP) of ATBF1 and RUNX3 was performed. Dual luciferase assays were performed by transfecting ATBF1 and RUNX3 with a p21{sup Waf1/Cip1} reporter vector. To investigate the nuclear translocation of endogenous ATBF1 and RUNX3 in response to TGF-{beta} signal, we examined the subcellular localization of ATBF1 and RUNX3 in gastric cancer cells treated with recombinant TGF-{beta}1 using confocal laser scanning microscopy. Results: Strong immunohistochemical nuclear staining of ATBF1 was observed in 37 (37.8%) of the gastric cancer tissue samples, and RUNX3 nuclear staining was observed in 15 (15.3%). There was a statistically significant correlation between ATBF1 and RUNX3 nuclear

  15. HeliCis: a DNA motif discovery tool for colocalized motif pairs with periodic spacing

    Directory of Open Access Journals (Sweden)

    Mostad Petter

    2007-10-01

    Full Text Available Abstract Background Correct temporal and spatial gene expression during metazoan development relies on combinatorial interactions between different transcription factors. As a consequence, cis-regulatory elements often colocalize in clusters termed cis-regulatory modules. These may have requirements on organizational features such as spacing, order and helical phasing (periodic spacing between binding sites. Due to the turning of the DNA helix, a small modification of the distance between a pair of sites may sometimes drastically disrupt function, while insertion of a full helical turn of DNA (10–11 bp between cis elements may cause functionality to be restored. Recently, de novo motif discovery methods which incorporate organizational properties such as colocalization and order preferences have been developed, but there are no tools which incorporate periodic spacing into the model. Results We have developed a web based motif discovery tool, HeliCis, which features a flexible model which allows de novo detection of motifs with periodic spacing. Depending on the parameter settings it may also be used for discovering colocalized motifs without periodicity or motifs separated by a fixed gap of known or unknown length. We show on simulated data that it can efficiently capture the synergistic effects of colocalization and periodic spacing to improve detection of weak DNA motifs. It provides a simple to use web interface which interactively visualizes the current settings and thereby makes it easy to understand the parameters and the model structure. Conclusion HeliCis provides simple and efficient de novo discovery of colocalized DNA motif pairs, with or without periodic spacing. Our evaluations show that it can detect weak periodic patterns which are not easily discovered using a sequential approach, i.e. first finding the binding sites and second analyzing the properties of their pairwise distances.

  16. STEME: a robust, accurate motif finder for large data sets.

    Directory of Open Access Journals (Sweden)

    John E Reid

    Full Text Available Motif finding is a difficult problem that has been studied for over 20 years. Some older popular motif finders are not suitable for analysis of the large data sets generated by next-generation sequencing. We recently published an efficient approximation (STEME to the EM algorithm that is at the core of many motif finders such as MEME. This approximation allows the EM algorithm to be applied to large data sets. In this work we describe several efficient extensions to STEME that are based on the MEME algorithm. Together with the original STEME EM approximation, these extensions make STEME a fully-fledged motif finder with similar properties to MEME. We discuss the difficulty of objectively comparing motif finders. We show that STEME performs comparably to existing prominent discriminative motif finders, DREME and Trawler, on 13 sets of transcription factor binding data in mouse ES cells. We demonstrate the ability of STEME to find long degenerate motifs which these discriminative motif finders do not find. As part of our method, we extend an earlier method due to Nagarajan et al. for the efficient calculation of motif E-values. STEME's source code is available under an open source license and STEME is available via a web interface.

  17. MINER: software for phylogenetic motif identification

    OpenAIRE

    La, David; Livesay, Dennis R.

    2005-01-01

    MINER is web-based software for phylogenetic motif (PM) identification. PMs are sequence regions (fragments) that conserve the overall familial phylogeny. PMs have been shown to correspond to a wide variety of catalytic regions, substrate-binding sites and protein interfaces, making them ideal functional site predictions. The MINER output provides an intuitive interface for interactive PM sequence analysis and structural visualization. The web implementation of MINER is freely available at . ...

  18. A novel cryptic binding motif, LRSKSRSFQVSDEQY, in the C-terminal fragment of MMP-3/7-cleaved osteopontin as a novel ligand for α9β1 integrin is involved in the anti-type II collagen antibody-induced arthritis.

    Directory of Open Access Journals (Sweden)

    Shigeyuki Kon

    Full Text Available Osteopontin (OPN is a multifunctional protein that has been linked to various intractable inflammatory diseases. One way by which OPN induces inflammation is the production of various functional fragments by enzyme cleavage. It has been well appreciated that OPN is cleaved by thrombin, and/or matrix metalloproteinase-3 and -7 (MMP-3/7. Although the function of thrombin-cleaved OPN is well characterized, little is known about the function of MMP-3/7-cleaved OPN. In this study, we found a novel motif, LRSKSRSFQVSDEQY, in the C-terminal fragment of MMP-3/7-cleaved mouse OPN binds to α9β1 integrin. Importantly, this novel motif is involved in the development of anti-type II collagen antibody-induced arthritis (CAIA. This study provides the first in vitro and in vivo evidence that OPN cleavage by MMP-3/7 is an important regulatory mechanism for CAIA.

  19. Assessing the effects of symmetry on motif discovery and modeling.

    Directory of Open Access Journals (Sweden)

    Lala M Motlhabi

    Full Text Available BACKGROUND: Identifying the DNA binding sites for transcription factors is a key task in modeling the gene regulatory network of a cell. Predicting DNA binding sites computationally suffers from high false positives and false negatives due to various contributing factors, including the inaccurate models for transcription factor specificity. One source of inaccuracy in the specificity models is the assumption of asymmetry for symmetric models. METHODOLOGY/PRINCIPAL FINDINGS: Using simulation studies, so that the correct binding site model is known and various parameters of the process can be systematically controlled, we test different motif finding algorithms on both symmetric and asymmetric binding site data. We show that if the true binding site is asymmetric the results are unambiguous and the asymmetric model is clearly superior to the symmetric model. But if the true binding specificity is symmetric commonly used methods can infer, incorrectly, that the motif is asymmetric. The resulting inaccurate motifs lead to lower sensitivity and specificity than would the correct, symmetric models. We also show how the correct model can be obtained by the use of appropriate measures of statistical significance. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that the most commonly used motif-finding approaches usually model symmetric motifs incorrectly, which leads to higher than necessary false prediction errors. It also demonstrates how alternative motif-finding methods can correct the problem, providing more accurate motif models and reducing the errors. Furthermore, it provides criteria for determining whether a symmetric or asymmetric model is the most appropriate for any experimental dataset.

  20. Structure of the HopA1(21-102)-ShcA chaperone-effector complex of Pseudomonas syringae reveals conservation of a virulence factor binding motif from animal to plant pathogens.

    Science.gov (United States)

    Janjusevic, Radmila; Quezada, Cindy M; Small, Jennifer; Stebbins, C Erec

    2013-02-01

    Pseudomonas syringae injects numerous bacterial proteins into host plant cells through a type 3 secretion system (T3SS). One of the first such bacterial effectors discovered, HopA1, is a protein that has unknown functions in the host cell but possesses close homologs that trigger the plant hypersensitive response in resistant strains. Like the virulence factors in many bacterial pathogens of animals, HopA1 depends upon a cognate chaperone in order to be effectively translocated by the P. syringae T3SS. Herein, we report the crystal structure of a complex of HopA1(21-102) with its chaperone, ShcA, determined to 1.56-Å resolution. The structure reveals that three key features of the chaperone-effector interactions found in animal pathogens are preserved in the Gram-negative pathogens of plants, namely, (i) the interaction of the chaperone with a nonglobular polypeptide of the effector, (ii) an interaction centered on the so-called β-motif, and (iii) the presence of a conserved hydrophobic patch in the chaperone that recognizes the β-motif. Structure-based mutagenesis and biochemical studies have established that the β-motif is critical for the stability of this complex. Overall, these results show that the β-motif interactions are broadly conserved in bacterial pathogens utilizing T3SSs, spanning an interkingdom host range.

  1. Effects of PBM in different energy densities and irradiance on maintaining cell viability and proliferation of pulp fibroblasts from human primary teeth.

    Science.gov (United States)

    Marques, Nádia Carolina Teixeira; Neto, Natalino Lourenço; Prado, Mariel Tavares Oliveira; Vitor, Luciana Lourenço Ribeiro; Oliveira, Rodrigo Cardoso; Sakai, Vivien Thiemy; Santos, Carlos Ferreira; Machado, Maria Aparecida Andrade Moreira; Oliveira, Thais Marchini

    2017-08-11

    This study aimed to compare the effects of photobiomodulation (PBM) in different energy densities and irradiances on maintaining cell viability, and proliferation of pulp fibroblasts from human primary teeth (HPF) were cultured in DMEM and used between the fourth and eighth passages. Then, HPF were irradiated with the following different energy densities: 1.25 J/cm(2) (a), 2.50 J/cm(2) (b), 3.75 J/cm(2) (c), 5.00 J/cm(2) (d), and 6.25 J/cm(2) (e); but varying either the time of irradiation (groups 1a-1e) or the output power (groups 2a-2e). Positive (groups 1f and 2f) and negative controls (groups 1g and 2g), respectively, comprised non-irradiated cells grown in regular nutritional conditions (10% fetal bovine serum [FBS]) and under nutritional deficit (1% FBS). Cell viability and proliferation were respectively assessed through MTT and crystal violet (CV) assays at 24, 48, and 72 h after irradiation. Statistical analysis was performed by two-way ANOVA, followed by Tukey test (P < 0.05). The negative controls showed significantly lower viability in relation to most of the corresponding subgroups, both for MTT and CV assays. For both assays, the intragroup comparison showed that the periods of 24 h exhibited lower viability than the periods of 48 and 72 h for most of the subgroups, except the negative controls with lower viability. The different irradiation protocols (equal energy densities applied with different irradiances) showed no statistically significant differences on cell viability and proliferation at the evaluated periods. The proposed PBM in different energy densities and irradiance did not affect the viability and proliferation of pulp fibroblasts from human primary teeth.

  2. BlockLogo: Visualization of peptide and sequence motif conservation

    DEFF Research Database (Denmark)

    Olsen, Lars Rønn; Kudahl, Ulrich Johan; Simon, Christian

    2013-01-01

    , selection of motif positions, type of sequence, and output format definition. The output has BlockLogo along with the sequence logo, and a table of motif frequencies. We deployed BlockLogo as an online application and have demonstrated its utility through examples that show visualization of T-cell epitopes...... and B-cell epitopes (both continuous and discontinuous). Our additional example shows a visualization and analysis of structural motifs that determine the specificity of peptide binding to HLA-DR molecules. The BlockLogo server also employs selected experimentally validated prediction algorithms...

  3. The effect of orthology and coregulation on detecting regulatory motifs.

    Directory of Open Access Journals (Sweden)

    Valerie Storms

    Full Text Available BACKGROUND: Computational de novo discovery of transcription factor binding sites is still a challenging problem. The growing number of sequenced genomes allows integrating orthology evidence with coregulation information when searching for motifs. Moreover, the more advanced motif detection algorithms explicitly model the phylogenetic relatedness between the orthologous input sequences and thus should be well adapted towards using orthologous information. In this study, we evaluated the conditions under which complementing coregulation with orthologous information improves motif detection for the class of probabilistic motif detection algorithms with an explicit evolutionary model. METHODOLOGY: We designed datasets (real and synthetic covering different degrees of coregulation and orthologous information to test how well Phylogibbs and Phylogenetic sampler, as representatives of the motif detection algorithms with evolutionary model performed as compared to MEME, a more classical motif detection algorithm that treats orthologs independently. RESULTS AND CONCLUSIONS: Under certain conditions detecting motifs in the combined coregulation-orthology space is indeed more efficient than using each space separately, but this is not always the case. Moreover, the difference in success rate between the advanced algorithms and MEME is still marginal. The success rate of motif detection depends on the complex interplay between the added information and the specificities of the applied algorithms. Insights in this relation provide information useful to both developers and users. All benchmark datasets are available at http://homes.esat.kuleuven.be/~kmarchal/Supplementary_Storms_Valerie_PlosONE.

  4. The distribution of RNA motifs in natural sequences.

    Science.gov (United States)

    Bourdeau, V; Ferbeyre, G; Pageau, M; Paquin, B; Cedergren, R

    1999-11-15

    Functional analysis of genome sequences has largely ignored RNA genes and their structures. We introduce here the notion of 'ribonomics' to describe the search for the distribution of and eventually the determination of the physiological roles of these RNA structures found in the sequence databases. The utility of this approach is illustrated here by the identification in the GenBank database of RNA motifs having known binding or chemical activity. The frequency of these motifs indicates that most have originated from evolutionary drift and are selectively neutral. On the other hand, their distribution among species and their location within genes suggest that the destiny of these motifs may be more elaborate. For example, the hammerhead motif has a skewed organismal presence, is phylogenetically stable and recent work on a schistosome version confirms its in vivo biological activity. The under-representation of the valine-binding motif and the Rev-binding element in GenBank hints at a detrimental effect on cell growth or viability. Data on the presence and the location of these motifs may provide critical guidance in the design of experiments directed towards the understanding and the manipulation of RNA complexes and activities in vivo.

  5. BayesMD: flexible biological modeling for motif discovery

    DEFF Research Database (Denmark)

    Tang, Man-Hung Eric; Krogh, Anders; Winther, Ole

    2008-01-01

    We present BayesMD, a Bayesian Motif Discovery model with several new features. Three different types of biological a priori knowledge are built into the framework in a modular fashion. A mixture of Dirichlets is used as prior over nucleotide probabilities in binding sites. It is trained on trans......We present BayesMD, a Bayesian Motif Discovery model with several new features. Three different types of biological a priori knowledge are built into the framework in a modular fashion. A mixture of Dirichlets is used as prior over nucleotide probabilities in binding sites. It is trained...

  6. MEME: discovering and analyzing DNA and protein sequence motifs.

    Science.gov (United States)

    Bailey, Timothy L; Williams, Nadya; Misleh, Chris; Li, Wilfred W

    2006-07-01

    MEME (Multiple EM for Motif Elicitation) is one of the most widely used tools for searching for novel 'signals' in sets of biological sequences. Applications include the discovery of new transcription factor binding sites and protein domains. MEME works by searching for repeated, ungapped sequence patterns that occur in the DNA or protein sequences provided by the user. Users can perform MEME searches via the web server hosted by the National Biomedical Computation Resource (http://meme.nbcr.net) and several mirror sites. Through the same web server, users can also access the Motif Alignment and Search Tool to search sequence databases for matches to motifs encoded in several popular formats. By clicking on buttons in the MEME output, users can compare the motifs discovered in their input sequences with databases of known motifs, search sequence databases for matches to the motifs and display the motifs in various formats. This article describes the freely accessible web server and its architecture, and discusses ways to use MEME effectively to find new sequence patterns in biological sequences and analyze their significance.

  7. Selection against spurious promoter motifs correlates withtranslational efficiency across bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Froula, Jeffrey L.; Francino, M. Pilar

    2007-05-01

    Because binding of RNAP to misplaced sites could compromise the efficiency of transcription, natural selection for the optimization of gene expression should regulate the distribution of DNA motifs capable of RNAP-binding across the genome. Here we analyze the distribution of the -10 promoter motifs that bind the {sigma}{sup 70} subunit of RNAP in 42 bacterial genomes. We show that selection on these motifs operates across the genome, maintaining an over-representation of -10 motifs in regulatory sequences while eliminating them from the nonfunctional and, in most cases, from the protein coding regions. In some genomes, however, -10 sites are over-represented in the coding sequences; these sites could induce pauses effecting regulatory roles throughout the length of a transcriptional unit. For nonfunctional sequences, the extent of motif under-representation varies across genomes in a manner that broadly correlates with the number of tRNA genes, a good indicator of translational speed and growth rate. This suggests that minimizing the time invested in gene transcription is an important selective pressure against spurious binding. However, selection against spurious binding is detectable in the reduced genomes of host-restricted bacteria that grow at slow rates, indicating that components of efficiency other than speed may also be important. Minimizing the number of RNAP molecules per cell required for transcription, and the corresponding energetic expense, may be most relevant in slow growers. These results indicate that genome-level properties affecting the efficiency of transcription and translation can respond in an integrated manner to optimize gene expression. The detection of selection against promoter motifs in nonfunctional regions also implies that no sequence may evolve free of selective constraints, at least in the relatively small and unstructured genomes of bacteria.

  8. MINER: software for phylogenetic motif identification.

    Science.gov (United States)

    La, David; Livesay, Dennis R

    2005-07-01

    MINER is web-based software for phylogenetic motif (PM) identification. PMs are sequence regions (fragments) that conserve the overall familial phylogeny. PMs have been shown to correspond to a wide variety of catalytic regions, substrate-binding sites and protein interfaces, making them ideal functional site predictions. The MINER output provides an intuitive interface for interactive PM sequence analysis and structural visualization. The web implementation of MINER is freely available at http://www.pmap.csupomona.edu/MINER/. Source code is available to the academic community on request.

  9. Exhaustive Search for Over-represented DNA Sequence Motifs with CisFinder

    Science.gov (United States)

    Sharov, Alexei A.; Ko, Minoru S.H.

    2009-01-01

    We present CisFinder software, which generates a comprehensive list of motifs enriched in a set of DNA sequences and describes them with position frequency matrices (PFMs). A new algorithm was designed to estimate PFMs directly from counts of n-mer words with and without gaps; then PFMs are extended over gaps and flanking regions and clustered to generate non-redundant sets of motifs. The algorithm successfully identified binding motifs for 12 transcription factors (TFs) in embryonic stem cells based on published chromatin immunoprecipitation sequencing data. Furthermore, CisFinder successfully identified alternative binding motifs of TFs (e.g. POU5F1, ESRRB, and CTCF) and motifs for known and unknown co-factors of genes associated with the pluripotent state of ES cells. CisFinder also showed robust performance in the identification of motifs that were only slightly enriched in a set of DNA sequences. PMID:19740934

  10. Effect of preparation procedure and nanostructuring on the thermoelectric properties of the lead telluride-based material system AgPbmBiTe2+m (BLST-m)

    Science.gov (United States)

    Falkenbach, Oliver; Schmitz, Andreas; Hartung, David; Dankwort, Torben; Koch, Guenter; Kienle, Lorenz; Klar, Peter J.; Mueller, Eckhard; Schlecht, Sabine

    2016-06-01

    We report on the preparation and thermoelectric properties of the quaternary system AgPbmBiTe2+m (Bismuth-Lead-Silver-Tellurium, BLST-m) that were nanostructured by mechanical alloying. Nanopowders of various compositions were compacted by three different methods: cold pressing/annealing, hot pressing, and short term sintering. The products are compared with respect to microstructure and sample density. The thermoelectric properties were measured: thermal conductivity in the temperature range from 300 K to 800 K and electrical conductivity and Seebeck coefficient between 100 K and 800 K. The compacting method and the composition had a substantial impact on carrier concentration and mobility as well as on the thermoelectric parameters. Room temperature Hall measurements yielded carrier concentrations in the order of 1019 cm-3, slightly increasing with increasing content of the additive silver bismuth telluride to the lead telluride base. ZT values close to the ones of bulk samples were achieved. X-ray diffraction and transmission electron microscopy (TEM) showed macroscopically homogeneous distributions of the constituting elements inside the nanopowders ensembles, indicating a solid solution. However, high resolution transmission electron microscopy (HRTEM) revealed disorder on the nanoscale inside individual nanopowders grains.

  11. PBM: a software package to create, display and manipulate interactively models of small molecules and proteins on IBM-compatible PCs.

    Science.gov (United States)

    Perrakis, A; Constantinides, C; Athanasiades, A; Hamodrakas, S J

    1995-04-01

    The PBM package was developed to create, display and conveniently manipulate protein and small molecule structures on IBM-compatible microcomputers. It consists of four modules: CREATE, SPHERE, RIBBON and CONVERT. CREATE includes commands to create or alter ('mutate') the primary and subsequently the tertiary structure of a given peptide or protein by defining phi and psi angles of residues at will, options to add, delete or alter atoms in a structure, utilities to choose easily between the most common rotamers of amino acid residue sidechains and options to analyse in various ways a protein conformation. SPHERE provides for an interactive manipulation of structures containing up to 2700 atoms which can belong up to six different molecules. All manipulations can be made with the use of an ordinary mouse, by choosing from a variety of pull-down menus. Three types of models can be implemented to display molecules on the computer screen or the plotter: skeletal, solid space-filling and wireframe space-filling models. RIBBON creates ribbon models of proteins and allows for a limited variety of interactive manipulations. CONVERT is a file converter, which is capable of converting files of atom coordinates of literally any format to Brookhaven Data Bank format files. The package produces very good results for protein molecules of reasonable sizes, both in terms of graphics quality and speed of operations, on an 80486 IBM PC-compatible machine equipped with a 1 MByte VGA display card and a colour VGA monitor, which is a recommended configuration.

  12. A speedup technique for (l, d-motif finding algorithms

    Directory of Open Access Journals (Sweden)

    Dinh Hieu

    2011-03-01

    Full Text Available Abstract Background The discovery of patterns in DNA, RNA, and protein sequences has led to the solution of many vital biological problems. For instance, the identification of patterns in nucleic acid sequences has resulted in the determination of open reading frames, identification of promoter elements of genes, identification of intron/exon splicing sites, identification of SH RNAs, location of RNA degradation signals, identification of alternative splicing sites, etc. In protein sequences, patterns have proven to be extremely helpful in domain identification, location of protease cleavage sites, identification of signal peptides, protein interactions, determination of protein degradation elements, identification of protein trafficking elements, etc. Motifs are important patterns that are helpful in finding transcriptional regulatory elements, transcription factor binding sites, functional genomics, drug design, etc. As a result, numerous papers have been written to solve the motif search problem. Results Three versions of the motif search problem have been proposed in the literature: Simple Motif Search (SMS, (l, d-motif search (or Planted Motif Search (PMS, and Edit-distance-based Motif Search (EMS. In this paper we focus on PMS. Two kinds of algorithms can be found in the literature for solving the PMS problem: exact and approximate. An exact algorithm identifies the motifs always and an approximate algorithm may fail to identify some or all of the motifs. The exact version of PMS problem has been shown to be NP-hard. Exact algorithms proposed in the literature for PMS take time that is exponential in some of the underlying parameters. In this paper we propose a generic technique that can be used to speedup PMS algorithms. Conclusions We present a speedup technique that can be used on any PMS algorithm. We have tested our speedup technique on a number of algorithms. These experimental results show that our speedup technique is indeed very

  13. Exhaustive Search for Over-represented DNA Sequence Motifs with CisFinder

    OpenAIRE

    Sharov, Alexei A; Minoru S.H. Ko

    2009-01-01

    We present CisFinder software, which generates a comprehensive list of motifs enriched in a set of DNA sequences and describes them with position frequency matrices (PFMs). A new algorithm was designed to estimate PFMs directly from counts of n-mer words with and without gaps; then PFMs are extended over gaps and flanking regions and clustered to generate non-redundant sets of motifs. The algorithm successfully identified binding motifs for 12 transcription factors (TFs) in embryonic stem cel...

  14. Minimal motif peptide structure of metzincin clan zinc peptidases in micelles.

    Science.gov (United States)

    Onoda, Akira; Suzuki, Takako; Ishizuka, Hiroaki; Sugiyama, Rumiko; Ariyasu, Shinya; Yamamura, Takeshi

    2009-12-01

    It is well known that the functions of metalloproteins generally originate from their metal-binding motifs. However, the intrinsic nature of individual motifs remains unknown, particularly the details about metal-binding effects on the folding of motifs; the converse is also unknown, although there is no doubt that the motif is the core of the reactivity for each metalloprotein. In this study, we focused our attention on the zinc-binding motif of the metzincin clan family, HEXXHXXGXXH; this family contains the general zinc-binding sequence His-Glu-Xaa-Xaa-His (HEXXH) and the extended GXXH region. We adopted the motif sequence of stromelysin-1 and investigated the folding properties of the Trp-labeled peptides WAHEIAHSLGLFHA (STR-W1), AWHEIAHSLGLFHA (STR-W2), AHEIAHSLGWFHA (STR-W11), and AHEIAHSLGLFHWA (STR-W14) in the presence and absence of zinc ions in hydrophobic micellar environments by circular dichroism (CD) measurements. We accessed successful incorporation of these zinc peptides into micelles using quenching of Trp fluorescence. Results of CD studies indicated that two of the Trp-incorporated peptides, STR-W1 and STR-W14, exhibited helical folding in the hydrophobic region of cetyltrimethylammonium chloride micelle. The NMR structural analysis of the apo STR-W14 revealed that the conformation in the C-terminus GXXH region significantly differred between the apo state in the micelle and the reported Zn-bound state of stromelysin-1 in crystal structures. The structural analyses of the qualitative Zn-binding properties of this motif peptide provide an interesting Zn-binding mechanism: the minimum consensus motif in the metzincin clan, a basic zinc-binding motif with an extended GXXH region, has the potential to serve as a preorganized Zn binding scaffold in a hydrophobic environment.

  15. Linear motif atlas for phosphorylation-dependent signaling

    DEFF Research Database (Denmark)

    Miller, Martin Lee; Jensen, LJ; Diella, F;

    2008-01-01

    Systematic and quantitative analysis of protein phosphorylation is revealing dynamic regulatory networks underlying cellular responses to environmental cues. However, matching these sites to the kinases that phosphorylate them and the phosphorylation-dependent binding domains that may subsequently...... sequence models of linear motifs. The atlas is available as a community resource (http://netphorest.info)....

  16. How curved membranes recruit amphipathic helices and protein anchoring motifs

    DEFF Research Database (Denmark)

    Hatzakis, Nikos; Bhatia, Vikram Kjøller; Larsen, Jannik;

    2009-01-01

    Lipids and several specialized proteins are thought to be able to sense the curvature of membranes (MC). Here we used quantitative fluorescence microscopy to measure curvature-selective binding of amphipathic motifs on single liposomes 50-700 nm in diameter. Our results revealed that sensing...

  17. RNA recognition motif (RRM)-containing proteins in Bombyx mori

    African Journals Online (AJOL)

    STORAGESEVER

    2009-03-20

    Mar 20, 2009 ... containing proteins in B. mori and may serve as a basis ... and domain structures, and then orthologous proteins were assigned with similar .... DQ648521. CG10466. RNA binding motif protein,. X-linked. 2. (RBMX2). 1RRM. 1 ... Polymerase delta ... tion or initiation, 8 in transcription, and 3 in apoptosis. For.

  18. Network motifs in music sequences

    CERN Document Server

    Zanette, Damian H

    2010-01-01

    In this note, I summarize ongoing research on motif distribution in networks built up out of symbolic sequences of Western musical origin. Their motif significance profiles exhibit remarkable consistency over different styles and periods, and define a class that cannot be identified with any of the four "superfamilies" to which most real networks seem to belong. Networks from music sequences possess an unusual abundance of bidirectional connections, due to the inherent reversibility of short musical note patterns. This property contributes to motif significance from both local and large-scale features of musical structure.

  19. Distinct recognition modes of FXXLF and LXXLL motifs by the androgen receptor.

    NARCIS (Netherlands)

    H.J. Dubbink (Erik Jan); R. Hersmus (Remko); C.S. Verma (Chandra); H.A.G.M. van der Korput (Hetty); C.A. Berrevoets (Cor); J. van Tol (Judith); A.C.J. Ziel-van der Made (Angelique); A.O. Brinkmann (Albert); A.C. Pike (Ashley); J. Trapman (Jan)

    2004-01-01

    textabstractAmong nuclear receptors, the androgen receptor (AR) is unique in that its ligand-binding domain (LBD) interacts with the FXXLF motif in the N-terminal domain, resembling coactivator LXXLL motifs. We compared AR- and estrogen receptor alpha-LBD interactions of the wild-t

  20. Fast and Accurate Discovery of Degenerate Linear Motifs in Protein Sequences

    Science.gov (United States)

    Levy, Emmanuel D.; Michnick, Stephen W.

    2014-01-01

    Linear motifs mediate a wide variety of cellular functions, which makes their characterization in protein sequences crucial to understanding cellular systems. However, the short length and degenerate nature of linear motifs make their discovery a difficult problem. Here, we introduce MotifHound, an algorithm particularly suited for the discovery of small and degenerate linear motifs. MotifHound performs an exact and exhaustive enumeration of all motifs present in proteins of interest, including all of their degenerate forms, and scores the overrepresentation of each motif based on its occurrence in proteins of interest relative to a background (e.g., proteome) using the hypergeometric distribution. To assess MotifHound, we benchmarked it together with state-of-the-art algorithms. The benchmark consists of 11,880 sets of proteins from S. cerevisiae; in each set, we artificially spiked-in one motif varying in terms of three key parameters, (i) number of occurrences, (ii) length and (iii) the number of degenerate or “wildcard” positions. The benchmark enabled the evaluation of the impact of these three properties on the performance of the different algorithms. The results showed that MotifHound and SLiMFinder were the most accurate in detecting degenerate linear motifs. Interestingly, MotifHound was 15 to 20 times faster at comparable accuracy and performed best in the discovery of highly degenerate motifs. We complemented the benchmark by an analysis of proteins experimentally shown to bind the FUS1 SH3 domain from S. cerevisiae. Using the full-length protein partners as sole information, MotifHound recapitulated most experimentally determined motifs binding to the FUS1 SH3 domain. Moreover, these motifs exhibited properties typical of SH3 binding peptides, e.g., high intrinsic disorder and evolutionary conservation, despite the fact that none of these properties were used as prior information. MotifHound is available (http://michnick.bcm.umontreal.ca or http

  1. A novel Bayesian DNA motif comparison method for clustering and retrieval.

    Directory of Open Access Journals (Sweden)

    Naomi Habib

    2008-02-01

    Full Text Available Characterizing the DNA-binding specificities of transcription factors is a key problem in computational biology that has been addressed by multiple algorithms. These usually take as input sequences that are putatively bound by the same factor and output one or more DNA motifs. A common practice is to apply several such algorithms simultaneously to improve coverage at the price of redundancy. In interpreting such results, two tasks are crucial: clustering of redundant motifs, and attributing the motifs to transcription factors by retrieval of similar motifs from previously characterized motif libraries. Both tasks inherently involve motif comparison. Here we present a novel method for comparing and merging motifs, based on Bayesian probabilistic principles. This method takes into account both the similarity in positional nucleotide distributions of the two motifs and their dissimilarity to the background distribution. We demonstrate the use of the new comparison method as a basis for motif clustering and retrieval procedures, and compare it to several commonly used alternatives. Our results show that the new method outperforms other available methods in accuracy and sensitivity. We incorporated the resulting motif clustering and retrieval procedures in a large-scale automated pipeline for analyzing DNA motifs. This pipeline integrates the results of various DNA motif discovery algorithms and automatically merges redundant motifs from multiple training sets into a coherent annotated library of motifs. Application of this pipeline to recent genome-wide transcription factor location data in S. cerevisiae successfully identified DNA motifs in a manner that is as good as semi-automated analysis reported in the literature. Moreover, we show how this analysis elucidates the mechanisms of condition-specific preferences of transcription factors.

  2. Identification of coupling DNA motif pairs on long-range chromatin interactions in human K562 cells

    KAUST Repository

    Wong, Ka-Chun

    2015-09-27

    Motivation: The protein-DNA interactions between transcription factors (TFs) and transcription factor binding sites (TFBSs, also known as DNA motifs) are critical activities in gene transcription. The identification of the DNA motifs is a vital task for downstream analysis. Unfortunately, the long-range coupling information between different DNA motifs is still lacking. To fill the void, as the first-of-its-kind study, we have identified the coupling DNA motif pairs on long-range chromatin interactions in human. Results: The coupling DNA motif pairs exhibit substantially higher DNase accessibility than the background sequences. Half of the DNA motifs involved are matched to the existing motif databases, although nearly all of them are enriched with at least one gene ontology term. Their motif instances are also found statistically enriched on the promoter and enhancer regions. Especially, we introduce a novel measurement called motif pairing multiplicity which is defined as the number of motifs that are paired with a given motif on chromatin interactions. Interestingly, we observe that motif pairing multiplicity is linked to several characteristics such as regulatory region type, motif sequence degeneracy, DNase accessibility and pairing genomic distance. Taken into account together, we believe the coupling DNA motif pairs identified in this study can shed lights on the gene transcription mechanism under long-range chromatin interactions. © The Author 2015. Published by Oxford University Press.

  3. Efficient motif finding algorithms for large-alphabet inputs

    Directory of Open Access Journals (Sweden)

    Pavlovic Vladimir

    2010-10-01

    Full Text Available Abstract Background We consider the problem of identifying motifs, recurring or conserved patterns, in the biological sequence data sets. To solve this task, we present a new deterministic algorithm for finding patterns that are embedded as exact or inexact instances in all or most of the input strings. Results The proposed algorithm (1 improves search efficiency compared to existing algorithms, and (2 scales well with the size of alphabet. On a synthetic planted DNA motif finding problem our algorithm is over 10× more efficient than MITRA, PMSPrune, and RISOTTO for long motifs. Improvements are orders of magnitude higher in the same setting with large alphabets. On benchmark TF-binding site problems (FNP, CRP, LexA we observed reduction in running time of over 12×, with high detection accuracy. The algorithm was also successful in rapidly identifying protein motifs in Lipocalin, Zinc metallopeptidase, and supersecondary structure motifs for Cadherin and Immunoglobin families. Conclusions Our algorithm reduces computational complexity of the current motif finding algorithms and demonstrate strong running time improvements over existing exact algorithms, especially in important and difficult cases of large-alphabet sequences.

  4. Discovering motifs in ranked lists of DNA sequences.

    Directory of Open Access Journals (Sweden)

    Eran Eden

    2007-03-01

    Full Text Available Computational methods for discovery of sequence elements that are enriched in a target set compared with a background set are fundamental in molecular biology research. One example is the discovery of transcription factor binding motifs that are inferred from ChIP-chip (chromatin immuno-precipitation on a microarray measurements. Several major challenges in sequence motif discovery still require consideration: (i the need for a principled approach to partitioning the data into target and background sets; (ii the lack of rigorous models and of an exact p-value for measuring motif enrichment; (iii the need for an appropriate framework for accounting for motif multiplicity; (iv the tendency, in many of the existing methods, to report presumably significant motifs even when applied to randomly generated data. In this paper we present a statistical framework for discovering enriched sequence elements in ranked lists that resolves these four issues. We demonstrate the implementation of this framework in a software application, termed DRIM (discovery of rank imbalanced motifs, which identifies sequence motifs in lists of ranked DNA sequences. We applied DRIM to ChIP-chip and CpG methylation data and obtained the following results. (i Identification of 50 novel putative transcription factor (TF binding sites in yeast ChIP-chip data. The biological function of some of them was further investigated to gain new insights on transcription regulation networks in yeast. For example, our discoveries enable the elucidation of the network of the TF ARO80. Another finding concerns a systematic TF binding enhancement to sequences containing CA repeats. (ii Discovery of novel motifs in human cancer CpG methylation data. Remarkably, most of these motifs are similar to DNA sequence elements bound by the Polycomb complex that promotes histone methylation. Our findings thus support a model in which histone methylation and CpG methylation are mechanistically linked

  5. Motif Yggdrasil: sampling sequence motifs from a tree mixture model.

    Science.gov (United States)

    Andersson, Samuel A; Lagergren, Jens

    2007-06-01

    In phylogenetic foot-printing, putative regulatory elements are found in upstream regions of orthologous genes by searching for common motifs. Motifs in different upstream sequences are subject to mutations along the edges of the corresponding phylogenetic tree, consequently taking advantage of the tree in the motif search is an appealing idea. We describe the Motif Yggdrasil sampler; the first Gibbs sampler based on a general tree that uses unaligned sequences. Previous tree-based Gibbs samplers have assumed a star-shaped tree or partially aligned upstream regions. We give a probabilistic model (MY model) describing upstream sequences with regulatory elements and build a Gibbs sampler with respect to this model. The model allows toggling, i.e., the restriction of a position to a subset of nucleotides, but does not require aligned sequences nor edge lengths, which may be difficult to come by. We apply the collapsing technique to eliminate the need to sample nuisance parameters, and give a derivation of the predictive update formula. We show that the MY model improves the modeling of difficult motif instances and that the use of the tree achieves a substantial increase in nucleotide level correlation coefficient both for synthetic data and 37 bacterial lexA genes. We investigate the sensitivity to errors in the tree and show that using random trees MY sampler still has a performance similar to the original version.

  6. High Affinity Heme Binding to a Heme Regulatory Motif on the Nuclear Receptor Rev-erbβ Leads to Its Degradation and Indirectly Regulates Its Interaction with Nuclear Receptor Corepressor.

    Science.gov (United States)

    Carter, Eric L; Gupta, Nirupama; Ragsdale, Stephen W

    2016-01-29

    Rev-erbα and Rev-erbβ are heme-binding nuclear receptors (NR) that repress the transcription of genes involved in regulating metabolism, inflammation, and the circadian clock. Previous gene expression and co-immunoprecipitation studies led to a model in which heme binding to Rev-erbα recruits nuclear receptor corepressor 1 (NCoR1) into an active repressor complex. However, in contradiction, biochemical and crystallographic studies have shown that heme decreases the affinity of the ligand-binding domain of Rev-erb NRs for NCoR1 peptides. One explanation for this discrepancy is that the ligand-binding domain and NCoR1 peptides used for in vitro studies cannot replicate the key features of the full-length proteins used in cellular studies. However, the combined in vitro and cellular results described here demonstrate that heme does not directly promote interactions between full-length Rev-erbβ (FLRev-erbβ) and an NCoR1 construct encompassing all three NR interaction domains. NCoR1 tightly binds both apo- and heme-replete FLRev-erbβ·DNA complexes; furthermore, heme, at high concentrations, destabilizes the FLRev-erbβ·NCoR1 complex. The interaction between FLRev-erbβ and NCoR1 as well as Rev-erbβ repression at the Bmal1 promoter appear to be modulated by another cellular factor(s), at least one of which is related to the ubiquitin-proteasome pathway. Our studies suggest that heme is involved in regulating the degradation of Rev-erbβ in a manner consistent with its role in circadian rhythm maintenance. Finally, the very slow rate constant (10(-6) s(-1)) of heme dissociation from Rev-erbβ rules out a prior proposal that Rev-erbβ acts as an intracellular heme sensor.

  7. Direct vs 2-stage approaches to structured motif finding

    Directory of Open Access Journals (Sweden)

    Federico Maria

    2012-08-01

    Full Text Available Abstract Background The notion of DNA motif is a mathematical abstraction used to model regions of the DNA (known as Transcription Factor Binding Sites, or TFBSs that are bound by a given Transcription Factor to regulate gene expression or repression. In turn, DNA structured motifs are a mathematical counterpart that models sets of TFBSs that work in concert in the gene regulations processes of higher eukaryotic organisms. Typically, a structured motif is composed of an ordered set of isolated (or simple motifs, separated by a variable, but somewhat constrained number of “irrelevant” base-pairs. Discovering structured motifs in a set of DNA sequences is a computationally hard problem that has been addressed by a number of authors using either a direct approach, or via the preliminary identification and successive combination of simple motifs. Results We describe a computational tool, named SISMA, for the de-novo discovery of structured motifs in a set of DNA sequences. SISMA is an exact, enumerative algorithm, meaning that it finds all the motifs conforming to the specifications. It does so in two stages: first it discovers all the possible component simple motifs, then combines them in a way that respects the given constraints. We developed SISMA mainly with the aim of understanding the potential benefits of such a 2-stage approach w.r.t. direct methods. In fact, no 2-stage software was available for the general problem of structured motif discovery, but only a few tools that solved restricted versions of the problem. We evaluated SISMA against other published tools on a comprehensive benchmark made of both synthetic and real biological datasets. In a significant number of cases, SISMA outperformed the competitors, exhibiting a good performance also in most of the cases in which it was inferior. Conclusions A reflection on the results obtained lead us to conclude that a 2-stage approach can be implemented with many advantages over direct

  8. Sequence Length Limits for Controlling False Positives in Discovering Nucleotide Sequence Motifs

    Institute of Scientific and Technical Information of China (English)

    CHEN Lei; QiAN Zi-liang

    2008-01-01

    In the study of motif discovery, especially the transcription factor DNA binding sites discovery, a too long input sequence would return non-informative motifs rather than those biological functional motifs. This paper gave theoretical analyses and computational experiments to suggest the length limits of the input sequence. When the sequence length exceeds a certain critical point, the probability of discovering the motif decreases sharply. The work not only gave an explanation on the unsatisfying results of the existed motif discovery problems that the input sequence length might be too long and exceed the point, but also provided an estimation of input sequence length we should accept to get more meaningful and reliable results in motif discovery.

  9. Methods for Identifying Ligands that Target Nucleic Acid Molecules and Nucleic Acid Structural Motifs

    Science.gov (United States)

    Disney, Matthew D. (Inventor); Childs-Disney, Jessica L. (Inventor)

    2017-01-01

    Disclosed are methods for identifying a nucleic acid (e.g., RNA, DNA, etc.) motif which interacts with a ligand. The method includes providing a plurality of ligands immobilized on a support, wherein each particular ligand is immobilized at a discrete location on the support; contacting the plurality of immobilized ligands with a nucleic acid motif library under conditions effective for one or more members of the nucleic acid motif library to bind with the immobilized ligands; and identifying members of the nucleic acid motif library that are bound to a particular immobilized ligand. Also disclosed are methods for selecting, from a plurality of candidate ligands, one or more ligands that have increased likelihood of binding to a nucleic acid molecule comprising a particular nucleic acid motif, as well as methods for identifying a nucleic acid which interacts with a ligand.

  10. Structure-Function Analysis of PPP1R3D, a Protein Phosphatase 1 Targeting Subunit, Reveals a Binding Motif for 14-3-3 Proteins which Regulates its Glycogenic Properties

    OpenAIRE

    Rubio-Villena, Carla; Sanz, Pascual; Garcia-Gimeno, Maria Adelaida

    2015-01-01

    Protein phosphatase 1 (PP1) is one of the major protein phosphatases in eukaryotic cells. It plays a key role in regulating glycogen synthesis, by dephosphorylating crucial enzymes involved in glycogen homeostasis such as glycogen synthase (GS) and glycogen phosphorylase (GP). To play this role, PP1 binds to specific glycogen targeting subunits that, on one hand recognize the substrates to be dephosphorylated and on the other hand recruit PP1 to glycogen particles. In this work we have analyz...

  11. TNFα modulates Fibroblast Growth Factor Receptor 2 gene expression through the pRB/E2F1 pathway: identification of a non-canonical E2F binding motif.

    Directory of Open Access Journals (Sweden)

    Sirio D'Amici

    Full Text Available Interactions between epithelium and mesenchyme during wound healing are not fully understood, but Fibroblast Growth Factors (FGFs and their receptors FGFRs are recognized as key elements. FGFR2 gene encodes for two splicing transcript variants, FGFR2-IIIb or Keratinocyte Growth Factor Receptor (KGFR and FGFR2-IIIc, which differ for tissue localization and ligand specificity. Proinflammatory cytokines play an essential role in the regulation of epithelial-mesenchymal interactions, and have been indicated to stimulate FGFs production. Here we demonstrated that upregulation of FGFR2 mRNA and protein expression is induced by the proinflammatory cytokines Tumor Necrosis Factor-α, Interleukin-1β and Interleukin 2. Furthermore, we found that TNFα determines FGFR2 transcriptional induction through activation of pRb, mediated by Raf and/or p38 pathways, and subsequent release of the transcription factor E2F1. Experiments based on FGFR2 promoter serial deletions and site-directed mutagenesis allowed us to identify a minimal responsive element that retains the capacity to be activated by E2F1. Computational analysis indicated that this element is a non-canonical E2F responsive motif. Thus far, the molecular mechanisms of FGFR2 upregulation during wound healing or in pathological events are not known. Our data suggest that FGFR2 expression can be modulated by local recruitment of inflammatory cytokines. Furthermore, since alterations in FGFR2 expression have been linked to the pathogenesis of certain human cancers, these findings could also provide elements for diagnosis and potential targets for novel therapeutic approaches.

  12. Structural fragment clustering reveals novel structural and functional motifs in α-helical transmembrane proteins

    Directory of Open Access Journals (Sweden)

    Vassilev Boris

    2010-04-01

    Full Text Available Abstract Background A large proportion of an organism's genome encodes for membrane proteins. Membrane proteins are important for many cellular processes, and several diseases can be linked to mutations in them. With the tremendous growth of sequence data, there is an increasing need to reliably identify membrane proteins from sequence, to functionally annotate them, and to correctly predict their topology. Results We introduce a technique called structural fragment clustering, which learns sequential motifs from 3D structural fragments. From over 500,000 fragments, we obtain 213 statistically significant, non-redundant, and novel motifs that are highly specific to α-helical transmembrane proteins. From these 213 motifs, 58 of them were assigned to function and checked in the scientific literature for a biological assessment. Seventy percent of the motifs are found in co-factor, ligand, and ion binding sites, 30% at protein interaction interfaces, and 12% bind specific lipids such as glycerol or cardiolipins. The vast majority of motifs (94% appear across evolutionarily unrelated families, highlighting the modularity of functional design in membrane proteins. We describe three novel motifs in detail: (1 a dimer interface motif found in voltage-gated chloride channels, (2 a proton transfer motif found in heme-copper oxidases, and (3 a convergently evolved interface helix motif found in an aspartate symporter, a serine protease, and cytochrome b. Conclusions Our findings suggest that functional modules exist in membrane proteins, and that they occur in completely different evolutionary contexts and cover different binding sites. Structural fragment clustering allows us to link sequence motifs to function through clusters of structural fragments. The sequence motifs can be applied to identify and characterize membrane proteins in novel genomes.

  13. Network motifs provide signatures that characterize metabolism†

    OpenAIRE

    Shellman, Erin R.; Burant, Charles F.; Schnell, Santiago

    2013-01-01

    Motifs are repeating patterns that determine the local properties of networks. In this work, we characterized all 3-node motifs using enzyme commission numbers of the International Union of Biochemistry and Molecular Biology to show that motif abundance is related to biochemical function. Further, we present a comparative analysis of motif distributions in the metabolic networks of 21 species across six kingdoms of life. We found the distribution of motif abundances to be similar between spec...

  14. A glycoconjugate antigen based on the recognition motif of a broadly neutralizing human immunodeficiency virus antibody, 2G12, is immunogenic but elicits antibodies unable to bind to the self glycans of gp120

    DEFF Research Database (Denmark)

    Astronomo, Rena D; Lee, Hing-Ken; Scanlan, Christopher N

    2008-01-01

    The glycan shield of human immunodeficiency virus type 1 (HIV-1) gp120 contributes to viral evasion from humoral immune responses. However, the shield is recognized by the HIV-1 broadly neutralizing antibody (Ab), 2G12, at a relatively conserved cluster of oligomannose glycans. The discovery of 2G...... of Man(9)GlcNAc(2) inhibits 2G12 binding to gp120 as efficiently as Man(9)GlcNAc(2) itself, indicating the potential use of Man(4) as a building block for creating immunogens. Here, we describe the development of neoglycoconjugates displaying variable copy numbers of Man(4) on bovine serum albumin (BSA...

  15. Identification of consensus binding sites clarifies FMRP binding determinants.

    Science.gov (United States)

    Anderson, Bart R; Chopra, Pankaj; Suhl, Joshua A; Warren, Stephen T; Bassell, Gary J

    2016-08-19

    Fragile X mental retardation protein (FMRP) is a multifunctional RNA-binding protein with crucial roles in neuronal development and function. Efforts aimed at elucidating how FMRP target mRNAs are selected have produced divergent sets of target mRNA and putative FMRP-bound motifs, and a clear understanding of FMRP's binding determinants has been lacking. To clarify FMRP's binding to its target mRNAs, we produced a shared dataset of FMRP consensus binding sequences (FCBS), which were reproducibly identified in two published FMRP CLIP sequencing datasets. This comparative dataset revealed that of the various sequence and structural motifs that have been proposed to specify FMRP binding, the short sequence motifs TGGA and GAC were corroborated, and a novel TAY motif was identified. In addition, the distribution of the FCBS set demonstrates that FMRP preferentially binds to the coding region of its targets but also revealed binding along 3' UTRs in a subset of target mRNAs. Beyond probing these putative motifs, the FCBS dataset of reproducibly identified FMRP binding sites is a valuable tool for investigating FMRP targets and function. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  16. Rice bZIP protein, REB, interacts with GCN4 motif in promoter of Waxy gene

    Institute of Scientific and Technical Information of China (English)

    程世军; 王宗阳; 洪孟民

    2002-01-01

    A bifactorial endosperm box (EB), which contains an endosperm motif (EM) and a GCN4 motif, was found in rice Wx promoter. EB was found in 5′ upstream region of many seed storage protein genes accounting for these genes expression exclusive in endosperm among various cereals. Many reports demonstrated that the bZIP transcription activators isolated from wheat, barley and maize, etc. regulate the gene expression through binding to the GCN4 motif. In this research, we showed that GCN4 sequence could be recognized by nuclear proteins extracted from immature rice seeds. Furthermore, a rice bZIP protein, REB was isolated by using PCR method and REB fusion protein was expressed in E. coli. The results of gel shift analysis showed that REB could recognize and bind to the GCN4 motif in the Wx gene in addition to binding to the target sequence in the promoter of α-globulin.

  17. Mechanism for activation of the growth factor-activated AGC kinases by turn motif phosphorylation

    DEFF Research Database (Denmark)

    Hauge, Camilla; Antal, Torben L; Hirschberg, Daniel

    2007-01-01

    investigated the role of the third, so-called turn motif phosphate, also located in the tail, in the AGC kinases PKB, S6K, RSK, MSK, PRK and PKC. We report cooperative action of the HM phosphate and the turn motif phosphate, because it binds a phosphoSer/Thr-binding site above the glycine-rich loop within...... the kinase domain, promoting zipper-like association of the tail with the kinase domain, serving to stabilize the HM in its kinase-activating binding site. We present a molecular model for allosteric activation of AGC kinases by the turn motif phosphate via HM-mediated stabilization of the alphaC helix. In S......6K and MSK, the turn motif phosphate thereby also protects the HM from dephosphorylation. Our results suggest that the mechanism described is a key feature in activation of upto 26 human AGC kinases....

  18. The endophytic fungus Piriformospora indica stimulates the expression of nitrate reductase and the starch-degrading enzyme glucan-water dikinase in tobacco and Arabidopsis roots through a homeodomain transcription factor that binds to a conserved motif in their promoters.

    Science.gov (United States)

    Sherameti, Irena; Shahollari, Bationa; Venus, Yvonne; Altschmied, Lothar; Varma, Ajit; Oelmüller, Ralf

    2005-07-15

    Piriformospora indica, an endophytic fungus of the Sebacinaceae family, promotes growth of Arabidopsis and tobacco seedlings and stimulates nitrogen accumulation and the expression of the genes for nitrate reductase and the starch-degrading enzyme glucan-water dikinase (SEX1) in roots. Neither growth promotion nor stimulation of the two enzymes requires heterotrimeric G proteins. P. indica also stimulates the expression of the uidA gene under the control of the Arabidopsis nitrate reductase (Nia2) promoter in transgenic tobacco seedlings. At least two regions (-470/-439 and -103/-89) are important for Nia2 promoter activity in tobacco roots. One of the regions contains an element, ATGATAGATAAT, that binds to a homeodomain transcription factor in vitro. The message for this transcription factor is up-regulated by P. indica. The transcription factor also binds to a CTGATAGATCT segment in the SEX1 promoter in vitro. We propose that the growth-promoting effect initiated by P. indica is accompanied by a co-regulated stimulation of enzymes involved in nitrate and starch metabolisms.

  19. Motif signatures of transcribed enhancers

    KAUST Repository

    Kleftogiannis, Dimitrios

    2017-09-14

    In mammalian cells, transcribed enhancers (TrEn) play important roles in the initiation of gene expression and maintenance of gene expression levels in spatiotemporal manner. One of the most challenging questions in biology today is how the genomic characteristics of enhancers relate to enhancer activities. This is particularly critical, as several recent studies have linked enhancer sequence motifs to specific functional roles. To date, only a limited number of enhancer sequence characteristics have been investigated, leaving space for exploring the enhancers genomic code in a more systematic way. To address this problem, we developed a novel computational method, TELS, aimed at identifying predictive cell type/tissue specific motif signatures. We used TELS to compile a comprehensive catalog of motif signatures for all known TrEn identified by the FANTOM5 consortium across 112 human primary cells and tissues. Our results confirm that distinct cell type/tissue specific motif signatures characterize TrEn. These signatures allow discriminating successfully a) TrEn from random controls, proxy of non-enhancer activity, and b) cell type/tissue specific TrEn from enhancers expressed and transcribed in different cell types/tissues. TELS codes and datasets are publicly available at http://www.cbrc.kaust.edu.sa/TELS.

  20. Peptide-binding motifs associated with MHC molecules common in Chinese rhesus macaques are analogous to those of human HLA supertypes and include HLA-B27-like alleles

    DEFF Research Database (Denmark)

    Mothé, Bianca R.; Southwood, Scott; Sidney, John

    2013-01-01

    and deciphering outcomes of infection and vaccine efficacy. In this study, we have provided detailed characterization of six prevalent Chinese rhesus macaque MHC class I alleles, yielding a combined phenotypic frequency of 29 %. The peptide-binding specificity of two of these alleles, Mamu-A2*01:02 and Mamu-B*010...... in humans. All six alleles characterized in the present study were found to have specificities analogous to HLA supertype alleles. These data contribute to the concept that Chinese rhesus macaque MHC immunogenetics is more similar to HLA than their Indian rhesus macaque counterparts and thereby warrants......Chinese rhesus macaques are of particular interest in simian immunodeficiency virus/human immunodeficiency virus (SIV/HIV) research as these animals have prolonged kinetics of disease progression to acquired immunodeficiency syndrome (AIDS), compared to their Indian counterparts, suggesting...

  1. Mapping of p140Cap phosphorylation sites: the EPLYA and EGLYA motifs have a key role in tyrosine phosphorylation and Csk binding, and are substrates of the Abl kinase.

    Directory of Open Access Journals (Sweden)

    Daniele Repetto

    Full Text Available Protein phosphorylation tightly regulates specific binding of effector proteins that control many diverse biological functions of cells (e. g. signaling, migration and proliferation. p140Cap is an adaptor protein, specifically expressed in brain, testis and epithelial cells, that undergoes phosphorylation and tunes its interactions with other regulatory molecules via post-translation modification. In this work, using mass spectrometry, we found that p140Cap is in vivo phosphorylated on tyrosine (Y within the peptide GEGLpYADPYGLLHEGR (from now on referred to as EGLYA as well as on three serine residues. Consistently, EGLYA has the highest score of in silico prediction of p140Cap phosphorylation. To further investigate the p140Cap function, we performed site specific mutagenesis on tyrosines inserted in EGLYA and EPLYA, a second sequence with the same highest score of phosphorylation. The mutant protein, in which both EPLYA/EGLYA tyrosines were converted to phenylalanine, was no longer tyrosine phosphorylated, despite the presence of other tyrosine residues in p140Cap sequence. Moreover, this mutant lost its ability to bind the C-terminal Src kinase (Csk, previously shown to interact with p140Cap by Far Western analysis. In addition, we found that in vitro and in HEK-293 cells, the Abelson kinase is the major kinase involved in p140Cap tyrosine phosphorylation on the EPLYA and EGLYA sequences. Overall, these data represent an original attempt to in vivo characterise phosphorylated residues of p140Cap. Elucidating the function of p140Cap will provide novel insights into its biological activity not only in normal cells, but also in tumors.

  2. Mapping of p140Cap phosphorylation sites: the EPLYA and EGLYA motifs have a key role in tyrosine phosphorylation and Csk binding, and are substrates of the Abl kinase.

    Science.gov (United States)

    Repetto, Daniele; Aramu, Simona; Boeri Erba, Elisabetta; Sharma, Nanaocha; Grasso, Silvia; Russo, Isabella; Jensen, Ole N; Cabodi, Sara; Turco, Emilia; Di Stefano, Paola; Defilippi, Paola

    2013-01-01

    Protein phosphorylation tightly regulates specific binding of effector proteins that control many diverse biological functions of cells (e. g. signaling, migration and proliferation). p140Cap is an adaptor protein, specifically expressed in brain, testis and epithelial cells, that undergoes phosphorylation and tunes its interactions with other regulatory molecules via post-translation modification. In this work, using mass spectrometry, we found that p140Cap is in vivo phosphorylated on tyrosine (Y) within the peptide GEGLpYADPYGLLHEGR (from now on referred to as EGLYA) as well as on three serine residues. Consistently, EGLYA has the highest score of in silico prediction of p140Cap phosphorylation. To further investigate the p140Cap function, we performed site specific mutagenesis on tyrosines inserted in EGLYA and EPLYA, a second sequence with the same highest score of phosphorylation. The mutant protein, in which both EPLYA/EGLYA tyrosines were converted to phenylalanine, was no longer tyrosine phosphorylated, despite the presence of other tyrosine residues in p140Cap sequence. Moreover, this mutant lost its ability to bind the C-terminal Src kinase (Csk), previously shown to interact with p140Cap by Far Western analysis. In addition, we found that in vitro and in HEK-293 cells, the Abelson kinase is the major kinase involved in p140Cap tyrosine phosphorylation on the EPLYA and EGLYA sequences. Overall, these data represent an original attempt to in vivo characterise phosphorylated residues of p140Cap. Elucidating the function of p140Cap will provide novel insights into its biological activity not only in normal cells, but also in tumors.

  3. The EDLL motif: a potent plant transcriptional activation domain from AP2/ERF transcription factors.

    Science.gov (United States)

    Tiwari, Shiv B; Belachew, Alemu; Ma, Siu Fong; Young, Melinda; Ade, Jules; Shen, Yu; Marion, Colleen M; Holtan, Hans E; Bailey, Adina; Stone, Jeffrey K; Edwards, Leslie; Wallace, Andreah D; Canales, Roger D; Adam, Luc; Ratcliffe, Oliver J; Repetti, Peter P

    2012-06-01

    In plants, the ERF/EREBP family of transcriptional regulators plays a key role in adaptation to various biotic and abiotic stresses. These proteins contain a conserved AP2 DNA-binding domain and several uncharacterized motifs. Here, we describe a short motif, termed 'EDLL', that is present in AtERF98/TDR1 and other clade members from the same AP2 sub-family. We show that the EDLL motif, which has a unique arrangement of acidic amino acids and hydrophobic leucines, functions as a strong activation domain. The motif is transferable to other proteins, and is active at both proximal and distal positions of target promoters. As such, the EDLL motif is able to partly overcome the repression conferred by the AtHB2 transcription factor, which contains an ERF-associated amphiphilic repression (EAR) motif. We further examined the activation potential of EDLL by analysis of the regulation of flowering time by NF-Y (nuclear factor Y) proteins. Genetic evidence indicates that NF-Y protein complexes potentiate the action of CONSTANS in regulation of flowering in Arabidopsis; we show that the transcriptional activation function of CONSTANS can be substituted by direct fusion of the EDLL activation motif to NF-YB subunits. The EDLL motif represents a potent plant activation domain that can be used as a tool to confer transcriptional activation potential to heterologous DNA-binding proteins.

  4. Advances in Simulation of Liquid-Liquid Two-phase Flow in Extraction Columns with CFD-PBM%萃取柱内液-液两相流CFD-PBM模拟研究进展

    Institute of Scientific and Technical Information of China (English)

    李少伟; 景山; 张琦; 吴秋林

    2012-01-01

    对萃取柱内CFD-PBM模拟研究进行了较详细的综述,包括其基本理论、不同的求解方法及模拟研究现状等.CFD-PBM模拟的基本方程包括流动方程和群体平衡方程,其相互耦合,群体平衡方程涉及破碎与聚并2个关键模型.群体平衡模型的求解方法包括直接离散化方法、矩量法、正交矩量法、直接正交矩量法、分段正交矩量法等,对这些方法的原理、优点和缺点进行了综述.目前国际上关于萃取柱内CFD模拟采用较多的是简单的欧拉-欧拉两相流模拟,考虑液滴尺寸分布和进一步的浓度分布的群体平衡模型应用较少.完善伴随传质的液-液分散体系的群体平衡模型,并将其应用于不同类型的萃取柱中,是萃取分离学科的重要任务.%A detailed review on the CFD-PBM research in the extraction columns is presented, including the fundamental theory, different solution methods, and status of the simulation research. The basic equations in the CFD-PBM simulation contain the flow equations and the population balance equation, which are coupled with each other. Coalescence and break-up models are two important models in the population equation. The methods to solve the population balance equation include the class method, the method of moments, the quadrature method of moments, the direct quadrature method of moments, and the sectional quadrature method of moments. The fundamental, advantages and disadvantaged of these methods were reviewed. The Eulerian-Eulerian method is mainly used in the CFD simulation of the two-phase flow in extraction columns to date. The PBM which considers the droplet size distribution and further the concentration distribution is relatively less used in extraction columns. To improve the PBM in a liquid-liquid dispersion system with mass transfer and to use the model in different types of extraction columns are important issues for extraction research.

  5. Genetic analysis of beta1 integrin "activation motifs" in mice

    DEFF Research Database (Denmark)

    Czuchra, Aleksandra; Meyer, Hannelore; Legate, Kyle R

    2006-01-01

    tails, leading to tail separation and integrin activation. We analyzed mice in which we mutated the tyrosines of the beta1 tail and the membrane-proximal aspartic acid required for the salt bridge. Tyrosine-to-alanine substitutions abolished beta1 integrin functions and led to a beta1 integrin......-null phenotype in vivo. Surprisingly, neither the substitution of the tyrosines with phenylalanine nor the aspartic acid with alanine resulted in an obvious defect. These data suggest that the NPXY motifs of the beta1 integrin tail are essential for beta1 integrin function, whereas tyrosine phosphorylation......Akey feature of integrins is their ability to regulate the affinity for ligands, a process termed integrin activation. The final step in integrin activation is talin binding to the NPXY motif of the integrin beta cytoplasmic domains. Talin binding disrupts the salt bridge between the alpha/beta...

  6. Genome-scale study of the importance of binding site context for transcription factor binding and gene regulation

    Directory of Open Access Journals (Sweden)

    Ronne Hans

    2008-11-01

    Full Text Available Abstract Background The rate of mRNA transcription is controlled by transcription factors that bind to specific DNA motifs in promoter regions upstream of protein coding genes. Recent results indicate that not only the presence of a motif but also motif context (for example the orientation of a motif or its location relative to the coding sequence is important for gene regulation. Results In this study we present ContextFinder, a tool that is specifically aimed at identifying cases where motif context is likely to affect gene regulation. We used ContextFinder to examine the role of motif context in S. cerevisiae both for DNA binding by transcription factors and for effects on gene expression. For DNA binding we found significant patterns of motif location bias, whereas motif orientations did not seem to matter. Motif context appears to affect gene expression even more than it affects DNA binding, as biases in both motif location and orientation were more frequent in promoters of co-expressed genes. We validated our results against data on nucleosome positioning, and found a negative correlation between preferred motif locations and nucleosome occupancy. Conclusion We conclude that the requirement for stable binding of transcription factors to DNA and their subsequent function in gene regulation can impose constraints on motif context.

  7. The Alzheimer's amyloid β-peptide (Aβ) binds a specific DNA Aβ-interacting domain (AβID) in the APP, BACE1, and APOE promoters in a sequence-specific manner: characterizing a new regulatory motif.

    Science.gov (United States)

    Maloney, Bryan; Lahiri, Debomoy K

    2011-11-15

    Deposition of extracellular plaques, primarily consisting of amyloid β peptide (Aβ), in the brain is the confirmatory diagnostic of Alzheimer's disease (AD); however, the physiological and pathological role of Aβ is not fully understood. Herein, we demonstrate novel Aβ activity as a putative transcription factor upon AD-associated genes. We used oligomers from 5'-flanking regions of the apolipoprotein E (APOE), Aβ-precursor protein (APP) and β-amyloid site cleaving enzyme-1 (BACE1) genes for electrophoretic mobility shift assay (EMSA) with different fragments of the Aβ peptide. Our results suggest that Aβ bound to an Aβ-interacting domain (AβID) with a consensus of "KGGRKTGGGG". This peptide-DNA interaction was sequence specific, and mutation of the first "G" of the decamer's terminal "GGGG" eliminated peptide-DNA interaction. Furthermore, the cytotoxic Aβ25-35 fragment had greatest DNA affinity. Such specificity of binding suggests that the AβID is worth of further investigation as a site wherein the Aβ peptide may act as a transcription factor. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. An integrative and applicable phylogenetic footprinting framework for cis-regulatory motifs identification in prokaryotic genomes.

    Science.gov (United States)

    Liu, Bingqiang; Zhang, Hanyuan; Zhou, Chuan; Li, Guojun; Fennell, Anne; Wang, Guanghui; Kang, Yu; Liu, Qi; Ma, Qin

    2016-08-09

    Phylogenetic footprinting is an important computational technique for identifying cis-regulatory motifs in orthologous regulatory regions from multiple genomes, as motifs tend to evolve slower than their surrounding non-functional sequences. Its application, however, has several difficulties for optimizing the selection of orthologous data and reducing the false positives in motif prediction. Here we present an integrative phylogenetic footprinting framework for accurate motif predictions in prokaryotic genomes (MP(3)). The framework includes a new orthologous data preparation procedure, an additional promoter scoring and pruning method and an integration of six existing motif finding algorithms as basic motif search engines. Specifically, we collected orthologous genes from available prokaryotic genomes and built the orthologous regulatory regions based on sequence similarity of promoter regions. This procedure made full use of the large-scale genomic data and taxonomy information and filtered out the promoters with limited contribution to produce a high quality orthologous promoter set. The promoter scoring and pruning is implemented through motif voting by a set of complementary predicting tools that mine as many motif candidates as possible and simultaneously eliminate the effect of random noise. We have applied the framework to Escherichia coli k12 genome and evaluated the prediction performance through comparison with seven existing programs. This evaluation was systematically carried out at the nucleotide and binding site level, and the results showed that MP(3) consistently outperformed other popular motif finding tools. We have integrated MP(3) into our motif identification and analysis server DMINDA, allowing users to efficiently identify and analyze motifs in 2,072 completely sequenced prokaryotic genomes. The performance evaluation indicated that MP(3) is effective for predicting regulatory motifs in prokaryotic genomes. Its application may enhance

  9. NestedMICA as an ab initio protein motif discovery tool

    Directory of Open Access Journals (Sweden)

    Down Thomas A

    2008-01-01

    Full Text Available Abstract Background Discovering overrepresented patterns in amino acid sequences is an important step in protein functional element identification. We adapted and extended NestedMICA, an ab initio motif finder originally developed for finding transcription binding site motifs, to find short protein signals, and compared its performance with another popular protein motif finder, MEME. NestedMICA, an open source protein motif discovery tool written in Java, is driven by a Monte Carlo technique called Nested Sampling. It uses multi-class sequence background models to represent different "uninteresting" parts of sequences that do not contain motifs of interest. In order to assess NestedMICA as a protein motif finder, we have tested it on synthetic datasets produced by spiking instances of known motifs into a randomly selected set of protein sequences. NestedMICA was also tested using a biologically-authentic test set, where we evaluated its performance with respect to varying sequence length. Results Generally NestedMICA recovered most of the short (3–9 amino acid long test protein motifs spiked into a test set of sequences at different frequencies. We showed that it can be used to find multiple motifs at the same time, too. In all the assessment experiments we carried out, its overall motif discovery performance was better than that of MEME. Conclusion NestedMICA proved itself to be a robust and sensitive ab initio protein motif finder, even for relatively short motifs that exist in only a small fraction of sequences. Availability NestedMICA is available under the Lesser GPL open-source license from: http://www.sanger.ac.uk/Software/analysis/nmica/

  10. Dietary Gluten-Induced Gut Dysbiosis Is Accompanied by Selective Upregulation of microRNAs with Intestinal Tight Junction and Bacteria-Binding Motifs in Rhesus Macaque Model of Celiac Disease.

    Science.gov (United States)

    Mohan, Mahesh; Chow, Cheryl-Emiliane T; Ryan, Caitlin N; Chan, Luisa S; Dufour, Jason; Aye, Pyone P; Blanchard, James; Moehs, Charles P; Sestak, Karol

    2016-10-28

    The composition of the gut microbiome reflects the overall health status of the host. In this study, stool samples representing the gut microbiomes from 6 gluten-sensitive (GS) captive juvenile rhesus macaques were compared with those from 6 healthy, age- and diet-matched peers. A total of 48 samples representing both groups were studied using V4 16S rRNA gene DNA analysis. Samples from GS macaques were further characterized based on type of diet administered: conventional monkey chow, i.e., wheat gluten-containing diet (GD), gluten-free diet (GFD), barley gluten-derived diet (BOMI) and reduced gluten barley-derived diet (RGB). It was hypothesized that the GD diet would lower the gut microbial diversity in GS macaques. This is the first report illustrating the reduction of gut microbial alpha-diversity (p gluten in GS macaques. Selected bacterial families (e.g., Streptococcaceae and Lactobacillaceae) were enriched in GS macaques while Coriobacteriaceae was enriched in healthy animals. Within several weeks after the replacement of the GD by the GFD diet, the composition (beta-diversity) of gut microbiome in GS macaques started to change (p = 0.011) towards that of a normal macaque. Significance for alpha-diversity however, was not reached by the day 70 when the feeding experiment ended. Several inflammation-associated microRNAs (miR-203, -204, -23a, -23b and -29b) were upregulated (p < 0.05) in jejunum of 4 biopsied GS macaques fed GD with predicted binding sites on 16S ribosomal RNA of Lactobacillus reuteri (accession number: NR_025911), Prevotella stercorea (NR_041364) and Streptococcus luteciae (AJ297218) that were overrepresented in feces. Additionally, claudin-1, a validated tight junction protein target of miR-29b was significantly downregulated in jejunal epithelium of GS macaques. Taken together, we predict that with the introduction of effective treatments in future studies the diversity of gut microbiomes in GS macaques will approach those of healthy

  11. Dietary Gluten-Induced Gut Dysbiosis Is Accompanied by Selective Upregulation of microRNAs with Intestinal Tight Junction and Bacteria-Binding Motifs in Rhesus Macaque Model of Celiac Disease

    Science.gov (United States)

    Mohan, Mahesh; Chow, Cheryl-Emiliane T.; Ryan, Caitlin N.; Chan, Luisa S.; Dufour, Jason; Aye, Pyone P.; Blanchard, James; Moehs, Charles P.; Sestak, Karol

    2016-01-01

    The composition of the gut microbiome reflects the overall health status of the host. In this study, stool samples representing the gut microbiomes from 6 gluten-sensitive (GS) captive juvenile rhesus macaques were compared with those from 6 healthy, age- and diet-matched peers. A total of 48 samples representing both groups were studied using V4 16S rRNA gene DNA analysis. Samples from GS macaques were further characterized based on type of diet administered: conventional monkey chow, i.e., wheat gluten-containing diet (GD), gluten-free diet (GFD), barley gluten-derived diet (BOMI) and reduced gluten barley-derived diet (RGB). It was hypothesized that the GD diet would lower the gut microbial diversity in GS macaques. This is the first report illustrating the reduction of gut microbial alpha-diversity (p < 0.05) following the consumption of dietary gluten in GS macaques. Selected bacterial families (e.g., Streptococcaceae and Lactobacillaceae) were enriched in GS macaques while Coriobacteriaceae was enriched in healthy animals. Within several weeks after the replacement of the GD by the GFD diet, the composition (beta-diversity) of gut microbiome in GS macaques started to change (p = 0.011) towards that of a normal macaque. Significance for alpha-diversity however, was not reached by the day 70 when the feeding experiment ended. Several inflammation-associated microRNAs (miR-203, -204, -23a, -23b and -29b) were upregulated (p < 0.05) in jejunum of 4 biopsied GS macaques fed GD with predicted binding sites on 16S ribosomal RNA of Lactobacillus reuteri (accession number: NR_025911), Prevotella stercorea (NR_041364) and Streptococcus luteciae (AJ297218) that were overrepresented in feces. Additionally, claudin-1, a validated tight junction protein target of miR-29b was significantly downregulated in jejunal epithelium of GS macaques. Taken together, we predict that with the introduction of effective treatments in future studies the diversity of gut microbiomes in GS

  12. Dietary Gluten-Induced Gut Dysbiosis Is Accompanied by Selective Upregulation of microRNAs with Intestinal Tight Junction and Bacteria-Binding Motifs in Rhesus Macaque Model of Celiac Disease

    Directory of Open Access Journals (Sweden)

    Mahesh Mohan

    2016-10-01

    Full Text Available The composition of the gut microbiome reflects the overall health status of the host. In this study, stool samples representing the gut microbiomes from 6 gluten-sensitive (GS captive juvenile rhesus macaques were compared with those from 6 healthy, age- and diet-matched peers. A total of 48 samples representing both groups were studied using V4 16S rRNA gene DNA analysis. Samples from GS macaques were further characterized based on type of diet administered: conventional monkey chow, i.e., wheat gluten-containing diet (GD, gluten-free diet (GFD, barley gluten-derived diet (BOMI and reduced gluten barley-derived diet (RGB. It was hypothesized that the GD diet would lower the gut microbial diversity in GS macaques. This is the first report illustrating the reduction of gut microbial alpha-diversity (p < 0.05 following the consumption of dietary gluten in GS macaques. Selected bacterial families (e.g., Streptococcaceae and Lactobacillaceae were enriched in GS macaques while Coriobacteriaceae was enriched in healthy animals. Within several weeks after the replacement of the GD by the GFD diet, the composition (beta-diversity of gut microbiome in GS macaques started to change (p = 0.011 towards that of a normal macaque. Significance for alpha-diversity however, was not reached by the day 70 when the feeding experiment ended. Several inflammation-associated microRNAs (miR-203, -204, -23a, -23b and -29b were upregulated (p < 0.05 in jejunum of 4 biopsied GS macaques fed GD with predicted binding sites on 16S ribosomal RNA of Lactobacillus reuteri (accession number: NR_025911, Prevotella stercorea (NR_041364 and Streptococcus luteciae (AJ297218 that were overrepresented in feces. Additionally, claudin-1, a validated tight junction protein target of miR-29b was significantly downregulated in jejunal epithelium of GS macaques. Taken together, we predict that with the introduction of effective treatments in future studies the diversity of gut microbiomes

  13. Genome-wide upstream motif analysis of Cryptosporidium parvum genes clustered by expression profile.

    Science.gov (United States)

    Oberstaller, Jenna; Joseph, Sandeep J; Kissinger, Jessica C

    2013-07-29

    There are very few molecular genetic tools available to study the apicomplexan parasite Cryptosporidium parvum. The organism is not amenable to continuous in vitro cultivation or transfection, and purification of intracellular developmental stages in sufficient numbers for most downstream molecular applications is difficult and expensive since animal hosts are required. As such, very little is known about gene regulation in C. parvum. We have clustered whole-genome gene expression profiles generated from a previous study of seven post-infection time points of 3,281 genes to identify genes that show similar expression patterns throughout the first 72 hours of in vitro epithelial cell culture. We used the algorithms MEME, AlignACE and FIRE to identify conserved, overrepresented DNA motifs in the upstream promoter region of genes with similar expression profiles. The most overrepresented motifs were E2F (5'-TGGCGCCA-3'); G-box (5'-G.GGGG-3'); a well-documented ApiAP2 binding motif (5'-TGCAT-3'), and an unknown motif (5'-[A/C] AACTA-3'). We generated a recombinant C. parvum DNA-binding protein domain from a putative ApiAP2 transcription factor [CryptoDB: cgd8_810] and determined its binding specificity using protein-binding microarrays. We demonstrate that cgd8_810 can putatively bind the overrepresented G-box motif, implicating this ApiAP2 in the regulation of many gene clusters. Several DNA motifs were identified in the upstream sequences of gene clusters that might serve as potential cis-regulatory elements. These motifs, in concert with protein DNA binding site data, establish for the first time the beginnings of a global C. parvum gene regulatory map that will contribute to our understanding of the development of this zoonotic parasite.

  14. How to find a leucine in a haystack? Structure, ligand recognition and regulation of leucine-aspartic acid (LD) motifs.

    Science.gov (United States)

    Alam, Tanvir; Alazmi, Meshari; Gao, Xin; Arold, Stefan T

    2014-06-15

    LD motifs (leucine-aspartic acid motifs) are short helical protein-protein interaction motifs that have emerged as key players in connecting cell adhesion with cell motility and survival. LD motifs are required for embryogenesis, wound healing and the evolution of multicellularity. LD motifs also play roles in disease, such as in cancer metastasis or viral infection. First described in the paxillin family of scaffolding proteins, LD motifs and similar acidic LXXLL interaction motifs have been discovered in several other proteins, whereas 16 proteins have been reported to contain LDBDs (LD motif-binding domains). Collectively, structural and functional analyses have revealed a surprising multivalency in LD motif interactions and a wide diversity in LDBD architectures. In the present review, we summarize the molecular basis for function, regulation and selectivity of LD motif interactions that has emerged from more than a decade of research. This overview highlights the intricate multi-level regulation and the inherently noisy and heterogeneous nature of signalling through short protein-protein interaction motifs.

  15. seeMotif: exploring and visualizing sequence motifs in 3D structures

    OpenAIRE

    2009-01-01

    Sequence motifs are important in the study of molecular biology. Motif discovery tools efficiently deliver many function related signatures of proteins and largely facilitate sequence annotation. As increasing numbers of motifs are detected experimentally or predicted computationally, characterizing the functional roles of motifs and identifying the potential synergetic relationships between them are important next steps. A good way to investigate novel motifs is to utilize the abundant 3D st...

  16. Detecting correlations among functional-sequence motifs

    Science.gov (United States)

    Pirino, Davide; Rigosa, Jacopo; Ledda, Alice; Ferretti, Luca

    2012-06-01

    Sequence motifs are words of nucleotides in DNA with biological functions, e.g., gene regulation. Identification of such words proceeds through rejection of Markov models on the expected motif frequency along the genome. Additional biological information can be extracted from the correlation structure among patterns of motif occurrences. In this paper a log-linear multivariate intensity Poisson model is estimated via expectation maximization on a set of motifs along the genome of E. coli K12. The proposed approach allows for excitatory as well as inhibitory interactions among motifs and between motifs and other genomic features like gene occurrences. Our findings confirm previous stylized facts about such types of interactions and shed new light on genome-maintenance functions of some particular motifs. We expect these methods to be applicable to a wider set of genomic features.

  17. Prediction of HIV-1 virus-host protein interactions using virus and host sequence motifs

    Directory of Open Access Journals (Sweden)

    Tozeren Aydin

    2009-05-01

    Full Text Available Abstract Background Host protein-protein interaction networks are altered by invading virus proteins, which create new interactions, and modify or destroy others. The resulting network topology favors excessive amounts of virus production in a stressed host cell network. Short linear peptide motifs common to both virus and host provide the basis for host network modification. Methods We focused our host-pathogen study on the binding and competing interactions of HIV-1 and human proteins. We showed that peptide motifs conserved across 70% of HIV-1 subtype B and C samples occurred in similar positions on HIV-1 proteins, and we documented protein domains that interact with these conserved motifs. We predicted which human proteins may be targeted by HIV-1 by taking pairs of human proteins that may interact via a motif conserved in HIV-1 and the corresponding interacting protein domain. Results Our predictions were enriched with host proteins known to interact with HIV-1 proteins ENV, NEF, and TAT (p-value Conclusion A list of host proteins highly enriched with those targeted by HIV-1 proteins can be obtained by searching for host protein motifs along virus protein sequences. The resulting set of host proteins predicted to be targeted by virus proteins will become more accurate with better annotations of motifs and domains. Nevertheless, our study validates the role of linear binding motifs shared by virus and host proteins as an important part of the crosstalk between virus and host.

  18. The MHC motif viewer: a visualization tool for MHC binding motifs

    DEFF Research Database (Denmark)

    Rapin, Nicolas; Hoof, Ilka; Lund, Ole

    2010-01-01

    In vertebrates, the onset of cellular immune reactions is controlled by presentation of peptides in complex with major histocompatibility complex (MHC) molecules to T cell receptors. In humans, MHCs are called human leukocyte antigens (HLAs). Different MHC molecules present different subsets...

  19. Statistical tests to compare motif count exceptionalities

    Directory of Open Access Journals (Sweden)

    Vandewalle Vincent

    2007-03-01

    Full Text Available Abstract Background Finding over- or under-represented motifs in biological sequences is now a common task in genomics. Thanks to p-value calculation for motif counts, exceptional motifs are identified and represent candidate functional motifs. The present work addresses the related question of comparing the exceptionality of one motif in two different sequences. Just comparing the motif count p-values in each sequence is indeed not sufficient to decide if this motif is significantly more exceptional in one sequence compared to the other one. A statistical test is required. Results We develop and analyze two statistical tests, an exact binomial one and an asymptotic likelihood ratio test, to decide whether the exceptionality of a given motif is equivalent or significantly different in two sequences of interest. For that purpose, motif occurrences are modeled by Poisson processes, with a special care for overlapping motifs. Both tests can take the sequence compositions into account. As an illustration, we compare the octamer exceptionalities in the Escherichia coli K-12 backbone versus variable strain-specific loops. Conclusion The exact binomial test is particularly adapted for small counts. For large counts, we advise to use the likelihood ratio test which is asymptotic but strongly correlated with the exact binomial test and very simple to use.

  20. A simple motif for protein recognition in DNA secondary structures.

    Science.gov (United States)

    Landt, Stephen G; Ramirez, Alejandro; Daugherty, Matthew D; Frankel, Alan D

    2005-09-02

    DNA in a single-stranded form (ssDNA) exists transiently within the cell and comprises the telomeres of linear chromosomes and the genomes of some DNA viruses. As with RNA, in the single-stranded state, some DNA sequences are able to fold into complex secondary and tertiary structures that may be recognized by proteins and participate in gene regulation. To better understand how such DNA elements might fold and interact with proteins, and to compare recognition features to those of a structured RNA, we used in vitro selection to identify ssDNAs that bind an RNA-binding peptide from the HIV Rev protein with high affinity and specificity. The large majority of selected binders contain a non-Watson-Crick G.T base-pair and an adjacent C:G base-pair and both are essential for binding. This GT motif can be presented in different DNA contexts, including a nearly perfect duplex and a branched three-helix structure, and appears to be recognized in large part by arginine residues separated by one turn of an alpha-helix. Interestingly, a very similar GT motif is necessary also for protein binding and function of a well-characterized model ssDNA regulatory element from the proenkephalin promoter.

  1. MEME-LaB: motif analysis in clusters.

    Science.gov (United States)

    Brown, Paul; Baxter, Laura; Hickman, Richard; Beynon, Jim; Moore, Jonathan D; Ott, Sascha

    2013-07-01

    Genome-wide expression analysis can result in large numbers of clusters of co-expressed genes. Although there are tools for ab initio discovery of transcription factor-binding sites, most do not provide a quick and easy way to study large numbers of clusters. To address this, we introduce a web tool called MEME-LaB. The tool wraps MEME (an ab initio motif finder), providing an interface for users to input multiple gene clusters, retrieve promoter sequences, run motif finding and then easily browse and condense the results, facilitating better interpretation of the results from large-scale datasets. MEME-LaB is freely accessible at: http://wsbc.warwick.ac.uk/wsbcToolsWebpage/. Supplementary data are available at Bioinformatics online.

  2. Sequence alignment reveals possible MAPK docking motifs on HIV proteins.

    Directory of Open Access Journals (Sweden)

    Perry Evans

    Full Text Available Over the course of HIV infection, virus replication is facilitated by the phosphorylation of HIV proteins by human ERK1 and ERK2 mitogen-activated protein kinases (MAPKs. MAPKs are known to phosphorylate their substrates by first binding with them at a docking site. Docking site interactions could be viable drug targets because the sequences guiding them are more specific than phosphorylation consensus sites. In this study we use multiple bioinformatics tools to discover candidate MAPK docking site motifs on HIV proteins known to be phosphorylated by MAPKs, and we discuss the possibility of targeting docking sites with drugs. Using sequence alignments of HIV proteins of different subtypes, we show that MAPK docking patterns previously described for human proteins appear on the HIV matrix, Tat, and Vif proteins in a strain dependent manner, but are absent from HIV Rev and appear on all HIV Nef strains. We revise the regular expressions of previously annotated MAPK docking patterns in order to provide a subtype independent motif that annotates all HIV proteins. One revision is based on a documented human variant of one of the substrate docking motifs, and the other reduces the number of required basic amino acids in the standard docking motifs from two to one. The proposed patterns are shown to be consistent with in silico docking between ERK1 and the HIV matrix protein. The motif usage on HIV proteins is sufficiently different from human proteins in amino acid sequence similarity to allow for HIV specific targeting using small-molecule drugs.

  3. Discovering novel sequence motifs with MEME.

    Science.gov (United States)

    Bailey, Timothy L

    2002-11-01

    This unit illustrates how to use MEME to discover motifs in a group of related nucleotide or peptide sequences. A MEME motif is a sequence pattern that occurs repeatedly in one or more sequences in the input group. MEME can be used to discover novel patterns because it bases its discoveries only on the input sequences, not on any prior knowledge (such as databases of known motifs). The input to MEME is a set of unaligned sequences of the same type (peptide or nucleotide). For each motif it discovers, MEME reports the occurrences (sites), consensus sequence, and the level of conservation (information content) at each position in the pattern. MEME also produces block diagrams showing where all of the discovered motifs occur in the training set sequences. MEME's hypertext (HTML) output also contains buttons that allow for the convenient use of the motifs in other searches.

  4. Identification of RNA binding motif proteins essential for cardiovascular development

    NARCIS (Netherlands)

    S. Maragh (Samantha); R.A. Miller (Ronald); S.L. Bessling (Seneca); D.M. McGaughey (David); M.W. Wessels (Marja); B.M. de Graaf (Bianca); E.A. Stone (Eric); A.M. Bertoli Avella (Aida); J.D. Gearhart (John); S. Fisher (Shannon); A.S. McCallion (Andrew)

    2011-01-01

    textabstractBackground: We recently identified Rbm24 as a novel gene expressed during mouse cardiac development. Due to its tightly restricted and persistent expression from formation of the cardiac crescent onwards and later in forming vasculature we posited it to be a key player in cardiogenesis w

  5. Identification of RNA binding motif proteins essential for cardiovascular development

    Directory of Open Access Journals (Sweden)

    Bertoli-Avella Aida M

    2011-10-01

    Full Text Available Abstract Background We recently identified Rbm24 as a novel gene expressed during mouse cardiac development. Due to its tightly restricted and persistent expression from formation of the cardiac crescent onwards and later in forming vasculature we posited it to be a key player in cardiogenesis with additional roles in vasculogenesis and angiogenesis. Results To determine the role of this gene in cardiac development, we have identified its zebrafish orthologs (rbm24a and rbm24b, and functionally evaluated them during zebrafish embryogenesis. Consistent with our underlying hypothesis, reduction in expression of either ortholog through injection of morpholino antisense oligonucleotides results in cardiogenic defects including cardiac looping and reduced circulation, leading to increasing pericardial edema over time. Additionally, morphant embryos for either ortholog display incompletely overlapping defects in the forming vasculature of the dorsal aorta (DA, posterior caudal vein (PCV and caudal vein (CV which are the first blood vessels to form in the embryo. Vasculogenesis and early angiogenesis in the trunk were similarly compromised in rbm24 morphant embryos at 48 hours post fertilization (hpf. Subsequent vascular maintenance was impaired in both rbm24 morphants with substantial vessel degradation noted at 72 hpf. Conclusion Taken collectively, our functional data support the hypothesis that rbm24a and rbm24b are key developmental cardiac genes with unequal roles in cardiovascular formation.

  6. Exon silencing by UAGG motifs in response to neuronal excitation.

    Directory of Open Access Journals (Sweden)

    Ping An

    2007-02-01

    Full Text Available Alternative pre-mRNA splicing plays fundamental roles in neurons by generating functional diversity in proteins associated with the communication and connectivity of the synapse. The CI cassette of the NMDA R1 receptor is one of a variety of exons that show an increase in exon skipping in response to cell excitation, but the molecular nature of this splicing responsiveness is not yet understood. Here we investigate the molecular basis for the induced changes in splicing of the CI cassette exon in primary rat cortical cultures in response to KCl-induced depolarization using an expression assay with a tight neuron-specific readout. In this system, exon silencing in response to neuronal excitation was mediated by multiple UAGG-type silencing motifs, and transfer of the motifs to a constitutive exon conferred a similar responsiveness by gain of function. Biochemical analysis of protein binding to UAGG motifs in extracts prepared from treated and mock-treated cortical cultures showed an increase in nuclear hnRNP A1-RNA binding activity in parallel with excitation. Evidence for the role of the NMDA receptor and calcium signaling in the induced splicing response was shown by the use of specific antagonists, as well as cell-permeable inhibitors of signaling pathways. Finally, a wider role for exon-skipping responsiveness is shown to involve additional exons with UAGG-related silencing motifs, and transcripts involved in synaptic functions. These results suggest that, at the post-transcriptional level, excitable exons such as the CI cassette may be involved in strategies by which neurons mount adaptive responses to hyperstimulation.

  7. How to find a leucine in a haystack? Structure, ligand recognition and regulation of leucine-aspartic acid (LD) motifs

    KAUST Repository

    Alam, Tanvir

    2014-05-29

    LD motifs (leucine-aspartic acidmotifs) are short helical protein-protein interaction motifs that have emerged as key players in connecting cell adhesion with cell motility and survival. LD motifs are required for embryogenesis, wound healing and the evolution of multicellularity. LD motifs also play roles in disease, such as in cancer metastasis or viral infection. First described in the paxillin family of scaffolding proteins, LD motifs and similar acidic LXXLL interaction motifs have been discovered in several other proteins, whereas 16 proteins have been reported to contain LDBDs (LD motif-binding domains). Collectively, structural and functional analyses have revealed a surprising multivalency in LD motif interactions and a wide diversity in LDBD architectures. In the present review, we summarize the molecular basis for function, regulation and selectivity of LD motif interactions that has emerged from more than a decade of research. This overview highlights the intricate multi-level regulation and the inherently noisy and heterogeneous nature of signalling through short protein-protein interaction motifs. © 2014 Biochemical Society.

  8. Disparate requirements for the Walker A and B ATPase motifs ofhuman RAD51D in homologous recombination

    Energy Technology Data Exchange (ETDEWEB)

    Wiese, Claudia; Hinz, John M.; Tebbs, Robert S.; Nham, Peter B.; Urbin, Salustra S.; Collins, David W.; Thompson, Larry H.; Schild, David

    2006-04-21

    In vertebrates, homologous recombinational repair (HRR) requires RAD51 and five RAD51 paralogs (XRCC2, XRCC3, RAD51B, RAD51C, and RAD51D) that all contain conserved Walker A and B ATPase motifs. In human RAD51D we examined the requirement for these motifs in interactions with XRCC2 and RAD51C, and for survival of cells in response to DNA interstrand crosslinks. Ectopic expression of wild type human RAD51D or mutants having a non-functional A or B motif was used to test for complementation of a rad51d knockout hamster CHO cell line. Although A-motif mutants complement very efficiently, B-motif mutants do not. Consistent with these results, experiments using the yeast two- and three-hybrid systems show that the interactions between RAD51D and its XRCC2 and RAD51C partners also require a functional RAD51D B motif, but not motif A. Similarly, hamster Xrcc2 is unable to bind to the non-complementing human RAD51D B-motif mutants in co-immunoprecipitation assays. We conclude that a functional Walker B motif, but not A motif, is necessary for RAD51D's interactions with other paralogs and for efficient HRR. We present a model in which ATPase sites are formed in a bipartite manner between RAD51D and other RAD51 paralogs.

  9. Motif-specific sampling of phosphoproteomes.

    Science.gov (United States)

    Ruse, Cristian I; McClatchy, Daniel B; Lu, Bingwen; Cociorva, Daniel; Motoyama, Akira; Park, Sung Kyu; Yates, John R

    2008-05-01

    Phosphoproteomics, the targeted study of a subfraction of the proteome which is modified by phosphorylation, has become an indispensable tool to study cell signaling dynamics. We described a methodology that linked phosphoproteome and proteome analysis based on Ba2+ binding properties of amino acids. This technology selected motif-specific phosphopeptides independent of the system under analysis. MudPIT (Multidimensional Identification Technology) identified 1037 precipitated phosphopeptides from as little as 250 microg of proteins. To extend coverage of the phosphoproteome, we sampled the nuclear extract of HeLa cells with three values of Ba2+ ions molarity. The presence of more than 70% of identified phosphoproteins was further substantiated by their nonmodified peptides. Upon isoproterenol stimulation of HEK cells, we identified an increasing number of phosphoproteins from MAPK cascades and AKAP signaling hubs. We quantified changes in both protein and phosphorylation levels of 197 phosphoproteins including a critical kinase, MAPK1. Integration of differential phosphorylation of MAPK1 with knowledge bases constructed modules that correlated well with its role as node in cross-talk of canonical pathways.

  10. Crystal structure of the G3BP2 NTF2-like domain in complex with a canonical FGDF motif peptide

    DEFF Research Database (Denmark)

    Kristensen, Ole

    2015-01-01

    The crystal structure of the NTF2-like domain of the human Ras GTPase SH3 Binding Protein (G3BP), isoform 2, was determined at a resolution of 2.75 Å in complex with a peptide containing a FGDF sequence motif. The overall structure of the protein is highly similar to the homodimeric N...... molecular modeling suggested that FGDF-motif containing peptides bind in an extended conformation into a hydrophobic groove on the surface of the G3BP NTF2-like domain in a manner similar to the known binding of FxFG nucleoporin repeats. The results in this paper provide evidence for a different binding...

  11. Characterization of protein hubs by inferring interacting motifs from protein interactions.

    Directory of Open Access Journals (Sweden)

    Ramon Aragues

    2007-09-01

    Full Text Available The characterization of protein interactions is essential for understanding biological systems. While genome-scale methods are available for identifying interacting proteins, they do not pinpoint the interacting motifs (e.g., a domain, sequence segments, a binding site, or a set of residues. Here, we develop and apply a method for delineating the interacting motifs of hub proteins (i.e., highly connected proteins. The method relies on the observation that proteins with common interaction partners tend to interact with these partners through a common interacting motif. The sole input for the method are binary protein interactions; neither sequence nor structure information is needed. The approach is evaluated by comparing the inferred interacting motifs with domain families defined for 368 proteins in the Structural Classification of Proteins (SCOP. The positive predictive value of the method for detecting proteins with common SCOP families is 75% at sensitivity of 10%. Most of the inferred interacting motifs were significantly associated with sequence patterns, which could be responsible for the common interactions. We find that yeast hubs with multiple interacting motifs are more likely to be essential than hubs with one or two interacting motifs, thus rationalizing the previously observed correlation between essentiality and the number of interacting partners of a protein. We also find that yeast hubs with multiple interacting motifs evolve slower than the average protein, contrary to the hubs with one or two interacting motifs. The proposed method will help us discover unknown interacting motifs and provide biological insights about protein hubs and their roles in interaction networks.

  12. Pipeline for the Analysis of ChIP-seq Data and New Motif Ranking Procedure

    KAUST Repository

    Ashoor, Haitham

    2011-06-01

    This thesis presents a computational methodology for ab-initio identification of transcription factor binding sites based on ChIP-seq data. This method consists of three main steps, namely ChIP-seq data processing, motif discovery and models selection. A novel method for ranking the models of motifs identified in this process is proposed. This method combines multiple factors in order to rank the provided candidate motifs. It combines the model coverage of the ChIP-seq fragments that contain motifs from which that model is built, the suitable background data made up of shuffled ChIP-seq fragments, and the p-value that resulted from evaluating the model on actual and background data. Two ChIP-seq datasets retrieved from ENCODE project are used to evaluate and demonstrate the ability of the method to predict correct TFBSs with high precision. The first dataset relates to neuron-restrictive silencer factor, NRSF, while the second one corresponds to growth-associated binding protein, GABP. The pipeline system shows high precision prediction for both datasets, as in both cases the top ranked motif closely resembles the known motifs for the respective transcription factors.

  13. Prevalent RNA recognition motif duplication in the human genome.

    Science.gov (United States)

    Tsai, Yihsuan S; Gomez, Shawn M; Wang, Zefeng

    2014-05-01

    The sequence-specific recognition of RNA by proteins is mediated through various RNA binding domains, with the RNA recognition motif (RRM) being the most frequent and present in >50% of RNA-binding proteins (RBPs). Many RBPs contain multiple RRMs, and it is unclear how each RRM contributes to the binding specificity of the entire protein. We found that RRMs within the same RBP (i.e., sibling RRMs) tend to have significantly higher similarity than expected by chance. Sibling RRM pairs from RBPs shared by multiple species tend to have lower similarity than those found only in a single species, suggesting that multiple RRMs within the same protein might arise from domain duplication followed by divergence through random mutations. This finding is exemplified by a recent RRM domain duplication in DAZ proteins and an ancient duplication in PABP proteins. Additionally, we found that different similarities between sibling RRMs are associated with distinct functions of an RBP and that the RBPs tend to contain repetitive sequences with low complexity. Taken together, this study suggests that the number of RBPs with multiple RRMs has expanded in mammals and that the multiple sibling RRMs may recognize similar target motifs in a cooperative manner.

  14. CircularLogo: A lightweight web application to visualize intra-motif dependencies.

    Science.gov (United States)

    Ye, Zhenqing; Ma, Tao; Kalmbach, Michael T; Dasari, Surendra; Kocher, Jean-Pierre A; Wang, Liguo

    2017-05-22

    The sequence logo has been widely used to represent DNA or RNA motifs for more than three decades. Despite its intelligibility and intuitiveness, the traditional sequence logo is unable to display the intra-motif dependencies and therefore is insufficient to fully characterize nucleotide motifs. Many methods have been developed to quantify the intra-motif dependencies, but fewer tools are available for visualization. We developed CircularLogo, a web-based interactive application, which is able to not only visualize the position-specific nucleotide consensus and diversity but also display the intra-motif dependencies. Applying CircularLogo to HNF6 binding sites and tRNA sequences demonstrated its ability to show intra-motif dependencies and intuitively reveal biomolecular structure. CircularLogo is implemented in JavaScript and Python based on the Django web framework. The program's source code and user's manual are freely available at http://circularlogo.sourceforge.net . CircularLogo web server can be accessed from http://bioinformaticstools.mayo.edu/circularlogo/index.html . CircularLogo is an innovative web application that is specifically designed to visualize and interactively explore intra-motif dependencies.

  15. Introducing Dunaliella LIP promoter containing light-inducible motifs improves transgenic expression in Chlamydomonas reinhardtii.

    Science.gov (United States)

    Baek, Kwangryul; Lee, Yew; Nam, Onyou; Park, Seunghye; Sim, Sang Jun; Jin, EonSeon

    2016-03-01

    Promoter of the light-inducible protein gene (LIP) of Dunaliella was recently isolated in our laboratory. The aim of this work is to find the light-inducible motif in the Dunaliella LIP promoter and verify its regulatory motif with a Gaussia luciferase reporter gene transformed in Chlamydomonas reinhardtii. 400 bp upstream to the translational start site of the Dunaliella LIP gene was gradually truncated and analyzed for the luciferase expression. Furthermore, this promoter comprising duplicated or triplicated light-responsive motifs was tested for its augmentation of light response. Two putative light-responsive motifs, GT-1 binding motif and sequences over-represented in light-repressed promoters (SORLIP) located in the 200 bp LIP promoter fragment were analyzed for their light responsibility. It is turned out that SORLIP was responsible for the light-inducible activity. With the copy number of SORLIP up to three showed stronger high light response compared with the native LIP promoter fragment. Therefore, we found a light-responsive DNA motif operating in Chlamydomonas and confirm a synthetic promoter including this motif displayed light inducibility in heterologously transformed green algae for the first time. This light-inducible expression system will be applied to various area of algal research including algal biotechnology.

  16. Identification of imine reductase-specific sequence motifs.

    Science.gov (United States)

    Fademrecht, Silvia; Scheller, Philipp N; Nestl, Bettina M; Hauer, Bernhard; Pleiss, Jürgen

    2016-05-01

    Chiral amines are valuable building blocks for the production of a variety of pharmaceuticals, agrochemicals and other specialty chemicals. Only recently, imine reductases (IREDs) were discovered which catalyze the stereoselective reduction of imines to chiral amines. Although several IREDs were biochemically characterized in the last few years, knowledge of the reaction mechanism and the molecular basis of substrate specificity and stereoselectivity is limited. To gain further insights into the sequence-function relationships, the Imine Reductase Engineering Database (www.IRED.BioCatNet.de) was established and a systematic analysis of 530 putative IREDs was performed. A standard numbering scheme based on R-IRED-Sk was introduced to facilitate the identification and communication of structurally equivalent positions in different proteins. A conservation analysis revealed a highly conserved cofactor binding region and a predominantly hydrophobic substrate binding cleft. Two IRED-specific motifs were identified, the cofactor binding motif GLGxMGx(5 )[ATS]x(4) Gx(4) [VIL]WNR[TS]x(2) [KR] and the active site motif Gx[DE]x[GDA]x[APS]x(3){K}x[ASL]x[LMVIAG]. Our results indicate a preference toward NADPH for all IREDs and explain why, despite their sequence similarity to β-hydroxyacid dehydrogenases (β-HADs), no conversion of β-hydroxyacids has been observed. Superfamily-specific conservations were investigated to explore the molecular basis of their stereopreference. Based on our analysis and previous experimental results on IRED mutants, an exclusive role of standard position 187 for stereoselectivity is excluded. Alternatively, two standard positions 139 and 194 were identified which are superfamily-specifically conserved and differ in R- and S-selective enzymes. © 2016 Wiley Periodicals, Inc.

  17. Interaction of Individual Structural Domains of hnRNP LL with the BCL2 Promoter i-Motif DNA.

    Science.gov (United States)

    Roy, Basab; Talukder, Poulami; Kang, Hyun-Jin; Tsuen, Shujian S; Alam, Mohammad P; Hurley, Laurence H; Hecht, Sidney M

    2016-08-31

    The recently discovered role of the BCL2 (B-cell lymphoma 2 gene) promoter i-motif DNA in modulation of gene expression via interaction with the ribonucleoprotein hnRNP L-like (hnRNP LL) has prompted a more detailed study of the nature of this protein-DNA interaction. The RNA recognition motifs (RRMs) of hnRNP LL were expressed individually, and both RRM1 and RRM2 were found to bind efficiently to the BCL2 i-motif DNA, as well as being critical for transcriptional activation, whereas RRM3-4 bound only weakly to this DNA. Binding was followed by unfolding of the DNA as monitored by changes in the CD spectrum. Mutational analysis of the i-motif DNA revealed that binding involved primarily the lateral loops of the i-motif. The kinetics of binding of the DNA with RRM1 was explored by recording CD spectra at predetermined times following admixture of the protein and DNA. The change in molar ellipticity was readily apparent after 30 s and largely complete within 1 min. A more detailed view of protein-DNA interaction was obtained by introducing the fluorescence donor 6-CNTrp in RRM1 at position 137, and the acceptor 4-aminobenzo[g]quinazoline-2-one (Cf) in lieu of cytidine22 in the i-motif DNA. The course of binding of the two species was monitored by FRET, which reflected a steady increase in energy transfer over a period of several minutes. The FRET signal could be diminished by the further addition of (unlabeled) RRM2, no doubt reflecting competition for binding to the i-motif DNA. These experiments using the individual RRM domains from hnRNP LL confirm the role of this transcription factor in activation of BCL2 transcription via the i-motif in the promoter element.

  18. Correlating overrepresented upstream motifs to gene expression a computational approach to regulatory element discovery in eukaryotes

    CERN Document Server

    Caselle, M; Provero, P

    2002-01-01

    Gene regulation in eukaryotes is mainly effected through transcription factors binding to rather short recognition motifs generally located upstream of the coding region. We present a novel computational method to identify regulatory elements in the upstream region of eukaryotic genes. The genes are grouped in sets sharing an overrepresented short motif in their upstream sequence. For each set, the average expression level from a microarray experiment is determined: If this level is significantly higher or lower than the average taken over the whole genome, then the overerpresented motif shared by the genes in the set is likely to play a role in their regulation. The method was tested by applying it to the genome of Saccharomyces cerevisiae, using the publicly available results of a DNA microarray experiment, in which expression levels for virtually all the genes were measured during the diauxic shift from fermentation to respiration. Several known motifs were correctly identified, and a new candidate regulat...

  19. Fitness for synchronization of network motifs

    DEFF Research Database (Denmark)

    Vega, Y.M.; Vázquez-Prada, M.; Pacheco, A.F.

    2004-01-01

    We study the synchronization of Kuramoto's oscillators in small parts of networks known as motifs. We first report on the system dynamics for the case of a scale-free network and show the existence of a non-trivial critical point. We compute the probability that network motifs synchronize, and fi...

  20. Present status of quinoxaline motifs: excellent pathfinders in therapeutic medicine.

    Science.gov (United States)

    Ajani, Olayinka Oyewale

    2014-10-01

    Quinoxalines belong to a class of excellent heterocyclic scaffolds owing to their wide biological properties and diverse therapeutic applications in medicinal research. They are complementary in shapes and charges to numerous biomolecules they interact with, thereby resulting in increased binding affinity. The pharmacokinetic properties of drugs bearing quinoxaline cores have shown them to be relatively easy to administer either as intramuscular solutions, oral capsules or rectal suppositories. This work deals with recent advances in the synthesis and pharmacological diversities of quinoxaline motifs which might pave ways for novel drugs development.

  1. EEVD motif of heat shock cognate protein 70 contributes to bacterial uptake by trophoblast giant cells

    Directory of Open Access Journals (Sweden)

    Kim Suk

    2009-12-01

    Full Text Available Abstract Background The uptake of abortion-inducing pathogens by trophoblast giant (TG cells is a key event in infectious abortion. However, little is known about phagocytic functions of TG cells against the pathogens. Here we show that heat shock cognate protein 70 (Hsc70 contributes to bacterial uptake by TG cells and the EEVD motif of Hsc70 plays an important role in this. Methods Brucella abortus and Listeria monocytogenes were used as the bacterial antigen in this study. Recombinant proteins containing tetratricopeptide repeat (TPR domains were constructed and confirmation of the binding capacity to Hsc70 was assessed by ELISA. The recombinant TPR proteins were used for investigation of the effect of TPR proteins on bacterial uptake by TG cells and on pregnancy in mice. Results The monoclonal antibody that inhibits bacterial uptake by TG cells reacted with the EEVD motif of Hsc70. Bacterial TPR proteins bound to the C-terminal of Hsc70 through its EEVD motif and this binding inhibited bacterial uptake by TG cells. Infectious abortion was also prevented by blocking the EEVD motif of Hsc70. Conclusions Our results demonstrate that surface located Hsc70 on TG cells mediates the uptake of pathogenic bacteria and proteins containing the TPR domain inhibit the function of Hsc70 by binding to its EEVD motif. These molecules may be useful in the development of methods for preventing infectious abortion.

  2. Helix-packing motifs in membrane proteins.

    Science.gov (United States)

    Walters, R F S; DeGrado, W F

    2006-09-12

    The fold of a helical membrane protein is largely determined by interactions between membrane-imbedded helices. To elucidate recurring helix-helix interaction motifs, we dissected the crystallographic structures of membrane proteins into a library of interacting helical pairs. The pairs were clustered according to their three-dimensional similarity (rmsd universe of common transmembrane helix-pairing motifs is relatively simple. The largest cluster, which comprises 29% of the library members, consists of an antiparallel motif with left-handed packing angles, and it is frequently stabilized by packing of small side chains occurring every seven residues in the sequence. Right-handed parallel and antiparallel structures show a similar tendency to segregate small residues to the helix-helix interface but spaced at four-residue intervals. Position-specific sequence propensities were derived for the most populated motifs. These structural and sequential motifs should be quite useful for the design and structural prediction of membrane proteins.

  3. Recognizing RNA structural motifs in HT-SELEX data for ribosomal protein S15.

    Science.gov (United States)

    Pei, Shermin; Slinger, Betty L; Meyer, Michelle M

    2017-06-06

    Proteins recognize many different aspects of RNA ranging from single stranded regions to discrete secondary or tertiary structures. High-throughput sequencing (HTS) of in vitro selected populations offers a large scale method to study RNA-proteins interactions. However, most existing analysis methods require that the binding motifs are enriched in the population relative to earlier rounds, and that motifs are found in a loop or single stranded region of the potential RNA secondary structure. Such methods do not generalize to all RNA-protein interaction as some RNA binding proteins specifically recognize more complex structures such as double stranded RNA. In this study, we use HT-SELEX derived populations to study the landscape of RNAs that interact with Geobacillus kaustophilus ribosomal protein S15. Our data show high sequence and structure diversity and proved intractable to existing methods. Conventional programs identified some sequence motifs, but these are found in less than 5-10% of the total sequence pool. Therefore, we developed a novel framework to analyze HT-SELEX data. Our process accounts for both sequence and structure components by abstracting the overall secondary structure into smaller substructures composed of a single base-pair stack, which allows us to leverage existing approaches already used in k-mer analysis to identify enriched motifs. By focusing on secondary structure motifs composed of specific two base-pair stacks, we identified significantly enriched or depleted structure motifs relative to earlier rounds. Discrete substructures are likely to be important to RNA-protein interactions, but they are difficult to elucidate. Substructures can help make highly diverse sequence data more tractable. The structure motifs provide limited accuracy in predicting enrichment suggesting that G. kaustophilus S15 can either recognize many different secondary structure motifs or some aspects of the interaction are not captured by the analysis. This

  4. Insertion of tetracysteine motifs into dopamine transporter extracellular domains.

    Directory of Open Access Journals (Sweden)

    Deanna M Navaroli

    Full Text Available The neuronal dopamine transporter (DAT is a major determinant of extracellular dopamine (DA levels and is the primary target for a variety of addictive and therapeutic psychoactive drugs. DAT is acutely regulated by protein kinase C (PKC activation and amphetamine exposure, both of which modulate DAT surface expression by endocytic trafficking. In order to use live imaging approaches to study DAT endocytosis, methods are needed to exclusively label the DAT surface pool. The use of membrane impermeant, sulfonated biarsenic dyes holds potential as one such approach, and requires introduction of an extracellular tetracysteine motif (tetraCys; CCPGCC to facilitate dye binding. In the current study, we took advantage of intrinsic proline-glycine (Pro-Gly dipeptides encoded in predicted DAT extracellular domains to introduce tetraCys motifs into DAT extracellular loops 2, 3, and 4. [(3H]DA uptake studies, surface biotinylation and fluorescence microscopy in PC12 cells indicate that tetraCys insertion into the DAT second extracellular loop results in a functional transporter that maintains PKC-mediated downregulation. Introduction of tetraCys into extracellular loops 3 and 4 yielded DATs with severely compromised function that failed to mature and traffic to the cell surface. This is the first demonstration of successful introduction of a tetracysteine motif into a DAT extracellular domain, and may hold promise for use of biarsenic dyes in live DAT imaging studies.

  5. Overlapping ETS and CRE Motifs (G/CCGGAAGTGACGTCA) Preferentially Bound by GABPα and CREB Proteins

    Science.gov (United States)

    Chatterjee, Raghunath; Zhao, Jianfei; He, Ximiao; Shlyakhtenko, Andrey; Mann, Ishminder; Waterfall, Joshua J.; Meltzer, Paul; Sathyanarayana, B. K.; FitzGerald, Peter C.; Vinson, Charles

    2012-01-01

    Previously, we identified 8-bps long DNA sequences (8-mers) that localize in human proximal promoters and grouped them into known transcription factor binding sites (TFBS). We now examine split 8-mers consisting of two 4-mers separated by 1-bp t