Sample records for promiscuous substrate recognition

  1. Substrate-Assisted Catalysis in the Reaction Catalyzed by Salicylic Acid Binding Protein 2 (SABP2), a Potential Mechanism of Substrate Discrimination for Some Promiscuous Enzymes.

    PubMed

    Yao, Jianzhuang; Guo, Haobo; Chaiprasongsuk, Minta; Zhao, Nan; Chen, Feng; Yang, Xiaohan; Guo, Hong

    2015-09-01

    Although one of an enzyme's hallmarks is the high specificity for their natural substrates, substrate promiscuity has been reported more frequently. It is known that promiscuous enzymes generally show different catalytic efficiencies to different substrates, but our understanding of the origin of such differences is still lacking. Here we report the results of quantum mechanical/molecular mechanical simulations and an experimental study of salicylic acid binding protein 2 (SABP2). SABP2 has promiscuous esterase activity toward a series of substrates but shows a high activity toward its natural substrate, methyl salicylate (MeSA). Our results demonstrate that this enzyme may use substrate-assisted catalysis involving the hydroxyl group from MeSA to enhance the activity and achieve substrate discrimination.

  2. Substrate-Assisted Catalysis in the Reaction Catalyzed by Salicylic Acid Binding Protein 2 (SABP2), a Potential Mechanism of Substrate Discrimination for Some Promiscuous Enzymes

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

    Yao, Jianzhuang; Guo, Haobo; Chaiprasongsuk, Minta

    Although one of an enzyme’s hallmarks is the high specificity for their natural substrates, substrate promiscuity has been reported more frequently. We know that promiscuous enzymes generally show different catalytic efficiencies to different substrates, but our understanding of the origin of such differences is still lacking. We report the results of quantum mechanical/molecular mechanical simulations and an experimental study of salicylic acid binding protein 2 (SABP2). SABP2 has promiscuous esterase activity toward a series of substrates but shows a high activity toward its natural substrate, methyl salicylate (MeSA). Finally, our results demonstrate that this enzyme may use substrate-assisted catalysis involvingmore » the hydroxyl group from MeSA to enhance the activity and achieve substrate discrimination.« less

  3. Substrate-Assisted Catalysis in the Reaction Catalyzed by Salicylic Acid Binding Protein 2 (SABP2), a Potential Mechanism of Substrate Discrimination for Some Promiscuous Enzymes

    DOE PAGES

    Yao, Jianzhuang; Guo, Haobo; Chaiprasongsuk, Minta; ...

    2015-08-05

    Although one of an enzyme’s hallmarks is the high specificity for their natural substrates, substrate promiscuity has been reported more frequently. We know that promiscuous enzymes generally show different catalytic efficiencies to different substrates, but our understanding of the origin of such differences is still lacking. We report the results of quantum mechanical/molecular mechanical simulations and an experimental study of salicylic acid binding protein 2 (SABP2). SABP2 has promiscuous esterase activity toward a series of substrates but shows a high activity toward its natural substrate, methyl salicylate (MeSA). Finally, our results demonstrate that this enzyme may use substrate-assisted catalysis involvingmore » the hydroxyl group from MeSA to enhance the activity and achieve substrate discrimination.« less

  4. "Multiple partial recognitions in dynamic equilibrium" in the binding sites of proteins form the molecular basis of promiscuous recognition of structurally diverse ligands.

    PubMed

    Kohda, Daisuke

    2018-04-01

    Promiscuous recognition of ligands by proteins is as important as strict recognition in numerous biological processes. In living cells, many short, linear amino acid motifs function as targeting signals in proteins to specify the final destination of the protein transport. In general, the target signal is defined by a consensus sequence containing wild-characters, and hence represented by diverse amino acid sequences. The classical lock-and-key or induced-fit/conformational selection mechanism may not cover all aspects of the promiscuous recognition. On the basis of our crystallographic and NMR studies on the mitochondrial Tom20 protein-presequence interaction, we proposed a new hypothetical mechanism based on "a rapid equilibrium of multiple states with partial recognitions". This dynamic, multiple recognition mode enables the Tom20 receptor to recognize diverse mitochondrial presequences with nearly equal affinities. The plant Tom20 is evolutionally unrelated to the animal Tom20 in our study, but is a functional homolog of the animal/fungal Tom20. NMR studies by another research group revealed that the presequence binding by the plant Tom20 was not fully explained by simple interaction modes, suggesting the presence of a similar dynamic, multiple recognition mode. Circumstantial evidence also suggested that similar dynamic mechanisms may be applicable to other promiscuous recognitions of signal peptides by the SRP54/Ffh and SecA proteins.

  5. Dynamics Govern Specificity of a Protein-Protein Interface: Substrate Recognition by Thrombin.

    PubMed

    Fuchs, Julian E; Huber, Roland G; Waldner, Birgit J; Kahler, Ursula; von Grafenstein, Susanne; Kramer, Christian; Liedl, Klaus R

    2015-01-01

    Biomolecular recognition is crucial in cellular signal transduction. Signaling is mediated through molecular interactions at protein-protein interfaces. Still, specificity and promiscuity of protein-protein interfaces cannot be explained using simplistic static binding models. Our study rationalizes specificity of the prototypic protein-protein interface between thrombin and its peptide substrates relying solely on binding site dynamics derived from molecular dynamics simulations. We find conformational selection and thus dynamic contributions to be a key player in biomolecular recognition. Arising entropic contributions complement chemical intuition primarily reflecting enthalpic interaction patterns. The paradigm "dynamics govern specificity" might provide direct guidance for the identification of specific anchor points in biomolecular recognition processes and structure-based drug design.

  6. Dynamics Govern Specificity of a Protein-Protein Interface: Substrate Recognition by Thrombin

    PubMed Central

    Fuchs, Julian E.; Huber, Roland G.; Waldner, Birgit J.; Kahler, Ursula; von Grafenstein, Susanne; Kramer, Christian; Liedl, Klaus R.

    2015-01-01

    Biomolecular recognition is crucial in cellular signal transduction. Signaling is mediated through molecular interactions at protein-protein interfaces. Still, specificity and promiscuity of protein-protein interfaces cannot be explained using simplistic static binding models. Our study rationalizes specificity of the prototypic protein-protein interface between thrombin and its peptide substrates relying solely on binding site dynamics derived from molecular dynamics simulations. We find conformational selection and thus dynamic contributions to be a key player in biomolecular recognition. Arising entropic contributions complement chemical intuition primarily reflecting enthalpic interaction patterns. The paradigm “dynamics govern specificity” might provide direct guidance for the identification of specific anchor points in biomolecular recognition processes and structure-based drug design. PMID:26496636

  7. Specificity of a protein-protein interface: local dynamics direct substrate recognition of effector caspases.

    PubMed

    Fuchs, Julian E; von Grafenstein, Susanne; Huber, Roland G; Wallnoefer, Hannes G; Liedl, Klaus R

    2014-04-01

    Proteases are prototypes of multispecific protein-protein interfaces. Proteases recognize and cleave protein and peptide substrates at a well-defined position in a substrate binding groove and a plethora of experimental techniques provide insights into their substrate recognition. We investigate the caspase family of cysteine proteases playing a key role in programmed cell death and inflammation, turning caspases into interesting drug targets. Specific ligand binding to one particular caspase is difficult to achieve, as substrate specificities of caspase isoforms are highly similar. In an effort to rationalize substrate specificity of two closely related caspases, we investigate the substrate promiscuity of the effector Caspases 3 and 7 by data mining (cleavage entropy) and by molecular dynamics simulations. We find a strong correlation between binding site rigidity and substrate readout for individual caspase subpockets explaining more stringent substrate readout of Caspase 7 via its narrower conformational space. Caspase 3 subpockets S3 and S4 show elevated local flexibility explaining the more unspecific substrate readout of that isoform in comparison to Caspase 7. We show by in silico exchange mutations in the S3 pocket of the proteases that a proline residue in Caspase 7 contributes to the narrowed conformational space of the binding site. These findings explain the substrate specificities of caspases via a mechanism of conformational selection and highlight the crucial importance of binding site local dynamics in substrate recognition of proteases. Proteins 2014; 82:546-555. © 2013 Wiley Periodicals, Inc. Copyright © 2013 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

  8. Exploiting the Substrate Promiscuity of Hydroxycinnamoyl-CoA:Shikimate Hydroxycinnamoyl Transferase to Reduce Lignin

    DOE PAGES

    Eudes, Aymerick; Pereira, Jose H.; Yogiswara, Sasha; ...

    2016-02-08

    Lignin poses a major challenge in the processing of plant biomass for agro-industrial applications. For bioengineering purposes, there is a pressing interest in identifying and characterizing the enzymes responsible for the biosynthesis of lignin. Hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase (HCT; EC 2.3.1.133) is a key metabolic entry point for the synthesis of the most important lignin monomers: coniferyl and sinapyl alcohols. In this study, we investigated the substrate promiscuity of HCT from a bryophyte (Physcomitrella) and from five representatives of vascular plants (Arabidopsis, poplar, switchgrass, pine and Selaginella) using a yeast expression system. We demonstrate for these HCTs a conserved capacity tomore » acylate with p-coumaroyl-CoA several phenolic compounds in addition to the canonical acceptor shikimate normally used during lignin biosynthesis. Using either recombinant HCT from switchgrass (PvHCT2a) or an Arabidopsis stem protein extract, we show evidence of the inhibitory effect of these phenolics on the synthesis of p-coumaroyl shikimate in vitro, which presumably occurs via a mechanism of competitive inhibition. A structural study of PvHCT2a confirmed the binding of a non-canonical acceptor in a similar manner to shikimate in the active site of the enzyme. Finally, we exploited in Arabidopsis the substrate flexibility of HCT to reduce lignin content and improve biomass saccharification by engineering transgenic lines that overproduce one of the HCT non-canonical acceptors. Our results demonstrate conservation of HCT substrate promiscuity and provide support for a new strategy for lignin reduction in the effort to improve the quality of plant biomass for forage and cellulosic biofuels.« less

  9. Statistical Profiling of One Promiscuous Protein Binding Site: Illustrated by Urokinase Catalytic Domain.

    PubMed

    Cerisier, Natacha; Regad, Leslie; Triki, Dhoha; Petitjean, Michel; Flatters, Delphine; Camproux, Anne-Claude

    2017-10-01

    While recent literature focuses on drug promiscuity, the characterization of promiscuous binding sites (ability to bind several ligands) remains to be explored. Here, we present a proteochemometric modeling approach to analyze diverse ligands and corresponding multiple binding sub-pockets associated with one promiscuous binding site to characterize protein-ligand recognition. We analyze both geometrical and physicochemical profile correspondences. This approach was applied to examine the well-studied druggable urokinase catalytic domain inhibitor binding site, which results in a large number of complex structures bound to various ligands. This approach emphasizes the importance of jointly characterizing pocket and ligand spaces to explore the impact of ligand diversity on sub-pocket properties and to establish their main profile correspondences. This work supports an interest in mining available 3D holo structures associated with a promiscuous binding site to explore its main protein-ligand recognition tendency. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Substrate interactions and promiscuity in a viral DNA packaging motor.

    PubMed

    Aathavan, K; Politzer, Adam T; Kaplan, Ariel; Moffitt, Jeffrey R; Chemla, Yann R; Grimes, Shelley; Jardine, Paul J; Anderson, Dwight L; Bustamante, Carlos

    2009-10-01

    The ASCE (additional strand, conserved E) superfamily of proteins consists of structurally similar ATPases associated with diverse cellular activities involving metabolism and transport of proteins and nucleic acids in all forms of life. A subset of these enzymes consists of multimeric ringed pumps responsible for DNA transport in processes including genome packaging in adenoviruses, herpesviruses, poxviruses and tailed bacteriophages. Although their mechanism of mechanochemical conversion is beginning to be understood, little is known about how these motors engage their nucleic acid substrates. Questions remain as to whether the motors contact a single DNA element, such as a phosphate or a base, or whether contacts are distributed over several parts of the DNA. Furthermore, the role of these contacts in the mechanochemical cycle is unknown. Here we use the genome packaging motor of the Bacillus subtilis bacteriophage varphi29 (ref. 4) to address these questions. The full mechanochemical cycle of the motor, in which the ATPase is a pentameric-ring of gene product 16 (gp16), involves two phases-an ATP-loading dwell followed by a translocation burst of four 2.5-base-pair (bp) steps triggered by hydrolysis product release. By challenging the motor with a variety of modified DNA substrates, we show that during the dwell phase important contacts are made with adjacent phosphates every 10-bp on the 5'-3' strand in the direction of packaging. As well as providing stable, long-lived contacts, these phosphate interactions also regulate the chemical cycle. In contrast, during the burst phase, we find that DNA translocation is driven against large forces by extensive contacts, some of which are not specific to the chemical moieties of DNA. Such promiscuous, nonspecific contacts may reflect common translocase-substrate interactions for both the nucleic acid and protein translocases of the ASCE superfamily.

  11. Substrate Interactions and Promiscuity in a Viral DNA Packaging Motor

    PubMed Central

    Aathavan, K.; Politzer, Adam T.; Kaplan, Ariel; Moffitt, Jeffrey R.; Chemla, Yann R.; Grimes, Shelley; Jardine, Paul J.; Anderson, Dwight L.; Bustamante, Carlos

    2009-01-01

    The ASCE superfamily of proteins consists of structurally similar ATPases associated with diverse cellular activities involving metabolism and transport of proteins and nucleic acids in all forms of life1. A subset of these enzymes are multimeric ringed pumps responsible for DNA transport in processes including genome packaging in adenoviruses, herpesviruses, poxviruses, and tailed bacteriophages2. While their mechanism of mechanochemical conversion is beginning to be understood3, little is known about how these motors engage their nucleic acid substrates. Do motors contact a single DNA element, such as a phosphate or a base, or are contacts distributed over multiple parts of the DNA? In addition, what role do these contacts play in the mechanochemical cycle? Here we use the genome packaging motor of the Bacillus subtilis bacteriophage φ294 to address these questions. The full mechanochemical cycle of the motor, whose ATPase is a pentameric-ring5 of gene product 16, involves two phases-- an ATP loading dwell followed by a translocation burst of four 2.5-bp steps6 triggered by hydrolysis product release7. By challenging the motor with a variety of modified DNA substrates, we find that during the dwell phase important contacts are made with adjacent phosphates every 10-bp on the 5’-3’ strand in the direction of packaging. In addition to providing stable, long-lived contacts, these phosphate interactions also regulate the chemical cycle. In contrast, during the burst phase, we find that DNA translocation is driven against large forces by extensive contacts, some of which are not specific to the chemical moieties of DNA. Such promiscuous, non-specific contacts may reflect common translocase-substrate interactions for both the nucleic acid and protein translocases of the ASCE superfamily1. PMID:19794496

  12. A Novel Semi-biosynthetic Route for Artemisinin Production Using Engineered Substrate-Promiscuous P450BM3

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

    Dietrich, Jeffrey; Yoshikuni, Yasuo; Fisher, Karl

    2009-11-30

    Production of fine heterologus pathways in microbial hosts is frequently hindered by insufficient knowledge of the native metabolic pathway and its cognate enzymes; often the pathway is unresolved and enzymes lack detailed characterization. An alternative paradigm to using native pathways is de novo pathway design using well-characterized, substrate-promiscuous enzymes. We demonstrate this concept using P450BM3 from Bacillus megaterium. Using a computer model, we illustrate how key P450BM3 activ site mutations enable binding of non-native substrate amorphadiene, incorporating these mutations into P450BM3 enabled the selective oxidation of amorphadiene arteminsinic-11s,12-epoxide, at titers of 250 mg L"1 in E. coli. We also demonstratemore » high-yeilding, selective transformations to dihydroartemisinic acid, the immediate precursor to the high value anti-malarial drug artemisinin.« less

  13. Promiscuity in the Enzymatic Catalysis of Phosphate and Sulfate Transfer

    PubMed Central

    2016-01-01

    The enzymes that facilitate phosphate and sulfate hydrolysis are among the most proficient natural catalysts known to date. Interestingly, a large number of these enzymes are promiscuous catalysts that exhibit both phosphatase and sulfatase activities in the same active site and, on top of that, have also been demonstrated to efficiently catalyze the hydrolysis of other additional substrates with varying degrees of efficiency. Understanding the factors that underlie such multifunctionality is crucial both for understanding functional evolution in enzyme superfamilies and for the development of artificial enzymes. In this Current Topic, we have primarily focused on the structural and mechanistic basis for catalytic promiscuity among enzymes that facilitate both phosphoryl and sulfuryl transfer in the same active site, while comparing this to how catalytic promiscuity manifests in other promiscuous phosphatases. We have also drawn on the large number of experimental and computational studies of selected model systems in the literature to explore the different features driving the catalytic promiscuity of such enzymes. Finally, on the basis of this comparative analysis, we probe the plausible origins and determinants of catalytic promiscuity in enzymes that catalyze phosphoryl and sulfuryl transfer. PMID:27187273

  14. Tracing the Repertoire of Promiscuous Enzymes along the Metabolic Pathways in Archaeal Organisms.

    PubMed

    Martínez-Núñez, Mario Alberto; Rodríguez-Escamilla, Zuemy; Rodríguez-Vázquez, Katya; Pérez-Rueda, Ernesto

    2017-07-13

    The metabolic pathways that carry out the biochemical transformations sustaining life depend on the efficiency of their associated enzymes. In recent years, it has become clear that promiscuous enzymes have played an important role in the function and evolution of metabolism. In this work we analyze the repertoire of promiscuous enzymes in 89 non-redundant genomes of the Archaea cellular domain. Promiscuous enzymes are defined as those proteins with two or more different Enzyme Commission (E.C.) numbers, according the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. From this analysis, it was found that the fraction of promiscuous enzymes is lower in Archaea than in Bacteria. A greater diversity of superfamily domains is associated with promiscuous enzymes compared to specialized enzymes, both in Archaea and Bacteria, and there is an enrichment of substrate promiscuity rather than catalytic promiscuity in the archaeal enzymes. Finally, the presence of promiscuous enzymes in the metabolic pathways was found to be heterogeneously distributed at the domain level and in the phyla that make up the Archaea. These analyses increase our understanding of promiscuous enzymes and provide additional clues to the evolution of metabolism in Archaea.

  15. Aptamer Recognition of Multiplexed Small-Molecule-Functionalized Substrates.

    PubMed

    Nakatsuka, Nako; Cao, Huan H; Deshayes, Stephanie; Melkonian, Arin Lucy; Kasko, Andrea M; Weiss, Paul S; Andrews, Anne M

    2018-05-31

    Aptamers are chemically synthesized oligonucleotides or peptides with molecular recognition capabilities. We investigated recognition of substrate-tethered small-molecule targets, using neurotransmitters as examples, and fluorescently labeled DNA aptamers. Substrate regions patterned via microfluidic channels with dopamine or L-tryptophan were selectively recognized by previously identified dopamine or L-tryptophan aptamers, respectively. The on-substrate dissociation constant determined for the dopamine aptamer was comparable to, though slightly greater than the previously determined solution dissociation constant. Using pre-functionalized neurotransmitter-conjugated oligo(ethylene glycol) alkanethiols and microfluidics patterning, we produced multiplexed substrates to capture and to sort aptamers. Substrates patterned with L-DOPA, L-DOPS, and L-5-HTP enabled comparison of the selectivity of the dopamine aptamer for different targets via simultaneous determination of in situ binding constants. Thus, beyond our previous demonstrations of recognition by protein binding partners (i.e., antibodies and G-protein-coupled receptors), strategically optimized small-molecule-functionalized substrates show selective recognition of nucleic acid binding partners. These substrates are useful for side-by-side target comparisons, and future identification and characterization of novel aptamers targeting neurotransmitters or other important small-molecules.

  16. Promiscuity, sexual selection, and genetic diversity: a reply to Spurgin.

    PubMed

    Lifjeld, Jan T; Gohli, Jostein; Johnsen, Arild

    2013-10-01

    We recently reported a positive association between female promiscuity and genetic diversity across passerine birds, and launched the hypothesis that female promiscuity acts as a balancing selection, pressure maintaining genetic diversity in populations (Gohli et al.2013). Spurgin (2013) questions both our analyses and interpretations. While we agree that the hypothesis needs more comprehensive empirical testing, we find his specific points of criticism unjustified. In a more general perspective, we call for a more explicit recognition of female mating preferences as mechanisms of selection in population genetics theory. © 2013 The Author(s). Evolution © 2013 The Society for the Study of Evolution.

  17. Structural basis of carbohydrate recognition by lectin II from Ulex europaeus, a protein with a promiscuous carbohydrate-binding site.

    PubMed

    Loris, R; De Greve, H; Dao-Thi, M H; Messens, J; Imberty, A; Wyns, L

    2000-08-25

    Protein-carbohydrate interactions are the language of choice for inter- cellular communication. The legume lectins form a large family of homologous proteins that exhibit a wide variety of carbohydrate specificities. The legume lectin family is therefore highly suitable as a model system to study the structural principles of protein-carbohydrate recognition. Until now, structural data are only available for two specificity families: Man/Glc and Gal/GalNAc. No structural data are available for any of the fucose or chitobiose specific lectins. The crystal structure of Ulex europaeus (UEA-II) is the first of a legume lectin belonging to the chitobiose specificity group. The complexes with N-acetylglucosamine, galactose and fucosylgalactose show a promiscuous primary binding site capable of accommodating both N-acetylglucos amine or galactose in the primary binding site. The hydrogen bonding network in these complexes can be considered suboptimal, in agreement with the low affinities of these sugars. In the complexes with chitobiose, lactose and fucosyllactose this suboptimal hydrogen bonding network is compensated by extensive hydrophobic interactions in a Glc/GlcNAc binding subsite. UEA-II thus forms the first example of a legume lectin with a promiscuous binding site and illustrates the importance of hydrophobic interactions in protein-carbohydrate complexes. Together with other known legume lectin crystal structures, it shows how different specificities can be grafted upon a conserved structural framework. Copyright 2000 Academic Press.

  18. PubChem promiscuity: a web resource for gathering compound promiscuity data from PubChem.

    PubMed

    Canny, Stephanie A; Cruz, Yasel; Southern, Mark R; Griffin, Patrick R

    2012-01-01

    Promiscuity counts allow for a better understanding of a compound's assay activity profile and drug potential. Although PubChem contains a vast amount of compound and assay data, it currently does not have a convenient or efficient method to obtain in-depth promiscuity counts for compounds. PubChem promiscuity fills this gap. It is a Java servlet that uses NCBI Entrez (eUtils) web services to interact with PubChem and provide promiscuity counts in a variety of categories along with compound descriptors, including PAINS-based functional group detection. http://chemutils.florida.scripps.edu/pcpromiscuity southern@scripps.edu

  19. PubChem promiscuity: a web resource for gathering compound promiscuity data from PubChem

    PubMed Central

    Canny, Stephanie A.; Cruz, Yasel; Southern, Mark R.; Griffin, Patrick R.

    2012-01-01

    Summary: Promiscuity counts allow for a better understanding of a compound's assay activity profile and drug potential. Although PubChem contains a vast amount of compound and assay data, it currently does not have a convenient or efficient method to obtain in-depth promiscuity counts for compounds. PubChem promiscuity fills this gap. It is a Java servlet that uses NCBI Entrez (eUtils) web services to interact with PubChem and provide promiscuity counts in a variety of categories along with compound descriptors, including PAINS-based functional group detection. Availability: http://chemutils.florida.scripps.edu/pcpromiscuity Contact: southern@scripps.edu PMID:22084255

  20. Substrate recognition by ribonucleoprotein ribonuclease MRP

    PubMed Central

    Esakova, Olga; Perederina, Anna; Quan, Chao; Berezin, Igor; Krasilnikov, Andrey S.

    2011-01-01

    The ribonucleoprotein complex ribonuclease (RNase) MRP is a site-specific endoribonuclease essential for the survival of the eukaryotic cell. RNase MRP closely resembles RNase P (a universal endoribonuclease responsible for the maturation of the 5′ ends of tRNA) but recognizes distinct substrates including pre-rRNA and mRNA. Here we report the results of an in vitro selection of Saccharomyces cerevisiae RNase MRP substrates starting from a pool of random sequences. The results indicate that RNase MRP cleaves single-stranded RNA and is sensitive to sequences in the immediate vicinity of the cleavage site requiring a cytosine at the position +4 relative to the cleavage site. Structural implications of the differences in substrate recognition by RNases P and MRP are discussed. PMID:21173200

  1. Substrate recognition by ribonucleoprotein ribonuclease MRP.

    PubMed

    Esakova, Olga; Perederina, Anna; Quan, Chao; Berezin, Igor; Krasilnikov, Andrey S

    2011-02-01

    The ribonucleoprotein complex ribonuclease (RNase) MRP is a site-specific endoribonuclease essential for the survival of the eukaryotic cell. RNase MRP closely resembles RNase P (a universal endoribonuclease responsible for the maturation of the 5' ends of tRNA) but recognizes distinct substrates including pre-rRNA and mRNA. Here we report the results of an in vitro selection of Saccharomyces cerevisiae RNase MRP substrates starting from a pool of random sequences. The results indicate that RNase MRP cleaves single-stranded RNA and is sensitive to sequences in the immediate vicinity of the cleavage site requiring a cytosine at the position +4 relative to the cleavage site. Structural implications of the differences in substrate recognition by RNases P and MRP are discussed.

  2. Female economic dependence and the morality of promiscuity.

    PubMed

    Price, Michael E; Pound, Nicholas; Scott, Isabel M

    2014-10-01

    In environments in which female economic dependence on a male mate is higher, male parental investment is more essential. In such environments, therefore, both sexes should value paternity certainty more and thus object more to promiscuity (because promiscuity undermines paternity certainty). We tested this theory of anti-promiscuity morality in two studies (N = 656 and N = 4,626) using U.S. samples. In both, we examined whether opposition to promiscuity was higher among people who perceived greater female economic dependence in their social network. In Study 2, we also tested whether economic indicators of female economic dependence (e.g., female income, welfare availability) predicted anti-promiscuity morality at the state level. Results from both studies supported the proposed theory. At the individual level, perceived female economic dependence explained significant variance in anti-promiscuity morality, even after controlling for variance explained by age, sex, religiosity, political conservatism, and the anti-promiscuity views of geographical neighbors. At the state level, median female income was strongly negatively related to anti-promiscuity morality and this relationship was fully mediated by perceived female economic dependence. These results were consistent with the view that anti-promiscuity beliefs may function to promote paternity certainty in circumstances where male parental investment is particularly important.

  3. Molecular mechanisms of substrate recognition and specificity of botulinum neurotoxin serotype F.

    PubMed

    Chen, Sheng; Wan, Hoi Ying

    2011-01-15

    BoNTs (botulinum neurotoxins) are both deadly neurotoxins and natural toxins that are widely used in protein therapies to treat numerous neurological disorders of dystonia and spinal spasticity. Understanding the mechanism of action and substrate specificity of BoNTs is a prerequisite to develop antitoxin and novel BoNT-derived protein therapy. To date, there is a lack of detailed information with regard to how BoNTs recognize and hydrolyse the substrate VAMP-2 (vesicle-associated membrane protein 2), even though it is known to be cleaved by four of the seven BoNT serotypes, B, D, F, G and TeNT (tetanus neurotoxin). In the present study we dissected the molecular mechanisms of VAMP-2 recognition by BoNT serotype F for the first time. The initial substrate recognition was mediated through sequential binding of VAMP-2 to the B1, B2 and B3 pockets in LC/F (light chain of BoNT serotype F), which directed VAMP-2 to the active site of LC/F and stabilized the active site substrate recognition, where the P2, P1' and P2' sites of VAMP-2 were specifically recognized by the S2, S1' and S2' pockets of LC/F to promote substrate hydrolysis. The understanding of the molecular mechanisms of LC/F substrate recognition provides insights into the development of antitoxins and engineering novel BoNTs to optimize current therapy and extend therapeutic interventions.

  4. Mechanism of substrate recognition by the novel Botulinum Neurotoxin subtype F5.

    PubMed

    Guo, Jiubiao; Chan, Edward Wai Chi; Chen, Sheng

    2016-01-22

    Botulinum Neurotoxins (BoNTs) are the causative agents of botulism, which act by potently inhibiting the neurotransmitter release in motor neurons. Seven serotypes of BoNTs designated as BoNT/A-G have been identified. Recently, two novel types of Botulinum neurotoxins, which cleave a novel scissile bond, L(54)-E(55), of VAMP-2 have been reported including BoNT/F subtype F5 and serotype H. However, little has been known on how these BoNTs recognize their substrates. The present study addressed for the first time the unique substrate recognition mechanism of LC/F5. Our data indicated that the optimal peptide required for efficient LC/F5 substrate cleavage is VAMP-2 (20-65). Interestingly, the overall mode of substrate recognition adopted by LC/F5 was similar to LC/F1, except that its recognition sites were shifted one helix toward the N-terminus of VAMP-2 when compared to that of LC/F1. The composition of LC/F5 pockets were found to have changed accordingly to facilitate specific recognition of these new sites of VAMP-2, including the P2', P1', P2, P3, B3, B2 and B1 sites. The study provides direct evidence of the evolutionary adaption of BoNT to recognize its substrate which is useful for effective antitoxin and inhibitor development.

  5. Steroid promiscuity: Diversity of enzyme action. Preface.

    PubMed

    Lathe, Richard; Kotelevtsev, Yuri; Mason, J Ian

    2015-07-01

    This Special Issue on the topic of Steroid and Sterol Signaling: Promiscuity and Diversity, dwells on the growing realization that the 'one ligand, one binding site' and 'one enzyme, one reaction' concepts are out of date. Focusing on cytochromes P450 (CYP), hydroxysteroid dehydrogenases (HSDs), and related enzymes, the Special Issue highlights that a single enzyme can bind to diverse substrates, and in different conformations, and can catalyze multiple different conversions (and in different directions), thereby, generating an unexpectedly wide spectrum of ligands that can have subtly different biological actions. This article is part of a Special Issue entitled 'Steroid/Sterol Signaling' . Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. A Measure of the Promiscuity of Proteins and Characteristics of Residues in the Vicinity of the Catalytic Site That Regulate Promiscuity

    PubMed Central

    Chakraborty, Sandeep; Rao, Basuthkar J.

    2012-01-01

    Promiscuity, the basis for the evolution of new functions through ‘tinkering’ of residues in the vicinity of the catalytic site, is yet to be quantitatively defined. We present a computational method Promiscuity Indices Estimator (PROMISE) - based on signatures derived from the spatial and electrostatic properties of the catalytic residues, to estimate the promiscuity (PromIndex) of proteins with known active site residues and 3D structure. PromIndex reflects the number of different active site signatures that have congruent matches in close proximity of its native catalytic site, the quality of the matches and difference in the enzymatic activity. Promiscuity in proteins is observed to follow a lognormal distribution (μ = 0.28, σ = 1.1 reduced chi-square = 3.0E-5). The PROMISE predicted promiscuous functions in any protein can serve as the starting point for directed evolution experiments. PROMISE ranks carboxypeptidase A and ribonuclease A amongst the more promiscuous proteins. We have also investigated the properties of the residues in the vicinity of the catalytic site that regulates its promiscuity. Linear regression establishes a weak correlation (R2∼0.1) between certain properties of the residues (charge, polar, etc) in the neighborhood of the catalytic residues and PromIndex. A stronger relationship states that most proteins with high promiscuity have high percentages of charged and polar residues within a radius of 3 Å of the catalytic site, which is validated using one-tailed hypothesis tests (P-values∼0.05). Since it is known that these characteristics are key factors in catalysis, their relationship with the promiscuity index cross validates the methodology of PROMISE. PMID:22359655

  7. Differential catalytic promiscuity of the alkaline phosphatase superfamily bimetallo core reveals mechanistic features underlying enzyme evolution

    DOE PAGES

    Sunden, Fanny; AlSadhan, Ishraq; Lyubimov, Artem; ...

    2017-10-25

    Members of enzyme superfamilies specialize in different reactions but often exhibit catalytic promiscuity for one another's reactions, consistent with catalytic promiscuity as an important driver in the evolution of new enzymes. Wanting to understand how catalytic promiscuity and other factors may influence evolution across a superfamily, we turned to the well-studied alkaline phosphatase (AP) superfamily, comparing three of its members, two evolutionarily distinct phosphatases and a phosphodiesterase. Here, we mutated distinguishing active-site residues to generate enzymes that had a common Zn 2+ bimetallo core but little sequence similarity and different auxiliary domains. We then tested the catalytic capabilities of thesemore » pruned enzymes with a series of substrates. A substantial rate enhancement of ~1011-fold for both phosphate mono- and diester hydrolysis by each enzyme indicated that the Zn 2+ bimetallo core is an effective mono/di-esterase generalist and that the bimetallo cores were not evolutionarily tuned to prefer their cognate reactions. In contrast, our pruned enzymes were ineffective sulfatases, and this limited promiscuity may have provided a driving force for founding the distinct one-metal-ion branch that contains all known AP superfamily sulfatases. Finally, our pruned enzymes exhibited 10 7–10 8-fold phosphotriesterase rate enhancements, despite absence of such enzymes within the AP superfamily. We speculate that the superfamily active-site architecture involved in nucleophile positioning prevents accommodation of the additional triester substituent. Overall, we suggest that catalytic promiscuity, and the ease or difficulty of remodeling and building onto existing protein scaffolds, have greatly influenced the course of enzyme evolution. Uncovering principles and properties of enzyme function, promiscuity, and repurposing provides lessons for engineering new enzymes.« less

  8. Differential catalytic promiscuity of the alkaline phosphatase superfamily bimetallo core reveals mechanistic features underlying enzyme evolution

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

    Sunden, Fanny; AlSadhan, Ishraq; Lyubimov, Artem

    Members of enzyme superfamilies specialize in different reactions but often exhibit catalytic promiscuity for one another's reactions, consistent with catalytic promiscuity as an important driver in the evolution of new enzymes. Wanting to understand how catalytic promiscuity and other factors may influence evolution across a superfamily, we turned to the well-studied alkaline phosphatase (AP) superfamily, comparing three of its members, two evolutionarily distinct phosphatases and a phosphodiesterase. Here, we mutated distinguishing active-site residues to generate enzymes that had a common Zn 2+ bimetallo core but little sequence similarity and different auxiliary domains. We then tested the catalytic capabilities of thesemore » pruned enzymes with a series of substrates. A substantial rate enhancement of ~1011-fold for both phosphate mono- and diester hydrolysis by each enzyme indicated that the Zn 2+ bimetallo core is an effective mono/di-esterase generalist and that the bimetallo cores were not evolutionarily tuned to prefer their cognate reactions. In contrast, our pruned enzymes were ineffective sulfatases, and this limited promiscuity may have provided a driving force for founding the distinct one-metal-ion branch that contains all known AP superfamily sulfatases. Finally, our pruned enzymes exhibited 10 7–10 8-fold phosphotriesterase rate enhancements, despite absence of such enzymes within the AP superfamily. We speculate that the superfamily active-site architecture involved in nucleophile positioning prevents accommodation of the additional triester substituent. Overall, we suggest that catalytic promiscuity, and the ease or difficulty of remodeling and building onto existing protein scaffolds, have greatly influenced the course of enzyme evolution. Uncovering principles and properties of enzyme function, promiscuity, and repurposing provides lessons for engineering new enzymes.« less

  9. Recognition of Nucleoside Monophosphate Substrates by Haemophilus influenzae Class C Acid Phosphatase

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

    Singh, Harkewal; Schuermann, Jonathan P.; Reilly, Thomas J.

    2010-12-08

    The e (P4) phosphatase from Haemophilus influenzae functions in a vestigial NAD{sup +} utilization pathway by dephosphorylating nicotinamide mononucleotide to nicotinamide riboside. P4 is also the prototype of class C acid phosphatases (CCAPs), which are nonspecific 5{prime},3{prime}-nucleotidases localized to the bacterial outer membrane. To understand substrate recognition by P4 and other class C phosphatases, we have determined the crystal structures of a substrate-trapping mutant P4 enzyme complexed with nicotinamide mononucleotide, 5{prime}-AMP, 3{prime}-AMP, and 2{prime}-AMP. The structures reveal an anchor-shaped substrate-binding cavity comprising a conserved hydrophobic box that clamps the nucleotide base, a buried phosphoryl binding site, and three solvent-filled pocketsmore » that contact the ribose and the hydrogen-bonding edge of the base. The span between the hydrophobic box and the phosphoryl site is optimal for recognizing nucleoside monophosphates, explaining the general preference for this class of substrate. The base makes no hydrogen bonds with the enzyme, consistent with an observed lack of base specificity. Two solvent-filled pockets flanking the ribose are key to the dual recognition of 5{prime}-nucleotides and 3{prime}-nucleotides. These pockets minimize the enzyme's direct interactions with the ribose and provide sufficient space to accommodate 5{prime} substrates in an anti conformation and 3{prime} substrates in a syn conformation. Finally, the structures suggest that class B acid phosphatases and CCAPs share a common strategy for nucleotide recognition.« less

  10. Insights into the evolution of enzyme substrate promiscuity after the discovery of (βα)₈ isomerase evolutionary intermediates from a diverse metagenome.

    PubMed

    Noda-García, Lianet; Juárez-Vázquez, Ana L; Ávila-Arcos, María C; Verduzco-Castro, Ernesto A; Montero-Morán, Gabriela; Gaytán, Paul; Carrillo-Tripp, Mauricio; Barona-Gómez, Francisco

    2015-06-10

    Current sequence-based approaches to identify enzyme functional shifts, such as enzyme promiscuity, have proven to be highly dependent on a priori functional knowledge, hampering our ability to reconstruct evolutionary history behind these mechanisms. Hidden Markov Model (HMM) profiles, broadly used to classify enzyme families, can be useful to distinguish between closely related enzyme families with different specificities. The (βα)8-isomerase HisA/PriA enzyme family, involved in L-histidine (HisA, mono-substrate) biosynthesis in most bacteria and plants, but also in L-tryptophan (HisA/TrpF or PriA, dual-substrate) biosynthesis in most Actinobacteria, has been used as model system to explore evolutionary hypotheses and therefore has a considerable amount of evolutionary, functional and structural knowledge available. We searched for functional evolutionary intermediates between the HisA and PriA enzyme families in order to understand the functional divergence between these families. We constructed a HMM profile that correctly classifies sequences of unknown function into the HisA and PriA enzyme sub-families. Using this HMM profile, we mined a large metagenome to identify plausible evolutionary intermediate sequences between HisA and PriA. These sequences were used to perform phylogenetic reconstructions and to identify functionally conserved amino acids. Biochemical characterization of one selected enzyme (CAM1) with a mutation within the functionally essential N-terminus phosphate-binding site, namely, an alanine instead of a glycine in HisA or a serine in PriA, showed that this evolutionary intermediate has dual-substrate specificity. Moreover, site-directed mutagenesis of this alanine residue, either backwards into a glycine or forward into a serine, revealed the robustness of this enzyme. None of these mutations, presumably upon functionally essential amino acids, significantly abolished its enzyme activities. A truncated version of this enzyme (CAM2

  11. Structures of Human Pumilio with Noncognate RNAs Reveal Molecular Mechanisms for Binding Promiscuity

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

    Gupta,Y.; Nair, D.; Wharton, R.

    2008-01-01

    Pumilio is a founder member of the evolutionarily conserved Puf family of RNA-binding proteins that control a number of physiological processes in eukaryotes. A structure of human Pumilio (hPum) Puf domain bound to a Drosophila regulatory sequence showed that each Puf repeat recognizes a single nucleotide. Puf domains in general bind promiscuously to a large set of degenerate sequences, but the structural basis for this promiscuity has been unclear. Here, we describe the structures of hPum Puf domain complexed to two noncognate RNAs, CycBreverse and Puf5. In each complex, one of the nucleotides is ejected from the binding surface, inmore » effect, acting as a 'spacer.' The complexes also reveal the plasticity of several Puf repeats, which recognize noncanonical nucleotides. Together, these complexes provide a molecular basis for recognition of degenerate binding sites, which significantly increases the number of mRNAs targeted for regulation by Puf proteins in vivo.« less

  12. Dynamics of Preferential Substrate Recognition in HIV-1 Protease: Redefining the Substrate Envelope

    PubMed Central

    Özen, Ayşegül; Haliloğlu, Türkan; Schiffer, Celia A.

    2011-01-01

    HIV-1 protease (PR) permits viral maturation by processing the Gag and Gag-Pro-Pol polyproteins. Though HIV-1 PR inhibitors (PIs) are used in combination antiviral therapy, the emergence of drug resistance has limited their efficacy. The rapid evolution of HIV-1 necessitates the consideration of drug resistance in novel drug-design strategies. Drug-resistant HIV-1 PR variants, while no longer efficiently inhibited, continue to efficiently hydrolyze the natural viral substrates. Though highly diverse in sequence, the HIV-1 PR substrates bind in a conserved three-dimensional shape we defined as the “substrate envelope”. We previously showed that resistance mutations arise where PIs protrude beyond the substrate envelope, as these regions are crucial for drug binding but not for substrate recognition. Here, we extend this model by considering the role of protein dynamics in the interaction of HIV-1 PR with its substrates. Seven molecular dynamics simulations of PR-substrate complexes were performed to estimate the conformational flexibility of substrates in their complexes. Interdependency of the substrate-protease interactions may compensate for the variations in cleavage-site sequences, and explain how a diverse set of sequences can be recognized as substrates by the same enzyme. This diversity may be essential for regulating sequential processing of substrates. We also define a dynamic substrate envelope as a more accurate representation of PR-substrate interactions. This dynamic substrate envelope, described by a probability distribution function, is a powerful tool for drug design efforts targeting ensembles of resistant HIV-1 PR variants with the aim of developing drugs that are less susceptible to resistance. PMID:21762811

  13. Mode of VAMP Substrate Recognition and Inhibition of Clostridium botulinum Neurotoxin F

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

    Agarwal, R.; Schmidt, J; Stafford, R

    2009-01-01

    Clostridium botulinum neurotoxins (BoNTs) cleave neuronal proteins responsible for neurotransmitter release, causing the neuroparalytic disease botulism. BoNT serotypes B, D, F and G cleave and inactivate vesicle-associated membrane protein (VAMP), each at a unique peptide bond. The specificity of BoNTs depends on the mode of substrate recognition. We have investigated the mechanism of substrate recognition of BoNT F by determining the crystal structures of its complex with two substrate-based inhibitors, VAMP 22-58/Gln58D-cysteine and 27-58/Gln58D-cysteine. The inhibitors bind to BoNT F in the canonical direction (as seen for BoNTs A and E substrates) but are positioned specifically via three major exositesmore » away from the active site. The cysteine sulfur of the inhibitors interacts with the zinc and exists as sulfinic acid in the inhibitor VAMP 27-58/Gln58D-cysteine. Arg133 and Arg171, which form part of two separate exosites, are crucial for substrate binding and catalysis.« less

  14. Site-directed mutagenesis maps interactions that enhance cognate and limit promiscuous catalysis by an alkaline phosphatase superfamily phosphodiesterase.

    PubMed

    Wiersma-Koch, Helen; Sunden, Fanny; Herschlag, Daniel

    2013-12-23

    Catalytic promiscuity, an evolutionary concept, also provides a powerful tool for gaining mechanistic insights into enzymatic reactions. Members of the alkaline phosphatase (AP) superfamily are highly amenable to such investigation, with several members having been shown to exhibit promiscuous activity for the cognate reactions of other superfamily members. Previous work has shown that nucleotide pyrophosphatase/phosphodiesterase (NPP) exhibits a >10⁶-fold preference for the hydrolysis of phosphate diesters over phosphate monoesters, and that the reaction specificity is reduced 10³-fold when the size of the substituent on the transferred phosphoryl group of phosphate diester substrates is reduced to a methyl group. Here we show additional specificity contributions from the binding pocket for this substituent (herein termed the R' substituent) that account for an additional ~250-fold differential specificity with the minimal methyl substituent. Removal of four hydrophobic side chains suggested on the basis of structural inspection to interact favorably with R' substituents decreases phosphate diester reactivity 10⁴-fold with an optimal diester substrate (R' = 5'-deoxythymidine) and 50-fold with a minimal diester substrate (R' = CH₃). These mutations also enhance the enzyme's promiscuous phosphate monoesterase activity by nearly an order of magnitude, an effect that is traced by mutation to the reduction of unfavorable interactions with the two residues closest to the nonbridging phosphoryl oxygen atoms. The quadruple R' pocket mutant exhibits the same activity toward phosphate diester and phosphate monoester substrates that have identical leaving groups, with substantial rate enhancements of ~10¹¹-fold. This observation suggests that the Zn²⁺ bimetallo core of AP superfamily enzymes, which is equipotent in phosphate monoester and diester catalysis, has the potential to become specialized for the hydrolysis of each class of phosphate esters via addition

  15. Molecular pharmacology of promiscuous seven transmembrane receptors sensing organic nutrients.

    PubMed

    Wellendorph, Petrine; Johansen, Lars Dan; Bräuner-Osborne, Hans

    2009-09-01

    A number of highly promiscuous seven transmembrane (7TM) receptors have been cloned and characterized within the last few years. It is noteworthy that many of these receptors are activated broadly by amino acids, proteolytic degradation products, carbohydrates, or free fatty acids and are expressed in taste tissue, the gastrointestinal tract, endocrine glands, adipose tissue, and/or kidney. These receptors thus hold the potential to act as sensors of food intake, regulating, for example, release of incretin hormones from the gut, insulin/glucagon from the pancreas, and leptin from adipose tissue. The promiscuous tendency in ligand recognition of these receptors is in contrast to the typical specific interaction with one physiological agonist seen for most receptors, which challenges the classic "lock-and-key" concept. We here review the molecular mechanisms of nutrient sensing of the calcium-sensing receptor, the G protein-coupled receptor family C, group 6, subtype A (GPRC6A), and the taste1 receptor T1R1/T1R3, which are sensing L-alpha-amino acids, the carbohydrate-sensing T1R2/T1R3 receptor, the proteolytic degradation product sensor GPR93 (also termed GPR92), and the free fatty acid (FFA) sensing receptors FFA1, FFA2, FFA3, GPR84, and GPR120. The involvement of the individual receptors in sensing of food intake has been validated to different degrees because of limited availability of specific pharmacological tools and/or receptor knockout mice. However, as a group, the receptors represent potential drug targets, to treat, for example, type II diabetes by mimicking food intake by potent agonists or positive allosteric modulators. The ligand-receptor interactions of the promiscuous receptors of organic nutrients thus remain an interesting subject of emerging functional importance.

  16. N-Carbamoyl-β-alanine amidohydrolase from Agrobacterium tumefaciens C58: a promiscuous enzyme for the production of amino acids.

    PubMed

    Martínez-Gómez, A I; Andújar-Sánchez, M; Clemente-Jiménez, J M; Neira, J L; Rodríguez-Vico, F; Martínez-Rodríguez, S; Las Heras-Vázquez, F J

    2011-11-01

    The availability of enzymes with a high promiscuity/specificity relationship permits the hydrolysis of several substrates with a view to obtaining a certain product or using one enzyme for several productive lines. N-Carbamoyl-β-alanine amidohydrolase from Agrobacterium tumefaciens (Atβcar) has shown high versatility to hydrolyze different N-carbamoyl-, N-acetyl- and N-formyl-amino acids to produce different α, β, γ and δ amino acids. We have calculated the promiscuity index for the enzyme, obtaining a value of 0.54, which indicates that it is a modestly promiscuous enzyme. Atβcar presented the highest probability of hydrolysis for N-carbamoyl-amino acids, being the enzyme more efficient for the production of α-amino acids. We have also demonstrated by mutagenesis, modelling, kinetic and binding experiments that W218 and A359 indirectly influence the plasticity of the enzyme due to interaction with the environment of R291, the key residue for catalytic activity. Copyright © 2011 Elsevier B.V. All rights reserved.

  17. Critical Determinants of Substrate Recognition by Cyclin-Dependent Kinase-like 5 (CDKL5).

    PubMed

    Katayama, Syouichi; Sueyoshi, Noriyuki; Kameshita, Isamu

    2015-05-19

    Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase known to be associated with X-linked neurodevelopmental disorders. In a previous study, we identified amphiphysin 1 (Amph1) as a potential substrate for CDKL5 and identified a single phosphorylation site at Ser-293. In this study, we investigated the molecular mechanisms of substrate recognition by CDKL5 using Amph1 as a model substrate. Amph1 served as an efficient CDKL5 substrate, whereas Amph2, a structurally related homologue of Amph1, was not phosphorylated by CDKL5. The sequence around the Amph1 phosphorylation site is RPR(293)SPSQ, while the corresponding sequence in Amph2 is IPK(332)SPSQ. To define the amino acid sequence specificity of the substrate, various point mutants of Amph1 and Amph2 were prepared and phosphorylated by CDKL5. Both Amph2(I329R) and Amph1 served as efficient CDKL5 substrates, but Amph1(R290I) did not, indicating that the arginyl residue at the P -3 position is critical for substrate recognition. With regard to prolyl residues around the phosphorylation site of Amph1, Pro-291 at the P -2 position, but not Pro-294 at the P +1 position, is indispensable for phosphorylation by CDKL5. Phosphorylation experiments using various deletion mutants of Amph1 revealed that the proline-rich domain (PRD) (amino acids 247-315) alone was not phosphorylated by CDKL5. In contrast, Amph1(247-385), which comprised the PRD and CLAP domains, served as an efficient CDKL5 substrate. These results, taken together, suggest that both the phosphorylation site sequence (RPXSX) and the CLAP domain structure in Amph1 play crucial roles in recognition and phosphorylation by CDKL5.

  18. Drug Promiscuity in PDB: Protein Binding Site Similarity Is Key.

    PubMed

    Haupt, V Joachim; Daminelli, Simone; Schroeder, Michael

    2013-01-01

    Drug repositioning applies established drugs to new disease indications with increasing success. A pre-requisite for drug repurposing is drug promiscuity (polypharmacology) - a drug's ability to bind to several targets. There is a long standing debate on the reasons for drug promiscuity. Based on large compound screens, hydrophobicity and molecular weight have been suggested as key reasons. However, the results are sometimes contradictory and leave space for further analysis. Protein structures offer a structural dimension to explain promiscuity: Can a drug bind multiple targets because the drug is flexible or because the targets are structurally similar or even share similar binding sites? We present a systematic study of drug promiscuity based on structural data of PDB target proteins with a set of 164 promiscuous drugs. We show that there is no correlation between the degree of promiscuity and ligand properties such as hydrophobicity or molecular weight but a weak correlation to conformational flexibility. However, we do find a correlation between promiscuity and structural similarity as well as binding site similarity of protein targets. In particular, 71% of the drugs have at least two targets with similar binding sites. In order to overcome issues in detection of remotely similar binding sites, we employed a score for binding site similarity: LigandRMSD measures the similarity of the aligned ligands and uncovers remote local similarities in proteins. It can be applied to arbitrary structural binding site alignments. Three representative examples, namely the anti-cancer drug methotrexate, the natural product quercetin and the anti-diabetic drug acarbose are discussed in detail. Our findings suggest that global structural and binding site similarity play a more important role to explain the observed drug promiscuity in the PDB than physicochemical drug properties like hydrophobicity or molecular weight. Additionally, we find ligand flexibility to have a minor

  19. Sexual promiscuity: knowledge of dangers in institutions of higher learning.

    PubMed

    Ebong, R D

    1994-06-01

    Knowledge of dangers of sexual promiscuity was assessed in 2 institutions of higher learning. The objectives were to find out the knowledge of medical and social consequences as well as the factors responsible for sexual promiscuity among Nigerian youths. The study also assessed the discrepancies in societal concept of sex norms for males and females. The result was used as an index to determine the need for sex education for Nigerian youths. A total of 200 students (100 from each school) was assessed by random selection and use of a questionnaire. The result showed that students had a fair knowledge of sexual promiscuity, although in terms of medical consequences the knowledge was low for both groups. On social consequences, the knowledge was fair for both groups. Students agreed that lack of financial support and of supervision from parents and teachers were among the causes of sexual promiscuity. Recommendations were made for Health Education in these areas in institutions of higher learning. Also, recommendations were made for parental education on how to bring up, and care for, their adolescents to reduce the problems of sexual promiscuity. It was also recommended that a compulsory course on sexual promiscuity should be included in the syllabus in institutions of higher learning.

  20. Specialized Dynamical Properties of Promiscuous Residues Revealed by Simulated Conformational Ensembles

    PubMed Central

    2013-01-01

    The ability to interact with different partners is one of the most important features in proteins. Proteins that bind a large number of partners (hubs) have been often associated with intrinsic disorder. However, many examples exist of hubs with an ordered structure, and evidence of a general mechanism promoting promiscuity in ordered proteins is still elusive. An intriguing hypothesis is that promiscuous binding sites have specific dynamical properties, distinct from the rest of the interface and pre-existing in the protein isolated state. Here, we present the first comprehensive study of the intrinsic dynamics of promiscuous residues in a large protein data set. Different computational methods, from coarse-grained elastic models to geometry-based sampling methods and to full-atom Molecular Dynamics simulations, were used to generate conformational ensembles for the isolated proteins. The flexibility and dynamic correlations of interface residues with a different degree of binding promiscuity were calculated and compared considering side chain and backbone motions, the latter both on a local and on a global scale. The study revealed that (a) promiscuous residues tend to be more flexible than nonpromiscuous ones, (b) this additional flexibility has a higher degree of organization, and (c) evolutionary conservation and binding promiscuity have opposite effects on intrinsic dynamics. Findings on simulated ensembles were also validated on ensembles of experimental structures extracted from the Protein Data Bank (PDB). Additionally, the low occurrence of single nucleotide polymorphisms observed for promiscuous residues indicated a tendency to preserve binding diversity at these positions. A case study on two ubiquitin-like proteins exemplifies how binding promiscuity in evolutionary related proteins can be modulated by the fine-tuning of the interface dynamics. The interplay between promiscuity and flexibility highlighted here can inspire new directions in protein

  1. Promiscuity and selectivity of bitter molecules and their receptors.

    PubMed

    Di Pizio, Antonella; Niv, Masha Y

    2015-07-15

    Bitter taste is essential for survival, as it protects against consuming poisonous compounds, which are often bitter. Bitter taste perception is mediated by bitter taste receptors (TAS2Rs), a subfamily of G-protein coupled receptors (GPCRs). The number of TAS2R subtypes is species-dependent, and varies from 3 in chicken to 50 in frog. TAS2Rs present an intriguing case for studying promiscuity: some of the receptors are still orphan, or have few known agonists, while others can be activated by numerous, structurally dissimilar compounds. The ligands also vary in the repertoire of TAS2Rs that they activate: some bitter compounds are selective toward a single TAS2R, while others activate multiple TAS2Rs. Selectivity/promiscuity profile of bitter taste receptors and their compounds was explored by a chemoinformatic approach. TAS2R-promiscuous and TAS2R-selective bitter molecules were found to differ in chemical features, such as AlogP, E-state, total charge, number of rings, globularity, and heavy atom count. This allowed the prediction of bitter ligand selectivity toward TAS2Rs. Interestingly, while promiscuous TAS2Rs are activated by both TAS2R-promiscuous and TAS2R-selective compounds, almost all selective TAS2Rs in human are activated by promiscuous compounds, which are recognized by other TAS2Rs anyway. Thus, unique ligands, that may have been the evolutionary driving force for development of selective TAS2Rs, still need to be unraveled. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Protein promiscuity: drug resistance and native functions--HIV-1 case.

    PubMed

    Fernández, Ariel; Tawfik, Dan S; Berkhout, Ben; Sanders, Rogier; Kloczkowski, Andrzej; Sen, Taner; Jernigan, Bob

    2005-06-01

    The association of a drug with its target protein has the effect of blocking the protein activity and is termed a promiscuous function to distinguish from the protein's native function (Tawfik and associates, Nat. Genet. 37, 73-6, 2005). Obviously, a protein has not evolved naturally for drug association or drug resistance. Promiscuous protein functions exhibit unique traits of evolutionary adaptability, or evolvability, which is dependent on the induction of novel phenotypic traits by a small number of mutations. These mutations might have small effects on native functions, but large effects on promiscuous function; for example, an evolving protein could become increasingly drug resistant while maintaining its original function. Ariel Fernandez, in his opinion piece, notes that drug-binding "promiscuity" can hardly be dissociated from native functions; a dominant approach to drug discovery is the protein-native-substrate transition-state mimetic strategy. Thus, man-made ligands (e.g. drugs) have been successfully crafted to restrain enzymatic activity by focusing on the very same structural features that determine the native function. Using the successful inhibition of HIV-1 protease as an example, Fernandez illustrates how drug designers have employed naturally evolved features of the protein to suppress its activity. Based on these arguments, he dismisses the notion that drug binding is quintessentially promiscuous, even though in principle, proteins did not evolve to associate with man made ligands. In short, Fernandez argues that there may not be separate protein domains that one could term promiscuous domains. While acknowledging that drugs may bind promiscuously or in a native-like manner a la Fernandez, Tawfik maintains the role of evolutionary adaptation, even when a drug binds native-like. In the case of HIV-1 protease, drugs bind natively, and the initial onset of mutations results in drug resistance in addition to a dramatic decline in enzymatic

  3. Expanding the Bioactive Chemical Space of Anthrabenzoxocinones through Engineering the Highly Promiscuous Biosynthetic Modification Steps.

    PubMed

    Mei, Xianyi; Yan, Xiaoli; Zhang, Hui; Yu, Mingjia; Shen, Guangqing; Zhou, Linjun; Deng, Zixin; Lei, Chun; Qu, Xudong

    2018-01-19

    Anthrabenzoxocinones (ABXs) including (-)-ABXs and (+)-ABXs are a group of bacterial FabF-specific inhibitors with potent antimicrobial activity of resistant strains. Optimization of their chemical structures is a promising method to develop potent antibiotics. Through biosynthetic investigation, we herein identified and characterized two highly promiscuous enzymes involved in the (-)-ABX structural modification. The promiscuous halogenase and methyltransferase can respectively introduce halogen-modifications into various positions of the ABX scaffolds and methylation to highly diverse substrates. Manipulation of their activity in both of the (-)-ABXs and (+)-ABXs biosyntheses led to the generation of 14 novel ABX analogues of both enantiomers. Bioactivity assessment revealed that a few of the analogues showed significantly improved antimicrobial activity, with the C3-hydroxyl and chlorine substitutions critical for their activity. This study enormously expands the bioactive chemical space of the ABX family and FabF-specific inhibitors. The disclosed broad-selective biosynthetic machineries and structure-activity relationship provide a solid basis for further generation of potent antimicrobial agents.

  4. The Role of Flexibility and Conformational Selection in the Binding Promiscuity of PDZ Domains

    PubMed Central

    Münz, Márton; Hein, Jotun; Biggin, Philip C.

    2012-01-01

    In molecular recognition, it is often the case that ligand binding is coupled to conformational change in one or both of the binding partners. Two hypotheses describe the limiting cases involved; the first is the induced fit and the second is the conformational selection model. The conformational selection model requires that the protein adopts conformations that are similar to the ligand-bound conformation in the absence of ligand, whilst the induced-fit model predicts that the ligand-bound conformation of the protein is only accessible when the ligand is actually bound. The flexibility of the apo protein clearly plays a major role in these interpretations. For many proteins involved in signaling pathways there is the added complication that they are often promiscuous in that they are capable of binding to different ligand partners. The relationship between protein flexibility and promiscuity is an area of active research and is perhaps best exemplified by the PDZ domain family of proteins. In this study we use molecular dynamics simulations to examine the relationship between flexibility and promiscuity in five PDZ domains: the human Dvl2 (Dishevelled-2) PDZ domain, the human Erbin PDZ domain, the PDZ1 domain of InaD (inactivation no after-potential D protein) from fruit fly, the PDZ7 domain of GRIP1 (glutamate receptor interacting protein 1) from rat and the PDZ2 domain of PTP-BL (protein tyrosine phosphatase) from mouse. We show that despite their high structural similarity, the PDZ binding sites have significantly different dynamics. Importantly, the degree of binding pocket flexibility was found to be closely related to the various characteristics of peptide binding specificity and promiscuity of the five PDZ domains. Our findings suggest that the intrinsic motions of the apo structures play a key role in distinguishing functional properties of different PDZ domains and allow us to make predictions that can be experimentally tested. PMID:23133356

  5. The lifestyle of prokaryotic organisms influences the repertoire of promiscuous enzymes.

    PubMed

    Martínez-Núñez, Mario Alberto; Rodríguez-Vázquez, Katya; Pérez-Rueda, Ernesto

    2015-09-01

    The metabolism of microbial organisms and its diversity are partly the result of an adaptation process to the characteristics of the environments that they inhabit. In this work, we analyze the influence of lifestyle on the content of promiscuous enzymes in 761 nonredundant bacterial and archaeal genomes. Promiscuous enzymes were defined as those proteins whose catalytic activities are defined by two or more different Enzyme Commission (E.C.) numbers. The genomes analyzed were categorized into four lifestyles for their exhaustive comparisons: free-living, extremophiles, pathogens, and intracellular. From these analyses we found that free-living organisms have larger genomes and an enrichment of promiscuous enzymes. In contrast, intracellular organisms showed smaller genomes and the lesser proportion of promiscuous enzymes. On the basis of our data, we show that the proportion of promiscuous enzymes in an organism is mainly influenced by the lifestyle, where fluctuating environments promote its emergence. Finally, we evidenced that duplication processes occur preferentially in metabolism of free-living and extremophiles species. © 2015 Wiley Periodicals, Inc.

  6. Spotting and designing promiscuous ligands for drug discovery.

    PubMed

    Schneider, P; Röthlisberger, M; Reker, D; Schneider, G

    2016-01-21

    The promiscuous binding behavior of bioactive compounds forms a mechanistic basis for understanding polypharmacological drug action. We present the development and prospective application of a computational tool for identifying potential promiscuous drug-like ligands. In combination with computational target prediction methods, the approach provides a working concept for rationally designing such molecular structures. We could confirm the multi-target binding of a de novo generated compound in a proof-of-concept study relying on the new method.

  7. Promiscuity and the evolution of sexual transmitted diseases

    NASA Astrophysics Data System (ADS)

    Gonçalves, Sebastián; Kuperman, Marcelo; Ferreira da Costa Gomes, Marcelo

    2003-09-01

    We study the relation between different social behaviors and the onset of epidemics in a model for the dynamics of sexual transmitted diseases. The model considers the society as a system of individual sexuated agents that can be organized in couples and interact with each other. The different social behaviors are incorporated assigning what we call a promiscuity value to each individual agent. The individual promiscuity is taken from a distribution and represents the daily probability of going out to look for a sexual partner, abandoning its eventual mate. In terms of this parameter we find a threshold for the epidemic which is much lower than the classical SIR model prediction, i.e., R0 (basic reproductive number)=1. Different forms for the distribution of the population promiscuity are considered showing that the threshold is weakly sensitive to them. We study the homosexual and the heterosexual case as well.

  8. Molecular Basis of Substrate Recognition and Degradation by Human Presequence Protease

    PubMed Central

    King, John V.; Liang, Wenguang G.; Scherpelz, Kathryn P.; Schilling, Alexander B.; Meredith, Stephen C.; Tang, Wei-Jen

    2014-01-01

    Summary Human Presequence Protease (hPreP) is an M16 metalloprotease localized in mitochondria. There, hPreP facilitates proteostasis by utilizing a ∼13,300Å3 catalytic chamber to degrade a diverse array of potentially toxic peptides, including mitochondrial presequences and amyloid-β (Aβ), the latter of which contributes to Alzheimer's disease pathogenesis. Here we report crystal structures for hPreP alone and in complex with Aβ, which show that hPreP uses size-exclusion and charge complementation for substrate recognition. These structures also reveal hPreP-specific features that permit a diverse array of peptides, with distinct distributions of charged and hydrophobic residues, to be specifically captured, cleaved, and their amyloidogenic features destroyed. SAXS analysis demonstrates that hPreP in solution exists in dynamic equilibrium between closed and open states, with the former being preferred. Furthermore, Aβ binding induces the closed state and hPreP dimerization. Together, these data reveal the molecular basis for flexible yet specific substrate recognition and degradation by hPreP. PMID:24931469

  9. Atomic substitution reveals the structural basis for substrate adenine recognition and removal by adenine DNA glycosylase

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

    Lee, Seongmin; Verdine, Gregory L.; Harvard)

    2010-01-14

    Adenine DNA glycosylase catalyzes the glycolytic removal of adenine from the promutagenic A {center_dot} oxoG base pair in DNA. The general features of DNA recognition by an adenine DNA glycosylase, Bacillus stearothermophilus MutY, have previously been revealed via the X-ray structure of a catalytically inactive mutant protein bound to an A:oxoG-containing DNA duplex. Although the structure revealed the substrate adenine to be, as expected, extruded from the DNA helix and inserted into an extrahelical active site pocket on the enzyme, the substrate adenine engaged in no direct contacts with active site residues. This feature was paradoxical, because other glycosylases havemore » been observed to engage their substrates primarily through direct contacts. The lack of direct contacts in the case of MutY suggested that either MutY uses a distinctive logic for substrate recognition or that the X-ray structure had captured a noncatalytically competent state in lesion recognition. To gain further insight into this issue, we crystallized wild-type MutY bound to DNA containing a catalytically inactive analog of 2'-deoxyadenosine in which a single 2'-H atom was replaced by fluorine. The structure of this fluorinated lesion-recognition complex (FLRC) reveals the substrate adenine buried more deeply into the active site pocket than in the prior structure and now engaged in multiple direct hydrogen bonding and hydrophobic interactions. This structure appears to capture the catalytically competent state of adenine DNA glycosylases, and it suggests a catalytic mechanism for this class of enzymes, one in which general acid-catalyzed protonation of the nucleobase promotes glycosidic bond cleavage.« less

  10. CYTOMEGALOVIRUS VECTORS VIOLATE CD8+ T CELL EPITOPE RECOGNITION PARADIGMS

    PubMed Central

    Hansen, Scott G.; Sacha, Jonah B.; Hughes, Colette M.; Ford, Julia C.; Burwitz, Benjamin J.; Scholz, Isabel; Gilbride, Roxanne M.; Lewis, Matthew S.; Gilliam, Awbrey N.; Ventura, Abigail B.; Malouli, Daniel; Xu, Guangwu; Richards, Rebecca; Whizin, Nathan; Reed, Jason S.; Hammond, Katherine B.; Fischer, Miranda; Turner, John M.; Legasse, Alfred W.; Axthelm, Michael K.; Edlefsen, Paul T.; Nelson, Jay A.; Lifson, Jeffrey D.; Früh, Klaus; Picker, Louis J.

    2013-01-01

    CD8+ T cell responses focus on a small fraction of pathogen- or vaccine-encoded peptides, and for some pathogens, these restricted recognition hierarchies limit the effectiveness of anti-pathogen immunity. We found that simian immunodeficiency virus (SIV) protein-expressing Rhesus Cytomegalovirus (RhCMV) vectors elicit SIV-specific CD8+ T cells that recognize unusual, diverse and highly promiscuous epitopes, including dominant responses to epitopes restricted by class II major histocompatibility complex (MHC) molecules. Induction of canonical SIV epitope-specific CD8+ T cell responses is suppressed by the RhCMV-encoded Rh189 (US11) gene, and the promiscuous MHC class I- and class II-restricted CD8+ T cell responses only occur in the absence of the Rh157.4-.6 (UL128-131) genes. Thus, CMV vectors can be genetically programmed to achieve distinct patterns of CD8+ T cell epitope recognition. PMID:23704576

  11. Predicting novel substrates for enzymes with minimal experimental effort with active learning

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

    Pertusi, Dante A.; Moura, Matthew E.; Jeffryes, James G.

    Enzymatic substrate promiscuity is more ubiquitous than previously thought, with significant consequences for understanding metabolism and its application to biocatalysis. This realization has given rise to the need for efficient characterization of enzyme promiscuity. Enzyme promiscuity is currently characterized with a limited number of human-selected compounds that may not be representative of the enzyme's versatility. While testing large numbers of compounds may be impractical, computational approaches can exploit existing data to determine the most informative substrates to test next, thereby more thoroughly exploring an enzyme's versatility. To demonstrate this, we used existing studies and tested compounds for four different enzymes,more » developed support vector machine (SVM) models using these datasets, and selected additional compounds for experiments using an active learning approach. SVMs trained on a chemically diverse set of compounds were discovered to achieve maximum accuracies of similar to 80% using similar to 33% fewer compounds than datasets based on all compounds tested in existing studies. Active learning-selected compounds for testing resolved apparent conflicts in the existing training data, while adding diversity to the dataset. The application of these algorithms to wide arrays of metabolic enzymes would result in a library of SVMs that can predict high-probability promiscuous enzymatic reactions and could prove a valuable resource for the design of novel metabolic pathways.« less

  12. Predicting novel substrates for enzymes with minimal experimental effort with active learning.

    PubMed

    Pertusi, Dante A; Moura, Matthew E; Jeffryes, James G; Prabhu, Siddhant; Walters Biggs, Bradley; Tyo, Keith E J

    2017-11-01

    Enzymatic substrate promiscuity is more ubiquitous than previously thought, with significant consequences for understanding metabolism and its application to biocatalysis. This realization has given rise to the need for efficient characterization of enzyme promiscuity. Enzyme promiscuity is currently characterized with a limited number of human-selected compounds that may not be representative of the enzyme's versatility. While testing large numbers of compounds may be impractical, computational approaches can exploit existing data to determine the most informative substrates to test next, thereby more thoroughly exploring an enzyme's versatility. To demonstrate this, we used existing studies and tested compounds for four different enzymes, developed support vector machine (SVM) models using these datasets, and selected additional compounds for experiments using an active learning approach. SVMs trained on a chemically diverse set of compounds were discovered to achieve maximum accuracies of ~80% using ~33% fewer compounds than datasets based on all compounds tested in existing studies. Active learning-selected compounds for testing resolved apparent conflicts in the existing training data, while adding diversity to the dataset. The application of these algorithms to wide arrays of metabolic enzymes would result in a library of SVMs that can predict high-probability promiscuous enzymatic reactions and could prove a valuable resource for the design of novel metabolic pathways. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  13. Promiscuous mating in the harem-roosting fruit bat, Cynopterus sphinx.

    PubMed

    Garg, Kritika M; Chattopadhyay, Balaji; Doss D, Paramanatha Swami; A K, Vinoth Kumar; Kandula, Sripathi; Ramakrishnan, Uma

    2012-08-01

    Observations on mating behaviours and strategies guide our understanding of mating systems and variance in reproductive success. However, the presence of cryptic strategies often results in situations where social mating system is not reflective of genetic mating system. We present such a study of the genetic mating system of a harem-forming bat Cynopterus sphinx where harems may not be true indicators of male reproductive success. This temporal study using data from six seasons on paternity reveals that social harem assemblages do not play a role in the mating system, and variance in male reproductive success is lower than expected assuming polygynous mating. Further, simulations reveal that the genetic mating system is statistically indistinguishable from promiscuity. Our results are in contrast to an earlier study that demonstrated high variance in male reproductive success. Although an outcome of behavioural mating patterns, standardized variance in male reproductive success (I(m)) affects the opportunity for sexual selection. To gain a better understanding of the evolutionary implications of promiscuity for mammals in general, we compared our estimates of I(m) and total opportunity for sexual selection (I(m) /I(f), where I(f) is standardized variance in female reproductive success) with those of other known promiscuous species. We observed a broad range of I(m) /I(f) values across known promiscuous species, indicating our poor understanding of the evolutionary implications of promiscuous mating. © 2012 Blackwell Publishing Ltd.

  14. Structural coupling of SH2-kinase domains links Fes and Abl substrate recognition and kinase activation.

    PubMed

    Filippakopoulos, Panagis; Kofler, Michael; Hantschel, Oliver; Gish, Gerald D; Grebien, Florian; Salah, Eidarus; Neudecker, Philipp; Kay, Lewis E; Turk, Benjamin E; Superti-Furga, Giulio; Pawson, Tony; Knapp, Stefan

    2008-09-05

    The SH2 domain of cytoplasmic tyrosine kinases can enhance catalytic activity and substrate recognition, but the molecular mechanisms by which this is achieved are poorly understood. We have solved the structure of the prototypic SH2-kinase unit of the human Fes tyrosine kinase, which appears specialized for positive signaling. In its active conformation, the SH2 domain tightly interacts with the kinase N-terminal lobe and positions the kinase alphaC helix in an active configuration through essential packing and electrostatic interactions. This interaction is stabilized by ligand binding to the SH2 domain. Our data indicate that Fes kinase activation is closely coupled to substrate recognition through cooperative SH2-kinase-substrate interactions. Similarly, we find that the SH2 domain of the active Abl kinase stimulates catalytic activity and substrate phosphorylation through a distinct SH2-kinase interface. Thus, the SH2 and catalytic domains of active Fes and Abl pro-oncogenic kinases form integrated structures essential for effective tyrosine kinase signaling.

  15. Promiscuous Feminisms for Troubling Times

    ERIC Educational Resources Information Center

    Voithofer, Rick

    2013-01-01

    Looking across the six articles in this issue, this paper argues that promiscuous uses of feminist methodologies offer a unique constellation of conceptual, pragmatic, material, and ethical strategies with which to understand and engage some of the social and cultural tensions that are occurring within and outside schools. It presents a…

  16. Promiscuity in mice is associated with increased vaginal bacterial diversity

    NASA Astrophysics Data System (ADS)

    Macmanes, Matthew David

    2011-11-01

    Differences in the number of sexual partners (i.e., mating system) have the potential to exert a strong influence on the bacterial communities present in reproductive structures like the vagina. Because this structure serves as a conduit for gametes, bacteria present there may have a pronounced, direct effect on host reproductive success. As a first step towards the identification of the relationship between sexual behavior and potentially pathogenic bacterial communities inhabiting vital reproductive structures, as well as their potential effects on fitness, I sought to quantify differences in bacterial diversity in a promiscuous and monogamous mammal species. To accomplish this, I used two sympatric species of Peromyscus rodents— Peromyscus californicus and Peromyscus maniculatus that differ with regard to the number of sexual partners per individual to test the hypothesis that bacterial diversity should be greater in the promiscuous P. maniculatus relative to the monogamous P. californicus. As predicted, phylogenetically controlled and operational taxonomic unit-based indices of bacterial diversity indicated that diversity is greater in the promiscuous species. These results provide important new insights into the effects of mating system on bacterial diversity in free-living vertebrates, and may suggest a potential cost of promiscuity.

  17. Evolution of domain promiscuity in eukaryotic genomes—a perspective from the inferred ancestral domain architectures†

    PubMed Central

    Cohen-Gihon, Inbar; Fong, Jessica H.; Sharan, Roded; Nussinov, Ruth

    2012-01-01

    Most eukaryotic proteins are composed of two or more domains. These assemble in a modular manner to create new proteins usually by the acquisition of one or more domains to an existing protein. Promiscuous domains which are found embedded in a variety of proteins and co-exist with many other domains are of particular interest and were shown to have roles in signaling pathways and mediating network communication. The evolution of domain promiscuity is still an open problem, mostly due to the lack of sequenced ancestral genomes. Here we use inferred domain architectures of ancestral genomes to trace the evolution of domain promiscuity in eukaryotic genomes. We find an increase in average promiscuity along many branches of the eukaryotic tree. Moreover, domain promiscuity can proceed at almost a steady rate over long evolutionary time or exhibit lineage-specific acceleration. We also observe that many signaling and regulatory domains gained domain promiscuity around the Bilateria divergence. In addition we show that those domains that played a role in the creation of two body axes and existed before the divergence of the bilaterians from fungi/metazoan achieve a boost in their promiscuities during the bilaterian evolution. PMID:21127809

  18. No genome barriers to promiscuous DNA

    NASA Astrophysics Data System (ADS)

    Lewin, R.

    1984-06-01

    Farrelly and Butow (1983) used the term 'promiscuous DNA' in their report of the apparent natural transfer of yeast mitochondrial DNA sequences into the nuclear genome. Ellis (1982) applied the same term in an editorial comment. It is pointed out since that time the subject of DNA's promiscuity has exploded with a series of reports. According to a report by Stern (1984), movement of DNA sequences between chloroplasts and mitochondria is not just a rare event but is a rampant process. It was recently concluded that 'the widespread presence of ctDNA sequences in plant mtDNA is best regarded as a dramatic demonstration of the dynamo nature of interactions between the chloroplast and the mitochondrion, similar to the ongoing process of interorganellar DNA transfer already documented between mitochondrion and nucleus and between chloroplast and nucleus'.

  19. Mutations, kataegis, and translocations in B lymphocytes: towards a mechanistic understanding of AID promiscuous activity

    PubMed Central

    Casellas, Rafael; Basu, Uttiya; Yewdell, William T.; Chaudhuri, Jayanta; Robbiani, Davide F.; Di Noia, Javier M.

    2016-01-01

    As B cells engage in the immune response they express the deaminase AID to initiate the hypermutation and recombination of immunoglobulin genes, which are crucial processes for the efficient recognition and disposal of pathogens, However, AID must be tightly controlled in B cells to minimize off-targeting mutations, which can drive chromosomal translocations and the development of B cell malignancies, such as lymphomas. Recent genomic and biochemical analyses have begun to unravel the crucial question of how AID-mediated deamination is targeted outside immunoglobulin genes. Here, we discuss the transcriptional and topological features that are emerging as key drivers of AID promiscuous activity. PMID:26898111

  20. Probing the mutational interplay between primary and promiscuous protein functions: a computational-experimental approach.

    PubMed

    Garcia-Seisdedos, Hector; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M

    2012-01-01

    Protein promiscuity is of considerable interest due its role in adaptive metabolic plasticity, its fundamental connection with molecular evolution and also because of its biotechnological applications. Current views on the relation between primary and promiscuous protein activities stem largely from laboratory evolution experiments aimed at increasing promiscuous activity levels. Here, on the other hand, we attempt to assess the main features of the simultaneous modulation of the primary and promiscuous functions during the course of natural evolution. The computational/experimental approach we propose for this task involves the following steps: a function-targeted, statistical coupling analysis of evolutionary data is used to determine a set of positions likely linked to the recruitment of a promiscuous activity for a new function; a combinatorial library of mutations on this set of positions is prepared and screened for both, the primary and the promiscuous activities; a partial-least-squares reconstruction of the full combinatorial space is carried out; finally, an approximation to the Pareto set of variants with optimal primary/promiscuous activities is derived. Application of the approach to the emergence of folding catalysis in thioredoxin scaffolds reveals an unanticipated scenario: diverse patterns of primary/promiscuous activity modulation are possible, including a moderate (but likely significant in a biological context) simultaneous enhancement of both activities. We show that this scenario can be most simply explained on the basis of the conformational diversity hypothesis, although alternative interpretations cannot be ruled out. Overall, the results reported may help clarify the mechanisms of the evolution of new functions. From a different viewpoint, the partial-least-squares-reconstruction/Pareto-set-prediction approach we have introduced provides the computational basis for an efficient directed-evolution protocol aimed at the simultaneous

  1. Probing the Mutational Interplay between Primary and Promiscuous Protein Functions: A Computational-Experimental Approach

    PubMed Central

    Garcia-Seisdedos, Hector; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M.

    2012-01-01

    Protein promiscuity is of considerable interest due its role in adaptive metabolic plasticity, its fundamental connection with molecular evolution and also because of its biotechnological applications. Current views on the relation between primary and promiscuous protein activities stem largely from laboratory evolution experiments aimed at increasing promiscuous activity levels. Here, on the other hand, we attempt to assess the main features of the simultaneous modulation of the primary and promiscuous functions during the course of natural evolution. The computational/experimental approach we propose for this task involves the following steps: a function-targeted, statistical coupling analysis of evolutionary data is used to determine a set of positions likely linked to the recruitment of a promiscuous activity for a new function; a combinatorial library of mutations on this set of positions is prepared and screened for both, the primary and the promiscuous activities; a partial-least-squares reconstruction of the full combinatorial space is carried out; finally, an approximation to the Pareto set of variants with optimal primary/promiscuous activities is derived. Application of the approach to the emergence of folding catalysis in thioredoxin scaffolds reveals an unanticipated scenario: diverse patterns of primary/promiscuous activity modulation are possible, including a moderate (but likely significant in a biological context) simultaneous enhancement of both activities. We show that this scenario can be most simply explained on the basis of the conformational diversity hypothesis, although alternative interpretations cannot be ruled out. Overall, the results reported may help clarify the mechanisms of the evolution of new functions. From a different viewpoint, the partial-least-squares-reconstruction/Pareto-set-prediction approach we have introduced provides the computational basis for an efficient directed-evolution protocol aimed at the simultaneous

  2. Highly Promiscuous Oxidases Discovered in the Bovine Rumen Microbiome.

    PubMed

    Ufarté, Lisa; Potocki-Veronese, Gabrielle; Cecchini, Davide; Tauzin, Alexandra S; Rizzo, Angeline; Morgavi, Diego P; Cathala, Bernard; Moreau, Céline; Cleret, Megane; Robe, Patrick; Klopp, Christophe; Laville, Elisabeth

    2018-01-01

    The bovine rumen hosts a diverse microbiota, which is highly specialized in the degradation of lignocellulose. Ruminal bacteria, in particular, are well equipped to deconstruct plant cell wall polysaccharides. Nevertheless, their potential role in the breakdown of the lignin network has never been investigated. In this study, we used functional metagenomics to identify bacterial redox enzymes acting on polyaromatic compounds. A new methodology was developed to explore the potential of uncultured microbes to degrade lignin derivatives, namely kraft lignin and lignosulfonate. From a fosmid library covering 0.7 Gb of metagenomic DNA, three hit clones were identified, producing enzymes able to oxidize a wide variety of polyaromatic compounds without the need for the addition of copper, manganese, or mediators. These promiscuous redox enzymes could thus be of potential interest both in plant biomass refining and dye remediation. The enzymes were derived from uncultured Clostridia, and belong to complex gene clusters involving proteins of different functional types, including hemicellulases, which likely work in synergy to produce substrate degradation.

  3. Molecular Recognition of Fluorine Impacts Substrate Selectivity in the Fluoroacetyl-CoA Thioesterase FlK

    PubMed Central

    2015-01-01

    The fluoroacetate-producing bacterium Streptomyces cattleya has evolved a fluoroacetyl-CoA thioesterase (FlK) that exhibits a remarkably high level of discrimination for its cognate substrate compared to the cellularly abundant analogue acetyl-CoA, which differs only by the absence of the fluorine substitution. A major determinant of FlK specificity derives from its ability to take advantage of the unique properties of fluorine to enhance the reaction rate, allowing fluorine discrimination under physiological conditions where both substrates are likely to be present at saturating concentrations. Using a combination of pH–rate profiles, pre-steady-state kinetic experiments, and Taft analysis of wild-type and mutant FlKs with a set of substrate analogues, we explore the role of fluorine in controlling the enzyme acylation and deacylation steps. Further analysis of chiral (R)- and (S)-[2H1]fluoroacetyl-CoA substrates demonstrates that a kinetic isotope effect (1.7 ± 0.2) is observed for only the (R)-2H1 isomer, indicating that deacylation requires recognition of the prochiral fluoromethyl group to position the α-carbon for proton abstraction. Taken together, the selectivity for the fluoroacetyl-CoA substrate appears to rely not only on the enhanced polarization provided by the electronegative fluorine substitution but also on molecular recognition of fluorine in both formation and breakdown of the acyl-enzyme intermediate to control active site reactivity. These studies provide insights into the basis of fluorine selectivity in a naturally occurring enzyme–substrate pair, with implications for drug design and the development of fluorine-selective biocatalysts. PMID:24635371

  4. Z linkage of female promiscuity genes in the moth Utetheisa ornatrix: support for the sexy-sperm hypothesis?

    PubMed

    Iyengar, Vikram K; Reeve, Hudson K

    2010-05-01

    Female preference genes for large males in the highly promiscuous moth Utetheisa ornatrix (Lepidoptera: Arctiidae) have previously been shown to be mostly Z-linked, in accordance with the hypothesis that ZZ-ZW sex chromosome systems should facilitate Fisherian sexual selection. We determined the heritability of both female and male promiscuity in the highly promiscuous moth U. ornatrix (Lepidoptera: Arctiidae) through parent-offspring and grandparent-offspring regression analyses. Our data show that male promiscuity is not sex-limited and either autosomal or sex-linked whereas female promiscuity is primarily determined by sex-limited, Z-linked genes. These data are consistent with the "sexy-sperm hypothesis," which posits that multiple-mating and sperm competitiveness coevolve through a Fisherian-like process in which female promiscuity is a kind of mate choice in which sperm-competitiveness is the trait favored in males. Such a Fisherian process should also be more potent when female preferences are Z-linked and sex-limited than when autosomal or not limited.

  5. SRB-2: a promiscuous rainbow aptamer for live-cell RNA imaging.

    PubMed

    Sunbul, Murat; Jäschke, Andres

    2018-06-21

    The SRB-2 aptamer originally selected against sulforhodamine B is shown here to promiscuously bind to various dyes with different colors. Binding of SRB-2 to these dyes results in either fluorescence increase or decrease, making them attractive for fluorescence microscopy and biological assays. By systematically varying fluorophore structural elements and measuring dissociation constants, the principles of fluorophore recognition by SRB-2 were analyzed. The obtained structure-activity relationships allowed us to rationally design a novel, bright, orange fluorescent turn-on probe (TMR-DN) with low background fluorescence, enabling no-wash live-cell RNA imaging. This new probe improved the signal-to-background ratio of fluorescence images by one order of magnitude over best previously known probe for this aptamer. The utility of TMR-DN is demonstrated by imaging ribosomal and messenger RNAs, allowing the observation of distinct localization patterns in bacteria and mammalian cells. The SRB-2 / TMR-DN system is found to be orthogonal to the Spinach/DFHBI and MG/Malachite green aptamer/dye systems.

  6. A PP2A-B55 recognition signal controls substrate dephosphorylation kinetics during mitotic exit

    PubMed Central

    Cundell, Michael J.; Holder, James

    2016-01-01

    PP2A-B55 is one of the major phosphatases regulating cell division. Despite its importance for temporal control during mitotic exit, how B55 substrates are recognized and differentially dephosphorylated is unclear. Using phosphoproteomics combined with kinetic modeling to extract B55-dependent rate constants, we have systematically identified B55 substrates and assigned their temporal order in mitotic exit. These substrates share a bipartite polybasic recognition determinant (BPR) flanking a Cdk1 phosphorylation site. Experiments and modeling show that dephosphorylation rate is encoded into B55 substrates, including its inhibitor ENSA, by cooperative action of basic residues within the BPR. A complementary acidic surface on B55 decodes this signal, supporting a cooperative electrostatic mechanism for substrate selection. A further level of specificity is encoded into B55 substrates because B55 displays selectivity for phosphothreonine. These simple biochemical properties, combined with feedback control of B55 activity by the phosphoserine-containing substrate/inhibitor ENSA, can help explain the temporal sequence of events during exit from mitosis. PMID:27551054

  7. The anchorless adhesin Eap (extracellular adherence protein) from Staphylococcus aureus selectively recognizes extracellular matrix aggregates but binds promiscuously to monomeric matrix macromolecules.

    PubMed

    Hansen, Uwe; Hussain, Muzaffar; Villone, Daniela; Herrmann, Mathias; Robenek, Horst; Peters, Georg; Sinha, Bhanu; Bruckner, Peter

    2006-05-01

    Besides a number of cell wall-anchored adhesins, the majority of Staphylococcus aureus strains produce anchorless, cell wall-associated proteins, such as Eap (extracellular adherence protein). Eap contains four to six tandem repeat (EAP)-domains. Eap mediates diverse biological functions, including adherence and immunomodulation, thus contributing to S. aureus pathogenesis. Eap binding to host macromolecules is unusually promiscuous and includes matrix or matricellular proteins as well as plasma proteins. The structural basis of this promiscuity is poorly understood. Here, we show that in spite of the preferential location of the binding epitopes within triple helical regions in some collagens there is a striking specificity of Eap binding to different collagen types. Collagen I, but not collagen II, is a binding substrate in monomolecular form. However, collagen I is virtually unrecognized by Eap when incorporated into banded fibrils. By contrast, microfibrils containing collagen VI as well as basement membrane-associated networks containing collagen IV, or aggregates containing fibronectin bound Eap as effectively as the monomeric proteins. Therefore, Eap-binding to extracellular matrix ligands is promiscuous at the molecular level but not indiscriminate with respect to supramolecular structures containing the same macromolecules. In addition, Eap bound to banded fibrils after their partial disintegration by matrix-degrading proteinases, including matrix metalloproteinase 1. Therefore, adherence to matrix suprastructures by S. aureus can be supported by inflammatory reactions.

  8. Allosteric Control of Substrate Specificity of the Escherichia coli ADP-glucose Pyrophosphorylase

    NASA Astrophysics Data System (ADS)

    Ebrecht, Ana C.; Solamen, Ligin; Hill, Benjamin L.; Iglesias, Alberto A.; Olsen, Kenneth W.; Ballicora, Miguel A.

    2017-06-01

    The substrate specificity of enzymes is crucial to control the fate of metabolites to different pathways. However, there is growing evidence that many enzymes can catalyze alternative reactions. This promiscuous behavior has important implications in protein evolution and the acquisition of new functions. The question is how the undesirable outcomes of in vivo promiscuity can be prevented. ADP-glucose pyrophosphorylase from Escherichia coli is an example of an enzyme that needs to select the correct substrate from a broad spectrum of alternatives. This selection will guide the flow of carbohydrate metabolism towards the synthesis of reserve polysaccharides. Here, we show that the allosteric activator fructose-1,6-bisphosphate plays a role in such selection by increasing the catalytic efficiency of the enzyme towards the use of ATP rather than other nucleotides. In the presence of fructose-1,6-bisphosphate, the kcat/S0.5 for ATP was near 600-fold higher that other nucleotides, whereas in the absence of activator was only 3-fold higher. We propose that the allosteric regulation of certain enzymes is an evolutionary mechanism of adaptation for the selection of specific substrates.

  9. Structural insights into the cofactor-assisted substrate recognition of yeast methylglyoxal/isovaleraldehyde reductase Gre2.

    PubMed

    Guo, Peng-Chao; Bao, Zhang-Zhi; Ma, Xiao-Xiao; Xia, Qingyou; Li, Wei-Fang

    2014-09-01

    Saccharomyces cerevisiae Gre2 (EC1.1.1.283) serves as a versatile enzyme that catalyzes the stereoselective reduction of a broad range of substrates including aliphatic and aromatic ketones, diketones, as well as aldehydes, using NADPH as the cofactor. Here we present the crystal structures of Gre2 from S. cerevisiae in an apo-form at 2.00Å and NADPH-complexed form at 2.40Å resolution. Gre2 forms a homodimer, each subunit of which contains an N-terminal Rossmann-fold domain and a variable C-terminal domain, which participates in substrate recognition. The induced fit upon binding to the cofactor NADPH makes the two domains shift toward each other, producing an interdomain cleft that better fits the substrate. Computational simulation combined with site-directed mutagenesis and enzymatic activity analysis enabled us to define a potential substrate-binding pocket that determines the stringent substrate stereoselectivity for catalysis. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Male mate choice influences female promiscuity in Soay sheep

    PubMed Central

    Preston, B.T.; Stevenson, I.R.; Pemberton, J.M.; Coltman, D.W.; Wilson, K.

    2005-01-01

    In most animal species, males are predicted to compete for reproductive opportunities, while females are expected to choose between potential mates. However, when males’ rate of reproduction is constrained, or females vary widely in ‘quality’, male mate choice is also predicted to occur. Such conditions exist in the promiscuous mating system of feral Soay sheep on St Kilda, Scotland, where a highly synchronized mating season, intense sperm competition and limitations on sperm production constrain males’ potential reproductive rate, and females vary substantially in their ability to produce successful offspring. We show that, consistent with predictions, competitive rams focus their mating activity and siring success towards heavier females with higher inclusive fitness. To our knowledge, this is the first time that male mate choice has been identified and shown to lead to assortative patterns of parentage in a natural mammalian system, and occurs despite fierce male–male competition for mates. An additional consequence of assortative mating in this population is that lighter females experience a series of unstable consorts with less adept rams, and hence are mated by a greater number of males during their oestrus. We have thus also identified a novel male-driven mechanism that generates variation in female promiscuity, which suggests that the high levels of female promiscuity in this system are not part of an adaptive female tactic to intensify post-copulatory competition between males. PMID:15734690

  11. Male mate choice influences female promiscuity in Soay sheep.

    PubMed

    Preston, B T; Stevenson, I R; Pemberton, J M; Coltman, D W; Wilson, K

    2005-02-22

    In most animal species, males are predicted to compete for reproductive opportunities, while females are expected to choose between potential mates. However, when males' rate of reproduction is constrained, or females vary widely in 'quality', male mate choice is also predicted to occur. Such conditions exist in the promiscuous mating system of feral Soay sheep on St Kilda, Scotland, where a highly synchronized mating season, intense sperm competition and limitations on sperm production constrain males' potential reproductive rate, and females vary substantially in their ability to produce successful offspring. We show that, consistent with predictions, competitive rams focus their mating activity and siring success towards heavier females with higher inclusive fitness. To our knowledge, this is the first time that male mate choice has been identified and shown to lead to assortative patterns of parentage in a natural mammalian system, and occurs despite fierce male-male competition for mates. An additional consequence of assortative mating in this population is that lighter females experience a series of unstable consorts with less adept rams, and hence are mated by a greater number of males during their oestrus. We have thus also identified a novel male-driven mechanism that generates variation in female promiscuity, which suggests that the high levels of female promiscuity in this system are not part of an adaptive female tactic to intensify post-copulatory competition between males.

  12. Simulation studies of substrate recognition by the exocellulase CelF from Clostridium cellulolyticum

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

    Chen, Mo; Himmel, Michael E.; Wilson, David B.

    Molecular dynamics (MD) simulations were used to study substrate recognition by the family 48 exocellulase CelF from Clostridium cellulolyticum. It was hypothesized that residues around the entrance of the active site tunnel of this enzyme might serve to recognize and bind the substrate through an affinity for the cellulose monomer repeat unit, ..beta..-d-glucopyranose. Simulations were conducted of the catalytic domain of this enzyme surrounded by a concentrated solution of ..beta..-d-glucopyranose, and the full three-dimensional probability distribution for finding sugar molecules adjacent to the enzyme was calculated from the trajectory. A significant probability of finding the sugar stacked against the planarmore » faces of Trp 310 and Trp 312 at the entrance of the active site tunnel was observed.« less

  13. Structural Determinants of Substrate Recognition in the HAD Superfamily Member D-glycero-D-manno-Heptose-1,7-bisphosphate Phosphatase (GmhB)

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

    Nguyen, H.; Wang, L; Huang, H

    2010-01-01

    The haloalkanoic acid dehalogenase (HAD) enzyme superfamily is the largest family of phosphohydrolases. In HAD members, the structural elements that provide the binding interactions that support substrate specificity are separated from those that orchestrate catalysis. For most HAD phosphatases, a cap domain functions in substrate recognition. However, for the HAD phosphatases that lack a cap domain, an alternate strategy for substrate selection must be operative. One such HAD phosphatase, GmhB of the HisB subfamily, was selected for structure-function analysis. Herein, the X-ray crystallographic structures of Escherichia coli GmhB in the apo form (1.6 {angstrom} resolution), in a complex with Mg{supmore » 2+} and orthophosphate (1.8 {angstrom} resolution), and in a complex with Mg{sup 2+} and D-glycero-D-manno-heptose 1{beta},7-bisphosphate (2.2 {angstrom} resolution) were determined, in addition to the structure of Bordetella bronchiseptica GmhB bound to Mg{sup 2+} and orthophosphate (1.7 {angstrom} resolution). The structures show that in place of a cap domain, the GmhB catalytic site is elaborated by three peptide inserts or loops that pack to form a concave, semicircular surface around the substrate leaving group. Structure-guided kinetic analysis of site-directed mutants was conducted in parallel with a bioinformatics study of sequence diversification within the HisB subfamily to identify loop residues that serve as substrate recognition elements and that distinguish GmhB from its subfamily counterpart, the histidinol-phosphate phosphatase domain of HisB. We show that GmhB and the histidinol-phosphate phosphatase domain use the same design of three substrate recognition loops inserted into the cap domain yet, through selective residue usage on the loops, have achieved unique substrate specificity and thus novel biochemical function.« less

  14. Investigation of the intermolecular recognition mechanism between the E3 ubiquitin ligase Keap1 and substrate based on multiple substrates analysis.

    PubMed

    Jiang, Zheng-Yu; Xu, Li-Li; Lu, Meng-Chen; Pan, Yang; Huang, Hao-Ze; Zhang, Xiao-Jin; Sun, Hao-Peng; You, Qi-Dong

    2014-12-01

    E3 ubiquitin ligases are attractive drug targets due to their specificity to the ubiquitin machinery. However, the development of E3 ligase inhibitors has proven challenging for the fact that they must disrupt protein-protein interactions (PPIs). The E3 ligase involved in interactome provide new hope for the discovery of the E3 ligase inhibitors. These currently known natural binding partners of the E3 ligase can benefit the discovery of other unknown substrates and also the E3 ligase inhibitors. Herein, we present a novel strategy that using multiple substrates to elucidate the molecular recognition mechanism of E3 ubiquitin ligase. Molecular dynamics simulation, molecular mechanics-generalized born surface area (MM-GBSA) binding energy calculation and energy decomposition scheme were incorporated to evaluate the quantitative contributions of sub-pocket and per-residue to binding. In this case, Kelch-like ECH-associated protein-1 (Keap1), a substrate adaptor component of the Cullin-RING ubiquitin ligases complex, is applied for the investigation of how it recognize its substrates, especially Nrf2, a master regulator of the antioxidant response. By analyzing multiple substrates binding determinants, we found that both the polar sub-pockets (P1 and P2) and the nonpolar sub-pockets (P4 and P5) of Keap1 can make remarkable contributions to intermolecular interactions. This finding stresses the requirement for substrates to interact with the polar and nonpolar sub-pockets simultaneously. The results discussed in this paper not only show the binding determinants of the Keap1 substrates but also provide valuable implications for both Keap1 substrate discovery and PPI inhibitor design.

  15. Investigation of the intermolecular recognition mechanism between the E3 ubiquitin ligase Keap1 and substrate based on multiple substrates analysis

    NASA Astrophysics Data System (ADS)

    Jiang, Zheng-Yu; Xu, Li-Li; Lu, Meng-Chen; Pan, Yang; Huang, Hao-Ze; Zhang, Xiao-Jin; Sun, Hao-Peng; You, Qi-Dong

    2014-12-01

    E3 ubiquitin ligases are attractive drug targets due to their specificity to the ubiquitin machinery. However, the development of E3 ligase inhibitors has proven challenging for the fact that they must disrupt protein-protein interactions (PPIs). The E3 ligase involved in interactome provide new hope for the discovery of the E3 ligase inhibitors. These currently known natural binding partners of the E3 ligase can benefit the discovery of other unknown substrates and also the E3 ligase inhibitors. Herein, we present a novel strategy that using multiple substrates to elucidate the molecular recognition mechanism of E3 ubiquitin ligase. Molecular dynamics simulation, molecular mechanics-generalized born surface area (MM-GBSA) binding energy calculation and energy decomposition scheme were incorporated to evaluate the quantitative contributions of sub-pocket and per-residue to binding. In this case, Kelch-like ECH-associated protein-1 (Keap1), a substrate adaptor component of the Cullin-RING ubiquitin ligases complex, is applied for the investigation of how it recognize its substrates, especially Nrf2, a master regulator of the antioxidant response. By analyzing multiple substrates binding determinants, we found that both the polar sub-pockets (P1 and P2) and the nonpolar sub-pockets (P4 and P5) of Keap1 can make remarkable contributions to intermolecular interactions. This finding stresses the requirement for substrates to interact with the polar and nonpolar sub-pockets simultaneously. The results discussed in this paper not only show the binding determinants of the Keap1 substrates but also provide valuable implications for both Keap1 substrate discovery and PPI inhibitor design.

  16. Allosteric response and substrate sensitivity in peptide binding of the signal recognition particle.

    PubMed

    Wang, Connie Y; Miller, Thomas F

    2014-10-31

    We characterize the conformational dynamics and substrate selectivity of the signal recognition particle (SRP) using a thermodynamic free energy cycle approach and microsecond timescale molecular dynamics simulations. The SRP is a central component of the co-translational protein targeting machinery that binds to the N-terminal signal peptide (SP) of nascent proteins. We determined the shift in relative conformational stability of the SRP upon substrate binding to quantify allosteric coupling between SRP domains. In particular, for dipeptidyl aminopeptidase, an SP that is recognized by the SRP for co-translational targeting, it is found that substrate binding induces substantial changes in the SRP toward configurations associated with targeting of the nascent protein, and it is found that the changes are modestly enhanced by a mutation that increases the hydrophobicity of the SP. However, for alkaline phosphatase, an SP that is recognized for post-translational targeting, substrate binding induces the reverse change in the SRP conformational distribution away from targeting configurations. Microsecond timescale trajectories reveal the intrinsic flexibility of the SRP conformational landscape and provide insight into recent single molecule studies by illustrating that 10-nm lengthscale changes between FRET pairs occur via the rigid-body movement of SRP domains connected by the flexible linker region. In combination, these results provide direct evidence for the hypothesis that substrate-controlled conformational switching in the SRP provides a mechanism for discriminating between different SPs and for connecting substrate binding to downstream steps in the protein targeting pathway. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Protein-Protein Interactions, Not Substrate Recognition, Dominate the Turnover of Chimeric Assembly Line Polyketide Synthases*

    PubMed Central

    Klaus, Maja; Ostrowski, Matthew P.; Austerjost, Jonas; Robbins, Thomas; Lowry, Brian; Cane, David E.; Khosla, Chaitan

    2016-01-01

    The potential for recombining intact polyketide synthase (PKS) modules has been extensively explored. Both enzyme-substrate and protein-protein interactions influence chimeric PKS activity, but their relative contributions are unclear. We now address this issue by studying a library of 11 bimodular and 8 trimodular chimeric PKSs harboring modules from the erythromycin, rifamycin, and rapamycin synthases. Although many chimeras yielded detectable products, nearly all had specific activities below 10% of the reference natural PKSs. Analysis of selected bimodular chimeras, each with the same upstream module, revealed that turnover correlated with the efficiency of intermodular chain translocation. Mutation of the acyl carrier protein (ACP) domain of the upstream module in one chimera at a residue predicted to influence ketosynthase-ACP recognition led to improved turnover. In contrast, replacement of the ketoreductase domain of the upstream module by a paralog that produced the enantiomeric ACP-bound diketide caused no changes in processing rates for each of six heterologous downstream modules compared with those of the native diketide. Taken together, these results demonstrate that protein-protein interactions play a larger role than enzyme-substrate recognition in the evolution or design of catalytically efficient chimeric PKSs. PMID:27246853

  18. Substrate promiscuity of a rosmarinic acid synthase from lavender (Lavandula angustifolia L.).

    PubMed

    Landmann, Christian; Hücherig, Stefanie; Fink, Barbara; Hoffmann, Thomas; Dittlein, Daniela; Coiner, Heather A; Schwab, Wilfried

    2011-08-01

    One of the most common types of modification of secondary metabolites is the acylation of oxygen- and nitrogen-containing substrates to produce esters and amides, respectively. Among the known acyltransferases, the members of the plant BAHD family are capable of acylating a wide variety of substrates. Two full-length acyltransferase cDNAs (LaAT1 and 2) were isolated from lavender flowers (Lavandula angustifolia L.) by reverse transcriptase-PCR using degenerate primers based on BAHD sequences. Recombinant LaAT1 exhibited a broad substrate tolerance accepting (hydroxy)cinnamoyl-CoAs as acyl donors and not only tyramine, tryptamine, phenylethylamine and anthranilic acid but also shikimic acid and 4-hydroxyphenyllactic acid as acceptors. Thus, LaLT1 forms esters and amides like its phylogenetic neighbors. In planta LaAT1 might be involved in the biosynthesis of rosmarinic acid, the ester of caffeic acid and 3,4-dihydroxyphenyllactic acid, a major constituent of lavender flowers. LaAT2 is one of three members of clade VI with unknown function.

  19. Nosology, ontology and promiscuous realism.

    PubMed

    Binney, Nicholas

    2015-06-01

    Medics may consider worrying about their metaphysics and ontology to be a waste of time. I will argue here that this is not the case. Promiscuous realism is a metaphysical position which holds that multiple, equally valid, classification schemes should be applied to objects (such as patients) to capture different aspects of their complex and heterogeneous nature. As medics at the bedside may need to capture different aspects of their patients' problems, they may need to use multiple classification schemes (multiple nosologies), and thus consider adopting a different metaphysics to the one commonly in use. © 2014 John Wiley & Sons, Ltd.

  20. The Polyomavirus BK Large T-Antigen-Derived Peptide Elicits an HLA-DR Promiscuous and Polyfunctional CD4+ T-Cell Response▿

    PubMed Central

    Ramaswami, Bala; Popescu, Iulia; Macedo, Camila; Luo, Chunqing; Shapiro, Ron; Metes, Diana; Chalasani, Geetha; Randhawa, Parmjeet S.

    2011-01-01

    BK virus (BKV) nephropathy and hemorrhagic cystitis are increasingly recognized causes of disease in renal and hematopoietic stem cell transplant recipients, respectively. Functional characterization of the immune response to BKV is important for clinical diagnosis, prognosis, and vaccine design. A peptide mix (PepMix) and overlapping (OPP) or random (RPP) peptide pools derived from BKV large T antigen (LTA) were used to restimulate 14-day-expanded peripheral blood mononuclear cells (PBMC) from 27 healthy control subjects in gamma interferon (IFN-γ)-specific enzyme-linked immunospot (ELISPOT) assays. A T-cell response to LTA PepMix was detected in 15/27 subjects. A response was frequently observed with peptides derived from the helicase domain (9/15 subjects), while the DNA binding and host range domains were immunologically inert (0/15 subjects). For all nine subjects who responded to LTA peptide pools, the immune response could be explained largely by a 15-mer peptide designated P313. P313-specific CD4+ T-cell clones demonstrated (i) stringent LTA peptide specificity; (ii) promiscuous recognition in the context of HLA-DR alleles; (iii) cross recognition of homologous peptides from the polyomavirus simian virus 40 (SV40); (iv) an effector memory phenotype, CD107a expression, and intracellular production of IFN-γ and tumor necrosis factor alpha (TNF-α); (v) cytotoxic activity in a chromium release assay; and (vi) the ability to directly present cognate antigen to autologous T cells. In conclusion, T-cell-mediated immunity to BKV in healthy subjects is associated with a polyfunctional population of CD4+ T cells with dual T-helper and T-cytotoxic properties. HLA class II promiscuity in antigen presentation makes the targeted LTA peptide sequence a suitable candidate for inclusion in immunotherapy protocols. PMID:21367979

  1. The polyomavirus BK large T-antigen-derived peptide elicits an HLA-DR promiscuous and polyfunctional CD4+ T-cell response.

    PubMed

    Ramaswami, Bala; Popescu, Iulia; Macedo, Camila; Luo, Chunqing; Shapiro, Ron; Metes, Diana; Chalasani, Geetha; Randhawa, Parmjeet S

    2011-05-01

    BK virus (BKV) nephropathy and hemorrhagic cystitis are increasingly recognized causes of disease in renal and hematopoietic stem cell transplant recipients, respectively. Functional characterization of the immune response to BKV is important for clinical diagnosis, prognosis, and vaccine design. A peptide mix (PepMix) and overlapping (OPP) or random (RPP) peptide pools derived from BKV large T antigen (LTA) were used to restimulate 14-day-expanded peripheral blood mononuclear cells (PBMC) from 27 healthy control subjects in gamma interferon (IFN-γ)-specific enzyme-linked immunospot (ELISPOT) assays. A T-cell response to LTA PepMix was detected in 15/27 subjects. A response was frequently observed with peptides derived from the helicase domain (9/15 subjects), while the DNA binding and host range domains were immunologically inert (0/15 subjects). For all nine subjects who responded to LTA peptide pools, the immune response could be explained largely by a 15-mer peptide designated P313. P313-specific CD4(+) T-cell clones demonstrated (i) stringent LTA peptide specificity; (ii) promiscuous recognition in the context of HLA-DR alleles; (iii) cross recognition of homologous peptides from the polyomavirus simian virus 40 (SV40); (iv) an effector memory phenotype, CD107a expression, and intracellular production of IFN-γ and tumor necrosis factor alpha (TNF-α); (v) cytotoxic activity in a chromium release assay; and (vi) the ability to directly present cognate antigen to autologous T cells. In conclusion, T-cell-mediated immunity to BKV in healthy subjects is associated with a polyfunctional population of CD4(+) T cells with dual T-helper and T-cytotoxic properties. HLA class II promiscuity in antigen presentation makes the targeted LTA peptide sequence a suitable candidate for inclusion in immunotherapy protocols.

  2. Promiscuous honey bee queens increase colony productivity by suppressing worker selfishness.

    PubMed

    Mattila, Heather R; Reeve, H Kern; Smith, Michael L

    2012-11-06

    Queen monogamy is ancestral among bees, ants, and wasps (Order Hymenoptera), and the close relatedness that it generates within colonies is considered key for the evolution of eusociality in these lineages. Paradoxically, queens of several eusocial species are extremely promiscuous, a derived behavior that decreases relatedness among workers and fitness gained from rearing siblings but benefits queens by enhancing colony productivity and inducing workers to rear queens' sons instead of less related worker-derived males. Selection for promiscuity would be especially strong if productivity in a singly inseminated queen's colony declined because selfish workers invested in personal reproduction at the expense of performing tasks that contribute to colony productivity. We show in honey bees that workers' ovaries are more developed when queens are singly rather than multiply inseminated and that increasing ovary activation is coupled with reductions in task performance by workers and colony-wide rates of foraging and waggle-dance recruitment. Increased investment in reproductive physiology by selfish workers might result from greater incentive for them to favor worker-derived males or because low mating frequency signals a queen's diminished quality or future fecundity. Either possibility fosters selection for queen promiscuity, revealing a novel benefit of it for eusocial insects. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. Directed divergent evolution of a thermostable D-tagatose epimerase towards improved activity for two hexose substrates.

    PubMed

    Bosshart, Andreas; Hee, Chee Seng; Bechtold, Matthias; Schirmer, Tilman; Panke, Sven

    2015-03-02

    Functional promiscuity of enzymes can often be harnessed as the starting point for the directed evolution of novel biocatalysts. Here we describe the divergent morphing of an engineered thermostable variant (Var8) of a promiscuous D-tagatose epimerase (DTE) into two efficient catalysts for the C3 epimerization of D-fructose to D-psicose and of L-sorbose to L-tagatose. Iterative single-site randomization and screening of 48 residues in the first and second shells around the substrate-binding site of Var8 yielded the eight-site mutant IDF8 (ninefold improved kcat for the epimerization of D-fructose) and the six-site mutant ILS6 (14-fold improved epimerization of L-sorbose), compared to Var8. Structure analysis of IDF8 revealed a charged patch at the entrance of its active site; this presumably facilitates entry of the polar substrate. The improvement in catalytic activity of variant ILS6 is thought to relate to subtle changes in the hydration of the bound substrate. The structures can now be used to select additional sites for further directed evolution of the ketohexose epimerase. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Structural Basis of Substrate Recognition by Hematopoietic Tyrosine Phosphatase (HePTP)

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

    Critton, D.; Tortajada, A; Stetson, G

    2008-01-01

    Hematopoietic tyrosine phosphatase (HePTP) is one of three members of the kinase interaction motif (KIM) phosphatase family which also includes STEP and PCPTP1. The KIM-PTPs are characterized by a 15 residue sequence, the KIM, which confers specific high-affinity binding to their only known substrates, the MAP kinases Erk and p38, an interaction which is critical for their ability to regulate processes such as T cell differentiation (HePTP) and neuronal signaling (STEP). The KIM-PTPs are also characterized by a unique set of residues in their PTP substrate binding loops, where 4 of the 13 residues are differentially conserved among the KIM-PTPsmore » as compared to more than 30 other class I PTPs. One of these residues, T106 in HePTP, is either an aspartate or asparagine in nearly every other PTP. Using multiple techniques, we investigate the role of these KIM-PTP specific residues in order to elucidate the molecular basis of substrate recognition by HePTP. First, we used NMR spectroscopy to show that Erk2-derived peptides interact specifically with HePTP at the active site. Next, to reveal the molecular details of this interaction, we solved the high-resolution three-dimensional structures of two distinct HePTP-Erk2 peptide complexes. Strikingly, we were only able to obtain crystals of these transient complexes using a KIM-PTP specific substrate-trapping mutant, in which the KIM-PTP specific residue T106 was mutated to an aspartic acid (T106D). The introduced aspartate side chain facilitates the coordination of the bound peptides, thereby stabilizing the active dephosphorylation complex. These structures establish the essential role of HePTP T106 in restricting HePTP specificity to only those substrates which are able to interact with KIM-PTPs via the KIM (e.g., Erk2, p38). Finally, we describe how this interaction of the KIM is sufficient for overcoming the otherwise weak interaction at the active site of KIM-PTPs.« less

  5. Promiscuous 2-aminothiazoles (PrATs): a frequent hitting scaffold.

    PubMed

    Devine, Shane M; Mulcair, Mark D; Debono, Cael O; Leung, Eleanor W W; Nissink, J Willem M; Lim, San Sui; Chandrashekaran, Indu R; Vazirani, Mansha; Mohanty, Biswaranjan; Simpson, Jamie S; Baell, Jonathan B; Scammells, Peter J; Norton, Raymond S; Scanlon, Martin J

    2015-02-12

    We have identified a class of molecules, known as 2-aminothiazoles (2-ATs), as frequent-hitting fragments in biophysical binding assays. This was exemplified by 4-phenylthiazol-2-amine being identified as a hit in 14/14 screens against a diverse range of protein targets, suggesting that this scaffold is a poor starting point for fragment-based drug discovery. This prompted us to analyze this scaffold in the context of an academic fragment library used for fragment-based drug discovery (FBDD) and two larger compound libraries used for high-throughput screening (HTS). This analysis revealed that such "promiscuous 2-aminothiazoles" (PrATs) behaved as frequent hitters under both FBDD and HTS settings, although the problem was more pronounced in the fragment-based studies. As 2-ATs are present in known drugs, they cannot necessarily be deemed undesirable, but the combination of their promiscuity and difficulties associated with optimizing them into a lead compound makes them, in our opinion, poor scaffolds for fragment libraries.

  6. Biosynthesis of nucleotide sugars by a promiscuous UDP-sugar pyrophosphorylase from Arabidopsis thaliana (AtUSP).

    PubMed

    Liu, Jun; Zou, Yang; Guan, Wanyi; Zhai, Yafei; Xue, Mengyang; Jin, Lan; Zhao, Xueer; Dong, Junkai; Wang, Wenjun; Shen, Jie; Wang, Peng George; Chen, Min

    2013-07-01

    Nucleotide sugars are activated forms of monosaccharides and key intermediates of carbohydrate metabolism in all organisms. The availability of structurally diverse nucleotide sugars is particularly important for the characterization of glycosyltransferases. Given that limited methods are available for preparation of nucleotide sugars, especially their useful non-natural derivatives, we introduced herein an efficient one-step three-enzyme catalytic system for the synthesis of nucleotide sugars from monosaccharides. In this study, a promiscuous UDP-sugar pyrophosphorylase (USP) from Arabidopsis thaliana (AtUSP) was used with a galactokinase from Streptococcus pneumoniae TIGR4 (SpGalK) and an inorganic pyrophosphatase (PPase) to effectively synthesize four UDP-sugars. AtUSP has better tolerance for C4-derivatives of Gal-1-P compared to UDP-glucose pyrophosphorylase from S. pneumoniae TIGR4 (SpGalU). Besides, the nucleotide substrate specificity and kinetic parameters of AtUSP were systematically studied. AtUSP exhibited considerable activity toward UTP, dUTP and dTTP, the yield of which was 87%, 85% and 84%, respectively. These results provide abundant information for better understanding of the relationship between substrate specificity and structural features of AtUSP. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. Unattractive, promiscuous and heavy drinkers: perceptions of women with tattoos.

    PubMed

    Swami, Viren; Furnham, Adrian

    2007-12-01

    This study examined social and physical perceptions of blonde and brunette women with different degrees of tattooing. Eighty-four female and 76 male undergraduates rated a series of 16 female line drawings that varied in 2 levels of hair colour and 8 levels of tattooing. Ratings were made for physical attractiveness and sexual promiscuity, as well as estimates of the number of alcohol units consumed on a typical night out. Results showed that tattooed women were rated as less physically attractive, more sexually promiscuous and heavier drinkers than untattooed women, with more negative ratings with increasing number of tattoos. There were also weak interactions between body art and hair colour, with blonde women in general rated more negatively than brunettes. Results are discussed in terms of stereotypes about women who have tattoos and the effects of such stereotypes on well-being.

  8. Neuroanatomical substrates involved in unrelated false facial recognition.

    PubMed

    Ronzon-Gonzalez, Eliane; Hernandez-Castillo, Carlos R; Pasaye, Erick H; Vaca-Palomares, Israel; Fernandez-Ruiz, Juan

    2017-11-22

    Identifying faces is a process central for social interaction and a relevant factor in eyewitness theory. False recognition is a critical mistake during an eyewitness's identification scenario because it can lead to a wrongful conviction. Previous studies have described neural areas related to false facial recognition using the standard Deese/Roediger-McDermott (DRM) paradigm, triggering related false recognition. Nonetheless, misidentification of faces without trying to elicit false memories (unrelated false recognition) in a police lineup could involve different cognitive processes, and distinct neural areas. To delve into the neural circuitry of unrelated false recognition, we evaluated the memory and response confidence of participants while watching faces photographs in an fMRI task. Functional activations of unrelated false recognition were identified by contrasting the activation on this condition vs. the activations related to recognition (hits) and correct rejections. The results identified the right precentral and cingulate gyri as areas with distinctive activations during false recognition events suggesting a conflict resulting in a dysfunction during memory retrieval. High confidence suggested that about 50% of misidentifications may be related to an unconscious process. These findings add to our understanding of the construction of facial memories and its biological basis, and the fallibility of the eyewitness testimony.

  9. Variation in promiscuity and sexual selection drives avian rate of Faster-Z evolution.

    PubMed

    Wright, Alison E; Harrison, Peter W; Zimmer, Fabian; Montgomery, Stephen H; Pointer, Marie A; Mank, Judith E

    2015-03-01

    Higher rates of coding sequence evolution have been observed on the Z chromosome relative to the autosomes across a wide range of species. However, despite a considerable body of theory, we lack empirical evidence explaining variation in the strength of the Faster-Z Effect. To assess the magnitude and drivers of Faster-Z Evolution, we assembled six de novo transcriptomes, spanning 90 million years of avian evolution. Our analysis combines expression, sequence and polymorphism data with measures of sperm competition and promiscuity. In doing so, we present the first empirical evidence demonstrating the positive relationship between Faster-Z Effect and measures of promiscuity, and therefore variance in male mating success. Our results from multiple lines of evidence indicate that selection is less effective on the Z chromosome, particularly in promiscuous species, and that Faster-Z Evolution in birds is due primarily to genetic drift. Our results reveal the power of mating system and sexual selection in shaping broad patterns in genome evolution. © 2015 John Wiley & Sons Ltd.

  10. Tetraterpene Synthase Substrate and Product Specificity in the Green Microalga Botryococcus braunii Race L.

    PubMed

    Thapa, Hem R; Tang, Su; Sacchettini, James C; Devarenne, Timothy P

    2017-09-15

    Recently, the biosynthetic pathway for lycopadiene, a C 40 tetraterpenoid hydrocarbon, was deciphered from the L race of Botryococcus braunii, an alga that produces hydrocarbon oils capable of being converted into combustible fuels. The lycopadiene pathway is initiated by the squalene synthase (SS)-like enzyme lycopaoctaene synthase (LOS), which catalyzes the head-to-head condensation of two C 20 geranylgeranyl diphosphate (GGPP) molecules to produce C 40 lycopaoctaene. LOS shows unusual substrate promiscuity for SS or SS-like enzymes by utilizing C 15 farnesyl diphosphate (FPP) and C 20 phytyl diphosphate in addition to GGPP as substrates. These three substrates can be combined by LOS individually or in combinations to produce six different hydrocarbons of C 30 , C 35 , and C 40 chain lengths. To understand LOS substrate and product specificity, rational mutagenesis experiments were conducted based on sequence alignment with several SS proteins as well as a structural comparison with the human SS (HSS) crystal structure. Characterization of the LOS mutants in vitro identified Ser276 and Ala288 in the LOS active site as key amino acids responsible for controlling substrate binding, and thus the promiscuity of this enzyme. Mutating these residues to those found in HSS largely converted LOS from lycopaoctaene production to C 30 squalene production. Furthermore, these studies were confirmed in vivo by expressing LOS in E. coli cells metabolically engineered to produce high FPP and GGPP levels. These studies also offer insights into tetraterpene hydrocarbon metabolism in B. braunii and provide a foundation for engineering LOS for robust production of specific hydrocarbons of a desired chain length.

  11. Fidelity and Promiscuity in an Ant-Plant Mutualism: A Case Study of Triplaris and Pseudomyrmex

    PubMed Central

    Sanchez, Adriana

    2015-01-01

    The association between the myrmecophyte Triplaris and ants of the genus Pseudomyrmex is an often-reported example of mutualism but no molecular studies have examined this association to date. In this study, the interspecific relationships of Triplaris were reconstructed using five molecular markers (two chloroplast and three nuclear), and the relationships of the associated Pseudomyrmex using two molecular regions (one mitochondrial and one nuclear). A data set including all known collections of plant hosts and resident ants was also compiled. The pattern of distribution of both organisms reveals that there are varying degrees of host specificity; most ants show broader host usage (promiscuous) but one species (P. dendroicus) is faithful to a single species of Triplaris. In most ant-plant interactions, host usage is not specific at the species level and preferences may result from geographical or ecological sorting. The specificity of P. dendroicus could be based on chemical recognition of the host they were raised on. PMID:26630384

  12. Structure of Human GIVD Cytosolic Phospholipase A2 Reveals Insights into Substrate Recognition

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

    Wang, Hui; Klein, Michael G.; Snell, Gyorgy

    Cytosolic phospholipases A2 (cPLA2s) consist of a family of calcium-sensitive enzymes that function to generate lipid second messengers through hydrolysis of membrane-associated glycerophospholipids. The GIVD cPLA2 (cPLA2δ) is a potential drug target for developing a selective therapeutic agent for the treatment of psoriasis. Here, we present two X-ray structures of human cPLA2δ, capturing an apo state, and in complex with a substrate-like inhibitor. Comparison of the apo and inhibitor-bound structures reveals conformational changes in a flexible cap that allows the substrate to access the relatively buried active site, providing new insight into the mechanism for substrate recognition. The cPLA2δ structuremore » reveals an unexpected second C2 domain that was previously unrecognized from sequence alignments, placing cPLA2δ into the class of membrane-associated proteins that contain a tandem pair of C2 domains. Furthermore, our structures elucidate novel inter-domain interactions and define three potential calcium-binding sites that are likely important for regulation and activation of enzymatic activity. These findings provide novel insights into the molecular mechanisms governing cPLA2's function in signal transduction.« less

  13. Structure of an XPF endonuclease with and without DNA suggests a model for substrate recognition

    PubMed Central

    Newman, Matthew; Murray-Rust, Judith; Lally, John; Rudolf, Jana; Fadden, Andrew; Knowles, Philip P; White, Malcolm F; McDonald, Neil Q

    2005-01-01

    The XPF/Mus81 structure-specific endonucleases cleave double-stranded DNA (dsDNA) within asymmetric branched DNA substrates and play an essential role in nucleotide excision repair, recombination and genome integrity. We report the structure of an archaeal XPF homodimer alone and bound to dsDNA. Superposition of these structures reveals a large domain movement upon binding DNA, indicating how the (HhH)2 domain and the nuclease domain are coupled to allow the recognition of double-stranded/single-stranded DNA junctions. We identify two nonequivalent DNA-binding sites and propose a model in which XPF distorts the 3′ flap substrate in order to engage both binding sites and promote strand cleavage. The model rationalises published biochemical data and implies a novel role for the ERCC1 subunit of eukaryotic XPF complexes. PMID:15719018

  14. TM0416, a Hyperthermophilic Promiscuous Nonphosphorylated Sugar Isomerase, Catalyzes Various C5 and C6 Epimerization Reactions

    PubMed Central

    Shin, Sun-Mi; Cao, Thinh-Phat; Choi, Jin Myung; Kim, Seong-Bo; Lee, Sang-Jae

    2017-01-01

    ABSTRACT There is currently little information on nonphosphorylated sugar epimerases, which are of potential interest for producing rare sugars. We found a gene (the TM0416 gene) encoding a putative d-tagatose-3-epimerase-related protein from the hyperthermophilic bacterium Thermotoga maritima. We overexpressed the TM0416 gene in Escherichia coli and purified the resulting recombinant protein for detailed characterization. Amino acid sequence alignment and a structural similarity search revealed that TM0416 is a putative nonphosphorylated sugar epimerase. The recombinant enzyme exhibited maximal C-3 epimerization of l-ribulose to l-xylulose at ∼80°C and pH 7 in the presence of 1 mM Mn2+. In addition, this enzyme showed unusually high activity for the epimerization of d-tagatose to d-sorbose, with a conversion yield of 20% after 6 h at 80°C. Remarkably, the enzyme catalyzed the isomerization of d-erythrose or d-threose to d-erythrulose significantly, with conversion yields of 71% and 54.5%, respectively, after 6 h at 80°C at pH 7. To further investigate the substrate specificity of TM0416, we determined its crystal structures in complex with divalent metal ions and l-erythrulose at resolutions of 1.5 and 1.6 Å. Detailed inspection of the structural features and biochemical data clearly demonstrated that this metalloenzyme, with a freely accessible substrate-binding site and neighboring hydrophobic residues, exhibits different and promiscuous substrate preferences, compared with its mesophilic counterparts. Therefore, this study suggests that TM0416 can be functionally classified as a novel type of l-ribulose 3-epimerase (R3E) with d-erythrose isomerase activity. IMPORTANCE Rare sugars, which occur naturally in small amounts, have attracted considerable attention in the food and drug industries. However, there is little information on nonphosphorylated sugar epimerases, which might potentially be applied for the production of rare sugars. This study describes

  15. TM0416, a Hyperthermophilic Promiscuous Nonphosphorylated Sugar Isomerase, Catalyzes Various C5 and C6 Epimerization Reactions.

    PubMed

    Shin, Sun-Mi; Cao, Thinh-Phat; Choi, Jin Myung; Kim, Seong-Bo; Lee, Sang-Jae; Lee, Sung Haeng; Lee, Dong-Woo

    2017-05-15

    There is currently little information on nonphosphorylated sugar epimerases, which are of potential interest for producing rare sugars. We found a gene (the TM0416 gene) encoding a putative d-tagatose-3-epimerase-related protein from the hyperthermophilic bacterium Thermotoga maritima We overexpressed the TM0416 gene in Escherichia coli and purified the resulting recombinant protein for detailed characterization. Amino acid sequence alignment and a structural similarity search revealed that TM0416 is a putative nonphosphorylated sugar epimerase. The recombinant enzyme exhibited maximal C-3 epimerization of l-ribulose to l-xylulose at ∼80°C and pH 7 in the presence of 1 mM Mn 2+ In addition, this enzyme showed unusually high activity for the epimerization of d-tagatose to d-sorbose, with a conversion yield of 20% after 6 h at 80°C. Remarkably, the enzyme catalyzed the isomerization of d-erythrose or d-threose to d-erythrulose significantly, with conversion yields of 71% and 54.5%, respectively, after 6 h at 80°C at pH 7. To further investigate the substrate specificity of TM0416, we determined its crystal structures in complex with divalent metal ions and l-erythrulose at resolutions of 1.5 and 1.6 Å. Detailed inspection of the structural features and biochemical data clearly demonstrated that this metalloenzyme, with a freely accessible substrate-binding site and neighboring hydrophobic residues, exhibits different and promiscuous substrate preferences, compared with its mesophilic counterparts. Therefore, this study suggests that TM0416 can be functionally classified as a novel type of l-ribulose 3-epimerase (R3E) with d-erythrose isomerase activity. IMPORTANCE Rare sugars, which occur naturally in small amounts, have attracted considerable attention in the food and drug industries. However, there is little information on nonphosphorylated sugar epimerases, which might potentially be applied for the production of rare sugars. This study describes the

  16. Tailoring Agility: Promiscuous Pair Story Authoring and Value Calculation

    NASA Astrophysics Data System (ADS)

    Tendon, Steve

    This chapter describes how a multi-national software organization created a business plan involving business units from eight countries that followed an agile way, after two previously failed attempts with traditional approaches. The case is told by the consultant who initiated implementation of agility into requirements gathering, estimation and planning processes in an international setting. The agile approach was inspired by XP, but then tailored to meet the peculiar requirements. Two innovations were critical. The first innovation was promiscuous pair story authoring, where user stories were written by two people (similarly to pair programming), and the pairing changed very often (as frequently as every 15-20 minutes) to achieve promiscuity and cater for diverse point of views. The second innovation was an economic value evaluation (and not the cost) which was attributed to stories. Continuous recalculation of the financial value of the stories allowed to assess the projects financial return. In this case implementation of agility in the international context allowed the involved team members to reach consensus and unanimity of decisions, vision and purpose.

  17. Probing protein flexibility reveals a mechanism for selective promiscuity

    PubMed Central

    Pabon, Nicolas A; Camacho, Carlos J

    2017-01-01

    Many eukaryotic regulatory proteins adopt distinct bound and unbound conformations, and use this structural flexibility to bind specifically to multiple partners. However, we lack an understanding of how an interface can select some ligands, but not others. Here, we present a molecular dynamics approach to identify and quantitatively evaluate the interactions responsible for this selective promiscuity. We apply this approach to the anticancer target PD-1 and its ligands PD-L1 and PD-L2. We discover that while unbound PD-1 exhibits a hard-to-drug hydrophilic interface, conserved specific triggers encoded in the cognate ligands activate a promiscuous binding pathway that reveals a flexible hydrophobic binding cavity. Specificity is then established by additional contacts that stabilize the PD-1 cavity into distinct bound-like modes. Collectively, our studies provide insight into the structural basis and evolution of multiple binding partners, and also suggest a biophysical approach to exploit innate binding pathways to drug seemingly undruggable targets. DOI: http://dx.doi.org/10.7554/eLife.22889.001 PMID:28432789

  18. Promiscuous binding by Hsp70 results in conformational heterogeneity and fuzzy chaperone-substrate ensembles

    PubMed Central

    Rosenzweig, Rina; Sekhar, Ashok; Nagesh, Jayashree; Kay, Lewis E

    2017-01-01

    The Hsp70 chaperone system is integrated into a myriad of biochemical processes that are critical for cellular proteostasis. Although detailed pictures of Hsp70 bound with peptides have emerged, correspondingly detailed structural information on complexes with folding-competent substrates remains lacking. Here we report a methyl-TROSY based solution NMR study showing that the Escherichia coli version of Hsp70, DnaK, binds to as many as four distinct sites on a small 53-residue client protein, hTRF1. A fraction of hTRF1 chains are also bound to two DnaK molecules simultaneously, resulting in a mixture of DnaK-substrate sub-ensembles that are structurally heterogeneous. The interactions of Hsp70 with a client protein at different sites results in a fuzzy chaperone-substrate ensemble and suggests a mechanism for Hsp70 function whereby the structural heterogeneity of released substrate molecules enables them to circumvent kinetic traps in their conformational free energy landscape and fold efficiently to the native state. DOI: http://dx.doi.org/10.7554/eLife.28030.001 PMID:28708484

  19. Systems-Wide Prediction of Enzyme Promiscuity Reveals a New Underground Alternative Route for Pyridoxal 5’-Phosphate Production in E. coli

    DOE PAGES

    Oberhardt, Matthew A.; Zarecki, Raphy; Reshef, Leah; ...

    2016-01-28

    Recent insights suggest that non-specific and/or promiscuous enzymes are common and active across life. Understanding the role of such enzymes is an important open question in biology. Here we develop a genome-wide method, PROPER, that uses a permissive PSI-BLAST approach to predict promiscuous activities of metabolic genes. Enzyme promiscuity is typically studied experimentally using multicopy suppression, in which over-expression of a promiscuous ‘replacer’ gene rescues lethality caused by inactivation of a ‘target’ gene. We use PROPER to predict multicopy suppression in Escherichia coli, achieving highly significant overlap with published cases (hypergeometric p = 4.4e-13). We then validate three novel predictedmore » target-replacer gene pairs in new multicopy suppression experiments. We next go beyond PROPER and develop a network-based approach, GEM-PROPER, that integrates PROPER with genome-scale metabolic modeling to predict promiscuous replacements via alternative metabolic pathways. GEM-PROPER predicts a new indirect replacer (thiG) for an essential enzyme (pdxB) in production of pyridoxal 5’-phosphate (the active form of Vitamin B 6), which we validate experimentally via multicopy suppression. Here, we perform a structural analysis of thiG to determine its potential promiscuous active site, which we validate experimentally by inactivating the pertaining residues and showing a loss of replacer activity. Thus, this study is a successful example where a computational investigation leads to a network-based identification of an indirect promiscuous replacement of a key metabolic enzyme, which would have been extremely difficult to identify directly.« less

  20. Retinoic acid biosynthesis catalyzed by retinal dehydrogenases relies on a rate-limiting conformational transition associated with substrate recognition

    PubMed Central

    Bchini, Raphaël; Vasiliou, Vasilis; Branlant, Guy; Talfournier, François; Rahuel-Clermont, Sophie

    2012-01-01

    Retinoic acid (RA), a metabolite of vitamin A, exerts pleiotropic effects throughout life in vertebrate organisms. Thus, RA action must be tightly regulated through the coordinated action of biosynthetic and degradating enzymes. The last step of retinoic acid biosynthesis is irreversibly catalyzed by the NAD-dependent retinal dehydrogenases (RALDH), which are members of the aldehyde dehydrogenase (ALDH) superfamily. Low intracellular retinal concentrations imply efficient substrate molecular recognition to ensure high affinity and specificity of RALDHs for retinal. This study addresses the molecular basis of retinal recognition in human ALDH1A1 (or RALDH1) and rat ALDH1A2 (or RALDH2), through the comparison of the catalytic behavior of retinal analogs and use of the fluorescence properties of retinol. We show that, in contrast to long chain unsaturated substrates, the rate-limiting step of retinal oxidation by RALDHs is associated with acylation. Use of the fluorescence resonance energy transfer upon retinol interaction with RALDHs provides evidence that retinal recognition occurs in two steps: binding into the substrate access channel, and a slower structural reorganization with a rate constant of the same magnitude as the kcat for retinal oxidation: 0.18 vs. 0.07 s−1 and 0.25 vs. 0.1 s−1 for ALDH1A1 and ALDH1A2, respectively. This suggests that the conformational transition of the RALDH-retinal complex significantly contributes to the rate-limiting step that controls the kinetics of retinal oxidation, as a prerequisite for the formation of a catalytically competent Michaelis complex. This conclusion is consistent with the general notion that structural flexibility within the active site of ALDH enzymes has been shown to be an integral component of catalysis. PMID:23220587

  1. Entering the 'big data' era in medicinal chemistry: molecular promiscuity analysis revisited.

    PubMed

    Hu, Ye; Bajorath, Jürgen

    2017-06-01

    The 'big data' concept plays an increasingly important role in many scientific fields. Big data involves more than unprecedentedly large volumes of data that become available. Different criteria characterizing big data must be carefully considered in computational data mining, as we discuss herein focusing on medicinal chemistry. This is a scientific discipline where big data is beginning to emerge and provide new opportunities. For example, the ability of many drugs to specifically interact with multiple targets, termed promiscuity, forms the molecular basis of polypharmacology, a hot topic in drug discovery. Compound promiscuity analysis is an area that is much influenced by big data phenomena. Different results are obtained depending on chosen data selection and confidence criteria, as we also demonstrate.

  2. Much More than Power: The Pedagogy of Promiscuous Black Feminism

    ERIC Educational Resources Information Center

    Huckaby, M. Francyne

    2013-01-01

    This paper explores promiscuous black feminism by juxtaposing black feminism, Foucualt's poststructuralism, and my grandmother. The tensions created by these juxtapositions illuminate the ways black feminism and poststructuralism are resources and challenges to each other, and how both offer understandings of the relations at play that shape…

  3. A parasitic selfish gene that affects host promiscuity.

    PubMed

    Giraldo-Perez, Paulina; Goddard, Matthew R

    2013-11-07

    Selfish genes demonstrate transmission bias and invade sexual populations despite conferring no benefit to their hosts. While the molecular genetics and evolutionary dynamics of selfish genes are reasonably well characterized, their effects on hosts are not. Homing endonuclease genes (HEGs) are one well-studied family of selfish genes that are assumed to be benign. However, we show that carrying HEGs is costly for Saccharomyces cerevisiae, demonstrating that these genetic elements are not necessarily benign but maybe parasitic. We estimate a selective load of approximately 1-2% in 'natural' niches. The second aspect we examine is the ability of HEGs to affect hosts' sexual behaviour. As all selfish genes critically rely on sex for spread, then any selfish gene correlated with increased host sexuality will enjoy a transmission advantage. While classic parasites are known to manipulate host behaviour, we are not aware of any evidence showing a selfish gene is capable of affecting host promiscuity. The data presented here show a selfish element may increase the propensity of its eukaryote host to undergo sex and along with increased rates of non-Mendelian inheritance, this may counterbalance mitotic selective load and promote spread. Demonstration that selfish genes are correlated with increased promiscuity in eukaryotes connects with ideas suggesting that selfish genes promoted the evolution of sex initially.

  4. Strand-specific Recognition of DNA Damages by XPD Provides Insights into Nucleotide Excision Repair Substrate Versatility*

    PubMed Central

    Buechner, Claudia N.; Heil, Korbinian; Michels, Gudrun; Carell, Thomas; Kisker, Caroline; Tessmer, Ingrid

    2014-01-01

    Recognition and removal of DNA damages is essential for cellular and organismal viability. Nucleotide excision repair (NER) is the sole mechanism in humans for the repair of carcinogenic UV irradiation-induced photoproducts in the DNA, such as cyclobutane pyrimidine dimers. The broad substrate versatility of NER further includes, among others, various bulky DNA adducts. It has been proposed that the 5′-3′ helicase XPD (xeroderma pigmentosum group D) protein plays a decisive role in damage verification. However, despite recent advances such as the identification of a DNA-binding channel and central pore in the protein, through which the DNA is threaded, as well as a dedicated lesion recognition pocket near the pore, the exact process of target site recognition and verification in eukaryotic NER still remained elusive. Our single molecule analysis by atomic force microscopy reveals for the first time that XPD utilizes different recognition strategies to verify structurally diverse lesions. Bulky fluorescein damage is preferentially detected on the translocated strand, whereas the opposite strand preference is observed for a cyclobutane pyrimidine dimer lesion. Both states, however, lead to similar conformational changes in the resulting specific complexes, indicating a merge to a “final” verification state, which may then trigger the recruitment of further NER proteins. PMID:24338567

  5. Genetic monogamy despite social promiscuity in the pot-bellied seahorse (Hippocampus abdominalis).

    PubMed

    Wilson, A B; Martin-Smith, K M

    2007-06-01

    Sexual selection theory predicts a positive correlation between relative parental investment and mate choice. In syngnathid fishes (seahorses and pipefish), males brood offspring in specialized brooding structures. While female-female mating competition has been demonstrated in some pipefishes, all seahorses (genus Hippocampus) studied to date have been found to have conventional sex roles with greater male-male competition for access to mates despite possessing the most complex brood structures in the family. Although multiple mating is common in pipefish, seahorses are again exceptional, exhibiting strict genetic monogamy. Both demographic and behavioural explanations have been offered to explain the lack of multiple mating in seahorse species, but these hypotheses have not yet been explicitly addressed. We investigated mating systems and brood parentage of the pot-bellied seahorse, Hippocampus abdominalis, a temperate-water species that is socially promiscuous with conventional sex roles in laboratory populations. We observed promiscuous courtship behaviour and sex-role reversal in high density, female-biased field populations of H. abdominalis. We hypothesize that sex roles are plastic in H. abdominalis, depending on local population density and sex ratio. Despite promiscuous courtship behaviour, all assayed male seahorses were genetically monogamous in both laboratory and wild populations. Physiological limitations associated with embryo incubation may explain the absence of multiple mating in seahorses and may have played an important role in the development of the unique reproductive behaviour typical in these species.

  6. Promiscuity is related to masculine and feminine body traits in both men and women: evidence from Brazilian and Czech samples.

    PubMed

    Varella, Marco Antonio Correa; Valentova, Jaroslava Varella; Pereira, Kamila Janaina; Bussab, Vera Silvia Raad

    2014-11-01

    One of the possible explanations for human within-sex variation in promiscuity stems from conditional strategies dependent on the level of body sex-dimorphism. There is some evidence that masculine men and feminine women are more promiscuous than their sex-atypical counterparts, although mixed results persist. Moreover, another line of evidence shows that more promiscuous women are rather sex-atypical. We tested whether diverse sex-dimorphic body measures (2D:4D, WHR/WSR, handgrip strength, and height and weight) influence sociosexual desires, attitudes, promiscuous behavior, and age of first intercourse in a sex-typical or sex-atypical direction. Participants were 185 young adults, 51 men and 54 women from Brazil, and 40 men and 40 women from the Czech Republic. In men stronger handgrip and more feminine 2D:4D predicted higher sociosexual behaviors, desires, and lower age of the first sexual intercourse. While in women, sociosexual desires were predicted by lower handgrip strength and more feminine 2D:4D. It thus seems that it is rather a mixture of masculine and feminine traits in men, and feminine traits in women that increase their sociosexuality. Masculine traits (height) predicting female promiscuous behavior were specific for only one population. In conclusion, a mosaic combination of sex-typical but also sex-atypical independent body traits can lead to higher promiscuity, particularly in men. Limitations, implications, and future directions for research are considered. This article is part of a Special Issue entitled: Neotropical Behaviour. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Characterizing the Promiscuity of LigAB, a Lignin Catabolite Degrading Extradiol Dioxygenase from Sphingomonas paucimobilis SYK-6

    PubMed Central

    Barry, Kevin P.; Taylor, Erika A.

    2014-01-01

    LigAB from Sphingomonas paucimobilis SYK-6 is the only structurally characterized dioxygenase of the largely uncharacterized superfamily of Type II extradiol dioxygenases (EDO). This enzyme catalyzes the oxidative ring-opening of protocatechuate (3,4-dihydroxybenzoic acid or PCA) in a pathway allowing the degradation of lignin derived aromatic compounds (LDACs). LigAB has also been shown to utilize two other LDACs from the same metabolic pathway as substrates, gallate, and 3-O-methyl gallate; however, kcat/KM had not been reported for any of these compounds. In order to assess the catalytic efficiency and get insights into the observed promiscuity of this enzyme, steady-state kinetic analyses were performed for LigAB with these and a library of related compounds. The dioxygenation of PCA by LigAB was highly efficient, with a kcat of 51 s−1 and a kcat/KM of 4.26 × 106 M−1s−1. LigAB demonstrated the ability to use a variety of catecholic molecules as substrates beyond the previously identified gallate and 3-O-methyl gallate, including 3,4-dihydroxybenzamide, homoprotocatechuate, catechol, and 3,4-dihydroxybenzonitrile. Interestingly, 3,4-dihydroxybenzamide (DHBAm) behaves in a manner similar to that of the preferred benzoic acid substrates, with a kcat/Km value only ~4-fold lower than that for gallate and ~10-fold higher than that for 3-O-methyl gallate. All of these most active substrates demonstrate mechanistic inactivation of LigAB. Additionally, DHBAm exhibits potent product inhibition that leads to an inactive enzyme, being more highly deactivating at lower substrate concentration, a phenomena that, to our knowledge, has not been reported for another dioxygenase substrate/product pair. These results provide valuable catalytic insight into the reactions catalyzed by LigAB and make it the first Type II EDO that is fully characterized both structurally and kinetically. PMID:23977959

  8. A Measure of the Broad Substrate Specificity of Enzymes Based on ‘Duplicate’ Catalytic Residues

    PubMed Central

    Chakraborty, Sandeep; Ásgeirsson, Bjarni; Rao, Basuthkar J.

    2012-01-01

    The ability of an enzyme to select and act upon a specific class of compounds with unerring precision and efficiency is an essential feature of life. Simultaneously, these enzymes often catalyze the reaction of a range of similar substrates of the same class, and also have promiscuous activities on unrelated substrates. Previously, we have established a methodology to quantify promiscuous activities in a wide range of proteins. In the current work, we quantitatively characterize the active site for the ability to catalyze distinct, yet related, substrates (BRASS). A protein with known structure and active site residues provides the framework for computing ‘duplicate’ residues, each of which results in slightly modified replicas of the active site scaffold. Such spatial congruence is supplemented by Finite difference Poisson Boltzmann analysis which filters out electrostatically unfavorable configurations. The congruent configurations are used to compute an index (BrassIndex), which reflects the broad substrate profile of the active site. We identify an acetylhydrolase and a methyltransferase as having the lowest and highest BrassIndex, respectively, from a set of non-homologous proteins extracted from the Catalytic Site Atlas. The acetylhydrolase, a regulatory enzyme, is known to be highly specific for platelet-activating factor. In the methyltransferase (PDB: 1QAM), various combinations of glycine (Gly38/40/42), asparagine (Asn101/11) and glutamic acid (Glu59/36) residues having similar spatial and electrostatic profiles with the specified scaffold (Gly38, Asn101 and Glu59) exemplifies the broad substrate profile such an active site may provide. ‘Duplicate’ residues identified by relaxing the spatial and/or electrostatic constraints can be the target of directed evolution methodologies, like saturation mutagenesis, for modulating the substrate specificity of proteins. PMID:23166637

  9. The Fanconi anemia associated protein FAAP24 uses two substrate specific binding surfaces for DNA recognition

    PubMed Central

    Wienk, Hans; Slootweg, Jack C.; Speerstra, Sietske; Kaptein, Robert; Boelens, Rolf; Folkers, Gert E.

    2013-01-01

    To maintain the integrity of the genome, multiple DNA repair systems exist to repair damaged DNA. Recognition of altered DNA, including bulky adducts, pyrimidine dimers and interstrand crosslinks (ICL), partially depends on proteins containing helix-hairpin-helix (HhH) domains. To understand how ICL is specifically recognized by the Fanconi anemia proteins FANCM and FAAP24, we determined the structure of the HhH domain of FAAP24. Although it resembles other HhH domains, the FAAP24 domain contains a canonical hairpin motif followed by distorted motif. The HhH domain can bind various DNA substrates; using nuclear magnetic resonance titration experiments, we demonstrate that the canonical HhH motif is required for double-stranded DNA (dsDNA) binding, whereas the unstructured N-terminus can interact with single-stranded DNA. Both DNA binding surfaces are used for binding to ICL-like single/double-strand junction-containing DNA substrates. A structural model for FAAP24 bound to dsDNA has been made based on homology with the translesion polymerase iota. Site-directed mutagenesis, sequence conservation and charge distribution support the dsDNA-binding model. Analogous to other HhH domain-containing proteins, we suggest that multiple FAAP24 regions together contribute to binding to single/double-strand junction, which could contribute to specificity in ICL DNA recognition. PMID:23661679

  10. A parasitic selfish gene that affects host promiscuity

    PubMed Central

    Giraldo-Perez, Paulina; Goddard, Matthew R.

    2013-01-01

    Selfish genes demonstrate transmission bias and invade sexual populations despite conferring no benefit to their hosts. While the molecular genetics and evolutionary dynamics of selfish genes are reasonably well characterized, their effects on hosts are not. Homing endonuclease genes (HEGs) are one well-studied family of selfish genes that are assumed to be benign. However, we show that carrying HEGs is costly for Saccharomyces cerevisiae, demonstrating that these genetic elements are not necessarily benign but maybe parasitic. We estimate a selective load of approximately 1–2% in ‘natural’ niches. The second aspect we examine is the ability of HEGs to affect hosts' sexual behaviour. As all selfish genes critically rely on sex for spread, then any selfish gene correlated with increased host sexuality will enjoy a transmission advantage. While classic parasites are known to manipulate host behaviour, we are not aware of any evidence showing a selfish gene is capable of affecting host promiscuity. The data presented here show a selfish element may increase the propensity of its eukaryote host to undergo sex and along with increased rates of non-Mendelian inheritance, this may counterbalance mitotic selective load and promote spread. Demonstration that selfish genes are correlated with increased promiscuity in eukaryotes connects with ideas suggesting that selfish genes promoted the evolution of sex initially. PMID:24048156

  11. Entering the ‘big data’ era in medicinal chemistry: molecular promiscuity analysis revisited

    PubMed Central

    Hu, Ye; Bajorath, Jürgen

    2017-01-01

    The ‘big data’ concept plays an increasingly important role in many scientific fields. Big data involves more than unprecedentedly large volumes of data that become available. Different criteria characterizing big data must be carefully considered in computational data mining, as we discuss herein focusing on medicinal chemistry. This is a scientific discipline where big data is beginning to emerge and provide new opportunities. For example, the ability of many drugs to specifically interact with multiple targets, termed promiscuity, forms the molecular basis of polypharmacology, a hot topic in drug discovery. Compound promiscuity analysis is an area that is much influenced by big data phenomena. Different results are obtained depending on chosen data selection and confidence criteria, as we also demonstrate. PMID:28670471

  12. Steroid signaling: ligand-binding promiscuity, molecular symmetry, and the need for gating.

    PubMed

    Lathe, Richard; Kotelevtsev, Yuri

    2014-04-01

    Steroid/sterol-binding receptors and enzymes are remarkably promiscuous in the range of ligands they can bind to and, in the case of enzymes, modify - raising the question of how specific receptor activation is achieved in vivo. Estrogen receptors (ER) are modulated by 27-hydroxycholesterol and 5α-androstane-3β,17β-diol (Adiol), in addition to estradiol (E2), and respond to diverse small molecules such as bisphenol A. Steroid-modifying enzymes are also highly promiscuous in ligand binding and metabolism. The specificity problem is compounded by the fact that the steroid core (hydrogenated cyclopentophenanthrene ring system) has several planes of symmetry. Ligand binding can be in symmetrical East-West (rotation) and North-South (inversion) orientations. Hydroxysteroid dehydrogenases (HSDs) can modify symmetrical 7 and 11, also 3 and 17/20, positions, exemplified here by yeast 3α,20β-HSD and mammalian 11β-HSD and 17β-HSD enzymes. Faced with promiscuity and symmetry, other strategies are clearly necessary to promote signaling selectivity in vivo. Gating regulates hormone access via enzymes that preferentially inactivate (or activate) a subclass of ligands, thereby governing which ligands gain receptor access - exemplified by 11β-HSD gating cortisol access to the mineralocorticoid receptor, and P450 CYP7B1 gating Adiol access to ER. Counter-intuitively, the specificity of steroid/sterol action is achieved not by intrinsic binding selectivity but by the combination of local metabolism and binding affinity. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Functional Trade-Offs in Promiscuous Enzymes Cannot Be Explained by Intrinsic Mutational Robustness of the Native Activity.

    PubMed

    Kaltenbach, Miriam; Emond, Stephane; Hollfelder, Florian; Tokuriki, Nobuhiko

    2016-10-01

    The extent to which an emerging new function trades off with the original function is a key characteristic of the dynamics of enzyme evolution. Various cases of laboratory evolution have unveiled a characteristic trend; a large increase in a new, promiscuous activity is often accompanied by only a mild reduction of the native, original activity. A model that associates weak trade-offs with "evolvability" was put forward, which proposed that enzymes possess mutational robustness in the native activity and plasticity in promiscuous activities. This would enable the acquisition of a new function without compromising the original one, reducing the benefit of early gene duplication and therefore the selection pressure thereon. Yet, to date, no experimental study has examined this hypothesis directly. Here, we investigate the causes of weak trade-offs by systematically characterizing adaptive mutations that occurred in two cases of evolutionary transitions in enzyme function: (1) from phosphotriesterase to arylesterase, and (2) from atrazine chlorohydrolase to melamine deaminase. Mutational analyses in various genetic backgrounds revealed that, in contrast to the prevailing model, the native activity is less robust to mutations than the promiscuous activity. For example, in phosphotriesterase, the deleterious effect of individual mutations on the native phosphotriesterase activity is much larger than their positive effect on the promiscuous arylesterase activity. Our observations suggest a revision of the established model: weak trade-offs are not caused by an intrinsic robustness of the native activity and plasticity of the promiscuous activity. We propose that upon strong adaptive pressure for the new activity without selection against the original one, selected mutations will lead to the largest possible increases in the new function, but whether and to what extent they decrease the old function is irrelevant, creating a bias towards initially weak trade-offs and the

  14. Engineering substrate promiscuity in halophilic alcohol dehydrogenase (HvADH2) by in silico design.

    PubMed

    Cassidy, Jennifer; Bruen, Larah; Rosini, Elena; Molla, Gianluca; Pollegioni, Loredano; Paradisi, Francesca

    2017-01-01

    An alcohol dehydrogenase from the halophilic archaeon Haloferax volcanii (HvADH2) has been engineered by rational design to broaden its substrate scope towards the conversion of a range of aromatic substrates, including flurbiprofenol, that is an intermediate of the non-steroidal anti-inflammatory drug, flurbiprofen. Wild-type HvADH2 showed minimal activity with flurbiprofenol (11.1 mU/mg). A homology model of HvADH2 was built and docking experiments with this substrate revealed that the biphenyl rings of flurbiprofenol formed strong interactions with residues F85 and F108, preventing its optimal binding in the active site. Mutations at position 85 however did not increase activity. Site directed mutagenesis at position F108 allowed the identification of three variants showing a significant (up to 2.3-fold) enhancement of activity towards flurbiprofenol, when compared to wild-type HvADH2. Interestingly, F108G variant did not show the classic inhibition in the presence of (R)-enantiomer when tested with rac-1-phenylethanol, underling its potential in racemic resolution of secondary alcohols.

  15. Monogamy has a fixation advantage based on fitness variance in an ideal promiscuity group.

    PubMed

    Garay, József; Móri, Tamás F

    2012-11-01

    We consider an ideal promiscuity group of females, which implies that all males have the same average mating success. If females have concealed ovulation, then the males' paternity chances are equal. We find that male-based monogamy will be fixed in females' promiscuity group when the stochastic Darwinian selection is described by a Markov chain.We point out that in huge populations the relative advantage (difference between average fitness of different strategies) determines primarily the end of evolution; in the case of neutrality (means are equal) the smallest variance guarantees fixation (absorption) advantage; when the means and variances are the same, then the higher third moment determines which types will be fixed in the Markov chains.

  16. Molecular Evolution of the CYP2D Subfamily in Primates: Purifying Selection on Substrate Recognition Sites without the Frequent or Long-Tract Gene Conversion

    PubMed Central

    Yasukochi, Yoshiki; Satta, Yoko

    2015-01-01

    The human cytochrome P450 (CYP) 2D6 gene is a member of the CYP2D gene subfamily, along with the CYP2D7P and CYP2D8P pseudogenes. Although the CYP2D6 enzyme has been studied extensively because of its clinical importance, the evolution of the CYP2D subfamily has not yet been fully understood. Therefore, the goal of this study was to reveal the evolutionary process of the human drug metabolic system. Here, we investigate molecular evolution of the CYP2D subfamily in primates by comparing 14 CYP2D sequences from humans to New World monkey genomes. Window analysis and statistical tests revealed that entire genomic sequences of paralogous genes were extensively homogenized by gene conversion during molecular evolution of CYP2D genes in primates. A neighbor-joining tree based on genomic sequences at the nonsubstrate recognition sites showed that CYP2D6 and CYP2D8 genes were clustered together due to gene conversion. In contrast, a phylogenetic tree using amino acid sequences at substrate recognition sites did not cluster the CYP2D6 and CYP2D8 genes, suggesting that the functional constraint on substrate specificity is one of the causes for purifying selection at the substrate recognition sites. Our results suggest that the CYP2D gene subfamily in primates has evolved to maintain the regioselectivity for a substrate hydroxylation activity between individual enzymes, even though extensive gene conversion has occurred across CYP2D coding sequences. PMID:25808902

  17. Predictors of Drug/Alcohol Abuse and Sexual Promiscuity of College Students.

    ERIC Educational Resources Information Center

    Nam, Jeong Sook; And Others

    This study examined the relationship between the individual's purpose in life, existential anxiety, powerlessness and use of alcohol/drugs and the tendency to be sexually promiscuous. The study is rooted in the work of Viktor E. Frankl, which suggested that a lack of meaning and purpose can cause socially deviant behavior and psychological…

  18. Reconstructed ancestral enzymes reveal that negative selection drove the evolution of substrate specificity in ADP-dependent kinases.

    PubMed

    Castro-Fernandez, Víctor; Herrera-Morande, Alejandra; Zamora, Ricardo; Merino, Felipe; Gonzalez-Ordenes, Felipe; Padilla-Salinas, Felipe; Pereira, Humberto M; Brandão-Neto, Jose; Garratt, Richard C; Guixe, Victoria

    2017-09-22

    One central goal in molecular evolution is to pinpoint the mechanisms and evolutionary forces that cause an enzyme to change its substrate specificity; however, these processes remain largely unexplored. Using the glycolytic ADP-dependent kinases of archaea, including the orders Thermococcales , Methanosarcinales , and Methanococcales , as a model and employing an approach involving paleoenzymology, evolutionary statistics, and protein structural analysis, we could track changes in substrate specificity during ADP-dependent kinase evolution along with the structural determinants of these changes. To do so, we studied five key resurrected ancestral enzymes as well as their extant counterparts. We found that a major shift in function from a bifunctional ancestor that could phosphorylate either glucose or fructose 6-phosphate (fructose-6-P) as a substrate to a fructose 6-P-specific enzyme was started by a single amino acid substitution resulting in negative selection with a ground-state mode against glucose and a subsequent 1,600-fold change in specificity of the ancestral protein. This change rendered the residual phosphorylation of glucose a promiscuous and physiologically irrelevant activity, highlighting how promiscuity may be an evolutionary vestige of ancestral enzyme activities, which have been eliminated over time. We also could reconstruct the evolutionary history of substrate utilization by using an evolutionary model of discrete binary characters, indicating that substrate uses can be discretely lost or acquired during enzyme evolution. These findings exemplify how negative selection and subtle enzyme changes can lead to major evolutionary shifts in function, which can subsequently generate important adaptive advantages, for example, in improving glycolytic efficiency in Thermococcales . © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Reprogramming caspase-7 specificity by regio-specific mutations and selection provides alternate solutions for substrate recognition

    DOE PAGES

    Hill, Maureen E.; MacPherson, Derek J.; Wu, Peng; ...

    2016-03-31

    The ability to routinely engineer protease specificity can allow us to better understand and modulate their biology for expanded therapeutic and industrial applications. In this paper, we report a new approach based on a caged green fluorescent protein (CA-GFP) reporter that allows for flow-cytometry-based selection in bacteria or other cell types enabling selection of intracellular protease specificity, regardless of the compositional complexity of the protease. Here, we apply this approach to introduce the specificity of caspase-6 into caspase-7, an intracellular cysteine protease important in cellular remodeling and cell death. We found that substitution of substrate-contacting residues from caspase-6 into caspase-7more » was ineffective, yielding an inactive enzyme, whereas saturation mutagenesis at these positions and selection by directed evolution produced active caspases. The process produced a number of nonobvious mutations that enabled conversion of the caspase-7 specificity to match caspase-6. The structures of the evolved-specificity caspase-7 (esCasp-7) revealed alternate binding modes for the substrate, including reorganization of an active site loop. Profiling the entire human proteome of esCasp-7 by N-terminomics demonstrated that the global specificity toward natural protein substrates is remarkably similar to that of caspase-6. Because the esCasp-7 maintained the core of caspase-7, we were able to identify a caspase-6 substrate, lamin C, that we predict relies on an exosite for substrate recognition. These reprogrammed proteases may be the first tool built with the express intent of distinguishing exosite dependent or independent substrates. Finally, this approach to specificity reprogramming should also be generalizable across a wide range of proteases.« less

  20. Reprogramming caspase-7 specificity by regio-specific mutations and selection provides alternate solutions for substrate recognition

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

    Hill, Maureen E.; MacPherson, Derek J.; Wu, Peng

    The ability to routinely engineer protease specificity can allow us to better understand and modulate their biology for expanded therapeutic and industrial applications. In this paper, we report a new approach based on a caged green fluorescent protein (CA-GFP) reporter that allows for flow-cytometry-based selection in bacteria or other cell types enabling selection of intracellular protease specificity, regardless of the compositional complexity of the protease. Here, we apply this approach to introduce the specificity of caspase-6 into caspase-7, an intracellular cysteine protease important in cellular remodeling and cell death. We found that substitution of substrate-contacting residues from caspase-6 into caspase-7more » was ineffective, yielding an inactive enzyme, whereas saturation mutagenesis at these positions and selection by directed evolution produced active caspases. The process produced a number of nonobvious mutations that enabled conversion of the caspase-7 specificity to match caspase-6. The structures of the evolved-specificity caspase-7 (esCasp-7) revealed alternate binding modes for the substrate, including reorganization of an active site loop. Profiling the entire human proteome of esCasp-7 by N-terminomics demonstrated that the global specificity toward natural protein substrates is remarkably similar to that of caspase-6. Because the esCasp-7 maintained the core of caspase-7, we were able to identify a caspase-6 substrate, lamin C, that we predict relies on an exosite for substrate recognition. These reprogrammed proteases may be the first tool built with the express intent of distinguishing exosite dependent or independent substrates. Finally, this approach to specificity reprogramming should also be generalizable across a wide range of proteases.« less

  1. Daphnia HR96 is a Promiscuous Xenobiotic and Endobiotic Nuclear Receptor

    PubMed Central

    Karimullina, Elina; Li, Yangchun; Ginjupalli, Gautam; Baldwin, William S.

    2012-01-01

    Daphnia pulex is the first crustacean to have its genome sequenced. The genome project provides new insight and data into how an aquatic crustacean may respond to environmental stressors, including toxicants. We cloned Daphnia pulex HR96 (DappuHR96), a nuclear receptor orthologous to the CAR/PXR/VDR group of nuclear receptors. In Drosophila melanogaster, (hormone receptor 96) HR96 responds to phenobarbital exposure and has been hypothesized as a toxicant receptor. Therefore, we set up a transactivation assay to test whether DappuHR96 is a promiscuous receptor activated by xenobiotics and endobiotics similar to the constitutive androstane receptor (CAR) and the pregnane X-receptor (PXR). Transactivation assays performed with a GAL4-HR96 chimera demonstrate that HR96 is a promiscuous toxicant receptor activated by a diverse set of chemicals such as pesticides, hormones, and fatty acids. Several environmental toxicants activate HR96 including estradiol, pyriproxyfen, chlorpyrifos, atrazine, and methane arsonate. We also observed repression of HR96 activity by chemicals such as triclosan, androstanol, and fluoxetine. Nearly 50% of the chemicals tested activated or inhibited HR96. Interestingly, unsaturated fatty acids were common activators or inhibitors of HR96 activity, indicating a link between diet and toxicant response. The omega-6 and omega-9 unsaturated fatty acids linoleic and oleic acid activated HR96, but the omega-3 unsaturated fatty acids alpha-linolenic acid and docosahexaenoic acid inhibited HR96, suggesting that these two distinct sets of lipids perform opposing roles in Daphnia physiology. This also provides a putative mechanism by which the ratio of dietary unsaturated fats may affect the ability of an organism to respond to a toxic insult. In summary, HR96 is a promiscuous nuclear receptor activated by numerous endo- and xenobiotics. PMID:22466357

  2. Promiscuity resolves constraints on social mate choice imposed by population viscosity.

    PubMed

    While, Geoffrey M; Uller, Tobias; Bordogna, Genevieve; Wapstra, Erik

    2014-02-01

    Population viscosity can have major consequences for adaptive evolution, in particular for phenotypes involved in social interactions. For example, population viscosity increases the probability of mating with close kin, resulting in selection for mechanisms that circumvent the potential negative consequences of inbreeding. Female promiscuity is often suggested to be one such mechanism. However, whether avoidance of genetically similar partners is a major selective force shaping patterns of promiscuity remains poorly supported by empirical data. Here, we show (i) that fine-scale genetic structure constrains social mate choice in a pair-bonding lizard, resulting in individuals pairing with genetically similar individuals, (ii) that these constraints are circumvented by multiple mating with less related individuals and (iii) that this results in increased heterozygosity of offspring. Despite this, we did not detect any significant effects of heterozygosity on offspring or adult fitness or a strong relationship between pair relatedness and female multiple mating. We discuss these results within the context of incorporating the genetic context dependence of mating strategies into a holistic understanding of mating system evolution. © 2013 John Wiley & Sons Ltd.

  3. Molecular evolution of the CYP2D subfamily in primates: purifying selection on substrate recognition sites without the frequent or long-tract gene conversion.

    PubMed

    Yasukochi, Yoshiki; Satta, Yoko

    2015-03-25

    The human cytochrome P450 (CYP) 2D6 gene is a member of the CYP2D gene subfamily, along with the CYP2D7P and CYP2D8P pseudogenes. Although the CYP2D6 enzyme has been studied extensively because of its clinical importance, the evolution of the CYP2D subfamily has not yet been fully understood. Therefore, the goal of this study was to reveal the evolutionary process of the human drug metabolic system. Here, we investigate molecular evolution of the CYP2D subfamily in primates by comparing 14 CYP2D sequences from humans to New World monkey genomes. Window analysis and statistical tests revealed that entire genomic sequences of paralogous genes were extensively homogenized by gene conversion during molecular evolution of CYP2D genes in primates. A neighbor-joining tree based on genomic sequences at the nonsubstrate recognition sites showed that CYP2D6 and CYP2D8 genes were clustered together due to gene conversion. In contrast, a phylogenetic tree using amino acid sequences at substrate recognition sites did not cluster the CYP2D6 and CYP2D8 genes, suggesting that the functional constraint on substrate specificity is one of the causes for purifying selection at the substrate recognition sites. Our results suggest that the CYP2D gene subfamily in primates has evolved to maintain the regioselectivity for a substrate hydroxylation activity between individual enzymes, even though extensive gene conversion has occurred across CYP2D coding sequences. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  4. Promiscuous activities of heterologous enzymes lead to unintended metabolic rerouting in Saccharomyces cerevisiae engineered to assimilate various sugars from renewable biomass.

    PubMed

    Yun, Eun Ju; Oh, Eun Joong; Liu, Jing-Jing; Yu, Sora; Kim, Dong Hyun; Kwak, Suryang; Kim, Kyoung Heon; Jin, Yong-Su

    2018-01-01

    Understanding the global metabolic network, significantly perturbed upon promiscuous activities of foreign enzymes and different carbon sources, is crucial for systematic optimization of metabolic engineering of yeast Saccharomyces cerevisiae . Here, we studied the effects of promiscuous activities of overexpressed enzymes encoded by foreign genes on rerouting of metabolic fluxes of an engineered yeast capable of assimilating sugars from renewable biomass by profiling intracellular and extracellular metabolites. Unbiased metabolite profiling of the engineered S. cerevisiae strain EJ4 revealed promiscuous enzymatic activities of xylose reductase and xylitol dehydrogenase on galactose and galactitol, respectively, resulting in accumulation of galactitol and tagatose during galactose fermentation. Moreover, during glucose fermentation, a trisaccharide consisting of glucose accumulated outside of the cells probably owing to the promiscuous and transglycosylation activity of β-glucosidase expressed for hydrolyzing cellobiose. Meanwhile, higher accumulation of fatty acids and secondary metabolites was observed during xylose and cellobiose fermentations, respectively. The heterologous enzymes functionally expressed in S. cerevisiae showed promiscuous activities that led to unintended metabolic rerouting in strain EJ4. Such metabolic rerouting could result in a low yield and productivity of a final product due to the formation of unexpected metabolites. Furthermore, the global metabolic network can be significantly regulated by carbon sources, thus yielding different patterns of metabolite production. This metabolomic study can provide useful information for yeast strain improvement and systematic optimization of yeast metabolism to manufacture bio-based products.

  5. Structural analysis of substrate recognition by glucose isomerase in Mn2+ binding mode at M2 site in S. rubiginosus.

    PubMed

    Bae, Ji-Eun; Hwang, Kwang Yeon; Nam, Ki Hyun

    2018-06-16

    Glucose isomerase (GI) catalyzes the reversible enzymatic isomerization of d-glucose and d-xylose to d-fructose and d-xylulose, respectively. This is one of the most important enzymes in the production of high-fructose corn syrup (HFCS) and biofuel. We recently determined the crystal structure of GI from S. rubiginosus (SruGI) complexed with a xylitol inhibitor in one metal binding mode. Although we assessed inhibitor binding at the M1 site, the metal binding at the M2 site and the substrate recognition mechanism for SruGI remains the unclear. Here, we report the crystal structure of the two metal binding modes of SruGI and its complex with glucose. This study provides a snapshot of metal binding at the SruGI M2 site in the presence of Mn 2+ , but not in the presence of Mg 2+ . Metal binding at the M2 site elicits a configuration change at the M1 site. Glucose molecule can only bind to the M1 site in presence of Mn 2+ at the M2 site. Glucose and Mn 2+ at the M2 site were bridged by water molecules using a hydrogen bonding network. The metal binding geometry of the M2 site indicates a distorted octahedral coordination with an angle of 55-110°, whereas the M1 site has a relatively stable octahedral coordination with an angle of 85-95°. We suggest a two-step sequential process for SruGI substrate recognition, in Mn 2+ binding mode, at the M2 site. Our results provide a better understanding of the molecular role of the M2 site in GI substrate recognition. Copyright © 2018. Published by Elsevier Inc.

  6. The structural basis for receptor recognition of human interleukin-18

    DOE PAGES

    Tsutsumi, Naotaka; Kimura, Takeshi; Arita, Kyohei; ...

    2014-12-15

    Interleukin (IL)-18 is a proinflammatory cytokine that belongs to the IL-1 family and plays an important role in inflammation. The uncontrolled release of this cytokine is associated with severe chronic inflammatory disease. IL-18 forms a signalling complex with the IL-18 receptor α (Rα) and β (Rβ) chains at the plasma membrane, which induces multiple inflammatory cytokines. Here, we present a crystal structure of human IL-18 bound to the two receptor extracellular domains. Generally, the receptors’ recognition mode for IL-18 is similar to IL-1β; however, certain notable differences were observed. The architecture of the IL-18 receptor second domain (D2) is uniquemore » among the other IL-1R family members, which presumably distinguishes them from the IL-1 receptors that exhibit a more promiscuous ligand recognition mode. The structures and associated biochemical and cellular data should aid in developing novel drugs to neutralize IL-8 activity.« less

  7. The structural basis for receptor recognition of human interleukin-18

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

    Tsutsumi, Naotaka; Kimura, Takeshi; Arita, Kyohei

    Interleukin (IL)-18 is a proinflammatory cytokine that belongs to the IL-1 family and plays an important role in inflammation. The uncontrolled release of this cytokine is associated with severe chronic inflammatory disease. IL-18 forms a signalling complex with the IL-18 receptor α (Rα) and β (Rβ) chains at the plasma membrane, which induces multiple inflammatory cytokines. Here, we present a crystal structure of human IL-18 bound to the two receptor extracellular domains. Generally, the receptors’ recognition mode for IL-18 is similar to IL-1β; however, certain notable differences were observed. The architecture of the IL-18 receptor second domain (D2) is uniquemore » among the other IL-1R family members, which presumably distinguishes them from the IL-1 receptors that exhibit a more promiscuous ligand recognition mode. The structures and associated biochemical and cellular data should aid in developing novel drugs to neutralize IL-8 activity.« less

  8. The effect of the promiscuity stereotype on opposition to gay rights.

    PubMed

    Pinsof, David; Haselton, Martie G

    2017-01-01

    Opposition to gay rights is prevalent in countries around the world. Recent correlational research suggests that opposition to gay rights may be driven by an interaction between one's own short-term mating orientation (i.e. willingness to engage in casual sex) and representations of gay people as sexually promiscuous. Here, we experimentally manipulated representations of gay men by randomly assigning participants to read one of two versions of a fictitious newspaper article, one of which contained faux scientific evidence confirming the stereotype that gay men are promiscuous, and the other containing faux scientific evidence refuting the stereotype. We found that the manipulation interacted with short-term mating orientation (STMO) to predict opposition to gay rights, such that low-STMO individuals (i.e. more averse to casual sex) exhibited more support for gay rights when assigned to read the stereotype-refuting article compared to the stereotype-confirming article, whereas high-STMO individuals (i.e. less averse to casual sex) were not significantly influenced by the manipulation. We discuss the implications of these findings for the study of antigay attitudes, as well as for recent societal changes in acceptance of homosexuality.

  9. The effect of the promiscuity stereotype on opposition to gay rights

    PubMed Central

    Haselton, Martie G.

    2017-01-01

    Opposition to gay rights is prevalent in countries around the world. Recent correlational research suggests that opposition to gay rights may be driven by an interaction between one’s own short-term mating orientation (i.e. willingness to engage in casual sex) and representations of gay people as sexually promiscuous. Here, we experimentally manipulated representations of gay men by randomly assigning participants to read one of two versions of a fictitious newspaper article, one of which contained faux scientific evidence confirming the stereotype that gay men are promiscuous, and the other containing faux scientific evidence refuting the stereotype. We found that the manipulation interacted with short-term mating orientation (STMO) to predict opposition to gay rights, such that low-STMO individuals (i.e. more averse to casual sex) exhibited more support for gay rights when assigned to read the stereotype-refuting article compared to the stereotype-confirming article, whereas high-STMO individuals (i.e. less averse to casual sex) were not significantly influenced by the manipulation. We discuss the implications of these findings for the study of antigay attitudes, as well as for recent societal changes in acceptance of homosexuality. PMID:28704375

  10. Testing whether Metazoan Tyrosine Loss Was Driven by Selection against Promiscuous Phosphorylation

    PubMed Central

    Pandya, Siddharth; Struck, Travis J.; Mannakee, Brian K.; Paniscus, Mary; Gutenkunst, Ryan N.

    2015-01-01

    Protein tyrosine phosphorylation is a key regulatory modification in metazoans, and the corresponding kinase enzymes have diversified dramatically. This diversification is correlated with a genome-wide reduction in protein tyrosine content, and it was recently suggested that this reduction was driven by selection to avoid promiscuous phosphorylation that might be deleterious. We tested three predictions of this intriguing hypothesis. 1) Selection should be stronger on residues that are more likely to be phosphorylated due to local solvent accessibility or structural disorder. 2) Selection should be stronger on proteins that are more likely to be promiscuously phosphorylated because they are abundant. We tested these predictions by comparing distributions of tyrosine within and among human and yeast orthologous proteins. 3) Selection should be stronger against mutations that create tyrosine versus remove tyrosine. We tested this prediction using human population genomic variation data. We found that all three predicted effects are modest for tyrosine when compared with the other amino acids, suggesting that selection against deleterious phosphorylation was not dominant in driving metazoan tyrosine loss. PMID:25312910

  11. Caulobacter crescentus Cell Cycle-Regulated DNA Methyltransferase Uses a Novel Mechanism for Substrate Recognition.

    PubMed

    Woodcock, Clayton B; Yakubov, Aziz B; Reich, Norbert O

    2017-08-01

    Caulobacter crescentus relies on DNA methylation by the cell cycle-regulated methyltransferase (CcrM) in addition to key transcription factors to control the cell cycle and direct cellular differentiation. CcrM is shown here to efficiently methylate its cognate recognition site 5'-GANTC-3' in single-stranded and hemimethylated double-stranded DNA. We report the K m , k cat , k methylation , and K d for single-stranded and hemimethylated substrates, revealing discrimination of 10 7 -fold for noncognate sequences. The enzyme also shows a similar discrimination against single-stranded RNA. Two independent assays clearly show that CcrM is highly processive with single-stranded and hemimethylated DNA. Collectively, the data provide evidence that CcrM and other DNA-modifying enzymes may use a new mechanism to recognize DNA in a key epigenetic process.

  12. Conformational Changes and Substrate Recognition in Pseudomonas aeruginosa d-Arginine Dehydrogenase

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

    Fu, Guoxing; Yuan, Hongling; Li, Congran

    2010-11-15

    DADH catalyzes the flavin-dependent oxidative deamination of D-amino acids to the corresponding {alpha}-keto acids and ammonia. Here we report the first X-ray crystal structures of DADH at 1.06 {angstrom} resolution and its complexes with iminoarginine (DADH{sub red}/iminoarginine) and iminohistidine (DADH{sub red}/iminohistidine) at 1.30 {angstrom} resolution. The DADH crystal structure comprises an unliganded conformation and a product-bound conformation, which is almost identical to the DADH{sub red}/iminoarginine crystal structure. The active site of DADH was partially occupied with iminoarginine product (30% occupancy) that interacts with Tyr53 in the minor conformation of a surface loop. This flexible loop forms an 'active site lid',more » similar to those seen in other enzymes, and may play an essential role in substrate recognition. The guanidinium side chain of iminoarginine forms a hydrogen bond interaction with the hydroxyl of Thr50 and an ionic interaction with Glu87. In the structure of DADH in complex with iminohistidine, two alternate conformations were observed for iminohistidine where the imidazole groups formed hydrogen bond interactions with the side chains of His48 and Thr50 and either Glu87 or Gln336. The different interactions and very distinct binding modes observed for iminoarginine and iminohistidine are consistent with the 1000-fold difference in k{sub cat}/K{sub m} values for D-arginine and D-histidine. Comparison of the kinetic data for the activity of DADH on different D-amino acids and the crystal structures in complex with iminoarginine and iminohistidine establishes that this enzyme is characterized by relatively broad substrate specificity, being able to oxidize positively charged and large hydrophobic D-amino acids bound within a flask-like cavity.« less

  13. Neural Substrates of Auditory Emotion Recognition Deficits in Schizophrenia.

    PubMed

    Kantrowitz, Joshua T; Hoptman, Matthew J; Leitman, David I; Moreno-Ortega, Marta; Lehrfeld, Jonathan M; Dias, Elisa; Sehatpour, Pejman; Laukka, Petri; Silipo, Gail; Javitt, Daniel C

    2015-11-04

    Deficits in auditory emotion recognition (AER) are a core feature of schizophrenia and a key component of social cognitive impairment. AER deficits are tied behaviorally to impaired ability to interpret tonal ("prosodic") features of speech that normally convey emotion, such as modulations in base pitch (F0M) and pitch variability (F0SD). These modulations can be recreated using synthetic frequency modulated (FM) tones that mimic the prosodic contours of specific emotional stimuli. The present study investigates neural mechanisms underlying impaired AER using a combined event-related potential/resting-state functional connectivity (rsfMRI) approach in 84 schizophrenia/schizoaffective disorder patients and 66 healthy comparison subjects. Mismatch negativity (MMN) to FM tones was assessed in 43 patients/36 controls. rsfMRI between auditory cortex and medial temporal (insula) regions was assessed in 55 patients/51 controls. The relationship between AER, MMN to FM tones, and rsfMRI was assessed in the subset who performed all assessments (14 patients, 21 controls). As predicted, patients showed robust reductions in MMN across FM stimulus type (p = 0.005), particularly to modulations in F0M, along with impairments in AER and FM tone discrimination. MMN source analysis indicated dipoles in both auditory cortex and anterior insula, whereas rsfMRI analyses showed reduced auditory-insula connectivity. MMN to FM tones and functional connectivity together accounted for ∼50% of the variance in AER performance across individuals. These findings demonstrate that impaired preattentive processing of tonal information and reduced auditory-insula connectivity are critical determinants of social cognitive dysfunction in schizophrenia, and thus represent key targets for future research and clinical intervention. Schizophrenia patients show deficits in the ability to infer emotion based upon tone of voice [auditory emotion recognition (AER)] that drive impairments in social cognition

  14. Enzyme-substrate relationships in the ubiquitin system: approaches for identifying substrates of ubiquitin ligases.

    PubMed

    O'Connor, Hazel F; Huibregtse, Jon M

    2017-09-01

    Protein ubiquitylation is an important post-translational modification, regulating aspects of virtually every biochemical pathway in eukaryotic cells. Hundreds of enzymes participate in the conjugation and deconjugation of ubiquitin, as well as the recognition, signaling functions, and degradation of ubiquitylated proteins. Regulation of ubiquitylation is most commonly at the level of recognition of substrates by E3 ubiquitin ligases. Characterization of the network of E3-substrate relationships is a major goal and challenge in the field, as this expected to yield fundamental biological insights and opportunities for drug development. There has been remarkable success in identifying substrates for some E3 ligases, in many instances using the standard protein-protein interaction techniques (e.g., two-hybrid screens and co-immunoprecipitations paired with mass spectrometry). However, some E3s have remained refractory to characterization, while others have simply not yet been studied due to the sheer number and diversity of E3s. This review will discuss the range of tools and techniques that can be used for substrate profiling of E3 ligases.

  15. Systematic Functional Analysis of Active-Site Residues in l-Threonine Dehydrogenase from Thermoplasma volcanium

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

    Desjardins, Morgan; Mak, Wai Shun; O’Brien, Terrence E.

    Enzymes have been through millions of years of evolution during which their active-site microenvironments are fine-tuned. Active-site residues are commonly conserved within protein families, indicating their importance for substrate recognition and catalysis. In this work, we systematically mutated active-site residues of l-threonine dehydrogenase from Thermoplasma volcanium and characterized the mutants against a panel of substrate analogs. Our results demonstrate that only a subset of these residues plays an essential role in substrate recognition and catalysis and that the native enzyme activity can be further enhanced roughly 4.6-fold by a single point mutation. Kinetic characterization of mutants on substrate analogs showsmore » that l-threonine dehydrogenase possesses promiscuous activities toward other chemically similar compounds not previously observed. Quantum chemical calculations on the hydride-donating ability of these substrates also reveal that this enzyme did not evolve to harness the intrinsic substrate reactivity for enzyme catalysis. Our analysis provides insights into connections between the details of enzyme active-site structure and specific function. Finally, these results are directly applicable to rational enzyme design and engineering.« less

  16. Systematic Functional Analysis of Active-Site Residues in l-Threonine Dehydrogenase from Thermoplasma volcanium

    DOE PAGES

    Desjardins, Morgan; Mak, Wai Shun; O’Brien, Terrence E.; ...

    2017-07-07

    Enzymes have been through millions of years of evolution during which their active-site microenvironments are fine-tuned. Active-site residues are commonly conserved within protein families, indicating their importance for substrate recognition and catalysis. In this work, we systematically mutated active-site residues of l-threonine dehydrogenase from Thermoplasma volcanium and characterized the mutants against a panel of substrate analogs. Our results demonstrate that only a subset of these residues plays an essential role in substrate recognition and catalysis and that the native enzyme activity can be further enhanced roughly 4.6-fold by a single point mutation. Kinetic characterization of mutants on substrate analogs showsmore » that l-threonine dehydrogenase possesses promiscuous activities toward other chemically similar compounds not previously observed. Quantum chemical calculations on the hydride-donating ability of these substrates also reveal that this enzyme did not evolve to harness the intrinsic substrate reactivity for enzyme catalysis. Our analysis provides insights into connections between the details of enzyme active-site structure and specific function. Finally, these results are directly applicable to rational enzyme design and engineering.« less

  17. Structures of the peptide-modifying radical SAM enzyme SuiB elucidate the basis of substrate recognition.

    PubMed

    Davis, Katherine M; Schramma, Kelsey R; Hansen, William A; Bacik, John P; Khare, Sagar D; Seyedsayamdost, Mohammad R; Ando, Nozomi

    2017-09-26

    Posttranslational modification of ribosomally synthesized peptides provides an elegant means for the production of biologically active molecules known as RiPPs (ribosomally synthesized and posttranslationally modified peptides). Although the leader sequence of the precursor peptide is often required for turnover, the exact mode of recognition by the modifying enzymes remains unclear for many members of this class of natural products. Here, we have used X-ray crystallography and computational modeling to examine the role of the leader peptide in the biosynthesis of a homolog of streptide, a recently identified peptide natural product with an intramolecular lysine-tryptophan cross-link, which is installed by the radical S -adenosylmethionine (SAM) enzyme, StrB. We present crystal structures of SuiB, a close ortholog of StrB, in various forms, including apo SuiB, SAM-bound SuiB, and a complex of SuiB with SAM and its peptide substrate, SuiA. Although the N-terminal domain of SuiB adopts a typical RRE (RiPP recognition element) motif, which has been implicated in precursor peptide recognition, we observe binding of the leader peptide in the catalytic barrel rather than the N-terminal domain. Computational simulations support a mechanism in which the leader peptide guides posttranslational modification by positioning the cross-linking residues of the precursor peptide within the active site. Together the results shed light onto binding of the precursor peptide and the associated conformational changes needed for the formation of the unique carbon-carbon cross-link in the streptide family of natural products.

  18. Activity-Based Profiling of a Physiologic Aglycone Library Reveals Sugar Acceptor Promiscuity of Family 1 UDP-Glucosyltransferases from Grape1[W

    PubMed Central

    Bönisch, Friedericke; Frotscher, Johanna; Stanitzek, Sarah; Rühl, Ernst; Wüst, Matthias; Bitz, Oliver; Schwab, Wilfried

    2014-01-01

    Monoterpenols serve various biological functions and accumulate in grape (Vitis vinifera), where a major fraction occurs as nonvolatile glycosides. We have screened the grape genome for sequences with similarity to terpene URIDINE DIPHOSPHATE GLYCOSYLTRANSFERASES (UGTs) from Arabidopsis (Arabidopsis thaliana). A ripening-related expression pattern was shown for three candidates by spatial and temporal expression analyses in five grape cultivars. Transcript accumulation correlated with the production of monoterpenyl β-d-glucosides in grape exocarp during ripening and was low in vegetative tissue. Targeted functional screening of the recombinant UGTs for their biological substrates was performed by activity-based metabolite profiling (ABMP) employing a physiologic library of aglycones built from glycosides isolated from grape. This approach led to the identification of two UDP-glucose:monoterpenol β-d-glucosyltransferases. Whereas VvGT14a glucosylated geraniol, R,S-citronellol, and nerol with similar efficiency, the three allelic forms VvGT15a, VvGT15b, and VvGT15c preferred geraniol over nerol. Kinetic resolution of R,S-citronellol and R,S-linalool was shown for VvGT15a and VvGT14a, respectively. ABMP revealed geraniol as the major biological substrate but also disclosed that these UGTs may add to the production of further glycoconjugates in planta. ABMP of aglycone libraries provides a versatile tool to uncover novel biologically relevant substrates of small-molecule glycosyltransferases that often show broad sugar acceptor promiscuity. PMID:25073706

  19. The promiscuous larvae: flexibility in the establishment of symbiosis in corals

    NASA Astrophysics Data System (ADS)

    Cumbo, V. R.; Baird, A. H.; van Oppen, M. J. H.

    2013-03-01

    Coral reefs thrive in part because of the symbiotic partnership between corals and Symbiodinium. While this partnership is one of the keys to the success of coral reef ecosystems, surprisingly little is known about many aspects of coral symbiosis, in particular the establishment and development of symbiosis in host species that acquire symbionts anew in each generation. More specifically, the point at which symbiosis is established (i.e., larva vs. juvenile) remains uncertain, as does the source of free-living Symbiodinium in the environment. In addition, the capacity of host and symbiont to form novel combinations is unknown. To explore patterns of initial association between host and symbiont, larvae of two species of Acropora were exposed to sediment collected from three locations on the Great Barrier Reef. A high proportion of larvae established symbiosis shortly after contact with sediments, and Acropora larvae were promiscuous, taking up multiple types of Symbiodinium. The Symbiodinium types acquired from the sediments reflected the symbiont assemblage within a wide range of cnidarian hosts at each of the three sites, suggesting potential regional differences in the free-living Symbiodinium assemblage. Coral larvae clearly have the capacity to take up Symbiodinium prior to settlement, and sediment is a likely source. Promiscuous larvae allow species to associate with Symbiodinium appropriate for potentially novel environments that may be experienced following dispersal.

  20. Recognition of facial emotions in neuropsychiatric disorders.

    PubMed

    Kohler, Christian G; Turner, Travis H; Gur, Raquel E; Gur, Ruben C

    2004-04-01

    Recognition of facial emotions represents an important aspect of interpersonal communication and is governed by select neural substrates. We present data on emotion recognition in healthy young adults utilizing a novel set of color photographs of evoked universal emotions. In addition, we review the recent literature on emotion recognition in psychiatric and neurologic disorders, and studies that compare different disorders.

  1. Exploiting the aglycon promiscuity of glycosyltransferase Bs-YjiC from Bacillus subtilis and its application in synthesis of glycosides.

    PubMed

    Dai, Longhai; Li, Jiao; Yao, Peiyuan; Zhu, Yueming; Men, Yan; Zeng, Yan; Yang, Jiangang; Sun, Yuanxia

    2017-04-20

    Glycosylation is a prominent biological mechanism for structural and functional diversity of natural products. Uridine diphosphate-dependent glycosyltransferases with aglycon promiscuity are generally recognised as effective biocatalysts for glycodiversification of natural products for practical applications. In this study, the aglycon promiscuity of glycosyltransferase Bs-YjiC from Bacillus subtilis 168 was explored. Bs-YjiC, with uridine diphosphate glucose (UDPG) as sugar donor, exhibited robust capabilities to glycosylate 19 structurally diverse types of drug-like scaffolds with regio- and stereospecificities and form O-, N- and S-linkage glycosides. Twenty-four glycosides of 17 aglycons were purified from scale-up reactions using Bs-YjiC as a biocatalyst, and their structures were confirmed by nuclear magnetic resonance spectra. Furthermore, a one-pot reaction by coupling Bs-YjiC to sucrose synthase from Arabidopsis thaliana was applied to glycosylate pterostilbene. Without adding the costly UDPG as sugar donor, 9mM (3.8g/L) pterostilbene 4'-O-β-glucoside was obtained by periodic feeding of pterostilbene. These results suggest the aglycon promiscuity of Bs-YjiC and demonstrate its significant application prospect in biosynthesis of valuable natural products. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Coexistence of unlimited bipartite and genuine multipartite entanglement: Promiscuous quantum correlations arising from discrete to continuous-variable systems

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

    Adesso, Gerardo; CNR-INFM Coherentia , Naples; Grup d'Informacio Quantica, Universitat Autonoma de Barcelona, E-08193 Bellaterra

    2007-08-15

    Quantum mechanics imposes 'monogamy' constraints on the sharing of entanglement. We show that, despite these limitations, entanglement can be fully 'promiscuous', i.e., simultaneously present in unlimited two-body and many-body forms in states living in an infinite-dimensional Hilbert space. Monogamy just bounds the divergence rate of the various entanglement contributions. This is demonstrated in simple families of N-mode (N{>=}4) Gaussian states of light fields or atomic ensembles, which therefore enable infinitely more freedom in the distribution of information, as opposed to systems of individual qubits. Such a finding is of importance for the quantification, understanding, and potential exploitation of shared quantummore » correlations in continuous variable systems. We discuss how promiscuity gradually arises when considering simple families of discrete variable states, with increasing Hilbert space dimension towards the continuous variable limit. Such models are somehow analogous to Gaussian states with asymptotically diverging, but finite, squeezing. In this respect, we find that non-Gaussian states (which in general are more entangled than Gaussian states) exhibit also the interesting feature that their entanglement is more shareable: in the non-Gaussian multipartite arena, unlimited promiscuity can be already achieved among three entangled parties, while this is impossible for Gaussian, even infinitely squeezed states.« less

  3. Replica exchange molecular dynamics simulations provide insight into substrate recognition by small heat shock proteins.

    PubMed

    Patel, Sunita; Vierling, Elizabeth; Tama, Florence

    2014-06-17

    The small heat shock proteins (sHSPs) are a virtually ubiquitous and diverse group of molecular chaperones that can bind and protect unfolding proteins from irreversible aggregation. It has been suggested that intrinsic disorder of the N-terminal arm (NTA) of sHSPs is important for substrate recognition. To investigate conformations of the NTA that could recognize substrates we performed replica exchange molecular dynamics simulations. Behavior at normal and stress temperatures of the dimeric building blocks of dodecameric HSPs from wheat (Ta16.9) and pea (Ps18.1) were compared because they display high sequence similarity, but Ps18.1 is more efficient in binding specific substrates. In our simulations, the NTAs of the dimer are flexible and dynamic; however, rather than exhibiting highly extended conformations they retain considerable α-helical character and contacts with the conserved α-crystallin domain (ACD). Network analysis and clustering methods reveal that there are two major conformational forms designated either "open" or "closed" based on the relative position of the two NTAs and their hydrophobic solvent accessible surface area. The equilibrium constant for the closed to open transition is significantly different for Ta16.9 and Ps18.1, with the latter showing more open conformations at elevated temperature correlated with its more effective chaperone activity. In addition, the Ps18.1 NTAs have more hydrophobic solvent accessible surface than those of Ta16.9. NTA hydrophobic patches are comparable in size to the area buried in many protein-protein interactions, which would enable sHSPs to bind early unfolding intermediates. Reduced interactions of the Ps18.1 NTAs with each other and with the ACD contribute to the differences in dynamics and hydrophobic surface area of the two sHSPs. These data support a major role for the conformational equilibrium of the NTA in substrate binding and indicate features of the NTA that contribute to sHSP chaperone

  4. Basis for substrate recognition and distinction by matrix metalloproteinases

    PubMed Central

    Ratnikov, Boris I.; Cieplak, Piotr; Gramatikoff, Kosi; Pierce, James; Eroshkin, Alexey; Igarashi, Yoshinobu; Kazanov, Marat; Sun, Qing; Godzik, Adam; Osterman, Andrei; Stec, Boguslaw; Strongin, Alex; Smith, Jeffrey W.

    2014-01-01

    Genomic sequencing and structural genomics produced a vast amount of sequence and structural data, creating an opportunity for structure–function analysis in silico [Radivojac P, et al. (2013) Nat Methods 10(3):221–227]. Unfortunately, only a few large experimental datasets exist to serve as benchmarks for function-related predictions. Furthermore, currently there are no reliable means to predict the extent of functional similarity among proteins. Here, we quantify structure–function relationships among three phylogenetic branches of the matrix metalloproteinase (MMP) family by comparing their cleavage efficiencies toward an extended set of phage peptide substrates that were selected from ∼64 million peptide sequences (i.e., a large unbiased representation of substrate space). The observed second-order rate constants [k(obs)] across the substrate space provide a distance measure of functional similarity among the MMPs. These functional distances directly correlate with MMP phylogenetic distance. There is also a remarkable and near-perfect correlation between the MMP substrate preference and sequence identity of 50–57 discontinuous residues surrounding the catalytic groove. We conclude that these residues represent the specificity-determining positions (SDPs) that allowed for the expansion of MMP proteolytic function during evolution. A transmutation of only a few selected SDPs proximal to the bound substrate peptide, and contributing the most to selectivity among the MMPs, is sufficient to enact a global change in the substrate preference of one MMP to that of another, indicating the potential for the rational and focused redesign of cleavage specificity in MMPs. PMID:25246591

  5. Structural Basis for Potency and Promiscuity in Poly(ADP-ribose) Polymerase (PARP) and Tankyrase Inhibitors.

    PubMed

    Thorsell, Ann-Gerd; Ekblad, Torun; Karlberg, Tobias; Löw, Mirjam; Pinto, Ana Filipa; Trésaugues, Lionel; Moche, Martin; Cohen, Michael S; Schüler, Herwig

    2017-02-23

    Selective inhibitors could help unveil the mechanisms by which inhibition of poly(ADP-ribose) polymerases (PARPs) elicits clinical benefits in cancer therapy. We profiled 10 clinical PARP inhibitors and commonly used research tools for their inhibition of multiple PARP enzymes. We also determined crystal structures of these compounds bound to PARP1 or PARP2. Veliparib and niraparib are selective inhibitors of PARP1 and PARP2; olaparib, rucaparib, and talazoparib are more potent inhibitors of PARP1 but are less selective. PJ34 and UPF1069 are broad PARP inhibitors; PJ34 inserts a flexible moiety into hydrophobic subpockets in various ADP-ribosyltransferases. XAV939 is a promiscuous tankyrase inhibitor and a potent inhibitor of PARP1 in vitro and in cells, whereas IWR1 and AZ-6102 are tankyrase selective. Our biochemical and structural analysis of PARP inhibitor potencies establishes a molecular basis for either selectivity or promiscuity and provides a benchmark for experimental design in assessment of PARP inhibitor effects.

  6. Some Surprising Implications of NMR-directed Simulations of Substrate Recognition and Binding by Cytochrome P450cam (CYP101A1).

    PubMed

    Asciutto, Eliana K; Pochapsky, Thomas C

    2018-04-27

    Cytochrome P450 cam (CYP101A1) catalyzes the stereospecific 5-exo hydroxylation of d-camphor by molecular oxygen. Previously, residual dipolar couplings measured for backbone amide 1 H- 15 N correlations in both substrate-free and bound forms of CYP101A1 were used as restraints in soft annealing molecular dynamic simulations in order to identify average conformations of the enzyme with and without substrate bound. Multiple substrate-dependent conformational changes remote from the enzyme active site were identified, and site-directed mutagenesis and activity assays confirmed the importance of these changes in substrate recognition. The current work makes use of perturbation response scanning (PRS) and umbrella sampling molecular dynamic of the residual dipolar coupling-derived CYP101A1 structures to probe the roles of remote structural features in enforcing the regio- and stereospecific nature of the hydroxylation reaction catalyzed by CYP101A1. An improper dihedral angle Ψ was defined and used to maintain substrate orientation in the CYP101A1 active site, and it was observed that different values of Ψ result in different PRS response maps. Umbrella sampling methods show that the free energy of the system is sensitive to Ψ, and bound substrate forms an important mechanical link in the transmission of mechanical coupling through the enzyme structure. Finally, a qualitative approach to interpreting PRS maps in terms of the roles of secondary structural features is proposed. Copyright © 2018 Elsevier Ltd. All rights reserved.

  7. Neural Substrates of View-Invariant Object Recognition Developed without Experiencing Rotations of the Objects

    PubMed Central

    Okamura, Jun-ya; Yamaguchi, Reona; Honda, Kazunari; Tanaka, Keiji

    2014-01-01

    One fails to recognize an unfamiliar object across changes in viewing angle when it must be discriminated from similar distractor objects. View-invariant recognition gradually develops as the viewer repeatedly sees the objects in rotation. It is assumed that different views of each object are associated with one another while their successive appearance is experienced in rotation. However, natural experience of objects also contains ample opportunities to discriminate among objects at each of the multiple viewing angles. Our previous behavioral experiments showed that after experiencing a new set of object stimuli during a task that required only discrimination at each of four viewing angles at 30° intervals, monkeys could recognize the objects across changes in viewing angle up to 60°. By recording activities of neurons from the inferotemporal cortex after various types of preparatory experience, we here found a possible neural substrate for the monkeys' performance. For object sets that the monkeys had experienced during the task that required only discrimination at each of four viewing angles, many inferotemporal neurons showed object selectivity covering multiple views. The degree of view generalization found for these object sets was similar to that found for stimulus sets with which the monkeys had been trained to conduct view-invariant recognition. These results suggest that the experience of discriminating new objects in each of several viewing angles develops the partially view-generalized object selectivity distributed over many neurons in the inferotemporal cortex, which in turn bases the monkeys' emergent capability to discriminate the objects across changes in viewing angle. PMID:25378169

  8. Bacterial protease uses distinct thermodynamic signatures for substrate recognition.

    PubMed

    Bezerra, Gustavo Arruda; Ohara-Nemoto, Yuko; Cornaciu, Irina; Fedosyuk, Sofiya; Hoffmann, Guillaume; Round, Adam; Márquez, José A; Nemoto, Takayuki K; Djinović-Carugo, Kristina

    2017-06-06

    Porphyromonas gingivalis and Porphyromonas endodontalis are important bacteria related to periodontitis, the most common chronic inflammatory disease in humans worldwide. Its comorbidity with systemic diseases, such as type 2 diabetes, oral cancers and cardiovascular diseases, continues to generate considerable interest. Surprisingly, these two microorganisms do not ferment carbohydrates; rather they use proteinaceous substrates as carbon and energy sources. However, the underlying biochemical mechanisms of their energy metabolism remain unknown. Here, we show that dipeptidyl peptidase 11 (DPP11), a central metabolic enzyme in these bacteria, undergoes a conformational change upon peptide binding to distinguish substrates from end products. It binds substrates through an entropy-driven process and end products in an enthalpy-driven fashion. We show that increase in protein conformational entropy is the main-driving force for substrate binding via the unfolding of specific regions of the enzyme ("entropy reservoirs"). The relationship between our structural and thermodynamics data yields a distinct model for protein-protein interactions where protein conformational entropy modulates the binding free-energy. Further, our findings provide a framework for the structure-based design of specific DPP11 inhibitors.

  9. Molecular recognition of pre-tRNA by Arabidopsis protein-only Ribonuclease P.

    PubMed

    Klemm, Bradley P; Karasik, Agnes; Kaitany, Kipchumba J; Shanmuganathan, Aranganathan; Henley, Matthew J; Thelen, Adam Z; Dewar, Allison J L; Jackson, Nathaniel D; Koutmos, Markos; Fierke, Carol A

    2017-12-01

    Protein-only ribonuclease P (PRORP) is an enzyme responsible for catalyzing the 5' end maturation of precursor transfer ribonucleic acids (pre-tRNAs) encoded by various cellular compartments in many eukaryotes. PRORPs from plants act as single-subunit enzymes and have been used as a model system for analyzing the function of the metazoan PRORP nuclease subunit, which requires two additional proteins for efficient catalysis. There are currently few molecular details known about the PRORP-pre-tRNA complex. Here, we characterize the determinants of substrate recognition by the single subunit Arabidopsis thaliana PRORP1 and PRORP2 using kinetic and thermodynamic experiments. The salt dependence of binding affinity suggests 4-5 contacts with backbone phosphodiester bonds on substrates, including a single phosphodiester contact with the pre-tRNA 5' leader, consistent with prior reports of short leader requirements. PRORPs contain an N-terminal pentatricopeptide repeat (PPR) domain, truncation of which results in a >30-fold decrease in substrate affinity. While most PPR-containing proteins have been implicated in single-stranded sequence-specific RNA recognition, we find that the PPR motifs of PRORPs recognize pre-tRNA substrates differently. Notably, the PPR domain residues most important for substrate binding in PRORPs do not correspond to positions involved in base recognition in other PPR proteins. Several of these residues are highly conserved in PRORPs from algae, plants, and metazoans, suggesting a conserved strategy for substrate recognition by the PRORP PPR domain. Furthermore, there is no evidence for sequence-specific interactions. This work clarifies molecular determinants of PRORP-substrate recognition and provides a new predictive model for the PRORP-substrate complex. © 2017 Klemm et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  10. The Structure and Specificity of the Type III Secretion System Effector NleC Suggest a DNA Mimicry Mechanism of Substrate Recognition

    PubMed Central

    2015-01-01

    Many pathogenic bacteria utilize the type III secretion system (T3SS) to translocate effector proteins directly into host cells, facilitating colonization. In enterohemmorhagic Escherichia coli (EHEC), a subset of T3SS effectors is essential for suppression of the inflammatory response in hosts, including humans. Identified as a zinc protease that cleaves NF-κB transcription factors, NleC is one such effector. Here, we investigate NleC substrate specificity, showing that four residues around the cleavage site in the DNA-binding loop of the NF-κB subunit RelA strongly influence the cleavage rate. Class I NF-κB subunit p50 is cleaved at a reduced rate consistent with conservation of only three of these four residues. However, peptides containing 10 residues on each side of the scissile bond were not efficiently cleaved by NleC, indicating that elements distal from the cleavage site are also important for substrate recognition. We present the crystal structure of NleC and show that it mimics DNA structurally and electrostatically. Consistent with this model, mutation of phosphate-mimicking residues in NleC reduces the level of RelA cleavage. We propose that global recognition of NF-κB subunits by DNA mimicry combined with a high sequence selectivity for the cleavage site results in exquisite NleC substrate specificity. The structure also shows that despite undetectable similarity of its sequence to those of other Zn2+ proteases beyond its conserved HExxH Zn2+-binding motif, NleC is a member of the Zincin protease superfamily, albeit divergent from its structural homologues. In particular, NleC displays a modified Ψ-loop motif that may be important for folding and refolding requirements implicit in T3SS translocation. PMID:25040221

  11. Cortical Networks for Visual Self-Recognition

    NASA Astrophysics Data System (ADS)

    Sugiura, Motoaki

    This paper briefly reviews recent developments regarding the brain mechanisms of visual self-recognition. A special cognitive mechanism for visual self-recognition has been postulated based on behavioral and neuropsychological evidence, but its neural substrate remains controversial. Recent functional imaging studies suggest that multiple cortical mechanisms play self-specific roles during visual self-recognition, reconciling the existing controversy. Respective roles for the left occipitotemporal, right parietal, and frontal cortices in symbolic, visuospatial, and conceptual aspects of self-representation have been proposed.

  12. Structural Basis for Substrate Specificity in Phosphate Binding (beta/alpha)8-Barrels: D-Allulose 6-Phosphate 3-Epimerase from Escherichia coli K-12

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

    Chan,K.; Fedorov, A.; Almo, S.

    2008-01-01

    Enzymes that share the ({beta}/{alpha})8-barrel fold catalyze a diverse range of reactions. Many utilize phosphorylated substrates and share a conserved C-terminal ({beta}/a)2-quarter barrel subdomain that provides a binding motif for the dianionic phosphate group. We recently reported functional and structural studies of d-ribulose 5-phosphate 3-epimerase (RPE) from Streptococcus pyogenes that catalyzes the equilibration of the pentulose 5-phosphates d-ribulose 5-phosphate and d-xylulose 5-phosphate in the pentose phosphate pathway [J. Akana, A. A. Fedorov, E. Fedorov, W. R. P. Novack, P. C. Babbitt, S. C. Almo, and J. A. Gerlt (2006) Biochemistry 45, 2493-2503]. We now report functional and structural studies ofmore » d-allulose 6-phosphate 3-epimerase (ALSE) from Escherichia coli K-12 that catalyzes the equilibration of the hexulose 6-phosphates d-allulose 6-phosphate and d-fructose 6-phosphate in a catabolic pathway for d-allose. ALSE and RPE prefer their physiological substrates but are promiscuous for each other's substrate. The active sites (RPE complexed with d-xylitol 5-phosphate and ALSE complexed with d-glucitol 6-phosphate) are superimposable (as expected from their 39% sequence identity), with the exception of the phosphate binding motif. The loop following the eighth {beta}-strand in ALSE is one residue longer than the homologous loop in RPE, so the binding site for the hexulose 6-phosphate substrate/product in ALSE is elongated relative to that for the pentulose 5-phosphate substrate/product in RPE. We constructed three single-residue deletion mutants of the loop in ALSE, ?T196, ?S197 and ?G198, to investigate the structural bases for the differing substrate specificities; for each, the promiscuity is altered so that d-ribulose 5-phosphate is the preferred substrate. The changes in kcat/Km are dominated by changes in kcat, suggesting that substrate discrimination results from differential transition state stabilization. In both ALSE and RPE, the

  13. Prediction and analysis of promiscuous T cell-epitopes derived from the vaccine candidate antigens of Leishmania donovani binding to MHC class-II alleles using in silico approach.

    PubMed

    Kashyap, Manju; Jaiswal, Varun; Farooq, Umar

    2017-09-01

    Visceral leishmaniasis is a dreadful infectious disease and caused by the intracellular protozoan parasites, Leishmania donovani and Leishmania infantum. Despite extensive efforts for developing effective prophylactic vaccine, still no vaccine is available against leishmaniasis. However, advancement in immunoinformatics methods generated new dimension in peptide based vaccine development. The present study was aimed to identify T-cell epitopes from the vaccine candidate antigens like Lipophosphogylcan-3(LPG-3) and Nucleoside hydrolase (NH) from the L. donovani using in silico methods. Available best tools were used for the identification of promiscuous peptides for MHC class-II alleles. A total of 34 promiscuous peptides from LPG-3, 3 from NH were identified on the basis of their 100% binding affinity towards all six HLA alleles, taken in this study. These peptides were further checked computationally to know their IFN-γ and IL4 inducing potential and nine peptides were identified. Peptide binding interactions with predominant HLA alleles were done by docking. Out of nine docked promiscuous peptides, only two peptides (QESRILRVIKKKLVR, RILRVIKKKLVRKTL), from LPG-3 and one peptide (FDKFWCLVIDALKRI) from NH showed lowest binding energy with all six alleles. These promiscuous T-cell epitopes were predicted on the basis of their antigenicity, hydrophobicity, potential immune response and docking scores. The immunogenicity of predicted promiscuous peptides might be used for subunit vaccine development with immune-modulating adjuvants. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Fractionating the Neural Substrates of Incidental Recognition Memory

    ERIC Educational Resources Information Center

    Greene, Ciara M.; Vidaki, Kleio; Soto, David

    2015-01-01

    Familiar stimuli are typically accompanied by decreases in neural response relative to the presentation of novel items, but these studies often include explicit instructions to discriminate old and new items; this creates difficulties in partialling out the contribution of top-down intentional orientation to the items based on recognition goals.…

  15. Symbiotic N nutrition, bradyrhizobial biodiversity and photosynthetic functioning of six inoculated promiscuous-nodulating soybean genotypes.

    PubMed

    Pule-Meulenberg, Flora; Gyogluu, Cynthia; Naab, Jesse; Dakora, Felix D

    2011-04-15

    Six promiscuous soybean genotypes were assessed for their ability to nodulate with indigenous root-nodule bacteria in Ghana, with Bradyrhizobium japonicum WB74 serving as positive control. Although the results revealed free nodulation of all six genotypes in both inoculated and uninoculated plots, there was a marked effect of inoculation on photosynthetic rates and whole-plant C. Inoculation also increased stomatal conductance in TGx1485-1D, TGx1448-2E, TGx1740-2F and TGx1445-3E, leading to significantly elevated transpiration rates in the last two genotypes, and a decrease in TGx1485-1D, TGx1440-1E and Salintuya-1, resulting in reduced leaf transpiration and decreased C accumulation. Nodulation, total plant biomass, plant N concentration and content also increased and ∂(15)N of the six genotypes, except for TGx1448-2E decreased. Significantly higher %Ndfa resulted in all the soybean genotypes tested (except for TGx1485-1D), and the symbiotic N yield in TGx1740-2F and TGx1448-2E doubled. PCR-RFLP revealed 18 distinct IGS types present in root nodules of the six promiscuous soybean genotypes, with IGS type II being isolated from all six genotypes, followed by IGS types X and XI from five out of the six genotypes. Marked differences in strain IGS type symbiotic efficiency were revealed. For example, as sole nodule occupant, IGS type XI produced high symbiotic N in TGx1445-3E, but low amounts in TGx1448-2E. Inoculated Salintuya-1, which trapped nine strain IGS types in its root nodules, was the most promiscuous genotype, but produced less symbiotic N compared to genotypes with fewer strains in their root nodules. Copyright © 2010 Elsevier GmbH. All rights reserved.

  16. Promiscuity in sand lizards (Lacerta agilis) and adder snakes (Vipera berus): causes and consequences.

    PubMed

    Olsson, M; Madsen, T

    2001-01-01

    We review postcopulatory phenomena in the Swedish sand lizard (Lacerta agilis) and adder (Vipera berus), and in particular, links between female promiscuity, determinants of paternity, and offspring viability. In both species, females mate multiply and exhibit a positive relationship between the number of partners and offspring viability. We conclude that this relationship is most likely the result of variable genetic compatibility between mates arising from postcopulatory phenomena, predominantly assortative fertilization with respect to parental genotypes. However, males who were more successful at mate acquisition were also more successful in situations of sperm competition, suggesting a possible link between male (diploid and haploid) genetic quality per se and probability of fertilization. Neither the number of partners nor the number of matings influenced the risk of infertility in sand lizards, suggesting that selection for reduced risk of infertility is not a sufficient explanation for maintaining female promiscuity in this population. Finally, we conclude that the relatively low genetic variability exhibited by our study populations may have facilitated detection of genetic benefits compared to more outbred ones. However, recent work derived from outbred populations in other taxa suggest a greater generality of the principles we discuss than previously may have been appreciated.

  17. Neural substrates of view-invariant object recognition developed without experiencing rotations of the objects.

    PubMed

    Okamura, Jun-Ya; Yamaguchi, Reona; Honda, Kazunari; Wang, Gang; Tanaka, Keiji

    2014-11-05

    One fails to recognize an unfamiliar object across changes in viewing angle when it must be discriminated from similar distractor objects. View-invariant recognition gradually develops as the viewer repeatedly sees the objects in rotation. It is assumed that different views of each object are associated with one another while their successive appearance is experienced in rotation. However, natural experience of objects also contains ample opportunities to discriminate among objects at each of the multiple viewing angles. Our previous behavioral experiments showed that after experiencing a new set of object stimuli during a task that required only discrimination at each of four viewing angles at 30° intervals, monkeys could recognize the objects across changes in viewing angle up to 60°. By recording activities of neurons from the inferotemporal cortex after various types of preparatory experience, we here found a possible neural substrate for the monkeys' performance. For object sets that the monkeys had experienced during the task that required only discrimination at each of four viewing angles, many inferotemporal neurons showed object selectivity covering multiple views. The degree of view generalization found for these object sets was similar to that found for stimulus sets with which the monkeys had been trained to conduct view-invariant recognition. These results suggest that the experience of discriminating new objects in each of several viewing angles develops the partially view-generalized object selectivity distributed over many neurons in the inferotemporal cortex, which in turn bases the monkeys' emergent capability to discriminate the objects across changes in viewing angle. Copyright © 2014 the authors 0270-6474/14/3415047-13$15.00/0.

  18. Enzymatic Detoxication, Conformational Selection, and the Role of Molten Globule Active Sites*

    PubMed Central

    Honaker, Matthew T.; Acchione, Mauro; Zhang, Wei; Mannervik, Bengt; Atkins, William M.

    2013-01-01

    The role of conformational ensembles in enzymatic reactions remains unclear. Discussion concerning “induced fit” versus “conformational selection” has, however, ignored detoxication enzymes, which exhibit catalytic promiscuity. These enzymes dominate drug metabolism and determine drug-drug interactions. The detoxication enzyme glutathione transferase A1–1 (GSTA1–1), exploits a molten globule-like active site to achieve remarkable catalytic promiscuity wherein the substrate-free conformational ensemble is broad with barrierless transitions between states. A quantitative index of catalytic promiscuity is used to compare engineered variants of GSTA1–1 and the catalytic promiscuity correlates strongly with characteristics of the thermodynamic partition function, for the substrate-free enzymes. Access to chemically disparate transition states is encoded by the substrate-free conformational ensemble. Pre-steady state catalytic data confirm an extension of the conformational selection model, wherein different substrates select different starting conformations. The kinetic liability of the conformational breadth is minimized by a smooth landscape. We propose that “local” molten globule behavior optimizes detoxication enzymes. PMID:23649628

  19. The neuro-immunological interface in an evolutionary perspective: the dynamic relationship between effector and recognition systems.

    PubMed

    Ottaviani, E; Valensin, S; Franceschi, C

    1998-04-16

    The evolutionary perspective indicates that an immune-neuroendocrine effector system integrating innate immunity, stress and inflammation is present in invertebrates. This defense network, centered on the macrophage and exerting primitive and highly promiscuous recognition units, is very effective, ancestral and appears to have been conserved throughout evolution from invertebrates to higher vertebrates. It would seem that there was a "big bang" in the recognition system of lower vertebrates, and T and B cell repertoires, MHC and antibodies suddenly appeared. We argue that this phenomenon is the counterpart of the increasing complexity of the internal circuitry and recognition units in the effector system. The immediate consequences were a progressive enlargement of the pathogen repertoire and new problems regarding self/not-self discrimination. Probably not by chance, a new organ appeared, capable of purging cells able of excessive self recognition. This organ, the thymus, appears to be the result of a well known evolutionary strategy of re-using pre-existing material (neuroendocrine cells and mediators constituting the thymic microenvironment). This bricolage at an organ level is similar to the effect we have already described at the level of molecules and functions of the defense network, and has a general counterpart at genetic level. Thus, in vertebrates, the conserved immune-neuroendocrine effector system remains of fundamental importance in defense against pathogens, while its efficiency has increased through synergy with the new, clonotipical recognition repertoire.

  20. The food colorant erythrosine is a promiscuous protein-protein interaction inhibitor.

    PubMed

    Ganesan, Lakshmi; Margolles-Clark, Emilio; Song, Yun; Buchwald, Peter

    2011-03-15

    Following our observation that erythrosine B (FD&C Red No. 3) is a relatively potent inhibitor of the TNF-R-TNFα and CD40-CD154 protein-protein interactions, we investigated whether this inhibitory activity extends to any other protein-protein interactions (PPI) as well as whether any other approved food colors possess such inhibitory activity. We found erythrosine, a poly-iodinated xanthene dye, to be a non-specific promiscuous inhibitor of a number of PPIs within the tumor necrosis factor superfamily (TNF-R-TNFα, CD40-CD154, BAFF-R-BAFF, RANK-RANKL, OX40-OX40L, 4-1BB-4-1BBL) as well as outside of it (EGF-R-EGF) with a remarkably consistent median inhibitory concentration (IC(50)) in the 2-20 μM (approximately 2-20mg/L) range. In agreement with this, erythrosine also showed cellular effects including clear cytotoxic effects around this concentration range (IC₅₀≈50 μM). Among the seven FDA-approved food colorants, only erythrosine showed consistent PPI inhibitory activity in the sub-100 μM range, which might also explain (at least partially) why it also has the lowest approved acceptable daily intake (ADI) (0.1 mg/kg body weight/day). Among a number of xanthene structural analogs of erythrosine tested for activity, rose Bengal, a food colorant approved in Japan, showed similar, maybe even more pronounced, promiscuous inhibitory activity, whereas fluorescein was inactive and gallein, phloxine, and eosin were somewhat active in some of the assays. Copyright © 2011 Elsevier Inc. All rights reserved.

  1. Recognition Imaging with a DNA Aptamer

    PubMed Central

    Lin, Liyun; Wang, Hongda; Liu, Yan; Yan, Hao; Lindsay, Stuart

    2006-01-01

    We have used a DNA-aptamer tethered to an atomic force microscope probe to carry out recognition imaging of IgE molecules attached to a mica substrate. The recognition was efficient (∼90%) and specific, being blocked by injection of IgE molecules in solution, and not being interfered with by high concentrations of a second protein. The signal/noise ratio of the recognition signal was better than that obtained with antibodies, despite the fact that the average force required to break the aptamer-protein bonds was somewhat smaller. PMID:16513776

  2. The Post-polyketide Synthase Steps in iso-Migrastatin Biosynthesis Featuring Tailoring Enzymes with Broad Substrate Specificity

    PubMed Central

    Ma, Ming; Kwong, Thomas; Lim, Si-Kyu; Ju, Jianhua; Lohman, Jeremy R.; Shen, Ben

    2013-01-01

    The iso-migrastatin (iso-MGS) biosynthetic gene cluster from Streptomyces platensis NRRL 18993 consists of 11 genes, featuring an acyltransferase (AT)-less type I polyketide synthase (PKS) and three tailoring enzymes MgsIJK. Systematic inactivation of mgsIJK in S. platensis enabled us to (i) identify two nascent products (10 and 13) of the iso-MGS AT-less type I PKS, establishing an unprecedented novel feature for AT-less type I PKSs, and (ii) account for the formation of all known post-PKS biosynthetic intermediates (10-17) generated by the three tailoring enzymes MgsIJK, which possessed significant substrate promiscuities. PMID:23394593

  3. Structural Basis of Substrate Recognition in Human Nicotinamide N-Methyltransferase

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

    Peng, Yi; Sartini, Davide; Pozzi, Valentina

    2012-05-02

    Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide, pyridines, and other analogues using S-adenosyl-L-methionine as donor. NNMT plays a significant role in the regulation of metabolic pathways and is expressed at markedly high levels in several kinds of cancers, presenting it as a potential molecular target for cancer therapy. We have determined the crystal structure of human NNMT as a ternary complex bound to both the demethylated donor S-adenosyl-L-homocysteine and the acceptor substrate nicotinamide, to 2.7 {angstrom} resolution. These studies reveal the structural basis for nicotinamide binding and highlight several residues in the active site which may play roles inmore » nicotinamide recognition and NNMT catalysis. The functional importance of these residues was probed by mutagenesis. Of three residues near the nicotinamide's amide group, substitution of S201 and S213 had no effect on enzyme activity while replacement of D197 dramatically decreased activity. Substitutions of Y20, whose side chain hydroxyl interacts with both the nicotinamide aromatic ring and AdoHcy carboxylate, also compromised activity. Enzyme kinetics analysis revealed k{sub cat}/K{sub m} decreases of 2-3 orders of magnitude for the D197A and Y20A mutants, confirming the functional importance of these active site residues. The mutants exhibited substantially increased K{sub m} for both NCA and AdoMet and modestly decreased k{sub cat}. MD simulations revealed long-range conformational effects which provide an explanation for the large increase in K{sub m}(AdoMet) for the D197A mutant, which interacts directly only with nicotinamide in the ternary complex crystal structure.« less

  4. Ligand-binding specificity and promiscuity of the main lignocellulolytic enzyme families as revealed by active-site architecture analysis.

    PubMed

    Tian, Li; Liu, Shijia; Wang, Shuai; Wang, Lushan

    2016-03-24

    Biomass can be converted into sugars by a series of lignocellulolytic enzymes, which belong to the glycoside hydrolase (GH) families summarized in CAZy databases. Here, using a structural bioinformatics method, we analyzed the active site architecture of the main lignocellulolytic enzyme families. The aromatic amino acids Trp/Tyr and polar amino acids Glu/Asp/Asn/Gln/Arg occurred at higher frequencies in the active site architecture than in the whole enzyme structure. And the number of potential subsites was significantly different among different families. In the cellulase and xylanase families, the conserved amino acids in the active site architecture were mostly found at the -2 to +1 subsites, while in β-glucosidase they were mainly concentrated at the -1 subsite. Families with more conserved binding amino acid residues displayed strong selectivity for their ligands, while those with fewer conserved binding amino acid residues often exhibited promiscuity when recognizing ligands. Enzymes with different activities also tended to bind different hydroxyl oxygen atoms on the ligand. These results may help us to better understand the common and unique structural bases of enzyme-ligand recognition from different families and provide a theoretical basis for the functional evolution and rational design of major lignocellulolytic enzymes.

  5. The promiscuous protein binding ability of erythrosine B studied by metachromasy (metachromasia).

    PubMed

    Ganesan, Lakshmi; Buchwald, Peter

    2013-04-01

    The present study aims to elucidate aspects of the protein binding ability of erythrosine B (ErB), a poly-iodinated xanthene dye and an FDA-approved food colorant (FD&C Red No. 3), which we have identified recently as a promiscuous inhibitor of protein-protein interactions (PPIs) with a remarkably consistent median inhibitory concentration (IC50 ) in the 5- to 30-μM range. Because ErB exhibits metachromasy, that is, color change upon binding to several proteins, we exploited this property to quantify its binding to proteins such as bovine serum albumin (BSA) and CD40L (CD154) and to determine the corresponding binding constants (Kd ) and stoichiometry (nb ) using spectrophotometric methods. Binding was reversible, and the estimated affinities for both protein targets obtained here (Kd values of 14 and 20 μM for BSA and CD40L, respectively) were in good agreement with that expected from the PPI inhibitory activity of ErB. A stoichiometry greater than one was observed both for CD40L and BSA binding (nb of 5-6 and 8-9 for BSA and CD40L, respectively), indicating the possibility of nonspecific binding of the flat and rigid ErB molecule at multiple sites, which could explain the promiscuous PPI inhibitory activity if some of these overlap with the binding site of the protein partner and interfere with the binding. Copyright © 2013 John Wiley & Sons, Ltd.

  6. The Promiscuous Protein Binding Ability of Erythrosine B Studied by Metachromasy (Metachromasia)

    PubMed Central

    Ganesan, Lakshmi; Buchwald, Peter

    2013-01-01

    The present study aims to elucidate aspects of the protein binding ability of erythrosine B (ErB), a poly-iodinated xanthene dye and an FDA-approved food colorant (FD&C Red No. 3), which we have identified recently as a promiscuous inhibitor of protein–protein interactions (PPI) with a remarkably consistent median inhibitory concentration (IC50) in the 5–30 µM range. Because ErB exhibits metachromasy, i.e., color change upon binding to several proteins, we exploited this property to quantify its binding to proteins such as bovine serum albumin (BSA) and CD40L (CD154) and to determine the corresponding binding constants (Kd) and stoichiometry (nb) using spectrophotometric methods. Binding was reversible and the estimated affinities for both protein targets obtained here (Kd values of 14 and 20 µM for BSA and CD40L, respectively) were in good agreement with that expected from the protein–protein interaction (PPI) inhibitory activity of ErB. A stoichiometry greater than one was observed both for CD40L and BSA binding (nb of 5–6 and 8–9 for BSA and CD40L, respectively) indicating the possibility of nonspecific binding of the flat an rigid ErB molecule at multiple sites, which could explain the promiscuous PPI inhibitory activity if some of these overlap with the binding site of the protein partner and interfere with the binding. PMID:23456742

  7. Delineation of xenobiotic substrate sites in rat glutathione S-transferase M1-1

    PubMed Central

    Hearne, Jennifer L.; Colman, Roberta F.

    2005-01-01

    Glutathione S-transferases catalyze the conjugation of glutathione with endogenous and exogenous xenobiotics. Hu and Colman (1995) proposed that there are two distinct substrate sites in rat GST M1-1, a 1-chloro-2,4-dintrobenzene (CDNB) substrate site located in the vicinity of tyrosine-115, and a monobromobimane (mBBr) substrate site. To determine whether the mBBr substrate site is distinguishable from the CDNB substrate site, we tested S-(hydroxyethyl)bimane, a nonreactive derivative of mBBr, for its ability to compete kinetically with the substrates. We find that S-(hydroxyethyl)bimane is a competitive inhibitor (KI = 0.36 μM) when mBBr is used as substrate, but not when CDNB is used as substrate, demonstrating that these two sites are distinct. Using site-directed mutagenesis, we have localized the mBBr substrate site to an area midway through α-helix 4 (residues 90–114) and have identified residues that are important in the enzymatic reaction. Substitution of alanine at positions along α-helix 4 reveals that mutations at positions 103, 104, and 109 exhibit a greater perturbation of the enzymatic reaction with mBBr than with CDNB as substrate. Various other substitutions at positions 103 and 104 reveal that a hydrophobic residue is necessary at each of these positions to maintain optimal affinity of the enzyme for mBBr and preserve the secondary structure of the enzyme. Substitutions at position 109 indicate that this residue is important in the enzyme’s affinity for mBBr but has a minimal effect on Vmax. These results demonstrate that the promiscuity of rat GST M1-1 is in part due to at least two distinct substrate sites. PMID:16195544

  8. Delineation of xenobiotic substrate sites in rat glutathione S-transferase M1-1.

    PubMed

    Hearne, Jennifer L; Colman, Roberta F

    2005-10-01

    Glutathione S-transferases catalyze the conjugation of glutathione with endogenous and exogenous xenobiotics. Hu and Colman (1995) proposed that there are two distinct substrate sites in rat GST M1-1, a 1-chloro-2,4-dintrobenzene (CDNB) substrate site located in the vicinity of tyrosine-115, and a monobromobimane (mBBr) substrate site. To determine whether the mBBr substrate site is distinguishable from the CDNB substrate site, we tested S-(hydroxyethyl)bimane, a nonreactive derivative of mBBr, for its ability to compete kinetically with the substrates. We find that S-(hydroxyethyl)bimane is a competitive inhibitor (K(I) = 0.36 microM) when mBBr is used as substrate, but not when CDNB is used as substrate, demonstrating that these two sites are distinct. Using site-directed mutagenesis, we have localized the mBBr substrate site to an area midway through alpha-helix 4 (residues 90-114) and have identified residues that are important in the enzymatic reaction. Substitution of alanine at positions along alpha-helix 4 reveals that mutations at positions 103, 104, and 109 exhibit a greater perturbation of the enzymatic reaction with mBBr than with CDNB as substrate. Various other substitutions at positions 103 and 104 reveal that a hydrophobic residue is necessary at each of these positions to maintain optimal affinity of the enzyme for mBBr and preserve the secondary structure of the enzyme. Substitutions at position 109 indicate that this residue is important in the enzyme's affinity for mBBr but has a minimal effect on Vmax. These results demonstrate that the promiscuity of rat GST M1-1 is in part due to at least two distinct substrate sites.

  9. Mechanism of Substrate Recognition And PLP-Induced Conformational Changes in II-Diaminopimelate Aminotransferase From Arabidopsis Thaliana

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

    Watanabe, N.; Clay, M.D.; Belkum, M.J.van

    2009-05-26

    LL-Diaminopimelate aminotransferase (LL-DAP-AT), a pyridoxal phosphate (PLP)-dependent enzyme in the lysine biosynthetic pathways of plants and Chlamydia, is a potential target for the development of herbicides or antibiotics. This homodimeric enzyme converts L-tetrahydrodipicolinic acid (THDP) directly to LL-DAP using L-glutamate as the source of the amino group. Earlier, we described the 3D structures of native and malate-bound LL-DAP-AT from Arabidopsis thaliana (AtDAP-AT). Seven additional crystal structures of AtDAP-AT and its variants are reported here as part of an investigation into the mechanism of substrate recognition and catalysis. Two structures are of AtDAP-AT with reduced external aldimine analogues: N-(5'-phosphopyridoxyl)-L-glutamate (PLP-Glu) andmore » N-(5'-phosphopyridoxyl)- LL-Diaminopimelate (PLP-DAP) bound in the active site. Surprisingly, they reveal that both L-glutamate and LL-DAP are recognized in a very similar fashion by the same sets of amino acid residues; both molecules adopt twisted V-shaped conformations. With both substrates, the {alpha}-carboxylates are bound in a salt bridge with Arg404, whereas the distal carboxylates are recognized via hydrogen bonds to the well-conserved side chains of Tyr37, Tyr125 and Lys129. The distal C{sup {var_epsilon}} amino group of LL-DAP is specifically recognized by several non-covalent interactions with residues from the other subunit (Asn309*, Tyr94*, Gly95*, and Glu97* (Amino acid designators followed by an asterisk (*) indicate that the residues originate in the other subunit of the dimer)) and by three bound water molecules. Two catalytically inactive variants of AtDAP-AT were created via site-directed mutagenesis of the active site lysine (K270N and K270Q). The structures of these variants permitted the observation of the unreduced external aldimines of PLP with L-glutamate and with LL-DAP in the active site, and revealed differences in the torsion angle about the PLP-substrate bond. Lastly

  10. Structure and molecular dynamics simulation of archaeal prefoldin: the molecular mechanism for binding and recognition of nonnative substrate proteins.

    PubMed

    Ohtaki, Akashi; Kida, Hiroshi; Miyata, Yusuke; Ide, Naoki; Yonezawa, Akihiro; Arakawa, Takatoshi; Iizuka, Ryo; Noguchi, Keiichi; Kita, Akiko; Odaka, Masafumi; Miki, Kunio; Yohda, Masafumi

    2008-02-29

    Prefoldin (PFD) is a heterohexameric molecular chaperone complex in the eukaryotic cytosol and archaea with a jellyfish-like structure containing six long coiled-coil tentacles. PFDs capture protein folding intermediates or unfolded polypeptides and transfer them to group II chaperonins for facilitated folding. Although detailed studies on the mechanisms for interaction with unfolded proteins or cooperation with chaperonins of archaeal PFD have been performed, it is still unclear how PFD captures the unfolded protein. In this study, we determined the X-ray structure of Pyrococcus horikoshii OT3 PFD (PhPFD) at 3.0 A resolution and examined the molecular mechanism for binding and recognition of nonnative substrate proteins by molecular dynamics (MD) simulation and mutation analyses. PhPFD has a jellyfish-like structure with six long coiled-coil tentacles and a large central cavity. Each subunit has a hydrophobic groove at the distal region where an unfolded substrate protein is bound. During MD simulation at 330 K, each coiled coil was highly flexible, enabling it to widen its central cavity and capture various nonnative proteins. Docking MD simulation of PhPFD with unfolded insulin showed that the beta subunit is essentially involved in substrate binding and that the alpha subunit modulates the shape and width of the central cavity. Analyses of mutant PhPFDs with amino acid replacement of the hydrophobic residues of the beta subunit in the hydrophobic groove have shown that beta Ile107 has a critical role in forming the hydrophobic groove.

  11. Galactokinase promiscuity: a question of flexibility?

    PubMed

    McAuley, Megan; Kristiansson, Helena; Huang, Meilan; Pey, Angel L; Timson, David J

    2016-02-01

    Galactokinase catalyses the first committed step of the Leloir pathway, i.e. the ATP-dependent phosphorylation of α-D-galactose at C1-OH. Reduced galactokinase activity results in the inherited metabolic disease type II galactosaemia. However, inhibition of galactokinase is considered a viable approach to treating more severe forms of galactosaemia (types I and III). Considerable progress has been made in the identification of high affinity, selective inhibitors. Although the structure of galactokinase from a variety of species is known, its catalytic mechanism remains uncertain. Although the bulk of evidence suggests that the reaction proceeds via an active site base mechanism, some experimental and theoretical studies contradict this. The enzyme has potential as a biocatalyst in the production of sugar 1-phosphates. This potential is limited by its high specificity. A variety of approaches have been taken to identify galactokinase variants which are more promiscuous. These have broadened galactokinase's specificity to include a wide range of D- and L-sugars. Initial studies suggest that some of these alterations result in increased flexibility at the active site. It is suggested that modulation of protein flexibility is at least as important as structural modifications in determining the success or failure of enzyme engineering. © 2016 Authors; published by Portland Press Limited.

  12. Rhizobial Diversity and Nodulation Characteristics of the Extremely Promiscuous Legume Sophora flavescens.

    PubMed

    Jiao, Yin Shan; Liu, Yuan Hui; Yan, Hui; Wang, En Tao; Tian, Chang Fu; Chen, Wen Xin; Guo, Bao Lin; Chen, Wen Feng

    2015-12-01

    In present study, we report our extensive survey on the diversity and biogeography of rhizobia associated with Sophora flavescens, a sophocarpidine (matrine)-containing medicinal legume. We additionally investigated the cross nodulation, infection pattern, light and electron microscopies of root nodule sections of S. flavescens infected by various rhizobia. Seventeen genospecies of rhizobia belonging to five genera with seven types of symbiotic nodC genes were found to nodulate S. flavescens in natural soils. In the cross-nodulation tests, most representative rhizobia in class α-Proteobacteria, whose host plants belong to different cross-nodulation groups, form effective indeterminate nodules, while representative rhizobia in class β-Proteobacteria form ineffective nodules on S. flavescens. Highly host-specific biovars of Rhizobium leguminosarum (bv. trifolii and bv. viciae) and Rhizobium etli bv. phaseoli could establish symbioses with S. flavescens, providing further evidence that S. flavescens is an extremely promiscuous legume and it does not have strict selectivity on either the symbiotic genes or the species-determining housekeeping genes of rhizobia. Root-hair infection is found as the pattern that rhizobia have gained entry into the curled root hairs. Electron microscopies of ultra-thin sections of S. flavescens root nodules formed by different rhizobia show that the bacteroids are regular or irregular rod shape and nonswollen types. Some bacteroids contain poly-β-hydroxybutyrate (PHB), while others do not, indicating the synthesis of PHB in bacteroids is rhizobia-dependent. The extremely promiscuous symbiosis between S. flavescens and different rhizobia provide us a basis for future studies aimed at understanding the molecular interactions of rhizobia and legumes.

  13. PAINS in the Assay: Chemical Mechanisms of Assay Interference and Promiscuous Enzymatic Inhibition Observed during a Sulfhydryl-Scavenging HTS

    PubMed Central

    2015-01-01

    Significant resources in early drug discovery are spent unknowingly pursuing artifacts and promiscuous bioactive compounds, while understanding the chemical basis for these adverse behaviors often goes unexplored in pursuit of lead compounds. Nearly all the hits from our recent sulfhydryl-scavenging high-throughput screen (HTS) targeting the histone acetyltransferase Rtt109 were such compounds. Herein, we characterize the chemical basis for assay interference and promiscuous enzymatic inhibition for several prominent chemotypes identified by this HTS, including some pan-assay interference compounds (PAINS). Protein mass spectrometry and ALARM NMR confirmed these compounds react covalently with cysteines on multiple proteins. Unfortunately, compounds containing these chemotypes have been published as screening actives in reputable journals and even touted as chemical probes or preclinical candidates. Our detailed characterization and identification of such thiol-reactive chemotypes should accelerate triage of nuisance compounds, guide screening library design, and prevent follow-up on undesirable chemical matter. PMID:25634295

  14. The Calcineurin Signaling Network Evolves Via Conserved Kinase–Phosphatase Modules That Transcend Substrate Identity

    PubMed Central

    Bodenmiller, Bernd; Wanka, Stefanie; Landry, Christian R.; Aebersold, Ruedi; Cyert, Martha S.

    2014-01-01

    Summary To define the first functional network for calcineurin, the conserved Ca2+/calmodulin-regulated phosphatase, we systematically identified its substrates in S. cerevisiae using phosphoproteomics and bioinformatics, followed by co-purification and dephosphorylation assays. This study establishes new calcineurin functions and reveals mechanisms that shape calcineurin network evolution. Analyses of closely related yeasts show that many proteins were recently recruited to the network by acquiring a calcineurin-recognition motif. Calcineurin substrates in yeast and mammals are distinct due to network rewiring but surprisingly are phosphorylated by similar kinases. We postulate that co-recognition of conserved substrate features, including phosphorylation and docking motifs, preserves calcineurin-kinase opposition during evolution. One example we document is a composite docking site that confers substrate recognition by both calcineurin and MAPK. We propose that conserved kinase-phosphatase pairs define the architecture of signaling networks and allow other connections between kinases and phosphatases to develop and establish common regulatory motifs in signaling networks. PMID:24930733

  15. Beyond perceptual expertise: revisiting the neural substrates of expert object recognition

    PubMed Central

    Harel, Assaf; Kravitz, Dwight; Baker, Chris I.

    2013-01-01

    Real-world expertise provides a valuable opportunity to understand how experience shapes human behavior and neural function. In the visual domain, the study of expert object recognition, such as in car enthusiasts or bird watchers, has produced a large, growing, and often-controversial literature. Here, we synthesize this literature, focusing primarily on results from functional brain imaging, and propose an interactive framework that incorporates the impact of high-level factors, such as attention and conceptual knowledge, in supporting expertise. This framework contrasts with the perceptual view of object expertise that has concentrated largely on stimulus-driven processing in visual cortex. One prominent version of this perceptual account has almost exclusively focused on the relation of expertise to face processing and, in terms of the neural substrates, has centered on face-selective cortical regions such as the Fusiform Face Area (FFA). We discuss the limitations of this face-centric approach as well as the more general perceptual view, and highlight that expert related activity is: (i) found throughout visual cortex, not just FFA, with a strong relationship between neural response and behavioral expertise even in the earliest stages of visual processing, (ii) found outside visual cortex in areas such as parietal and prefrontal cortices, and (iii) modulated by the attentional engagement of the observer suggesting that it is neither automatic nor driven solely by stimulus properties. These findings strongly support a framework in which object expertise emerges from extensive interactions within and between the visual system and other cognitive systems, resulting in widespread, distributed patterns of expertise-related activity across the entire cortex. PMID:24409134

  16. Structure and Function of the 26S Proteasome.

    PubMed

    Bard, Jared A M; Goodall, Ellen A; Greene, Eric R; Jonsson, Erik; Dong, Ken C; Martin, Andreas

    2018-06-20

    As the endpoint for the ubiquitin-proteasome system, the 26S proteasome is the principal proteolytic machine responsible for regulated protein degradation in eukaryotic cells. The proteasome's cellular functions range from general protein homeostasis and stress response to the control of vital processes such as cell division and signal transduction. To reliably process all the proteins presented to it in the complex cellular environment, the proteasome must combine high promiscuity with exceptional substrate selectivity. Recent structural and biochemical studies have shed new light on the many steps involved in proteasomal substrate processing, including recognition, deubiquitination, and ATP-driven translocation and unfolding. In addition, these studies revealed a complex conformational landscape that ensures proper substrate selection before the proteasome commits to processive degradation. These advances in our understanding of the proteasome's intricate machinery set the stage for future studies on how the proteasome functions as a major regulator of the eukaryotic proteome.

  17. Cleavage Entropy as Quantitative Measure of Protease Specificity

    PubMed Central

    Fuchs, Julian E.; von Grafenstein, Susanne; Huber, Roland G.; Margreiter, Michael A.; Spitzer, Gudrun M.; Wallnoefer, Hannes G.; Liedl, Klaus R.

    2013-01-01

    A purely information theory-guided approach to quantitatively characterize protease specificity is established. We calculate an entropy value for each protease subpocket based on sequences of cleaved substrates extracted from the MEROPS database. We compare our results with known subpocket specificity profiles for individual proteases and protease groups (e.g. serine proteases, metallo proteases) and reflect them quantitatively. Summation of subpocket-wise cleavage entropy contributions yields a measure for overall protease substrate specificity. This total cleavage entropy allows ranking of different proteases with respect to their specificity, separating unspecific digestive enzymes showing high total cleavage entropy from specific proteases involved in signaling cascades. The development of a quantitative cleavage entropy score allows an unbiased comparison of subpocket-wise and overall protease specificity. Thus, it enables assessment of relative importance of physicochemical and structural descriptors in protease recognition. We present an exemplary application of cleavage entropy in tracing substrate specificity in protease evolution. This highlights the wide range of substrate promiscuity within homologue proteases and hence the heavy impact of a limited number of mutations on individual substrate specificity. PMID:23637583

  18. The “gating” residues Ile199 and Tyr326 in human monoamine oxidase B function in substrate and inhibitor recognition

    PubMed Central

    Milczek, Erika M.; Binda, Claudia; Rovida, Stefano; Mattevi, Andrea; Edmondson, Dale E.

    2011-01-01

    Summary The major structural difference between human monoamine oxidases A (MAO A) and B (MAO B) is that MAO A has a monopartite substrate cavity of ~550 Å3 volume and MAO B contains a dipartite cavity structure with volumes of ~290 Å3 (entrance cavity) and ~400 Å3 (substrate cavity). Ile199 and Tyr326 side chains separate these two cavities in MAO B. To probe the function of these gating residues, Ile199Ala and Ile199Ala Tyr326Ala mutant forms of MAO B were investigated. Structural data on the Ile199Ala MAO B mutant show no alterations in active site geometries compared to WT enzyme while the Ile199Ala-Tyr326Ala MAO B mutant exhibits alterations in residues 100–103 which are part of the loop gating the entrance to the active site. Both mutant enzymes exhibit catalytic properties with increased amine KM but unaltered kcat values. The altered KM values on mutation are attributed to the influence of the cavity structure in the binding and subsequent deprotonation of the amine substrate. Both mutant enzymes exhibit weaker binding affinities relative to WT enzyme for small reversible inhibitors. Ile199Ala MAO B exhibits an increase in binding affinity for reversible MAO B specific inhibitors which bridge both cavities. The Ile199Ala-Tyr326Ala double mutant exhibits inhibitor binding properties more similar to those of MAO A than to MAO B. These results demonstrate the bipartite cavity structure in MAO B plays an important role in substrate and inhibitor recognition to distinguish its specificities from those of MAO A and provides insights into specific reversible inhibitor design for these membrane-bound enzymes. PMID:21978362

  19. Structural basis of a histidine-DNA nicking/joining mechanism for gene transfer and promiscuous spread of antibiotic resistance

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

    Pluta, Radoslaw; Boer, D. Roeland; Lorenzo-Diaz, Fabian

    Relaxases are metal-dependent nucleases that break and join DNA for the initiation and completion of conjugative bacterial gene transfer. Conjugation is the main process through which antibiotic resistance spreads among bacteria, with multidrug-resistant staphylococci and streptococci infections posing major threats to human health. The MOB V family of relaxases accounts for approximately 85% of all relaxases found in Staphylococcus aureus isolates. Here, we present six structures of the MOB V relaxase MobM from the promiscuous plasmid pMV158 in complex with several origin of transfer DNA fragments. A combined structural, biochemical, and computational approach reveals that MobM follows a previously uncharacterizedmore » histidine/metal-dependent DNA processing mechanism, which involves the formation of a covalent phosphoramidate histidine-DNA adduct for cell-to-cell transfer. In conclusion, we discuss how the chemical features of the high-energy phosphorus-nitrogen bond shape the dominant position of MOB V histidine relaxases among small promiscuous plasmids and their preference toward Gram-positive bacteria.« less

  20. Structural basis of a histidine-DNA nicking/joining mechanism for gene transfer and promiscuous spread of antibiotic resistance

    DOE PAGES

    Pluta, Radoslaw; Boer, D. Roeland; Lorenzo-Diaz, Fabian; ...

    2017-07-24

    Relaxases are metal-dependent nucleases that break and join DNA for the initiation and completion of conjugative bacterial gene transfer. Conjugation is the main process through which antibiotic resistance spreads among bacteria, with multidrug-resistant staphylococci and streptococci infections posing major threats to human health. The MOB V family of relaxases accounts for approximately 85% of all relaxases found in Staphylococcus aureus isolates. Here, we present six structures of the MOB V relaxase MobM from the promiscuous plasmid pMV158 in complex with several origin of transfer DNA fragments. A combined structural, biochemical, and computational approach reveals that MobM follows a previously uncharacterizedmore » histidine/metal-dependent DNA processing mechanism, which involves the formation of a covalent phosphoramidate histidine-DNA adduct for cell-to-cell transfer. In conclusion, we discuss how the chemical features of the high-energy phosphorus-nitrogen bond shape the dominant position of MOB V histidine relaxases among small promiscuous plasmids and their preference toward Gram-positive bacteria.« less

  1. Associations between dopamine D4 receptor gene variation with both infidelity and sexual promiscuity.

    PubMed

    Garcia, Justin R; MacKillop, James; Aller, Edward L; Merriwether, Ann M; Wilson, David Sloan; Lum, J Koji

    2010-11-30

    Human sexual behavior is highly variable both within and between populations. While sex-related characteristics and sexual behavior are central to evolutionary theory (sexual selection), little is known about the genetic bases of individual variation in sexual behavior. The variable number tandem repeats (VNTR) polymorphism in exon III of the human dopamine D4 receptor gene (DRD4) has been correlated with an array of behavioral phenotypes and may be predicatively responsible for variation in motivating some sexual behaviors, particularly promiscuity and infidelity. We administered an anonymous survey on personal history of sexual behavior and intimate relationships to 181 young adults. We also collected buccal wash samples and genotyped the DRD4 VNTR. Here we show that individuals with at least one 7-repeat allele (7R+) report a greater categorical rate of promiscuous sexual behavior (i.e., having ever had a "one-night stand") and report a more than 50% increase in instances of sexual infidelity. DRD4 VNTR genotype varies considerably within and among populations and has been subject to relatively recent, local selective pressures. Individual differences in sexual behavior are likely partially mediated by individual genetic variation in genes coding for motivation and reward in the brain. Conceptualizing these findings in terms of r/K selection theory suggests a mechanism for selective pressure for and against the 7R+ genotype that may explain the considerable global allelic variation for this polymorphism.

  2. Associations between Dopamine D4 Receptor Gene Variation with Both Infidelity and Sexual Promiscuity

    PubMed Central

    Garcia, Justin R.; MacKillop, James; Aller, Edward L.; Merriwether, Ann M.; Wilson, David Sloan; Lum, J. Koji

    2010-01-01

    Background Human sexual behavior is highly variable both within and between populations. While sex-related characteristics and sexual behavior are central to evolutionary theory (sexual selection), little is known about the genetic bases of individual variation in sexual behavior. The variable number tandem repeats (VNTR) polymorphism in exon III of the human dopamine D4 receptor gene (DRD4) has been correlated with an array of behavioral phenotypes and may be predicatively responsible for variation in motivating some sexual behaviors, particularly promiscuity and infidelity. Methodology/Principal Findings We administered an anonymous survey on personal history of sexual behavior and intimate relationships to 181 young adults. We also collected buccal wash samples and genotyped the DRD4 VNTR. Here we show that individuals with at least one 7-repeat allele (7R+) report a greater categorical rate of promiscuous sexual behavior (i.e., having ever had a “one-night stand”) and report a more than 50% increase in instances of sexual infidelity. Conclusions/Significance DRD4 VNTR genotype varies considerably within and among populations and has been subject to relatively recent, local selective pressures. Individual differences in sexual behavior are likely partially mediated by individual genetic variation in genes coding for motivation and reward in the brain. Conceptualizing these findings in terms of r/K selection theory suggests a mechanism for selective pressure for and against the 7R+ genotype that may explain the considerable global allelic variation for this polymorphism. PMID:21152404

  3. Engineering a Promiscuous Tautomerase into a More Efficient Aldolase for Self-Condensations of Linear Aliphatic Aldehydes.

    PubMed

    Rahimi, Mehran; van der Meer, Jan-Ytzen; Geertsema, Edzard M; Poelarends, Gerrit J

    2017-07-18

    The enzyme 4-oxalocrotonate tautomerase (4-OT) from Pseudomonas putida mt-2 takes part in a catabolic pathway for aromatic hydrocarbons, where it catalyzes the conversion of 2hydroxyhexa-2,4-dienedioate into 2-oxohexa-3-enedioate. This tautomerase can also promiscuously catalyze carbon-carbon bond-forming reactions, including various types of aldol reactions, by using its amino-terminal proline as a key catalytic residue. Here, we used systematic mutagenesis to identify two hotspots in 4-OT (Met45 and Phe50) at which single mutations give marked improvements in aldolase activity for the self-condensation of propanal. Activity screening of a focused library in which these two hotspots were varied led to the discovery of a 4-OT variant (M45Y/F50V) with strongly enhanced aldolase activity in the self-condensation of linear aliphatic aldehydes, such as acetaldehyde, propanal, and butanal, to yield α,β-unsaturated aldehydes. With both propanal and benzaldehyde, this double mutant, unlike the previously constructed single mutant F50A, mainly catalyzes the self-condensation of propanal rather than the cross-condensation of propanal and benzaldehyde, thus indicating that it indeed has altered substrate specificity. This variant could serve as a template to create new biocatalysts that lack dehydration activity and possess further enhanced aldolase activity, thus enabling the efficient enzymatic self-coupling of aliphatic aldehydes. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  4. Simple and rapid mercury ion selective electrode based on 1-undecanethiol assembled Au substrate and its recognition mechanism.

    PubMed

    Li, Xian-Qing; Liang, Hai-Qing; Cao, Zhong; Xiao, Qing; Xiao, Zhong-Liang; Song, Liu-Bin; Chen, Dan; Wang, Fu-Liang

    2017-03-01

    A simple and rapid mercury ion selective electrode based on 1-undecanethiol (1-UDT) assembled Au substrate (Au/1-UDT) has been well constructed. 1-UDT was for the purpose of generating self-assembled monolayer on gold surface to recognize Hg 2+ in aqueous solution, which had a working concentration range of 1.0×10 -8 -1.0×10 -4 molL -1 , with a Nernst response slope of 28.83±0.4mV/-pC, a detection limit of 4.5×10 -9 molL -1 , and a good selectivity over the other tested cations. Also, the Au/1-UDT possessed good reproducibility, stability, and short response time. The recovery obtained for the determination of mercury ion in practical tremella samples was in the range of 99.8-103.4%. Combined electrochemical analysis and X-ray photoelectron spectroscopy (XPS) with quantum chemical computation, the probable recognition mechanism of the electrode for selective recognition of Hg 2+ has been investigated. The covalent bond formed between mercury and sulfur is stronger than the one between gold and sulfur and thus prevents the adsorption of 1-UDT molecules on the gold surface. The quantum chemical computation with density functional theory further demonstrates that the strong interaction between the mercury atom and the sulfur atom on the gold surface leads to the gold sulfur bond ruptured and the gold mercury metallophilic interaction. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Substrate Scope of O-Methyltransferase from Streptomyces peucetius for Biosynthesis of Diverse Natural Products Methoxides.

    PubMed

    Parajuli, Prakash; Pandey, Ramesh Prasad; Nguyen, Thi Huyen Trang; Dhakal, Dipesh; Sohng, Jae Kyung

    2018-04-01

    Methylation is a common post-modification reaction that is observed during the biosynthesis of secondary metabolites produced by plants and microorganisms. Based on the sequence information from Streptomyces peucetius ATCC27952, a putative O-methyltransferase (OMT) gene SpOMT7740 was polymerase chain reaction amplified and cloned into E. coli BL21 (DE3) host to test the substrate promiscuity and conduct functional characterization. In vitro and in vivo reaction assays were carried out over various classes of substrates: flavonoids (flavonol, flavones, and isoflavonoid), chalcones, anthraquinones, anthracyclines, and sterol molecules, and the applications in synthesizing diverse classes of O-methoxy natural products were also illustrated. SpOMT7740 catalyzed the O-methylation reaction to form various natural and non-natural O-methoxides, includes 7-hydroxy-8-O-methoxy flavone, 3-O-methoxy flavone, three mono-, di-, and tri-O-methoxy genistein, mono-O-methoxy phloretin, mono-O-methoxy luteolin, 3-O-methoxy β-sitosterol, and O-methoxy anthraquinones (emodin and aloe emodin) and O-methoxy anthracycline (daunorubicin) exhibiting diverse substrate flexibility. Daunorubicin is a native secondary metabolite of S. peucetius. Among the compounds tested, 7,8-dihydroxyflavone was the best substrate for bioconversion to 7-hydroxy-8-O-methoxy flavone, and it was structurally elucidated. This enzyme showed a flexible catalysis over the given ranges of temperature, pH, and divalent cationic conditions for O-methylation.

  6. Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1

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

    Rashid, Fahad; Harris, Paul D.; Zaher, Manal S.

    Human flap endonuclease 1 (FEN1) and related structure-specific 5’nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5’nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually ‘locks’ protein and DNA conformation and enables substrate verification with extreme precision. Strikingly, FEN1 never missesmore » cleavage of its cognate substrate while blocking probable formation of catalytically competent interactions with noncognate substrates and fostering their pre-incision dissociation. These findings establish FEN1 has practically perfect precision and that separate control of induced-fit substrate recognition sets up the catalytic selectivity of the nuclease active site for genome stability.« less

  7. Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1

    DOE PAGES

    Rashid, Fahad; Harris, Paul D.; Zaher, Manal S.; ...

    2017-02-23

    Human flap endonuclease 1 (FEN1) and related structure-specific 5’nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5’nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually ‘locks’ protein and DNA conformation and enables substrate verification with extreme precision. Strikingly, FEN1 never missesmore » cleavage of its cognate substrate while blocking probable formation of catalytically competent interactions with noncognate substrates and fostering their pre-incision dissociation. These findings establish FEN1 has practically perfect precision and that separate control of induced-fit substrate recognition sets up the catalytic selectivity of the nuclease active site for genome stability.« less

  8. Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1

    PubMed Central

    Rashid, Fahad; Harris, Paul D; Zaher, Manal S; Sobhy, Mohamed A; Joudeh, Luay I; Yan, Chunli; Piwonski, Hubert; Tsutakawa, Susan E; Ivanov, Ivaylo; Tainer, John A; Habuchi, Satoshi; Hamdan, Samir M

    2017-01-01

    Human flap endonuclease 1 (FEN1) and related structure-specific 5’nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5’nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually ‘locks’ protein and DNA conformation and enables substrate verification with extreme precision. Strikingly, FEN1 never misses cleavage of its cognate substrate while blocking probable formation of catalytically competent interactions with noncognate substrates and fostering their pre-incision dissociation. These findings establish FEN1 has practically perfect precision and that separate control of induced-fit substrate recognition sets up the catalytic selectivity of the nuclease active site for genome stability. DOI: http://dx.doi.org/10.7554/eLife.21884.001 PMID:28230529

  9. A promiscuous intermediate underlies the evolution of LEAFY DNA binding specificity.

    PubMed

    Sayou, Camille; Monniaux, Marie; Nanao, Max H; Moyroud, Edwige; Brockington, Samuel F; Thévenon, Emmanuel; Chahtane, Hicham; Warthmann, Norman; Melkonian, Michael; Zhang, Yong; Wong, Gane Ka-Shu; Weigel, Detlef; Parcy, François; Dumas, Renaud

    2014-02-07

    Transcription factors (TFs) are key players in evolution. Changes affecting their function can yield novel life forms but may also have deleterious effects. Consequently, gene duplication events that release one gene copy from selective pressure are thought to be the common mechanism by which TFs acquire new activities. Here, we show that LEAFY, a major regulator of flower development and cell division in land plants, underwent changes to its DNA binding specificity, even though plant genomes generally contain a single copy of the LEAFY gene. We examined how these changes occurred at the structural level and identify an intermediate LEAFY form in hornworts that appears to adopt all different specificities. This promiscuous intermediate could have smoothed the evolutionary transitions, thereby allowing LEAFY to evolve new binding specificities while remaining a single-copy gene.

  10. Design and isolation of ribozyme-substrate pairs using RNase P-based ribozymes containing altered substrate binding sites.

    PubMed Central

    Mobley, E M; Pan, T

    1999-01-01

    Substrate recognition and cleavage by the bacterial RNase P RNA requires two domains, a specificity domain, or S-domain, and a catalytic domain, or C-domain. The S-domain binds the T stem-loop region in a pre-tRNA substrate to confer specificity for tRNA substrates. In this work, the entire S-domain of the Bacillus subtilis RNase P RNA is replaced with an artificial substrate binding module. New RNA substrates are isolated by in vitro selection using two libraries containing random regions of 60 nt. At the end of the selection, the cleavage rates of the substrate library are approximately 0.7 min(-1)in 10 mM MgCl(2)at 37 degrees C, approximately 4-fold better than the cleavage of a pre-tRNA substrate by the wild-type RNase P RNA under the same conditions. The contribution of the S-domain replacement to the catalytic efficiency is from 6- to 22 000-fold. Chemical and nuclease mapping of two ribozyme-product complexes shows that this contribution correlates with direct interactions between the S-domain replacement and the selected substrate. These results demonstrate the feasibility of design and isolation of RNase P-based, matching ribozyme-substrate pairs without prior knowledge of the sequence or structure of the interactive modules in the ribozyme or substrate. PMID:10518624

  11. Promiscuity drives sexual selection in a socially monogamous bird.

    PubMed

    Webster, Michael S; Tarvin, Keith A; Tuttle, Elaina M; Pruett-Jones, Stephen

    2007-09-01

    Many socially monogamous species paradoxically show signs of strong sexual selection, suggesting cryptic sources of sexual competition among males. Darwin argued that sexual selection could operate in monogamous systems if breeding sex ratios are biased or if some males attract highly fecund females. Alternatively, sexual selection might result from promiscuous copulations outside the pair bond, although several recent studies have cast doubt on this possibility, in particular by showing that variance in apparent male reproductive success (number of social young) differs little from variance in actual male reproductive success (number of young sired). Our results from a long-term study of the socially monogamous splendid fairy-wren (Malurus splendens) demonstrate that such comparisons are misleading and do not adequately assess the effects of extra-pair paternity (EPP). By partitioning the opportunity for selection and calculating Bateman gradients, we show that EPP has a strong effect on male annual and lifetime fitness, whereas other proposed mechanisms of sexual selection do not. Thus, EPP drives sexual selection in this, and possibly other, socially monogamous species.

  12. Pirating conserved phage mechanisms promotes promiscuous staphylococcal pathogenicity island transfer.

    PubMed

    Bowring, Janine; Neamah, Maan M; Donderis, Jorge; Mir-Sanchis, Ignacio; Alite, Christian; Ciges-Tomas, J Rafael; Maiques, Elisa; Medmedov, Iltyar; Marina, Alberto; Penadés, José R

    2017-08-08

    Targeting conserved and essential processes is a successful strategy to combat enemies. Remarkably, the clinically important Staphylococcus aureus pathogenicity islands (SaPIs) use this tactic to spread in nature. SaPIs reside passively in the host chromosome, under the control of the SaPI-encoded master repressor, Stl. It has been assumed that SaPI de-repression is effected by specific phage proteins that bind to Stl, initiating the SaPI cycle. Different SaPIs encode different Stl repressors, so each targets a specific phage protein for its de-repression. Broadening this narrow vision, we report here that SaPIs ensure their promiscuous transfer by targeting conserved phage mechanisms. This is accomplished because the SaPI Stl repressors have acquired different domains to interact with unrelated proteins, encoded by different phages, but in all cases performing the same conserved function. This elegant strategy allows intra- and inter-generic SaPI transfer, highlighting these elements as one of nature's most fascinating subcellular parasites.

  13. Butterfly genome reveals promiscuous exchange of mimicry adaptations among species

    PubMed Central

    Dasmahapatra, Kanchon K; Walters, James R.; Briscoe, Adriana D.; Davey, John W.; Whibley, Annabel; Nadeau, Nicola J.; Zimin, Aleksey V.; Hughes, Daniel S. T.; Ferguson, Laura C.; Martin, Simon H.; Salazar, Camilo; Lewis, James J.; Adler, Sebastian; Ahn, Seung-Joon; Baker, Dean A.; Baxter, Simon W.; Chamberlain, Nicola L.; Chauhan, Ritika; Counterman, Brian A.; Dalmay, Tamas; Gilbert, Lawrence E.; Gordon, Karl; Heckel, David G.; Hines, Heather M.; Hoff, Katharina J.; Holland, Peter W.H.; Jacquin-Joly, Emmanuelle; Jiggins, Francis M.; Jones, Robert T.; Kapan, Durrell D.; Kersey, Paul; Lamas, Gerardo; Lawson, Daniel; Mapleson, Daniel; Maroja, Luana S.; Martin, Arnaud; Moxon, Simon; Palmer, William J.; Papa, Riccardo; Papanicolaou, Alexie; Pauchet, Yannick; Ray, David A.; Rosser, Neil; Salzberg, Steven L.; Supple, Megan A.; Surridge, Alison; Tenger-Trolander, Ayse; Vogel, Heiko; Wilkinson, Paul A.; Wilson, Derek; Yorke, James A.; Yuan, Furong; Balmuth, Alexi L.; Eland, Cathlene; Gharbi, Karim; Thomson, Marian; Gibbs, Richard A.; Han, Yi; Jayaseelan, Joy C.; Kovar, Christie; Mathew, Tittu; Muzny, Donna M.; Ongeri, Fiona; Pu, Ling-Ling; Qu, Jiaxin; Thornton, Rebecca L.; Worley, Kim C.; Wu, Yuan-Qing; Linares, Mauricio; Blaxter, Mark L.; Constant, Richard H. ffrench; Joron, Mathieu; Kronforst, Marcus R.; Mullen, Sean P.; Reed, Robert D.; Scherer, Steven E.; Richards, Stephen; Mallet, James; McMillan, W. Owen; Jiggins, Chris D.

    2012-01-01

    The evolutionary importance of hybridization and introgression has long been debated1. We used genomic tools to investigate introgression in Heliconius, a rapidly radiating genus of neotropical butterflies widely used in studies of ecology, behaviour, mimicry and speciation2-5 . We sequenced the genome of Heliconius melpomene and compared it with other taxa to investigate chromosomal evolution in Lepidoptera and gene flow among multiple Heliconius species and races. Among 12,657 predicted genes for Heliconius, biologically important expansions of families of chemosensory and Hox genes are particularly noteworthy. Chromosomal organisation has remained broadly conserved since the Cretaceous, when butterflies split from the silkmoth lineage. Using genomic resequencing, we show hybrid exchange of genes between three co-mimics, H. melpomene, H. timareta, and H. elevatus, especially at two genomic regions that control mimicry pattern. Closely related Heliconius species clearly exchange protective colour pattern genes promiscuously, implying a major role for hybridization in adaptive radiation. PMID:22722851

  14. Rpn1 provides adjacent receptor sites for substrate binding and deubiquitination by the proteasome

    PubMed Central

    Shi, Yuan; Chen, Xiang; Elsasser, Suzanne; Stocks, Bradley B.; Tian, Geng; Lee, Byung-Hoon; Shi, Yanhong; Zhang, Naixia; de Poot, Stefanie A. H.; Tuebing, Fabian; Sun, Shuangwu; Vannoy, Jacob; Tarasov, Sergey G.; Engen, John R.; Finley, Daniel; Walters, Kylie J.

    2016-01-01

    Structured Abstract INTRODUCTION The ubiquitin-proteasome system comprises hundreds of distinct pathways of degradation, which converge at the step of ubiquitin recognition by the proteasome. Five proteasomal ubiquitin receptors have been identified, two that are intrinsic to the proteasome (Rpn10 and Rpn13) and three reversibly associated proteasomal ubiquitin receptors (Rad23, Dsk2, and Ddi1). RATIONALE We found that the five known proteasomal ubiquitin receptors of yeast are collectively nonessential for ubiquitin recognition by the proteasome. We therefore screened for additional ubiquitin receptors in the proteasome and identified subunit Rpn1 as a candidate. We used nuclear magnetic resonance (NMR) spectroscopy to characterize the structure of the binding site within Rpn1, which we term the T1 site. Mutational analysis of this site showed its functional importance within the context of intact proteasomes. T1 binds both ubiquitin and ubiquitin-like (UBL) proteins, in particular the substrate-delivering shuttle factor Rad23. A second site within the Rpn1 toroid, T2, recognizes the UBL domain of deubiquitinating enzyme Ubp6, as determined by hydrogen-deuterium exchange mass spectrometry analysis and validated by amino acid substitution and functional assays. The Rpn1 toroid thus serves a critical scaffolding role within the proteasome, helping to assemble multiple proteasome cofactors as well as substrates. RESULTS Our results indicate that proteasome subunit Rpn1 can recognize both ubiquitin and UBL domains of substrate shuttling factors that themselves bind ubiquitin and function as reversibly-associated proteasomal ubiquitin receptors. Recognition is mediated by the T1 site within the Rpn1 toroid, which supports proteasome function in vivo. We found that the capacity of T1 to recognize both ubiquitin and UBL proteins was shared with Rpn10 and Rpn13. The surprising multiplicity of ubiquitin-recognition domains within the proteasome may promote enhanced

  15. Males and females gain differentially from sociality in a promiscuous fruit bat Cynopterus sphinx.

    PubMed

    Garg, Kritika M; Chattopadhyay, Balaji; Swami Doss, D P; Kumar, A K Vinoth; Kandula, Sripathi; Ramakrishnan, Uma

    2015-01-01

    Sociality emerges when the benefits of group living outweigh its costs. While both males and females are capable of strong social ties, the evolutionary drivers for sociality and the benefits accrued maybe different for each sex. In this study, we investigate the differential reproductive success benefits of group membership that males and females might obtain in the promiscuous fruit bat Cynopterus sphinx. Individuals of this species live in flexible social groups called colonies. These colonies are labile and there is high turnover of individuals. However, colony males sire more offspring within the colony suggesting that being part of a colony may result in reproductive benefits for males. This also raises the possibility that long-term loyalty towards the colony may confer additional advantage in terms of higher reproductive success. We used ten seasons of genetic parentage data to estimate reproductive success and relatedness of individuals in the colony. We used recapture data to identify long and short-term residents in the colony as well as to obtain rates of recapture for males and females. Our results reveal that males have a significantly higher chance of becoming long-term residents (than females), and these long-term resident males gain twice the reproductive success compared to short-term resident males. We also observed that long-term resident females are related to each other and also achieve higher reproductive success than short-term resident females. In contrast, long-term resident males do not differ from short-term resident males in their levels of relatedness. Our results re-iterate the benefits of sociality even in species that are promiscuous and socially labile and possible benefits of maintaining a colony.

  16. Molecular recognition in protein modification with rhodium metallopeptides

    PubMed Central

    Ball, Zachary T.

    2015-01-01

    Chemical manipulation of natural, unengineered proteins is a daunting challenge which tests the limits of reaction design. By combining transition-metal or other catalysts with molecular recognition ideas, it is possible to achieve site-selective protein reactivity without the need for engineered recognition sequences or reactive sites. Some recent examples in this area have used ruthenium photocatalysis, pyridine organocatalysis, and rhodium(II) metallocarbene catalysis, indicating that the fundamental ideas provide opportunities for using diverse reactivity on complex protein substrates and in complex cell-like environments. PMID:25588960

  17. Legume-rhizobia signal exchange: promiscuity and environmental effects.

    PubMed

    Lira, Mario A; Nascimento, Luciana R S; Fracetto, Giselle G M

    2015-01-01

    Although signal exchange between legumes and their rhizobia is among the best-known examples of this biological process, most of the more characterized data comes from just a few legume species and environmental stresses. Although a relative wealth of information is available for some model legumes and some of the major pulses such as soybean, little is known about tropical legumes. This relative disparity in current knowledge is also apparent in the research on the effects of environmental stress on signal exchange; cool-climate stresses, such as low-soil temperature, comprise a relatively large body of research, whereas high-temperature stresses and drought are not nearly as well understood. Both tropical legumes and their environmental stress-induced effects are increasingly important due to global population growth (the demand for protein), climate change (increasing temperatures and more extreme climate behavior), and urbanization (and thus heavy metals). This knowledge gap for both legumes and their environmental stresses is compounded because whereas most temperate legume-rhizobia symbioses are relatively specific and cultivated under relatively stable environments, the converse is true for tropical legumes, which tend to be promiscuous, and grow in highly variable conditions. This review will clarify some of this missing information and highlight fields in which further research would benefit our current knowledge.

  18. Phytochemicals perturb membranes and promiscuously alter protein function.

    PubMed

    Ingólfsson, Helgi I; Thakur, Pratima; Herold, Karl F; Hobart, E Ashley; Ramsey, Nicole B; Periole, Xavier; de Jong, Djurre H; Zwama, Martijn; Yilmaz, Duygu; Hall, Katherine; Maretzky, Thorsten; Hemmings, Hugh C; Blobel, Carl; Marrink, Siewert J; Koçer, Armağan; Sack, Jon T; Andersen, Olaf S

    2014-08-15

    A wide variety of phytochemicals are consumed for their perceived health benefits. Many of these phytochemicals have been found to alter numerous cell functions, but the mechanisms underlying their biological activity tend to be poorly understood. Phenolic phytochemicals are particularly promiscuous modifiers of membrane protein function, suggesting that some of their actions may be due to a common, membrane bilayer-mediated mechanism. To test whether bilayer perturbation may underlie this diversity of actions, we examined five bioactive phenols reported to have medicinal value: capsaicin from chili peppers, curcumin from turmeric, EGCG from green tea, genistein from soybeans, and resveratrol from grapes. We find that each of these widely consumed phytochemicals alters lipid bilayer properties and the function of diverse membrane proteins. Molecular dynamics simulations show that these phytochemicals modify bilayer properties by localizing to the bilayer/solution interface. Bilayer-modifying propensity was verified using a gramicidin-based assay, and indiscriminate modulation of membrane protein function was demonstrated using four proteins: membrane-anchored metalloproteases, mechanosensitive ion channels, and voltage-dependent potassium and sodium channels. Each protein exhibited similar responses to multiple phytochemicals, consistent with a common, bilayer-mediated mechanism. Our results suggest that many effects of amphiphilic phytochemicals are due to cell membrane perturbations, rather than specific protein binding.

  19. Phytochemicals Perturb Membranes and Promiscuously Alter Protein Function

    PubMed Central

    2015-01-01

    A wide variety of phytochemicals are consumed for their perceived health benefits. Many of these phytochemicals have been found to alter numerous cell functions, but the mechanisms underlying their biological activity tend to be poorly understood. Phenolic phytochemicals are particularly promiscuous modifiers of membrane protein function, suggesting that some of their actions may be due to a common, membrane bilayer-mediated mechanism. To test whether bilayer perturbation may underlie this diversity of actions, we examined five bioactive phenols reported to have medicinal value: capsaicin from chili peppers, curcumin from turmeric, EGCG from green tea, genistein from soybeans, and resveratrol from grapes. We find that each of these widely consumed phytochemicals alters lipid bilayer properties and the function of diverse membrane proteins. Molecular dynamics simulations show that these phytochemicals modify bilayer properties by localizing to the bilayer/solution interface. Bilayer-modifying propensity was verified using a gramicidin-based assay, and indiscriminate modulation of membrane protein function was demonstrated using four proteins: membrane-anchored metalloproteases, mechanosensitive ion channels, and voltage-dependent potassium and sodium channels. Each protein exhibited similar responses to multiple phytochemicals, consistent with a common, bilayer-mediated mechanism. Our results suggest that many effects of amphiphilic phytochemicals are due to cell membrane perturbations, rather than specific protein binding. PMID:24901212

  20. Beyond promiscuity: mate-choice commitments in social breeding

    PubMed Central

    Boomsma, Jacobus J.

    2013-01-01

    Obligate eusociality with distinct caste phenotypes has evolved from strictly monogamous sub-social ancestors in ants, some bees, some wasps and some termites. This implies that no lineage reached the most advanced form of social breeding, unless helpers at the nest gained indirect fitness values via siblings that were identical to direct fitness via offspring. The complete lack of re-mating promiscuity equalizes sex-specific variances in reproductive success. Later, evolutionary developments towards multiple queen-mating retained lifetime commitment between sexual partners, but reduced male variance in reproductive success relative to female's, similar to the most advanced vertebrate cooperative breeders. Here, I (i) discuss some of the unique and highly peculiar mating system adaptations of eusocial insects; (ii) address ambiguities that remained after earlier reviews and extend the monogamy logic to the evolution of soldier castes; (iii) evaluate the evidence for indirect fitness benefits driving the dynamics of (in)vertebrate cooperative breeding, while emphasizing the fundamental differences between obligate eusociality and cooperative breeding; (iv) infer that lifetime commitment is a major driver towards higher levels of organization in bodies, colonies and mutualisms. I argue that evolutionary informative definitions of social systems that separate direct and indirect fitness benefits facilitate transparency when testing inclusive fitness theory. PMID:23339241

  1. Substrate-Tuned Catalysis of the Radical S-Adenosyl-L-Methionine Enzyme NosL Involved in Nosiheptide Biosynthesis.

    PubMed

    Ji, Xinjian; Li, Yongzhen; Ding, Wei; Zhang, Qi

    2015-07-27

    NosL is a radical S-adenosyl-L-methionine (SAM) enzyme that converts L-Trp to 3-methyl-2-indolic acid, a key intermediate in the biosynthesis of a thiopeptide antibiotic nosiheptide. In this work we investigated NosL catalysis by using a series of Trp analogues as the molecular probes. Using a benzofuran substrate 2-amino-3-(benzofuran-3-yl)propanoic acid (ABPA), we clearly demonstrated that the 5'-deoxyadenosyl (dAdo) radical-mediated hydrogen abstraction in NosL catalysis is not from the indole nitrogen but likely from the amino group of L-Trp. Unexpectedly, the major product of ABPA is a decarboxylated compound, indicating that NosL was transformed to a novel decarboxylase by an unnatural substrate. Furthermore, we showed that, for the first time to our knowledge, the dAdo radical-mediated hydrogen abstraction can occur from an alcohol hydroxy group. Our study demonstrates the intriguing promiscuity of NosL catalysis and highlights the potential of engineering radical SAM enzymes for novel activities. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Substrate degradation by the proteasome: a single-molecule kinetic analysis

    PubMed Central

    Lu, Ying; Lee, Byung-hoon; King, Randall W; Finley, Daniel; Kirschner, Marc W

    2015-01-01

    To address how the configuration of conjugated ubiquitins determines the recognition of substrates by the proteasome, we analyzed the degradation kinetics of substrates with chemically defined ubiquitin configurations. Contrary to the view that a tetraubiquitin chain is the minimal signal for efficient degradation, we find that distributing the ubiquitins as diubiquitin chains provides a more efficient signal. To understand how the proteasome actually discriminates among ubiquitin configurations, we developed single-molecule assays that distinguished intermediate steps of degradation kinetically. The level of ubiquitin on a substrate drives proteasome-substrate interaction, whereas the chain structure of ubiquitin affects translocation into the axial channel on the proteasome. Together these two features largely determine the susceptibility of substrates for proteasomal degradation. PMID:25859050

  3. The structural neuroanatomy of music emotion recognition: Evidence from frontotemporal lobar degeneration

    PubMed Central

    Omar, Rohani; Henley, Susie M.D.; Bartlett, Jonathan W.; Hailstone, Julia C.; Gordon, Elizabeth; Sauter, Disa A.; Frost, Chris; Scott, Sophie K.; Warren, Jason D.

    2011-01-01

    Despite growing clinical and neurobiological interest in the brain mechanisms that process emotion in music, these mechanisms remain incompletely understood. Patients with frontotemporal lobar degeneration (FTLD) frequently exhibit clinical syndromes that illustrate the effects of breakdown in emotional and social functioning. Here we investigated the neuroanatomical substrate for recognition of musical emotion in a cohort of 26 patients with FTLD (16 with behavioural variant frontotemporal dementia, bvFTD, 10 with semantic dementia, SemD) using voxel-based morphometry. On neuropsychological evaluation, patients with FTLD showed deficient recognition of canonical emotions (happiness, sadness, anger and fear) from music as well as faces and voices compared with healthy control subjects. Impaired recognition of emotions from music was specifically associated with grey matter loss in a distributed cerebral network including insula, orbitofrontal cortex, anterior cingulate and medial prefrontal cortex, anterior temporal and more posterior temporal and parietal cortices, amygdala and the subcortical mesolimbic system. This network constitutes an essential brain substrate for recognition of musical emotion that overlaps with brain regions previously implicated in coding emotional value, behavioural context, conceptual knowledge and theory of mind. Musical emotion recognition may probe the interface of these processes, delineating a profile of brain damage that is essential for the abstraction of complex social emotions. PMID:21385617

  4. Parents' Divorce Is More Strongly Related to the Self-Perceived Promiscuity and Drinking Behavior of Male than of Female College Students

    ERIC Educational Resources Information Center

    Stringfellow, Erica L.; McAndrew, Francis T.

    2010-01-01

    A study of 357 students (112 males, 245 females) responding to an online survey at a Midwestern liberal arts college revealed that males and children from divorced families perceived themselves as more promiscuous and drank more than did students from intact families. However, a significant interaction between the gender of the students and the…

  5. Helix-length compensation studies reveal the adaptability of the VS ribozyme architecture.

    PubMed

    Lacroix-Labonté, Julie; Girard, Nicolas; Lemieux, Sébastien; Legault, Pascale

    2012-03-01

    Compensatory mutations in RNA are generally regarded as those that maintain base pairing, and their identification forms the basis of phylogenetic predictions of RNA secondary structure. However, other types of compensatory mutations can provide higher-order structural and evolutionary information. Here, we present a helix-length compensation study for investigating structure-function relationships in RNA. The approach is demonstrated for stem-loop I and stem-loop V of the Neurospora VS ribozyme, which form a kissing-loop interaction important for substrate recognition. To rapidly characterize the substrate specificity (k(cat)/K(M)) of several substrate/ribozyme pairs, a procedure was established for simultaneous kinetic characterization of multiple substrates. Several active substrate/ribozyme pairs were identified, indicating the presence of limited substrate promiscuity for stem Ib variants and helix-length compensation between stems Ib and V. 3D models of the I/V interaction were generated that are compatible with the kinetic data. These models further illustrate the adaptability of the VS ribozyme architecture for substrate cleavage and provide global structural information on the I/V kissing-loop interaction. By exploring higher-order compensatory mutations in RNA our approach brings a deeper understanding of the adaptability of RNA structure, while opening new avenues for RNA research.

  6. Structural basis for the substrate selectivity of a HAD phosphatase from Thermococcus onnurineus NA1.

    PubMed

    Ngo, Tri Duc; Van Le, Binh; Subramani, Vinod Kumar; Thi Nguyen, Chi My; Lee, Hyun Sook; Cho, Yona; Kim, Kyeong Kyu; Hwang, Hye-Yeon

    2015-05-22

    Proteins in the haloalkaloic acid dehalogenase (HAD) superfamily, which is one of the largest enzyme families, is generally composed of a catalytic core domain and a cap domain. Although proteins in this family show broad substrate specificities, the mechanisms of their substrate recognition are not well understood. In this study, we identified a new substrate binding motif of HAD proteins from structural and functional analyses, and propose that this motif might be crucial for interacting with hydrophobic rings of substrates. The crystal structure of TON_0338, one of the 17 putative HAD proteins identified in a hyperthermophilic archaeon, Thermococcus onnurineus NA1, was determined as an apo-form at 2.0 Å resolution. In addition, we determined the crystal structure TON_0338 in complex with Mg(2+) or N-cyclohexyl-2-aminoethanesulfonic acid (CHES) at 1.7 Å resolution. Examination of the apo-form and CHES-bound structures revealed that CHES is sandwiched between Trp58 and Trp61, suggesting that this Trp sandwich might function as a substrate recognition motif. In the phosphatase assay, TON_0338 was shown to have high activity for flavin mononucleotide (FMN), and the docking analysis suggested that the flavin of FMN may interact with Trp58 and Trp61 in a way similar to that observed in the crystal structure. Moreover, the replacement of these tryptophan residues significantly reduced the phosphatase activity for FMN. Our results suggest that WxxW may function as a substrate binding motif in HAD proteins, and expand the diversity of their substrate recognition mode. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Emotion recognition impairment and apathy after subthalamic nucleus stimulation in Parkinson's disease have separate neural substrates.

    PubMed

    Drapier, D; Péron, J; Leray, E; Sauleau, P; Biseul, I; Drapier, S; Le Jeune, F; Travers, D; Bourguignon, A; Haegelen, C; Millet, B; Vérin, M

    2008-09-01

    To test the hypothesis that emotion recognition and apathy share the same functional circuit involving the subthalamic nucleus (STN). A consecutive series of 17 patients with advanced Parkinson's disease (PD) was assessed 3 months before (M-3) and 3 months (M+3) after STN deep brain stimulation (DBS). Mean (+/-S.D.) age at surgery was 56.9 (8.7) years. Mean disease duration at surgery was 11.8 (2.6) years. Apathy was measured using the Apathy Evaluation Scale (AES) at both M-3 and M3. Patients were also assessed using a computerised paradigm of facial emotion recognition [Ekman, P., & Friesen, W. V. (1976). Pictures of facial affect. Palo Alto: Consulting Psychologist Press] before and after STN DBS. Prior to this, the Benton Facial Recognition Test was used to check that the ability to perceive faces was intact. Apathy had significantly worsened at M3 (42.5+/-8.9, p=0.006) after STN-DBS, in relation to the preoperative assessment (37.2+/-5.5). There was also a significant reduction in recognition percentages for facial expressions of fear (43.1%+/-22.9 vs. 61.6%+/-21.4, p=0.022) and sadness (52.7%+/-19.1 vs. 67.6%+/-22.8, p=0.031) after STN DBS. However, the postoperative worsening of apathy and emotion recognition impairment were not correlated. Our results confirm that the STN is involved in both the apathy and emotion recognition networks. However, the absence of any correlation between apathy and emotion recognition impairment suggests that the worsening of apathy following surgery could not be explained by a lack of facial emotion recognition and that its behavioural and cognitive components should therefore also be taken into consideration.

  8. HIV-1 protease-substrate coevolution in nelfinavir resistance.

    PubMed

    Kolli, Madhavi; Ozen, Ayşegül; Kurt-Yilmaz, Nese; Schiffer, Celia A

    2014-07-01

    Resistance to various human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. The virus accumulates mutations within the protease (PR) that render the PIs less potent. Occasionally, Gag sequences also coevolve with mutations at PR cleavage sites contributing to drug resistance. In this study, we investigated the structural basis of coevolution of the p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations by determining crystal structures of wild-type and NFV-resistant HIV-1 protease in complex with p1-p6 substrate peptide variants with L449F and/or S451N. Alterations of residue 30's interaction with the substrate are compensated by the coevolving L449F and S451N cleavage site mutations. This interdependency in the PR-p1-p6 interactions enhances intermolecular contacts and reinforces the overall fit of the substrate within the substrate envelope, likely enabling coevolution to sustain substrate recognition and cleavage in the presence of PR resistance mutations. Resistance to human immunodeficiency virus type 1 (HIV-1) protease inhibitors challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. Mutations in HIV-1 protease selected under the pressure of protease inhibitors render the inhibitors less potent. Occasionally, Gag sequences also mutate and coevolve with protease, contributing to maintenance of viral fitness and to drug resistance. In this study, we investigated the structural basis of coevolution at the Gag p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations. Our structural analysis reveals the interdependency of protease-substrate interactions and how coevolution may restore substrate recognition and cleavage in the presence of protease drug resistance mutations. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  9. Mechanistic diversity in the RuBisCO superfamily: RuBisCO from Rhodospirillum rubrum is not promiscuous for reactions catalyzed by RuBisCO-like proteins.

    PubMed

    Warlick, Benjamin P E; Imker, Heidi J; Sriram, Jaya; Tabita, F Robert; Gerlt, John A

    2012-11-27

    d-Ribulose 1,5-bisphosphate carboxylase/oxygenases (RuBisCOs) are promiscuous, catalyzing not only carboxylation and oxygenation of d-ribulose 1,5-bisphosphate but also other promiscuous, presumably nonphysiological, reactions initiated by abstraction of the 3-proton of d-ribulose 1,5-bisphosphate. Also, RuBisCO has homologues that do not catalyze carboxylation; these are designated RuBisCO-like proteins or RLPs. Members of the two families of RLPs catalyze reactions in the recycling of 5'-methylthioadenosine (MTA) generated by polyamine synthesis: (1) the 2,3-diketo-5-methylthiopentane 1-phosphate (DK-MTP 1-P) "enolase" reaction in the well-known "methionine salvage" pathway in Bacillus sp. and (2) the 5-methylthio-d-ribulose 1-phosphate (MTRu 1-P) 1,3-isomerase reaction in the recently discovered "MTA-isoprenoid shunt" that generates 1-deoxy-d-xylulose 5-phosphate for nonmevalonate isoprene synthesis in Rhodospirillum rubrum. We first studied the structure and reactivity of DK-MTP 1-P that was reported to decompose rapidly [Ashida, H., Saito, Y., Kojima, C., and Yokota, A. (2008) Biosci., Biotechnol., Biochem. 72, 959-967]. The 2-carbonyl group of DK-MTP 1-P is rapidly hydrated and can undergo enolization both nonenzymatically and enzymatically via the small amount of unhydrated material that is present. We then examined the ability of RuBisCO from R. rubrum to catalyze both of the RLP-catalyzed reactions. Contrary to a previous report [Ashida, H., Saito, Y., Kojima, C., Kobayashi, K., Ogasawara, N., and Yokota, A. (2003) Science 302, 286-290], we were unable to confirm that this RuBisCO catalyzes the DK-MTP 1-P "enolase" reaction either in vitro or in vivo. We also determined that this RuBisCO does not catalyze the MTRu 1-P 1,3-isomerase reaction in vitro. Thus, although RuBisCOs can be functionally promiscuous, RuBisCO from R. rubrum is not promiscuous for either of the known RLP-catalyzed reactions.

  10. [Face recognition in patients with autism spectrum disorders].

    PubMed

    Kita, Yosuke; Inagaki, Masumi

    2012-07-01

    The present study aimed to review previous research conducted on face recognition in patients with autism spectrum disorders (ASD). Face recognition is a key question in the ASD research field because it can provide clues for elucidating the neural substrates responsible for the social impairment of these patients. Historically, behavioral studies have reported low performance and/or unique strategies of face recognition among ASD patients. However, the performance and strategy of ASD patients is comparable to those of the control group, depending on the experimental situation or developmental stage, suggesting that face recognition of ASD patients is not entirely impaired. Recent brain function studies, including event-related potential and functional magnetic resonance imaging studies, have investigated the cognitive process of face recognition in ASD patients, and revealed impaired function in the brain's neural network comprising the fusiform gyrus and amygdala. This impaired function is potentially involved in the diminished preference for faces, and in the atypical development of face recognition, eliciting symptoms of unstable behavioral characteristics in these patients. Additionally, face recognition in ASD patients is examined from a different perspective, namely self-face recognition, and facial emotion recognition. While the former topic is intimately linked to basic social abilities such as self-other discrimination, the latter is closely associated with mentalizing. Further research on face recognition in ASD patients should investigate the connection between behavioral and neurological specifics in these patients, by considering developmental changes and the spectrum clinical condition of ASD.

  11. A Promiscuous De Novo Retro-Aldolase Catalyzes Asymmetric Michael Additions via Schiff Base Intermediates.

    PubMed

    Garrabou, Xavier; Beck, Tobias; Hilvert, Donald

    2015-05-04

    Recent advances in computational design have enabled the development of primitive enzymes for a range of mechanistically distinct reactions. Here we show that the rudimentary active sites of these catalysts can give rise to useful chemical promiscuity. Specifically, RA95.5-8, designed and evolved as a retro-aldolase, also promotes asymmetric Michael additions of carbanions to unsaturated ketones with high rates and selectivities. The reactions proceed by amine catalysis, as indicated by mutagenesis and X-ray data. The inherent flexibility and tunability of this catalyst should make it a versatile platform for further optimization and/or mechanistic diversification by directed evolution. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Psychosocial predictors of human papillomavirus vaccination intentions for young women 18 to 26: religiosity, morality, promiscuity, and cancer worry.

    PubMed

    Krakow, Melinda M; Jensen, Jakob D; Carcioppolo, Nick; Weaver, Jeremy; Liu, Miao; Guntzviller, Lisa M

    2015-01-01

    To determine whether five psychosocial variables, namely, religiosity, morality, perceived promiscuity, cancer worry frequency, and cancer worry severity, predict young women's intentions to receive the human papillomavirus (HPV) vaccination. Female undergraduate students (n=408) completed an online survey. Questions pertaining to hypothesized predictors were analyzed through bivariate correlations and hierarchical regression equations. Regressions examined whether the five psychosocial variables of interest predicted intentions to vaccinate above and beyond controls. Proposed interactions among predictor variables were also tested. Study findings supported cancer worry as a direct predictor of HPV vaccination intention, and religiosity and sexual experience as moderators of the relationship between concerns of promiscuity reputation and intentions to vaccinate. One dimension of cancer worry (severity) emerged as a particularly robust predictor for this population. This study provides support for several important, yet understudied, factors contributing to HPV vaccination intentions among college-aged women: cancer worry severity and religiosity. Future research should continue to assess the predictive contributions of these variables and evaluate how messages and campaigns to increase HPV vaccination uptake can utilize religious involvement and worry about cancer to promote more effectively HPV vaccination as a cancer prevention strategy. Copyright © 2015 Jacobs Institute of Women's Health. Published by Elsevier Inc. All rights reserved.

  13. Structural basis for substrate recognition by the human N-terminal methyltransferase 1

    DOE PAGES

    Dong, Cheng; Mao, Yunfei; Tempel, Wolfram; ...

    2015-11-05

    α-N-terminal methylation represents a highly conserved and prevalent post-translational modification, yet its biological function has remained largely speculative. The recent discovery of α-N-terminal methyltransferase 1 (NTMT1) and its physiological substrates propels the elucidation of a general role of α-N-terminal methylation in mediating DNA-binding ability of the modified proteins. The phenotypes, observed from both NTMT1 knockdown in breast cancer cell lines and knockout mouse models, suggest the potential involvement of α-N-terminal methylation in DNA damage response and cancer development. In this study, we report the first crystal structures of human NTMT1 in complex with cofactor S-adenosyl-L-homocysteine (SAH) and six substrate peptides,more » respectively, and reveal that NTMT1 contains two characteristic structural elements (a β hairpin and an N-terminal extension) that contribute to its substrate specificity. Our complex structures, coupled with mutagenesis, binding, and enzymatic studies, also present the key elements involved in locking the consensus substrate motif XPK (X indicates any residue type other than D/E) into the catalytic pocket for α-N-terminal methylation and explain why NTMT1 prefers an XPK sequence motif. We propose a catalytic mechanism for α-N-terminal methylation. Overall, this study gives us the first glimpse of the molecular mechanism of α-N-terminal methylation and potentially contributes to the advent of therapeutic agents for human diseases associated with deregulated α-N-terminal methylation.« less

  14. Peptidoglycan recognition protein genes and their roles in the innate immune pathways of the red flour beetle, Tribolium castaneum.

    PubMed

    Koyama, Hiroaki; Kato, Daiki; Minakuchi, Chieka; Tanaka, Toshiharu; Yokoi, Kakeru; Miura, Ken

    2015-11-01

    We have previously demonstrated that the functional Toll and IMD innate immune pathways indeed exist in the model beetle, Tribolium castaneum while the beetle's pathways have broader specificity in terms of microbial activation than that of Drosophila. To elucidate the molecular basis of this broad microbial activation, we here focused on potential upstream sensors of the T. castaneum innate immune pathways, peptidoglycan recognition proteins (PGRPs). Our phenotype analyses utilizing RNA interference-based comprehensive gene knockdown followed by bacterial challenge suggested: PGRP-LA functions as a pivotal sensor of the IMD pathway for both Gram-negative and Gram-positive bacteria; PGRP-LC acts as an IMD pathway-associated sensor mainly for Gram-negative bacteria; PGRP-LE also has some roles in Gram-negative bacterial recognition of the IMD pathway. On the other hand, we did not obtain clear phenotype changes by gene knockdown of short-type PGRP genes, probably because of highly inducible nature of these genes. Our results may collectively account for the promiscuous bacterial activation of the T. castaneum innate immune pathways at least in part. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. The structural neuroanatomy of music emotion recognition: evidence from frontotemporal lobar degeneration.

    PubMed

    Omar, Rohani; Henley, Susie M D; Bartlett, Jonathan W; Hailstone, Julia C; Gordon, Elizabeth; Sauter, Disa A; Frost, Chris; Scott, Sophie K; Warren, Jason D

    2011-06-01

    Despite growing clinical and neurobiological interest in the brain mechanisms that process emotion in music, these mechanisms remain incompletely understood. Patients with frontotemporal lobar degeneration (FTLD) frequently exhibit clinical syndromes that illustrate the effects of breakdown in emotional and social functioning. Here we investigated the neuroanatomical substrate for recognition of musical emotion in a cohort of 26 patients with FTLD (16 with behavioural variant frontotemporal dementia, bvFTD, 10 with semantic dementia, SemD) using voxel-based morphometry. On neuropsychological evaluation, patients with FTLD showed deficient recognition of canonical emotions (happiness, sadness, anger and fear) from music as well as faces and voices compared with healthy control subjects. Impaired recognition of emotions from music was specifically associated with grey matter loss in a distributed cerebral network including insula, orbitofrontal cortex, anterior cingulate and medial prefrontal cortex, anterior temporal and more posterior temporal and parietal cortices, amygdala and the subcortical mesolimbic system. This network constitutes an essential brain substrate for recognition of musical emotion that overlaps with brain regions previously implicated in coding emotional value, behavioural context, conceptual knowledge and theory of mind. Musical emotion recognition may probe the interface of these processes, delineating a profile of brain damage that is essential for the abstraction of complex social emotions. Copyright © 2011 Elsevier Inc. All rights reserved.

  16. An Ecological Analysis of the Effects of Deviant Peer Clustering on Sexual Promiscuity, Problem Behavior, and Childbearing from Early Adolescence to Adulthood: An Enhancement of the Life History Framework

    PubMed Central

    Dishion, Thomas J.; Ha, Thao; Véronneau, Marie-Hélène

    2012-01-01

    This study proposes the inclusion of peer relationships in a life history perspective on adolescent problem behavior. Longitudinal analyses were used to examine deviant peer clustering as the mediating link between attenuated family ties, peer marginalization, and social disadvantage in early adolescence and sexual promiscuity in middle adolescence and childbearing by early adulthood. Specifically, 998 youth and their families were assessed at age 11 years and periodically through age 24 years. Structural equation modeling revealed that the peer-enhanced life history model provided a good fit to the longitudinal data, with deviant peer clustering strongly predicting adolescent sexual promiscuity and other correlated problem behaviors. Sexual promiscuity, as expected, also strongly predicted the number of children by age 22–24 years. Consistent with a life history perspective, family social disadvantage directly predicted deviant peer clustering and number of children in early adulthood, controlling for all other variables in the model. These data suggest that deviant peer clustering is a core dimension of a fast life history strategy, with strong links to sexual activity and childbearing. The implications of these findings are discussed with respect to the need to integrate an evolutionary-based model of self-organized peer groups in developmental and intervention science. PMID:22409765

  17. An ecological analysis of the effects of deviant peer clustering on sexual promiscuity, problem behavior, and childbearing from early adolescence to adulthood: an enhancement of the life history framework.

    PubMed

    Dishion, Thomas J; Ha, Thao; Véronneau, Marie-Hélène

    2012-05-01

    The authors propose that peer relationships should be included in a life history perspective on adolescent problem behavior. Longitudinal analyses were used to examine deviant peer clustering as the mediating link between attenuated family ties, peer marginalization, and social disadvantage in early adolescence and sexual promiscuity in middle adolescence and childbearing by early adulthood. Specifically, 998 youths, along with their families, were assessed at age 11 years and periodically through age 24 years. Structural equation modeling revealed that the peer-enhanced life history model provided a good fit to the longitudinal data, with deviant peer clustering strongly predicting adolescent sexual promiscuity and other correlated problem behaviors. Sexual promiscuity, as expected, also strongly predicted the number of children by ages 22-24 years. Consistent with a life history perspective, family social disadvantage directly predicted deviant peer clustering and number of children in early adulthood, controlling for all other variables in the model. These data suggest that deviant peer clustering is a core dimension of a fast life history strategy, with strong links to sexual activity and childbearing. The implications of these findings are discussed with respect to the need to integrate an evolutionary-based model of self-organized peer groups in developmental and intervention science.

  18. Promiscuous Girls, Good Wives, and Cheating Husbands: Gender Inequality, Transitions to Marriage, and Infidelity in Southeastern Nigeria.

    PubMed

    Smith, Daniel Jordan

    2010-01-01

    The transition from premarital sexual relationships and courtship to marriage and parenthood in southeastern Nigeria involves particularly dramatic adjustments for young women who have absorbed changing ideas about sexuality, marriage, and gender equality, and who have had active premarital sexual lives. In the eyes of society, these women must transform from being promiscuous girls to good wives. This paper examines these adjustments and, specifically, how young married women's lives are affected by the reality of male infidelity and a persistent gendered double standard regarding the acceptability of extramarital sex.

  19. Promiscuous Girls, Good Wives, and Cheating Husbands: Gender Inequality, Transitions to Marriage, and Infidelity in Southeastern Nigeria

    PubMed Central

    Smith, Daniel Jordan

    2013-01-01

    The transition from premarital sexual relationships and courtship to marriage and parenthood in southeastern Nigeria involves particularly dramatic adjustments for young women who have absorbed changing ideas about sexuality, marriage, and gender equality, and who have had active premarital sexual lives. In the eyes of society, these women must transform from being promiscuous girls to good wives. This paper examines these adjustments and, specifically, how young married women’s lives are affected by the reality of male infidelity and a persistent gendered double standard regarding the acceptability of extramarital sex. PMID:24259752

  20. Substrate-based inhibitors exhibiting excellent protective and therapeutic effects against Botulinum Neurotoxin A intoxication.

    PubMed

    Guo, Jiubiao; Wang, Jinglin; Gao, Shan; Ji, Bin; Waichi Chan, Edward; Chen, Sheng

    2015-11-20

    Potent inhibitors to reverse Botulinum neurotoxins (BoNTs) activity in neuronal cells are currently not available. A better understanding of the substrate recognition mechanism of BoNTs enabled us to design a novel class of peptide inhibitors which were derivatives of the BoNT/A substrate, SNAP25. Through a combination of in vitro, cellular based, and in vivo mouse assays, several potent inhibitors of approximately one nanomolar inhibitory strength both in vitro and in vivo have been identified. These compounds represent the first set of inhibitors that exhibited full protection against BoNT/A intoxication in mice model with undetectable toxicity. Our findings validated the hypothesis that a peptide inhibitor targeting the two BoNT structural regions which were responsible for substrate recognition and cleavage respectively could exhibit excellent inhibitory effect, thereby providing insight on future development of more potent inhibitors against BoNTs.

  1. Metabolic Noise, Vestigial Metabolites or the Raw Material of Ecological Adaptation? Opportunitistic Enzymes, Catalytic Promiscuity and the Evolution of Chemodiversity in Nature (2010 JGI User Meeting)

    ScienceCinema

    Noel, Joseph

    2018-04-26

    Joseph Noel from the Salk Institute on "Metabolic Noise, Vestigial Metabolites or the Raw Material of Ecological Adaptation? Enzymes, Catalytic Promiscuity and the Evolution of Chemodiversity in Nature" on March 26, 2010 at the 5th Annual DOE JGI User Meeting.

  2. [A new mechanism of ubiquitin-dependent proteolytic pathway--polyubiquitin chain recognition and proteasomal targeting].

    PubMed

    Kawahara, Hiroyuki; Yokosawa, Hideyoshi

    2008-01-01

    The RPN10 subunit of 26S proteasome and several UBA domain proteins can bind to the polyubiquitin chain and play a role as ubiquitin receptors of the 26S proteasome. Although it was thought that substrate recognition is an essential step in the proteasome-mediated protein degradation, deletion of rpn10 genes in yeast does not influence the viability of cells but instead causes only a mild phenotype, suggesting that the above ubiquitin receptors are redundantly involved in substrate delivery to the proteasome. However, their functional difference is still enigmatic. In this review, we summarize recent advances in polyubiquitin chain recognition/delivery system and provide potential applications to modulate this system as a probable target for drug development.

  3. Employing the promiscuity of lantibiotic biosynthetic machineries to produce novel antimicrobials.

    PubMed

    Montalbán-López, Manuel; van Heel, Auke J; Kuipers, Oscar P

    2017-01-01

    As the number of new antibiotics that reach the market is decreasing and the demand for them is rising, alternative sources of novel antimicrobials are needed. Lantibiotics are potent peptide antimicrobials that are ribosomally synthesized and stabilized by post-translationally introduced lanthionine rings. Their ribosomal synthesis and enzymatic modifications provide excellent opportunities to design and engineer a large variety of novel antimicrobial compounds. The research conducted in this area demonstrates that the modularity present in both the peptidic rings as well as in the combination of promiscuous modification enzymes can be exploited to further increase the diversity of lantibiotics. Various approaches, where the modifying enzymes and corresponding leader peptides are decoupled from their natural core peptide and integrated in designed plug-and-play production systems, enable the production of modified peptides that are either derived from vast genomic data or designed using functional parts from a wide diversity of core peptides. These approaches constitute a powerful discovery platform to develop novel antimicrobials with high therapeutic potential. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  4. The protein kinase promiscuities in the cancer-preventive mechanisms of NSAIDs

    PubMed Central

    Norvaisas, Povilas; Chan, Diana; Yokoi, Kenji; Dave, Bhuvanesh

    2016-01-01

    NSAIDs have been observed to have cancer-preventive properties, but the actual mechanism is elusive. We hypothesize that NSAIDs might have an effect through common pathways and targets of anticancer drugs by exploiting promiscuities of anticancer drug targets. Here, we have explored NSAIDs by their structural and pharmacophoric similarities with small anticancer molecules. In-silico analyses have shown a strong similarity between NSAIDs and protein kinase (PK) inhibitors. The calculated affinities of NSAIDs were found to be lower than the affinities of anticancer drugs, but higher than the affinities of compounds that are not specific to PKs. The competitive inhibition model suggests that PK might be inhibited by around 10%, which was confirmed by biochemical screening of some NSAIDs against PKs. NSAIDs did not affect all PKs universally, but had specificities for certain sets of PKs, which differed according to the NSAID. The study revealed potentially new features and mechanisms of NSAIDs that are useful in explaining their role in cancer prevention, which might lead to clinically significant breakthroughs in the future. PMID:25714784

  5. Structural properties of the promiscuous VP16 activation domain.

    PubMed

    Jonker, Hendrik R A; Wechselberger, Rainer W; Boelens, Rolf; Folkers, Gert E; Kaptein, Rob

    2005-01-25

    Herpes simplex virion protein 16 (VP16) contains two strong activation regions that can independently and cooperatively activate transcription in vivo. We have identified the regions and residues involved in the interaction with the human transcriptional coactivator positive cofactor 4 (PC4) and the general transcription factor TFIIB. NMR and biochemical experiments revealed that both VP16 activation regions are required for the interaction and undergo a conformational transition from random coil to alpha-helix upon binding to its target PC4. The interaction is strongly electrostatically driven and the binding to PC4 is enhanced by the presence of its amino-terminal domain. We propose models for binding of VP16 to the core domains of PC4 and TFIIB that are based on two independent docking approaches using NMR chemical shift changes observed in titration experiments. The models are consistent with results from site-directed mutagenesis and provide an explanation for the contribution of both acidic and hydrophobic residues for transcriptional activation by VP16. Both intrinsically unstructured activation domains are attracted to their interaction partner by electrostatic interactions, and adopt an alpha-helical conformation around the important hydrophobic residues. The models showed multiple distinct binding surfaces upon interaction with various partners, providing an explanation for the promiscuous properties, cooperativity, and the high activity of this activation domain.

  6. In situ imaging and proteome profiling indicate andrographolide is a highly promiscuous compound

    NASA Astrophysics Data System (ADS)

    Li, Lin; Wijaya, Hadhi; Samanta, Sanjay; Lam, Yulin; Yao, Shao Q.

    2015-06-01

    Natural products represent an enormous source of pharmacologically useful compounds, and are often used as the starting point in modern drug discovery. Many biologically interesting natural products are however not being pursued as potential drug candidates, partly due to a lack of well-defined mechanism-of-action. Traditional in vitro methods for target identification of natural products based on affinity protein enrichment from crude cellular lysates cannot faithfully recapitulate protein-drug interactions in living cells. Reported herein are dual-purpose probes inspired by the natural product andrographolide, capable of both reaction-based, real-time bioimaging and in situ proteome profiling/target identification in live mammalian cells. Our results confirm that andrographolide is a highly promiscuous compound and engaged in covalent interactions with numerous previously unknown cellular targets in cell type-specific manner. We caution its potential therapeutic effects should be further investigated in detail.

  7. Model Communities Hint at Promiscuous Metabolic Linkages between Ubiquitous Free-Living Freshwater Bacteria

    PubMed Central

    Buck, Moritz; Hamilton, Joshua J.; Wurzbacher, Christian; Grossart, Hans-Peter; Eiler, Alexander

    2018-01-01

    ABSTRACT Genome streamlining is frequently observed in free-living aquatic microorganisms and results in physiological dependencies between microorganisms. However, we know little about the specificity of these microbial associations. In order to examine the specificity and extent of these associations, we established mixed cultures from three different freshwater environments and analyzed the cooccurrence of organisms using a metagenomic time series. Free-living microorganisms with streamlined genomes lacking multiple biosynthetic pathways showed no clear recurring pattern in their interaction partners. Free-living freshwater bacteria form promiscuous cooperative associations. This notion contrasts with the well-documented high specificities of interaction partners in host-associated bacteria. Considering all data together, we suggest that highly abundant free-living bacterial lineages are functionally versatile in their interactions despite their distinct streamlining tendencies at the single-cell level. This metabolic versatility facilitates interactions with a variable set of community members. PMID:29848762

  8. Structure of a [NiFe] hydrogenase maturation protease HycI provides insights into its substrate selectivity.

    PubMed

    Kwon, Sunghark; Nishitani, Yuichi; Hirao, Yoshinori; Kanai, Tamotsu; Atomi, Haruyuki; Miki, Kunio

    2018-04-15

    The immature large subunit of [NiFe] hydrogenases undergoes C-terminal cleavage by a specific protease in the final step of the post-translational process before assembly with other subunits. It has been reported that the [NiFe] hydrogenase maturation protease HycI from Thermococcus kodakarensis (TkHycI) has the catalytic ability to target the membrane-bound hydrogenase large subunit MbhL from T. kodakarensis. However, the detailed mechanism of its substrate recognition remains elusive. We determined the crystal structure of TkHycI at 1.59 Å resolution to clarify how TkHycI recognizes its own substrate MbhL. Although the overall structure of TkHycI is similar to that of its homologous protease TkHybD, TkHycI adopts a larger loop than TkHybD, thereby creating a broad and deep cleft. We analyzed the structural properties of the TkHycI cleft probably involved in its substrate recognition. Our findings provide novel and profound insights into the substrate selectivity of TkHycI. Copyright © 2018 Elsevier Inc. All rights reserved.

  9. Recognition of Acyl Carrier Proteins by Ketoreductases in Assembly Line Polyketide Synthases

    PubMed Central

    Ostrowski, Matthew P.; Cane, David E.; Khosla, Chaitan

    2016-01-01

    Ketoreductases (KRs) are the most widespread tailoring domains found in individual modules of assembly line polyketide synthases (PKSs), and are responsible for controlling the configurations of both the α-methyl and β-hydroxyl stereogenic centers in the growing polyketide chain. Because they recognize substrates that are covalently bound to acyl carrier proteins (ACPs) within the same PKS module, we sought to quantify the extent to which protein-protein recognition contributes to the turnover of these oxidoreductive enzymes using stand-alone domains from the 6-deoxyerythronolide B synthase (DEBS). Reduced 2-methyl-3-hydroxyacyl-ACP substrates derived from two enantiomeric acyl chains and four distinct ACP domains were synthesized and presented to four distinct KR domains. Two KRs, from DEBS modules 2 and 5, displayed little preference for oxidation of substrates tethered to their cognate ACP domains over those attached to the other ACP domains tested. In contrast, the KR from DEBS module 1 showed a ca. 10-50-fold preference for substrate attached to its native ACP domain, whereas the KR from DEBS module 6 actually displayed a ca. 10-fold preference for the ACP from DEBS module 5. Our findings suggest that recognition of the ACP by a KR domain is unlikely to affect the rate of native assembly line polyketide biosynthesis. In some cases, however, unfavorable KR-ACP interactions may suppress the rate of substrate processing when KR domains are swapped to construct hybrid PKS modules. PMID:27118242

  10. Molecular Basis of Substrate Promiscuity for the SAM-Dependent O-Methyltransferase NcsB1, Involved in the Biosynthesis of the Enediyne Antitumor Antibiotic Neocarzinostatin

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

    Cooke, H.; Guenther, E; Luo, Y

    2009-01-01

    The small molecule component of chromoprotein enediyne antitumor antibiotics is biosynthesized through a convergent route, incorporating amino acid, polyketide, and carbohydrate building blocks around a central enediyne hydrocarbon core. The naphthoic acid moiety of the enediyne neocarzinostatin plays key roles in the biological activity of the natural product by interacting with both the carrier protein and duplex DNA at the site of action. We have previously described the in vitro characterization of an S-adenosylmethionine-dependent O-methyltransferase (NcsB1) in the neocarzinostatin biosynthetic pathway [Luo, Y., Lin, S., Zhang, J., Cooke, H. A., Bruner, S. D., and Shen, B. (2008) J. Biol. Chem.more » 283, 14694-14702]. Here we provide a structural basis for NcsB1 activity, illustrating that the enzyme shares an overall architecture with a large family of S-adenosylmethionine-dependent proteins. In addition, NcsB1 represents the first enzyme to be structurally characterized in the biosynthetic pathway of neocarzinostatin. By cocrystallizing the enzyme with various combinations of the cofactor and substrate analogues, details of the active site structure have been established. Changes in subdomain orientation were observed via comparison of structures in the presence and absence of substrate, suggesting that reorientation of the enzyme is involved in binding of the substrate. In addition, residues important for substrate discrimination were predicted and probed through site-directed mutagenesis and in vitro biochemical characterization.« less

  11. Functional and computational analysis of amino acid patterns predictive of type III secretion system substrates in Pseudomonas syringae

    USDA-ARS?s Scientific Manuscript database

    Bacterial type III secretion systems (T3SSs) deliver proteins called effectors into eukaryotic cells. Although N-terminal amino acid sequences are required for translocation, the mechanism of substrate recognition by the T3SS is unknown. Almost all actively deployed T3SS substrates in the plant path...

  12. Molecular recognition on a cavitand-functionalized silicon surface.

    PubMed

    Biavardi, Elisa; Favazza, Maria; Motta, Alessandro; Fragalà, Ignazio L; Massera, Chiara; Prodi, Luca; Montalti, Marco; Melegari, Monica; Condorelli, Guglielmo G; Dalcanale, Enrico

    2009-06-03

    A Si(100) surface featuring molecular recognition properties was obtained by covalent functionalization with a tetraphosphonate cavitand (Tiiii), able to complex positively charged species. Tiiii cavitand was grafted onto the Si by photochemical hydrosilylation together with 1-octene as a spatial spectator. The recognition properties of the Si-Tiiii surface were demonstrated through two independent analytical techniques, namely XPS and fluorescence spectroscopy, during the course of reversible complexation-guest exchange-decomplexation cycles with specifically designed ammonium and pyridinium salts. Control experiments employing a Si(100) surface functionalized with a structurally similar, but complexation inactive, tetrathiophosphonate cavitand (TSiiii) demonstrated no recognition events. This provides evidence for the complexation properties of the Si-Tiiii surface, ruling out the possibility of nonspecific interactions between the substrate and the guests. The residual Si-O(-) terminations on the surface replace the guests' original counterions, thus stabilizing the complex ion pairs. These results represent a further step toward the control of self-assembly of complex supramolecular architectures on surfaces.

  13. Structural Basis for Substrate Recognition by the Ankyrin Repeat Domain of Human DHHC17 Palmitoyltransferase

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

    Verardi, Raffaello; Kim, Jin-Sik; Ghirlando, Rodolfo

    DHHC enzymes catalyze palmitoylation, a major post-translational modification that regulates a number of key cellular processes. There are up to 24 DHHCs in mammals and hundreds of substrate proteins that get palmitoylated. However, how DHHC enzymes engage with their substrates is still poorly understood. There is currently no structural information about the interaction between any DHHC enzyme and protein substrates. In this study we have investigated the structural and thermodynamic bases of interaction between the ankyrin repeat domain of human DHHC17 (ANK17) and Snap25b. We solved a high-resolution crystal structure of the complex between ANK17 and a peptide fragment ofmore » Snap25b. Through structure-guided mutagenesis, we discovered key residues in DHHC17 that are critically important for interaction with Snap25b. We further extended our finding by showing that the same residues are also crucial for the interaction of DHHC17 with Huntingtin, one of its most physiologically relevant substrates.« less

  14. Structural insights into the difference in substrate recognition of two mannoside phosphorylases from two GH130 subfamilies.

    PubMed

    Ye, Yuxin; Saburi, Wataru; Odaka, Rei; Kato, Koji; Sakurai, Naofumi; Komoda, Keisuke; Nishimoto, Mamoru; Kitaoka, Motomitsu; Mori, Haruhide; Yao, Min

    2016-03-01

    In Ruminococcus albus, 4-O-β-D-mannosyl-D-glucose phosphorylase (RaMP1) and β-(1,4)-mannooligosaccharide phosphorylase (RaMP2) belong to two subfamilies of glycoside hydrolase family 130. The two enzymes phosphorolyze β-mannosidic linkages at the nonreducing ends of their substrates, and have substantially diverse substrate specificity. The differences in their mechanism of substrate binding have not yet been fully clarified. In the present study, we report the crystal structures of RaMP1 with/without 4-O-β-D-mannosyl-d-glucose and RaMP2 with/without β-(1→4)-mannobiose. The structures of the two enzymes differ at the +1 subsite of the substrate-binding pocket. Three loops are proposed to determine the different substrate specificities. One of these loops is contributed from the adjacent molecule of the oligomer structure. In RaMP1, His245 of loop 3 forms a hydrogen-bond network with the substrate through a water molecule, and is indispensible for substrate binding. © 2016 Federation of European Biochemical Societies.

  15. Chemically mediated species recognition in closely related Podarcis wall lizards.

    PubMed

    Barbosa, Diana; Font, Enrique; Desfilis, Ester; Carretero, Miguel A

    2006-07-01

    In many animals, chemical signals play an important role in species recognition and may contribute to reproductive isolation and speciation. The Iberian lizards of the genus Podarcis, with up to nine currently recognized lineages that are often sympatric, are highly chemosensory and provide an excellent model for the study of chemically mediated species recognition in closely related taxa. In this study, we tested the ability of male and female lizards of two sister species with widely overlapping distribution ranges (Podarcis bocagei and P. hispanica type 1) to discriminate between conspecific and heterospecific mates by using only substrate-borne chemical cues. We scored the number of tongue flicks directed at the paper substrate by each individual in a terrarium previously occupied by a conspecific or a heterospecific lizard of the opposite sex. Results show that males of P. bocagei and P. hispanica type 1 are capable of discriminating chemically between conspecifics and heterospecifics of the opposite sex, but females are not. These results suggest that differences in female, but not male, chemical cues may underlie species recognition and contribute to reproductive isolation in these species. The apparent inability of females to discriminate conspecific from heterospecific males, which is not because of reduced baseline exploration rates, is discussed in the context of sexual selection theory and species discrimination.

  16. Exosites in the substrate specificity of blood coagulation reactions.

    PubMed

    Bock, P E; Panizzi, P; Verhamme, I M A

    2007-07-01

    The specificity of blood coagulation proteinases for substrate, inhibitor, and effector recognition is mediated by exosites on the surfaces of the catalytic domains, physically separated from the catalytic site. Some thrombin ligands bind specifically to either exosite I or II, while others engage both exosites. The involvement of different, overlapping constellations of exosite residues enables binding of structurally diverse ligands. The flexibility of the thrombin structure is central to the mechanism of complex formation and the specificity of exosite interactions. Encounter complex formation is driven by electrostatic ligand-exosite interactions, followed by conformational rearrangement to a stable complex. Exosites on some zymogens are in low affinity proexosite states and are expressed concomitant with catalytic site activation. The requirement for exosite expression controls the specificity of assembly of catalytic complexes on the coagulation pathway, such as the membrane-bound factor Xa*factor Va (prothrombinase) complex, and prevents premature assembly. Substrate recognition by prothrombinase involves a two-step mechanism with initial docking of prothrombin to exosites, followed by a conformational change to engage the FXa catalytic site. Prothrombin and its activation intermediates bind prothrombinase in two alternative conformations determined by the zymogen to proteinase transition that are hypothesized to involve prothrombin (pro)exosite I interactions with FVa, which underpin the sequential activation pathway. The role of exosites as the major source of substrate specificity has stimulated development of exosite-targeted anticoagulants for treatment of thrombosis.

  17. Molecular mechanism of substrate recognition and transport by the AtSWEET13 sugar transporter.

    PubMed

    Han, Lei; Zhu, Yongping; Liu, Min; Zhou, Ye; Lu, Guangyuan; Lan, Lan; Wang, Xianping; Zhao, Yongfang; Zhang, Xuejun C

    2017-09-19

    Sugar Will Eventually be Exported Transporters (SWEETs) are recently identified sugar transporters that can discriminate and transport di- or monosaccharides across a membrane following the concentration gradient. SWEETs play key roles in plant biological processes, such as pollen nutrition, nectar secretion, seed filling, and phloem loading. SWEET13 from Arabidopsis thaliana (AtSWEET13) is an important sucrose transporter in pollen development. Here, we report the 2.8-Å resolution crystal structure of AtSWEET13 in the inward-facing conformation with a substrate analog, 2'-deoxycytidine 5'-monophosphate, bound in the central cavity. In addition, based on the results of an in-cell transport activity assay and single-molecule Förster resonance energy transfer analysis, we suggest a mechanism for substrate selectivity based on the size of the substrate-binding pocket. Furthermore, AtSWEET13 appears to form a higher order structure presumably related to its function.

  18. Interactions between Casein Kinase Iε (CKIε) and Two Substrates from Disparate Signaling Pathways Reveal Mechanisms for Substrate-Kinase Specificity

    PubMed Central

    Dahlberg, Caroline Lund; Nguyen, Elizabeth Z.; Goodlett, David; Kimelman, David

    2009-01-01

    Background Members of the Casein Kinase I (CKI) family of serine/threonine kinases regulate diverse biological pathways. The seven mammalian CKI isoforms contain a highly conserved kinase domain and divergent amino- and carboxy-termini. Although they share a preferred target recognition sequence and have overlapping expression patterns, individual isoforms often have specific substrates. In an effort to determine how substrates recognize differences between CKI isoforms, we have examined the interaction between CKIε and two substrates from different signaling pathways. Methodology/Principal Findings CKIε, but not CKIα, binds to and phosphorylates two proteins: Period, a transcriptional regulator of the circadian rhythms pathway, and Disheveled, an activator of the planar cell polarity pathway. We use GST-pull-down assays data to show that two key residues in CKIα's kinase domain prevent Disheveled and Period from binding. We also show that the unique C-terminus of CKIε does not determine Dishevelled's and Period's preference for CKIε nor is it essential for binding, but instead plays an auxillary role in stabilizing the interactions of CKIε with its substrates. We demonstrate that autophosphorylation of CKIε's C-terminal tail prevents substrate binding, and use mass spectrometry and chemical crosslinking to reveal how a phosphorylation-dependent interaction between the C-terminal tail and the kinase domain prevents substrate phosphorylation and binding. Conclusions/Significance The biochemical interactions between CKIε and Disheveled, Period, and its own C-terminus lead to models that explain CKIε's specificity and regulation. PMID:19274088

  19. Facial emotion recognition in patients with focal and diffuse axonal injury.

    PubMed

    Yassin, Walid; Callahan, Brandy L; Ubukata, Shiho; Sugihara, Genichi; Murai, Toshiya; Ueda, Keita

    2017-01-01

    Facial emotion recognition impairment has been well documented in patients with traumatic brain injury. Studies exploring the neural substrates involved in such deficits have implicated specific grey matter structures (e.g. orbitofrontal regions), as well as diffuse white matter damage. Our study aims to clarify whether different types of injuries (i.e. focal vs. diffuse) will lead to different types of impairments on facial emotion recognition tasks, as no study has directly compared these patients. The present study examined performance and response patterns on a facial emotion recognition task in 14 participants with diffuse axonal injury (DAI), 14 with focal injury (FI) and 22 healthy controls. We found that, overall, participants with FI and DAI performed more poorly than controls on the facial emotion recognition task. Further, we observed comparable emotion recognition performance in participants with FI and DAI, despite differences in the nature and distribution of their lesions. However, the rating response pattern between the patient groups was different. This is the first study to show that pure DAI, without gross focal lesions, can independently lead to facial emotion recognition deficits and that rating patterns differ depending on the type and location of trauma.

  20. Rational engineering of the Neurospora VS ribozyme to allow substrate recognition via different kissing-loop interactions.

    PubMed

    Lacroix-Labonté, Julie; Girard, Nicolas; Dagenais, Pierre; Legault, Pascale

    2016-08-19

    The Neurospora VS ribozyme is a catalytic RNA that has the unique ability to specifically recognize and cleave a stem-loop substrate through formation of a highly stable kissing-loop interaction (KLI). In order to explore the engineering potential of the VS ribozyme to cleave alternate substrates, we substituted the wild-type KLI by other known KLIs using an innovative engineering method that combines rational and combinatorial approaches. A bioinformatic search of the protein data bank was initially performed to identify KLIs that are structurally similar to the one found in the VS ribozyme. Next, substrate/ribozyme (S/R) pairs that incorporate these alternative KLIs were kinetically and structurally characterized. Interestingly, several of the resulting S/R pairs allowed substrate cleavage with substantial catalytic efficiency, although with reduced activity compared to the reference S/R pair. Overall, this study describes an innovative approach for RNA engineering and establishes that the KLI of the trans VS ribozyme can be adapted to cleave other folded RNA substrates. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

    NASA Astrophysics Data System (ADS)

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

    2017-01-01

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

  2. Two unexpected promiscuous activities of the iron-sulfur protein IspH in production of isoprene and isoamylene.

    PubMed

    Ge, Deyong; Xue, Yanfen; Ma, Yanhe

    2016-05-11

    Bacillus species, possessing the methylerythritol phosphate (MEP) pathway for the synthesis of isoprenoid feedstock, are the highest producers of isoprene among bacteria; however, the enzyme responsible for isoprene synthesis has not been identified. The iron-sulfur protein IspH is the final enzyme of the MEP pathway and catalyses the reductive dehydration of (E)-4-hydroxy-3-methyl-2-butenyl diphosphate (HMBPP) to form isopentenyl diphosphate and dimethylallyl diphosphate (DMAPP). In this study, we demonstrated two unexpected promiscuous activities of IspH from alkaliphilic Bacillus sp. N16-5, which can produce high levels of isoprene. Bacillus sp. N16-5 IspH could catalyse the formation of isoprene from HMBPP and the conversion of DMAPP into a mixture of 2-methyl-2-butene and 3-methyl-1-butene. Both reactions require an electron transfer system, such as that used for HMBPP dehydration. Isoprene and isoamylene synthesis in Bacillus sp. N16-5 was investigated and the reaction system was reconstituted in vitro, including IspH, ferredoxin and ferredoxin-NADP(+)-reductase proteins and NADPH. The roles of specific IspH protein residues were also investigated by site-directed mutagenesis experiments; two variants (H131N and E133Q) were found to have lost the HMBPP reductase activity but could still catalyse the formation of isoprene. Overexpression of IspH H131N in Bacillus sp. N16-5 resulted in a twofold enhancement of isoprene production, and the yield of isoprene from the strain expressing E133Q was increased 300% compared with the wild-type strain. IspH from Bacillus sp. N16-5 is a promiscuous enzyme that can catalyse formation of isoprene and isoamylene. This enzyme, especially the H131N and E133Q variants, could be used for the production of isoprene from HMBPP.

  3. Facial Emotion Recognition Impairments are Associated with Brain Volume Abnormalities in Individuals with HIV

    PubMed Central

    Clark, Uraina S.; Walker, Keenan A.; Cohen, Ronald A.; Devlin, Kathryn N.; Folkers, Anna M.; Pina, Mathew M.; Tashima, Karen T.

    2015-01-01

    Impaired facial emotion recognition abilities in HIV+ patients are well documented, but little is known about the neural etiology of these difficulties. We examined the relation of facial emotion recognition abilities to regional brain volumes in 44 HIV-positive (HIV+) and 44 HIV-negative control (HC) adults. Volumes of structures implicated in HIV− associated neuropathology and emotion recognition were measured on MRI using an automated segmentation tool. Relative to HC, HIV+ patients demonstrated emotion recognition impairments for fearful expressions, reduced anterior cingulate cortex (ACC) volumes, and increased amygdala volumes. In the HIV+ group, fear recognition impairments correlated significantly with ACC, but not amygdala volumes. ACC reductions were also associated with lower nadir CD4 levels (i.e., greater HIV-disease severity). These findings extend our understanding of the neurobiological substrates underlying an essential social function, facial emotion recognition, in HIV+ individuals and implicate HIV-related ACC atrophy in the impairment of these abilities. PMID:25744868

  4. Essential differences in ligand presentation and T cell epitope recognition among HLA molecules of the HLA-B44 supertype.

    PubMed

    Hillen, Nina; Mester, Gabor; Lemmel, Claudia; Weinzierl, Andreas O; Müller, Margret; Wernet, Dorothee; Hennenlotter, Jörg; Stenzl, Arnulf; Rammensee, Hans-Georg; Stevanović, Stefan

    2008-11-01

    Human leukocyte antigens (HLA) have long been grouped into supertypes to facilitate peptide-based immunotherapy. Analysis of several hundreds of peptides presented by all nine antigens of the HLA-B44 supertype (HLA-B*18, B*37, B*40, B*41, B*44, B*45, B*47, B*49 and B*50) revealed unique peptide motifs for each of them. Taking all supertype members into consideration only 25 out of 670 natural ligands were found on more than one HLA molecule. Further direct comparisons by two mass spectrometric methods--isotope labeling as well as a label-free approach--consistently demonstrated only minute overlaps of below 3% between the ligandomes of different HLA antigens. In addition, T cell reactions of healthy donors against immunodominant HLA-B*44 and HLA-B*40 epitopes from EBV lacked promiscuous T-cell recognition within the HLA-B44 supertype. Taken together, these results challenge the common paradigm of broadly presented epitopes within this supertype.

  5. Mechanism and Substrate Recognition of Human Holo ACP Synthase

    PubMed Central

    Bunkoczi, Gabor; Pasta, Saloni; Joshi, Anil; Wu, Xiaoqiu; Kavanagh, Kathryn L.; Smith, Stuart; Oppermann, Udo

    2007-01-01

    Summary Mammals utilize a single phosphopantetheinyl transferase for the posttranslational modification of at least three different apoproteins: the carrier protein components of cytosolic and mitochondrial fatty acid synthases and the aminoadipate semialdehyde reductase involved in lysine degradation. We determined the crystal structure of the human phosphopantetheinyl transferase, a eukaryotic phosphopantetheinyl transferase characterized, complexed with CoA and Mg2+, and in ternary complex with CoA and ACP. The involvement of key residues in ligand binding and catalysis was confirmed by mutagenesis and kinetic analysis. Human phosphopantetheinyl transferase exhibits an α/β fold and 2-fold pseudosymmetry similar to the Sfp phosphopantetheinyl transferase from Bacillus subtilis. Although the bound ACP exhibits a typical four-helix structure, its binding is unusual in that it is facilitated predominantly by hydrophobic interactions. A detailed mechanism is proposed describing the substrate binding and catalytic process. PMID:18022563

  6. Structural modeling of glucanase-substrate complexes suggests a conserved tyrosine is involved in carbohydrate recognition in plant 1,3-1,4-β- d-glucanases

    NASA Astrophysics Data System (ADS)

    Tsai, Li-Chu; Chen, Yi-Ning; Shyur, Lie-Fen

    2008-12-01

    Glycosyl hydrolase family 16 (GHF16) truncated Fibrobacter succinogenes (TFs) and GHF17 barley 1,3-1,4-β- d-glucanases (β-glucanases) possess different structural folds, β-jellyroll and (β/α)8, although they both catalyze the specific hydrolysis of β-1,4 glycosidic bonds adjacent to β-1,3 linkages in mixed β-1,3 and β-1,4 β- d-glucans or lichenan. Differences in the active site region residues of TFs β-glucanase and barley β-glucanase create binding site topographies that require different substrate conformations. In contrast to barley β-glucanase, TFs β-glucanase possesses a unique and compact active site. The structural analysis results suggest that the tyrosine residue, which is conserved in all known 1,3-1,4-β- d-glucanases, is involved in the recognition of mixed β-1,3 and β-1,4 linked polysaccharide.

  7. Recognition intent and visual word recognition.

    PubMed

    Wang, Man-Ying; Ching, Chi-Le

    2009-03-01

    This study adopted a change detection task to investigate whether and how recognition intent affects the construction of orthographic representation in visual word recognition. Chinese readers (Experiment 1-1) and nonreaders (Experiment 1-2) detected color changes in radical components of Chinese characters. Explicit recognition demand was imposed in Experiment 2 by an additional recognition task. When the recognition was implicit, a bias favoring the radical location informative of character identity was found in Chinese readers (Experiment 1-1), but not nonreaders (Experiment 1-2). With explicit recognition demands, the effect of radical location interacted with radical function and word frequency (Experiment 2). An estimate of identification performance under implicit recognition was derived in Experiment 3. These findings reflect the joint influence of recognition intent and orthographic regularity in shaping readers' orthographic representation. The implication for the role of visual attention in word recognition was also discussed.

  8. DNA recognition by an RNA-guided bacterial Argonaute

    PubMed Central

    Doudna, Jennifer A.

    2017-01-01

    Argonaute (Ago) proteins are widespread in prokaryotes and eukaryotes and share a four-domain architecture capable of RNA- or DNA-guided nucleic acid recognition. Previous studies identified a prokaryotic Argonaute protein from the eubacterium Marinitoga piezophila (MpAgo), which binds preferentially to 5′-hydroxylated guide RNAs and cleaves single-stranded RNA (ssRNA) and DNA (ssDNA) targets. Here we present a 3.2 Å resolution crystal structure of MpAgo bound to a 21-nucleotide RNA guide and a complementary 21-nucleotide ssDNA substrate. Comparison of this ternary complex to other target-bound Argonaute structures reveals a unique orientation of the N-terminal domain, resulting in a straight helical axis of the entire RNA-DNA heteroduplex through the central cleft of the protein. Additionally, mismatches introduced into the heteroduplex reduce MpAgo cleavage efficiency with a symmetric profile centered around the middle of the helix. This pattern differs from the canonical mismatch tolerance of other Argonautes, which display decreased cleavage efficiency for substrates bearing sequence mismatches to the 5′ region of the guide strand. This structural analysis of MpAgo bound to a hybrid helix advances our understanding of the diversity of target recognition mechanisms by Argonaute proteins. PMID:28520746

  9. Substrate- and isoform-specific proteome stability in normal and stressed cardiac mitochondria.

    PubMed

    Lau, Edward; Wang, Ding; Zhang, Jun; Yu, Hongxiu; Lam, Maggie P Y; Liang, Xiangbo; Zong, Nobel; Kim, Tae-Young; Ping, Peipei

    2012-04-27

    Mitochondrial protein homeostasis is an essential component of the functions and oxidative stress responses of the heart. To determine the specificity and efficiency of proteome turnover of the cardiac mitochondria by endogenous and exogenous proteolytic mechanisms. Proteolytic degradation of the murine cardiac mitochondria was assessed by 2-dimensional differential gel electrophoresis and liquid chromatography-tandem mass spectrometry. Mitochondrial proteases demonstrated a substrate preference for basic protein variants, which indicates a possible recognition mechanism based on protein modifications. Endogenous mitochondrial proteases and the cytosolic 20S proteasome exhibited different substrate specificities. The cardiac mitochondrial proteome contains low amounts of proteases and is remarkably stable in isolation. Oxidative damage lowers the proteolytic capacity of cardiac mitochondria and reduces substrate availability for mitochondrial proteases. The 20S proteasome preferentially degrades specific substrates in the mitochondria and may contribute to cardiac mitochondrial proteostasis.

  10. Directing enzyme devolution for biosynthesis of alkanols and 1,n-alkanediols from natural polyhydroxy compounds.

    PubMed

    Dai, Lu; Tao, Fei; Tang, Hongzhi; Guo, Yali; Shen, Yaling; Xu, Ping

    2017-11-01

    Primordial enzymes are proposed to possess broad specificities. Through divergence and evolution, enzymes have been refined to exhibit specificity towards one reaction or substrate, and are thus commonly assumed as "specialists". However, some enzymes are "generalists" that catalyze a range of substrates and reactions. This property has been defined as enzyme promiscuity and is of great importance for the evolution of new functions. The promiscuities of two enzymes, namely glycerol dehydratase and diol dehydratase, were herein exploited for catalyzing long-chain polyols, including 1,2-butanediol, 1,2,4-butanetriol, erythritol, 1,2-pentanediol, 1,2,5-pentanetriol, and 1,2,6-hexanetriol. The specific activities required for catalyzing these six long-chain polyols were studied via in vitro enzyme assays, and the catalytic efficiencies were increased through protein engineering. The promiscuous functions were subsequently applied in vivo to establish 1,4-butanediol pathways from lignocellulose derived compounds, including xylose and erythritol. In addition, a pathway for 1-pentanol production from 1,2-pentanediol was also constructed. The results suggest that exploiting enzyme promiscuity is promising for exploring new catalysts, which would expand the repertoire of genetic elements available to synthetic biology and may provide a starting point for designing and engineering novel pathways for valuable chemicals. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  11. Self-face recognition in children with autism spectrum disorders: a near-infrared spectroscopy study.

    PubMed

    Kita, Yosuke; Gunji, Atsuko; Inoue, Yuki; Goto, Takaaki; Sakihara, Kotoe; Kaga, Makiko; Inagaki, Masumi; Hosokawa, Toru

    2011-06-01

    It is assumed that children with autism spectrum disorders (ASD) have specificities for self-face recognition, which is known to be a basic cognitive ability for social development. In the present study, we investigated neurological substrates and potentially influential factors for self-face recognition of ASD patients using near-infrared spectroscopy (NIRS). The subjects were 11 healthy adult men, 13 normally developing boys, and 10 boys with ASD. Their hemodynamic activities in the frontal area and their scanning strategies (eye-movement) were examined during self-face recognition. Other factors such as ASD severities and self-consciousness were also evaluated by parents and patients, respectively. Oxygenated hemoglobin levels were higher in the regions corresponding to the right inferior frontal gyrus than in those corresponding to the left inferior frontal gyrus. In two groups of children these activities reflected ASD severities, such that the more serious ASD characteristics corresponded with lower activity levels. Moreover, higher levels of public self-consciousness intensified the activities, which were not influenced by the scanning strategies. These findings suggest that dysfunction in the right inferior frontal gyrus areas responsible for self-face recognition is one of the crucial neural substrates underlying ASD characteristics, which could potentially be used to evaluate psychological aspects such as public self-consciousness. Copyright © 2010 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

  12. Probing the Catalytic Promiscuity of a Regio- and Stereospecific C-Glycosyltransferase from Mangifera indica.

    PubMed

    Chen, Dawei; Chen, Ridao; Wang, Ruishan; Li, Jianhua; Xie, Kebo; Bian, Chuancai; Sun, Lili; Zhang, Xiaolin; Liu, Jimei; Yang, Lin; Ye, Fei; Yu, Xiaoming; Dai, Jungui

    2015-10-19

    The catalytic promiscuity of the novel benzophenone C-glycosyltransferase, MiCGT, which is involved in the biosynthesis of mangiferin from Mangifera indica, was explored. MiCGT exhibited a robust capability to regio- and stereospecific C-glycosylation of 35 structurally diverse druglike scaffolds and simple phenolics with UDP-glucose, and also formed O- and N-glycosides. Moreover, MiCGT was able to generate C-xylosides with UDP-xylose. The OGT-reversibility of MiCGT was also exploited to generate C-glucosides with simple sugar donor. Three aryl-C-glycosides exhibited potent SGLT2 inhibitory activities with IC50  values of 2.6×, 7.6×, and 7.6×10(-7)  M, respectively. These findings demonstrate for the first time the significant potential of an enzymatic approach to diversification through C-glycosidation of bioactive natural and unnatural products in drug discovery. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Experimental evolution under hyper-promiscuity in Drosophila melanogaster.

    PubMed

    Perry, Jennifer C; Joag, Richa; Hosken, David J; Wedell, Nina; Radwan, Jacek; Wigby, Stuart

    2016-06-16

    The number of partners that individuals mate with over their lifetime is a defining feature of mating systems, and variation in mate number is thought to be a major driver of sexual evolution. Although previous research has investigated the evolutionary consequences of reductions in the number of mates, we know little about the costs and benefits of increased numbers of mates. Here, we use a genetic manipulation of mating frequency in Drosophila melanogaster to create a novel, highly promiscuous mating system. We generated D. melanogaster populations in which flies were deficient for the sex peptide receptor (SPR) gene - resulting in SPR- females that mated more frequently - and genetically-matched control populations, and allowed them to evolve for 55 generations. At several time-points during this experimental evolution, we assayed behavioural, morphological and transcriptional reproductive phenotypes expected to evolve in response to increased population mating frequencies. We found that males from the high mating frequency SPR- populations evolved decreased ability to inhibit the receptivity of their mates and decreased copulation duration, in line with predictions of decreased per-mating investment with increased sperm competition. Unexpectedly, SPR- population males also evolved weakly increased sex peptide (SP) gene expression. Males from SPR- populations initially (i.e., before experimental evolution) exhibited more frequent courtship and faster time until mating relative to controls, but over evolutionary time these differences diminished or reversed. In response to experimentally increased mating frequency, SPR- males evolved behavioural responses consistent with decreased male post-copulatory investment at each mating and decreased overall pre-copulatory performance. The trend towards increased SP gene expression might plausibly relate to functional differences in the two domains of the SP protein. Our study highlights the utility of genetic

  14. An Ecological Analysis of the Effects of Deviant Peer Clustering on Sexual Promiscuity, Problem Behavior, and Childbearing from Early Adolescence to Adulthood: An Enhancement of the Life History Framework

    ERIC Educational Resources Information Center

    Dishion, Thomas J.; Ha, Thao; Veronneau, Marie-Helene

    2012-01-01

    The authors propose that peer relationships should be included in a life history perspective on adolescent problem behavior. Longitudinal analyses were used to examine deviant peer clustering as the mediating link between attenuated family ties, peer marginalization, and social disadvantage in early adolescence and sexual promiscuity in middle…

  15. Structural Basis for Prereceptor Modulation of Plant Hormones by GH3 Proteins

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

    Westfall, Corey S.; Zubieta, Chloe; Herrmann, Jonathan

    Acyl acid amido synthetases of the GH3 family act as critical prereceptor modulators of plant hormone action; however, the molecular basis for their hormone selectivity is unclear. Here, we report the crystal structures of benzoate-specific Arabidopsis thaliana AtGH3.12/PBS3 and jasmonic acid-specific AtGH3.11/JAR1. These structures, combined with biochemical analysis, define features for the conjugation of amino acids to diverse acyl acid substrates and highlight the importance of conformational changes in the carboxyl-terminal domain for catalysis. We also identify residues forming the acyl acid binding site across the GH3 family and residues critical for amino acid recognition. Our results demonstrate how amore » highly adaptable three-dimensional scaffold is used for the evolution of promiscuous activity across an enzyme family for modulation of plant signaling molecules.« less

  16. Access channels to the buried active site control substrate specificity in CYP1A P450 enzymes.

    PubMed

    Urban, Philippe; Truan, Gilles; Pompon, Denis

    2015-04-01

    A cytochrome P450 active site is buried within the protein molecule and several channels connect the catalytic cavity to the protein surface. Their role in P450 catalysis is still matter of debate. The aim of this study was to understand the possible relations existing between channels and substrate specificity. Time course studies were carried out with a collection of polycyclic substrates of increasing sizes assayed with a library of wild-type and chimeric CYP1A enzymes. This resulted in a matrix of activities sufficiently large to allow statistical analysis. Multivariate statistical tools were used to decipher the correlation between observed activity shifts and sequence segment swaps. The global kinetic behavior of CYP1A enzymes toward polycyclic substrates is significantly different depending on the size of the substrate. Mutations which are close or lining the P450 channels significantly affect this discrimination, whereas mutations distant from the P450 channels do not. Size discrimination is taking place for polycyclic substrates at the entrance of the different P450 access channels. It is thus hypothesized that channels differentiate small from large substrates in CYP1A enzymes, implying that residues located at the surface of the protein may be implied in this differential recognition. Catalysis thus occurs after a two-step recognition process, one at the surface of the protein and the second within the catalytic cavity in enzymes with a buried active site. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Discrete Neural Correlates for the Recognition of Negative Emotions: Insights from Frontotemporal Dementia

    PubMed Central

    Kumfor, Fiona; Irish, Muireann; Hodges, John R.; Piguet, Olivier

    2013-01-01

    Patients with frontotemporal dementia have pervasive changes in emotion recognition and social cognition, yet the neural changes underlying these emotion processing deficits remain unclear. The multimodal system model of emotion proposes that basic emotions are dependent on distinct brain regions, which undergo significant pathological changes in frontotemporal dementia. As such, this syndrome may provide important insight into the impact of neural network degeneration upon the innate ability to recognise emotions. This study used voxel-based morphometry to identify discrete neural correlates involved in the recognition of basic emotions (anger, disgust, fear, sadness, surprise and happiness) in frontotemporal dementia. Forty frontotemporal dementia patients (18 behavioural-variant, 11 semantic dementia, 11 progressive nonfluent aphasia) and 27 healthy controls were tested on two facial emotion recognition tasks: The Ekman 60 and Ekman Caricatures. Although each frontotemporal dementia group showed impaired recognition of negative emotions, distinct associations between emotion-specific task performance and changes in grey matter intensity emerged. Fear recognition was associated with the right amygdala; disgust recognition with the left insula; anger recognition with the left middle and superior temporal gyrus; and sadness recognition with the left subcallosal cingulate, indicating that discrete neural substrates are necessary for emotion recognition in frontotemporal dementia. The erosion of emotion-specific neural networks in neurodegenerative disorders may produce distinct profiles of performance that are relevant to understanding the neurobiological basis of emotion processing. PMID:23805313

  18. Structural insight into mechanism and diverse substrate selection strategy of L-ribulokinase

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

    Agarwal R.; Swaminathan S.; Burley, S. K.

    2012-01-01

    The araBAD operon encodes three different enzymes required for catabolism of L-arabinose, which is one of the most abundant monosaccharides in nature. L-ribulokinase, encoded by the araB gene, catalyzes conversion of L-ribulose to L-ribulose-5-phosphate, the second step in the catabolic pathway. Unlike other kinases, ribulokinase exhibits diversity in substrate selectivity and catalyzes phosphorylation of all four 2-ketopentose sugars with comparable k{sub cat} values. To understand ribulokinase recognition and phosphorylation of a diverse set of substrates, we have determined the X-ray structure of ribulokinase from Bacillus halodurans bound to L-ribulose and investigated its substrate and ATP co-factor binding properties. The polypeptidemore » chain is folded into two domains, one small and the other large, with a deep cleft in between. By analogy with related sugar kinases, we identified {sup 447}{und GG}LPQ{und K}{sup 452} as the ATP-binding motif within the smaller domain. L-ribulose binds in the cleft between the two domains via hydrogen bonds with the side chains of highly conserved Trp126, Lys208, Asp274, and Glu329 and the main chain nitrogen of Ala96. The interaction of L-ribulokinase with L-ribulose reveals versatile structural features that help explain recognition of various 2-ketopentose substrates and competitive inhibition by L-erythrulose. Comparison of our structure to that of the structures of other sugar kinases revealed conformational variations that suggest domain-domain closure movements are responsible for establishing the observed active site environment.« less

  19. Resveratrol serves as a protein-substrate interaction stabilizer in human SIRT1 activation

    NASA Astrophysics Data System (ADS)

    Hou, Xuben; Rooklin, David; Fang, Hao; Zhang, Yingkai

    2016-11-01

    Resveratrol is a natural compound found in red wine that has been suggested to exert its potential health benefit through the activation of SIRT1, a crucial member of the mammalian NAD+-dependent deacetylases. SIRT1 has emerged as an attractive therapeutic target for many aging related diseases, however, how its activity can only be activated toward some specific substrates by resveratrol has been poorly understood. Herein, by employing extensive molecular dynamics simulations as well as fragment-centric topographical mapping of binding interfaces, we have clarified current controversies in the literature and elucidated that resveratrol plays an important activation role by stabilizing SIRT1/peptide interactions in a substrate-specific manner. This new mechanism highlights the importance of the N-terminal domain in substrate recognition, explains the activity restoration role of resveratrol toward some “loose-binding” substrates of SIRT1, and has significant implications for the rational design of new substrate-specific SIRT1 modulators.

  20. Resveratrol serves as a protein-substrate interaction stabilizer in human SIRT1 activation.

    PubMed

    Hou, Xuben; Rooklin, David; Fang, Hao; Zhang, Yingkai

    2016-11-30

    Resveratrol is a natural compound found in red wine that has been suggested to exert its potential health benefit through the activation of SIRT1, a crucial member of the mammalian NAD + -dependent deacetylases. SIRT1 has emerged as an attractive therapeutic target for many aging related diseases, however, how its activity can only be activated toward some specific substrates by resveratrol has been poorly understood. Herein, by employing extensive molecular dynamics simulations as well as fragment-centric topographical mapping of binding interfaces, we have clarified current controversies in the literature and elucidated that resveratrol plays an important activation role by stabilizing SIRT1/peptide interactions in a substrate-specific manner. This new mechanism highlights the importance of the N-terminal domain in substrate recognition, explains the activity restoration role of resveratrol toward some "loose-binding" substrates of SIRT1, and has significant implications for the rational design of new substrate-specific SIRT1 modulators.

  1. Substrate-Driven Mapping of the Degradome by Comparison of Sequence Logos

    PubMed Central

    Fuchs, Julian E.; von Grafenstein, Susanne; Huber, Roland G.; Kramer, Christian; Liedl, Klaus R.

    2013-01-01

    Sequence logos are frequently used to illustrate substrate preferences and specificity of proteases. Here, we employed the compiled substrates of the MEROPS database to introduce a novel metric for comparison of protease substrate preferences. The constructed similarity matrix of 62 proteases can be used to intuitively visualize similarities in protease substrate readout via principal component analysis and construction of protease specificity trees. Since our new metric is solely based on substrate data, we can engraft the protease tree including proteolytic enzymes of different evolutionary origin. Thereby, our analyses confirm pronounced overlaps in substrate recognition not only between proteases closely related on sequence basis but also between proteolytic enzymes of different evolutionary origin and catalytic type. To illustrate the applicability of our approach we analyze the distribution of targets of small molecules from the ChEMBL database in our substrate-based protease specificity trees. We observe a striking clustering of annotated targets in tree branches even though these grouped targets do not necessarily share similarity on protein sequence level. This highlights the value and applicability of knowledge acquired from peptide substrates in drug design of small molecules, e.g., for the prediction of off-target effects or drug repurposing. Consequently, our similarity metric allows to map the degradome and its associated drug target network via comparison of known substrate peptides. The substrate-driven view of protein-protein interfaces is not limited to the field of proteases but can be applied to any target class where a sufficient amount of known substrate data is available. PMID:24244149

  2. Emotion recognition deficits associated with ventromedial prefrontal cortex lesions are improved by gaze manipulation.

    PubMed

    Wolf, Richard C; Pujara, Maia; Baskaya, Mustafa K; Koenigs, Michael

    2016-09-01

    Facial emotion recognition is a critical aspect of human communication. Since abnormalities in facial emotion recognition are associated with social and affective impairment in a variety of psychiatric and neurological conditions, identifying the neural substrates and psychological processes underlying facial emotion recognition will help advance basic and translational research on social-affective function. Ventromedial prefrontal cortex (vmPFC) has recently been implicated in deploying visual attention to the eyes of emotional faces, although there is mixed evidence regarding the importance of this brain region for recognition accuracy. In the present study of neurological patients with vmPFC damage, we used an emotion recognition task with morphed facial expressions of varying intensities to determine (1) whether vmPFC is essential for emotion recognition accuracy, and (2) whether instructed attention to the eyes of faces would be sufficient to improve any accuracy deficits. We found that vmPFC lesion patients are impaired, relative to neurologically healthy adults, at recognizing moderate intensity expressions of anger and that recognition accuracy can be improved by providing instructions of where to fixate. These results suggest that vmPFC may be important for the recognition of facial emotion through a role in guiding visual attention to emotionally salient regions of faces. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Nucleophile Promiscuity of Natural and Engineered Aldolases.

    PubMed

    Clapes, Pere; Hernández, Karel; Szekrenyi, Anna

    2018-04-12

    Asymmetric aldol addition reaction mediated by aldolases is recognized as a green and sustainable way for carbon-carbon bond formation. Research in this line has unveiled their unprecedented synthetic potentiality toward diverse new chemical structures, novel product families and even as a technology for industrial manufacturing processes. Despite that, aldolases have long been regarded as strictly selective catalysts, particularly for the nucleophilic substrate, limiting their broad applicability. In recent years, the advances in screening technologies and metagenomics uncovered novel C-C biocatalysts from superfamilies of widely known lyases. Moreover, protein engineering revealed the extraordinary malleability of different carboligases, offering a toolbox of biocatalysts active towards a large structural diversity of nucleophile substrates. In this paper, the nucleophile ambiguity of native and engineered aldolases is discussed with recent examples proving this novel concept. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Deciphering kinase-substrate relationships by analysis of domain-specific phosphorylation network.

    PubMed

    Damle, Nikhil Prakash; Mohanty, Debasisa

    2014-06-15

    In silico prediction of site-specific kinase-substrate relationships (ssKSRs) is crucial for deciphering phosphorylation networks by linking kinomes to phosphoproteomes. However, currently available predictors for ssKSRs give rise to a large number of false-positive results because they use only a short sequence stretch around phosphosite as determinants of kinase specificity and do not consider the biological context of kinase-substrate recognition. Based on the analysis of domain-specific kinase-substrate relationships, we have constructed a domain-level phosphorylation network that implicitly incorporates various contextual factors. It reveals preferential phosphorylation of specific domains by certain kinases. These novel correlations have been implemented in PhosNetConstruct, an automated program for predicting target kinases for a substrate protein. PhosNetConstruct distinguishes cognate kinase-substrate pairs from a large number of non-cognate combinations. Benchmarking on independent datasets using various statistical measures demonstrates the superior performance of PhosNetConstruct over ssKSR-based predictors. PhosNetConstruct is freely available at http://www.nii.ac.in/phosnetconstruct.html. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  5. Crystal structure of the human cytosolic sialidase Neu2. Evidence for the dynamic nature of substrate recognition.

    PubMed

    Chavas, Leonard M G; Tringali, Cristina; Fusi, Paola; Venerando, Bruno; Tettamanti, Guido; Kato, Ryuichi; Monti, Eugenio; Wakatsuki, Soichi

    2005-01-07

    Gangliosides play key roles in cell differentiation, cell-cell interactions, and transmembrane signaling. Sialidases hydrolyze sialic acids to produce asialo compounds, which is the first step of degradation processes of glycoproteins and gangliosides. Sialidase involvement has been implicated in some lysosomal storage disorders such as sialidosis and galactosialidosis. Neu2 is a recently identified human cytosolic sialidase. Here we report the first high resolution x-ray structures of mammalian sialidase, human Neu2, in its apo form and in complex with an inhibitor, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA). The structure shows the canonical six-blade beta-propeller observed in viral and bacterial sialidases with its active site in a shallow crevice. In the complex structure, the inhibitor lies in the catalytic crevice surrounded by ten amino acids. In particular, the arginine triad, conserved among sialidases, aids in the proper positioning of the carboxylate group of DANA within the active site region. The tyrosine residue, Tyr(334), conserved among mammalian and bacterial sialidases as well as in viral neuraminidases, facilitates the enzymatic reaction by stabilizing a putative carbonium ion in the transition state. The loops containing Glu(111) and the catalytic aspartate Asp(46) are disordered in the apo form but upon binding of DANA become ordered to adopt two short alpha-helices to cover the inhibitor, illustrating the dynamic nature of substrate recognition. The N-acetyl and glycerol moieties of DANA are recognized by Neu2 residues not shared by bacterial sialidases and viral neuraminidases, which can be regarded as a key structural difference for potential drug design against bacteria, influenza, and other viruses.

  6. Computer-assisted prediction of HLA-DR binding and experimental analysis for human promiscuous Th1-cell peptides in the 24 kDa secreted lipoprotein (LppX) of Mycobacterium tuberculosis.

    PubMed

    Al-Attiyah, R; Mustafa, A S

    2004-01-01

    The secreted 24 kDa lipoprotein (LppX) is an antigen that is specific for Mycobacterium tuberculosis complex and M. leprae. The present study was carried out to identify the promiscuous T helper 1 (Th1)-cell epitopes of the M. tuberculosis LppX (MT24, Rv2945c) antigen by using 15 overlapping synthetic peptides (25 mers overlapping by 10 residues) covering the sequence of the complete protein. The analysis of Rv2945c sequence for binding to 51 alleles of nine serologically defined HLA-DR molecules, by using a virtual matrix-based prediction program (propred), showed that eight of the 15 peptides of Rv2945c were predicted to bind promiscuously to >/=10 alleles from more than or equal to three serologically defined HLA-DR molecules. The Th1-cell reactivity of all the peptides was assessed in antigen-induced proliferation and interferon-gamma (IFN-gamma)-secretion assays with peripheral blood mononuclear cells (PBMCs) from 37 bacille Calmette-Guérin (BCG)-vaccinated healthy subjects. The results showed that 17 of the 37 donors, which represented an HLA-DR-heterogeneous group, responded to one or more peptides of Rv2945c in the Th1-cell assays. Although each peptide stimulated PBMCs from one or more donors in the above assays, the best positive responses (12/17 (71%) responders) were observed with the peptide p14 (aa 196-220). This suggested a highly promiscuous presentation of p14 to Th1 cells. In addition, the sequence of p14 is completely identical among the LppX of M. tuberculosis, M. bovis and M. leprae, which further supports the usefulness of Rv2945c and p14 in the subunit vaccine design against both tuberculosis and leprosy.

  7. Mechanistic insights into metal ion activation and operator recognition by the ferric uptake regulator

    NASA Astrophysics Data System (ADS)

    Deng, Zengqin; Wang, Qing; Liu, Zhao; Zhang, Manfeng; Machado, Ana Carolina Dantas; Chiu, Tsu-Pei; Feng, Chong; Zhang, Qi; Yu, Lin; Qi, Lei; Zheng, Jiangge; Wang, Xu; Huo, Xinmei; Qi, Xiaoxuan; Li, Xiaorong; Wu, Wei; Rohs, Remo; Li, Ying; Chen, Zhongzhou

    2015-07-01

    Ferric uptake regulator (Fur) plays a key role in the iron homeostasis of prokaryotes, such as bacterial pathogens, but the molecular mechanisms and structural basis of Fur-DNA binding remain incompletely understood. Here, we report high-resolution structures of Magnetospirillum gryphiswaldense MSR-1 Fur in four different states: apo-Fur, holo-Fur, the Fur-feoAB1 operator complex and the Fur-Pseudomonas aeruginosa Fur box complex. Apo-Fur is a transition metal ion-independent dimer whose binding induces profound conformational changes and confers DNA-binding ability. Structural characterization, mutagenesis, biochemistry and in vivo data reveal that Fur recognizes DNA by using a combination of base readout through direct contacts in the major groove and shape readout through recognition of the minor-groove electrostatic potential by lysine. The resulting conformational plasticity enables Fur binding to diverse substrates. Our results provide insights into metal ion activation and substrate recognition by Fur that suggest pathways to engineer magnetotactic bacteria and antipathogenic drugs.

  8. Structural basis of substrate discrimination and integrin binding by autotaxin

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

    Hausmann, Jens; Kamtekar, Satwik; Christodoulou, Evangelos

    2013-09-25

    Autotaxin (ATX, also known as ectonucleotide pyrophosphatase/phosphodiesterase-2, ENPP2) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemoattractant for many cell types. ATX-LPA signaling is involved in various pathologies including tumor progression and inflammation. However, the molecular basis of substrate recognition and catalysis by ATX and the mechanism by which it interacts with target cells are unclear. Here, we present the crystal structure of ATX, alone and in complex with a small-molecule inhibitor. We have identified a hydrophobic lipid-binding pocket and mapped key residues for catalysis and selection between nucleotide and phospholipid substrates.more » We have shown that ATX interacts with cell-surface integrins through its N-terminal somatomedin B-like domains, using an atypical mechanism. Our results define determinants of substrate discrimination by the ENPP family, suggest how ATX promotes localized LPA signaling and suggest new approaches for targeting ATX with small-molecule therapeutic agents.« less

  9. Substrate-induced ubiquitylation and endocytosis of yeast amino acid permeases.

    PubMed

    Ghaddar, Kassem; Merhi, Ahmad; Saliba, Elie; Krammer, Eva-Maria; Prévost, Martine; André, Bruno

    2014-12-01

    Many plasma membrane transporters are downregulated by ubiquitylation, endocytosis, and delivery to the lysosome in response to various stimuli. We report here that two amino acid transporters of Saccharomyces cerevisiae, the general amino acid permease (Gap1) and the arginine-specific permease (Can1), undergo ubiquitin-dependent downregulation in response to their substrates and that this downregulation is not due to intracellular accumulation of the transported amino acids but to transport catalysis itself. Following an approach based on permease structural modeling, mutagenesis, and kinetic parameter analysis, we obtained evidence that substrate-induced endocytosis requires transition of the permease to a conformational state preceding substrate release into the cell. Furthermore, this transient conformation must be stable enough, and thus sufficiently populated, for the permease to undergo efficient downregulation. Additional observations, including the constitutive downregulation of two active Gap1 mutants altered in cytosolic regions, support the model that the substrate-induced conformational transition inducing endocytosis involves remodeling of cytosolic regions of the permeases, thereby promoting their recognition by arrestin-like adaptors of the Rsp5 ubiquitin ligase. Similar mechanisms might control many other plasma membrane transporters according to the external concentrations of their substrates. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  10. The Promiscuity of [beta]-Strand Pairing Allows for Rational Design of [beta]-Sheet Face Inversion

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

    Makabe, Koki; Koide, Shohei

    2009-06-17

    Recent studies suggest the dominant role of main-chain H-bond formation in specifying {beta}-sheet topology. Its essentially sequence-independent nature implies a large degree of freedom in designing {beta}-sheet-based nanomaterials. Here we show rational design of {beta}-sheet face inversions by incremental deletions of {beta}-strands from the single-layer {beta}-sheet of Borrelia outer surface protein A. We show that a {beta}-sheet structure can be maintained when a large number of native contacts are removed and that one can design large-scale conformational transitions of a {beta}-sheet such as face inversion by exploiting the promiscuity of strand-strand interactions. High-resolution X-ray crystal structures confirmed the success ofmore » the design and supported the importance of main-chain H-bonds in determining {beta}-sheet topology. This work suggests a simple but effective strategy for designing and controlling nanomaterials based on {beta}-rich peptide self-assemblies.« less

  11. 8 CFR 1292.2 - Organizations qualified for recognition; requests for recognition; withdrawal of recognition...

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ...; requests for recognition; withdrawal of recognition; accreditation of representatives; roster. 1292.2...; requests for recognition; withdrawal of recognition; accreditation of representatives; roster. (a) Qualifications of organizations. A non-profit religious, charitable, social service, or similar organization...

  12. Long-range electrostatic complementarity governs substrate recognition by human chymotrypsin C, a key regulator of digestive enzyme activation.

    PubMed

    Batra, Jyotica; Szabó, András; Caulfield, Thomas R; Soares, Alexei S; Sahin-Tóth, Miklós; Radisky, Evette S

    2013-04-05

    Human chymotrypsin C (CTRC) is a pancreatic serine protease that regulates activation and degradation of trypsinogens and procarboxypeptidases by targeting specific cleavage sites within their zymogen precursors. In cleaving these regulatory sites, which are characterized by multiple flanking acidic residues, CTRC shows substrate specificity that is distinct from that of other isoforms of chymotrypsin and elastase. Here, we report the first crystal structure of active CTRC, determined at 1.9-Å resolution, revealing the structural basis for binding specificity. The structure shows human CTRC bound to the small protein protease inhibitor eglin c, which binds in a substrate-like manner filling the S6-S5' subsites of the substrate binding cleft. Significant binding affinity derives from burial of preferred hydrophobic residues at the P1, P4, and P2' positions of CTRC, although acidic P2' residues can also be accommodated by formation of an interfacial salt bridge. Acidic residues may also be specifically accommodated in the P6 position. The most unique structural feature of CTRC is a ring of intense positive electrostatic surface potential surrounding the primarily hydrophobic substrate binding site. Our results indicate that long-range electrostatic attraction toward substrates of concentrated negative charge governs substrate discrimination, which explains CTRC selectivity in regulating active digestive enzyme levels.

  13. A conserved loop-wedge motif moderates reaction site search and recognition by FEN1.

    PubMed

    Thompson, Mark J; Gotham, Victoria J B; Ciani, Barbara; Grasby, Jane A

    2018-06-07

    DNA replication and repair frequently involve intermediate two-way junction structures with overhangs, or flaps, that must be promptly removed; a task performed by the essential enzyme flap endonuclease 1 (FEN1). We demonstrate a functional relationship between two intrinsically disordered regions of the FEN1 protein, which recognize opposing sides of the junction and order in response to the requisite substrate. Our results inform a model in which short-range translocation of FEN1 on DNA facilitates search for the annealed 3'-terminus of a primer strand, which is recognized by breaking the terminal base pair to generate a substrate with a single nucleotide 3'-flap. This recognition event allosterically signals hydrolytic removal of the 5'-flap through reaction in the opposing junction duplex, by controlling access of the scissile phosphate diester to the active site. The recognition process relies on a highly-conserved 'wedge' residue located on a mobile loop that orders to bind the newly-unpaired base. The unanticipated 'loop-wedge' mechanism exerts control over substrate selection, rate of reaction and reaction site precision, and shares features with other enzymes that recognize irregular DNA structures. These new findings reveal how FEN1 precisely couples 3'-flap verification to function.

  14. Promiscuous interactions and protein disaggregases determine the material state of stress-inducible RNP granules

    PubMed Central

    Kroschwald, Sonja; Maharana, Shovamayee; Mateju, Daniel; Malinovska, Liliana; Nüske, Elisabeth; Poser, Ina; Richter, Doris; Alberti, Simon

    2015-01-01

    RNA-protein (RNP) granules have been proposed to assemble by forming solid RNA/protein aggregates or through phase separation into a liquid RNA/protein phase. Which model describes RNP granules in living cells is still unclear. In this study, we analyze P bodies in budding yeast and find that they have liquid-like properties. Surprisingly, yeast stress granules adopt a different material state, which is reminiscent of solid protein aggregates and controlled by protein disaggregases. By using an assay to ectopically nucleate RNP granules, we further establish that RNP granule formation does not depend on amyloid-like aggregation but rather involves many promiscuous interactions. Finally, we show that stress granules have different properties in mammalian cells, where they show liquid-like behavior. Thus, we propose that the material state of RNP granules is flexible and that the solid state of yeast stress granules is an adaptation to extreme environments, made possible by the presence of a powerful disaggregation machine. DOI: http://dx.doi.org/10.7554/eLife.06807.001 PMID:26238190

  15. The neural substrates of recognition memory for verbal information: spanning the divide between short- and long-term memory.

    PubMed

    Buchsbaum, Bradley R; Padmanabhan, Aarthi; Berman, Karen Faith

    2011-04-01

    One of the classic categorical divisions in the history of memory research is that between short-term and long-term memory. Indeed, because memory for the immediate past (a few seconds) and memory for the relatively more remote past (several seconds and beyond) are assumed to rely on distinct neural systems, more often than not, memory research has focused either on short- (or "working memory") or on long-term memory. Using an auditory-verbal continuous recognition paradigm designed for fMRI, we examined how the neural signatures of recognition memory change across an interval of time (from 2.5 to 30 sec) that spans this hypothetical division between short- and long-term memory. The results revealed that activity during successful auditory-verbal item recognition in inferior parietal cortex and the posterior superior temporal lobe was maximal for early lags, whereas, conversely, activity in the left inferior frontal gyrus increased as a function of lag. Taken together, the results reveal that as the interval between item repetitions increases, there is a shift in the distribution of memory-related activity that moves from posterior temporo-parietal cortex (lags 1-4) to inferior frontal regions (lags 5-10), indicating that as time advances, the burden of recognition memory is increasingly placed on top-down retrieval mechanisms that are mediated by structures in inferior frontal cortex.

  16. Specific Impairments in the Recognition of Emotional Facial Expressions in Parkinson’s Disease

    PubMed Central

    Clark, Uraina S.; Neargarder, Sandy; Cronin-Golomb, Alice

    2008-01-01

    Studies investigating the ability to recognize emotional facial expressions in non-demented individuals with Parkinson’s disease (PD) have yielded equivocal findings. A possible reason for this variability may lie in the confounding of emotion recognition with cognitive task requirements, a confound arising from the lack of a control condition using non-emotional stimuli. The present study examined emotional facial expression recognition abilities in 20 non-demented patients with PD and 23 control participants relative to their performances on a non-emotional landscape categorization test with comparable task requirements. We found that PD participants were normal on the control task but exhibited selective impairments in the recognition of facial emotion, specifically for anger (driven by those with right hemisphere pathology) and surprise (driven by those with left hemisphere pathology), even when controlling for depression level. Male but not female PD participants further displayed specific deficits in the recognition of fearful expressions. We suggest that the neural substrates that may subserve these impairments include the ventral striatum, amygdala, and prefrontal cortices. Finally, we observed that in PD participants, deficiencies in facial emotion recognition correlated with higher levels of interpersonal distress, which calls attention to the significant psychosocial impact that facial emotion recognition impairments may have on individuals with PD. PMID:18485422

  17. Long-range Electrostatic Complementarity Governs Substrate Recognition by Human Chymotrypsin C, a Key Regulator of Digestive Enzyme Activation*

    PubMed Central

    Batra, Jyotica; Szabó, András; Caulfield, Thomas R.; Soares, Alexei S.; Sahin-Tóth, Miklós; Radisky, Evette S.

    2013-01-01

    Human chymotrypsin C (CTRC) is a pancreatic serine protease that regulates activation and degradation of trypsinogens and procarboxypeptidases by targeting specific cleavage sites within their zymogen precursors. In cleaving these regulatory sites, which are characterized by multiple flanking acidic residues, CTRC shows substrate specificity that is distinct from that of other isoforms of chymotrypsin and elastase. Here, we report the first crystal structure of active CTRC, determined at 1.9-Å resolution, revealing the structural basis for binding specificity. The structure shows human CTRC bound to the small protein protease inhibitor eglin c, which binds in a substrate-like manner filling the S6-S5′ subsites of the substrate binding cleft. Significant binding affinity derives from burial of preferred hydrophobic residues at the P1, P4, and P2′ positions of CTRC, although acidic P2′ residues can also be accommodated by formation of an interfacial salt bridge. Acidic residues may also be specifically accommodated in the P6 position. The most unique structural feature of CTRC is a ring of intense positive electrostatic surface potential surrounding the primarily hydrophobic substrate binding site. Our results indicate that long-range electrostatic attraction toward substrates of concentrated negative charge governs substrate discrimination, which explains CTRC selectivity in regulating active digestive enzyme levels. PMID:23430245

  18. Conserved Sequence Preferences Contribute to Substrate Recognition by the Proteasome*

    PubMed Central

    Yu, Houqing; Singh Gautam, Amit K.; Wilmington, Shameika R.; Wylie, Dennis; Martinez-Fonts, Kirby; Kago, Grace; Warburton, Marie; Chavali, Sreenivas; Inobe, Tomonao; Finkelstein, Ilya J.; Babu, M. Madan

    2016-01-01

    The proteasome has pronounced preferences for the amino acid sequence of its substrates at the site where it initiates degradation. Here, we report that modulating these sequences can tune the steady-state abundance of proteins over 2 orders of magnitude in cells. This is the same dynamic range as seen for inducing ubiquitination through a classic N-end rule degron. The stability and abundance of His3 constructs dictated by the initiation site affect survival of yeast cells and show that variation in proteasomal initiation can affect fitness. The proteasome's sequence preferences are linked directly to the affinity of the initiation sites to their receptor on the proteasome and are conserved between Saccharomyces cerevisiae, Schizosaccharomyces pombe, and human cells. These findings establish that the sequence composition of unstructured initiation sites influences protein abundance in vivo in an evolutionarily conserved manner and can affect phenotype and fitness. PMID:27226608

  19. Integrating the protein and metabolic engineering toolkits for next-generation chemical biosynthesis.

    PubMed

    Pirie, Christopher M; De Mey, Marjan; Jones Prather, Kristala L; Ajikumar, Parayil Kumaran

    2013-04-19

    Through microbial engineering, biosynthesis has the potential to produce thousands of chemicals used in everyday life. Metabolic engineering and synthetic biology are fields driven by the manipulation of genes, genetic regulatory systems, and enzymatic pathways for developing highly productive microbial strains. Fundamentally, it is the biochemical characteristics of the enzymes themselves that dictate flux through a biosynthetic pathway toward the product of interest. As metabolic engineers target sophisticated secondary metabolites, there has been little recognition of the reduced catalytic activity and increased substrate/product promiscuity of the corresponding enzymes compared to those of central metabolism. Thus, fine-tuning these enzymatic characteristics through protein engineering is paramount for developing high-productivity microbial strains for secondary metabolites. Here, we describe the importance of protein engineering for advancing metabolic engineering of secondary metabolism pathways. This pathway integrated enzyme optimization can enhance the collective toolkit of microbial engineering to shape the future of chemical manufacturing.

  20. Mapping substrate interactions of the human membrane-associated neuraminidase, NEU3, using STD NMR.

    PubMed

    Albohy, Amgad; Richards, Michele R; Cairo, Christopher W

    2015-03-01

    Saturation transfer difference (STD) nuclear magnetic resonance (NMR) is a powerful technique which can be used to investigate interactions between proteins and their substrates. The method identifies specific sites of interaction found on a small molecule ligand when in complex with a protein. The ability of STD NMR to provide specific insight into binding interactions in the absence of other structural data is an attractive feature for its use with membrane proteins. We chose to employ STD NMR in our ongoing investigations of the human membrane-associated neuraminidase NEU3 and its interaction with glycolipid substrates (e.g., GM3). In order to identify critical substrate-enzyme interactions, we performed STD NMR with a catalytically inactive form of the enzyme, NEU3(Y370F), containing an N-terminal maltose-binding protein (MBP)-affinity tag. In the absence of crystallographic data on the enzyme, these data represent a critical experimental test of proposed homology models, as well as valuable new structural data. To aid interpretation of the STD NMR data, we compared the results with molecular dynamics (MD) simulations of the enzyme-substrate complexes. We find that the homology model is able to predict essential features of the experimental data, including close contact of the hydrophobic aglycone and the Neu5Ac residue with the enzyme. Additionally, the model and STD NMR data agree on the facial recognition of the galactose and glucose residues of the GM3-analog studied. We conclude that the homology model of NEU3 can be used to predict substrate recognition, but our data indicate that unstructured portions of the NEU3 model may require further refinement. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  1. Understanding Substrate Selectivity of Human UDP-glucuronosyltransferases through QSAR modeling and analysis of homologous enzymes

    PubMed Central

    Dong, Dong; Ako, Roland; Hu, Ming; Wu, Baojian

    2015-01-01

    The UDP-glucuronosyltransferase (UGT) enzyme catalyzes the glucuronidation reaction which is a major metabolic and detoxification pathway in humans. Understanding the mechanisms for substrate recognition by UGT assumes great importance in an attempt to predict its contribution to xenobiotic/drug disposition in vivo. Spurred on by this interest, 2D/3D-quantitative structure activity relationships (QSAR) and pharmacophore models have been established in the absence of a complete mammalian UGT crystal structure. This review discusses the recent progress in modeling human UGT substrates including those with multiple sites of glucuronidation. A better understanding of UGT active site contributing to substrate selectivity (and regioselectivity) from the homologous enzymes (i.e., plant and bacterial UGTs, all belong to family 1 of glycosyltransferase (GT1)) is also highlighted, as these enzymes share a common catalytic mechanism and/or overlapping substrate selectivity. PMID:22385482

  2. DNA/RNA hybrid substrates modulate the catalytic activity of purified AID.

    PubMed

    Abdouni, Hala S; King, Justin J; Ghorbani, Atefeh; Fifield, Heather; Berghuis, Lesley; Larijani, Mani

    2018-01-01

    Activation-induced cytidine deaminase (AID) converts cytidine to uridine at Immunoglobulin (Ig) loci, initiating somatic hypermutation and class switching of antibodies. In vitro, AID acts on single stranded DNA (ssDNA), but neither double-stranded DNA (dsDNA) oligonucleotides nor RNA, and it is believed that transcription is the in vivo generator of ssDNA targeted by AID. It is also known that the Ig loci, particularly the switch (S) regions targeted by AID are rich in transcription-generated DNA/RNA hybrids. Here, we examined the binding and catalytic behavior of purified AID on DNA/RNA hybrid substrates bearing either random sequences or GC-rich sequences simulating Ig S regions. If substrates were made up of a random sequence, AID preferred substrates composed entirely of DNA over DNA/RNA hybrids. In contrast, if substrates were composed of S region sequences, AID preferred to mutate DNA/RNA hybrids over substrates composed entirely of DNA. Accordingly, AID exhibited a significantly higher affinity for binding DNA/RNA hybrid substrates composed specifically of S region sequences, than any other substrates composed of DNA. Thus, in the absence of any other cellular processes or factors, AID itself favors binding and mutating DNA/RNA hybrids composed of S region sequences. AID:DNA/RNA complex formation and supporting mutational analyses suggest that recognition of DNA/RNA hybrids is an inherent structural property of AID. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. CD94-NKG2A recognition of human leukocyte antigen (HLA)-E bound to an HLA class I leader sequence.

    PubMed

    Petrie, Emma J; Clements, Craig S; Lin, Jie; Sullivan, Lucy C; Johnson, Darryl; Huyton, Trevor; Heroux, Annie; Hoare, Hilary L; Beddoe, Travis; Reid, Hugh H; Wilce, Matthew C J; Brooks, Andrew G; Rossjohn, Jamie

    2008-03-17

    The recognition of human leukocyte antigen (HLA)-E by the heterodimeric CD94-NKG2 natural killer (NK) receptor family is a central innate mechanism by which NK cells monitor the expression of other HLA molecules, yet the structural basis of this highly specific interaction is unclear. Here, we describe the crystal structure of CD94-NKG2A in complex with HLA-E bound to a peptide derived from the leader sequence of HLA-G. The CD94 subunit dominated the interaction with HLA-E, whereas the NKG2A subunit was more peripheral to the interface. Moreover, the invariant CD94 subunit dominated the peptide-mediated contacts, albeit with poor surface and chemical complementarity. This unusual binding mode was consistent with mutagenesis data at the CD94-NKG2A-HLA-E interface. There were few conformational changes in either CD94-NKG2A or HLA-E upon ligation, and such a "lock and key" interaction is typical of innate receptor-ligand interactions. Nevertheless, the structure also provided insight into how this interaction can be modulated by subtle changes in the peptide ligand or by the pairing of CD94 with other members of the NKG2 family. Differences in the docking strategies used by the NKG2D and CD94-NKG2A receptors provided a basis for understanding the promiscuous nature of ligand recognition by NKG2D compared with the fidelity of the CD94-NKG2 receptors.

  4. Multi-parameter optimization of aza-follow-ups to BI 207524, a thumb pocket 1 HCV NS5B polymerase inhibitor. Part 2: Impact of lipophilicity on promiscuity and in vivo toxicity.

    PubMed

    Beaulieu, Pierre L; Bolger, Gordon; Deon, Dan; Duplessis, Martin; Fazal, Gulrez; Gagnon, Alexandre; Garneau, Michel; LaPlante, Steven; Stammers, Timothy; Kukolj, George; Duan, Jianmin

    2015-03-01

    We describe our efforts to identify analogs of thumb pocket 1 HCV NS5B inhibitor 1 (aza-analog of BI 207524) with improved plasma to liver partitioning and a predicted human half-life consistent with achieving a strong antiviral effect at a reasonable dose in HCV-infected patients. Compounds 3 and 7 were identified that met these criteria but exhibited off-target promiscuity in an in vitro pharmacology screen and in vivo toxicity in rats. High lipophilicity in this class was found to correlate with increased probability for promiscuous behavior and toxicity. The synthesis of an 8×11 matrix of analogs allowed the identification of C3, an inhibitor that displayed comparable potency to 1, improved partitioning to the liver and reduced lipophilicity. Although C3 displayed reduced propensity for in vitro off-target inhibition and the toxicity profile in rats was improved, the predicted human half-life of this compound was short, resulting in unacceptable dosing requirements to maintain a strong antiviral effect in patients. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Exploring a recognition-induced recognition decrement

    PubMed Central

    Dopkins, Stephen; Ngo, Catherine Trinh; Sargent, Jesse

    2007-01-01

    Four experiments explored a recognition decrement that is associated with the recognition of a word from a short list. The stimulus material for demonstrating the phenomenon was a list of words of different syntactic types. A word from the list was recognized less well following a decision that a word of the same type had occurred in the list than following a decision that such a word had not occurred in the list. A recognition decrement did not occur for a word of a given type following a positive recognition decision to a word of a different type. A recognition decrement did not occur when the list consisted exclusively of nouns. It was concluded that the phenomenon may reflect a criterion shift but probably does not reflect a list strength effect, suppression, or familiarity attribution consequent to a perceived discrepancy between actual and expected fluency. PMID:17063915

  6. Miscoding-induced stalling of substrate translocation on the bacterial ribosome.

    PubMed

    Alejo, Jose L; Blanchard, Scott C

    2017-10-10

    Directional transit of the ribosome along the messenger RNA (mRNA) template is a key determinant of the rate and processivity of protein synthesis. Imaging of the multistep translocation mechanism using single-molecule FRET has led to the hypothesis that substrate movements relative to the ribosome resolve through relatively long-lived late intermediates wherein peptidyl-tRNA enters the P site of the small ribosomal subunit via reversible, swivel-like motions of the small subunit head domain within the elongation factor G (GDP)-bound ribosome complex. Consistent with translocation being rate-limited by recognition and productive engagement of peptidyl-tRNA within the P site, we now show that base-pairing mismatches between the peptidyl-tRNA anticodon and the mRNA codon dramatically delay this rate-limiting, intramolecular process. This unexpected relationship between aminoacyl-tRNA decoding and translocation suggests that miscoding antibiotics may impact protein synthesis by impairing the recognition of peptidyl-tRNA in the small subunit P site during EF-G-catalyzed translocation. Strikingly, we show that elongation factor P (EF-P), traditionally known to alleviate ribosome stalling at polyproline motifs, can efficiently rescue translocation defects arising from miscoding. These findings help reveal the nature and origin of the rate-limiting steps in substrate translocation on the bacterial ribosome and indicate that EF-P can aid in resuming translation elongation stalled by miscoding errors.

  7. Miscoding-induced stalling of substrate translocation on the bacterial ribosome

    PubMed Central

    Alejo, Jose L.; Blanchard, Scott C.

    2017-01-01

    Directional transit of the ribosome along the messenger RNA (mRNA) template is a key determinant of the rate and processivity of protein synthesis. Imaging of the multistep translocation mechanism using single-molecule FRET has led to the hypothesis that substrate movements relative to the ribosome resolve through relatively long-lived late intermediates wherein peptidyl-tRNA enters the P site of the small ribosomal subunit via reversible, swivel-like motions of the small subunit head domain within the elongation factor G (GDP)-bound ribosome complex. Consistent with translocation being rate-limited by recognition and productive engagement of peptidyl-tRNA within the P site, we now show that base-pairing mismatches between the peptidyl-tRNA anticodon and the mRNA codon dramatically delay this rate-limiting, intramolecular process. This unexpected relationship between aminoacyl-tRNA decoding and translocation suggests that miscoding antibiotics may impact protein synthesis by impairing the recognition of peptidyl-tRNA in the small subunit P site during EF-G–catalyzed translocation. Strikingly, we show that elongation factor P (EF-P), traditionally known to alleviate ribosome stalling at polyproline motifs, can efficiently rescue translocation defects arising from miscoding. These findings help reveal the nature and origin of the rate-limiting steps in substrate translocation on the bacterial ribosome and indicate that EF-P can aid in resuming translation elongation stalled by miscoding errors. PMID:28973849

  8. Toward a unified model of face and object recognition in the human visual system

    PubMed Central

    Wallis, Guy

    2013-01-01

    Our understanding of the mechanisms and neural substrates underlying visual recognition has made considerable progress over the past 30 years. During this period, accumulating evidence has led many scientists to conclude that objects and faces are recognised in fundamentally distinct ways, and in fundamentally distinct cortical areas. In the psychological literature, in particular, this dissociation has led to a palpable disconnect between theories of how we process and represent the two classes of object. This paper follows a trend in part of the recognition literature to try to reconcile what we know about these two forms of recognition by considering the effects of learning. Taking a widely accepted, self-organizing model of object recognition, this paper explains how such a system is affected by repeated exposure to specific stimulus classes. In so doing, it explains how many aspects of recognition generally regarded as unusual to faces (holistic processing, configural processing, sensitivity to inversion, the other-race effect, the prototype effect, etc.) are emergent properties of category-specific learning within such a system. Overall, the paper describes how a single model of recognition learning can and does produce the seemingly very different types of representation associated with faces and objects. PMID:23966963

  9. Cuticular Hydrocarbons of Tribolium confusum Larvae Mediate Trail Following and Host Recognition in the Ectoparasitoid Holepyris sylvanidis.

    PubMed

    Fürstenau, Benjamin; Hilker, Monika

    2017-09-01

    Parasitic wasps which attack insects infesting processed stored food need to locate their hosts hidden inside these products. Their host search is well-known to be guided by host kairomones, perceived via olfaction or contact. Among contact kairomones, host cuticular hydrocarbons (CHCs) may provide reliable information for a parasitoid. However, the chemistry of CHC profiles of hosts living in processed stored food products is largely unknown. Here we showed that the ectoparasitoid Holepyris sylvanidis uses CHCs of its host Tribolium confusum, a worldwide stored product pest, as kairomones for host location and recognition at short range. Chemical analysis of T. confusum larval extracts by gas chromatography coupled with mass spectrometry revealed a rich blend of long-chain (C25-C30) hydrocarbons, including n-alkanes, mono-, and dimethylalkanes. We further studied whether host larvae leave sufficient CHCs on a substrate where they walk along, thus allowing parasitoids to perceive a CHC trail and follow it to their host larvae. We detected 18 CHCs on a substrate that had been exposed to host larvae. These compounds were also found in crude extracts of host larvae and made up about a fifth of the CHC amount extracted. Behavioral assays showed that trails of host CHCs were followed by the parasitoids and reduced their searching time until successful host recognition. Host CHC trails deposited on different substrates were persistent for about a day. Hence, the parasitoid H. sylvanidis exploits CHCs of T. confusum larvae for host finding by following host CHC trails and for host recognition by direct contact with host larvae.

  10. Quantitative Photochemical Immobilization of Biomolecules on Planar and Corrugated Substrates: A Versatile Strategy for Creating Functional Biointerfaces

    PubMed Central

    Martin, Teresa A.; Herman, Christine T.; Limpoco, Francis T.; Michael, Madeline C.; Potts, Gregory K.; Bailey, Ryan C.

    2014-01-01

    Methods for the generation of substrates presenting biomolecules in a spatially controlled manner are enabling tools for applications in biosensor systems, microarray technologies, fundamental biological studies and biointerface science. We have implemented a method to create biomolecular patterns by using light to control the direct covalent immobilization of biomolecules onto benzophenone-modified glass substrates. We have generated substrates presenting up to three different biomolecules patterned in sequence, and demonstrate biomolecular photopatterning on corrugated substrates. The chemistry of the underlying monolayer was optimized to incorporate poly(ethylene glycol) to enable adhesive cell adhesion onto patterned extracellular matrix proteins. Substrates were characterized with contact angle goniometry, AFM, and immunofluorescence microscopy. Importantly, radioimmunoassays were performed to quantify the site density of immobilized biomolecules on photopatterned substrates. Retention of function of photopatterned proteins was demonstrated both by native ligand recognition and cell adhesion to photopatterned substrates, revealing that substrates generated with this method are suitable for probing specific cell receptor-ligand interactions. This molecularly general photochemical patterning method is an enabling tool that will allow the creation of substrates presenting both biochemical and topographical variation, which is an important feature of many native biointerfaces. PMID:21793535

  11. The first murine zygotic transcription is promiscuous and uncoupled from splicing and 3′ processing

    PubMed Central

    Abe, Ken-ichiro; Yamamoto, Ryoma; Franke, Vedran; Cao, Minjun; Suzuki, Yutaka; Suzuki, Masataka G; Vlahovicek, Kristian; Svoboda, Petr; Schultz, Richard M; Aoki, Fugaku

    2015-01-01

    Initiation of zygotic transcription in mammals is poorly understood. In mice, zygotic transcription is first detected shortly after pronucleus formation in 1-cell embryos, but the identity of the transcribed loci and mechanisms regulating their expression are not known. Using total RNA-Seq, we have found that transcription in 1-cell embryos is highly promiscuous, such that intergenic regions are extensively expressed and thousands of genes are transcribed at comparably low levels. Striking is that transcription can occur in the absence of defined core-promoter elements. Furthermore, accumulation of translatable zygotic mRNAs is minimal in 1-cell embryos because of inefficient splicing and 3′ processing of nascent transcripts. These findings provide novel insights into regulation of gene expression in 1-cell mouse embryos that may confer a protective mechanism against precocious gene expression that is the product of a relaxed chromatin structure present in 1-cell embryos. The results also suggest that the first zygotic transcription itself is an active component of chromatin remodeling in 1-cell embryos. PMID:25896510

  12. 8 CFR 292.2 - Organizations qualified for recognition; requests for recognition; withdrawal of recognition...

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ...; requests for recognition; withdrawal of recognition; accreditation of representatives; roster. 292.2...; withdrawal of recognition; accreditation of representatives; roster. (a) Qualifications of organizations. A non-profit religious, charitable, social service, or similar organization established in the United...

  13. Characterization of cyclo-Acetoacetyl-L-Tryptophan Dimethylallyltransferase in Cyclopiazonic Acid Biosynthesis: Substrate Promiscuity and Site Directed Mutagenesis Studies

    PubMed Central

    Liu, Xinyu; Walsh, Christopher T.

    2009-01-01

    The fungal neurotoxin α-cyclopiazonic acid (CPA), a nanomolar inhibitor of Ca2+-ATPase with a unique pentacyclic indole tetramic acid scaffold is assembled by a three enzyme pathway CpaS, CpaD and CpaO in Aspergillus sp. We recently characterized the first pathway-specific enzyme CpaS, a hybrid two module polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) that generates cyclo-acetoacetyl-L-tryptophan (cAATrp). Here we report the characterization of the second pathway-specific enzyme CpaD that regiospecifically dimethylallylates cAATrp to form β-cyclopiazonic acid. By exploring the tryptophan and tetramate moieties of cAATrp, we demonstrate that CpaD discriminates against free Trp but accepts tryptophan-containing thiohydantoins, diketopiperazines and linear peptides as substrates for C4-prenylation and also acts as regiospecific O-dimethylallyltransferase (DMAT) on a tyrosine-derived tetramic acid. Comparative evaluation of CpaDs from A. oryzae RIB40 and A. flavus NRRL3357 indicated the importance of the N-terminal region for its activity. Sequence alignment of CpaD with eleven homologous fungal Trp-DMATs revealed five regions of conservation suggesting the presense of critical motifs that could be diagonostic for discovering additional Trp-DMATs. Subsequent site-directed mutagenesis studies identified five polar/charged residues and five tyrosine residues within these motifs that are critical for CpaD activity. This motif characerization will enable a gene probe-based approach to discover additional biosynthetic Trp-DMATs. PMID:19877600

  14. X-ray structures of the Pseudomonas cichorii D-tagatose 3-epimerase mutant form C66S recognizing deoxy sugars as substrates.

    PubMed

    Yoshida, Hiromi; Yoshihara, Akihide; Ishii, Tomohiko; Izumori, Ken; Kamitori, Shigehiro

    2016-12-01

    Pseudomonas cichorii D-tagatose 3-epimerase (PcDTE), which has a broad substrate specificity, efficiently catalyzes the epimerization of not only D-tagatose to D-sorbose but also D-fructose to D-psicose (D-allulose) and also recognizes the deoxy sugars as substrates. In an attempt to elucidate the substrate recognition and catalytic reaction mechanisms of PcDTE for deoxy sugars, the X-ray structures of the PcDTE mutant form with the replacement of Cys66 by Ser (PcDTE_C66S) in complexes with deoxy sugars were determined. These X-ray structures showed that substrate recognition by the enzyme at the 1-, 2-, and 3-positions is responsible for enzymatic activity and that substrate-enzyme interactions at the 4-, 5-, and 6-positions are not essential for the catalytic reaction of the enzyme leading to the broad substrate specificity of PcDTE. They also showed that the epimerization site of 1-deoxy 3-keto D-galactitol is shifted from C3 to C4 and that 1-deoxy sugars may bind to the catalytic site in the inhibitor-binding mode. The hydrophobic groove that acts as an accessible surface for substrate binding is formed through the dimerization of PcDTE. In PcDTE_C66S/deoxy sugar complex structures, bound ligand molecules in both the linear and ring forms were detected in the hydrophobic groove, while bound ligand molecules in the catalytic site were in the linear form. This result suggests that the sugar-ring opening of a substrate may occur in the hydrophobic groove and also that the narrow channel of the passageway to the catalytic site allows a substrate in the linear form to pass through.

  15. Substrate Specificity and Possible Heterologous Targets of Phytaspase, a Plant Cell Death Protease*

    PubMed Central

    Galiullina, Raisa A.; Kasperkiewicz, Paulina; Chichkova, Nina V.; Szalek, Aleksandra; Serebryakova, Marina V.; Poreba, Marcin; Drag, Marcin; Vartapetian, Andrey B.

    2015-01-01

    Plants lack aspartate-specific cell death proteases homologous to animal caspases. Instead, a subtilisin-like serine-dependent plant protease named phytaspase shown to be involved in the accomplishment of programmed death of plant cells is able to hydrolyze a number of peptide-based caspase substrates. Here, we determined the substrate specificity of rice (Oryza sativa) phytaspase by using the positional scanning substrate combinatorial library approach. Phytaspase was shown to display an absolute specificity of hydrolysis after an aspartic acid residue. The preceding amino acid residues, however, significantly influence the efficiency of hydrolysis. Efficient phytaspase substrates demonstrated a remarkable preference for an aromatic amino acid residue in the P3 position. The deduced optimum phytaspase recognition motif has the sequence IWLD and is strikingly hydrophobic. The established pattern was confirmed through synthesis and kinetic analysis of cleavage of a set of optimized peptide substrates. An amino acid motif similar to the phytaspase cleavage site is shared by the human gastrointestinal peptide hormones gastrin and cholecystokinin. In agreement with the established enzyme specificity, phytaspase was shown to hydrolyze gastrin-1 and cholecystokinin at the predicted sites in vitro, thus destroying the active moieties of the hormones. PMID:26283788

  16. Convenience polyandry or convenience polygyny? Costly sex under female control in a promiscuous primate.

    PubMed

    Huchard, Elise; Canale, Cindy I; Le Gros, Chloé; Perret, Martine; Henry, Pierre-Yves; Kappeler, Peter M

    2012-04-07

    Classic sex roles depict females as choosy, but polyandry is widespread. Empirical attempts to understand the evolution of polyandry have often focused on its adaptive value to females, whereas 'convenience polyandry' might simply decrease the costs of sexual harassment. We tested whether constraint-free female strategies favour promiscuity over mating selectivity through an original experimental design. We investigated variation in mating behaviour in response to a reversible alteration of sexual dimorphism in body mass in the grey mouse lemur, a small primate where female brief sexual receptivity allows quantifying polyandry. We manipulated body condition in captive females, predicting that convenience polyandry would increase when females are weaker than males, thus less likely to resist their solicitations. Our results rather support the alternative hypothesis of 'adaptive polyandry': females in better condition are more polyandrous. Furthermore, we reveal that multiple mating incurs significant energetic costs, which are strikingly symmetrical between the sexes. Our study shows that mouse lemur females exert tight control over mating and actively seek multiple mates. The benefits of remating are nevertheless not offset by its costs in low-condition females, suggesting that polyandry is a flexible strategy yielding moderate fitness benefits in this small mammal.

  17. Social cues of sperm competition influence accessory reproductive gland size in a promiscuous mammal.

    PubMed

    Lemaître, Jean-François; Ramm, Steven A; Hurst, Jane L; Stockley, Paula

    2011-04-22

    Theory predicts that males should increase overall investment in ejaculate expenditure with increasing levels of sperm competition. Since ejaculate production is costly, we may expect males to tailor their reproductive investment according to anticipated levels of sperm competition. Here, we investigate plasticity in ejaculate investment in response to cues of population average levels of sperm competition in a promiscuous mammal, the bank vole (Myodes glareolus). We manipulated the social experience of experimental subjects during sexual development via differential exposure to the odour of rival males, to simulate conditions associated with relatively high or low average levels of sperm competition. Males exposed to a high level of competition developed larger major accessory reproductive glands (seminal vesicles) than those that experienced a low level of competition, suggesting that an increased investment in the production of copulatory plugs and/or mating rate may be beneficial at relatively high sperm competition levels. However, investment in sperm production, testis size and sperm motility were not altered according to social experience. Our findings emphasize the importance of non-sperm components of the ejaculate in mammalian postcopulatory sexual selection, and add to the growing evidence linking plasticity in reproductive traits to social cues of sperm competition.

  18. Direct Role for Proliferating Cell Nuclear Antigen in Substrate Recognition by the E3 Ubiquitin Ligase CRL4Cdt2*

    PubMed Central

    Havens, Courtney G.; Shobnam, Nadia; Guarino, Estrella; Centore, Richard C.; Zou, Lee; Kearsey, Stephen E.; Walter, Johannes C.

    2012-01-01

    The E3 ubiquitin ligase Cullin-ring ligase 4-Cdt2 (CRL4Cdt2) is emerging as an important cell cycle regulator that targets numerous proteins for destruction in S phase and after DNA damage, including Cdt1, p21, and Set8. CRL4Cdt2 substrates contain a “PIP degron,” which consists of a canonical proliferating cell nuclear antigen (PCNA) interaction motif (PIP box) and an adjacent basic amino acid. Substrates use their PIP box to form a binary complex with PCNA on chromatin and the basic residue to recruit CRL4Cdt2 for substrate ubiquitylation. Using Xenopus egg extracts, we identify an acidic residue in PCNA that is essential to support destruction of all CRL4Cdt2 substrates. This PCNA residue, which adjoins the basic amino acid of the bound PIP degron, is dispensable for substrate binding to PCNA but essential for CRL4Cdt2 recruitment to chromatin. Our data show that the interaction of CRL4Cdt2 with substrates requires molecular determinants not only in the substrate degron but also on PCNA. The results illustrate a potentially general mechanism by which E3 ligases can couple ubiquitylation to the formation of protein-protein interactions. PMID:22303007

  19. Deficits in facial affect recognition among antisocial populations: a meta-analysis.

    PubMed

    Marsh, Abigail A; Blair, R J R

    2008-01-01

    Individuals with disorders marked by antisocial behavior frequently show deficits in recognizing displays of facial affect. Antisociality may be associated with specific deficits in identifying fearful expressions, which would implicate dysfunction in neural structures that subserve fearful expression processing. A meta-analysis of 20 studies was conducted to assess: (a) if antisocial populations show any consistent deficits in recognizing six emotional expressions; (b) beyond any generalized impairment, whether specific fear recognition deficits are apparent; and (c) if deficits in fear recognition are a function of task difficulty. Results show a robust link between antisocial behavior and specific deficits in recognizing fearful expressions. This impairment cannot be attributed solely to task difficulty. These results suggest dysfunction among antisocial individuals in specified neural substrates, namely the amygdala, involved in processing fearful facial affect.

  20. Programmable RNA recognition and cleavage by CRISPR/Cas9.

    PubMed

    O'Connell, Mitchell R; Oakes, Benjamin L; Sternberg, Samuel H; East-Seletsky, Alexandra; Kaplan, Matias; Doudna, Jennifer A

    2014-12-11

    The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA-DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms, but it has been thought to be incapable of targeting RNA. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags.

  1. Programmable RNA recognition and cleavage by CRISPR/Cas9

    PubMed Central

    O’Connell, Mitchell R.; Oakes, Benjamin L.; Sternberg, Samuel H.; East-Seletsky, Alexandra; Kaplan, Matias; Doudna, Jennifer A.

    2014-01-01

    The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA:DNA complementarity to identify target sites for sequence-specific doublestranded DNA (dsDNA) cleavage1-5. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, the protospacer adjacent motif (PAM), next to and on the strand opposite the 20-nucleotide target site in dsDNA4-7. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in many cell types and organisms8, but it has been thought to be incapable of targeting RNA5. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalyzed DNA cleavage7. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous mRNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable and tagless transcript recognition. PMID:25274302

  2. Brain correlates of musical and facial emotion recognition: evidence from the dementias.

    PubMed

    Hsieh, S; Hornberger, M; Piguet, O; Hodges, J R

    2012-07-01

    The recognition of facial expressions of emotion is impaired in semantic dementia (SD) and is associated with right-sided brain atrophy in areas known to be involved in emotion processing, notably the amygdala. Whether patients with SD also experience difficulty recognizing emotions conveyed by other media, such as music, is unclear. Prior studies have used excerpts of known music from classical or film repertoire but not unfamiliar melodies designed to convey distinct emotions. Patients with SD (n = 11), Alzheimer's disease (n = 12) and healthy control participants (n = 20) underwent tests of emotion recognition in two modalities: unfamiliar musical tunes and unknown faces as well as volumetric MRI. Patients with SD were most impaired with the recognition of facial and musical emotions, particularly for negative emotions. Voxel-based morphometry showed that the labelling of emotions, regardless of modality, correlated with the degree of atrophy in the right temporal pole, amygdala and insula. The recognition of musical (but not facial) emotions was also associated with atrophy of the left anterior and inferior temporal lobe, which overlapped with regions correlating with standardized measures of verbal semantic memory. These findings highlight the common neural substrates supporting the processing of emotions by facial and musical stimuli but also indicate that the recognition of emotions from music draws upon brain regions that are associated with semantics in language. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. Distinct parietal sites mediate the influences of mood, arousal, and their interaction on human recognition memory.

    PubMed

    Greene, Ciara M; Flannery, Oliver; Soto, David

    2014-12-01

    The two dimensions of emotion, mood valence and arousal, have independent effects on recognition memory. At present, however, it is not clear how those effects are reflected in the human brain. Previous research in this area has generally dealt with memory for emotionally valenced or arousing stimuli, but the manner in which interacting mood and arousal states modulate responses in memory substrates remains poorly understood. We investigated memory for emotionally neutral items while independently manipulating mood valence and arousal state by means of music exposure. Four emotional conditions were created: positive mood/high arousal, positive mood/low arousal, negative mood/high arousal, and negative mood/low arousal. We observed distinct effects of mood valence and arousal in parietal substrates of recognition memory. Positive mood increased activity in ventral posterior parietal cortex (PPC) and orbitofrontal cortex, whereas arousal condition modulated activity in dorsal PPC and the posterior cingulate. An interaction between valence and arousal was observed in left ventral PPC, notably in a parietal area distinct from the those identified for the main effects, with a stronger effect of mood on recognition memory responses here under conditions of relative high versus low arousal. We interpreted the PPC activations in terms of the attention-to-memory hypothesis: Increased arousal may lead to increased top-down control of memory, and hence dorsal PPC activation, whereas positive mood valence may result in increased activity in ventral PPC regions associated with bottom-up attention to memory. These findings indicate that distinct parietal sites mediate the influences of mood, arousal, and their interplay during recognition memory.

  4. Evolution of hormone signaling in elasmobranchs by exploitation of promiscuous receptors.

    PubMed

    Carroll, Sean Michael; Bridgham, Jamie T; Thornton, Joseph W

    2008-12-01

    Specific interactions among proteins, nucleic acids, and metabolites drive virtually all cellular functions and underlie phenotypic complexity and diversity. Despite the fundamental importance of interactions, the mechanisms and dynamics by which they evolve are poorly understood. Here we describe novel interactions between a lineage-specific hormone and its receptors in elasmobranchs, a subclass of cartilaginous fishes, and infer how these associations evolved using phylogenetic and protein structural analyses. The hormone 1alpha-hydroxycorticosterone (1alpha-B) is a physiologically important steroid synthesized only in elasmobranchs. We show that 1alpha-B modulates gene expression in vitro by activating two paralogous intracellular transcription factors, the mineralocorticoid receptor (MR) and glucocorticoid receptor (GR), in the little skate Leucoraja erinacea; MR serves as a high-sensitivity and GR as a low-sensitivity receptor. Using functional analysis of extant and resurrected ancestral proteins, we show that receptor sensitivity to 1alpha-B evolved millions of years before the hormone itself evolved. The 1alpha-B differs from more ancient corticosteroids only by the addition of a hydroxyl group; the three-dimensional structure of the ancestral receptor shows that the ligand pocket contained ample unoccupied space to accommodate this moiety. Our findings indicate that the interactions between 1alpha-B and elasmobranch GR and MR proteins evolved by molecular exploitation: a novel hormone recruited into new functional partnerships two ancient receptors that had previously interacted with other ligands. The ancestral receptor's promiscuous capacity to fortuitously bind compounds that are slight structural variants of its original ligands set the stage for the evolution of this new interaction.

  5. A Bottom-Up Proteomic Approach to Identify Substrate Specificity of Outer-Membrane Protease OmpT.

    PubMed

    Wood, Sarah E; Sinsinbar, Gaurav; Gudlur, Sushanth; Nallani, Madhavan; Huang, Che-Fan; Liedberg, Bo; Mrksich, Milan

    2017-12-22

    Identifying peptide substrates that are efficiently cleaved by proteases gives insights into substrate recognition and specificity, guides development of inhibitors, and improves assay sensitivity. Peptide arrays and SAMDI mass spectrometry were used to identify a tetrapeptide substrate exhibiting high activity for the bacterial outer-membrane protease (OmpT). Analysis of protease activity for the preferred residues at the cleavage site (P1, P1') and nearest-neighbor positions (P2, P2') and their positional interdependence revealed FRRV as the optimal peptide with the highest OmpT activity. Substituting FRRV into a fragment of LL37, a natural substrate of OmpT, led to a greater than 400-fold improvement in OmpT catalytic efficiency, with a k cat /K m value of 6.1×10 6  L mol -1  s -1 . Wild-type and mutant OmpT displayed significant differences in their substrate specificities, demonstrating that even modest mutants may not be suitable substitutes for the native enzyme. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Substrate scope of a dehydrogenase from Sphingomonas species A1 and its potential application in the synthesis of rare sugars and sugar derivatives.

    PubMed

    Beer, Barbara; Pick, André; Döring, Manuel; Lommes, Petra; Sieber, Volker

    2018-07-01

    Rare sugars and sugar derivatives that can be obtained from abundant sugars are of great interest to biochemical and pharmaceutical research. Here, we describe the substrate scope of a short-chain dehydrogenase/reductase from Sphingomonas species A1 (SpsADH) in the oxidation of aldonates and polyols. The resulting products are rare uronic acids and rare sugars respectively. We provide insight into the substrate recognition of SpsADH using kinetic analyses, which show that the configuration of the hydroxyl groups adjacent to the oxidized carbon is crucial for substrate recognition. Furthermore, the specificity is demonstrated by the oxidation of d-sorbitol leading to l-gulose as sole product instead of a mixture of d-glucose and l-gulose. Finally, we applied the enzyme to the synthesis of l-gulose from d-sorbitol in an in vitro system using a NADH oxidase for cofactor recycling. This study shows the usefulness of exploring the substrate scope of enzymes to find new enzymatic reaction pathways from renewable resources to value-added compounds. © 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  7. Super Spy variants implicate flexibility in chaperone action.

    PubMed

    Quan, Shu; Wang, Lili; Petrotchenko, Evgeniy V; Makepeace, Karl At; Horowitz, Scott; Yang, Jianyi; Zhang, Yang; Borchers, Christoph H; Bardwell, James Ca

    2014-01-01

    Experimental study of the role of disorder in protein function is challenging. It has been proposed that proteins utilize disordered regions in the adaptive recognition of their various binding partners. However apart from a few exceptions, defining the importance of disorder in promiscuous binding interactions has proven to be difficult. In this paper, we have utilized a genetic selection that links protein stability to antibiotic resistance to isolate variants of the newly discovered chaperone Spy that show an up to 7 fold improved chaperone activity against a variety of substrates. These "Super Spy" variants show tighter binding to client proteins and are generally more unstable than is wild type Spy and show increases in apparent flexibility. We establish a good relationship between the degree of their instability and the improvement they show in their chaperone activity. Our results provide evidence for the importance of disorder and flexibility in chaperone function. DOI: http://dx.doi.org/10.7554/eLife.01584.001.

  8. Super Spy variants implicate flexibility in chaperone action

    PubMed Central

    Quan, Shu; Wang, Lili; Petrotchenko, Evgeniy V; Makepeace, Karl AT; Horowitz, Scott; Yang, Jianyi; Zhang, Yang; Borchers, Christoph H; Bardwell, James CA

    2014-01-01

    Experimental study of the role of disorder in protein function is challenging. It has been proposed that proteins utilize disordered regions in the adaptive recognition of their various binding partners. However apart from a few exceptions, defining the importance of disorder in promiscuous binding interactions has proven to be difficult. In this paper, we have utilized a genetic selection that links protein stability to antibiotic resistance to isolate variants of the newly discovered chaperone Spy that show an up to 7 fold improved chaperone activity against a variety of substrates. These “Super Spy” variants show tighter binding to client proteins and are generally more unstable than is wild type Spy and show increases in apparent flexibility. We establish a good relationship between the degree of their instability and the improvement they show in their chaperone activity. Our results provide evidence for the importance of disorder and flexibility in chaperone function. DOI: http://dx.doi.org/10.7554/eLife.01584.001 PMID:24497545

  9. Structural Basis for the Recognition of Mycolic Acid Precursors by KasA, a Condensing Enzyme and Drug Target from Mycobacterium Tuberculosis *

    PubMed Central

    Schiebel, Johannes; Kapilashrami, Kanishk; Fekete, Agnes; Bommineni, Gopal R.; Schaefer, Christin M.; Mueller, Martin J.; Tonge, Peter J.; Kisker, Caroline

    2013-01-01

    The survival of Mycobacterium tuberculosis depends on mycolic acids, very long α-alkyl-β-hydroxy fatty acids comprising 60–90 carbon atoms. However, despite considerable efforts, little is known about how enzymes involved in mycolic acid biosynthesis recognize and bind their hydrophobic fatty acyl substrates. The condensing enzyme KasA is pivotal for the synthesis of very long (C38–42) fatty acids, the precursors of mycolic acids. To probe the mechanism of substrate and inhibitor recognition by KasA, we determined the structure of this protein in complex with a mycobacterial phospholipid and with several thiolactomycin derivatives that were designed as substrate analogs. Our structures provide consecutive snapshots along the reaction coordinate for the enzyme-catalyzed reaction and support an induced fit mechanism in which a wide cavity is established through the concerted opening of three gatekeeping residues and several α-helices. The stepwise characterization of the binding process provides mechanistic insights into the induced fit recognition in this system and serves as an excellent foundation for the development of high affinity KasA inhibitors. PMID:24108128

  10. Complete abolition of reading and writing ability with a third ventricle colloid cyst: implications for surgical intervention and proposed neural substrates of visual recognition and visual imaging ability.

    PubMed

    Barker, Lynne Ann; Morton, Nicholas; Romanowski, Charles A J; Gosden, Kevin

    2013-10-24

    We report a rare case of a patient unable to read (alexic) and write (agraphic) after a mild head injury. He had preserved speech and comprehension, could spell aloud, identify words spelt aloud and copy letter features. He was unable to visualise letters but showed no problems with digits. Neuropsychological testing revealed general visual memory, processing speed and imaging deficits. Imaging data revealed an 8 mm colloid cyst of the third ventricle that splayed the fornix. Little is known about functions mediated by fornical connectivity, but this region is thought to contribute to memory recall. Other regions thought to mediate letter recognition and letter imagery, visual word form area and visual pathways were intact. We remediated reading and writing by multimodal letter retraining. The study raises issues about the neural substrates of reading, role of fornical tracts to selective memory in the absence of other pathology, and effective remediation strategies for selective functional deficits.

  11. CD94-NKG2A recognition of human leukocyte antigen (HLA)-E bound to an HLA class I leader sequence

    PubMed Central

    Petrie, Emma J.; Clements, Craig S.; Lin, Jie; Sullivan, Lucy C.; Johnson, Darryl; Huyton, Trevor; Heroux, Annie; Hoare, Hilary L.; Beddoe, Travis; Reid, Hugh H.; Wilce, Matthew C.J.; Brooks, Andrew G.; Rossjohn, Jamie

    2008-01-01

    The recognition of human leukocyte antigen (HLA)-E by the heterodimeric CD94-NKG2 natural killer (NK) receptor family is a central innate mechanism by which NK cells monitor the expression of other HLA molecules, yet the structural basis of this highly specific interaction is unclear. Here, we describe the crystal structure of CD94-NKG2A in complex with HLA-E bound to a peptide derived from the leader sequence of HLA-G. The CD94 subunit dominated the interaction with HLA-E, whereas the NKG2A subunit was more peripheral to the interface. Moreover, the invariant CD94 subunit dominated the peptide-mediated contacts, albeit with poor surface and chemical complementarity. This unusual binding mode was consistent with mutagenesis data at the CD94-NKG2A–HLA-E interface. There were few conformational changes in either CD94-NKG2A or HLA-E upon ligation, and such a “lock and key” interaction is typical of innate receptor–ligand interactions. Nevertheless, the structure also provided insight into how this interaction can be modulated by subtle changes in the peptide ligand or by the pairing of CD94 with other members of the NKG2 family. Differences in the docking strategies used by the NKG2D and CD94-NKG2A receptors provided a basis for understanding the promiscuous nature of ligand recognition by NKG2D compared with the fidelity of the CD94-NKG2 receptors. PMID:18332182

  12. The Structural Basis of Substrate Recognition in an exo-b-d-glucosaminidase Involved in Chitosan Hydrolysis

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

    Van Bueren, A.; Ghinet, M; Gregg, K

    2009-01-01

    Family 2 of the glycoside hydrolase classification is one of the largest families. Structurally characterized members of this family include enzymes with ?-galactosidase activity (Escherichia coli LacZ), ?-glucuronidase activity (Homo sapiens GusB), and ?-mannosidase activity (Bacteroides thetaiotaomicron BtMan2A). Here, we describe the structure of a family 2 glycoside hydrolase, CsxA, from Amycolatopsis orientalis that has exo-?-d-glucosaminidase (exo-chitosanase) activity. Analysis of a product complex (1.85 A resolution) reveals a unique negatively charged pocket that specifically accommodates the nitrogen of nonreducing end glucosamine residues, allowing this enzyme to discriminate between glucose and glucosamine. This also provides structural evidence for the role ofmore » E541 as the catalytic nucleophile and D469 as the catalytic acid/base. The structures of an E541A mutant in complex with a natural ?-1,4-d-glucosamine tetrasaccharide substrate and both E541A and D469A mutants in complex with a pNP-?-d-glucosaminide synthetic substrate provide insight into interactions in the + 1 subsite of this enzyme. Overall, a comparison with the active sites of other GH2 enzymes highlights the unique architecture of the CsxA active site, which imparts specificity for its cationic substrate.« less

  13. The Structural Basis of Substrate Recognition in an exo-beta-d-Glucosaminidase Involved in Chitosan Hydrolysis

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

    Lammerts van Bueren, A.; Ghinet, M; Gregg, K

    2009-01-01

    Family 2 of the glycoside hydrolase classification is one of the largest families. Structurally characterized members of this family include enzymes with beta-galactosidase activity (Escherichia coli LacZ), beta-glucuronidase activity (Homo sapiens GusB), and beta-mannosidase activity (Bacteroides thetaiotaomicron BtMan2A). Here, we describe the structure of a family 2 glycoside hydrolase, CsxA, from Amycolatopsis orientalis that has exo-beta-D-glucosaminidase (exo-chitosanase) activity. Analysis of a product complex (1.85 A resolution) reveals a unique negatively charged pocket that specifically accommodates the nitrogen of nonreducing end glucosamine residues, allowing this enzyme to discriminate between glucose and glucosamine. This also provides structural evidence for the role ofmore » E541 as the catalytic nucleophile and D469 as the catalytic acid/base. The structures of an E541A mutant in complex with a natural beta-1,4-D-glucosamine tetrasaccharide substrate and both E541A and D469A mutants in complex with a pNP-beta-D-glucosaminide synthetic substrate provide insight into interactions in the +1 subsite of this enzyme. Overall, a comparison with the active sites of other GH2 enzymes highlights the unique architecture of the CsxA active site, which imparts specificity for its cationic substrate.« less

  14. Illuminating structure and acyl donor sites of a physiological transglutaminase substrate from Streptomyces mobaraensis.

    PubMed

    Juettner, Norbert E; Schmelz, Stefan; Bogen, Jan P; Happel, Dominic; Fessner, Wolf-Dieter; Pfeifer, Felicitas; Fuchsbauer, Hans-Lothar; Scrima, Andrea

    2018-05-01

    Transglutaminase from Streptomyces mobaraensis (MTG) has become a powerful tool to covalently and highly specifically link functional amines to glutamine donor sites of therapeutic proteins. However, details regarding the mechanism of substrate recognition and interaction of the enzyme with proteinaceous substrates still remain mostly elusive. We have determined the crystal structure of the Streptomyces papain inhibitory protein (SPI p ), a substrate of MTG, to study the influence of various substrate amino acids on positioning glutamine to the active site of MTG. SPI p exhibits a rigid, thermo-resistant double-psi-beta-barrel fold that is stabilized by two cysteine bridges. Incorporation of biotin cadaverine identified Gln-6 as the only amine acceptor site on SPI p accessible for MTG. Substitution of Lys-7 demonstrated that small and hydrophobic residues in close proximity to Gln-6 favor MTG-mediated modification and are likely to facilitate introduction of the substrate into the front vestibule of MTG. Moreover, exchange of various surface residues of SPI p for arginine and glutamate/aspartate outside the glutamine donor region influences the efficiency of modification by MTG. These results suggest the occurrence of charged contact areas between MTG and the acyl donor substrates beyond the front vestibule, and pave the way for protein engineering approaches to improve the properties of artificial MTG-substrates used in biomedical applications. © 2018 The Protein Society.

  15. Structural basis of redox-dependent substrate binding of protein disulfide isomerase

    PubMed Central

    Yagi-Utsumi, Maho; Satoh, Tadashi; Kato, Koichi

    2015-01-01

    Protein disulfide isomerase (PDI) is a multidomain enzyme, operating as an essential folding catalyst, in which the b′ and a′ domains provide substrate binding sites and undergo an open–closed domain rearrangement depending on the redox states of the a′ domain. Despite the long research history of this enzyme, three-dimensional structural data remain unavailable for its ligand-binding mode. Here we characterize PDI substrate recognition using α-synuclein (αSN) as the model ligand. Our nuclear magnetic resonance (NMR) data revealed that the substrate-binding domains of PDI captured the αSN segment Val37–Val40 only in the oxidized form. Furthermore, we determined the crystal structure of an oxidized form of the b′–a′ domains in complex with an undecapeptide corresponding to this segment. The peptide-binding mode observed in the crystal structure with NMR validation, was characterized by hydrophobic interactions on the b′ domain in an open conformation. Comparison with the previously reported crystal structure indicates that the a′ domain partially masks the binding surface of the b′ domain, causing steric hindrance against the peptide in the reduced form of the b′–a′ domains that exhibits a closed conformation. These findings provide a structural basis for the mechanism underlying the redox-dependent substrate binding of PDI. PMID:26350503

  16. Interdependence of Inhibitor Recognition in HIV-1 Protease.

    PubMed

    Paulsen, Janet L; Leidner, Florian; Ragland, Debra A; Kurt Yilmaz, Nese; Schiffer, Celia A

    2017-05-09

    Molecular recognition is a highly interdependent process. Subsite couplings within the active site of proteases are most often revealed through conditional amino acid preferences in substrate recognition. However, the potential effect of these couplings on inhibition and thus inhibitor design is largely unexplored. The present study examines the interdependency of subsites in HIV-1 protease using a focused library of protease inhibitors, to aid in future inhibitor design. Previously a series of darunavir (DRV) analogs was designed to systematically probe the S1' and S2' subsites. Co-crystal structures of these analogs with HIV-1 protease provide the ideal opportunity to probe subsite interdependency. All-atom molecular dynamics simulations starting from these structures were performed and systematically analyzed in terms of atomic fluctuations, intermolecular interactions, and water structure. These analyses reveal that the S1' subsite highly influences other subsites: the extension of the hydrophobic P1' moiety results in 1) reduced van der Waals contacts in the P2' subsite, 2) more variability in the hydrogen bond frequencies with catalytic residues and the flap water, and 3) changes in the occupancy of conserved water sites both proximal and distal to the active site. In addition, one of the monomers in this homodimeric enzyme has atomic fluctuations more highly correlated with DRV than the other monomer. These relationships intricately link the HIV-1 protease subsites and are critical to understanding molecular recognition and inhibitor binding. More broadly, the interdependency of subsite recognition within an active site requires consideration in the selection of chemical moieties in drug design; this strategy is in contrast to what is traditionally done with independent optimization of chemical moieties of an inhibitor.

  17. Role of the Anterior Cingulate Cortex in the Retrieval of Novel Object Recognition Memory after a Long Delay

    ERIC Educational Resources Information Center

    Pezze, Marie A.; Marshall, Hayley J.; Fone, Kevin C. F.; Cassaday, Helen J.

    2017-01-01

    Previous in vivo electrophysiological studies suggest that the anterior cingulate cortex (ACgx) is an important substrate of novel object recognition (NOR) memory. However, intervention studies are needed to confirm this conclusion and permanent lesion studies cannot distinguish effects on encoding and retrieval. The interval between encoding and…

  18. Conformational plasticity of RepB, the replication initiator protein of promiscuous streptococcal plasmid pMV158

    PubMed Central

    Boer, D. Roeland; Ruiz-Masó, José Angel; Rueda, Manuel; Petoukhov, Maxim V.; Machón, Cristina; Svergun, Dmitri I.; Orozco, Modesto; del Solar, Gloria; Coll, Miquel

    2016-01-01

    DNA replication initiation is a vital and tightly regulated step in all replicons and requires an initiator factor that specifically recognizes the DNA replication origin and starts replication. RepB from the promiscuous streptococcal plasmid pMV158 is a hexameric ring protein evolutionary related to viral initiators. Here we explore the conformational plasticity of the RepB hexamer by i) SAXS, ii) sedimentation experiments, iii) molecular simulations and iv) X-ray crystallography. Combining these techniques, we derive an estimate of the conformational ensemble in solution showing that the C-terminal oligomerisation domains of the protein form a rigid cylindrical scaffold to which the N-terminal DNA-binding/catalytic domains are attached as highly flexible appendages, featuring multiple orientations. In addition, we show that the hinge region connecting both domains plays a pivotal role in the observed plasticity. Sequence comparisons and a literature survey show that this hinge region could exists in other initiators, suggesting that it is a common, crucial structural element for DNA binding and manipulation. PMID:26875695

  19. 16p11.2 Deletion Mice Display Cognitive Deficits in Touchscreen Learning and Novelty Recognition Tasks

    ERIC Educational Resources Information Center

    Yang, Mu; Lewis, Freeman C.; Sarvi, Michael S.; Foley, Gillian M.; Crawley, Jacqueline N.

    2015-01-01

    Chromosomal 16p11.2 deletion syndrome frequently presents with intellectual disabilities, speech delays, and autism. Here we investigated the Dolmetsch line of 16p11.2 heterozygous (+/-) mice on a range of cognitive tasks with different neuroanatomical substrates. Robust novel object recognition deficits were replicated in two cohorts of 16p11.2…

  20. Identification of a Degradation Signal Sequence within Substrates of the Mitochondrial i-AAA Protease.

    PubMed

    Rampello, Anthony J; Glynn, Steven E

    2017-03-24

    The i-AAA protease is a component of the mitochondrial quality control machinery that regulates respiration, mitochondrial dynamics, and protein import. The protease is required to select specific substrates for degradation from among the diverse complement of proteins present in mitochondria, yet the rules that govern this selection are unclear. Here, we reconstruct the yeast i-AAA protease, Yme1p, to examine the in vitro degradation of two intermembrane space chaperone subunits, Tim9 and Tim10. Yme1p degrades Tim10 more rapidly than Tim9 despite high sequence and structural similarity, and loss of Tim10 is accelerated by the disruption of conserved disulfide bonds within the substrate. An unstructured N-terminal region of Tim10 is necessary and sufficient to target the substrate to the protease through recognition of a short phenylalanine-rich motif, and the presence of similar motifs in other small Tim proteins predicts robust degradation by the protease. Together, these results identify the first specific degron sequence within a native i-AAA protease substrate. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Substrate Specificity and Possible Heterologous Targets of Phytaspase, a Plant Cell Death Protease.

    PubMed

    Galiullina, Raisa A; Kasperkiewicz, Paulina; Chichkova, Nina V; Szalek, Aleksandra; Serebryakova, Marina V; Poreba, Marcin; Drag, Marcin; Vartapetian, Andrey B

    2015-10-09

    Plants lack aspartate-specific cell death proteases homologous to animal caspases. Instead, a subtilisin-like serine-dependent plant protease named phytaspase shown to be involved in the accomplishment of programmed death of plant cells is able to hydrolyze a number of peptide-based caspase substrates. Here, we determined the substrate specificity of rice (Oryza sativa) phytaspase by using the positional scanning substrate combinatorial library approach. Phytaspase was shown to display an absolute specificity of hydrolysis after an aspartic acid residue. The preceding amino acid residues, however, significantly influence the efficiency of hydrolysis. Efficient phytaspase substrates demonstrated a remarkable preference for an aromatic amino acid residue in the P3 position. The deduced optimum phytaspase recognition motif has the sequence IWLD and is strikingly hydrophobic. The established pattern was confirmed through synthesis and kinetic analysis of cleavage of a set of optimized peptide substrates. An amino acid motif similar to the phytaspase cleavage site is shared by the human gastrointestinal peptide hormones gastrin and cholecystokinin. In agreement with the established enzyme specificity, phytaspase was shown to hydrolyze gastrin-1 and cholecystokinin at the predicted sites in vitro, thus destroying the active moieties of the hormones. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. The "parts and wholes" of face recognition: A review of the literature.

    PubMed

    Tanaka, James W; Simonyi, Diana

    2016-10-01

    It has been claimed that faces are recognized as a "whole" rather than by the recognition of individual parts. In a paper published in the Quarterly Journal of Experimental Psychology in 1993, Martha Farah and I attempted to operationalize the holistic claim using the part/whole task. In this task, participants studied a face and then their memory presented in isolation and in the whole face. Consistent with the holistic view, recognition of the part was superior when tested in the whole-face condition compared to when it was tested in isolation. The "whole face" or holistic advantage was not found for faces that were inverted, or scrambled, nor for non-face objects, suggesting that holistic encoding was specific to normal, intact faces. In this paper, we reflect on the part/whole paradigm and how it has contributed to our understanding of what it means to recognize a face as a "whole" stimulus. We describe the value of part/whole task for developing theories of holistic and non-holistic recognition of faces and objects. We discuss the research that has probed the neural substrates of holistic processing in healthy adults and people with prosopagnosia and autism. Finally, we examine how experience shapes holistic face recognition in children and recognition of own- and other-race faces in adults. The goal of this article is to summarize the research on the part/whole task and speculate on how it has informed our understanding of holistic face processing.

  3. An enhanced feature set for pattern recognition based contrast enhancement of contact-less captured latent fingerprints in digitized crime scene forensics

    NASA Astrophysics Data System (ADS)

    Hildebrandt, Mario; Kiltz, Stefan; Dittmann, Jana; Vielhauer, Claus

    2014-02-01

    In crime scene forensics latent fingerprints are found on various substrates. Nowadays primarily physical or chemical preprocessing techniques are applied for enhancing the visibility of the fingerprint trace. In order to avoid altering the trace it has been shown that contact-less sensors offer a non-destructive acquisition approach. Here, the exploitation of fingerprint or substrate properties and the utilization of signal processing techniques are an essential requirement to enhance the fingerprint visibility. However, especially the optimal sensory is often substrate-dependent. An enhanced generic pattern recognition based contrast enhancement approach for scans of a chromatic white light sensor is introduced in Hildebrandt et al.1 using statistical, structural and Benford's law2 features for blocks of 50 micron. This approach achieves very good results for latent fingerprints on cooperative, non-textured, smooth substrates. However, on textured and structured substrates the error rates are very high and the approach thus unsuitable for forensic use cases. We propose the extension of the feature set with semantic features derived from known Gabor filter based exemplar fingerprint enhancement techniques by suggesting an Epsilon-neighborhood of each block in order to achieve an improved accuracy (called fingerprint ridge orientation semantics). Furthermore, we use rotation invariant Hu moments as an extension of the structural features and two additional preprocessing methods (separate X- and Y Sobel operators). This results in a 408-dimensional feature space. In our experiments we investigate and report the recognition accuracy for eight substrates, each with ten latent fingerprints: white furniture surface, veneered plywood, brushed stainless steel, aluminum foil, "Golden-Oak" veneer, non-metallic matte car body finish, metallic car body finish and blued metal. In comparison to Hildebrandt et al.,1 our evaluation shows a significant reduction of the error rates

  4. Self-face recognition in social context.

    PubMed

    Sugiura, Motoaki; Sassa, Yuko; Jeong, Hyeonjeong; Wakusawa, Keisuke; Horie, Kaoru; Sato, Shigeru; Kawashima, Ryuta

    2012-06-01

    The concept of "social self" is often described as a representation of the self-reflected in the eyes or minds of others. Although the appearance of one's own face has substantial social significance for humans, neuroimaging studies have failed to link self-face recognition and the likely neural substrate of the social self, the medial prefrontal cortex (MPFC). We assumed that the social self is recruited during self-face recognition under a rich social context where multiple other faces are available for comparison of social values. Using functional magnetic resonance imaging (fMRI), we examined the modulation of neural responses to the faces of the self and of a close friend in a social context. We identified an enhanced response in the ventral MPFC and right occipitoparietal sulcus in the social context specifically for the self-face. Neural response in the right lateral parietal and inferior temporal cortices, previously claimed as self-face-specific, was unaffected for the self-face but unexpectedly enhanced for the friend's face in the social context. Self-face-specific activation in the pars triangularis of the inferior frontal gyrus, and self-face-specific reduction of activation in the left middle temporal gyrus and the right supramarginal gyrus, replicating a previous finding, were not subject to such modulation. Our results thus demonstrated the recruitment of a social self during self-face recognition in the social context. At least three brain networks for self-face-specific activation may be dissociated by different patterns of response-modulation in the social context, suggesting multiple dynamic self-other representations in the human brain. Copyright © 2011 Wiley-Liss, Inc.

  5. Convenience polyandry or convenience polygyny? Costly sex under female control in a promiscuous primate

    PubMed Central

    Huchard, Elise; Canale, Cindy I.; Le Gros, Chloé; Perret, Martine; Henry, Pierre-Yves; Kappeler, Peter M.

    2012-01-01

    Classic sex roles depict females as choosy, but polyandry is widespread. Empirical attempts to understand the evolution of polyandry have often focused on its adaptive value to females, whereas ‘convenience polyandry’ might simply decrease the costs of sexual harassment. We tested whether constraint-free female strategies favour promiscuity over mating selectivity through an original experimental design. We investigated variation in mating behaviour in response to a reversible alteration of sexual dimorphism in body mass in the grey mouse lemur, a small primate where female brief sexual receptivity allows quantifying polyandry. We manipulated body condition in captive females, predicting that convenience polyandry would increase when females are weaker than males, thus less likely to resist their solicitations. Our results rather support the alternative hypothesis of ‘adaptive polyandry’: females in better condition are more polyandrous. Furthermore, we reveal that multiple mating incurs significant energetic costs, which are strikingly symmetrical between the sexes. Our study shows that mouse lemur females exert tight control over mating and actively seek multiple mates. The benefits of remating are nevertheless not offset by its costs in low-condition females, suggesting that polyandry is a flexible strategy yielding moderate fitness benefits in this small mammal. PMID:21976684

  6. Optical Pattern Recognition

    NASA Astrophysics Data System (ADS)

    Yu, Francis T. S.; Jutamulia, Suganda

    2008-10-01

    Contributors; Preface; 1. Pattern recognition with optics Francis T. S. Yu and Don A. Gregory; 2. Hybrid neural networks for nonlinear pattern recognition Taiwei Lu; 3. Wavelets, optics, and pattern recognition Yao Li and Yunglong Sheng; 4. Applications of the fractional Fourier transform to optical pattern recognition David Mendlovic, Zeev Zalesky and Haldum M. Oxaktas; 5. Optical implementation of mathematical morphology Tien-Hsin Chao; 6. Nonlinear optical correlators with improved discrimination capability for object location and recognition Leonid P. Yaroslavsky; 7. Distortion-invariant quadratic filters Gregory Gheen; 8. Composite filter synthesis as applied to pattern recognition Shizhou Yin and Guowen Lu; 9. Iterative procedures in electro-optical pattern recognition Joseph Shamir; 10. Optoelectronic hybrid system for three-dimensional object pattern recognition Guoguang Mu, Mingzhe Lu and Ying Sun; 11. Applications of photrefractive devices in optical pattern recognition Ziangyang Yang; 12. Optical pattern recognition with microlasers Eung-Gi Paek; 13. Optical properties and applications of bacteriorhodopsin Q. Wang Song and Yu-He Zhang; 14. Liquid-crystal spatial light modulators Aris Tanone and Suganda Jutamulia; 15. Representations of fully complex functions on real-time spatial light modulators Robert W. Cohn and Laurence G. Hassbrook; Index.

  7. An Exquisitely Specific PDZ/Target Recognition Revealed by the Structure of INAD PDZ3 in Complex with TRP Channel Tail.

    PubMed

    Ye, Fei; Liu, Wei; Shang, Yuan; Zhang, Mingjie

    2016-03-01

    The vast majority of PDZ domains are known to bind to a few C-terminal tail residues of target proteins with modest binding affinities and specificities. Such promiscuous PDZ/target interactions are not compatible with highly specific physiological functions of PDZ domain proteins and their targets. Here, we report an unexpected PDZ/target binding occurring between the scaffold protein inactivation no afterpotential D (INAD) and transient receptor potential (TRP) channel in Drosophila photoreceptors. The C-terminal 15 residues of TRP are required for the specific interaction with INAD PDZ3. The INAD PDZ3/TRP peptide complex structure reveals that only the extreme C-terminal Leu of TRP binds to the canonical αB/βB groove of INAD PDZ3. The rest of the TRP peptide, by forming a β hairpin structure, binds to a surface away from the αB/βB groove of PDZ3 and contributes to the majority of the binding energy. Thus, the INAD PDZ3/TRP channel interaction is exquisitely specific and represents a new mode of PDZ/target recognitions. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. A new selective developmental deficit: Impaired object recognition with normal face recognition.

    PubMed

    Germine, Laura; Cashdollar, Nathan; Düzel, Emrah; Duchaine, Bradley

    2011-05-01

    Studies of developmental deficits in face recognition, or developmental prosopagnosia, have shown that individuals who have not suffered brain damage can show face recognition impairments coupled with normal object recognition (Duchaine and Nakayama, 2005; Duchaine et al., 2006; Nunn et al., 2001). However, no developmental cases with the opposite dissociation - normal face recognition with impaired object recognition - have been reported. The existence of a case of non-face developmental visual agnosia would indicate that the development of normal face recognition mechanisms does not rely on the development of normal object recognition mechanisms. To see whether a developmental variant of non-face visual object agnosia exists, we conducted a series of web-based object and face recognition tests to screen for individuals showing object recognition memory impairments but not face recognition impairments. Through this screening process, we identified AW, an otherwise normal 19-year-old female, who was then tested in the lab on face and object recognition tests. AW's performance was impaired in within-class visual recognition memory across six different visual categories (guns, horses, scenes, tools, doors, and cars). In contrast, she scored normally on seven tests of face recognition, tests of memory for two other object categories (houses and glasses), and tests of recall memory for visual shapes. Testing confirmed that her impairment was not related to a general deficit in lower-level perception, object perception, basic-level recognition, or memory. AW's results provide the first neuropsychological evidence that recognition memory for non-face visual object categories can be selectively impaired in individuals without brain damage or other memory impairment. These results indicate that the development of recognition memory for faces does not depend on intact object recognition memory and provide further evidence for category-specific dissociations in visual

  9. Recognition and Resistance in TEM [superscript beta]-Lactamase

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

    Wang, Xiaojun; Minasov, George; Blazquez, Jesus

    Developing antimicrobials that are less likely to engender resistance has become an important design criterion as more and more drugs fall victim to resistance mutations. One hypothesis is that the more closely an inhibitor resembles a substrate, the more difficult it will be to develop resistant mutations that can at once disfavor the inhibitor and still recognize the substrate. To investigate this hypothesis, 10 transition-state analogues, of greater or lesser similarity to substrates, were tested for inhibition of TEM-1 beta-lactamase, the most widespread resistance enzyme to penicillin antibiotics. The inhibitors were also tested against four characteristic mutant enzymes: TEM-30, TEM-32,more » TEM-52, and TEM-64. The inhibitor most similar to the substrate, compound 10, was the most potent inhibitor of the WT enzyme, with a K(i) value of 64 nM. Conversely, compound 10 was the most susceptible to the TEM-30 (R244S) mutant, for which inhibition dropped by over 100-fold. The other inhibitors were relatively impervious to the TEM-30 mutant enzyme. To understand recognition and resistance to these transition-state analogues, the structures of four of these inhibitors in complex with TEM-1 were determined by X-ray crystallography. These structures suggest a structural basis for distinguishing inhibitors that mimic the acylation transition state and those that mimic the deacylation transition state; they also suggest how TEM-30 reduces the affinity of compound 10. In cell culture, this inhibitor reversed the resistance of bacteria to ampicillin, reducing minimum inhibitory concentrations of this penicillin by between 4- and 64-fold, depending on the strain of bacteria. Notwithstanding this activity, the resistance of TEM-30, which is already extant in the clinic, suggests that there can be resistance liabilities with substrate-based design.« less

  10. Complementary and partially complementary DNA duplexes tethered to a functionalized substrate: a molecular dynamics approach to biosensing.

    PubMed

    Monti, Susanna; Cacelli, Ivo; Ferretti, Alessandro; Prampolini, Giacomo; Barone, Vincenzo

    2011-07-21

    Molecular dynamics simulations (90 ns) of different DNA complexes attached to a functionalized substrate in solution were performed in order to clarify the behavior of mismatched DNA sequences captured by a tethered DNA probe (biochip). Examination of the trajectories revealed that the substrate influence and a series of cooperative events, including recognition, reorientation and reorganization of the bases, could induce the formation of stable duplexes having non-canonical arrangements. Major adjustment of the structures was observed when the mutated base was located in the end region of the chain close to the surface. This journal is © the Owner Societies 2011

  11. Novel Substrate-Based Inhibitors of Human Glutamate Carboxypeptidase II with Enhanced Lipophilicity

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

    Plechanovová, Anna; Byun, Youngjoo; Alquicer, Glenda

    2012-10-09

    Virtually all low molecular weight inhibitors of human glutamate carboxypeptidase II (GCPII) are highly polar compounds that have limited use in settings where more lipophilic molecules are desired. Here we report the identification and characterization of GCPII inhibitors with enhanced liphophilicity that are derived from a series of newly identified dipeptidic GCPII substrates featuring nonpolar aliphatic side chains at the C-terminus. To analyze the interactions governing the substrate recognition by GCPII, we determined crystal structures of the inactive GCPII(E424A) mutant in complex with selected dipeptides and complemented the structural data with quantum mechanics/molecular mechanics calculations. Results reveal the importance ofmore » nonpolar interactions governing GCPII affinity toward novel substrates as well as formerly unnoticed plasticity of the S1' specificity pocket. On the basis of those data, we designed, synthesized, and evaluated a series of novel GCPII inhibitors with enhanced lipophilicity, with the best candidates having low nanomolar inhibition constants and clogD > -0.3. Our findings offer new insights into the design of more lipophilic inhibitors targeting GCPII.« less

  12. A Tail of Two Sites: A Bipartite Mechanism for Recognition of Notch Ligands by Mind Bomb E3 Ligases

    PubMed Central

    McMillan, Brian J.; Schnute, Björn; Ohlenhard, Nadja; Zimmerman, Brandon; Miles, Laura; Beglova, Natalia; Klein, Thomas; Blacklow, Stephen C.

    2015-01-01

    Summary Mind bomb (Mib) proteins are large, multi-domain E3 ligases that promote ubiquitination of the cytoplasmic tails of Notch ligands. This ubiquitination step marks the ligand proteins for epsin-dependent endocytosis, which is critical for in vivo Notch receptor activation. We present here crystal structures of the substrate recognition domains of Mib1, both in isolation and in complex with peptides derived from Notch ligands. The structures, in combination with biochemical, cellular and in vivo assays, show that Mib1 contains two independent substrate recognition domains that engage two distinct epitopes from the cytoplasmic tail of the ligand Jagged1, one in the intracellular membrane proximal region and the other near the C-terminus. Together, these studies provide new insights into the mechanism of ubiquitin transfer by Mind bomb E3 ligases, illuminate a key event in ligand-induced activation of Notch receptors, and identify a potential new target for therapeutic modulation of Notch signal transduction in disease. PMID:25747658

  13. A Tail of Two Sites: A Bipartite Mechanism for Recognition of Notch Ligands by Mind Bomb E3 Ligases

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

    McMillan, Brian J.; Schnute, Björn; Ohlenhard, Nadja

    Mind bomb (Mib) proteins are large, multi-domain E3 ligases that promote ubiquitination of the cytoplasmic tails of Notch ligands. This ubiquitination step marks the ligand proteins for epsin-dependent endocytosis, which is critical for in vivo Notch receptor activation. Here we present crystal structures of the substrate recognition domains of Mib1, both in isolation and in complex with peptides derived from Notch ligands. The structures, in combination with biochemical, cellular, and in vivo assays, show that Mib1 contains two independent substrate recognition domains that engage two distinct epitopes from the cytoplasmic tail of the ligand Jagged1, one in the intracellular membranemore » proximal region and the other near the C terminus. Together, these studies provide insights into the mechanism of ubiquitin transfer by Mind bomb E3 ligases, illuminate a key event in ligand-induced activation of Notch receptors, and identify a potential target for therapeutic modulation of Notch signal transduction in disease.« less

  14. A Tail of Two Sites: A Bipartite Mechanism for Recognition of Notch Ligands by Mind Bomb E3 Ligases

    DOE PAGES

    McMillan, Brian J.; Schnute, Björn; Ohlenhard, Nadja; ...

    2015-03-05

    Mind bomb (Mib) proteins are large, multi-domain E3 ligases that promote ubiquitination of the cytoplasmic tails of Notch ligands. This ubiquitination step marks the ligand proteins for epsin-dependent endocytosis, which is critical for in vivo Notch receptor activation. Here we present crystal structures of the substrate recognition domains of Mib1, both in isolation and in complex with peptides derived from Notch ligands. The structures, in combination with biochemical, cellular, and in vivo assays, show that Mib1 contains two independent substrate recognition domains that engage two distinct epitopes from the cytoplasmic tail of the ligand Jagged1, one in the intracellular membranemore » proximal region and the other near the C terminus. Together, these studies provide insights into the mechanism of ubiquitin transfer by Mind bomb E3 ligases, illuminate a key event in ligand-induced activation of Notch receptors, and identify a potential target for therapeutic modulation of Notch signal transduction in disease.« less

  15. Breaking the Dogma of Aldolase Specificity: Simple Aliphatic Ketones and Aldehydes are Nucleophiles for Fructose-6-phosphate Aldolase.

    PubMed

    Roldán, Raquel; Sanchez-Moreno, Israel; Scheidt, Thomas; Hélaine, Virgil; Lemaire, Marielle; Parella, Teodor; Clapés, Pere; Fessner, Wolf-Dieter; Guérard-Hélaine, Christine

    2017-04-11

    d-Fructose-6-phosphate aldolase (FSA) was probed for extended nucleophile promiscuity by using a series of fluorogenic substrates to reveal retro-aldol activity. Four nucleophiles ethanal, propanone, butanone, and cyclopentanone were subsequently confirmed to be non-natural substrates in the synthesis direction using the wild-type enzyme and its D6H variant. This exceptional widening of the nucleophile substrate scope offers a rapid entry, in good yields and high stereoselectivity, to less oxygenated alkyl ketones and aldehydes, which was hitherto impossible. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Interdependence of Inhibitor Recognition in HIV-1 Protease

    PubMed Central

    2017-01-01

    Molecular recognition is a highly interdependent process. Subsite couplings within the active site of proteases are most often revealed through conditional amino acid preferences in substrate recognition. However, the potential effect of these couplings on inhibition and thus inhibitor design is largely unexplored. The present study examines the interdependency of subsites in HIV-1 protease using a focused library of protease inhibitors, to aid in future inhibitor design. Previously a series of darunavir (DRV) analogs was designed to systematically probe the S1′ and S2′ subsites. Co-crystal structures of these analogs with HIV-1 protease provide the ideal opportunity to probe subsite interdependency. All-atom molecular dynamics simulations starting from these structures were performed and systematically analyzed in terms of atomic fluctuations, intermolecular interactions, and water structure. These analyses reveal that the S1′ subsite highly influences other subsites: the extension of the hydrophobic P1′ moiety results in 1) reduced van der Waals contacts in the P2′ subsite, 2) more variability in the hydrogen bond frequencies with catalytic residues and the flap water, and 3) changes in the occupancy of conserved water sites both proximal and distal to the active site. In addition, one of the monomers in this homodimeric enzyme has atomic fluctuations more highly correlated with DRV than the other monomer. These relationships intricately link the HIV-1 protease subsites and are critical to understanding molecular recognition and inhibitor binding. More broadly, the interdependency of subsite recognition within an active site requires consideration in the selection of chemical moieties in drug design; this strategy is in contrast to what is traditionally done with independent optimization of chemical moieties of an inhibitor. PMID:28358514

  17. In silico engineering of aggregation-prone recombinant proteins for substrate recognition by the chaperonin GroEL.

    PubMed

    Kumar, Vipul; Punetha, Ankita; Sundar, Durai; Chaudhuri, Tapan K

    2012-01-01

    Molecular chaperones appear to have been evolved to facilitate protein folding in the cell through entrapment of folding intermediates on the interior of a large cavity formed between GroEL and its co-chaperonin GroES. They bind newly synthesized or non-native polypeptides through hydrophobic interactions and prevent their aggregation. Some proteins do not interact with GroEL, hence even though they are aggregation prone, cannot be assisted by GroEL for their folding. In this study, we have attempted to engineer these non-substrate proteins to convert them as the substrate for GroEL, without compromising on their function. We have used a computational biology approach to generate mutants of the selected proteins by selectively mutating residues in the hydrophobic patch, similar to GroES mobile loop region that are responsible for interaction with GroEL, and compared with the wild counterparts for calculation of their instability and aggregation propensities. The energies of the newly designed mutants were computed through molecular dynamics simulations. We observed increased aggregation propensity of some of the mutants formed after replacing charged amino acid residues with hydrophobic ones in the well defined hydrophobic patch, raising the possibility of their binding ability to GroEL. The newly generated mutants may provide potential substrates for Chaperonin GroEL, which can be experimentally generated and tested for their tendency of aggregation, interactions with GroEL and the possibility of chaperone-assisted folding to produce functional proteins.

  18. Substrate-binding specificity of chitinase and chitosanase as revealed by active-site architecture analysis.

    PubMed

    Liu, Shijia; Shao, Shangjin; Li, Linlin; Cheng, Zhi; Tian, Li; Gao, Peiji; Wang, Lushan

    2015-12-11

    Chitinases and chitosanases, referred to as chitinolytic enzymes, are two important categories of glycoside hydrolases (GH) that play a key role in degrading chitin and chitosan, two naturally abundant polysaccharides. Here, we investigate the active site architecture of the major chitosanase (GH8, GH46) and chitinase families (GH18, GH19). Both charged (Glu, His, Arg, Asp) and aromatic amino acids (Tyr, Trp, Phe) are observed with higher frequency within chitinolytic active sites as compared to elsewhere in the enzyme structure, indicating significant roles related to enzyme function. Hydrogen bonds between chitinolytic enzymes and the substrate C2 functional groups, i.e. amino groups and N-acetyl groups, drive substrate recognition, while non-specific CH-π interactions between aromatic residues and substrate mainly contribute to tighter binding and enhanced processivity evident in GH8 and GH18 enzymes. For different families of chitinolytic enzymes, the number, type, and position of substrate atoms bound in the active site vary, resulting in different substrate-binding specificities. The data presented here explain the synergistic action of multiple enzyme families at a molecular level and provide a more reasonable method for functional annotation, which can be further applied toward the practical engineering of chitinases and chitosanases. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Comprehensive structural and substrate specificity classification of the Saccharomyces cerevisiae methyltransferome.

    PubMed

    Wlodarski, Tomasz; Kutner, Jan; Towpik, Joanna; Knizewski, Lukasz; Rychlewski, Leszek; Kudlicki, Andrzej; Rowicka, Maga; Dziembowski, Andrzej; Ginalski, Krzysztof

    2011-01-01

    Methylation is one of the most common chemical modifications of biologically active molecules and it occurs in all life forms. Its functional role is very diverse and involves many essential cellular processes, such as signal transduction, transcriptional control, biosynthesis, and metabolism. Here, we provide further insight into the enzymatic methylation in S. cerevisiae by conducting a comprehensive structural and functional survey of all the methyltransferases encoded in its genome. Using distant homology detection and fold recognition, we found that the S. cerevisiae methyltransferome comprises 86 MTases (53 well-known and 33 putative with unknown substrate specificity). Structural classification of their catalytic domains shows that these enzymes may adopt nine different folds, the most common being the Rossmann-like. We also analyzed the domain architecture of these proteins and identified several new domain contexts. Interestingly, we found that the majority of MTase genes are periodically expressed during yeast metabolic cycle. This finding, together with calculated isoelectric point, fold assignment and cellular localization, was used to develop a novel approach for predicting substrate specificity. Using this approach, we predicted the general substrates for 24 of 33 putative MTases and confirmed these predictions experimentally in both cases tested. Finally, we show that, in S. cerevisiae, methylation is carried out by 34 RNA MTases, 32 protein MTases, eight small molecule MTases, three lipid MTases, and nine MTases with still unknown substrate specificity.

  20. Improved microautoradiographic method to determine individual microorganisms active in substrate uptake in natural waters.

    PubMed

    Tabor, P S; Neihof, R A

    1982-10-01

    We report a method which combines epifluorescence microscopy and microautoradiography to determine both the total number of microorganisms in natural water populations and those individual organisms active in the uptake of specific substrates. After incubation with H-labeled substrate, the sample is filtered and, while still on the filter, mounted directly in a film of autoradiographic emulsion on a microscope slide. The microautoradiogram is processed and stained with acridine orange, and, subsequently, the filter is removed before microscopic observation. This novel preparation resulted in increased accuracy in direct counts made from the autoradiogram, improved sensitivity in the recognition of uptake-active (H-labeled) organisms, and enumeration of a significantly greater number of labeled organisms compared with corresponding samples prepared by a previously reported method.

  1. Crystal structure of a four-copper laccase complexed with an arylamine: insights into substrate recognition and correlation with kinetics.

    PubMed

    Bertrand, Thomas; Jolivalt, Claude; Briozzo, Pierre; Caminade, Eliane; Joly, Nathalie; Madzak, Catherine; Mougin, Christian

    2002-06-11

    Laccases are multicopper oxidases that catalyze the oxidation of a wide range of phenols or arylamines, and their use in industrial oxidative processes is increasing. We purified from the white rot fungus Trametes versicolor a laccase that exists as five different isozymes, depending on glycosylation. The 2.4 A resolution structure of the most abundant isozyme of the glycosylated enzyme was solved. The four copper atoms are present, and it is the first crystal structure of a laccase in its active form. The crystallized enzyme binds 2,5-xylidine, which was used as a laccase inducer in the fungus culture. This arylamine is a very weak reducing substrate of the enzyme. The cavity enclosing 2,5-xylidine is rather wide, allowing the accommodation of substrates of various sizes. Several amino acid residues make hydrophobic interactions with the aromatic ring of the ligand. In addition, two charged or polar residues interact with its amino group. The first one is an histidine that also coordinates the copper that functions as the primary electron acceptor. The second is an aspartate conserved among fungal laccases. The purified enzyme can oxidize various hydroxylated compounds of the phenylurea family of herbicides that we synthesized. These phenolic substrates have better affinities at pH 5 than at pH 3, which could be related to the 2,5-xylidine binding by the aspartate. This is the first high-resolution structure of a multicopper oxidase complexed to a reducing substrate. It provides a model for engineering laccases that are either more efficient or with a wider substrate specificity.

  2. Promiscuous survivin peptide induces robust CD4+ T-cell responses in the majority of vaccinated cancer patients.

    PubMed

    Widenmeyer, Melanie; Griesemann, Heinrich; Stevanović, Stefan; Feyerabend, Susan; Klein, Reinhild; Attig, Sebastian; Hennenlotter, Jörg; Wernet, Dorothee; Kuprash, Dmitri V; Sazykin, Alexei Y; Pascolo, Steve; Stenzl, Arnulf; Gouttefangeas, Cécile; Rammensee, Hans-Georg

    2012-07-01

    CD4(+) T cells have been shown to be crucial for the induction and maintenance of cytotoxic T cell responses and to be also capable of mediating direct tumor rejection. Therefore, the anticancer therapeutic efficacy of peptide-based vaccines may be improved by addition of HLA class II epitopes to stimulate T helper cells. Survivin is an apoptosis inhibiting protein frequently overexpressed in tumors. Here we describe the first immunological evaluation of a survivin-derived CD4(+) T cell epitope in a multipeptide immunotherapy trial for prostate carcinoma patients. The survivin peptide is promiscuously presented by several human HLA-DRB1 molecules and, most importantly, is naturally processed by dendritic cells. In vaccinated patients, it was able to induce frequent, robust and multifunctional CD4(+) T cell responses, as monitored by IFN-γ ELISPOT and intracellular cytokine staining. Thus, this HLA-DR restricted epitope is broadly immunogenic and should be valuable for stimulating T helper cells in patients suffering from a wide range of tumors. Copyright © 2011 UICC.

  3. Conformational Plasticity of an Enzyme during Catalysis: Intricate Coupling between Cyclophilin A Dynamics and Substrate Turnover

    PubMed Central

    McGowan, Lauren C.; Hamelberg, Donald

    2013-01-01

    Enzyme catalysis is central to almost all biochemical processes, speeding up rates of reactions to biological relevant timescales. Enzymes make use of a large ensemble of conformations in recognizing their substrates and stabilizing the transition states, due to the inherent dynamical nature of biomolecules. The exact role of these diverse enzyme conformations and the interplay between enzyme conformational dynamics and catalysis is, according to the literature, not well understood. Here, we use molecular dynamics simulations to study human cyclophilin A (CypA), in order to understand the role of enzyme motions in the catalytic mechanism and recognition. Cyclophilin A is a tractable model system to study using classical simulation methods, because catalysis does not involve bond formation or breakage. We show that the conformational dynamics of active site residues of substrate-bound CypA is inherent in the substrate-free enzyme. CypA interacts with its substrate via conformational selection as the configurations of the substrate changes during catalysis. We also show that, in addition to tight intermolecular hydrophobic interactions between CypA and the substrate, an intricate enzyme-substrate intermolecular hydrogen-bonding network is extremely sensitive to the configuration of the substrate. These enzyme-substrate intermolecular interactions are loosely formed when the substrate is in the reactant and product states and become well formed and reluctant to break when the substrate is in the transition state. Our results clearly suggest coupling among enzyme-substrate intermolecular interactions, the dynamics of the enzyme, and the chemical step. This study provides further insights into the mechanism of peptidyl-prolyl cis/trans isomerases and the general interplay between enzyme conformational dynamics and catalysis. PMID:23332074

  4. Structures of a Na+-coupled, substrate-bound MATE multidrug transporter

    PubMed Central

    Lu, Min; Symersky, Jindrich; Radchenko, Martha; Koide, Akiko; Guo, Yi; Nie, Rongxin; Koide, Shohei

    2013-01-01

    Multidrug transporters belonging to the multidrug and toxic compound extrusion (MATE) family expel dissimilar lipophilic and cationic drugs across cell membranes by dissipating a preexisting Na+ or H+ gradient. Despite its clinical relevance, the transport mechanism of MATE proteins remains poorly understood, largely owing to a lack of structural information on the substrate-bound transporter. Here we report crystal structures of a Na+-coupled MATE transporter NorM from Neisseria gonorrheae in complexes with three distinct translocation substrates (ethidium, rhodamine 6G, and tetraphenylphosphonium), as well as Cs+ (a Na+ congener), all captured in extracellular-facing and drug-bound states. The structures revealed a multidrug-binding cavity festooned with four negatively charged amino acids and surprisingly limited hydrophobic moieties, in stark contrast to the general belief that aromatic amino acids play a prominent role in multidrug recognition. Furthermore, we discovered an uncommon cation–π interaction in the Na+-binding site located outside the drug-binding cavity and validated the biological relevance of both the substrate- and cation-binding sites by conducting drug resistance and transport assays. Additionally, we uncovered potential rearrangement of at least two transmembrane helices upon Na+-induced drug export. Based on our structural and functional analyses, we suggest that Na+ triggers multidrug extrusion by inducing protein conformational changes rather than by directly competing for the substrate-binding amino acids. This scenario is distinct from the canonical antiport mechanism, in which both substrate and counterion compete for a shared binding site in the transporter. Collectively, our findings provide an important step toward a detailed and mechanistic understanding of multidrug transport. PMID:23341609

  5. Legume-rhizobium symbiotic promiscuity and effectiveness do not affect plant invasiveness.

    PubMed

    Keet, Jan-Hendrik; Ellis, Allan G; Hui, Cang; Le Roux, Johannes J

    2017-06-01

    The ability to fix atmospheric nitrogen is thought to play an important role in the invasion success of legumes. Interactions between legumes and nitrogen-fixing bacteria (rhizobia) span a continuum of specialization, and promiscuous legumes are thought to have higher chances of forming effective symbioses in novel ranges. Using Australian Acacia species in South Africa, it was hypothesized that widespread and highly invasive species will be more generalist in their rhizobial symbiotic requirements and more effective in fixing atmospheric nitrogen compared with localized and less invasive species. To test these hypotheses, eight localized and 11 widespread acacias were examined using next-generation sequencing data for the nodulation gene, nodC , to compare the identity, species richness, diversity and compositional similarity of rhizobia associated with these acacias. Stable isotope analysis was also used to determine levels of nitrogen obtained from the atmosphere via symbiotic nitrogen fixation. No differences were found in richness, diversity and community composition between localized and widespread acacias. Similarly, widespread and localized acacias did not differ in their ability to fix atmospheric nitrogen. However, for some species by site comparisons, significant differences in δ15N isotopic signatures were found, indicating differential symbiotic effectiveness between these species at specific localities. Overall, the results support recent findings that root nodule rhizobial diversity and community composition do not differ between acacias that vary in their invasiveness. Differential invasiveness of acacias in South Africa is probably linked to attributes such as differences in propagule pressure, reasons for (e.g. forestry vs. ornamental) and extent of, plantings in the country. © The Author 2017. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com

  6. Crystal structures of the structure-selective nuclease Mus81-Eme1 bound to flap DNA substrates

    PubMed Central

    Gwon, Gwang Hyeon; Jo, Aera; Baek, Kyuwon; Jin, Kyeong Sik; Fu, Yaoyao; Lee, Jong-Bong; Kim, YoungChang; Cho, Yunje

    2014-01-01

    The Mus81-Eme1 complex is a structure-selective endonuclease with a critical role in the resolution of recombination intermediates during DNA repair after interstrand cross-links, replication fork collapse, or double-strand breaks. To explain the molecular basis of 3′ flap substrate recognition and cleavage mechanism by Mus81-Eme1, we determined crystal structures of human Mus81-Eme1 bound to various flap DNA substrates. Mus81-Eme1 undergoes gross substrate-induced conformational changes that reveal two key features: (i) a hydrophobic wedge of Mus81 that separates pre- and post-nick duplex DNA and (ii) a “5′ end binding pocket” that hosts the 5′ nicked end of post-nick DNA. These features are crucial for comprehensive protein-DNA interaction, sharp bending of the 3′ flap DNA substrate, and incision strand placement at the active site. While Mus81-Eme1 unexpectedly shares several common features with members of the 5′ flap nuclease family, the combined structural, biochemical, and biophysical analyses explain why Mus81-Eme1 preferentially cleaves 3′ flap DNA substrates with 5′ nicked ends. PMID:24733841

  7. Pheromone Recognition and Selectivity by ComR Proteins among Streptococcus Species

    PubMed Central

    Morrison, Donald A.; Talagas, Antoine; Nessler, Sylvie; Federle, Michael J.; Prehna, Gerd

    2016-01-01

    Natural transformation, or competence, is an ability inherent to bacteria for the uptake of extracellular DNA. This process is central to bacterial evolution and allows for the rapid acquirement of new traits, such as antibiotic resistance in pathogenic microorganisms. For the Gram-positive bacteria genus Streptococcus, genes required for competence are under the regulation of quorum sensing (QS) mediated by peptide pheromones. One such system, ComRS, consists of a peptide (ComS) that is processed (XIP), secreted, and later imported into the cytoplasm, where it binds and activates the transcription factor ComR. ComR then engages in a positive feedback loop for the expression of ComS and the alternative sigma-factor SigX. Although ComRS are present in the majority of Streptococcus species, the sequence of both ComS/XIP and ComR diverge significantly, suggesting a mechanism for species-specific communication. To study possible cross-talk between streptococcal species in the regulation of competence, and to explore in detail the molecular interaction between ComR and XIP we undertook an interdisciplinary approach. We developed a ‘test-bed’ assay to measure the activity of different ComR proteins in response to cognate and heterologous XIP peptides in vivo, revealing distinct ComR classes of strict, intermediate, and promiscuous specificity among species. We then solved an X-ray crystal structure of ComR from S. suis to further understand the interaction with XIP and to search for structural features in ComR proteins that may explain XIP recognition. Using the structure as a guide, we probed the apo conformation of the XIP-binding pocket by site-directed mutagenesis, both in test-bed cultures and biochemically in vitro. In alignments with ComR proteins from other species, we find that the pocket is lined by a variable and a conserved face, where residues of the conserved face contribute to ligand binding and the variable face discriminate among XIP peptides

  8. Promiscuity and selectivity of small-molecule inhibitors across TAM receptor tyrosine kinases in pediatric leukemia.

    PubMed

    Liu, Mao-Hua; Chen, Shi-Bing; Yu, Juan; Liu, Cheng-Jun; Zhang, Xiao-Jing

    2017-08-01

    The TAM receptor tyrosine kinase family member Mer has been recognized as an attractive therapeutic target for pediatric leukemia. Beside Mer the family contains other two kinases, namely, Tyro3 and Axl, which are highly homologues with Mer and thus most existing small-molecule inhibitors show moderate or high promiscuity across the three kinases. Here, the structural basis and energetic property of selective binding of small-molecule inhibitors to the three kinases were investigated at molecular level. It is found that the selectivity is primarily determined by the size, shape and configuration of kinase's ATP-binding site; the Mer and Axl possess a small, closed active pocket as compared to the bulky, open pocket of Tyro3. The location and conformation of active-site residues of Mer and Axl are highly consistent, suggesting that small-molecule inhibitors generally have a low Mer-over-Axl selectivity and a high Mer-over-Tyro3 selectivity. We demonstrated that the difference in ATP binding potency to the three kinases is also responsible for inhibitor selectivity. We also found that the long-range interactions and allosteric effect arising from rest of the kinase's active site can indirectly influence inhibitor binding and selectivity. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Decoding the patterns of ubiquitin recognition by ubiquitin-associated domains from free energy simulations.

    PubMed

    Bouvier, Benjamin

    2014-01-07

    Ubiquitin is a highly conserved, highly represented protein acting as a regulating signal in numerous cellular processes. It leverages a single hydrophobic binding patch to recognize and bind a large variety of protein domains with remarkable specificity, but can also self-assemble into chains of poly-diubiquitin units in which these interfaces are sequestered, profoundly altering the individual monomers' recognition characteristics. Despite numerous studies, the origins of this varied specificity and the competition between substrates for the binding of the ubiquitin interface patch remain under heated debate. This study uses enhanced sampling all-atom molecular dynamics to simulate the unbinding of complexes of mono- or K48-linked diubiquitin bound to several ubiquitin-associated domains, providing insights into the mechanism and free energetics of ubiquitin recognition and binding. The implications for the subtle tradeoff between the stability of the polyubiquitin signal and its easy recognition by target protein assemblies are discussed, as is the enhanced affinity of the latter for long polyubiquitin chains compared to isolated mono- or diubiquitin.

  10. Use of the recognition heuristic depends on the domain's recognition validity, not on the recognition validity of selected sets of objects.

    PubMed

    Pohl, Rüdiger F; Michalkiewicz, Martha; Erdfelder, Edgar; Hilbig, Benjamin E

    2017-07-01

    According to the recognition-heuristic theory, decision makers solve paired comparisons in which one object is recognized and the other not by recognition alone, inferring that recognized objects have higher criterion values than unrecognized ones. However, success-and thus usefulness-of this heuristic depends on the validity of recognition as a cue, and adaptive decision making, in turn, requires that decision makers are sensitive to it. To this end, decision makers could base their evaluation of the recognition validity either on the selected set of objects (the set's recognition validity), or on the underlying domain from which the objects were drawn (the domain's recognition validity). In two experiments, we manipulated the recognition validity both in the selected set of objects and between domains from which the sets were drawn. The results clearly show that use of the recognition heuristic depends on the domain's recognition validity, not on the set's recognition validity. In other words, participants treat all sets as roughly representative of the underlying domain and adjust their decision strategy adaptively (only) with respect to the more general environment rather than the specific items they are faced with.

  11. Minimal sulfated carbohydrates for recognition by L-selectin and the MECA-79 antibody.

    PubMed

    Bruehl, R E; Bertozzi, C R; Rosen, S D

    2000-10-20

    Sulfated forms of sialyl-Le(X) containing Gal-6-SO(4) or GlcNAc-6-SO(4) have been implicated as potential recognition determinants on high endothelial venule ligands for L-selectin. The optimal configuration of sulfate esters on the N-acetyllactosamine (Galbeta1-->4GlcNAc) core of sulfosialyl-Le(X), however, remains unsettled. Using a panel of sulfated lactose (Galbeta1-->4Glc) neoglycolipids as substrates in direct binding assays, we found that 6',6-disulfolactose was the preferred structure for L-selectin, although significant binding to 6'- and 6-sulfolactose was observed as well. Binding was EDTA-sensitive and blocked by L-selectin-specific monoclonal antibodies. Surprisingly, 6', 6-disulfolactose was poorly recognized by MECA-79, a carbohydrate- and sulfate-dependent monoclonal antibody that binds competitively to L-selectin ligands. Instead, MECA-79 bound preferentially to 6-sulfolactose. The difference in preferred substrates between L-selectin and MECA-79 may explain the variable activity of MECA-79 as an inhibitor of lymphocyte adhesion to high endothelial venules in lymphoid organs. Our results suggest that both Gal-6-SO(4) and GlcNAc-6-SO(4) may contribute to L-selectin recognition, either as components of sulfosialyl-Le(X) capping groups or in internal structures. By contrast, only GlcNAc-6-SO(4) appears to contribute to MECA-79 binding.

  12. Chlorella virus DNA ligase: nick recognition and mutational analysis.

    PubMed

    Sriskanda, V; Shuman, S

    1998-01-15

    Chlorella virus PBCV-1 DNA ligase seals nicked DNA substrates consisting of a 5'-phosphate-terminated strand and a 3'-hydroxyl-terminated strand annealed to a bridging DNA template strand. The enzyme discriminates at the DNA binding step between substrates containing a 5'-phosphate versus a 5'-hydroxyl at the nick. Mutational analysis of the active site motif KxDGxR (residues 27-32) illuminates essential roles for the conserved Lys, Asp and Arg moieties at different steps of the ligase reaction. Mutant K27A is unable to form the covalent ligase-(Lys-straightepsilonN-P)-adenylate intermediate and hence cannot activate a nicked DNA substrate via formation of the DNA-adenylate intermediate. Nonetheless, K27A catalyzes phosphodiester bond formation at a pre-adenylated nick. This shows that the active site lysine is not required for the strand closure reaction. K27A binds to nicked DNA-adenylate, but not to a standard DNA nick. This suggests that occupancy of the AMP binding pocket of DNA ligase is important for nick recognition. Mutant D29A is active in enzyme-adenylate formation and binds readily to nicked DNA, but is inert in DNA-adenylate formation. R32A is unable to catalyze any of the three reactions of the ligation pathway and does not bind to nicked DNA.

  13. Improved Microautoradiographic Method to Determine Individual Microorganisms Active in Substrate Uptake in Natural Waters

    PubMed Central

    Tabor, Paul S.; Neihof, Rex A.

    1982-01-01

    We report a method which combines epifluorescence microscopy and microautoradiography to determine both the total number of microorganisms in natural water populations and those individual organisms active in the uptake of specific substrates. After incubation with 3H-labeled substrate, the sample is filtered and, while still on the filter, mounted directly in a film of autoradiographic emulsion on a microscope slide. The microautoradiogram is processed and stained with acridine orange, and, subsequently, the filter is removed before microscopic observation. This novel preparation resulted in increased accuracy in direct counts made from the autoradiogram, improved sensitivity in the recognition of uptake-active (3H-labeled) organisms, and enumeration of a significantly greater number of labeled organisms compared with corresponding samples prepared by a previously reported method. Images PMID:16346120

  14. Conformational plasticity of an enzyme during catalysis: intricate coupling between cyclophilin A dynamics and substrate turnover.

    PubMed

    McGowan, Lauren C; Hamelberg, Donald

    2013-01-08

    Enzyme catalysis is central to almost all biochemical processes, speeding up rates of reactions to biological relevant timescales. Enzymes make use of a large ensemble of conformations in recognizing their substrates and stabilizing the transition states, due to the inherent dynamical nature of biomolecules. The exact role of these diverse enzyme conformations and the interplay between enzyme conformational dynamics and catalysis is, according to the literature, not well understood. Here, we use molecular dynamics simulations to study human cyclophilin A (CypA), in order to understand the role of enzyme motions in the catalytic mechanism and recognition. Cyclophilin A is a tractable model system to study using classical simulation methods, because catalysis does not involve bond formation or breakage. We show that the conformational dynamics of active site residues of substrate-bound CypA is inherent in the substrate-free enzyme. CypA interacts with its substrate via conformational selection as the configurations of the substrate changes during catalysis. We also show that, in addition to tight intermolecular hydrophobic interactions between CypA and the substrate, an intricate enzyme-substrate intermolecular hydrogen-bonding network is extremely sensitive to the configuration of the substrate. These enzyme-substrate intermolecular interactions are loosely formed when the substrate is in the reactant and product states and become well formed and reluctant to break when the substrate is in the transition state. Our results clearly suggest coupling among enzyme-substrate intermolecular interactions, the dynamics of the enzyme, and the chemical step. This study provides further insights into the mechanism of peptidyl-prolyl cis/trans isomerases and the general interplay between enzyme conformational dynamics and catalysis. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  15. Structural determinants of species-selective substrate recognition in human and Drosophila serotonin transporters revealed through computational docking studies

    PubMed Central

    Kaufmann, Kristian W.; Dawson, Eric S.; Henry, L. Keith; Field, Julie R.; Blakely, Randy D.; Meiler, Jens

    2009-01-01

    To identify potential determinants of substrate selectivity in serotonin (5-HT) transporters (SERT), models of human and Drosophila serotonin transporters (hSERT, dSERT) were built based on the leucine transporter (LeuTAa) structure reported by Yamashita et al. (Nature 2005;437:215–223), PBDID 2A65. Although the overall amino acid identity between SERTs and the LeuTAa is only 17%, it increases to above 50% in the first shell of the putative 5-HT binding site, allowing de novo computational docking of tryptamine derivatives in atomic detail. Comparison of hSERT and dSERT complexed with substrates pinpoints likely structural determinants for substrate binding. Forgoing the use of experimental transport and binding data of tryptamine derivatives for construction of these models enables us to cHitically assess and validate their predictive power: A single 5-HT binding mode was identified that retains the amine placement observed in the LeuTAa structure, matches site-directed mutagenesis and substituted cysteine accessibility method (SCAM) data, complies with support vector machine derived relations activity relations, and predicts computational binding energies for 5-HT analogs with a significant correlation coefficient (R = 0.72). This binding mode places 5-HT deep in the binding pocket of the SERT with the 5-position near residue hSERT A169/dSERT D164 in transmembrane helix 3, the indole nitrogen next to residue Y176/Y171, and the ethylamine tail under residues F335/F327 and S336/S328 within 4 Å of residue D98. Our studies identify a number of potential contacts whose contribution to substrate binding and transport was previously unsuspected. PMID:18704946

  16. The “parts and wholes” of face recognition: a review of the literature

    PubMed Central

    Tanaka, James W.; Simonyi, Diana

    2016-01-01

    It has been claimed that faces are recognized as a “whole” rather than the recognition of individual parts. In a paper published in the Quarterly Journal of Experimental Psychology in 1993, Martha Farah and I attempted to operationalize the holistic claim using the part/whole task. In this task, participants studied a face and then their memory presented in isolation and in the whole face. Consistent with the holistic view, recognition of the part was superior when tested in the whole-face condition compared to when it was tested in isolation. The “whole face” or holistic advantage was not found for faces that were inverted, or scrambled, nor for non-face objects suggesting that holistic encoding was specific to normal, intact faces. In this paper, we reflect on the part/whole paradigm and how it has contributed to our understanding of what it means to recognize a face as a “whole” stimulus. We describe the value of part/whole task for developing theories of holistic and non-holistic recognition of faces and objects. We discuss the research that has probed the neural substrates of holistic processing in healthy adults and people with prosopagnosia and autism. Finally, we examine how experience shapes holistic face recognition in children and recognition of own- and other-race faces in adults. The goal of this article is to summarize the research on the part/whole task and speculate on how it has informed our understanding of holistic face processing. PMID:26886495

  17. The trimethylammonium headgroup of choline is a major determinant for substrate binding and specificity in choline oxidase.

    PubMed

    Gadda, Giovanni; Powell, Nichole L N; Menon, Prashanthi

    2004-10-15

    Choline oxidase catalyzes the oxidation of choline to glycine betaine via two sequential flavin-linked transfers of hydride equivalents to molecular oxygen and formation of a betaine aldehyde intermediate. In the present study, choline and glycine betaine analogs were used as substrates and inhibitors for the enzyme to investigate the structural determinants that are relevant for substrate recognition and specificity. Competitive inhibition patterns with respect to choline were determined for a number of substituted amines at pH 6.5 and 25 degrees C. The Kis values for the carboxylate-containing ligands glycine betaine, N,N-dimethylglycine, and N-methylglycine increased monotonically with decreasing number of methyl groups, consistent with the trimethylammonium portion of the ligand being important for binding. In contrast, the acetate portion of glycine betaine did not contribute to binding, as suggested by lack of changes in the Kis values upon substituting glycine betaine with inhibitors containing methyl, ethyl, allyl, and 2-amino-ethyl side chains. In agreement with the inhibition data, the specificity of the enzyme for the organic substrate (kcat/Km value) decreased when N,N-dimethylethanolamine, N-methylethanolamine, and the isosteric substrate 3,3-dimethyl-1-butanol were used as substrate instead of choline; a contribution of approximately 7 kcal mol(-1) toward substrate discrimination was estimated for the interaction of the trimethylammonium portion of the substrate with the active site of choline oxidase.

  18. Alteration in substrate specificity of horse liver alcohol dehydrogenase by an acyclic nicotinamide analog of NAD(+).

    PubMed

    Malver, Olaf; Sebastian, Mina J; Oppenheimer, Norman J

    2014-11-01

    A new, acyclic NAD-analog, acycloNAD(+) has been synthesized where the nicotinamide ribosyl moiety has been replaced by the nicotinamide (2-hydroxyethoxy)methyl moiety. The chemical properties of this analog are comparable to those of β-NAD(+) with a redox potential of -324mV and a 341nm λmax for the reduced form. Both yeast alcohol dehydrogenase (YADH) and horse liver alcohol dehydrogenase (HLADH) catalyze the reduction of acycloNAD(+) by primary alcohols. With HLADH 1-butanol has the highest Vmax at 49% that of β-NAD(+). The primary deuterium kinetic isotope effect is greater than 3 indicating a significant contribution to the rate limiting step from cleavage of the carbon-hydrogen bond. The stereochemistry of the hydride transfer in the oxidation of stereospecifically deuterium labeled n-butanol is identical to that for the reaction with β-NAD(+). In contrast to the activity toward primary alcohols there is no detectable reduction of acycloNAD(+) by secondary alcohols with HLADH although these alcohols serve as competitive inhibitors. The net effect is that acycloNAD(+) has converted horse liver ADH from a broad spectrum alcohol dehydrogenase, capable of utilizing either primary or secondary alcohols, into an exclusively primary alcohol dehydrogenase. This is the first example of an NAD analog that alters the substrate specificity of a dehydrogenase and, like site-directed mutagenesis of proteins, establishes that modifications of the coenzyme distance from the active site can be used to alter enzyme function and substrate specificity. These and other results, including the activity with α-NADH, clearly demonstrate the promiscuity of the binding interactions between dehydrogenases and the riboside phosphate of the nicotinamide moiety, thus greatly expanding the possibilities for the design of analogs and inhibitors of specific dehydrogenases. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Structure of the carboxypeptidase B complex with N-sulfamoyl-L-phenylalanine - a transition state analog of non-specific substrate.

    PubMed

    Akparov, Valery; Timofeev, Vladimir; Khaliullin, Ilyas; Švedas, Vytas; Kuranova, Inna

    2018-03-01

    Carboxypeptidase B (EC 3.4.17.2) (CPB) is commonly used in the industrial insulin production and as a template for drug design. However, its ability to discriminate substrates with hydrophobic, hydrophilic, and charged side chains is not well understood. We report structure of CPB complex with a transition state analog N-sulfamoyl-L-phenylalanine solved at 1.74Å. The study provided an insight into structural basis of CPB substrate specificity. Ligand binding is affected by structure-depended conformational changes of Asp255 in S1'-subsite, interactions with Asn144 and Arg145 in C-terminal binding subsite, and Glu270 in the catalytic center. Side chain of the non-specific substrate analog SPhe in comparison with that of specific substrate analog SArg (reported earlier) not only loses favorable electrostatic interactions and two hydrogen bonds with Asp255 and three fixed water molecules, but is forced to be in the unfavorable hydrophilic environment. Thus, Ser207, Gly253, Tyr248, and Asp255 residues play major role in the substrate recognition by S1'-subsite.

  20. Power electronics substrate for direct substrate cooling

    DOEpatents

    Le, Khiet [Mission Viejo, CA; Ward, Terence G [Redondo Beach, CA; Mann, Brooks S [Redondo Beach, CA; Yankoski, Edward P [Corona, CA; Smith, Gregory S [Woodland Hills, CA

    2012-05-01

    Systems and apparatus are provided for power electronics substrates adapted for direct substrate cooling. A power electronics substrate comprises a first surface configured to have electrical circuitry disposed thereon, a second surface, and a plurality of physical features on the second surface. The physical features are configured to promote a turbulent boundary layer in a coolant impinged upon the second surface.

  1. The emerging role of promiscuous 7TM receptors as chemosensors for food intake.

    PubMed

    Wellendorph, Petrine; Johansen, Lars Dan; Bräuner-Osborne, Hans

    2010-01-01

    In recent years, several highly promiscuous seven transmembrane (7TM) receptors have been cloned and characterized of which many are activated broadly by amino acids, proteolytic degradation products, carbohydrates, or free fatty acids (FFAs) and are expressed in taste tissue, the gastrointestinal (GI) tract, endocrine glands, adipose tissue, and/or kidney. This has led to the hypothesis that these receptors may act as sensors of food intake modulating, for example, release of incretin hormones from the gut, insulin/glucagon from the pancreas, and leptin from adipose tissue. In the present review, we describe the molecular mechanisms of nutrient-sensing of the calcium-sensing receptor (CaR), the G protein-coupled receptor family C, group 6, subtype A (GPRC6A), and the taste1 receptor T1R1/T1R3-sensing L-α-amino acids; the carbohydrate-sensing T1R2/T1R3 receptor; the proteolytic degradation product sensor GPR93 (also termed GPR92); and the FFA sensing receptors FFA1, FFA2, FFA3, GPR84, and GPR120. Due to their omnipresent nature, the natural ligands have had limited usability in pharmacological/physiological studies which has hampered the elucidation of the physiological function and therapeutic prospect of their receptors. However, an increasing number of subtype-selective ligands and/or receptor knockout mice are being developed which at least for some of the receptors have validated them as promising drug targets in, for example, type II diabetes. Copyright © 2010 Elsevier Inc. All rights reserved.

  2. Darwin revisited: The vagus nerve is a causal element in controlling recognition of other's emotions.

    PubMed

    Colzato, Lorenza S; Sellaro, Roberta; Beste, Christian

    2017-07-01

    Charles Darwin proposed that via the vagus nerve, the tenth cranial nerve, emotional facial expressions are evolved, adaptive and serve a crucial communicative function. In line with this idea, the later-developed polyvagal theory assumes that the vagus nerve is the key phylogenetic substrate that regulates emotional and social behavior. The polyvagal theory assumes that optimal social interaction, which includes the recognition of emotion in faces, is modulated by the vagus nerve. So far, in humans, it has not yet been demonstrated that the vagus plays a causal role in emotion recognition. To investigate this we employed transcutaneous vagus nerve stimulation (tVNS), a novel non-invasive brain stimulation technique that modulates brain activity via bottom-up mechanisms. A sham/placebo-controlled, randomized cross-over within-subjects design was used to infer a causal relation between the stimulated vagus nerve and the related ability to recognize emotions as indexed by the Reading the Mind in the Eyes Test in 38 healthy young volunteers. Active tVNS, compared to sham stimulation, enhanced emotion recognition for easy items, suggesting that it promoted the ability to decode salient social cues. Our results confirm that the vagus nerve is causally involved in emotion recognition, supporting Darwin's argumentation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Structures of Saccharomyces cerevisiae D-arabinose dehydrogenase Ara1 and its complex with NADPH: implications for cofactor-assisted substrate recognition.

    PubMed

    Hu, Xiao-Qian; Guo, Peng-Chao; Ma, Jin-Di; Li, Wei-Fang

    2013-11-01

    The primary role of yeast Ara1, previously mis-annotated as a D-arabinose dehydrogenase, is to catalyze the reduction of a variety of toxic α,β-dicarbonyl compounds using NADPH as a cofactor at physiological pH levels. Here, crystal structures of Ara1 in apo and NADPH-complexed forms are presented at 2.10 and 2.00 Å resolution, respectively. Ara1 exists as a homodimer, each subunit of which adopts an (α/β)8-barrel structure and has a highly conserved cofactor-binding pocket. Structural comparison revealed that induced fit upon NADPH binding yielded an intact active-site pocket that recognizes the substrate. Moreover, the crystal structures combined with computational simulation defined an open substrate-binding site to accommodate various substrates that possess a dicarbonyl group.

  4. Key Mutations Alter the Cytochrome P450 BM3 Conformational Landscape and Remove Inherent Substrate Bias*

    PubMed Central

    Butler, Christopher F.; Peet, Caroline; Mason, Amy E.; Voice, Michael W.; Leys, David; Munro, Andrew W.

    2013-01-01

    Cytochrome P450 monooxygenases (P450s) have enormous potential in the production of oxychemicals, due to their unparalleled regio- and stereoselectivity. The Bacillus megaterium P450 BM3 enzyme is a key model system, with several mutants (many distant from the active site) reported to alter substrate selectivity. It has the highest reported monooxygenase activity of the P450 enzymes, and this catalytic efficiency has inspired protein engineering to enable its exploitation for biotechnologically relevant oxidations with structurally diverse substrates. However, a structural rationale is lacking to explain how these mutations have such effects in the absence of direct change to the active site architecture. Here, we provide the first crystal structures of BM3 mutants in complex with a human drug substrate, the proton pump inhibitor omeprazole. Supported by solution data, these structures reveal how mutation alters the conformational landscape and decreases the free energy barrier for transition to the substrate-bound state. Our data point to the importance of such “gatekeeper” mutations in enabling major changes in substrate recognition. We further demonstrate that these mutants catalyze the same 5-hydroxylation reaction as performed by human CYP2C19, the major human omeprazole-metabolizing P450 enzyme. PMID:23828198

  5. Rotation-invariant neural pattern recognition system with application to coin recognition.

    PubMed

    Fukumi, M; Omatu, S; Takeda, F; Kosaka, T

    1992-01-01

    In pattern recognition, it is often necessary to deal with problems to classify a transformed pattern. A neural pattern recognition system which is insensitive to rotation of input pattern by various degrees is proposed. The system consists of a fixed invariance network with many slabs and a trainable multilayered network. The system was used in a rotation-invariant coin recognition problem to distinguish between a 500 yen coin and a 500 won coin. The results show that the approach works well for variable rotation pattern recognition.

  6. "Off-On"switching electrochemiluminescence biosensor for mercury(II) detection based on molecular recognition technology.

    PubMed

    Cheng, Lin; Wei, BingGuo; He, Ling Ling; Mao, Ling; Zhang, Jie; Ceng, JinXiang; Kong, DeRong; Chen, ChaDan; Cui, HanFeng; Hong, Nian; Fan, Hao

    2017-02-01

    A novel "off-On" electrogenerated chemiluminescence (ECL) biosensor has been developed for the detection of mercury(II) based on molecular recognition technology. The ECL mercury(II) biosensor comprises two main parts: an ECL substrate and an ECL intensity switch. The ECL substrate was made by modifying the complex of Ruthenium(II) tris-(bipyridine)(Ru(bpy) 3 2+ )/Cyclodextrins-Au nanoparticles(CD-AuNps)/Nafion on the surface of glass carbon electrode (GCE), and the ECL intensity switch is the single hairpin DNA probe designed according to the "molecular recognition" strategy which was functionalized with ferrocene tag at one end and attached to Cyclodextrins (CD) on modified GCE through supramolecular noncovalent interaction. We demonstrated that, in the absence of Hg(II) ion, the probe keeps single hairpin structure and resulted in a quenching of ECL of Ru(bpy) 3 2+ . Whereas, in the presence of Hg(II) ion, the probe prefers to form the T-Hg(II)-T complex and lead to an obvious recovery of ECL of Ru(bpy) 3 2+ , which provided a sensing platform for the detection of Hg(II) ion. Using this sensing platform, a simple, rapid and selective "off-On" ECL biosensor for the detection of mercury(II) with a detection limit of 0.1 nM has been developed. Copyright © 2016. Published by Elsevier Inc.

  7. A model of EcoRII restriction endonuclease action: the active complex is most likely formed by one protein subunit and one DNA recognition site

    NASA Technical Reports Server (NTRS)

    Karpova, E. A.; Kubareva, E. A.; Shabarova, Z. A.

    1999-01-01

    To elucidate the mechanism of interaction of restriction endonuclease EcoRII with DNA, we studied by native gel electrophoresis the binding of this endonuclease to a set of synthetic DNA-duplexes containing the modified or canonical recognition sequence 5'-d(CCA/TGG)-3'. All binding substrate or substrate analogues tested could be divided into two major groups: (i) duplexes that, at the interaction with endonuclease EcoRII, form two types of stable complexes on native gel in the absence of Mg2+ cofactor; (ii) duplexes that form only one type of complex, observed both in the presence and absence of Mg2+. Unlike the latter, duplexes under the first group can be hydrolyzed by endonuclease. Data obtained suggest that the active complex is most likely formed by one protein subunit and one DNA recognition sequence. A model of EcoRII endonuclease action is presented.

  8. Nucleosome Recognition by the Piccolo NuA4 Histone Acetyltransferase Complex†

    PubMed Central

    Berndsen, Christopher E.; Selleck, William; McBryant, Steven J.; Hansen, Jeffrey C.; Tan, Song; Demi, John M.

    2007-01-01

    The mechanisms by which multisubunit histone acetyltransferase (HAT) complexes recognize and perform efficient acetylation on nucleosome substrates are largely unknown. Here, we use a variety of biochemical approaches and compare histone-based substrates of increasing complexity to determine the critical components of nucleosome recognition by the MOZ, Ybf2/Sas3, Sas2, Tip60 family HAT complex, Piccolo NuA4 (picNuA4). We find the histone tails to be dispensable for binding to both nucleosomes and free histones and that the H2A, H3, and H2B tails do not influence the ability of picNuA4 to tetra-acetylate the H4 tail within the nucleosome. Most notably, we discovered that the histone-fold domain (HFD) regions of histones, particularly residues 21–52 of H4, are critical for tight binding and efficient tail acetylation. Presented evidence suggests that picNuA4 recognizes the open surface of the nucleosome on which the HFD of H4 is located. This binding mechanism serves to direct substrate access to the tails of H4 and H2A and allows the enzyme to be “tethered”, thereby increasing the effective concentration of the histone tail and permitting successive cycles of H4 tail acetylation. PMID:17274630

  9. A promiscuous liaison between IL-15 receptor and Axl receptor tyrosine kinase in cell death control

    PubMed Central

    Budagian, Vadim; Bulanova, Elena; Orinska, Zane; Thon, Lutz; Mamat, Uwe; Bellosta, Paola; Basilico, Claudio; Adam, Dieter; Paus, Ralf; Bulfone-Paus, Silvia

    2005-01-01

    Discrimination between cytokine receptor and receptor tyrosine kinase (RTK) signaling pathways is a central paradigm in signal transduction research. Here, we report a ‘promiscuous liaison' between both receptors that enables interleukin (IL)-15 to transactivate the signaling pathway of a tyrosine kinase. IL-15 protects murine L929 fibroblasts from tumor necrosis factor α (TNFα)-induced cell death, but fails to rescue them upon targeted depletion of the RTK, Axl; however, Axl-overexpressing fibroblasts are TNFα-resistant. IL-15Rα and Axl colocalize on the cell membrane and co-immunoprecipitate even in the absence of IL-15, whereby the extracellular part of Axl proved to be essential for Axl/IL-15Rα interaction. Most strikingly, IL-15 treatment mimics stimulation by the Axl ligand, Gas6, resulting in a rapid tyrosine phosphorylation of both Axl and IL-15Rα, and activation of the phosphatidylinositol 3-kinase/Akt pathway. This is also seen in mouse embryonic fibroblasts from wild-type but not Axl−/− or IL-15Rα−/− mice. Thus, IL-15-induced protection from TNFα-mediated cell death involves a hitherto unknown IL-15 receptor complex, consisting of IL-15Rα and Axl RTK, and requires their reciprocal activation initiated by ligand-induced IL-15Rα. PMID:16308569

  10. A Plasmodium Promiscuous T Cell Epitope Delivered within the Ad5 Hexon Protein Enhances the Protective Efficacy of a Protein Based Malaria Vaccine.

    PubMed

    Fonseca, Jairo Andres; Cabrera-Mora, Monica; Kashentseva, Elena A; Villegas, John Paul; Fernandez, Alejandra; Van Pelt, Amelia; Dmitriev, Igor P; Curiel, David T; Moreno, Alberto

    2016-01-01

    A malaria vaccine is a public health priority. In order to produce an effective vaccine, a multistage approach targeting both the blood and the liver stage infection is desirable. The vaccine candidates also need to induce balanced immune responses including antibodies, CD4+ and CD8+ T cells. Protein-based subunit vaccines like RTS,S are able to induce strong antibody response but poor cellular reactivity. Adenoviral vectors have been effective inducing protective CD8+ T cell responses in several models including malaria; nonetheless this vaccine platform exhibits a limited induction of humoral immune responses. Two approaches have been used to improve the humoral immunogenicity of recombinant adenovirus vectors, the use of heterologous prime-boost regimens with recombinant proteins or the genetic modification of the hypervariable regions (HVR) of the capsid protein hexon to express B cell epitopes of interest. In this study, we describe the development of capsid modified Ad5 vectors that express a promiscuous Plasmodium yoelii T helper epitope denominated PyT53 within the hexon HVR2 region. Several regimens were tested in mice to determine the relevance of the hexon modification in enhancing protective immune responses induced by the previously described protein-based multi-stage experimental vaccine PyCMP. A heterologous prime-boost immunization regime that combines a hexon modified vector with transgenic expression of PyCMP followed by protein immunizations resulted in the induction of robust antibody and cellular immune responses in comparison to a similar regimen that includes a vector with unmodified hexon. These differences in immunogenicity translated into a better protective efficacy against both the hepatic and red blood cell stages of P. yoelii. To our knowledge, this is the first time that a hexon modification is used to deliver a promiscuous T cell epitope. Our data support the use of such modification to enhance the immunogenicity and protective

  11. Investigating biomolecular recognition at the cell surface using atomic force microscopy.

    PubMed

    Wang, Congzhou; Yadavalli, Vamsi K

    2014-05-01

    Probing the interaction forces that drive biomolecular recognition on cell surfaces is essential for understanding diverse biological processes. Force spectroscopy has been a widely used dynamic analytical technique, allowing measurement of such interactions at the molecular and cellular level. The capabilities of working under near physiological environments, combined with excellent force and lateral resolution make atomic force microscopy (AFM)-based force spectroscopy a powerful approach to measure biomolecular interaction forces not only on non-biological substrates, but also on soft, dynamic cell surfaces. Over the last few years, AFM-based force spectroscopy has provided biophysical insight into how biomolecules on cell surfaces interact with each other and induce relevant biological processes. In this review, we focus on describing the technique of force spectroscopy using the AFM, specifically in the context of probing cell surfaces. We summarize recent progress in understanding the recognition and interactions between macromolecules that may be found at cell surfaces from a force spectroscopy perspective. We further discuss the challenges and future prospects of the application of this versatile technique. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Placing a Disrupted Degradation Motif at the C Terminus of Proteasome Substrates Attenuates Degradation without Impairing Ubiquitylation*

    PubMed Central

    Alfassy, Omri S.; Cohen, Itamar; Reiss, Yuval; Tirosh, Boaz; Ravid, Tommer

    2013-01-01

    Protein elimination by the ubiquitin-proteasome system requires the presence of a cis-acting degradation signal. Efforts to discern degradation signals of misfolded proteasome substrates thus far revealed a general mechanism whereby the exposure of cryptic hydrophobic motifs provides a degradation determinant. We have previously characterized such a determinant, employing the yeast kinetochore protein Ndc10 as a model substrate. Ndc10 is essentially a stable protein that is rapidly degraded upon exposure of a hydrophobic motif located at the C-terminal region. The degradation motif comprises two distinct and essential elements: DegA, encompassing two amphipathic helices, and DegB, a hydrophobic sequence within the loosely structured C-terminal tail of Ndc10. Here we show that the hydrophobic nature of DegB is irrelevant for the ubiquitylation of substrates containing the Ndc10 degradation motif, but is essential for proteasomal degradation. Mutant DegB, in which the hydrophobic sequence was disrupted, acted as a dominant degradation inhibitory element when expressed at the C-terminal regions of ubiquitin-dependent and -independent substrates of the 26S proteasome. This mutant stabilized substrates in both yeast and mammalian cells, indicative of a modular recognition moiety. The dominant function of the mutant DegB provides a powerful experimental tool for evaluating the physiological implications of stabilization of specific proteasome substrates in intact cells and for studying the associated pathological effects. PMID:23519465

  13. Familiar Person Recognition: Is Autonoetic Consciousness More Likely to Accompany Face Recognition Than Voice Recognition?

    NASA Astrophysics Data System (ADS)

    Barsics, Catherine; Brédart, Serge

    2010-11-01

    Autonoetic consciousness is a fundamental property of human memory, enabling us to experience mental time travel, to recollect past events with a feeling of self-involvement, and to project ourselves in the future. Autonoetic consciousness is a characteristic of episodic memory. By contrast, awareness of the past associated with a mere feeling of familiarity or knowing relies on noetic consciousness, depending on semantic memory integrity. Present research was aimed at evaluating whether conscious recollection of episodic memories is more likely to occur following the recognition of a familiar face than following the recognition of a familiar voice. Recall of semantic information (biographical information) was also assessed. Previous studies that investigated the recall of biographical information following person recognition used faces and voices of famous people as stimuli. In this study, the participants were presented with personally familiar people's voices and faces, thus avoiding the presence of identity cues in the spoken extracts and allowing a stricter control of frequency exposure with both types of stimuli (voices and faces). In the present study, the rate of retrieved episodic memories, associated with autonoetic awareness, was significantly higher from familiar faces than familiar voices even though the level of overall recognition was similar for both these stimuli domains. The same pattern was observed regarding semantic information retrieval. These results and their implications for current Interactive Activation and Competition person recognition models are discussed.

  14. The external gate of the human and Drosophila serotonin transporters requires a basic/acidic amino acid pair for 3,4-methylenedioxymethamphetamine (MDMA) translocation and the induction of substrate efflux.

    PubMed

    Sealover, Natalie R; Felts, Bruce; Kuntz, Charles P; Jarrard, Rachel E; Hockerman, Gregory H; Lamb, Patrick W; Barker, Eric L; Henry, L Keith

    2016-11-15

    The substituted amphetamine, 3,4-methylenedioxy-methamphetamine (MDMA, ecstasy), is a widely used drug of abuse that induces non-exocytotic release of serotonin, dopamine, and norepinephrine through their cognate transporters as well as blocking the reuptake of neurotransmitter by the same transporters. The resulting dramatic increase in volume transmission and signal duration of neurotransmitters leads to psychotropic, stimulant, and entactogenic effects. The mechanism by which amphetamines drive reverse transport of the monoamines remains largely enigmatic, however, promising outcomes for the therapeutic utility of MDMA for post-traumatic stress disorder and the long-time use of the dopaminergic and noradrenergic-directed amphetamines in treatment of attention-deficit hyperactivity disorder and narcolepsy increases the importance of understanding this phenomenon. Previously, we identified functional differences between the human and Drosophila melanogaster serotonin transporters (hSERT and dSERT, respectively) revealing that MDMA is an effective substrate for hSERT but not dSERT even though serotonin is a potent substrate for both transporters. Chimeric dSERT/hSERT transporters revealed that the molecular components necessary for recognition of MDMA as a substrate was linked to regions of the protein flanking transmembrane domains (TM) V through IX. Here, we performed species-scanning mutagenesis of hSERT, dSERT and C. elegans SERT (ceSERT) along with biochemical and electrophysiological analysis and identified a single amino acid in TM10 (Glu394, hSERT; Asn484, dSERT, Asp517, ceSERT) that is primarily responsible for the differences in MDMA recognition. Our findings reveal that an acidic residue is necessary at this position for MDMA recognition as a substrate and serotonin releaser. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Catalytic Function and Substrate Specificity of the Papain-Like Protease Domain of nsp3 from the Middle East Respiratory Syndrome Coronavirus

    PubMed Central

    Báez-Santos, Yahira M.; Mielech, Anna M.; Deng, Xufang; Baker, Susan

    2014-01-01

    ABSTRACT The papain-like protease (PLpro) domain from the deadly Middle East respiratory syndrome coronavirus (MERS-CoV) was overexpressed and purified. MERS-CoV PLpro constructs with and without the putative ubiquitin-like (UBL) domain at the N terminus were found to possess protease, deubiquitinating, deISGylating, and interferon antagonism activities in transfected HEK293T cells. The quaternary structure and substrate preferences of MERS-CoV PLpro were determined and compared to those of severe acute respiratory syndrome coronavirus (SARS-CoV) PLpro, revealing prominent differences between these closely related enzymes. Steady-state kinetic analyses of purified MERS-CoV and SARS-CoV PLpros uncovered significant differences in their rates of hydrolysis of 5-aminomethyl coumarin (AMC) from C-terminally labeled peptide, ubiquitin, and ISG15 substrates, as well as in their rates of isopeptide bond cleavage of K48- and K63-linked polyubiquitin chains. MERS-CoV PLpro was found to have 8-fold and 3,500-fold higher catalytic efficiencies for hydrolysis of ISG15-AMC than for hydrolysis of the Ub-AMC and Z-RLRGG-AMC substrates, respectively. A similar trend was observed for SARS-CoV PLpro, although it was much more efficient than MERS-CoV PLpro toward ISG15-AMC and peptide-AMC substrates. MERS-CoV PLpro was found to process K48- and K63-linked polyubiquitin chains at similar rates and with similar debranching patterns, producing monoubiquitin species. However, SARS-CoV PLpro much preferred K48-linked polyubiquitin chains to K63-linked chains, and it rapidly produced di-ubiquitin molecules from K48-linked chains. Finally, potent inhibitors of SARS-CoV PLpro were found to have no effect on MERS-CoV PLpro. A homology model of the MERS-CoV PLpro structure was generated and compared to the X-ray structure of SARS-CoV PLpro to provide plausible explanations for differences in substrate and inhibitor recognition. IMPORTANCE Unlocking the secrets of how coronavirus (CoV) papain

  16. Structure and substrate-binding mechanism of human Ap4A hydrolase.

    PubMed

    Swarbrick, James D; Buyya, Smrithi; Gunawardana, Dilantha; Gayler, Kenwyn R; McLennan, Alexander G; Gooley, Paul R

    2005-03-04

    Asymmetric diadenosine 5',5'''-P(1),P(4)-tetraphosphate (Ap(4)A) hydrolases play a major role in maintaining homeostasis by cleaving the metabolite diadenosine tetraphosphate (Ap(4)A) back into ATP and AMP. The NMR solution structures of the 17-kDa human asymmetric Ap(4)A hydrolase have been solved in both the presence and absence of the product ATP. The adenine moiety of the nucleotide predominantly binds in a ring stacking arrangement equivalent to that observed in the x-ray structure of the homologue from Caenorhabditis elegans. The binding site is, however, markedly divergent to that observed in the plant/pathogenic bacteria class of enzymes, opening avenues for the exploration of specific therapeutics. Binding of ATP induces substantial conformational and dynamic changes that were not observed in the C. elegans structure. In contrast to the C. elegans homologue, important side chains that play a major role in substrate binding do not have to reorient to accommodate the ligand. This may have important implications in the mechanism of substrate recognition in this class of enzymes.

  17. Structure of conjugated polyketone reductase from Candida parapsilosis IFO 0708 reveals conformational changes for substrate recognition upon NADPH binding.

    PubMed

    Qin, Hui-Min; Yamamura, Akihiro; Miyakawa, Takuya; Kataoka, Michihiko; Nagai, Takahiro; Kitamura, Nahoko; Urano, Nobuyuki; Maruoka, Shintaro; Ohtsuka, Jun; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

    2014-01-01

    Conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708, identified as a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ketopantoyl lactone reductase, belongs to the aldo-keto reductase superfamily. This enzyme reduces ketopantoyl lactone to D-pantoyl lactone in a strictly stereospecific manner. To elucidate the structural basis of the substrate specificity, we determined the crystal structures of the apo CPR-C2 and CPR-C2/NADPH complex at 1.70 and 1.80 Å resolutions, respectively. CPR-C2 adopted a triose-phosphate isomerase barrel fold at the core of the structure. Binding with the cofactor NADPH induced conformational changes in which Thr27 and Lys28 moved 15 and 5.0 Å, respectively, in the close vicinity of the adenosine 2'-phosphate group of NADPH to form hydrogen bonds. Based on the comparison of the CPR-C2/NADPH structure with 3-α-hydroxysteroid dehydrogenase and mutation analyses, we constructed substrate binding models with ketopantoyl lactone, which provided insight into the substrate specificity by the cofactor-induced structure. The results will be useful for the rational design of CPR-C2 mutants targeted for use in the industrial manufacture of ketopantoyl lactone.

  18. Ribbed electrode substrates

    DOEpatents

    Breault, Richard D.; Goller, Glen J.

    1983-01-01

    A ribbed substrate for an electrochemical cell electrode is made from a mixture of carbon fibers and carbonizable resin and has a mean pore size in the ribs which is 60-75% of the mean pore size of the web portions of the substrate which interconnect the ribs. Preferably the mean pore size of the web portion is 25-45 microns; and, if the substrate includes edge seals parallel to the ribs, the edge seals preferably have a mean pore size no greater than about ten microns. Most preferably the substrate has the same ratio of carbon fibers to polymeric carbon in all areas, including the ribs, webs, and edge seals. A substrate according to the present invention will have better overall performance than prior art substrates and minimizes the substrate thickness required for the substrate to perform all its functions well.

  19. [Prosopagnosia and facial expression recognition].

    PubMed

    Koyama, Shinichi

    2014-04-01

    This paper reviews clinical neuropsychological studies that have indicated that the recognition of a person's identity and the recognition of facial expressions are processed by different cortical and subcortical areas of the brain. The fusiform gyrus, especially the right fusiform gyrus, plays an important role in the recognition of identity. The superior temporal sulcus, amygdala, and medial frontal cortex play important roles in facial-expression recognition. Both facial recognition and facial-expression recognition are highly intellectual processes that involve several regions of the brain.

  20. Computer Recognition of Facial Profiles

    DTIC Science & Technology

    1974-08-01

    facial recognition 20. ABSTRACT (Continue on reverse side It necessary and Identify by block number) A system for the recognition of human faces from...21 2.6 Classification Algorithms ........... ... 32 III FACIAL RECOGNITION AND AUTOMATIC TRAINING . . . 37 3.1 Facial Profile Recognition...provide a fair test of the classification system. The work of Goldstein, Harmon, and Lesk [81 indicates, however, that for facial recognition , a ten class

  1. Pupil dilation during recognition memory: Isolating unexpected recognition from judgment uncertainty.

    PubMed

    Mill, Ravi D; O'Connor, Akira R; Dobbins, Ian G

    2016-09-01

    Optimally discriminating familiar from novel stimuli demands a decision-making process informed by prior expectations. Here we demonstrate that pupillary dilation (PD) responses during recognition memory decisions are modulated by expectations, and more specifically, that pupil dilation increases for unexpected compared to expected recognition. Furthermore, multi-level modeling demonstrated that the time course of the dilation during each individual trial contains separable early and late dilation components, with the early amplitude capturing unexpected recognition, and the later trailing slope reflecting general judgment uncertainty or effort. This is the first demonstration that the early dilation response during recognition is dependent upon observer expectations and that separate recognition expectation and judgment uncertainty components are present in the dilation time course of every trial. The findings provide novel insights into adaptive memory-linked orienting mechanisms as well as the general cognitive underpinnings of the pupillary index of autonomic nervous system activity. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. The active site of O-GlcNAc transferase imposes constraints on substrate sequence

    PubMed Central

    Rafie, Karim; Blair, David E.; Borodkin, Vladimir S.; Albarbarawi, Osama; van Aalten, Daan M. F.

    2016-01-01

    O-GlcNAc transferase (OGT) glycosylates a diverse range of intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc), an essential and dynamic post-translational modification in metazoa. Although this enzyme modifies hundreds of proteins with O-GlcNAc, it is not understood how OGT achieves substrate specificity. In this study, we describe the application of a high-throughput OGT assay on a library of peptides. The sites of O-GlcNAc modification were mapped by ETD-mass spectrometry, and found to correlate with previously detected O-GlcNAc sites. Crystal structures of four acceptor peptides in complex with human OGT suggest that a combination of size and conformational restriction defines sequence specificity in the −3 to +2 subsites. This work reveals that while the N-terminal TPR repeats of hOGT may play a role in substrate recognition, the sequence restriction imposed by the peptide-binding site makes a significant contribution to O-GlcNAc site specificity. PMID:26237509

  3. Purification and characterization of Taenia crassiceps cysticerci thioredoxin: insight into thioredoxin-glutathione-reductase (TGR) substrate recognition.

    PubMed

    Martínez-González, J J; Guevara-Flores, A; Rendón, J L; Sosa-Peinado, A; Del Arenal Mena, I P

    2015-04-01

    Thioredoxin (Trx) is an oxidoreductase central to redox homeostasis in cells and is involved in the regulation of protein activity through thiol/disulfide exchanges. Based on these facts, our goal was to purify and characterize cytosolic thioredoxin from Taenia crassiceps cysticerci, as well as to study its behavior as a substrate of thioredoxin-glutathione reductase (TGR). The enzyme was purified >133-fold with a total yield of 9.7%. A molecular mass of 11.7kDa and a pI of 4.84 were measured. Native electrophoresis was used to identify the oxidized and reduced forms of the monomer as well as the presence of a homodimer. In addition to the catalytic site cysteines, cysticerci thioredoxin contains Cys28 and Cys65 residues conserved in previously sequenced cestode thioredoxins. The following kinetic parameters were obtained for the substrate of TGR: a Km of 3.1μM, a kcat of 10s(-1) and a catalytic efficiency of 3.2×10(6)M(-1)s(-1). The negative patch around the α3-helix of Trx is involved in the interaction with TGR and suggests variable specificity and catalytic efficiency of the reductase toward thioredoxins of different origins. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  4. Self-recognition in corals facilitates deep-sea habitat engineering

    USGS Publications Warehouse

    Hennige, Sebastian J; Morrison, Cheryl L.; Form, Armin U.; Buscher, Janina; Kamenos, Nicholas A.; Roberts, J. Murray

    2014-01-01

    The ability of coral reefs to engineer complex three-dimensional habitats is central to their success and the rich biodiversity they support. In tropical reefs, encrusting coralline algae bind together substrates and dead coral framework to make continuous reef structures, but beyond the photic zone, the cold-water coral Lophelia pertusa also forms large biogenic reefs, facilitated by skeletal fusion. Skeletal fusion in tropical corals can occur in closely related or juvenile individuals as a result of non-aggressive skeletal overgrowth or allogeneic tissue fusion, but contact reactions in many species result in mortality if there is no ‘self-recognition’ on a broad species level. This study reveals areas of ‘flawless’ skeletal fusion in Lophelia pertusa, potentially facilitated by allogeneic tissue fusion, are identified as having small aragonitic crystals or low levels of crystal organisation, and strong molecular bonding. Regardless of the mechanism, the recognition of ‘self’ between adjacent L. pertusa colonies leads to no observable mortality, facilitates ecosystem engineering and reduces aggression-related energetic expenditure in an environment where energy conservation is crucial. The potential for self-recognition at a species level, and subsequent skeletal fusion in framework-forming cold-water corals is an important first step in understanding their significance as ecological engineers in deep-seas worldwide.

  5. Unexpected expansion of tRNA substrate recognition by the yeast m1G9 methyltransferase Trm10.

    PubMed

    Swinehart, William E; Henderson, Jeremy C; Jackman, Jane E

    2013-08-01

    N-1 Methylation of the nearly invariant purine residue found at position 9 of tRNA is a nucleotide modification found in multiple tRNA species throughout Eukarya and Archaea. First discovered in Saccharomyces cerevisiae, the tRNA methyltransferase Trm10 is a highly conserved protein both necessary and sufficient to catalyze all known instances of m1G9 modification in yeast. Although there are 19 unique tRNA species that contain a G at position 9 in yeast, and whose fully modified sequence is known, only 9 of these tRNA species are modified with m1G9 in wild-type cells. The elements that allow Trm10 to distinguish between structurally similar tRNA species are not known, and sequences that are shared between all substrate or all nonsubstrate tRNAs have not been identified. Here, we demonstrate that the in vitro methylation activity of yeast Trm10 is not sufficient to explain the observed pattern of modification in vivo, as additional tRNA species are substrates for Trm10 m1G9 methyltransferase activity. Similarly, overexpression of Trm10 in yeast yields m1G9 containing tRNA species that are ordinarily unmodified in vivo. Thus, yeast Trm10 has a significantly broader tRNA substrate specificity than is suggested by the observed pattern of modification in wild-type yeast. These results may shed light onto the suggested involvement of Trm10 in other pathways in other organisms, particularly in higher eukaryotes that contain up to three different genes with sequence similarity to the single TRM10 gene in yeast, and where these other enzymes have been implicated in pathways beyond tRNA processing.

  6. Exploring the transferase activity of Ffase from Schwanniomyces occidentalis, a β-fructofuranosidase showing high fructosyl-acceptor promiscuity.

    PubMed

    Piedrabuena, David; Míguez, Noa; Poveda, Ana; Plou, Francisco J; Fernández-Lobato, María

    2016-10-01

    The β-fructofuranosidase from the yeast Schwanniomyces occidentalis (Ffase) produces the prebiotic sugars 6-kestose and 1-kestose by transfructosylation of sucrose, which makes it of biotechnological interest. In this study, the hydrolase and transferase activity of this enzyme was kinetically characterized and its potential to synthesize new fructosylated products explored. A total of 40 hydroxylated compounds were used as potential fructosyl-acceptor alternatives to sucrose. Only 17 of them, including some monosaccharides, disaccharides, and oligosaccharides as well as alditols and glycosides were fructosylated. The best alternative acceptors were the alditols. The major transfer product of the reaction including mannitol was purified and characterized as 1-O-β-D-fructofuranosyl-D-mannitol, whose maximum concentration reached 44 g/L, representing about 7.3 % of total compounds in the mixture and 89 % of all products generated by transfructosylation. The reactions including erythritol produced 35 g/L of an isomer mixture comprising 1- and 4-O-β-D-fructofuranosyl-D-erythritol. In addition, Ffase produced 24 g/L of the disaccharide blastose by direct fructosylation of glucose, which makes it the first enzyme characterized from yeast showing this ability. Thus, novel fructosylated compounds with potential applications in food and pharmaceutical industries can be obtained due to the Ffase fructosyl-acceptor promiscuity.

  7. A multistep damage recognition mechanism for global genomic nucleotide excision repair

    PubMed Central

    Sugasawa, Kaoru; Okamoto, Tomoko; Shimizu, Yuichiro; Masutani, Chikahide; Iwai, Shigenori; Hanaoka, Fumio

    2001-01-01

    A mammalian nucleotide excision repair (NER) factor, the XPC–HR23B complex, can specifically bind to certain DNA lesions and initiate the cell-free repair reaction. Here we describe a detailed analysis of its binding specificity using various DNA substrates, each containing a single defined lesion. A highly sensitive gel mobility shift assay revealed that XPC–HR23B specifically binds a small bubble structure with or without damaged bases, whereas dual incision takes place only when damage is present in the bubble. This is evidence that damage recognition for NER is accomplished through at least two steps; XPC–HR23B first binds to a site that has a DNA helix distortion, and then the presence of injured bases is verified prior to dual incision. Cyclobutane pyrimidine dimers (CPDs) were hardly recognized by XPC–HR23B, suggesting that additional factors may be required for CPD recognition. Although the presence of mismatched bases opposite a CPD potentiated XPC–HR23B binding, probably due to enhancement of the helix distortion, cell-free excision of such compound lesions was much more efficient than expected from the observed affinity for XPC–HR23B. This also suggests that additional factors and steps are required for the recognition of some types of lesions. A multistep mechanism of this sort may provide a molecular basis for ensuring the high level of damage discrimination that is required for global genomic NER. PMID:11238373

  8. A multistep damage recognition mechanism for global genomic nucleotide excision repair.

    PubMed

    Sugasawa, K; Okamoto, T; Shimizu, Y; Masutani, C; Iwai, S; Hanaoka, F

    2001-03-01

    A mammalian nucleotide excision repair (NER) factor, the XPC-HR23B complex, can specifically bind to certain DNA lesions and initiate the cell-free repair reaction. Here we describe a detailed analysis of its binding specificity using various DNA substrates, each containing a single defined lesion. A highly sensitive gel mobility shift assay revealed that XPC-HR23B specifically binds a small bubble structure with or without damaged bases, whereas dual incision takes place only when damage is present in the bubble. This is evidence that damage recognition for NER is accomplished through at least two steps; XPC-HR23B first binds to a site that has a DNA helix distortion, and then the presence of injured bases is verified prior to dual incision. Cyclobutane pyrimidine dimers (CPDs) were hardly recognized by XPC-HR23B, suggesting that additional factors may be required for CPD recognition. Although the presence of mismatched bases opposite a CPD potentiated XPC-HR23B binding, probably due to enhancement of the helix distortion, cell-free excision of such compound lesions was much more efficient than expected from the observed affinity for XPC-HR23B. This also suggests that additional factors and steps are required for the recognition of some types of lesions. A multistep mechanism of this sort may provide a molecular basis for ensuring the high level of damage discrimination that is required for global genomic NER.

  9. Profiles of Discourse Recognition

    ERIC Educational Resources Information Center

    Singer, Murray

    2013-01-01

    A discourse recognition theory derived from more general memory formulations would be broad in its psychological implications. This study compared discourse recognition with some established profiles of item recognition. Participants read 10 stories either once or twice each. They then rated their confidence in recognizing explicit, paraphrased,…

  10. Sudden Event Recognition: A Survey

    PubMed Central

    Suriani, Nor Surayahani; Hussain, Aini; Zulkifley, Mohd Asyraf

    2013-01-01

    Event recognition is one of the most active research areas in video surveillance fields. Advancement in event recognition systems mainly aims to provide convenience, safety and an efficient lifestyle for humanity. A precise, accurate and robust approach is necessary to enable event recognition systems to respond to sudden changes in various uncontrolled environments, such as the case of an emergency, physical threat and a fire or bomb alert. The performance of sudden event recognition systems depends heavily on the accuracy of low level processing, like detection, recognition, tracking and machine learning algorithms. This survey aims to detect and characterize a sudden event, which is a subset of an abnormal event in several video surveillance applications. This paper discusses the following in detail: (1) the importance of a sudden event over a general anomalous event; (2) frameworks used in sudden event recognition; (3) the requirements and comparative studies of a sudden event recognition system and (4) various decision-making approaches for sudden event recognition. The advantages and drawbacks of using 3D images from multiple cameras for real-time application are also discussed. The paper concludes with suggestions for future research directions in sudden event recognition. PMID:23921828

  11. AlkB Dioxygenase Preferentially Repairs Protonated Substrates

    PubMed Central

    Maciejewska, Agnieszka M.; Poznański, Jarosław; Kaczmarska, Zuzanna; Krowisz, Beata; Nieminuszczy, Jadwiga; Polkowska-Nowakowska, Agnieszka; Grzesiuk, Elżbieta; Kuśmierek, Jarosław T.

    2013-01-01

    Efficient repair by Escherichia coli AlkB dioxygenase of exocyclic DNA adducts 3,N4-ethenocytosine, 1,N6-ethenoadenine, 3,N4-α-hydroxyethanocytosine, and reported here for the first time 3,N4-α-hydroxypropanocytosine requires higher Fe(II) concentration than the reference 3-methylcytosine. The pH optimum for the repair follows the order of pKa values for protonation of the adduct, suggesting that positively charged substrates favorably interact with the negatively charged carboxylic group of Asp-135 side chain in the enzyme active center. This interaction is supported by molecular modeling, indicating that 1,N6-ethenoadenine and 3,N4-ethenocytosine are bound to AlkB more favorably in their protonated cationic forms. An analysis of the pattern of intermolecular interactions that stabilize the location of the ligand points to a role of Asp-135 in recognition of the adduct in its protonated form. Moreover, ab initio calculations also underline the role of substrate protonation in lowering the free energy barrier of the transition state of epoxidation of the etheno adducts studied. The observed time courses of repair of mixtures of stereoisomers of 3,N4-α-hydroxyethanocytosine or 3,N4-α-hydroxypropanocytosine are unequivocally two-exponential curves, indicating that the respective isomers are repaired by AlkB with different efficiencies. Molecular modeling of these adducts bound by AlkB allowed evaluation of the participation of their possible conformational states in the enzymatic reaction. PMID:23148216

  12. Examining the Relationships among Item Recognition, Source Recognition, and Recall from an Individual Differences Perspective

    ERIC Educational Resources Information Center

    Unsworth, Nash; Brewer, Gene A.

    2009-01-01

    The authors of the current study examined the relationships among item-recognition, source-recognition, free recall, and other memory and cognitive ability tasks via an individual differences analysis. Two independent sources of variance contributed to item-recognition and source-recognition performance, and these two constructs related…

  13. Recognition mechanism of p63 by the E3 ligase Itch

    PubMed Central

    Bellomaria, Alessia; Barbato, Gaetano; Melino, Gerry; Paci, Maurizio; Melino, Sonia

    2012-01-01

    The HECT-containing E3 ubiquitin ligase Itch mediates the degradation of several proteins, including p63 and p73, involved in cell specification and fate. Itch contains four WW domains, which are essential for recognition on the target substrate, which contains a short proline-rich sequence. Several signaling complexes containing these domains have been associated with human diseases such as muscular dystrophy, Alzheimer’s or Huntington’s diseases. To gain further insight into the structural determinants of the Itch-WW2 domain, we investigated its interaction with p63. We assigned, by 3D heteronuclear NMR experiments, the backbone and side chains of the uniformly ¹³C-¹⁵N-labeled Itch-WW2. In vitro interaction of Itch-WW2 domain with p63 was studied using its interactive p63 peptide, pep63. Pep63 is an 18-mer peptide corresponding to the region from 534–551 residue of p63, encompassing the PPxY motif that interacts with the Itch-WW domains, and we identified the residues involved in this molecular recognition. Moreover, here, a strategy of stabilization of the conformation of the PPxY peptide has been adopted, increasing the WW-ligand binding. We demonstrated that cyclization of pep63 leads to an increase of both the biological stability of the peptide and of the WW-ligand complex. Stable metal-binding complexes of the pep63 have been also obtained, and localized oxidative damage on Itch-WW2 domain has been induced, demonstrating the possibility of use of metal-pep63 complexes as models for the design of metal drugs to inhibit the Itch-WW-p63 recognition in vivo. Thus, our data suggest a novel strategy to study and inhibit the recognition mechanism of Itch E3-ligase. PMID:22935697

  14. Nonspecific Symbiosis Between Sophora flavescens and Different Rhizobia.

    PubMed

    Liu, Yuan Hui; Jiao, Yin Shan; Liu, Li Xue; Wang, Dan; Tian, Chang Fu; Wang, En Tao; Wang, Lei; Chen, Wen Xin; Wu, Shang Ying; Guo, Bao Lin; Guan, Zha Gen; Poinsot, Véréna; Chen, Wen Feng

    2018-02-01

    We explored the genetic basis of the promiscuous symbiosis of Sophora flavescens with diverse rhizobia. To determine the impact of Nod factors (NFs) on the symbiosis of S. flavescens, nodulation-related gene mutants of representative rhizobial strains were generated. Strains with mutations in common nodulation genes (nodC, nodM, and nodE) failed to nodulate S. flavescens, indicating that the promiscuous nodulation of this plant is strictly dependent on the basic NF structure. Mutations of the NF decoration genes nodH, nodS, nodZ, and noeI did not affect the nodulation of S. flavescens, but these mutations affected the nitrogen-fixation efficiency of nodules. Wild-type Bradyrhizobium diazoefficiens USDA110 cannot nodulate S. flavescens, but we obtained 14 Tn5 mutants of B. diazoefficiens that nodulated S. flavescens. This suggested that the mutations had disrupted a negative regulator that prevents nodulation of S. flavescens, leading to nonspecific nodulation. For Ensifer fredii CCBAU 45436 mutants, the minimal NF structure was sufficient for nodulation of soybean and S. flavescens. In summary, the mechanism of promiscuous symbiosis of S. flavescens with rhizobia might be related to its nonspecific recognition of NF structures, and the host specificity of rhizobia may also be controlled by currently unknown nodulation-related genes.

  15. One-pot multienzyme (OPME) systems for chemoenzymatic synthesis of carbohydrates.

    PubMed

    Yu, Hai; Chen, Xi

    2016-03-14

    Glycosyltransferase-catalyzed enzymatic and chemoenzymatic syntheses are powerful approaches for the production of oligosaccharides, polysaccharides, glycoconjugates, and their derivatives. Enzymes involved in the biosynthesis of sugar nucleotide donors can be combined with glycosyltransferases in one pot for efficient production of the target glycans from simple monosaccharides and acceptors. The identification of enzymes involved in the salvage pathway of sugar nucleotide generation has greatly facilitated the development of simplified and efficient one-pot multienzyme (OPME) systems for synthesizing major glycan epitopes in mammalian glycomes. The applications of OPME methods are steadily gaining popularity mainly due to the increasing availability of wild-type and engineered enzymes. Substrate promiscuity of these enzymes and their mutants allows OPME synthesis of carbohydrates with naturally occurring post-glycosylational modifications (PGMs) and their non-natural derivatives using modified monosaccharides as precursors. The OPME systems can be applied in sequence for synthesizing complex carbohydrates. The sequence of the sequential OPME processes, the glycosyltransferase used, and the substrate specificities of the glycosyltransferases define the structures of the products. The OPME and sequential OPME strategies can be extended to diverse glycans in other glycomes when suitable enzymes with substrate promiscuity become available. This Perspective summarizes the work of the authors and collaborators on the development of glycosyltransferase-based OPME systems for carbohydrate synthesis. Future directions are also discussed.

  16. One-pot multienzyme (OPME) systems for chemoenzymatic synthesis of carbohydrates

    PubMed Central

    Yu, Hai; Chen, Xi

    2016-01-01

    Glycosyltransferase-catalyzed enzymatic and chemoenzymatic syntheses are powerful approaches for the production of oligosaccharides, polysaccharides, glycoconjugates, and their derivatives. Enzymes involved in the biosynthesis of sugar nucleotide donors can be combined with the glycosyltransferases in one pot for efficient production of target glycans from simple monosaccharides and accpetors. The identification of enzymes involved in the salvage pathway of sugar nucleotide generation has greatly facilitate the development of simplified and efficient one-pot multienzyme (OPME) systems for synthesizing major glycan epitopes in mammalian glycomes. The applications of OPME methods are steadily gaining popularity mainly due to the increasing availability of wild-type and engineered enzymes. Substrate promiscuity of these enzymes and their mutants allows OPME synthesis of carbohydrates with naturally occurring post-glycosylational modificiation (PGMs) and their non-natural derivatives using modified monosaccharides as precursors. The OPME systems can be applied in sequential for synthesizing complex carbohydrates. The sequence of the sequential OPME processes, the glycosyltransferase used, and the substrate specificities of glycosyltransferasese define the structures of the products. The OPME and sequential OPME strategies can be extended to diverse glycans in other glycomes when suitable enzymes with substrate promiscuity become available. The Perspective summariezes the work of the authors and collaborators on the development of glycosyltransferase-based OPME systems for carbohydrate synthesis. Future directions are also discussed. PMID:26881499

  17. The Legal Recognition of Sign Languages

    ERIC Educational Resources Information Center

    De Meulder, Maartje

    2015-01-01

    This article provides an analytical overview of the different types of explicit legal recognition of sign languages. Five categories are distinguished: constitutional recognition, recognition by means of general language legislation, recognition by means of a sign language law or act, recognition by means of a sign language law or act including…

  18. Conformational states and recognition of amyloidogenic peptides of human insulin-degrading enzyme.

    PubMed

    McCord, Lauren A; Liang, Wenguang G; Dowdell, Evan; Kalas, Vasilios; Hoey, Robert J; Koide, Akiko; Koide, Shohei; Tang, Wei-Jen

    2013-08-20

    Insulin-degrading enzyme (IDE) selectively degrades the monomer of amyloidogenic peptides and contributes to clearance of amyloid β (Aβ). Thus, IDE retards the progression of Alzheimer's disease. IDE possesses an enclosed catalytic chamber that engulfs and degrades its peptide substrates; however, the molecular mechanism of IDE function, including substrate access to the chamber and recognition, remains elusive. Here, we captured a unique IDE conformation by using a synthetic antibody fragment as a crystallization chaperone. An unexpected displacement of a door subdomain creates an ~18-Å opening to the chamber. This swinging-door mechanism permits the entry of short peptides into the catalytic chamber and disrupts the catalytic site within IDE door subdomain. Given the propensity of amyloidogenic peptides to convert into β-strands for their polymerization into amyloid fibrils, they also use such β-strands to stabilize the disrupted catalytic site resided at IDE door subdomain for their degradation by IDE. Thus, action of the swinging door allows IDE to recognize amyloidogenicity by substrate-induced stabilization of the IDE catalytic cleft. Small angle X-ray scattering (SAXS) analysis revealed that IDE exists as a mixture of closed and open states. These open states, which are distinct from the swinging door state, permit entry of larger substrates (e.g., Aβ, insulin) to the chamber and are preferred in solution. Mutational studies confirmed the critical roles of the door subdomain and hinge loop joining the N- and C-terminal halves of IDE for catalysis. Together, our data provide insights into the conformational changes of IDE that govern the selective destruction of amyloidogenic peptides.

  19. Toward open set recognition.

    PubMed

    Scheirer, Walter J; de Rezende Rocha, Anderson; Sapkota, Archana; Boult, Terrance E

    2013-07-01

    To date, almost all experimental evaluations of machine learning-based recognition algorithms in computer vision have taken the form of "closed set" recognition, whereby all testing classes are known at training time. A more realistic scenario for vision applications is "open set" recognition, where incomplete knowledge of the world is present at training time, and unknown classes can be submitted to an algorithm during testing. This paper explores the nature of open set recognition and formalizes its definition as a constrained minimization problem. The open set recognition problem is not well addressed by existing algorithms because it requires strong generalization. As a step toward a solution, we introduce a novel "1-vs-set machine," which sculpts a decision space from the marginal distances of a 1-class or binary SVM with a linear kernel. This methodology applies to several different applications in computer vision where open set recognition is a challenging problem, including object recognition and face verification. We consider both in this work, with large scale cross-dataset experiments performed over the Caltech 256 and ImageNet sets, as well as face matching experiments performed over the Labeled Faces in the Wild set. The experiments highlight the effectiveness of machines adapted for open set evaluation compared to existing 1-class and binary SVMs for the same tasks.

  20. Effect of Ca2+ on the promiscuous target-protein binding of calmodulin.

    PubMed

    Westerlund, Annie M; Delemotte, Lucie

    2018-04-01

    Calmodulin (CaM) is a calcium sensing protein that regulates the function of a large number of proteins, thus playing a crucial part in many cell signaling pathways. CaM has the ability to bind more than 300 different target peptides in a Ca2+-dependent manner, mainly through the exposure of hydrophobic residues. How CaM can bind a large number of targets while retaining some selectivity is a fascinating open question. Here, we explore the mechanism of CaM selective promiscuity for selected target proteins. Analyzing enhanced sampling molecular dynamics simulations of Ca2+-bound and Ca2+-free CaM via spectral clustering has allowed us to identify distinct conformational states, characterized by interhelical angles, secondary structure determinants and the solvent exposure of specific residues. We searched for indicators of conformational selection by mapping solvent exposure of residues in these conformational states to contacts in structures of CaM/target peptide complexes. We thereby identified CaM states involved in various binding classes arranged along a depth binding gradient. Binding Ca2+ modifies the accessible hydrophobic surface of the two lobes and allows for deeper binding. Apo CaM indeed shows shallow binding involving predominantly polar and charged residues. Furthermore, binding to the C-terminal lobe of CaM appears selective and involves specific conformational states that can facilitate deep binding to target proteins, while binding to the N-terminal lobe appears to happen through a more flexible mechanism. Thus the long-ranged electrostatic interactions of the charged residues of the N-terminal lobe of CaM may initiate binding, while the short-ranged interactions of hydrophobic residues in the C-terminal lobe of CaM may account for selectivity. This work furthers our understanding of the mechanism of CaM binding and selectivity to different target proteins and paves the way towards a comprehensive model of CaM selectivity.

  1. Effect of Ca2+ on the promiscuous target-protein binding of calmodulin

    PubMed Central

    Westerlund, Annie M.

    2018-01-01

    Calmodulin (CaM) is a calcium sensing protein that regulates the function of a large number of proteins, thus playing a crucial part in many cell signaling pathways. CaM has the ability to bind more than 300 different target peptides in a Ca2+-dependent manner, mainly through the exposure of hydrophobic residues. How CaM can bind a large number of targets while retaining some selectivity is a fascinating open question. Here, we explore the mechanism of CaM selective promiscuity for selected target proteins. Analyzing enhanced sampling molecular dynamics simulations of Ca2+-bound and Ca2+-free CaM via spectral clustering has allowed us to identify distinct conformational states, characterized by interhelical angles, secondary structure determinants and the solvent exposure of specific residues. We searched for indicators of conformational selection by mapping solvent exposure of residues in these conformational states to contacts in structures of CaM/target peptide complexes. We thereby identified CaM states involved in various binding classes arranged along a depth binding gradient. Binding Ca2+ modifies the accessible hydrophobic surface of the two lobes and allows for deeper binding. Apo CaM indeed shows shallow binding involving predominantly polar and charged residues. Furthermore, binding to the C-terminal lobe of CaM appears selective and involves specific conformational states that can facilitate deep binding to target proteins, while binding to the N-terminal lobe appears to happen through a more flexible mechanism. Thus the long-ranged electrostatic interactions of the charged residues of the N-terminal lobe of CaM may initiate binding, while the short-ranged interactions of hydrophobic residues in the C-terminal lobe of CaM may account for selectivity. This work furthers our understanding of the mechanism of CaM binding and selectivity to different target proteins and paves the way towards a comprehensive model of CaM selectivity. PMID:29614072

  2. Substrate recognition by the hetero-octameric ATP phosphoribosyltransferase from Lactococcus lactis†

    PubMed Central

    Champagne, Karen S.; Piscitelli, Elise; Francklyn, Christopher S.

    2008-01-01

    Two families of ATP phosphoribosyl transferases (ATP-PRT) join ATP and 5-phosphoribosyl-1 pyrophosphate (PRPP) in the first reaction of histidine biosynthesis. These consist of a homohexameric form found in all three kingdoms, and a hetero-octameric form largely restricted to bacteria. Hetero-octameric ATP-PRTs consist of four HisGS catalytic subunits related to periplasmic binding proteins, and four HisZ regulatory subunits that resemble histidyl-tRNA synthetases. To clarify the relationship between the two families of ATP-PRTs, and among phosphoribosyltransferases in general, we determined the steady state kinetics for the hetero-octameric form, and characterized the active site by mutagenesis. The Km PRPP (18.4 ± 3.5 μM) and kcat (2.7 ± 0.3 sec−1) values for the PRPP substrate are similar to those of hexameric ATP-PRTs, but the Km for ATP (2.7 ± 0.3 mM) is 4-fold higher, suggestive of tighter regulation by energy charge. Histidine and AMP were determined to be non-competitive (Ki = 81.1 μM) and competitive (Ki= 1.44 mM) inhibitors, respectively, with values that approximate their intracellular concentrations. Mutagenesis experiments investigating the side chains recognizing PRPP showed that 5′ phosphate contacts (T159A and T162A) had the largest (25- and 155-fold) decreases in kcat/Km, while smaller decreases were seen with mutants making cross subunit contacts (K50A and K8A) to the pyrophosphate moiety, or contacts to the 2′ OH. Despite their markedly different quaternary structures, hexameric and hetero-octameric ATRP-PRTs exhibit similar functional parameters, and employ mechanistic strategies reminiscent of the broader PRT superfamily. PMID:17154531

  3. The first mammalian aldehyde oxidase crystal structure: insights into substrate specificity.

    PubMed

    Coelho, Catarina; Mahro, Martin; Trincão, José; Carvalho, Alexandra T P; Ramos, Maria João; Terao, Mineko; Garattini, Enrico; Leimkühler, Silke; Romão, Maria João

    2012-11-23

    Aldehyde oxidases have pharmacological relevance, and AOX3 is the major drug-metabolizing enzyme in rodents. The crystal structure of mouse AOX3 with kinetics and molecular docking studies provides insights into its enzymatic characteristics. Differences in substrate and inhibitor specificities can be rationalized by comparing the AOX3 and xanthine oxidase structures. The first aldehyde oxidase structure represents a major advance for drug design and mechanistic studies. Aldehyde oxidases (AOXs) are homodimeric proteins belonging to the xanthine oxidase family of molybdenum-containing enzymes. Each 150-kDa monomer contains a FAD redox cofactor, two spectroscopically distinct [2Fe-2S] clusters, and a molybdenum cofactor located within the protein active site. AOXs are characterized by broad range substrate specificity, oxidizing different aldehydes and aromatic N-heterocycles. Despite increasing recognition of its role in the metabolism of drugs and xenobiotics, the physiological function of the protein is still largely unknown. We have crystallized and solved the crystal structure of mouse liver aldehyde oxidase 3 to 2.9 Å. This is the first mammalian AOX whose structure has been solved. The structure provides important insights into the protein active center and further evidence on the catalytic differences characterizing AOX and xanthine oxidoreductase. The mouse liver aldehyde oxidase 3 three-dimensional structure combined with kinetic, mutagenesis data, molecular docking, and molecular dynamics studies make a decisive contribution to understand the molecular basis of its rather broad substrate specificity.

  4. Multienzyme kinetics and sequential metabolism.

    PubMed

    Wienkers, Larry C; Rock, Brooke

    2014-01-01

    Enzymes are the catalysts of biological systems and are extremely efficient. A typical enzyme accelerates the rate of a reaction by factors of at least a million compared to the rate of the same reaction in the absence of the enzyme. In contrast to traditional catalytic enzymes, the family of cytochrome P450 (CYP) enzymes are catalytically promiscuous, and thus they possess remarkable versatility in substrates. The great diversity of reactions catalyzed by CYP enzymes appears to be based on two unique properties of these heme proteins, the ability of their iron to exist under multiple oxidation states with different reactivities and a flexible active site that can accommodate a wide variety of substrates. Herein is a discussion of two distinct types of kinetics observed with CYP enzymes. The first example is of CYP complex kinetic profiles when multiple CYP enzymes form the sample product. The second is sequential metabolism, in other words, the formation of multiple products from one CYP enzyme. Given the degree of CYP enzyme promiscuity, it is hardly surprising that there is also a high degree of complex kinetic profiles generated during the catalytic cycle.

  5. Transcutaneous vagus nerve stimulation (tVNS) enhances recognition of emotions in faces but not bodies.

    PubMed

    Sellaro, Roberta; de Gelder, Beatrice; Finisguerra, Alessandra; Colzato, Lorenza S

    2018-02-01

    The polyvagal theory suggests that the vagus nerve is the key phylogenetic substrate enabling optimal social interactions, a crucial aspect of which is emotion recognition. A previous study showed that the vagus nerve plays a causal role in mediating people's ability to recognize emotions based on images of the eye region. The aim of this study is to verify whether the previously reported causal link between vagal activity and emotion recognition can be generalized to situations in which emotions must be inferred from images of whole faces and bodies. To this end, we employed transcutaneous vagus nerve stimulation (tVNS), a novel non-invasive brain stimulation technique that causes the vagus nerve to fire by the application of a mild electrical stimulation to the auricular branch of the vagus nerve, located in the anterior protuberance of the outer ear. In two separate sessions, participants received active or sham tVNS before and while performing two emotion recognition tasks, aimed at indexing their ability to recognize emotions from facial and bodily expressions. Active tVNS, compared to sham stimulation, enhanced emotion recognition for whole faces but not for bodies. Our results confirm and further extend recent observations supporting a causal relationship between vagus nerve activity and the ability to infer others' emotional state, but restrict this association to situations in which the emotional state is conveyed by the whole face and/or by salient facial cues, such as eyes. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Super-recognition in development: A case study of an adolescent with extraordinary face recognition skills.

    PubMed

    Bennetts, Rachel J; Mole, Joseph; Bate, Sarah

    2017-09-01

    Face recognition abilities vary widely. While face recognition deficits have been reported in children, it is unclear whether superior face recognition skills can be encountered during development. This paper presents O.B., a 14-year-old female with extraordinary face recognition skills: a "super-recognizer" (SR). O.B. demonstrated exceptional face-processing skills across multiple tasks, with a level of performance that is comparable to adult SRs. Her superior abilities appear to be specific to face identity: She showed an exaggerated face inversion effect and her superior abilities did not extend to object processing or non-identity aspects of face recognition. Finally, an eye-movement task demonstrated that O.B. spent more time than controls examining the nose - a pattern previously reported in adult SRs. O.B. is therefore particularly skilled at extracting and using identity-specific facial cues, indicating that face and object recognition are dissociable during development, and that super recognition can be detected in adolescence.

  7. Polished polymide substrate

    DOEpatents

    Farah, John; Sudarshanam, Venkatapuram S.

    2003-05-13

    Polymer substrates, in particular polyimide substrates, and polymer laminates for optical applications are described. Polyimide substrates are polished on one or both sides depending on their thickness, and single-layer or multi-layer waveguide structures are deposited on the polished polyimide substrates. Optical waveguide devices are machined by laser ablation using a combination of IR and UV lasers. A waveguide-fiber coupler with a laser-machined groove for retaining the fiber is also disclosed.

  8. A motivational determinant of facial emotion recognition: regulatory focus affects recognition of emotions in faces.

    PubMed

    Sassenrath, Claudia; Sassenberg, Kai; Ray, Devin G; Scheiter, Katharina; Jarodzka, Halszka

    2014-01-01

    Two studies examined an unexplored motivational determinant of facial emotion recognition: observer regulatory focus. It was predicted that a promotion focus would enhance facial emotion recognition relative to a prevention focus because the attentional strategies associated with promotion focus enhance performance on well-learned or innate tasks - such as facial emotion recognition. In Study 1, a promotion or a prevention focus was experimentally induced and better facial emotion recognition was observed in a promotion focus compared to a prevention focus. In Study 2, individual differences in chronic regulatory focus were assessed and attention allocation was measured using eye tracking during the facial emotion recognition task. Results indicated that the positive relation between a promotion focus and facial emotion recognition is mediated by shorter fixation duration on the face which reflects a pattern of attention allocation matched to the eager strategy in a promotion focus (i.e., striving to make hits). A prevention focus did not have an impact neither on perceptual processing nor on facial emotion recognition. Taken together, these findings demonstrate important mechanisms and consequences of observer motivational orientation for facial emotion recognition.

  9. Implicit phonological priming during visual word recognition.

    PubMed

    Wilson, Lisa B; Tregellas, Jason R; Slason, Erin; Pasko, Bryce E; Rojas, Donald C

    2011-03-15

    Phonology is a lower-level structural aspect of language involving the sounds of a language and their organization in that language. Numerous behavioral studies utilizing priming, which refers to an increased sensitivity to a stimulus following prior experience with that or a related stimulus, have provided evidence for the role of phonology in visual word recognition. However, most language studies utilizing priming in conjunction with functional magnetic resonance imaging (fMRI) have focused on lexical-semantic aspects of language processing. The aim of the present study was to investigate the neurobiological substrates of the automatic, implicit stages of phonological processing. While undergoing fMRI, eighteen individuals performed a lexical decision task (LDT) on prime-target pairs including word-word homophone and pseudoword-word pseudohomophone pairs with a prime presentation below perceptual threshold. Whole-brain analyses revealed several cortical regions exhibiting hemodynamic response suppression due to phonological priming including bilateral superior temporal gyri (STG), middle temporal gyri (MTG), and angular gyri (AG) with additional region of interest (ROI) analyses revealing response suppression in the left lateralized supramarginal gyrus (SMG). Homophone and pseudohomophone priming also resulted in different patterns of hemodynamic responses relative to one another. These results suggest that phonological processing plays a key role in visual word recognition. Furthermore, enhanced hemodynamic responses for unrelated stimuli relative to primed stimuli were observed in midline cortical regions corresponding to the default-mode network (DMN) suggesting that DMN activity can be modulated by task requirements within the context of an implicit task. Copyright © 2010 Elsevier Inc. All rights reserved.

  10. The Promiscuous sumA Missense Suppressor from Salmonella enterica Has an Intriguing Mechanism of Action

    PubMed Central

    Cole, Ashley E.; Hani, Fatmah M.; Altman, Ronni; Meservy, Megan; Roth, John R.; Altman, Elliot

    2017-01-01

    While most missense suppressors have very narrow specificities and only suppress the allele against which they were isolated, the sumA missense suppressor from Salmonella enterica serovar Typhimurium is a promiscuous or broad-acting missense suppressor that suppresses numerous missense mutants. The sumA missense suppressor was identified as a glyV tRNA Gly3(GAU/C) missense suppressor that can recognize GAU or GAC aspartic acid codons and insert a glycine amino acid instead of aspartic acid. In addition to rescuing missense mutants caused by glycine to aspartic acid changes as expected, sumA could also rescue a number of other missense mutants as well by changing a neighboring (contacting) aspartic acid to glycine, which compensated for the other amino acid change. Thus the ability of sumA to rescue numerous missense mutants was due in part to the large number of glycine codons in genes that can be mutated to an aspartic acid codon and in part to the general tolerability and/or preference for glycine amino acids in proteins. Because the glyV tRNA Gly3(GAU/C) missense suppressor has also been extensively characterized in Escherichia coli as the mutA mutator, we demonstrated that all gain-of-function mutants isolated in a glyV tRNA Gly3(GAU/C) missense suppressor are transferable to a wild-type background and thus the increased mutation rates, which occur in glyV tRNA Gly3(GAU/C) missense suppressors, are not due to the suppression of these mutants. PMID:27974497

  11. Challenging ocular image recognition

    NASA Astrophysics Data System (ADS)

    Pauca, V. Paúl; Forkin, Michael; Xu, Xiao; Plemmons, Robert; Ross, Arun A.

    2011-06-01

    Ocular recognition is a new area of biometric investigation targeted at overcoming the limitations of iris recognition performance in the presence of non-ideal data. There are several advantages for increasing the area beyond the iris, yet there are also key issues that must be addressed such as size of the ocular region, factors affecting performance, and appropriate corpora to study these factors in isolation. In this paper, we explore and identify some of these issues with the goal of better defining parameters for ocular recognition. An empirical study is performed where iris recognition methods are contrasted with texture and point operators on existing iris and face datasets. The experimental results show a dramatic recognition performance gain when additional features are considered in the presence of poor quality iris data, offering strong evidence for extending interest beyond the iris. The experiments also highlight the need for the direct collection of additional ocular imagery.

  12. Method of forming fluorine-bearing diamond layer on substrates, including tool substrates

    DOEpatents

    Chang, R. P. H.; Grannen, Kevin J.

    2002-01-01

    A method of forming a fluorine-bearing diamond layer on non-diamond substrates, especially on tool substrates comprising a metal matrix and hard particles, such as tungsten carbide particles, in the metal matrix. The substrate and a fluorine-bearing plasma or other gas are then contacted under temperature and pressure conditions effective to nucleate fluorine-bearing diamond on the substrate. A tool insert substrate is treated prior to the diamond nucleation and growth operation by etching both the metal matrix and the hard particles using suitable etchants.

  13. Genetic specificity of face recognition.

    PubMed

    Shakeshaft, Nicholas G; Plomin, Robert

    2015-10-13

    Specific cognitive abilities in diverse domains are typically found to be highly heritable and substantially correlated with general cognitive ability (g), both phenotypically and genetically. Recent twin studies have found the ability to memorize and recognize faces to be an exception, being similarly heritable but phenotypically substantially uncorrelated both with g and with general object recognition. However, the genetic relationships between face recognition and other abilities (the extent to which they share a common genetic etiology) cannot be determined from phenotypic associations. In this, to our knowledge, first study of the genetic associations between face recognition and other domains, 2,000 18- and 19-year-old United Kingdom twins completed tests assessing their face recognition, object recognition, and general cognitive abilities. Results confirmed the substantial heritability of face recognition (61%), and multivariate genetic analyses found that most of this genetic influence is unique and not shared with other cognitive abilities.

  14. Preparation and Characterization of Biofunctionalized Inorganic Substrates.

    PubMed

    Dugger, Jason W; Webb, Lauren J

    2015-09-29

    Integrating the function of biological molecules into traditional inorganic materials and substrates couples biologically relevant function to synthetic devices and generates new materials and capabilities by combining biological and inorganic functions. At this so-called "bio/abio interface," basic biological functions such as ligand binding and catalysis can be co-opted to detect analytes with exceptional sensitivity or to generate useful molecules with chiral specificity under entirely benign reaction conditions. Proteins function in dynamic, complex, and crowded environments (the living cell) and are therefore appropriate for integrating into multistep, multiscale, multimaterial devices such as integrated circuits and heterogeneous catalysts. However, the goal of reproducing the highly specific activities of biomolecules in the perturbed chemical and electrostatic environment at an inorganic interface while maintaining their native conformations is challenging to achieve. Moreover, characterizing protein structure and function at a surface is often difficult, particularly if one wishes to compare the activity of the protein to that of the dilute, aqueous solution phase. Our laboratory has developed a general strategy to address this challenge by taking advantage of the structural and chemical properties of alkanethiol self-assembled monolayers (SAMs) on gold surfaces that are functionalized with covalently tethered peptides. These surface-bound peptides then act as the chemical recognition element for a target protein, generating a biomimetic surface in which protein orientation, structure, density, and function are controlled and variable. Herein we discuss current research and future directions related to generating a chemically tunable biofunctionalization strategy that has potential to successfully incorporate the highly specialized functions of proteins onto inorganic substrates.

  15. Moreland Recognition Program.

    ERIC Educational Resources Information Center

    Moreland Elementary School District, San Jose, CA.

    THE FOLLOWING IS THE FULL TEXT OF THIS DOCUMENT: Recognition for special effort and achievement has been noted as a component of effective schools. Schools in the Moreland School District have effectively improved standards of discipline and achievement by providing forty-six different ways for children to receive positive recognition. Good…

  16. Diversity in the architecture of ATLs, a family of plant ubiquitin-ligases, leads to recognition and targeting of substrates in different cellular environments.

    PubMed

    Aguilar-Hernández, Victor; Aguilar-Henonin, Laura; Guzmán, Plinio

    2011-01-01

    Ubiquitin-ligases or E3s are components of the ubiquitin proteasome system (UPS) that coordinate the transfer of ubiquitin to the target protein. A major class of ubiquitin-ligases consists of RING-finger domain proteins that include the substrate recognition sequences in the same polypeptide; these are known as single-subunit RING finger E3s. We are studying a particular family of RING finger E3s, named ATL, that contain a transmembrane domain and the RING-H2 finger domain; none of the member of the family contains any other previously described domain. Although the study of a few members in A. thaliana and O. sativa has been reported, the role of this family in the life cycle of a plant is still vague. To provide tools to advance on the functional analysis of this family we have undertaken a phylogenetic analysis of ATLs in twenty-four plant genomes. ATLs were found in all the 24 plant species analyzed, in numbers ranging from 20-28 in two basal species to 162 in soybean. Analysis of ATLs arrayed in tandem indicates that sets of genes are expanding in a species-specific manner. To get insights into the domain architecture of ATLs we generated 75 pHMM LOGOs from 1815 ATLs, and unraveled potential protein-protein interaction regions by means of yeast two-hybrid assays. Several ATLs were found to interact with DSK2a/ubiquilin through a region at the amino-terminal end, suggesting that this is a widespread interaction that may assist in the mode of action of ATLs; the region was traced to a distinct sequence LOGO. Our analysis provides significant observations on the evolution and expansion of the ATL family in addition to information on the domain structure of this class of ubiquitin-ligases that may be involved in plant adaptation to environmental stress.

  17. Activity Augmentation of Amphioxus Peptidoglycan Recognition Protein BbtPGRP3 via Fusion with a Chitin Binding Domain

    PubMed Central

    Wang, Wen-Jie; Cheng, Wang; Luo, Ming; Yan, Qingyu; Yu, Hong-Mei; Li, Qiong; Cao, Dong-Dong; Huang, Shengfeng; Xu, Anlong; Mariuzza, Roy A.; Chen, Yuxing; Zhou, Cong-Zhao

    2015-01-01

    Peptidoglycan recognition proteins (PGRPs), which have been identified in most animals, are pattern recognition molecules that involve antimicrobial defense. Resulting from extraordinary expansion of innate immune genes, the amphioxus encodes many PGRPs of diverse functions. For instance, three isoforms of PGRP encoded by Branchiostoma belcheri tsingtauense, termed BbtPGRP1~3, are fused with a chitin binding domain (CBD) at the N-terminus. Here we report the 2.7 Å crystal structure of BbtPGRP3, revealing an overall structure of an N-terminal hevein-like CBD followed by a catalytic PGRP domain. Activity assays combined with site-directed mutagenesis indicated that the individual PGRP domain exhibits amidase activity towards both DAP-type and Lys-type peptidoglycans (PGNs), the former of which is favored. The N-terminal CBD not only has the chitin-binding activity, but also enables BbtPGRP3 to gain a five-fold increase of amidase activity towards the Lys-type PGNs, leading to a significantly broadened substrate spectrum. Together, we propose that modular evolution via domain shuffling combined with gene horizontal transfer makes BbtPGRP1~3 novel PGRPs of augmented catalytic activity and broad recognition spectrum. PMID:26479246

  18. Voice Recognition in Face-Blind Patients

    PubMed Central

    Liu, Ran R.; Pancaroglu, Raika; Hills, Charlotte S.; Duchaine, Brad; Barton, Jason J. S.

    2016-01-01

    Right or bilateral anterior temporal damage can impair face recognition, but whether this is an associative variant of prosopagnosia or part of a multimodal disorder of person recognition is an unsettled question, with implications for cognitive and neuroanatomic models of person recognition. We assessed voice perception and short-term recognition of recently heard voices in 10 subjects with impaired face recognition acquired after cerebral lesions. All 4 subjects with apperceptive prosopagnosia due to lesions limited to fusiform cortex had intact voice discrimination and recognition. One subject with bilateral fusiform and anterior temporal lesions had a combined apperceptive prosopagnosia and apperceptive phonagnosia, the first such described case. Deficits indicating a multimodal syndrome of person recognition were found only in 2 subjects with bilateral anterior temporal lesions. All 3 subjects with right anterior temporal lesions had normal voice perception and recognition, 2 of whom performed normally on perceptual discrimination of faces. This confirms that such lesions can cause a modality-specific associative prosopagnosia. PMID:25349193

  19. Acquired prosopagnosia without word recognition deficits.

    PubMed

    Susilo, Tirta; Wright, Victoria; Tree, Jeremy J; Duchaine, Bradley

    2015-01-01

    It has long been suggested that face recognition relies on specialized mechanisms that are not involved in visual recognition of other object categories, including those that require expert, fine-grained discrimination at the exemplar level such as written words. But according to the recently proposed many-to-many theory of object recognition (MTMT), visual recognition of faces and words are carried out by common mechanisms [Behrmann, M., & Plaut, D. C. ( 2013 ). Distributed circuits, not circumscribed centers, mediate visual recognition. Trends in Cognitive Sciences, 17, 210-219]. MTMT acknowledges that face and word recognition are lateralized, but posits that the mechanisms that predominantly carry out face recognition still contribute to word recognition and vice versa. MTMT makes a key prediction, namely that acquired prosopagnosics should exhibit some measure of word recognition deficits. We tested this prediction by assessing written word recognition in five acquired prosopagnosic patients. Four patients had lesions limited to the right hemisphere while one had bilateral lesions with more pronounced lesions in the right hemisphere. The patients completed a total of seven word recognition tasks: two lexical decision tasks and five reading aloud tasks totalling more than 1200 trials. The performances of the four older patients (3 female, age range 50-64 years) were compared to those of 12 older controls (8 female, age range 56-66 years), while the performances of the younger prosopagnosic (male, 31 years) were compared to those of 14 younger controls (9 female, age range 20-33 years). We analysed all results at the single-patient level using Crawford's t-test. Across seven tasks, four prosopagnosics performed as quickly and accurately as controls. Our results demonstrate that acquired prosopagnosia can exist without word recognition deficits. These findings are inconsistent with a key prediction of MTMT. They instead support the hypothesis that face

  20. Examining ERP correlates of recognition memory: Evidence of accurate source recognition without recollection

    PubMed Central

    Addante, Richard, J.; Ranganath, Charan; Yonelinas, Andrew, P.

    2012-01-01

    Recollection is typically associated with high recognition confidence and accurate source memory. However, subjects sometimes make accurate source memory judgments even for items that are not confidently recognized, and it is not known whether these responses are based on recollection or some other memory process. In the current study, we measured event related potentials (ERPs) while subjects made item and source memory confidence judgments in order to determine whether recollection supported accurate source recognition responses for items that were not confidently recognized. In line with previous studies, we found that recognition memory was associated with two ERP effects: an early on-setting FN400 effect, and a later parietal old-new effect [Late Positive Component (LPC)], which have been associated with familiarity and recollection, respectively. The FN400 increased gradually with item recognition confidence, whereas the LPC was only observed for highly confident recognition responses. The LPC was also related to source accuracy, but only for items that had received a high confidence item recognition response; accurate source judgments to items that were less confidently recognized did not exhibit the typical ERP correlate of recollection or familiarity, but rather showed a late, broadly distributed negative ERP difference. The results indicate that accurate source judgments of episodic context can occur even when recollection fails. PMID:22548808

  1. Interactive object recognition assistance: an approach to recognition starting from target objects

    NASA Astrophysics Data System (ADS)

    Geisler, Juergen; Littfass, Michael

    1999-07-01

    Recognition of target objects in remotely sensed imagery required detailed knowledge about the target object domain as well as about mapping properties of the sensing system. The art of object recognition is to combine both worlds appropriately and to provide models of target appearance with respect to sensor characteristics. Common approaches to support interactive object recognition are either driven from the sensor point of view and address the problem of displaying images in a manner adequate to the sensing system. Or they focus on target objects and provide exhaustive encyclopedic information about this domain. Our paper discusses an approach to assist interactive object recognition based on knowledge about target objects and taking into account the significance of object features with respect to characteristics of the sensed imagery, e.g. spatial and spectral resolution. An `interactive recognition assistant' takes the image analyst through the interpretation process by indicating step-by-step the respectively most significant features of objects in an actual set of candidates. The significance of object features is expressed by pregenerated trees of significance, and by the dynamic computation of decision relevance for every feature at each step of the recognition process. In the context of this approach we discuss the question of modeling and storing the multisensorial/multispectral appearances of target objects and object classes as well as the problem of an adequate dynamic human-machine-interface that takes into account various mental models of human image interpretation.

  2. Structural Basis for Antigenic Peptide Recognition and Processing by Endoplasmic Reticulum (ER) Aminopeptidase 2.

    PubMed

    Mpakali, Anastasia; Giastas, Petros; Mathioudakis, Nikolas; Mavridis, Irene M; Saridakis, Emmanuel; Stratikos, Efstratios

    2015-10-23

    Endoplasmic reticulum (ER) aminopeptidases process antigenic peptide precursors to generate epitopes for presentation by MHC class I molecules and help shape the antigenic peptide repertoire and cytotoxic T-cell responses. To perform this function, ER aminopeptidases have to recognize and process a vast variety of peptide sequences. To understand how these enzymes recognize substrates, we determined crystal structures of ER aminopeptidase 2 (ERAP2) in complex with a substrate analogue and a peptidic product to 2.5 and 2.7 Å, respectively, and compared them to the apo-form structure determined to 3.0 Å. The peptides were found within the internal cavity of the enzyme with no direct access to the outside solvent. The substrate analogue extends away from the catalytic center toward the distal end of the internal cavity, making interactions with several shallow pockets along the path. A similar configuration was evident for the peptidic product, although decreasing electron density toward its C terminus indicated progressive disorder. Enzymatic analysis confirmed that visualized interactions can either positively or negatively impact in vitro trimming rates. Opportunistic side-chain interactions and lack of deep specificity pockets support a limited-selectivity model for antigenic peptide processing by ERAP2. In contrast to proposed models for the homologous ERAP1, no specific recognition of the peptide C terminus by ERAP2 was evident, consistent with functional differences in length selection and self-activation between these two enzymes. Our results suggest that ERAP2 selects substrates by sequestering them in its internal cavity and allowing opportunistic interactions to determine trimming rates, thus combining substrate permissiveness with sequence bias. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Word Recognition in Auditory Cortex

    ERIC Educational Resources Information Center

    DeWitt, Iain D. J.

    2013-01-01

    Although spoken word recognition is more fundamental to human communication than text recognition, knowledge of word-processing in auditory cortex is comparatively impoverished. This dissertation synthesizes current models of auditory cortex, models of cortical pattern recognition, models of single-word reading, results in phonetics and results in…

  4. Shared neural substrates for song discrimination in parental and parasitic songbirds.

    PubMed

    Louder, Matthew I M; Voss, Henning U; Manna, Thomas J; Carryl, Sophia S; London, Sarah E; Balakrishnan, Christopher N; Hauber, Mark E

    2016-05-27

    In many social animals, early exposure to conspecific stimuli is critical for the development of accurate species recognition. Obligate brood parasitic songbirds, however, forego parental care and young are raised by heterospecific hosts in the absence of conspecific stimuli. Having evolved from non-parasitic, parental ancestors, how brood parasites recognize their own species remains unclear. In parental songbirds (e.g. zebra finch Taeniopygia guttata), the primary and secondary auditory forebrain areas are known to be critical in the differential processing of conspecific vs. heterospecific songs. Here we demonstrate that the same auditory brain regions underlie song discrimination in adult brood parasitic pin-tailed whydahs (Vidua macroura), a close relative of the zebra finch lineage. Similar to zebra finches, whydahs showed stronger behavioral responses during conspecific vs. heterospecific song and tone pips as well as increased neural responses within the auditory forebrain, as measured by both functional magnetic resonance imaging (fMRI) and immediate early gene (IEG) expression. Given parallel behavioral and neuroanatomical patterns of song discrimination, our results suggest that the evolutionary transition to brood parasitism from parental songbirds likely involved an "evolutionary tinkering" of existing proximate mechanisms, rather than the wholesale reworking of the neural substrates of species recognition. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  5. Substrate system for spray forming

    DOEpatents

    Chu, Men G.; Chernicoff, William P.

    2000-01-01

    A substrate system for receiving a deposit of sprayed metal droplets including a movable outer substrate on which the sprayed metal droplets are deposited. The substrate system also includes an inner substrate disposed adjacent the outer substrate where the sprayed metal droplets are deposited on the outer substrate. The inner substrate includes zones of differing thermal conductivity to resist substrate layer porosity and to resist formation of large grains and coarse constituent particles in a bulk layer of the metal droplets which have accumulated on the outer substrate. A spray forming apparatus and associated method of spray forming a molten metal to form a metal product using the substrate system of the invention is also provided.

  6. Substrate system for spray forming

    DOEpatents

    Chu, Men G.; Chernicoff, William P.

    2002-01-01

    A substrate system for receiving a deposit of sprayed metal droplets including a movable outer substrate on which the sprayed metal droplets are deposited. The substrate system also includes an inner substrate disposed adjacent the outer substrate where the sprayed metal droplets are deposited on the outer substrate. The inner substrate includes zones of differing thermal conductivity to resist substrate layer porosity and to resist formation of large grains and coarse constituent particles in a bulk layer of the metal droplets which have accumulated on the outer substrate. A spray forming apparatus and associated method of spray forming a molten metal to form a metal product using the substrate system of the invention is also provided.

  7. [Comparative studies of face recognition].

    PubMed

    Kawai, Nobuyuki

    2012-07-01

    Every human being is proficient in face recognition. However, the reason for and the manner in which humans have attained such an ability remain unknown. These questions can be best answered-through comparative studies of face recognition in non-human animals. Studies in both primates and non-primates show that not only primates, but also non-primates possess the ability to extract information from their conspecifics and from human experimenters. Neural specialization for face recognition is shared with mammals in distant taxa, suggesting that face recognition evolved earlier than the emergence of mammals. A recent study indicated that a social insect, the golden paper wasp, can distinguish their conspecific faces, whereas a closely related species, which has a less complex social lifestyle with just one queen ruling a nest of underlings, did not show strong face recognition for their conspecifics. Social complexity and the need to differentiate between one another likely led humans to evolve their face recognition abilities.

  8. Genetic specificity of face recognition

    PubMed Central

    Shakeshaft, Nicholas G.; Plomin, Robert

    2015-01-01

    Specific cognitive abilities in diverse domains are typically found to be highly heritable and substantially correlated with general cognitive ability (g), both phenotypically and genetically. Recent twin studies have found the ability to memorize and recognize faces to be an exception, being similarly heritable but phenotypically substantially uncorrelated both with g and with general object recognition. However, the genetic relationships between face recognition and other abilities (the extent to which they share a common genetic etiology) cannot be determined from phenotypic associations. In this, to our knowledge, first study of the genetic associations between face recognition and other domains, 2,000 18- and 19-year-old United Kingdom twins completed tests assessing their face recognition, object recognition, and general cognitive abilities. Results confirmed the substantial heritability of face recognition (61%), and multivariate genetic analyses found that most of this genetic influence is unique and not shared with other cognitive abilities. PMID:26417086

  9. Coated substrates and process

    DOEpatents

    Chu, Wei-kan; Childs, Charles B.

    1991-01-01

    Disclosed herein is a coated substrate and a process for forming films on substrates and for providing a particularly smooth film on a substrate. The method of this invention involves subjecting a surface of a substrate to contact with a stream of ions of an inert gas having sufficient force and energy to substantially change the surface characteristics of said substrate, and then exposing a film-forming material to a stream of ions of an inert gas having sufficient energy to vaporize the atoms of said film-forming material and to transmit the vaporized atoms to the substrate surface with sufficient force to form a film bonded to the substrate. This process is particularly useful commercially because it forms strong bonds at room temperature. This invention is particularly useful for adhering a gold film to diamond and forming ohmic electrodes on diamond, but also can be used to bond other films to substrates.

  10. Is having similar eye movement patterns during face learning and recognition beneficial for recognition performance? Evidence from hidden Markov modeling.

    PubMed

    Chuk, Tim; Chan, Antoni B; Hsiao, Janet H

    2017-12-01

    The hidden Markov model (HMM)-based approach for eye movement analysis is able to reflect individual differences in both spatial and temporal aspects of eye movements. Here we used this approach to understand the relationship between eye movements during face learning and recognition, and its association with recognition performance. We discovered holistic (i.e., mainly looking at the face center) and analytic (i.e., specifically looking at the two eyes in addition to the face center) patterns during both learning and recognition. Although for both learning and recognition, participants who adopted analytic patterns had better recognition performance than those with holistic patterns, a significant positive correlation between the likelihood of participants' patterns being classified as analytic and their recognition performance was only observed during recognition. Significantly more participants adopted holistic patterns during learning than recognition. Interestingly, about 40% of the participants used different patterns between learning and recognition, and among them 90% switched their patterns from holistic at learning to analytic at recognition. In contrast to the scan path theory, which posits that eye movements during learning have to be recapitulated during recognition for the recognition to be successful, participants who used the same or different patterns during learning and recognition did not differ in recognition performance. The similarity between their learning and recognition eye movement patterns also did not correlate with their recognition performance. These findings suggested that perceptuomotor memory elicited by eye movement patterns during learning does not play an important role in recognition. In contrast, the retrieval of diagnostic information for recognition, such as the eyes for face recognition, is a better predictor for recognition performance. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Substrate quality alters microbial mineralization of added substrate and soil organic carbon

    NASA Astrophysics Data System (ADS)

    Jagadamma, S.; Mayes, M. A.; Steinweg, J. M.; Schaeffer, S. M.

    2014-03-01

    The rate and extent of decomposition of soil organic carbon (SOC) is dependent on substrate chemistry and microbial dynamics. Our objectives were to understand the influence of substrate chemistry on microbial processing of carbon (C), and to use model fitting to quantify differences in pool sizes and mineralization rates. We conducted an incubation experiment for 270 days using four uniformly-labeled 14C substrates (glucose, starch, cinnamic acid and stearic acid) on four different soils (a temperate Mollisol, a tropical Ultisol, a sub-arctic Andisol, and an arctic Gelisol). The 14C labeling enabled us to separate CO2 respired from added substrates and from native SOC. Microbial gene copy numbers were quantified at days 4, 30 and 270 using quantitative polymerase chain reaction (qPCR). Substrate C respiration was always higher for glucose than other substrates. Soils with cinnamic and stearic acid lost more native SOC than glucose- and starch-amended soils, despite an initial delay in respiration. Cinnamic and stearic acid amendments also exhibited higher fungal gene copy numbers at the end of incubation compared to unamended soils. We found that 270 days was sufficient to model decomposition of simple substrates (glucose and starch) with three pools, but was insufficient for more complex substrates (cinnamic and stearic acid) and native SOC. This study reveals that substrate quality imparts considerable control on microbial decomposition of newly added and native SOC, and demonstrates the need for multi-year incubation experiments to constrain decomposition parameters for the most recalcitrant fractions of SOC and added substrates.

  12. BDNF Expression in Perirhinal Cortex is Associated with Exercise-Induced Improvement in Object Recognition Memory

    PubMed Central

    Hopkins, Michael E.; Bucci, David J.

    2010-01-01

    Physical exercise induces widespread neurobiological adaptations and improves learning and memory. Most research in this field has focused on hippocampus-based spatial tasks and changes in brain-derived neurotrophic factor (BDNF) as a putative substrate underlying exercise-induced cognitive improvements. Chronic exercise can also be anxiolytic and causes adaptive changes in stress reactivity. The present study employed a perirhinal cortex-dependent object recognition task as well as the elevated plus maze to directly test for interactions between the cognitive and anxiolytic effects of exercise in male Long Evans rats. Hippocampal and perirhinal cortex tissue was collected to determine whether the relationship between BDNF and cognitive performance extends to this non-spatial and non-hippocampal-dependent task. We also examined whether the cognitive improvements persisted once the exercise regimen was terminated. Our data indicate that 4 weeks of voluntary exercise every-other-day improved object recognition memory. Importantly, BDNF expression in the perirhinal cortex of exercising rats was strongly correlated with object recognition memory. Exercise also decreased anxiety-like behavior, however there was no evidence to support a relationship between anxiety-like behavior and performance on the novel object recognition task. There was a trend for a negative relationship between anxiety-like behavior and hippocampal BDNF. Neither the cognitive improvements nor the relationship between cognitive function and perirhinal BDNF levels persisted after 2 weeks of inactivity. These are the first data demonstrating that region-specific changes in BDNF protein levels are correlated with exercise-induced improvements in non-spatial memory, mediated by structures outside the hippocampus and are consistent with the theory that, with regard to object recognition, the anxiolytic and cognitive effects of exercise may be mediated through separable mechanisms. PMID:20601027

  13. Transfer-appropriate processing in recognition memory: perceptual and conceptual effects on recognition memory depend on task demands.

    PubMed

    Parks, Colleen M

    2013-07-01

    Research examining the importance of surface-level information to familiarity in recognition memory tasks is mixed: Sometimes it affects recognition and sometimes it does not. One potential explanation of the inconsistent findings comes from the ideas of dual process theory of recognition and the transfer-appropriate processing framework, which suggest that the extent to which perceptual fluency matters on a recognition test depends in large part on the task demands. A test that recruits perceptual processing for discrimination should show greater perceptual effects and smaller conceptual effects than standard recognition, similar to the pattern of effects found in perceptual implicit memory tasks. This idea was tested in the current experiment by crossing a levels of processing manipulation with a modality manipulation on a series of recognition tests that ranged from conceptual (standard recognition) to very perceptually demanding (a speeded recognition test with degraded stimuli). Results showed that the levels of processing effect decreased and the effect of modality increased when tests were made perceptually demanding. These results support the idea that surface-level features influence performance on recognition tests when they are made salient by the task demands. PsycINFO Database Record (c) 2013 APA, all rights reserved.

  14. Molecular dynamics and binding selectivity of nucleotides and polynucleotide substrates with EIF2C2/Ago2 PAZ domain.

    PubMed

    Kandeel, Mahmoud; Kitade, Yukio

    2018-02-01

    RNA interference (RNAi) constitutes a major target in drug discovery. Recently, we reported that the Argonaute protein 2 (Ago2) PAZ domain selectively binds with all ribonucleotides except adenine and poorly recognizes deoxyribonucleotides. The binding properties of the PAZ domain with polynucleotides and the molecular mechanisms of substrates' selectivity remains unclear. In this study, the binding potencies of polynucleotides and the associated conformational and dynamic changes in PAZ domain are investigated. Coinciding with nucleotides' binding profile with the PAZ domain, polyuridylate (PolyU) and polycytidylate (PolyC) were potent binders. However, K dPolyU and K dPolyC were 15.8 and 9.3μM, respectively. In contrast, polyadenylate (PolyA) binding was not detectable. Molecular dynamics (MD) simulation revealed the highest change in root mean square deviation (RMSD) with ApoPAZ or PAZ domain bound with experimentally approved, low affinity substrates, whereas stronger binding substrates such as UMP or PolyU showed minimal RMSD changes. The loop between α3 and β5 in the β-hairpin subdomain showed the most responsive change in RMSD, being highly movable in the ApoPAZ and PAZ-AMP complex. Favorable substrate recognition was associate with moderate change in secondary structure content. In conclusion, the PAZ domain retains differential substrate selectivity associated with corresponding dynamic and structural changes upon binding. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Molecular recognition of 7-(2-octadecyloxycarbonylethyl)guanine to cytidine at the air/water interface and LB film studied by Fourier transform infrared spectroscopy.

    PubMed

    Miao, Wangen; Luo, Xuzhong; Liang, Yingqiu

    2003-03-15

    Monolayer behavior of a nucleolipid amphiphile, 7-(2-octadecyloxycarbonylethyl)guanine (ODCG), on aqueous cytidine solution was investigated by means of surface-molecular area (pi-A) isotherms. It indicates that molecular recognition by hydrogen bonding is present between ODCG monolayer and the cytidine in subphase. The Fourier transform infrared (FTIR) transmission spectroscopic result indicates that the cytidine molecules in the subphase can be transferred onto solid substrates by Langmuir-Blodgett (LB) technique as a result of the formation of Watson-Crick base-pairing at the air/water interface. Investigation by rotating polarized FTIR transmission also suggests that the headgroup recognition of this amphiphile to the dissolved cytidine influence the orientation of the tailchains. Copyright 2002 Elsevier Science B.V.

  16. Molecular recognition of 7-(2-octadecyloxycarbonylethyl)guanine to cytidine at the air/water interface and LB film studied by Fourier transform infrared spectroscopy

    NASA Astrophysics Data System (ADS)

    Miao, Wangen; Luo, Xuzhong; Liang, Yingqiu

    2003-03-01

    Monolayer behavior of a nucleolipid amphiphile, 7-(2-octadecyloxycarbonylethyl)guanine (ODCG), on aqueous cytidine solution was investigated by means of surface-molecular area ( π- A) isotherms. It indicates that molecular recognition by hydrogen bonding is present between ODCG monolayer and the cytidine in subphase. The Fourier transform infrared (FTIR) transmission spectroscopic result indicates that the cytidine molecules in the subphase can be transferred onto solid substrates by Langmuir-Blodgett (LB) technique as a result of the formation of Watson-Crick base-pairing at the air/water interface. Investigation by rotating polarized FTIR transmission also suggests that the headgroup recognition of this amphiphile to the dissolved cytidine influence the orientation of the tailchains.

  17. Characterization and identification of ubiquitin conjugation sites with E3 ligase recognition specificities.

    PubMed

    Nguyen, Van-Nui; Huang, Kai-Yao; Huang, Chien-Hsun; Chang, Tzu-Hao; Bretaña, Neil; Lai, K; Weng, Julia; Lee, Tzong-Yi

    2015-01-01

    In eukaryotes, ubiquitin-conjugation is an important mechanism underlying proteasome-mediated degradation of proteins, and as such, plays an essential role in the regulation of many cellular processes. In the ubiquitin-proteasome pathway, E3 ligases play important roles by recognizing a specific protein substrate and catalyzing the attachment of ubiquitin to a lysine (K) residue. As more and more experimental data on ubiquitin conjugation sites become available, it becomes possible to develop prediction models that can be scaled to big data. However, no development that focuses on the investigation of ubiquitinated substrate specificities has existed. Herein, we present an approach that exploits an iteratively statistical method to identify ubiquitin conjugation sites with substrate site specificities. In this investigation, totally 6259 experimentally validated ubiquitinated proteins were obtained from dbPTM. After having filtered out homologous fragments with 40% sequence identity, the training data set contained 2658 ubiquitination sites (positive data) and 5532 non-ubiquitinated sites (negative data). Due to the difficulty in characterizing the substrate site specificities of E3 ligases by conventional sequence logo analysis, a recursively statistical method has been applied to obtain significant conserved motifs. The profile hidden Markov model (profile HMM) was adopted to construct the predictive models learned from the identified substrate motifs. A five-fold cross validation was then used to evaluate the predictive model, achieving sensitivity, specificity, and accuracy of 73.07%, 65.46%, and 67.93%, respectively. Additionally, an independent testing set, completely blind to the training data of the predictive model, was used to demonstrate that the proposed method could provide a promising accuracy (76.13%) and outperform other ubiquitination site prediction tool. A case study demonstrated the effectiveness of the characterized substrate motifs for

  18. Supporting Quality Teachers with Recognition

    ERIC Educational Resources Information Center

    Andrews, Hans A.

    2011-01-01

    Value has been found in providing recognition and awards programs for excellent teachers. Research has also found a major lack of these programs in both the USA and in Australia. Teachers receiving recognition and awards for their teaching have praised recognition programs as providing motivation for them to continue high-level instruction.…

  19. PREFACE: Cell-substrate interactions Cell-substrate interactions

    NASA Astrophysics Data System (ADS)

    Gardel, Margaret; Schwarz, Ulrich

    2010-05-01

    One of the most striking achievements of evolution is the ability to build cellular systems that are both robust and dynamic. Taken by themselves, both properties are obvious requirements: robustness reflects the fact that cells are there to survive, and dynamics is required to adapt to changing environments. However, it is by no means trivial to understand how these two requirements can be implemented simultaneously in a physical system. The long and difficult quest to build adaptive materials is testimony to the inherent difficulty of this goal. Here materials science can learn a lot from nature, because cellular systems show that robustness and dynamics can be achieved in a synergetic fashion. For example, the capabilities of tissues to repair and regenerate are still unsurpassed in the world of synthetic materials. One of the most important aspects of the way biological cells adapt to their environment is their adhesive interaction with the substrate. Numerous aspects of the physiology of metazoan cells, including survival, proliferation, differentiation and migration, require the formation of adhesions to the cell substrate, typically an extracellular matrix protein. Adhesions guide these diverse processes both by mediating force transmission from the cell to the substrate and by controlling biochemical signaling pathways. While the study of cell-substrate adhesions is a mature field in cell biology, a quantitative biophysical understanding of how the interactions of the individual molecular components give rise to the rich dynamics and mechanical behaviors observed for cell-substrate adhesions has started to emerge only over the last decade or so. The recent growth of research activities on cell-substrate interactions was strongly driven by the introduction of new physical techniques for surface engineering into traditional cell biological work with cell culture. For example, microcontact printing of adhesive patterns was used to show that cell fate depends

  20. 16p11.2 Deletion mice display cognitive deficits in touchscreen learning and novelty recognition tasks.

    PubMed

    Yang, Mu; Lewis, Freeman C; Sarvi, Michael S; Foley, Gillian M; Crawley, Jacqueline N

    2015-12-01

    Chromosomal 16p11.2 deletion syndrome frequently presents with intellectual disabilities, speech delays, and autism. Here we investigated the Dolmetsch line of 16p11.2 heterozygous (+/-) mice on a range of cognitive tasks with different neuroanatomical substrates. Robust novel object recognition deficits were replicated in two cohorts of 16p11.2+/- mice, confirming previous findings. A similarly robust deficit in object location memory was discovered in +/-, indicating impaired spatial novelty recognition. Generalizability of novelty recognition deficits in +/- mice extended to preference for social novelty. Robust learning deficits and cognitive inflexibility were detected using Bussey-Saksida touchscreen operant chambers. During acquisition of pairwise visual discrimination, +/- mice required significantly more training trials to reach criterion than wild-type littermates (+/+), and made more errors and correction errors than +/+. In the reversal phase, all +/+ reached criterion, whereas most +/- failed to reach criterion by the 30-d cutoff. Contextual and cued fear conditioning were normal in +/-. These cognitive phenotypes may be relevant to some aspects of cognitive impairments in humans with 16p11.2 deletion, and support the use of 16p11.2+/- mice as a model system for discovering treatments for cognitive impairments in 16p11.2 deletion syndrome. © 2015 Yang et al.; Published by Cold Spring Harbor Laboratory Press.

  1. Document Form and Character Recognition using SVM

    NASA Astrophysics Data System (ADS)

    Park, Sang-Sung; Shin, Young-Geun; Jung, Won-Kyo; Ahn, Dong-Kyu; Jang, Dong-Sik

    2009-08-01

    Because of development of computer and information communication, EDI (Electronic Data Interchange) has been developing. There is OCR (Optical Character Recognition) of Pattern recognition technology for EDI. OCR contributed to changing many manual in the past into automation. But for the more perfect database of document, much manual is needed for excluding unnecessary recognition. To resolve this problem, we propose document form based character recognition method in this study. Proposed method is divided into document form recognition part and character recognition part. Especially, in character recognition, change character into binarization by using SVM algorithm and extract more correct feature value.

  2. Ependymin as a substrate for outgrowth of axons from cultured explants of goldfish retina.

    PubMed

    Schmidt, J T; Schmidt, R; Lin, W C; Jian, X Y; Stuermer, C A

    1991-01-01

    Ependymin, a prominent protein of the brain's extracellular fluid (ECF) was previously implicated in the consolidation of memory and in the activity-driven sharpening of the retinotectal projection. Because both these phenomena probably involve the growth and elaboration of appropriate synapses, we have tested whether ependymin can serve as a substrate for the growth of axons from goldfish retinal ganglion cells in a culture assay. Ependymin (Ep), laminin (LAM), polylysine (PL), and Concanavalin A (Con A) were plated on glass coverslips either uniformly or in striped patterns. Ep alone, either soluble or partly polymerized (by dropping calcium concentration and pH), was a good substrate for axonal outgrowth, as good or better than PL and Con A, but not as good as LAM. Neurites grew faster on LAM (71 microns/h) than on Ep (32 microns/h) or on PL (22 microns/h). Fasciculation was low on LAM, intermediate on Ep, and highest on PL. In exclusive side-by-side stripe assays, axons preferred LAM over Ep, but gave weak or no preference for Ep over Con A or PL. With stripes of LAM + Ep alongside pure LAM, the axons preferred the mixture of LAM + Ep. When antibodies to Ep were plated in stripes over continuous Ep substrate, the axons avoided the antibody-blocked stripes and grew on the Ep stripes. Antibodies to Ep did not, however, block growth on laminin substrates, nor did antibodies to LAM block growth on Ep. Dot blots and western blots showed very little cross recognition between the antibodies. Ependymin is a good substrate for neurite outgrowth, which is normally present in ECF, and adhesion to Ep is independent of LAM and possibly additive to it.

  3. The medial prefrontal cortex-lateral entorhinal cortex circuit is essential for episodic-like memory and associative object-recognition.

    PubMed

    Chao, Owen Y; Huston, Joseph P; Li, Jay-Shake; Wang, An-Li; de Souza Silva, Maria A

    2016-05-01

    The prefrontal cortex directly projects to the lateral entorhinal cortex (LEC), an important substrate for engaging item-associated information and relaying the information to the hippocampus. Here we ask to what extent the communication between the prefrontal cortex and LEC is critically involved in the processing of episodic-like memory. We applied a disconnection procedure to test whether the interaction between the medial prefrontal cortex (mPFC) and LEC is essential for the expression of recognition memory. It was found that male rats that received unilateral NMDA lesions of the mPFC and LEC in the same hemisphere, exhibited intact episodic-like (what-where-when) and object-recognition memories. When these lesions were placed in the opposite hemispheres (disconnection), episodic-like and associative memories for object identity, location and context were impaired. However, the disconnection did not impair the components of episodic memory, namely memory for novel object (what), object place (where) and temporal order (when), per se. Thus, the present findings suggest that the mPFC and LEC are a critical part of a neural circuit that underlies episodic-like and associative object-recognition memory. © 2015 Wiley Periodicals, Inc.

  4. Structural and mutational analyses of dipeptidyl peptidase 11 from Porphyromonas gingivalis reveal the molecular basis for strict substrate specificity

    PubMed Central

    Sakamoto, Yasumitsu; Suzuki, Yoshiyuki; Iizuka, Ippei; Tateoka, Chika; Roppongi, Saori; Fujimoto, Mayu; Inaka, Koji; Tanaka, Hiroaki; Yamada, Mitsugu; Ohta, Kazunori; Gouda, Hiroaki; Nonaka, Takamasa; Ogasawara, Wataru; Tanaka, Nobutada

    2015-01-01

    The dipeptidyl peptidase 11 from Porphyromonas gingivalis (PgDPP11) belongs to the S46 family of serine peptidases and preferentially cleaves substrates with Asp/Glu at the P1 position. The molecular mechanism underlying the substrate specificity of PgDPP11, however, is unknown. Here, we report the crystal structure of PgDPP11. The enzyme contains a catalytic domain with a typical double β-barrel fold and a recently identified regulatory α-helical domain. Crystal structure analyses, docking studies, and biochemical studies revealed that the side chain of Arg673 in the S1 subsite is essential for recognition of the Asp/Glu side chain at the P1 position of the bound substrate. Because S46 peptidases are not found in mammals and the Arg673 is conserved among DPP11s, we anticipate that DPP11s could be utilised as targets for antibiotics. In addition, the present structure analyses could be useful templates for the design of specific inhibitors of DPP11s from pathogenic organisms. PMID:26057589

  5. Superficial Priming in Episodic Recognition

    ERIC Educational Resources Information Center

    Dopkins, Stephen; Sargent, Jesse; Ngo, Catherine T.

    2010-01-01

    We explored the effect of superficial priming in episodic recognition and found it to be different from the effect of semantic priming in episodic recognition. Participants made recognition judgments to pairs of items, with each pair consisting of a prime item and a test item. Correct positive responses to the test item were impeded if the prime…

  6. Academic Recognition: Status and Challenges

    ERIC Educational Resources Information Center

    Bergan, Sjur

    2009-01-01

    The Council of Europe/UNESCO Recognition Convention (also known as the Lisbon Recognition Convention) provides the legal framework for academic recognition in Europe, and it serves a double purpose: as a legal text and as a guide to good practice. The ENIC and NARIC Networks promote the implementation of the Convention and seek to develop a better…

  7. An Acrobatic Substrate Metamorphosis Reveals a Requirement for Substrate Conformational Dynamics in Trypsin Proteolysis*

    PubMed Central

    Kayode, Olumide; Wang, Ruiying; Pendlebury, Devon F.; Cohen, Itay; Henin, Rachel D.; Hockla, Alexandra; Soares, Alexei S.; Papo, Niv; Caulfield, Thomas R.; Radisky, Evette S.

    2016-01-01

    The molecular basis of enzyme catalytic power and specificity derives from dynamic interactions between enzyme and substrate during catalysis. Although considerable effort has been devoted to understanding how conformational dynamics within enzymes affect catalysis, the role of conformational dynamics within protein substrates has not been addressed. Here, we examine the importance of substrate dynamics in the cleavage of Kunitz-bovine pancreatic trypsin inhibitor protease inhibitors by mesotrypsin, finding that the varied conformational dynamics of structurally similar substrates can profoundly impact the rate of catalysis. A 1.4-Å crystal structure of a mesotrypsin-product complex formed with a rapidly cleaved substrate reveals a dramatic conformational change in the substrate upon proteolysis. By using long all-atom molecular dynamics simulations of acyl-enzyme intermediates with proteolysis rates spanning 3 orders of magnitude, we identify global and local dynamic features of substrates on the nanosecond-microsecond time scale that correlate with enzymatic rates and explain differential susceptibility to proteolysis. By integrating multiple enhanced sampling methods for molecular dynamics, we model a viable conformational pathway between substrate-like and product-like states, linking substrate dynamics on the nanosecond-microsecond time scale with large collective substrate motions on the much slower time scale of catalysis. Our findings implicate substrate flexibility as a critical determinant of catalysis. PMID:27810896

  8. A biomolecular recognition approach for the functionalization of cellulose with gold nanoparticles.

    PubMed

    Almeida, A; Rosa, A M M; Azevedo, A M; Prazeres, D M F

    2017-09-01

    Materials with new and improved functionalities can be obtained by modifying cellulose with gold nanoparticles (AuNPs) via the in situ reduction of a gold precursor or the deposition or covalent immobilization of pre-synthesized AuNPs. Here, we present an alternative biomolecular recognition approach to functionalize cellulose with biotin-AuNPs that relies on a complex of 2 recognition elements: a ZZ-CBM3 fusion that combines a carbohydrate-binding module (CBM) with the ZZ fragment of the staphylococcal protein A and an anti-biotin antibody. Paper and cellulose microparticles with AuNPs immobilized via the ZZ-CBM3:anti-biotin IgG supramolecular complex displayed an intense red color, whereas essentially no color was detected when AuNPs were deposited over the unmodified materials. Scanning electron microscopy analysis revealed a homogeneous distribution of AuNPs when immobilized via ZZ-CBM3:anti-biotin IgG complexes and aggregation of AuNPs when deposited over paper, suggesting that color differences are due to interparticle plasmon coupling effects. The approach could be used to functionalize paper substrates and cellulose nanocrystals with AuNPs. More important, however, is the fact that the occurrence of a biomolecular recognition event between the CBM-immobilized antibody and its specific, AuNP-conjugated antigen is signaled by red color. This opens up the way for the development of simple and straightforward paper/cellulose-based tests where detection of a target analyte can be made by direct use of color signaling. Copyright © 2017 John Wiley & Sons, Ltd.

  9. Social emotion recognition, social functioning, and attempted suicide in late-life depression.

    PubMed

    Szanto, Katalin; Dombrovski, Alexandre Y; Sahakian, Barbara J; Mulsant, Benoit H; Houck, Patricia R; Reynolds, Charles F; Clark, Luke

    2012-03-01

    : Lack of feeling connected and poor social problem solving have been described in suicide attempters. However, cognitive substrates of this apparent social impairment in suicide attempters remain unknown. One possible deficit, the inability to recognize others' complex emotional states has been observed not only in disorders characterized by prominent social deficits (autism-spectrum disorders and frontotemporal dementia) but also in depression and normal aging. This study assessed the relationship between social emotion recognition, problem solving, social functioning, and attempted suicide in late-life depression. : There were 90 participants: 24 older depressed suicide attempters, 38 nonsuicidal depressed elders, and 28 comparison subjects with no psychiatric history. We compared performance on the Reading the Mind in the Eyes test and measures of social networks, social support, social problem solving, and chronic interpersonal difficulties in these three groups. : Suicide attempters committed significantly more errors in social emotion recognition and showed poorer global cognitive performance than elders with no psychiatric history. Attempters had restricted social networks: they were less likely to talk to their children, had fewer close friends, and did not engage in volunteer activities, compared to nonsuicidal depressed elders and those with no psychiatric history. They also reported a pattern of struggle against others and hostility in relationships, felt a lack of social support, perceived social problems as impossible to resolve, and displayed a careless/impulsive approach to problems. : Suicide attempts in depressed elders were associated with poor social problem solving, constricted social networks, and disruptive interpersonal relationships. Impaired social emotion recognition in the suicide attempter group was related.

  10. Infant visual attention and object recognition.

    PubMed

    Reynolds, Greg D

    2015-05-15

    This paper explores the role visual attention plays in the recognition of objects in infancy. Research and theory on the development of infant attention and recognition memory are reviewed in three major sections. The first section reviews some of the major findings and theory emerging from a rich tradition of behavioral research utilizing preferential looking tasks to examine visual attention and recognition memory in infancy. The second section examines research utilizing neural measures of attention and object recognition in infancy as well as research on brain-behavior relations in the early development of attention and recognition memory. The third section addresses potential areas of the brain involved in infant object recognition and visual attention. An integrated synthesis of some of the existing models of the development of visual attention is presented which may account for the observed changes in behavioral and neural measures of visual attention and object recognition that occur across infancy. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Infant Visual Attention and Object Recognition

    PubMed Central

    Reynolds, Greg D.

    2015-01-01

    This paper explores the role visual attention plays in the recognition of objects in infancy. Research and theory on the development of infant attention and recognition memory are reviewed in three major sections. The first section reviews some of the major findings and theory emerging from a rich tradition of behavioral research utilizing preferential looking tasks to examine visual attention and recognition memory in infancy. The second section examines research utilizing neural measures of attention and object recognition in infancy as well as research on brain-behavior relations in the early development of attention and recognition memory. The third section addresses potential areas of the brain involved in infant object recognition and visual attention. An integrated synthesis of some of the existing models of the development of visual attention is presented which may account for the observed changes in behavioral and neural measures of visual attention and object recognition that occur across infancy. PMID:25596333

  12. Analysis and Recognition of Curve Type as The Basis of Object Recognition in Image

    NASA Astrophysics Data System (ADS)

    Nugraha, Nurma; Madenda, Sarifuddin; Indarti, Dina; Dewi Agushinta, R.; Ernastuti

    2016-06-01

    An object in an image when analyzed further will show the characteristics that distinguish one object with another object in an image. Characteristics that are used in object recognition in an image can be a color, shape, pattern, texture and spatial information that can be used to represent objects in the digital image. The method has recently been developed for image feature extraction on objects that share characteristics curve analysis (simple curve) and use the search feature of chain code object. This study will develop an algorithm analysis and the recognition of the type of curve as the basis for object recognition in images, with proposing addition of complex curve characteristics with maximum four branches that will be used for the process of object recognition in images. Definition of complex curve is the curve that has a point of intersection. By using some of the image of the edge detection, the algorithm was able to do the analysis and recognition of complex curve shape well.

  13. Immunologic hierarchy, class II MHC promiscuity, and epitope spreading of a melanoma helper peptide vaccine.

    PubMed

    Hu, Yinin; Petroni, Gina R; Olson, Walter C; Czarkowski, Andrea; Smolkin, Mark E; Grosh, William W; Chianese-Bullock, Kimberly A; Slingluff, Craig L

    2014-08-01

    Immunization with a combination melanoma helper peptide (6MHP) vaccine has been shown to induce CD4(+) T cell responses, which are associated with patient survival. In the present study, we define the relative immunogenicity and HLA allele promiscuity of individual helper peptides and identify helper peptide-mediated augmentation of specific CD8(+) T cell responses. Thirty-seven participants with stage IIIB-IV melanoma were vaccinated with 6MHP in incomplete Freund's adjuvant. The 6MHP vaccine is comprised of 6 peptides representing melanocytic differentiation proteins gp100, tyrosinase, Melan-A/MART-1, and cancer testis antigens from the MAGE family. CD4(+) and CD8(+) T cell responses were assessed in peripheral blood and in sentinel immunized nodes (SIN) by thymidine uptake after exposure to helper peptides and by direct interferon-γ ELIspot assay against 14 MHC class I-restricted peptides. Vaccine-induced CD4(+) T cell responses to individual epitopes were detected in the SIN of 63 % (22/35) and in the peripheral blood of 38 % (14/37) of participants for an overall response rate of 65 % (24/37). The most frequently immunogenic peptides were MAGE-A3281-295 (49 %) and tyrosinase386-406 (32 %). Responses were not limited to HLA restrictions originally described. Vaccine-associated CD8(+) T cell responses against class I-restricted peptides were observed in 45 % (5/11) of evaluable participants. The 6MHP vaccine induces both CD4(+) and CD8(+) T cell responses against melanoma antigens. CD4(+) T cell responses were detected beyond reported HLA-DR restrictions. Induction of CD8(+) T cell responses suggests epitope spreading and systemic activity mediated at the tumor site.

  14. Binding-induced Folding of Prokaryotic Ubiquitin-like Protein on the Mycobacterium Proteasomal ATPase Targets Substrates for Degradation

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

    T Wang; K Heran Darwin; H Li

    2011-12-31

    Mycobacterium tuberculosis uses a proteasome system that is analogous to the eukaryotic ubiquitin-proteasome pathway and is required for pathogenesis. However, the bacterial analog of ubiquitin, prokaryotic ubiquitin-like protein (Pup), is an intrinsically disordered protein that bears little sequence or structural resemblance to the highly structured ubiquitin. Thus, it was unknown how pupylated proteins were recruited to the proteasome. Here, we show that the Mycobacterium proteasomal ATPase (Mpa) has three pairs of tentacle-like coiled coils that recognize Pup. Mpa bound unstructured Pup through hydrophobic interactions and a network of hydrogen bonds, leading to the formation of an {alpha}-helix in Pup. Ourmore » work describes a binding-induced folding recognition mechanism in the Pup-proteasome system that differs mechanistically from substrate recognition in the ubiquitin-proteasome system. This key difference between the prokaryotic and eukaryotic systems could be exploited for the development of a small molecule-based treatment for tuberculosis.« less

  15. Binding-induced folding of prokaryotic ubiquitin-like protein on the mycobacterium proteasomal ATPase targets substrates for degradation

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

    Wang, T.; Li, H.; Darwin, K. H.

    2010-11-01

    Mycobacterium tuberculosis uses a proteasome system that is analogous to the eukaryotic ubiquitin-proteasome pathway and is required for pathogenesis. However, the bacterial analog of ubiquitin, prokaryotic ubiquitin-like protein (Pup), is an intrinsically disordered protein that bears little sequence or structural resemblance to the highly structured ubiquitin. Thus, it was unknown how pupylated proteins were recruited to the proteasome. Here, we show that the Mycobacterium proteasomal ATPase (Mpa) has three pairs of tentacle-like coiled coils that recognize Pup. Mpa bound unstructured Pup through hydrophobic interactions and a network of hydrogen bonds, leading to the formation of an {alpha}-helix in Pup. Ourmore » work describes a binding-induced folding recognition mechanism in the Pup-proteasome system that differs mechanistically from substrate recognition in the ubiquitin-proteasome system. This key difference between the prokaryotic and eukaryotic systems could be exploited for the development of a small molecule-based treatment for tuberculosis.« less

  16. Overt and covert competition in a promiscuous mammal: the importance of weaponry and testes size to male reproductive success.

    PubMed

    Preston, B T; Stevenson, I R; Pemberton, J M; Coltman, D W; Wilson, K

    2003-03-22

    Male contests for access to receptive females are thought to have selected for the larger male body size and conspicuous weaponry frequently observed in mammalian species. However, when females copulate with multiple males within an oestrus, male reproductive success is a function of both pre- and postcopulatory strategies. The relative importance of these overt and covert forms of sexual competition has rarely been assessed in wild populations. The Soay sheep mating system is characterized by male contests for mating opportunities and high female promiscuity. We find that greater horn length, body size and good condition each independently influence a male's ability to monopolize receptive females. For males with large horns at least, this behavioural success translates into greater siring success. Consistent with sperm-competition theory, we also find that larger testes are independently associated with both higher copulation rates and increased siring success. This advantage of larger testes emerges, and strengthens, as the number of oestrous females increases, as dominant males can no longer control access to them all. Our results thus provide direct quantitative evidence that male reproductive success in wild populations of mammals is dependent upon the relative magnitude of both overt contest competition and covert sperm competition.

  17. [Neural mechanisms of facial recognition].

    PubMed

    Nagai, Chiyoko

    2007-01-01

    We review recent researches in neural mechanisms of facial recognition in the light of three aspects: facial discrimination and identification, recognition of facial expressions, and face perception in itself. First, it has been demonstrated that the fusiform gyrus has a main role of facial discrimination and identification. However, whether the FFA (fusiform face area) is really a special area for facial processing or not is controversial; some researchers insist that the FFA is related to 'becoming an expert' for some kinds of visual objects, including faces. Neural mechanisms of prosopagnosia would be deeply concerned to this issue. Second, the amygdala seems to be very concerned to recognition of facial expressions, especially fear. The amygdala, connected with the superior temporal sulcus and the orbitofrontal cortex, appears to operate the cortical function. The amygdala and the superior temporal sulcus are related to gaze recognition, which explains why a patient with bilateral amygdala damage could not recognize only a fear expression; the information from eyes is necessary for fear recognition. Finally, even a newborn infant can recognize a face as a face, which is congruent with the innate hypothesis of facial recognition. Some researchers speculate that the neural basis of such face perception is the subcortical network, comprised of the amygdala, the superior colliculus, and the pulvinar. This network would relate to covert recognition that prosopagnosic patients have.

  18. Probabilistic Open Set Recognition

    NASA Astrophysics Data System (ADS)

    Jain, Lalit Prithviraj

    Real-world tasks in computer vision, pattern recognition and machine learning often touch upon the open set recognition problem: multi-class recognition with incomplete knowledge of the world and many unknown inputs. An obvious way to approach such problems is to develop a recognition system that thresholds probabilities to reject unknown classes. Traditional rejection techniques are not about the unknown; they are about the uncertain boundary and rejection around that boundary. Thus traditional techniques only represent the "known unknowns". However, a proper open set recognition algorithm is needed to reduce the risk from the "unknown unknowns". This dissertation examines this concept and finds existing probabilistic multi-class recognition approaches are ineffective for true open set recognition. We hypothesize the cause is due to weak adhoc assumptions combined with closed-world assumptions made by existing calibration techniques. Intuitively, if we could accurately model just the positive data for any known class without overfitting, we could reject the large set of unknown classes even under this assumption of incomplete class knowledge. For this, we formulate the problem as one of modeling positive training data by invoking statistical extreme value theory (EVT) near the decision boundary of positive data with respect to negative data. We provide a new algorithm called the PI-SVM for estimating the unnormalized posterior probability of class inclusion. This dissertation also introduces a new open set recognition model called Compact Abating Probability (CAP), where the probability of class membership decreases in value (abates) as points move from known data toward open space. We show that CAP models improve open set recognition for multiple algorithms. Leveraging the CAP formulation, we go on to describe the novel Weibull-calibrated SVM (W-SVM) algorithm, which combines the useful properties of statistical EVT for score calibration with one-class and binary

  19. An insight to the dynamics of conserved water molecular triad in IMPDH II (human): recognition of cofactor and substrate to catalytic Arg 322.

    PubMed

    Bairagya, Hridoy R; Mukhopadhyay, Bishnu P; Sekar, K

    2009-10-01

    Inosine 5' monophosphate dehydrogenase (IMPDH II) is a key enzyme involved in the de novo biosynthesis pathway of purine nucleotides and is also considered to be an excellent target for cancer inhibitor design. The conserve R 322 residue (in human) is thought to play some role in the recognition of inhibitor and cofactor through the catalytic D 364 and N 303. The 15 ns simulation and the water dynamics of the three different PDB structures (1B3O, 1NF7, and 1NFB) of human IMPDH by CHARMM force field have clearly indicated the involvement of three conserved water molecules (W(L), W(M), and W(C)) in the recognition of catalytic residues (R 322, D 364, and N 303) to inhibitor and cofactor. Both the guanidine nitrogen atoms (NH1 and NH 2) of the R 322 have anchored the di- and mono-nucleotide (cofactor and inhibitor) binding domains via the conserved W(C) and W(L) water molecules. Another conserved water molecule WM seems to bridge the two domains including the R 322 and also the W(C) and W(L) through seven centers H-bonding coordination. The conserved water molecular triad (W(C)-W(M)-W(L)) in the protein complex may thought to play some important role in the recognition of inhibitor and cofactor to the protein through R 322 residue.

  20. Hemispheric lateralization of linguistic prosody recognition in comparison to speech and speaker recognition.

    PubMed

    Kreitewolf, Jens; Friederici, Angela D; von Kriegstein, Katharina

    2014-11-15

    Hemispheric specialization for linguistic prosody is a controversial issue. While it is commonly assumed that linguistic prosody and emotional prosody are preferentially processed in the right hemisphere, neuropsychological work directly comparing processes of linguistic prosody and emotional prosody suggests a predominant role of the left hemisphere for linguistic prosody processing. Here, we used two functional magnetic resonance imaging (fMRI) experiments to clarify the role of left and right hemispheres in the neural processing of linguistic prosody. In the first experiment, we sought to confirm previous findings showing that linguistic prosody processing compared to other speech-related processes predominantly involves the right hemisphere. Unlike previous studies, we controlled for stimulus influences by employing a prosody and speech task using the same speech material. The second experiment was designed to investigate whether a left-hemispheric involvement in linguistic prosody processing is specific to contrasts between linguistic prosody and emotional prosody or whether it also occurs when linguistic prosody is contrasted against other non-linguistic processes (i.e., speaker recognition). Prosody and speaker tasks were performed on the same stimulus material. In both experiments, linguistic prosody processing was associated with activity in temporal, frontal, parietal and cerebellar regions. Activation in temporo-frontal regions showed differential lateralization depending on whether the control task required recognition of speech or speaker: recognition of linguistic prosody predominantly involved right temporo-frontal areas when it was contrasted against speech recognition; when contrasted against speaker recognition, recognition of linguistic prosody predominantly involved left temporo-frontal areas. The results show that linguistic prosody processing involves functions of both hemispheres and suggest that recognition of linguistic prosody is based on

  1. Recognition memory in developmental prosopagnosia: electrophysiological evidence for abnormal routes to face recognition

    PubMed Central

    Burns, Edwin J.; Tree, Jeremy J.; Weidemann, Christoph T.

    2014-01-01

    Dual process models of recognition memory propose two distinct routes for recognizing a face: recollection and familiarity. Recollection is characterized by the remembering of some contextual detail from a previous encounter with a face whereas familiarity is the feeling of finding a face familiar without any contextual details. The Remember/Know (R/K) paradigm is thought to index the relative contributions of recollection and familiarity to recognition performance. Despite researchers measuring face recognition deficits in developmental prosopagnosia (DP) through a variety of methods, none have considered the distinct contributions of recollection and familiarity to recognition performance. The present study examined recognition memory for faces in eight individuals with DP and a group of controls using an R/K paradigm while recording electroencephalogram (EEG) data at the scalp. Those with DP were found to produce fewer correct “remember” responses and more false alarms than controls. EEG results showed that posterior “remember” old/new effects were delayed and restricted to the right posterior (RP) area in those with DP in comparison to the controls. A posterior “know” old/new effect commonly associated with familiarity for faces was only present in the controls whereas individuals with DP exhibited a frontal “know” old/new effect commonly associated with words, objects and pictures. These results suggest that individuals with DP do not utilize normal face-specific routes when making face recognition judgments but instead process faces using a pathway more commonly associated with objects. PMID:25177283

  2. An Acrobatic Substrate Metamorphosis Reveals a Requirement for Substrate Conformational Dynamics in Trypsin Proteolysis.

    PubMed

    Kayode, Olumide; Wang, Ruiying; Pendlebury, Devon F; Cohen, Itay; Henin, Rachel D; Hockla, Alexandra; Soares, Alexei S; Papo, Niv; Caulfield, Thomas R; Radisky, Evette S

    2016-12-16

    The molecular basis of enzyme catalytic power and specificity derives from dynamic interactions between enzyme and substrate during catalysis. Although considerable effort has been devoted to understanding how conformational dynamics within enzymes affect catalysis, the role of conformational dynamics within protein substrates has not been addressed. Here, we examine the importance of substrate dynamics in the cleavage of Kunitz-bovine pancreatic trypsin inhibitor protease inhibitors by mesotrypsin, finding that the varied conformational dynamics of structurally similar substrates can profoundly impact the rate of catalysis. A 1.4-Å crystal structure of a mesotrypsin-product complex formed with a rapidly cleaved substrate reveals a dramatic conformational change in the substrate upon proteolysis. By using long all-atom molecular dynamics simulations of acyl-enzyme intermediates with proteolysis rates spanning 3 orders of magnitude, we identify global and local dynamic features of substrates on the nanosecond-microsecond time scale that correlate with enzymatic rates and explain differential susceptibility to proteolysis. By integrating multiple enhanced sampling methods for molecular dynamics, we model a viable conformational pathway between substrate-like and product-like states, linking substrate dynamics on the nanosecond-microsecond time scale with large collective substrate motions on the much slower time scale of catalysis. Our findings implicate substrate flexibility as a critical determinant of catalysis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Substrate recognition and catalysis by GH47 α-mannosidases involved in Asn-linked glycan maturation in the mammalian secretory pathway

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

    Xiang, Yong; Karaveg, Khanita; Moremen, Kelley W.

    2016-11-17

    Asn-linked glycosylation of newly synthesized polypeptides occurs in the endoplasmic reticulum of eukaryotic cells. Glycan structures are trimmed and remodeled as they transit the secretory pathway, and processing intermediates play various roles as ligands for folding chaperones and signals for quality control and intracellular transport. Key steps for the generation of these trimmed intermediates are catalyzed by glycoside hydrolase family 47 (GH47) α-mannosidases that selectively cleave α1,2-linked mannose residues. Despite the sequence and structural similarities among the GH47 enzymes, the molecular basis for residue-specific cleavage remains obscure. The present studies reveal enzyme–substrate complex structures for two related GH47 α-mannosidases andmore » provide insights into how these enzymes recognize the same substrates differently and catalyze the complementary glycan trimming reactions necessary for glycan maturation.« less

  4. Shifts in Host Range of a Promiscuous Plasmid through Parallel Evolution of its Replication Initiation Protein

    PubMed Central

    Sota, Masahiro; Yano, Hirokazu; Hughes, Julie; Daughdrill, Gary W.; Abdo, Zaid; Forney, Larry J.; Top, Eva M.

    2011-01-01

    The ability of bacterial plasmids to adapt to novel hosts and thereby shift their host range is key to their long-term persistence in bacterial communities. Promiscuous plasmids of the IncP-1 group can colonize a wide range of hosts, but it is not known if and how they can contract, shift or further expand their host range. To understand the evolutionary mechanisms of host range shifts of IncP-1 plasmids, an IncP-1β mini-replicon was experimentally evolved in four hosts wherein it was initially unstable. After 1000 generations in serial batch cultures under antibiotic selection for plasmid maintenance (kanamycin resistance), the stability of the mini-plasmid had dramatically improved in all coevolved hosts. However, only plasmids evolved in Shewanella oneidensis showed improved stability in the ancestor, indicating that adaptive mutations had occurred in the plasmid itself. Complete genome sequence analysis of nine independently evolved plasmids showed seven unique plasmid genotypes that had various kinds of single mutations at one locus, namely the N-terminal region of the replication initiation protein TrfA. Such parallel evolution indicates that this region was under strong selection. In five of the seven evolved plasmids these trfA mutations resulted in a significantly higher plasmid copy number. Evolved plasmids were found to be stable in four other naïve hosts, but could no longer replicate in Pseudomonas aeruginosa. This study demonstrates that plasmids can specialize to a novel host through trade-offs between improved stability in the new host and the ability to replicate in a previously permissive host. PMID:20520653

  5. Promiscuous activity of the LXR antagonist GSK2033 in a mouse model of fatty liver disease

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

    Griffett, Kristine; Burris, Thomas P., E-mail: burristp@slu.edu

    The liver X receptor (LXR) functions as a receptor for oxysterols and plays a critical role in the regulation of glucose and lipid metabolism. We recently described a synthetic LXR inverse agonist that displayed efficacy in treatment of hepatic steatosis in a mouse model of non-alcoholic fatty liver disease (NAFLD). This compound, SR9238, was designed to display liver specificity so as to avoid potential detrimental effects on reverse cholesterol transport in peripheral tissues. Here, we examined the effects of a LXR antagonist/inverse agonist, GSK2033, which displays systemic exposure. Although GSK2033 performed as expected in cell-based models as a LXR inversemore » agonist, it displayed unexpected activity in the mouse NAFLD model. The expression of lipogenic enzyme genes such as fatty acid synthase and sterol regulatory binding protein 1c were induced rather than suppressed and no effect on hepatic steatosis was found. Further characterization of the specificity of GSK2033 revealed that it displayed a significant degree of promiscuity, targeting a number of other nuclear receptors that could clearly alter hepatic gene expression. - Highlights: • The LXR antagonist GSK2033 suppresses the expression of lipogenic genes FASN and SREBF1 in HepG2 cells. • GSK2033 exhibits sufficient exposure to perform animal experiments targeting the liver. • GSK2033 has fails to suppress hepatic Fasn and Srebf1 expression in an animal model of non-alcoholic fatty liver disease. • GSK2033 may regulate the activity of several nuclear receptors.« less

  6. Escherichia coli twin arginine (Tat) mutant translocases possessing relaxed signal peptide recognition specificities.

    PubMed

    Kreutzenbeck, Peter; Kröger, Carsten; Lausberg, Frank; Blaudeck, Natascha; Sprenger, Georg A; Freudl, Roland

    2007-03-16

    The twin arginine (Tat) secretion pathway allows the translocation of folded proteins across the cytoplasmic membrane of bacteria. Tat-specific signal peptides contain a characteristic amino acid motif ((S/T)RRXFLK) including two highly conserved consecutive arginine residues that are thought to be involved in the recognition of the signal peptides by the Tat translocase. Here, we have analyzed the specificity of Tat signal peptide recognition by using a genetic approach. Replacement of the two arginine residues in a Tat-specific precursor protein by lysine-glutamine resulted in an export-defective mutant precursor that was no longer accepted by the wild-type translocase. Selection for restored export allowed for the isolation of Tat translocases possessing single mutations in either the amino-terminal domain of TatB or the first cytosolic domain of TatC. The mutant Tat translocases still efficiently accepted the unaltered precursor protein, indicating that the substrate specificity of the translocases was not strictly changed; rather, the translocases showed an increased tolerance toward variations of the amino acids occupying the positions of the twin arginine residues in the consensus motif of a Tat signal peptide.

  7. New approach for logo recognition

    NASA Astrophysics Data System (ADS)

    Chen, Jingying; Leung, Maylor K. H.; Gao, Yongsheng

    2000-03-01

    The problem of logo recognition is of great interest in the document domain, especially for document database. By recognizing the logo we obtain semantic information about the document which may be useful in deciding whether or not to analyze the textual components. In order to develop a logo recognition method that is efficient to compute and product intuitively reasonable results, we investigate the Line Segment Hausdorff Distance on logo recognition. Researchers apply Hausdorff Distance to measure the dissimilarity of two point sets. It has been extended to match two sets of line segments. The new approach has the advantage to incorporate structural and spatial information to compute the dissimilarity. The added information can conceptually provide more and better distinctive capability for recognition. The proposed technique has been applied on line segments of logos with encouraging results that support the concept experimentally. This might imply a new way for logo recognition.

  8. Substrate quality alters the microbial mineralization of added substrate and soil organic carbon

    NASA Astrophysics Data System (ADS)

    Jagadamma, S.; Mayes, M. A.; Steinweg, J. M.; Schaeffer, S. M.

    2014-09-01

    The rate and extent of decomposition of soil organic carbon (SOC) is dependent, among other factors, on substrate chemistry and microbial dynamics. Our objectives were to understand the influence of substrate chemistry on microbial decomposition of carbon (C), and to use model fitting to quantify differences in pool sizes and mineralization rates. We conducted an incubation experiment for 270 days using four uniformly labeled 14C substrates (glucose, starch, cinnamic acid and stearic acid) on four different soils (a temperate Mollisol, a tropical Ultisol, a sub-arctic Andisol, and an arctic Gelisol). The 14C labeling enabled us to separate CO2 respired from added substrates and from native SOC. Microbial gene copy numbers were quantified at days 4, 30 and 270 using quantitative polymerase chain reaction (qPCR). Substrate C respiration was always higher for glucose than other substrates. Soils with cinnamic and stearic acid lost more native SOC than glucose- and starch-amended soils. Cinnamic and stearic acid amendments also exhibited higher fungal gene copy numbers at the end of incubation compared to unamended soils. We found that 270 days were sufficient to model the decomposition of simple substrates (glucose and starch) with three pools, but were insufficient for more complex substrates (cinnamic and stearic acid) and native SOC. This study reveals that substrate quality exerts considerable control on the microbial decomposition of newly added and native SOC, and demonstrates the need for multi-year incubation experiments to constrain decomposition parameters for the most recalcitrant fractions of SOC and complex substrates.

  9. An Acrobatic Substrate Metamorphosis Reveals a Requirement for Substrate Conformational Dynamics in Trypsin Proteolysis

    DOE PAGES

    Kayode, Olumide; Wang, Ruiying; Pendlebury, Devon F.; ...

    2016-11-03

    The molecular basis of enzyme catalytic power and specificity derives from dynamic interactions between enzyme and substrate during catalysis. While considerable effort has been devoted to understanding how conformational dynamics within enzymes affect catalysis, the role of conformational dynamics within protein substrates has not been addressed. Here in this paper, we examine the importance of substrate dynamics in the cleavage of Kunitz-BPTI protease inhibitors by mesotrypsin, finding that the varied conformational dynamics of structurally similar substrates can profoundly impact the rate of catalysis. A 1.4 Å crystal structure of a mesotrypsin-product complex formed with a rapidly cleaved substrate reveals amore » dramatic conformational change in the substrate upon proteolysis. Using long all-atom molecular dynamics simulations of acyl-enzyme intermediates with proteolysis rates spanning three orders of magnitude, we identify global and local dynamic features of substrates on the ns-μs timescale that correlate with enzymatic rates and explain differential susceptibility to proteolysis. By integrating multiple enhanced sampling methods for molecular dynamics, we model a viable conformational pathway between substratelike and product-like states, linking substrate dynamics on the ns-μs timescale with large collective substrate motions on the much slower timescale of catalysis. Our findings implicate substrate flexibility as a critical determinant of catalysis.« less

  10. Page Recognition: Quantum Leap In Recognition Technology

    NASA Astrophysics Data System (ADS)

    Miller, Larry

    1989-07-01

    No milestone has proven as elusive as the always-approaching "year of the LAN," but the "year of the scanner" might claim the silver medal. Desktop scanners have been around almost as long as personal computers. And everyone thinks they are used for obvious desktop-publishing and business tasks like scanning business documents, magazine articles and other pages, and translating those words into files your computer understands. But, until now, the reality fell far short of the promise. Because it's true that scanners deliver an accurate image of the page to your computer, but the software to recognize this text has been woefully disappointing. Old optical-character recognition (OCR) software recognized such a limited range of pages as to be virtually useless to real users. (For example, one OCR vendor specified 12-point Courier font from an IBM Selectric typewriter: the same font in 10-point, or from a Diablo printer, was unrecognizable!) Computer dealers have told me the chasm between OCR expectations and reality is so broad and deep that nine out of ten prospects leave their stores in disgust when they learn the limitations. And this is a very important, very unfortunate gap. Because the promise of recognition -- what people want it to do -- carries with it tremendous improvements in our productivity and ability to get tons of written documents into our computers where we can do real work with it. The good news is that a revolutionary new development effort has led to the new technology of "page recognition," which actually does deliver the promise we've always wanted from OCR. I'm sure every reader appreciates the breakthrough represented by the laser printer and page-makeup software, a combination so powerful it created new reasons for buying a computer. A similar breakthrough is happening right now in page recognition: the Macintosh (and, I must admit, other personal computers) equipped with a moderately priced scanner and OmniPage software (from Caere

  11. Bidirectional Modulation of Recognition Memory

    PubMed Central

    Ho, Jonathan W.; Poeta, Devon L.; Jacobson, Tara K.; Zolnik, Timothy A.; Neske, Garrett T.; Connors, Barry W.

    2015-01-01

    Perirhinal cortex (PER) has a well established role in the familiarity-based recognition of individual items and objects. For example, animals and humans with perirhinal damage are unable to distinguish familiar from novel objects in recognition memory tasks. In the normal brain, perirhinal neurons respond to novelty and familiarity by increasing or decreasing firing rates. Recent work also implicates oscillatory activity in the low-beta and low-gamma frequency bands in sensory detection, perception, and recognition. Using optogenetic methods in a spontaneous object exploration (SOR) task, we altered recognition memory performance in rats. In the SOR task, normal rats preferentially explore novel images over familiar ones. We modulated exploratory behavior in this task by optically stimulating channelrhodopsin-expressing perirhinal neurons at various frequencies while rats looked at novel or familiar 2D images. Stimulation at 30–40 Hz during looking caused rats to treat a familiar image as if it were novel by increasing time looking at the image. Stimulation at 30–40 Hz was not effective in increasing exploration of novel images. Stimulation at 10–15 Hz caused animals to treat a novel image as familiar by decreasing time looking at the image, but did not affect looking times for images that were already familiar. We conclude that optical stimulation of PER at different frequencies can alter visual recognition memory bidirectionally. SIGNIFICANCE STATEMENT Recognition of novelty and familiarity are important for learning, memory, and decision making. Perirhinal cortex (PER) has a well established role in the familiarity-based recognition of individual items and objects, but how novelty and familiarity are encoded and transmitted in the brain is not known. Perirhinal neurons respond to novelty and familiarity by changing firing rates, but recent work suggests that brain oscillations may also be important for recognition. In this study, we showed that

  12. Nitrification in a zeoponic substrate

    NASA Technical Reports Server (NTRS)

    McGilloway, R. L.; Weaver, R. W.; Ming, D. W.; Gruener, J. E.

    2003-01-01

    Clinoptilolite is a zeolite mineral with high cation exchange capacity used in zeoponic substrates that have been proposed as a solid medium for growing plants or as a fertilizer material. The kinetics of nitrification has not been measured for NH4+ saturated zeoponic substrate. Experiments were conducted to evaluate the production of NO2- and NO3-, and nitrifier populations in zeoponic substrates. Small columns were filled with zeoponic substrate inoculated with a commercial inoculum or soil enrichment culture of nitrifying bacteria. In addition to column studies, a growth chamber study was conducted to evaluate the kinetics of nitrification in zeoponic substrates used to grow radishes (Raphanus sativus L.). The zeoponic substrate provided a readily available source of NH4+, and nitrifying bacteria were active in the substrate. Ammonium oxidation rates in column studies ranged from 5 to 10 micrograms N g-1 substrate h-1, and NO2- oxidation rates were 2 to 9.5 micrograms N g-1 substrate h-1. Rates determined from the growth chamber study were approximately 1.2 micrograms N g-1 substrate h-1. Quantities of NH4+ oxidized to NO2- and NO3- in inoculated zeoponic substrate were in excess of plant up-take. Acidification as a result of NH4+ oxidation resulted in a pH decline, and the zeoponic substrate showed limited buffering capacity.

  13. Marked Object Recognition Multitouch Screen Printed Touchpad for Interactive Applications

    PubMed Central

    Nunes, Jivago Serrado; Castro, Nelson; Pereira, Nélson; Correia, Vitor; Lanceros-Mendez, Senentxu

    2017-01-01

    The market for interactive platforms is rapidly growing, and touchscreens have been incorporated in an increasing number of devices. Thus, the area of smart objects and devices is strongly increasing by adding interactive touch and multimedia content, leading to new uses and capabilities. In this work, a flexible screen printed sensor matrix is fabricated based on silver ink in a polyethylene terephthalate (PET) substrate. Diamond shaped capacitive electrodes coupled with conventional capacitive reading electronics enables fabrication of a highly functional capacitive touchpad, and also allows for the identification of marked objects. For the latter, the capacitive signatures are identified by intersecting points and distances between them. Thus, this work demonstrates the applicability of a low cost method using royalty-free geometries and technologies for the development of flexible multitouch touchpads for the implementation of interactive and object recognition applications. PMID:29194414

  14. Marked Object Recognition Multitouch Screen Printed Touchpad for Interactive Applications.

    PubMed

    Nunes, Jivago Serrado; Castro, Nelson; Gonçalves, Sergio; Pereira, Nélson; Correia, Vitor; Lanceros-Mendez, Senentxu

    2017-12-01

    The market for interactive platforms is rapidly growing, and touchscreens have been incorporated in an increasing number of devices. Thus, the area of smart objects and devices is strongly increasing by adding interactive touch and multimedia content, leading to new uses and capabilities. In this work, a flexible screen printed sensor matrix is fabricated based on silver ink in a polyethylene terephthalate (PET) substrate. Diamond shaped capacitive electrodes coupled with conventional capacitive reading electronics enables fabrication of a highly functional capacitive touchpad, and also allows for the identification of marked objects. For the latter, the capacitive signatures are identified by intersecting points and distances between them. Thus, this work demonstrates the applicability of a low cost method using royalty-free geometries and technologies for the development of flexible multitouch touchpads for the implementation of interactive and object recognition applications.

  15. Amy63, a novel type of marine bacterial multifunctional enzyme possessing amylase, agarase and carrageenase activities

    PubMed Central

    Liu, Ge; Wu, Shimei; Jin, Weihua; Sun, Chaomin

    2016-01-01

    A multifunctional enzyme is one that performs multiple physiological functions, thus benefiting the organism. Characterization of multifunctional enzymes is important for researchers to understand how organisms adapt to different environmental challenges. In the present study, we report the discovery of a novel multifunctional enzyme Amy63 produced by marine bacterium Vibrio alginolyticus 63. Remarkably, Amy63 possesses amylase, agarase and carrageenase activities. Amy63 is a substrate promiscuous α-amylase, with the substrate priority order of starch, carrageenan and agar. Amy63 maintains considerable amylase, carrageenase and agarase activities and stabilities at wide temperature and pH ranges, and optimum activities are detected at temperature of 60 °C and pH of 6.0, respectively. Moreover, the heteroexpression of Amy63 dramatically enhances the ability of E. coli to degrade starch, carrageenan and agar. Motif searching shows three continuous glycosyl hydrolase 70 (GH70) family homologs existed in Amy63 encoding sequence. Combining serial deletions and phylogenetic analysis of Amy63, the GH70 homologs are proposed as the determinants of enzyme promiscuity. Notably, such enzymes exist in all kingdoms of life, thus providing an expanded perspective on studies of multifunctional enzymes. To our knowledge, this is the first report of an amylase having additional agarase and carrageenase activities. PMID:26725302

  16. Online handwritten mathematical expression recognition

    NASA Astrophysics Data System (ADS)

    Büyükbayrak, Hakan; Yanikoglu, Berrin; Erçil, Aytül

    2007-01-01

    We describe a system for recognizing online, handwritten mathematical expressions. The system is designed with a user-interface for writing scientific articles, supporting the recognition of basic mathematical expressions as well as integrals, summations, matrices etc. A feed-forward neural network recognizes symbols which are assumed to be single-stroke and a recursive algorithm parses the expression by combining neural network output and the structure of the expression. Preliminary results show that writer-dependent recognition rates are very high (99.8%) while writer-independent symbol recognition rates are lower (75%). The interface associated with the proposed system integrates the built-in recognition capabilities of the Microsoft's Tablet PC API for recognizing textual input and supports conversion of hand-drawn figures into PNG format. This enables the user to enter text, mathematics and draw figures in a single interface. After recognition, all output is combined into one LATEX code and compiled into a PDF file.

  17. Using GOMS and Bayesian plan recognition to develop recognition models of operator behavior

    NASA Astrophysics Data System (ADS)

    Zaientz, Jack D.; DeKoven, Elyon; Piegdon, Nicholas; Wood, Scott D.; Huber, Marcus J.

    2006-05-01

    Trends in combat technology research point to an increasing role for uninhabited vehicles in modern warfare tactics. To support increased span of control over these vehicles human responsibilities need to be transformed from tedious, error-prone and cognition intensive operations into tasks that are more supervisory and manageable, even under intensely stressful conditions. The goal is to move away from only supporting human command of low-level system functions to intention-level human-system dialogue about the operator's tasks and situation. A critical element of this process is developing the means to identify when human operators need automated assistance and to identify what assistance they need. Toward this goal, we are developing an unmanned vehicle operator task recognition system that combines work in human behavior modeling and Bayesian plan recognition. Traditionally, human behavior models have been considered generative, meaning they describe all possible valid behaviors. Basing behavior recognition on models designed for behavior generation can offers advantages in improved model fidelity and reuse. It is not clear, however, how to reconcile the structural differences between behavior recognition and behavior modeling approaches. Our current work demonstrates that by pairing a cognitive psychology derived human behavior modeling approach, GOMS, with a Bayesian plan recognition engine, ASPRN, we can translate a behavior generation model into a recognition model. We will discuss the implications for using human performance models in this manner as well as suggest how this kind of modeling may be used to support the real-time control of multiple, uninhabited battlefield vehicles and other semi-autonomous systems.

  18. [Neurological disease and facial recognition].

    PubMed

    Kawamura, Mitsuru; Sugimoto, Azusa; Kobayakawa, Mutsutaka; Tsuruya, Natsuko

    2012-07-01

    To discuss the neurological basis of facial recognition, we present our case reports of impaired recognition and a review of previous literature. First, we present a case of infarction and discuss prosopagnosia, which has had a large impact on face recognition research. From a study of patient symptoms, we assume that prosopagnosia may be caused by unilateral right occipitotemporal lesion and right cerebral dominance of facial recognition. Further, circumscribed lesion and degenerative disease may also cause progressive prosopagnosia. Apperceptive prosopagnosia is observed in patients with posterior cortical atrophy (PCA), pathologically considered as Alzheimer's disease, and associative prosopagnosia in frontotemporal lobar degeneration (FTLD). Second, we discuss face recognition as part of communication. Patients with Parkinson disease show social cognitive impairments, such as difficulty in facial expression recognition and deficits in theory of mind as detected by the reading the mind in the eyes test. Pathological and functional imaging studies indicate that social cognitive impairment in Parkinson disease is possibly related to damages in the amygdalae and surrounding limbic system. The social cognitive deficits can be observed in the early stages of Parkinson disease, and even in the prodromal stage, for example, patients with rapid eye movement (REM) sleep behavior disorder (RBD) show impairment in facial expression recognition. Further, patients with myotonic dystrophy type 1 (DM 1), which is a multisystem disease that mainly affects the muscles, show social cognitive impairment similar to that of Parkinson disease. Our previous study showed that facial expression recognition impairment of DM 1 patients is associated with lesion in the amygdalae and insulae. Our study results indicate that behaviors and personality traits in DM 1 patients, which are revealed by social cognitive impairment, are attributable to dysfunction of the limbic system.

  19. Simple Learned Weighted Sums of Inferior Temporal Neuronal Firing Rates Accurately Predict Human Core Object Recognition Performance

    PubMed Central

    Hong, Ha; Solomon, Ethan A.; DiCarlo, James J.

    2015-01-01

    To go beyond qualitative models of the biological substrate of object recognition, we ask: can a single ventral stream neuronal linking hypothesis quantitatively account for core object recognition performance over a broad range of tasks? We measured human performance in 64 object recognition tests using thousands of challenging images that explore shape similarity and identity preserving object variation. We then used multielectrode arrays to measure neuronal population responses to those same images in visual areas V4 and inferior temporal (IT) cortex of monkeys and simulated V1 population responses. We tested leading candidate linking hypotheses and control hypotheses, each postulating how ventral stream neuronal responses underlie object recognition behavior. Specifically, for each hypothesis, we computed the predicted performance on the 64 tests and compared it with the measured pattern of human performance. All tested hypotheses based on low- and mid-level visually evoked activity (pixels, V1, and V4) were very poor predictors of the human behavioral pattern. However, simple learned weighted sums of distributed average IT firing rates exactly predicted the behavioral pattern. More elaborate linking hypotheses relying on IT trial-by-trial correlational structure, finer IT temporal codes, or ones that strictly respect the known spatial substructures of IT (“face patches”) did not improve predictive power. Although these results do not reject those more elaborate hypotheses, they suggest a simple, sufficient quantitative model: each object recognition task is learned from the spatially distributed mean firing rates (100 ms) of ∼60,000 IT neurons and is executed as a simple weighted sum of those firing rates. SIGNIFICANCE STATEMENT We sought to go beyond qualitative models of visual object recognition and determine whether a single neuronal linking hypothesis can quantitatively account for core object recognition behavior. To achieve this, we designed a

  20. Simple Learned Weighted Sums of Inferior Temporal Neuronal Firing Rates Accurately Predict Human Core Object Recognition Performance.

    PubMed

    Majaj, Najib J; Hong, Ha; Solomon, Ethan A; DiCarlo, James J

    2015-09-30

    To go beyond qualitative models of the biological substrate of object recognition, we ask: can a single ventral stream neuronal linking hypothesis quantitatively account for core object recognition performance over a broad range of tasks? We measured human performance in 64 object recognition tests using thousands of challenging images that explore shape similarity and identity preserving object variation. We then used multielectrode arrays to measure neuronal population responses to those same images in visual areas V4 and inferior temporal (IT) cortex of monkeys and simulated V1 population responses. We tested leading candidate linking hypotheses and control hypotheses, each postulating how ventral stream neuronal responses underlie object recognition behavior. Specifically, for each hypothesis, we computed the predicted performance on the 64 tests and compared it with the measured pattern of human performance. All tested hypotheses based on low- and mid-level visually evoked activity (pixels, V1, and V4) were very poor predictors of the human behavioral pattern. However, simple learned weighted sums of distributed average IT firing rates exactly predicted the behavioral pattern. More elaborate linking hypotheses relying on IT trial-by-trial correlational structure, finer IT temporal codes, or ones that strictly respect the known spatial substructures of IT ("face patches") did not improve predictive power. Although these results do not reject those more elaborate hypotheses, they suggest a simple, sufficient quantitative model: each object recognition task is learned from the spatially distributed mean firing rates (100 ms) of ∼60,000 IT neurons and is executed as a simple weighted sum of those firing rates. Significance statement: We sought to go beyond qualitative models of visual object recognition and determine whether a single neuronal linking hypothesis can quantitatively account for core object recognition behavior. To achieve this, we designed a

  1. Face Recognition From One Example View.

    DTIC Science & Technology

    1995-09-01

    Proceedings, International Workshop on Automatic Face- and Gesture-Recognition, pages 248{253, Zurich, 1995. [32] Yael Moses, Shimon Ullman, and Shimon...recognition. Journal of Cognitive Neuroscience, 3(1):71{86, 1991. [49] Shimon Ullman and Ronen Basri. Recognition by linear combinations of models

  2. Automatic face recognition in HDR imaging

    NASA Astrophysics Data System (ADS)

    Pereira, Manuela; Moreno, Juan-Carlos; Proença, Hugo; Pinheiro, António M. G.

    2014-05-01

    The gaining popularity of the new High Dynamic Range (HDR) imaging systems is raising new privacy issues caused by the methods used for visualization. HDR images require tone mapping methods for an appropriate visualization on conventional and non-expensive LDR displays. These visualization methods might result in completely different visualization raising several issues on privacy intrusion. In fact, some visualization methods result in a perceptual recognition of the individuals, while others do not even show any identity. Although perceptual recognition might be possible, a natural question that can rise is how computer based recognition will perform using tone mapping generated images? In this paper, a study where automatic face recognition using sparse representation is tested with images that result from common tone mapping operators applied to HDR images. Its ability for the face identity recognition is described. Furthermore, typical LDR images are used for the face recognition training.

  3. Pedestal substrate for coated optics

    DOEpatents

    Hale, Layton C.; Malsbury, Terry N.; Patterson, Steven R.

    2001-01-01

    A pedestal optical substrate that simultaneously provides high substrate dynamic stiffness, provides low surface figure sensitivity to mechanical mounting hardware inputs, and constrains surface figure changes caused by optical coatings to be primarily spherical in nature. The pedestal optical substrate includes a disk-like optic or substrate section having a top surface that is coated, a disk-like base section that provides location at which the substrate can be mounted, and a connecting cylindrical section between the base and optics or substrate sections. The connecting cylindrical section may be attached via three spaced legs or members. However, the pedestal optical substrate can be manufactured from a solid piece of material to form a monolith, thus avoiding joints between the sections, or the disk-like base can be formed separately and connected to the connecting section. By way of example, the pedestal optical substrate may be utilized in the fabrication of optics for an extreme ultraviolet (EUV) lithography imaging system, or in any optical system requiring coated optics and substrates with reduced sensitivity to mechanical mounts.

  4. Document recognition serving people with disabilities

    NASA Astrophysics Data System (ADS)

    Fruchterman, James R.

    2007-01-01

    Document recognition advances have improved the lives of people with print disabilities, by providing accessible documents. This invited paper provides perspectives on the author's career progression from document recognition professional to social entrepreneur applying this technology to help people with disabilities. Starting with initial thoughts about optical character recognition in college, it continues with the creation of accurate omnifont character recognition that did not require training. It was difficult to make a reading machine for the blind in a commercial setting, which led to the creation of a nonprofit social enterprise to deliver these devices around the world. This network of people with disabilities scanning books drove the creation of Bookshare.org, an online library of scanned books. Looking forward, the needs for improved document recognition technology to further lower the barriers to reading are discussed. Document recognition professionals should be proud of the positive impact their work has had on some of society's most disadvantaged communities.

  5. Metallic coatings on silicon substrates, and methods of forming metallic coatings on silicon substrates

    DOEpatents

    Branagan, Daniel J [Idaho Falls, ID; Hyde, Timothy A [Idaho Falls, ID; Fincke, James R [Los Alamos, NM

    2008-03-11

    The invention includes methods of forming a metallic coating on a substrate which contains silicon. A metallic glass layer is formed over a silicon surface of the substrate. The invention includes methods of protecting a silicon substrate. The substrate is provided within a deposition chamber along with a deposition target. Material from the deposition target is deposited over at least a portion of the silicon substrate to form a protective layer or structure which contains metallic glass. The metallic glass comprises iron and one or more of B, Si, P and C. The invention includes structures which have a substrate containing silicon and a metallic layer over the substrate. The metallic layer contains less than or equal to about 2 weight % carbon and has a hardness of at least 9.2 GPa. The metallic layer can have an amorphous microstructure or can be devitrified to have a nanocrystalline microstructure.

  6. Infant Visual Recognition Memory

    ERIC Educational Resources Information Center

    Rose, Susan A.; Feldman, Judith F.; Jankowski, Jeffery J.

    2004-01-01

    Visual recognition memory is a robust form of memory that is evident from early infancy, shows pronounced developmental change, and is influenced by many of the same factors that affect adult memory; it is surprisingly resistant to decay and interference. Infant visual recognition memory shows (a) modest reliability, (b) good discriminant…

  7. Mapping protease substrates using a biotinylated phage substrate library.

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

    Scholle, M. D.; Kriplani, U.; Pabon, A.

    We describe a bacteriophage M13 substrate library encoding the AviTag (BirA substrate) and combinatorial heptamer peptides displayed at the N terminus of the mature form of capsid protein III. Phages are biotinylated efficiently (> or = 50%) when grown in E. coli cells coexpressing BirA, and such viral particles can be immobilized on a streptavidin-coated support and released by protease cleavage within the combinatorial peptide. We have used this library to map the specificity of human Factor Xa and a neuropeptidase, neurolysin (EC3.4.24.16). Validation by analysis of isolated peptide substrates has revealed that neurolysin recognizes the motif hydrophobic-X-Pro-Arg-hydrophobic, where Arg-hydrophobicmore » is the scissile bond.« less

  8. Experimental study on GMM-based speaker recognition

    NASA Astrophysics Data System (ADS)

    Ye, Wenxing; Wu, Dapeng; Nucci, Antonio

    2010-04-01

    Speaker recognition plays a very important role in the field of biometric security. In order to improve the recognition performance, many pattern recognition techniques have be explored in the literature. Among these techniques, the Gaussian Mixture Model (GMM) is proved to be an effective statistic model for speaker recognition and is used in most state-of-the-art speaker recognition systems. The GMM is used to represent the 'voice print' of a speaker through modeling the spectral characteristic of speech signals of the speaker. In this paper, we implement a speaker recognition system, which consists of preprocessing, Mel-Frequency Cepstrum Coefficients (MFCCs) based feature extraction, and GMM based classification. We test our system with TIDIGITS data set (325 speakers) and our own recordings of more than 200 speakers; our system achieves 100% correct recognition rate. Moreover, we also test our system under the scenario that training samples are from one language but test samples are from a different language; our system also achieves 100% correct recognition rate, which indicates that our system is language independent.

  9. The development of object recognition memory in rhesus macaques with neonatal lesions of the perirhinal cortex.

    PubMed

    Zeamer, Alyson; Richardson, Rebecca L; Weiss, Alison R; Bachevalier, Jocelyne

    2015-02-01

    To investigate the role of the perirhinal cortex on the development of recognition measured by the visual paired-comparison (VPC) task, infant monkeys with neonatal perirhinal lesions and sham-operated controls were tested at 1.5, 6, 18, and 48 months of age on the VPC task with color stimuli and intermixed delays of 10 s, 30 s, 60 s, and 120 s. Monkeys with neonatal perirhinal lesions showed an increase in novelty preference between 1.5 and 6 months of age similar to controls, although at these two ages, performance remained significantly poorer than that of control animals. With age, performance in animals with neonatal perirhinal lesions deteriorated as compared to that of controls. In contrast to the lack of novelty preference in monkeys with perirhinal lesions acquired in adulthood, novelty preference in the neonatally operated animals remained above chance at all delays and all ages. The data suggest that, although incidental recognition memory processes can be supported by the perirhinal cortex in early infancy, other temporal cortical areas may support these processes in the absence of a functional perirhinal cortex early in development. The neural substrates mediating incidental recognition memory processes appear to be more widespread in early infancy than in adulthood. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  10. Quantum-Limited Image Recognition

    DTIC Science & Technology

    1989-12-01

    J. S. Bomba ,’Alpha-numeric character recognition using local operations,’ Fall Joint Comput. Conf., 218-224 (1959). 53. D. Barnea and H. Silverman...for Chapter 6 1. J. S. Bomba ,’Alpha-numeric character recognition using local operations,’ Fall Joint Comput. Conf., 218-224 (1959). 2. D. Bamea and H

  11. Famous face recognition, face matching, and extraversion.

    PubMed

    Lander, Karen; Poyarekar, Siddhi

    2015-01-01

    It has been previously established that extraverts who are skilled at interpersonal interaction perform significantly better than introverts on a face-specific recognition memory task. In our experiment we further investigate the relationship between extraversion and face recognition, focusing on famous face recognition and face matching. Results indicate that more extraverted individuals perform significantly better on an upright famous face recognition task and show significantly larger face inversion effects. However, our results did not find an effect of extraversion on face matching or inverted famous face recognition.

  12. Kazakh Traditional Dance Gesture Recognition

    NASA Astrophysics Data System (ADS)

    Nussipbekov, A. K.; Amirgaliyev, E. N.; Hahn, Minsoo

    2014-04-01

    Full body gesture recognition is an important and interdisciplinary research field which is widely used in many application spheres including dance gesture recognition. The rapid growth of technology in recent years brought a lot of contribution in this domain. However it is still challenging task. In this paper we implement Kazakh traditional dance gesture recognition. We use Microsoft Kinect camera to obtain human skeleton and depth information. Then we apply tree-structured Bayesian network and Expectation Maximization algorithm with K-means clustering to calculate conditional linear Gaussians for classifying poses. And finally we use Hidden Markov Model to detect dance gestures. Our main contribution is that we extend Kinect skeleton by adding headwear as a new skeleton joint which is calculated from depth image. This novelty allows us to significantly improve the accuracy of head gesture recognition of a dancer which in turn plays considerable role in whole body gesture recognition. Experimental results show the efficiency of the proposed method and that its performance is comparable to the state-of-the-art system performances.

  13. Fine-grained recognition of plants from images.

    PubMed

    Šulc, Milan; Matas, Jiří

    2017-01-01

    Fine-grained recognition of plants from images is a challenging computer vision task, due to the diverse appearance and complex structure of plants, high intra-class variability and small inter-class differences. We review the state-of-the-art and discuss plant recognition tasks, from identification of plants from specific plant organs to general plant recognition "in the wild". We propose texture analysis and deep learning methods for different plant recognition tasks. The methods are evaluated and compared them to the state-of-the-art. Texture analysis is only applied to images with unambiguous segmentation (bark and leaf recognition), whereas CNNs are only applied when sufficiently large datasets are available. The results provide an insight in the complexity of different plant recognition tasks. The proposed methods outperform the state-of-the-art in leaf and bark classification and achieve very competitive results in plant recognition "in the wild". The results suggest that recognition of segmented leaves is practically a solved problem, when high volumes of training data are available. The generality and higher capacity of state-of-the-art CNNs makes them suitable for plant recognition "in the wild" where the views on plant organs or plants vary significantly and the difficulty is increased by occlusions and background clutter.

  14. A rodent model for the study of invariant visual object recognition

    PubMed Central

    Zoccolan, Davide; Oertelt, Nadja; DiCarlo, James J.; Cox, David D.

    2009-01-01

    The human visual system is able to recognize objects despite tremendous variation in their appearance on the retina resulting from variation in view, size, lighting, etc. This ability—known as “invariant” object recognition—is central to visual perception, yet its computational underpinnings are poorly understood. Traditionally, nonhuman primates have been the animal model-of-choice for investigating the neuronal substrates of invariant recognition, because their visual systems closely mirror our own. Meanwhile, simpler and more accessible animal models such as rodents have been largely overlooked as possible models of higher-level visual functions, because their brains are often assumed to lack advanced visual processing machinery. As a result, little is known about rodents' ability to process complex visual stimuli in the face of real-world image variation. In the present work, we show that rats possess more advanced visual abilities than previously appreciated. Specifically, we trained pigmented rats to perform a visual task that required them to recognize objects despite substantial variation in their appearance, due to changes in size, view, and lighting. Critically, rats were able to spontaneously generalize to previously unseen transformations of learned objects. These results provide the first systematic evidence for invariant object recognition in rats and argue for an increased focus on rodents as models for studying high-level visual processing. PMID:19429704

  15. Evaporation of liquid droplets on solid substrates. II. Periodic substrates with moving contact lines

    NASA Astrophysics Data System (ADS)

    Amini, Amirhossein; Homsy, G. M.

    2017-04-01

    Experiments on evaporating droplets on structured surfaces have shown that the contact line does not move with constant speed, but rather in a steplike "stick-slip" fashion. As a first step in understanding such behavior, we study the evaporation of a two-dimensional volatile liquid droplet on a nonplanar heated solid substrate with a moving contact line and fixed contact angle. The model for the flat case is adapted to include curved substrates, numerical solutions are achieved for various periodic and quasiperiodic substrate profiles, and the dynamics of the contact line and the apparent contact angle are studied. In contrast with our results for a flat substrate, for which the contact line recedes in a nearly constant speed, we observe that the contact line speed and position show significant time variation and that the contact line moves in an approximate steplike fashion on relatively steep substrates. For the simplest case of a periodic substrate, we find that the apparent contact angle is periodic in time. For doubly periodic substrates, we find that the apparent contact angle is periodic and that the problem exhibits a phase-locking behavior. For multimode quasiperiodic substrates, we find the contact line behavior to be temporally complex and not only limited to a stick-slip motion. In all cases, we find that the overall evaporation is increased relative to the flat substrate.

  16. Recovery of EUVL substrates

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

    Vernon, S.P.; Baker, S.L.

    1995-01-19

    Mo/Si multilayers, were removed from superpolished zerodur and fused silica substrates with a dry etching process that, under suitable processing conditions, produces negligible change in either the substrate surface figure or surface roughness. Full recovery of the initial normal incidence extreme ultra-violet (EUV) reflectance response has been demonstrated on reprocessed substrates.

  17. Transfer-Appropriate Processing in Recognition Memory: Perceptual and Conceptual Effects on Recognition Memory Depend on Task Demands

    ERIC Educational Resources Information Center

    Parks, Colleen M.

    2013-01-01

    Research examining the importance of surface-level information to familiarity in recognition memory tasks is mixed: Sometimes it affects recognition and sometimes it does not. One potential explanation of the inconsistent findings comes from the ideas of dual process theory of recognition and the transfer-appropriate processing framework, which…

  18. Automated cassette-to-cassette substrate handling system

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

    Kraus, Joseph Arthur; Boyer, Jeremy James; Mack, Joseph

    2014-03-18

    An automated cassette-to-cassette substrate handling system includes a cassette storage module for storing a plurality of substrates in cassettes before and after processing. A substrate carrier storage module stores a plurality of substrate carriers. A substrate carrier loading/unloading module loads substrates from the cassette storage module onto the plurality of substrate carriers and unloads substrates from the plurality of substrate carriers to the cassette storage module. A transport mechanism transports the plurality of substrates between the cassette storage module and the plurality of substrate carriers and transports the plurality of substrate carriers between the substrate carrier loading/unloading module and amore » processing chamber. A vision system recognizes recesses in the plurality of substrate carriers corresponding to empty substrate positions in the substrate carrier. A processor receives data from the vision system and instructs the transport mechanism to transport substrates to positions on the substrate carrier in response to the received data.« less

  19. Brain catechol-o-methyltransferase (COMT) inhibition by tolcapone counteracts recognition memory deficits in normal and chronic phencyclidine-treated rats and in COMT-Val transgenic mice

    PubMed Central

    Detrait, E.R.; Carr, G.V.; Ferraille, S.; Weinberger, D.R.; Lamberty, Y.

    2015-01-01

    The critical involvement of dopamine in cognitive processes has been well established, suggesting therapies targeting dopamine metabolism may alleviate cognitive dysfunction. COMT is a catecholamine-degrading enzyme, the substrates of which include dopamine, epinephrine, and norepinephrine. The present work illustrates the potential therapeutic efficacy of COMT inhibition for alleviating cognitive impairment. A brain penetrant COMT inhibitor, tolcapone, was tested in normal and phencyclidine (PCP)-treated rats and COMT–Val transgenic mice. In a Novel Object Recognition (NOR) procedure, tolcapone counteracted a 24h-dependent forgetting of a familiar object and counteracted PCP-induced recognition deficits in the rats at doses ranging from 7.5 to 30 mg/kg. In contrast, entacapone, a COMT inhibitor which does not readily cross the blood-brain barrier failed to show efficacy at doses up to 30mg/kg. Tolcapone at a dose of 30 mg/kg also improved NOR performance in the transgenic mice, which showed clear recognition deficits. Complementing earlier studies, our results indicate that central inhibition of COMT positively impacts recognition memory processes and might constitute an appealing treatment for cognitive dysfunction related to neuropsychiatric disorders. PMID:26919286

  20. Relevance feedback-based building recognition

    NASA Astrophysics Data System (ADS)

    Li, Jing; Allinson, Nigel M.

    2010-07-01

    Building recognition is a nontrivial task in computer vision research which can be utilized in robot localization, mobile navigation, etc. However, existing building recognition systems usually encounter the following two problems: 1) extracted low level features cannot reveal the true semantic concepts; and 2) they usually involve high dimensional data which require heavy computational costs and memory. Relevance feedback (RF), widely applied in multimedia information retrieval, is able to bridge the gap between the low level visual features and high level concepts; while dimensionality reduction methods can mitigate the high-dimensional problem. In this paper, we propose a building recognition scheme which integrates the RF and subspace learning algorithms. Experimental results undertaken on our own building database show that the newly proposed scheme appreciably enhances the recognition accuracy.