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  1. Structure of the Hantavirus Nucleoprotein Provides Insights into the Mechanism of RNA Encapsidation.

    PubMed

    Olal, Daniel; Daumke, Oliver

    2016-03-08

    Hantaviruses are etiological agents of life-threatening hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome. The nucleoprotein (N) of hantavirus is essential for viral transcription and replication, thus representing an attractive target for therapeutic intervention. We have determined the crystal structure of hantavirus N to 3.2 Å resolution. The structure reveals a two-lobed, mostly α-helical structure that is distantly related to that of orthobunyavirus Ns. A basic RNA binding pocket is located at the intersection between the two lobes. We provide evidence that oligomerization is mediated by amino- and C-terminal arms that bind to the adjacent monomers. Based on these findings, we suggest a model for the oligomeric ribonucleoprotein (RNP) complex. Our structure provides mechanistic insights into RNA encapsidation in the genus Hantavirus and constitutes a template for drug discovery efforts aimed at combating hantavirus infections.

  2. Crystal structure of Streptococcus pneumoniae pneumolysin provides key insights into early steps of pore formation

    PubMed Central

    Lawrence, Sara L.; Feil, Susanne C.; Morton, Craig J.; Farrand, Allison J.; Mulhern, Terrence D.; Gorman, Michael A.; Wade, Kristin R.; Tweten, Rodney K.; Parker, Michael W.

    2015-01-01

    Pore-forming proteins are weapons often used by bacterial pathogens to breach the membrane barrier of target cells. Despite their critical role in infection important structural aspects of the mechanism of how these proteins assemble into pores remain unknown. Streptococcus pneumoniae is the world’s leading cause of pneumonia, meningitis, bacteremia and otitis media. Pneumolysin (PLY) is a major virulence factor of S. pneumoniae and a target for both small molecule drug development and vaccines. PLY is a member of the cholesterol-dependent cytolysins (CDCs), a family of pore-forming toxins that form gigantic pores in cell membranes. Here we present the structure of PLY determined by X-ray crystallography and, in solution, by small-angle X-ray scattering. The crystal structure reveals PLY assembles as a linear oligomer that provides key structural insights into the poorly understood early monomer-monomer interactions of CDCs at the membrane surface. PMID:26403197

  3. Structure of sulfamidase provides insight into the molecular pathology of mucopolysaccharidosis IIIA

    SciTech Connect

    Sidhu, Navdeep S.; Schreiber, Kathrin; Pröpper, Kevin; Becker, Stefan; Usón, Isabel; Sheldrick, George M.; Gärtner, Jutta; Krätzner, Ralph Steinfeld, Robert

    2014-05-01

    Mucopolysaccharidosis IIIA is a fatal neurodegenerative disease that typically manifests itself in childhood and is caused by mutations in the gene for the lysosomal enzyme sulfamidase. The first structure of this enzyme is presented, which provides insight into the molecular basis of disease-causing mutations, and the enzymatic mechanism is proposed. Mucopolysaccharidosis type IIIA (Sanfilippo A syndrome), a fatal childhood-onset neurodegenerative disease with mild facial, visceral and skeletal abnormalities, is caused by an inherited deficiency of the enzyme N-sulfoglucosamine sulfohydrolase (SGSH; sulfamidase). More than 100 mutations in the SGSH gene have been found to reduce or eliminate its enzymatic activity. However, the molecular understanding of the effect of these mutations has been confined by a lack of structural data for this enzyme. Here, the crystal structure of glycosylated SGSH is presented at 2 Å resolution. Despite the low sequence identity between this unique N-sulfatase and the group of O-sulfatases, they share a similar overall fold and active-site architecture, including a catalytic formylglycine, a divalent metal-binding site and a sulfate-binding site. However, a highly conserved lysine in O-sulfatases is replaced in SGSH by an arginine (Arg282) that is positioned to bind the N-linked sulfate substrate. The structure also provides insight into the diverse effects of pathogenic mutations on SGSH function in mucopolysaccharidosis type IIIA and convincing evidence for the molecular consequences of many missense mutations. Further, the molecular characterization of SGSH mutations will lay the groundwork for the development of structure-based drug design for this devastating neurodegenerative disorder.

  4. Biophysical studies suggest a new structural arrangement of crotoxin and provide insights into its toxic mechanism.

    PubMed

    Fernandes, Carlos A H; Pazin, Wallance M; Dreyer, Thiago R; Bicev, Renata N; Cavalcante, Walter L G; Fortes-Dias, Consuelo L; Ito, Amando S; Oliveira, Cristiano L P; Fernandez, Roberto Morato; Fontes, Marcos R M

    2017-03-03

    Crotoxin (CTX) is the main neurotoxin found in Crotalus durissus rattlesnake venoms being composed by a nontoxic and non-enzymatic component (CA) and a toxic phospholipase A2 (CB). Previous crystallographic structures of CTX and CB provided relevant insights: (i) CTX structure showed a 1:1 molecular ratio between CA and CB, presenting three tryptophan residues in the CA/CB interface and one exposed to solvent; (ii) CB structure displayed a tetrameric conformation. This study aims to provide further information on the CTX mechanism of action by several biophysical methods. Our data show that isolated CB can in fact form tetramers in solution; however, these tetramers can be dissociated by CA titration. Furthermore, CTX exhibits a strong reduction in fluorescence intensity and lifetime compared with isolated CA and CB, suggesting that all tryptophan residues in CTX may be hidden by the CA/CB interface. By companying spectroscopy fluorescence and SAXS data, we obtained a new structural model for the CTX heterodimer in which all tryptophans are located in the interface, and the N-terminal region of CB is largely exposed to the solvent. Based on this model, we propose a toxic mechanism of action for CTX, involving the interaction of N-terminal region of CB with the target before CA dissociation.

  5. Biophysical studies suggest a new structural arrangement of crotoxin and provide insights into its toxic mechanism

    PubMed Central

    Fernandes, Carlos A. H.; Pazin, Wallance M.; Dreyer, Thiago R.; Bicev, Renata N.; Cavalcante, Walter L. G.; Fortes-Dias, Consuelo L.; Ito, Amando S.; Oliveira, Cristiano L. P.; Fernandez, Roberto Morato; Fontes, Marcos R. M.

    2017-01-01

    Crotoxin (CTX) is the main neurotoxin found in Crotalus durissus rattlesnake venoms being composed by a nontoxic and non-enzymatic component (CA) and a toxic phospholipase A2 (CB). Previous crystallographic structures of CTX and CB provided relevant insights: (i) CTX structure showed a 1:1 molecular ratio between CA and CB, presenting three tryptophan residues in the CA/CB interface and one exposed to solvent; (ii) CB structure displayed a tetrameric conformation. This study aims to provide further information on the CTX mechanism of action by several biophysical methods. Our data show that isolated CB can in fact form tetramers in solution; however, these tetramers can be dissociated by CA titration. Furthermore, CTX exhibits a strong reduction in fluorescence intensity and lifetime compared with isolated CA and CB, suggesting that all tryptophan residues in CTX may be hidden by the CA/CB interface. By companying spectroscopy fluorescence and SAXS data, we obtained a new structural model for the CTX heterodimer in which all tryptophans are located in the interface, and the N-terminal region of CB is largely exposed to the solvent. Based on this model, we propose a toxic mechanism of action for CTX, involving the interaction of N-terminal region of CB with the target before CA dissociation. PMID:28256632

  6. Structures of parasite calreticulins provide insights into their flexibility and dual carbohydrate/peptide-binding properties

    PubMed Central

    Moreau, Christophe; Cioci, Gianluca; Iannello, Marina; Laffly, Emmanuelle; Chouquet, Anne; Ferreira, Arturo; Thielens, Nicole M.; Gaboriaud, Christine

    2016-01-01

    Calreticulin (CRT) is a multifaceted protein, initially discovered as an endoplasmic reticulum (ER) chaperone protein, that is essential in calcium metabolism. Various implications in cancer, early development and immunology have been discovered more recently for CRT, as well as its role as a dominant ‘eat-me’ prophagocytic signal. Intriguingly, cell-surface exposure/secretion of CRT is among the infective strategies used by parasites such as Trypanosoma cruzi, Entamoeba histolytica, Taenia solium, Leishmania donovani and Schistosoma mansoni. Because of the inherent flexibility of CRTs, their analysis by X-ray crystallography requires the design of recombinant constructs suitable for crystallization, and thus only the structures of two very similar mammalian CRT lectin domains are known. With the X-ray structures of two distant parasite CRTs, insights into species structural determinants that might be harnessed to fight against the parasites without affecting the functions of the host CRT are now provided. Moreover, although the hypothesis that CRT can exhibit both open and closed conformations has been proposed in relation to its chaperone function, only the open conformation has so far been observed in crystal structures. The first evidence is now provided of a complex conformational transition with the junction reoriented towards P-domain closure. SAXS experiments also provided additional information about the flexibility of T. cruzi CRT in solution, thus complementing crystallographic data on the open conformation. Finally, regarding the conserved lectin-domain structure and chaperone function, evidence is provided of its dual carbohydrate/protein specificity and a new scheme is proposed to interpret such unusual substrate-binding properties. These fascinating features are fully consistent with previous experimental observations, as discussed considering the broad spectrum of CRT sequence conservations and differences. PMID:27840680

  7. Crystal structure of the Epithiospecifier Protein, ESP from Arabidopsis thaliana provides insights into its product specificity.

    PubMed

    Zhang, Weiwei; Wang, Wenhe; Liu, Zihe; Xie, Yongchao; Wang, Hao; Mu, Yajuan; Huang, Yao; Feng, Yue

    2016-09-16

    Specifier proteins are important components of the glucosinolate-myrosinase system, which mediate plant defense against herbivory and pathogen attacks. Upon tissue disruption, glucosinolates are hydrolyzed to instable aglucones by myrosinases, and then aglucones will rearrange to form defensive isothiocyanates. Specifier proteins can redirect this reaction to form other products, such as simple nitriles, epithionitriles and organic thiocyanates instead of isothiocyanates based on the side chain structure of glucosinolate and the type of the specifier proteins. Nevertheless, the molecular mechanism underlying the different product spectrums of various specifier proteins was not fully understood. Here in this study, we solved the crystal structure of the Epithiospecifier Protein, ESP from Arabidopsis thaliana (AtESP) at 2.3 Å resolution. Structural comparisons with the previously solved structure of thiocyanate forming protein, TFP from Thlaspi arvense (TaTFP) reveal that AtESP shows a dimerization pattern different from TaTFP. Moreover, AtESP harbors a slightly larger active site pocket than TaTFP and several residues around the active site are different between the two proteins, which might account for the different product spectrums of the two proteins. Together, our structural study provides important insights into the molecular mechanisms of specifier proteins and shed light on the basis of their different product spectrums.

  8. Structure of sulfamidase provides insight into the molecular pathology of mucopolysaccharidosis IIIA.

    PubMed

    Sidhu, Navdeep S; Schreiber, Kathrin; Pröpper, Kevin; Becker, Stefan; Usón, Isabel; Sheldrick, George M; Gärtner, Jutta; Krätzner, Ralph; Steinfeld, Robert

    2014-05-01

    Mucopolysaccharidosis type IIIA (Sanfilippo A syndrome), a fatal childhood-onset neurodegenerative disease with mild facial, visceral and skeletal abnormalities, is caused by an inherited deficiency of the enzyme N-sulfoglucosamine sulfohydrolase (SGSH; sulfamidase). More than 100 mutations in the SGSH gene have been found to reduce or eliminate its enzymatic activity. However, the molecular understanding of the effect of these mutations has been confined by a lack of structural data for this enzyme. Here, the crystal structure of glycosylated SGSH is presented at 2 Å resolution. Despite the low sequence identity between this unique N-sulfatase and the group of O-sulfatases, they share a similar overall fold and active-site architecture, including a catalytic formylglycine, a divalent metal-binding site and a sulfate-binding site. However, a highly conserved lysine in O-sulfatases is replaced in SGSH by an arginine (Arg282) that is positioned to bind the N-linked sulfate substrate. The structure also provides insight into the diverse effects of pathogenic mutations on SGSH function in mucopolysaccharidosis type IIIA and convincing evidence for the molecular consequences of many missense mutations. Further, the molecular characterization of SGSH mutations will lay the groundwork for the development of structure-based drug design for this devastating neurodegenerative disorder.

  9. Crystal structure of Manduca sexta prophenoloxidase provides insights into the mechanism of type 3 copper enzymes

    SciTech Connect

    Li, Yongchao; Wang, Yang; Jiang, Haobo; Deng, Junpeng

    2010-02-22

    Arthropod phenoloxidase (PO) generates quinones and other toxic compounds to sequester and kill pathogens during innate immune responses. It is also involved in wound healing and other physiological processes. Insect PO is activated from its inactive precursor, prophenoloxidase (PPO), by specific proteolysis via a serine protease cascade. Here, we report the crystal structure of PPO from a lepidopteran insect at a resolution of 1.97 {angstrom}, which is the initial structure for a PPO from the type 3 copper protein family. Manduca sexta PPO is a heterodimer consisting of 2 homologous polypeptide chains, PPO1 and PPO2. The active site of each subunit contains a canonical type 3 di-nuclear copper center, with each copper ion coordinated with 3 structurally conserved histidines. The acidic residue Glu-395 located at the active site of PPO2 may serve as a general base for deprotonation of monophenolic substrates, which is key to the ortho-hydroxylase activity of PO. The structure provides unique insights into the mechanism by which type 3 copper proteins differ in their enzymatic activities, albeit sharing a common active center. A drastic change in electrostatic surface induced on cleavage at Arg-51 allows us to propose a model for localized PPO activation in insects.

  10. Human acid sphingomyelinase structures provide insight to molecular basis of Niemann–Pick disease

    PubMed Central

    Zhou, Yan-Feng; Metcalf, Matthew C.; Garman, Scott C.; Edmunds, Tim; Qiu, Huawei; Wei, Ronnie R.

    2016-01-01

    Acid sphingomyelinase (ASM) hydrolyzes sphingomyelin to ceramide and phosphocholine, essential components of myelin in neurons. Genetic alterations in ASM lead to ASM deficiency (ASMD) and have been linked to Niemann–Pick disease types A and B. Olipudase alfa, a recombinant form of human ASM, is being developed as enzyme replacement therapy to treat the non-neurological manifestations of ASMD. Here we present the human ASM holoenzyme and product bound structures encompassing all of the functional domains. The catalytic domain has a metallophosphatase fold, and two zinc ions and one reaction product phosphocholine are identified in a histidine-rich active site. The structures reveal the underlying catalytic mechanism, in which two zinc ions activate a water molecule for nucleophilic attack of the phosphodiester bond. Docking of sphingomyelin provides a model that allows insight into the selectivity of the enzyme and how the ASM domains collaborate to complete hydrolysis. Mapping of known mutations provides a basic understanding on correlations between enzyme dysfunction and phenotypes observed in ASMD patients. PMID:27725636

  11. Human acid sphingomyelinase structures provide insight to molecular basis of Niemann–Pick disease

    SciTech Connect

    Zhou, Yan-Feng; Metcalf, Matthew C.; Garman, Scott C.; Edmunds, Tim; Qiu, Huawei; Wei, Ronnie R.

    2016-10-26

    Acid sphingomyelinase (ASM) hydrolyzes sphingomyelin to ceramide and phosphocholine, essential components of myelin in neurons. Genetic alterations in ASM lead to ASM deficiency (ASMD) and have been linked to Niemann–Pick disease types A and B. Olipudase alfa, a recombinant form of human ASM, is being developed as enzyme replacement therapy to treat the non-neurological manifestations of ASMD. Here we present the human ASM holoenzyme and product bound structures encompassing all of the functional domains. The catalytic domain has a metallophosphatase fold, and two zinc ions and one reaction product phosphocholine are identified in a histidine-rich active site. The structures reveal the underlying catalytic mechanism, in which two zinc ions activate a water molecule for nucleophilic attack of the phosphodiester bond. Docking of sphingomyelin provides a model that allows insight into the selectivity of the enzyme and how the ASM domains collaborate to complete hydrolysis. Mapping of known mutations provides a basic understanding on correlations between enzyme dysfunction and phenotypes observed in ASMD patients.

  12. Crystal structures of enterovirus 71 (EV71) recombinant virus particles provide insights into vaccine design.

    PubMed

    Lyu, Ke; Wang, Guang-Chuan; He, Ya-Ling; Han, Jian-Feng; Ye, Qing; Qin, Cheng-Feng; Chen, Rong

    2015-02-06

    Hand-foot-and-mouth disease (HFMD) remains a major health concern in the Asia-Pacific regions, and its major causative agents include human enterovirus 71 (EV71) and coxsackievirus A16. A desirable vaccine against HFMD would be multivalent and able to elicit protective responses against multiple HFMD causative agents. Previously, we have demonstrated that a thermostable recombinant EV71 vaccine candidate can be produced by the insertion of a foreign peptide into the BC loop of VP1 without affecting viral replication. Here we present crystal structures of two different naturally occurring empty particles, one from a clinical C4 strain EV71 and the other from its recombinant virus containing an insertion in the VP1 BC loop. Crystal structure analysis demonstrated that the inserted foreign peptide is well exposed on the particle surface without significant structural changes in the capsid. Importantly, such insertions do not seem to affect the virus uncoating process as illustrated by the conformational similarity between an uncoating intermediate of another recombinant virus and that of EV71. Especially, at least 18 residues from the N terminus of VP1 are transiently externalized. Altogether, our study provides insights into vaccine development against HFMD.

  13. Structure-function analysis of peroxidasin provides insight into the mechanism of collagen IV crosslinking.

    PubMed

    Lázár, Enikő; Péterfi, Zalán; Sirokmány, Gábor; Kovács, Hajnal A; Klement, Eva; Medzihradszky, Katalin F; Geiszt, Miklós

    2015-06-01

    Basement membranes provide structural support and convey regulatory signals to cells in diverse tissues. Assembly of collagen IV into a sheet-like network is a fundamental mechanism during the formation of basement membranes. Peroxidasin (PXDN) was recently described to catalyze crosslinking of collagen IV through the formation of sulfilimine bonds. Despite the significance of this pathway in tissue genesis, our understanding of PXDN function is far from complete. In this work we demonstrate that collagen IV crosslinking is a physiological function of mammalian PXDN. Moreover, we carried out structure-function analysis of PXDN to gain a better insight into its role in collagen IV synthesis. We identify conserved cysteines in PXDN that mediate the oligomerization of the protein into a trimeric complex. We also demonstrate that oligomerization is not an absolute requirement for enzymatic activity, but optimal collagen IV coupling is only catalyzed by the PXDN trimers. Localization experiments of different PXDN mutants in two different cell models revealed that PXDN oligomers, but not monomers, adhere on the cell surface in "hot spots," which represent previously unknown locations of collagen IV crosslinking.

  14. Crystal structures of nitric oxide reductases provide key insights into functional conversion of respiratory enzymes.

    PubMed

    Tosha, Takehiko; Shiro, Yoshitsugu

    2013-03-01

    Respiration is an essential biological process to get bioenergy, ATP, for all kingdoms of life. Cytochrome c oxidase (COX) plays central role in aerobic respiration, catalyzing the reduction of O(2) coupled with pumping proton across the biological membrane. Nitric oxide reductase (NOR) involved in anaerobic nitrate respiration is suggested to be evolutionary related to COX and share the same progenitor with COX, on the basis of the amino acid sequence homology. Contrary to COX, NOR catalyzes the reduction of nitric oxide and shows no proton pumping ability. Thus, the respiratory enzyme acquires (or loses) proton pumping ability in addition to the conversion of the catalytic property along with the environmental change on earth. Recently, we solved the structures of two types of NORs, which provides novel insights into the functional conversion of the respiratory enzymes. In this review, we focus on the structural similarities and differences between COXs and NORs and discuss possible mechanism for the functional conversion of these enzymes during molecular evolution.

  15. Trehalulose synthase native and carbohydrate complexed structures provide insights into sucrose isomerization.

    PubMed

    Ravaud, Stéphanie; Robert, Xavier; Watzlawick, Hildegard; Haser, Richard; Mattes, Ralf; Aghajari, Nushin

    2007-09-21

    Various diseases related to the overconsumption of sugar make a growing need for sugar substitutes. Because sucrose is an inexpensive and readily available d-glucose donor, the industrial potential for enzymatic synthesis of the sucrose isomers trehalulose and/or isomaltulose from sucrose is large. The product specificity of sucrose isomerases that catalyze this reaction depends essentially on the possibility for tautomerization of sucrose, which is required for trehalulose formation. For optimal use of the enzyme, targeting controlled synthesis of these functional isomers, it is necessary to minimize the side reactions. This requires an extensive analysis of substrate binding modes and of the specificity-determining sites in the structure. The 1.6-2.2-A resolution three-dimensional structures of native and mutant complexes of a trehalulose synthase from Pseudomonas mesoacidophila MX-45 mimic successive states of the enzyme reaction. Combined with mutagenesis studies they give for the first time thorough insights into substrate recognition and processing and reaction specificities of these enzymes. Among the important outcomes of this study is the revelation of an aromatic clamp defined by Phe(256) and Phe(280) playing an essential role in substrate recognition and in controlling the reaction specificity, which is further supported by mutagenesis studies. Furthermore, this study highlights essential residues for binding the glucosyl and fructosyl moieties. The introduction of subtle changes informed by comparative three-dimensional structural data observed within our study can lead to fundamental modifications in the mode of action of sucrose isomerases and hence provide a template for industrial catalysts.

  16. Crystal Structure of Human Myotubularin-Related Protein 1 Provides Insight into the Structural Basis of Substrate Specificity.

    PubMed

    Bong, Seoung Min; Son, Kka-bi; Yang, Seung-Won; Park, Jae-Won; Cho, Jea-Won; Kim, Kyung-Tae; Kim, Hackyoung; Kim, Seung Jun; Kim, Young Jun; Lee, Byung Il

    2016-01-01

    Myotubularin-related protein 1 (MTMR1) is a phosphatase that belongs to the tyrosine/dual-specificity phosphatase superfamily. MTMR1 has been shown to use phosphatidylinositol 3-monophosphate (PI(3)P) and/or phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) as substrates. Here, we determined the crystal structure of human MTMR1. The refined model consists of the Pleckstrin homology (PH)-GRAM and phosphatase (PTP) domains. The overall structure was highly similar to the previously reported MTMR2 structure. Interestingly, two phosphate molecules were coordinated by strictly conserved residues located in the C(X)5R motif of the active site. Additionally, our biochemical studies confirmed the substrate specificity of MTMR1 for PI(3)P and PI(3,5)P2 over other phosphatidylinositol phosphates. Our structural and enzymatic analyses provide insight into the catalytic mechanism and biochemical properties of MTMR1.

  17. Structural studies of Pseudomonas and Chromobacterium ω-aminotransferases provide insights into their differing substrate specificity

    SciTech Connect

    Sayer, Christopher; Isupov, Michail N.; Westlake, Aaron; Littlechild, Jennifer A.

    2013-04-01

    The X-ray structures of two ω-aminotransferases from P. aeruginosa and C. violaceum in complex with an inhibitor offer the first detailed insight into the structural basis of the substrate specificity of these industrially important enzymes. The crystal structures and inhibitor complexes of two industrially important ω-aminotransferase enzymes from Pseudomonas aeruginosa and Chromobacterium violaceum have been determined in order to understand the differences in their substrate specificity. The two enzymes share 30% sequence identity and use the same amino acceptor, pyruvate; however, the Pseudomonas enzyme shows activity towards the amino donor β-alanine, whilst the Chromobacterium enzyme does not. Both enzymes show activity towards S-α-methylbenzylamine (MBA), with the Chromobacterium enzyme having a broader substrate range. The crystal structure of the P. aeruginosa enzyme has been solved in the holo form and with the inhibitor gabaculine bound. The C. violaceum enzyme has been solved in the apo and holo forms and with gabaculine bound. The structures of the holo forms of both enzymes are quite similar. There is little conformational difference observed between the inhibitor complex and the holoenzyme for the P. aeruginosa aminotransferase. In comparison, the crystal structure of the C. violaceum gabaculine complex shows significant structural rearrangements from the structures of both the apo and holo forms of the enzyme. It appears that the different rigidity of the protein scaffold contributes to the substrate specificity observed for the two ω-aminotransferases.

  18. Lactone-bound structures of cyclohexanone monooxygenase provide insight into the stereochemistry of catalysis.

    PubMed

    Yachnin, Brahm J; McEvoy, Michelle B; MacCuish, Roderick J D; Morley, Krista L; Lau, Peter C K; Berghuis, Albert M

    2014-12-19

    The Baeyer-Villiger monooxygenases (BVMOs) are microbial enzymes that catalyze the synthetically useful Baeyer-Villiger oxidation reaction. The available BVMO crystal structures all lack a substrate or product bound in a position that would determine the substrate specificity and stereospecificity of the enzyme. Here, we report two crystal structures of cyclohexanone monooxygenase (CHMO) with its product, ε-caprolactone, bound: the CHMO(Tight) and CHMO(Loose) structures. The CHMO(Tight) structure represents the enzyme state in which substrate acceptance and stereospecificity is determined, providing a foundation for engineering BVMOs with altered substrate spectra and/or stereospecificity. The CHMO(Loose) structure is the first structure where the product is solvent accessible. This structure represents the enzyme state upon binding and release of the substrate and product. In addition, the role of the invariant Arg329 in chaperoning the substrate/product during the catalytic cycle is highlighted. Overall, these data provide a structural framework for the engineering of BVMOs with altered substrate spectra and/or stereospecificity.

  19. Structure of a Bimodular Botulinum Neurotoxin Complex Provides Insights into Its Oral Toxicity

    PubMed Central

    Jin, Lei; Le, Thi Tuc Nghi; Cheng, Luisa W.; Strotmeier, Jasmin; Kruel, Anna Magdalena; Yao, Guorui; Perry, Kay; Rummel, Andreas; Jin, Rongsheng

    2013-01-01

    Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum and cause the fatal disease botulism, a flaccid paralysis of the muscle. BoNTs are released together with several auxiliary proteins as progenitor toxin complexes (PTCs) to become highly potent oral poisons. Here, we report the structure of a ∼760 kDa 14-subunit large PTC of serotype A (L-PTC/A) and reveal insight into its absorption mechanism. Using a combination of X-ray crystallography, electron microscopy, and functional studies, we found that L-PTC/A consists of two structurally and functionally independent sub-complexes. A hetero-dimeric 290 kDa complex protects BoNT, while a hetero-dodecameric 470 kDa complex facilitates its absorption in the harsh environment of the gastrointestinal tract. BoNT absorption is mediated by nine glycan-binding sites on the dodecameric sub-complex that forms multivalent interactions with carbohydrate receptors on intestinal epithelial cells. We identified monosaccharides that blocked oral BoNT intoxication in mice, which suggests a new strategy for the development of preventive countermeasures for BoNTs based on carbohydrate receptor mimicry. PMID:24130488

  20. Crystal Structures of the Histidine Acid Phosphatase from Francisella tularensis Provide Insight into Substrate Recognition

    SciTech Connect

    Singh, Harkewal; Felts, Richard L.; Schuermann, Jonathan P.; Reilly, Thomas J.; Tanner, John J.

    2009-12-01

    Histidine acid phosphatases catalyze the transfer of a phosphoryl group from phosphomonoesters to water at acidic pH using an active-site histidine. The histidine acid phosphatase from the category A pathogen Francisella tularensis (FtHAP) has been implicated in intramacrophage survival and virulence, motivating interest in understanding the structure and mechanism of this enzyme. Here, we report a structure-based study of ligand recognition by FtHAP. The 1.70-{angstrom}-resolution structure of FtHAP complexed with the competitive inhibitor L(+)-tartrate was solved using single-wavelength anomalous diffraction phasing. Structures of the ligand-free enzyme and the complex with inorganic phosphate were determined at resolutions of 1.85 and 1.70 {angstrom}, respectively. The structure of the Asp261Ala mutant enzyme complexed with the substrate 3'-AMP was determined at 1.50 {angstrom} resolution to gain insight into substrate recognition. FtHAP exhibits a two-domain fold similar to that of human prostatic acid phosphatase, consisting of an {alpha}/{beta} core domain and a smaller domain that caps the core domain. The structures show that the core domain supplies the phosphoryl binding site, catalytic histidine (His17), and an aspartic acid residue (Asp261) that protonates the leaving group, while the cap domain contributes residues that enforce substrate preference. FtHAP and human prostatic acid phosphatase differ in the orientation of the crucial first helix of the cap domain, implying differences in the substrate preferences of the two enzymes. 3'-AMP binds in one end of a 15-{angstrom}-long tunnel, with the adenine clamped between Phe23 and Tyr135, and the ribose 2'-hydroxyl interacting with Gln132. The importance of the clamp is confirmed with site-directed mutagenesis; mutation of Phe23 and Tyr135 individually to Ala increases K{sub m} by factors of 7 and 10, respectively. The structural data are consistent with a role for FtHAP in scavenging phosphate from small

  1. Crystal Structures of Phosphite Dehydrogenase Provide Insights into Nicotinamide Cofactor Regeneration

    SciTech Connect

    Zou, Yaozhong; Zhang, Houjin; Brunzelle, Joseph S.; Johannes, Tyler W.; Woodyer, Ryan; Hung, John E.; Nair, Nikhil; van der Donk, Wilfred A.; Zhao, Huimin; Nair, Satish K.

    2012-08-21

    The enzyme phosphite dehydrogenase (PTDH) catalyzes the NAD{sup +}-dependent conversion of phosphite to phosphate and represents the first biological catalyst that has been shown to conduct the enzymatic oxidation of phosphorus. Despite investigation for more than a decade into both the mechanism of its unusual reaction and its utility in cofactor regeneration, there has been a lack of any structural data for PTDH. Here we present the cocrystal structure of an engineered thermostable variant of PTDH bound to NAD{sup +} (1.7 {angstrom} resolution), as well as four other cocrystal structures of thermostable PTDH and its variants with different ligands (all between 1.85 and 2.3 {angstrom} resolution). These structures provide a molecular framework for understanding prior mutational analysis and point to additional residues, located in the active site, that may contribute to the enzymatic activity of this highly unusual catalyst.

  2. Structures of the four subfamilies of phosphodiesterase-4 provide insight into the selectivity of their inhibitors.

    PubMed

    Wang, Huanchen; Peng, Ming-Sheng; Chen, Yi; Geng, Jie; Robinson, Howard; Houslay, Miles D; Cai, Jiwen; Ke, Hengming

    2007-12-01

    PDE4 (phosphodiesterase-4)-selective inhibitors have attracted much attention as potential therapeutics for the treatment of both depression and major inflammatory diseases, but their practical application has been compromised by side effects. A possible cause for the side effects is that current PDE4-selective inhibitors similarly inhibit isoforms from all four PDE4 subfamilies. The development of PDE4 subfamily-selective inhibitors has been hampered by a lack of structural information. In the present study, we rectify this by providing the crystal structures of the catalytic domains of PDE4A, PDE4B and PDE4D in complex with the PDE4 inhibitor NVP {4-[8-(3-nitrophenyl)-[1,7]naphthyridin-6-yl]benzoic acid} as well as the unliganded PDE4C structure. NVP binds in the same conformation to the deep cAMP substrate pocket and interacts with the same residues in each instance. However, detailed structural comparison reveals significant conformational differences. Although the active sites of PDE4B and PDE4D are mostly comparable, PDE4A shows significant displacements of the residues next to the invariant glutamine residue that is critical for substrate and inhibitor binding. PDE4C appears to be more distal from other PDE4 subfamilies, with certain key residues being disordered. Our analyses provide the first structural basis for the development of PDE4 subfamily-selective inhibitors.

  3. Structures of the Four Subfamilies of Phosphodiesterase-4 Provide Insight into the Selectivity of Their Inhibitors

    SciTech Connect

    Wang, H.; Peng, M; Chen , Y; Geng, J; Robinson, H; Houslay , M; Cai, J; Ke, H

    2007-01-01

    PDE4 (phosphodiesterase-4)-selective inhibitors have attracted much attention as potential therapeutics for the treatment of both depression and major inflammatory diseases, but their practical application has been compromised by side effects. A possible cause for the side effects is that current PDE4-selective inhibitors similarly inhibit isoforms from all four PDE4 subfamilies. The development of PDE4 subfamily-selective inhibitors has been hampered by a lack of structural information. In the present study, we rectify this by providing the crystal structures of the catalytic domains of PDE4A, PDE4B and PDE4D in complex with the PDE4 inhibitor NVP 4-[8-(3-nitrophenyl)-[1,7]naphthyridin-6-yl]benzoic acid as well as the unliganded PDE4C structure. NVP binds in the same conformation to the deep cAMP substrate pocket and interacts with the same residues in each instance. However, detailed structural comparison reveals significant conformational differences. Although the active sites of PDE4B and PDE4D are mostly comparable, PDE4A shows significant displacements of the residues next to the invariant glutamine residue that is critical for substrate and inhibitor binding. PDE4C appears to be more distal from other PDE4 subfamilies, with certain key residues being disordered. Our analyses provide the first structural basis for the development of PDE4 subfamily-selective inhibitors.

  4. Genomic organization of the crested ibis MHC provides new insight into ancestral avian MHC structure

    PubMed Central

    Chen, Li-Cheng; Lan, Hong; Sun, Li; Deng, Yan-Li; Tang, Ke-Yi; Wan, Qiu-Hong

    2015-01-01

    The major histocompatibility complex (MHC) plays an important role in immune response. Avian MHCs are not well characterized, only reporting highly compact Galliformes MHCs and extensively fragmented zebra finch MHC. We report the first genomic structure of an endangered Pelecaniformes (crested ibis) MHC containing 54 genes in three regions spanning ~500 kb. In contrast to the loose BG (26 loci within 265 kb) and Class I (11 within 150) genomic structures, the Core Region is condensed (17 within 85). Furthermore, this Region exhibits a COL11A2 gene, followed by four tandem MHC class II αβ dyads retaining two suites of anciently duplicated “αβ” lineages. Thus, the crested ibis MHC structure is entirely different from the known avian MHC architectures but similar to that of mammalian MHCs, suggesting that the fundamental structure of ancestral avian class II MHCs should be “COL11A2-IIαβ1-IIαβ2.” The gene structures, residue characteristics, and expression levels of the five class I genes reveal inter-locus functional divergence. However, phylogenetic analysis indicates that these five genes generate a well-supported intra-species clade, showing evidence for recent duplications. Our analyses suggest dramatic structural variation among avian MHC lineages, help elucidate avian MHC evolution, and provide a foundation for future conservation studies. PMID:25608659

  5. Crystal Structure of the Herpesvirus Nuclear Egress Complex Provides Insights into Inner Nuclear Membrane Remodeling.

    PubMed

    Zeev-Ben-Mordehai, Tzviya; Weberruß, Marion; Lorenz, Michael; Cheleski, Juliana; Hellberg, Teresa; Whittle, Cathy; El Omari, Kamel; Vasishtan, Daven; Dent, Kyle C; Harlos, Karl; Franzke, Kati; Hagen, Christoph; Klupp, Barbara G; Antonin, Wolfram; Mettenleiter, Thomas C; Grünewald, Kay

    2015-12-29

    Although nucleo-cytoplasmic transport is typically mediated through nuclear pore complexes, herpesvirus capsids exit the nucleus via a unique vesicular pathway. Together, the conserved herpesvirus proteins pUL31 and pUL34 form the heterodimeric nuclear egress complex (NEC), which, in turn, mediates the formation of tight-fitting membrane vesicles around capsids at the inner nuclear membrane. Here, we present the crystal structure of the pseudorabies virus NEC. The structure revealed that a zinc finger motif in pUL31 and an extensive interaction network between the two proteins stabilize the complex. Comprehensive mutational analyses, characterized both in situ and in vitro, indicated that the interaction network is not redundant but rather complementary. Fitting of the NEC crystal structure into the recently determined cryoEM-derived hexagonal lattice, formed in situ by pUL31 and pUL34, provided details on the molecular basis of NEC coat formation and inner nuclear membrane remodeling.

  6. Crystal structure of class III chitinase from pomegranate provides the insight into its metal storage capacity.

    PubMed

    Masuda, Taro; Zhao, Guanghua; Mikami, Bunzo

    2015-01-01

    Chitinase hydrolyzes the β-1,4-glycosidic bond in chitin. In higher plants, this enzyme has been regarded as a pathogenesis-related protein. Recently, we identified a class III chitinase, which functions as a calcium storage protein in pomegranate (Punica granatum) seed (PSC, pomegranate seed chitinase). Here, we solved a crystal structure of PSC at 1.6 Å resolution. Although its overall structure, including the structure of catalytic site and non-proline cis-peptides, was closely similar to those of other class III chitinases, PSC had some unique structural characteristics. First, there were some metal-binding sites with coordinated water molecules on the surface of PSC. Second, many unconserved aspartate residues were present in the PSC sequence which rendered the surface of PSC negatively charged. This acidic electrostatic property is in contrast to that of hevamine, well-characterized plant class III chitinase, which has rather a positively charged surface. Thus, the crystal structure provides a clue for metal association property of PSC.

  7. Stable isotopes provide insight into population structure and segregation in eastern North Atlantic sperm whales.

    PubMed

    Borrell, Asunción; Velásquez Vacca, Adriana; Pinela, Ana M; Kinze, Carl; Lockyer, Christina H; Vighi, Morgana; Aguilar, Alex

    2013-01-01

    In pelagic species inhabiting large oceans, genetic differentiation tends to be mild and populations devoid of structure. However, large cetaceans have provided many examples of structuring. Here we investigate whether the sperm whale, a pelagic species with large population sizes and reputedly highly mobile, shows indication of structuring in the eastern North Atlantic, an ocean basin in which a single population is believed to occur. To do so, we examined stable isotope values in sequential growth layer groups of teeth from individuals sampled in Denmark and NW Spain. In each layer we measured oxygen- isotope ratios (δ(18)O) in the inorganic component (hydroxyapatite), and nitrogen and carbon isotope ratios (δ(15)N: δ(13)C) in the organic component (primarily collagenous). We found significant differences between Denmark and NW Spain in δ(15)N and δ(18)O values in the layer deposited at age 3, considered to be the one best representing the baseline of the breeding ground, in δ(15)N, δ(13)C and δ(18)O values in the period up to age 20, and in the ontogenetic variation of δ(15)N and δ(18)O values. These differences evidence that diet composition, use of habitat and/or migratory destinations are dissimilar between whales from the two regions and suggest that the North Atlantic population of sperm whales is more structured than traditionally accepted.

  8. Crystal Structure of the Herpesvirus Nuclear Egress Complex Provides Insights into Inner Nuclear Membrane Remodeling

    PubMed Central

    Zeev-Ben-Mordehai, Tzviya; Weberruß, Marion; Lorenz, Michael; Cheleski, Juliana; Hellberg, Teresa; Whittle, Cathy; El Omari, Kamel; Vasishtan, Daven; Dent, Kyle C.; Harlos, Karl; Franzke, Kati; Hagen, Christoph; Klupp, Barbara G.; Antonin, Wolfram; Mettenleiter, Thomas C.; Grünewald, Kay

    2015-01-01

    Summary Although nucleo-cytoplasmic transport is typically mediated through nuclear pore complexes, herpesvirus capsids exit the nucleus via a unique vesicular pathway. Together, the conserved herpesvirus proteins pUL31 and pUL34 form the heterodimeric nuclear egress complex (NEC), which, in turn, mediates the formation of tight-fitting membrane vesicles around capsids at the inner nuclear membrane. Here, we present the crystal structure of the pseudorabies virus NEC. The structure revealed that a zinc finger motif in pUL31 and an extensive interaction network between the two proteins stabilize the complex. Comprehensive mutational analyses, characterized both in situ and in vitro, indicated that the interaction network is not redundant but rather complementary. Fitting of the NEC crystal structure into the recently determined cryoEM-derived hexagonal lattice, formed in situ by pUL31 and pUL34, provided details on the molecular basis of NEC coat formation and inner nuclear membrane remodeling. PMID:26711332

  9. A Near-Atomic Structure of the Dark Apoptosome Provides Insight into Assembly and Activation.

    PubMed

    Cheng, Tat Cheung; Akey, Ildikó V; Yuan, Shujun; Yu, Zhiheng; Ludtke, Steven J; Akey, Christopher W

    2017-01-03

    In Drosophila, the Apaf-1-related killer (Dark) forms an apoptosome that activates procaspases. To investigate function, we have determined a near-atomic structure of Dark double rings using cryo-electron microscopy. We then built a nearly complete model of the apoptosome that includes 7- and 8-blade β-propellers. We find that the preference for dATP during Dark assembly may be governed by Ser325, which is in close proximity to the 2' carbon of the deoxyribose ring. Interestingly, β-propellers in V-shaped domains of the Dark apoptosome are more widely separated, relative to these features in the Apaf-1 apoptosome. This wider spacing may be responsible for the lack of cytochrome c binding to β-propellers in the Dark apoptosome. Our structure also highlights the roles of two loss-of-function mutations that may block Dark assembly. Finally, the improved model provides a framework to understand apical procaspase activation in the intrinsic cell death pathway.

  10. Crystal structure of Anoxybacillus α-amylase provides insights into maltose binding of a new glycosyl hydrolase subclass

    PubMed Central

    Chai, Kian Piaw; Othman, Noor Farhan Binti; Teh, Aik-Hong; Ho, Kok Lian; Chan, Kok-Gan; Shamsir, Mohd Shahir; Goh, Kian Mau; Ng, Chyan Leong

    2016-01-01

    A new subfamily of glycosyl hydrolase family GH13 was recently proposed for α-amylases from Anoxybacillus species (ASKA and ADTA), Geobacillus thermoleovorans (GTA, Pizzo, and GtamyII), Bacillus aquimaris (BaqA), and 95 other putative protein homologues. To understand this new GH13 subfamily, we report crystal structures of truncated ASKA (TASKA). ASKA is a thermostable enzyme capable of producing high levels of maltose. Unlike GTA, biochemical analysis showed that Ca2+ ion supplementation enhances the catalytic activities of ASKA and TASKA. The crystal structures reveal the presence of four Ca2+ ion binding sites, with three of these binding sites are highly conserved among Anoxybacillus α-amylases. This work provides structural insights into this new GH13 subfamily both in the apo form and in complex with maltose. Furthermore, structural comparison of TASKA and GTA provides an overview of the conformational changes accompanying maltose binding at each subsite. PMID:26975884

  11. Crystal structure of Anoxybacillus α-amylase provides insights into maltose binding of a new glycosyl hydrolase subclass.

    PubMed

    Chai, Kian Piaw; Othman, Noor Farhan Binti; Teh, Aik-Hong; Ho, Kok Lian; Chan, Kok-Gan; Shamsir, Mohd Shahir; Goh, Kian Mau; Ng, Chyan Leong

    2016-03-15

    A new subfamily of glycosyl hydrolase family GH13 was recently proposed for α-amylases from Anoxybacillus species (ASKA and ADTA), Geobacillus thermoleovorans (GTA, Pizzo, and GtamyII), Bacillus aquimaris (BaqA), and 95 other putative protein homologues. To understand this new GH13 subfamily, we report crystal structures of truncated ASKA (TASKA). ASKA is a thermostable enzyme capable of producing high levels of maltose. Unlike GTA, biochemical analysis showed that Ca(2+) ion supplementation enhances the catalytic activities of ASKA and TASKA. The crystal structures reveal the presence of four Ca(2+) ion binding sites, with three of these binding sites are highly conserved among Anoxybacillus α-amylases. This work provides structural insights into this new GH13 subfamily both in the apo form and in complex with maltose. Furthermore, structural comparison of TASKA and GTA provides an overview of the conformational changes accompanying maltose binding at each subsite.

  12. Geographic variation in the structure of oak hybrid zones provides insights into the dynamics of speciation.

    PubMed

    Zeng, Yan-Fei; Liao, Wan-Jin; Petit, Rémy J; Zhang, Da-Yong

    2011-12-01

    Studying geographic variation in the rate of hybridization between closely related species could provide a useful window on the evolution of reproductive isolation. Reinforcement theory predicts greater prezygotic isolation in areas of prolonged contact between recently diverged species than in areas of recent contact, which implies that old contact zones would be dominated by parental phenotypes with few hybrids (bimodal hybrid zones), whereas recent contact zones would be characterized by hybrid swarms (unimodal hybrid zones). Here, we investigate how the hybrid zones of two closely related Chinese oaks, Quercus mongolica and Q. liaotungensis, are structured geographically using both nuclear and chloroplast markers. We found that populations of Q. liaotungensis located around the Changbai Mountains in Northeast China, an inferred glacial refugium, were introgressed by genes from Q. mongolica, suggesting historical contact between the two species in this region. However, these introgressed populations form sharp bimodal hybrid zones with Q. mongolica. In contrast, populations of Q. liaotungensis located in North China, which show no sign of ancient introgression with Q. mongolica, form unimodal hybrid zones with Q. mongolica. These results are consistent with the hypothesis that selection against hybrids has had sufficient time to reinforce the reproductive barriers between Q. liaotungensis and Q. mongolica in Northeast China but not in North China.

  13. Quantitative analysis of glycerol in dicarboxylic acid-rich cutins provides insights into Arabidopsis cutin structure.

    PubMed

    Yang, Weili; Pollard, Mike; Li-Beisson, Yonghua; Ohlrogge, John

    2016-10-01

    Cutin is an extracellular lipid polymer that contributes to protective cuticle barrier functions against biotic and abiotic stresses in land plants. Glycerol has been reported as a component of cutin, contributing up to 14% by weight of total released monomers. Previous studies using partial hydrolysis of cuticle-enriched preparations established the presence of oligomers with glycerol-aliphatic ester links. Furthermore, glycerol-3-phosphate 2-O-acyltransferases (sn-2-GPATs) are essential for cutin biosynthesis. However, precise roles of glycerol in cutin assembly and structure remain uncertain. Here, a stable isotope-dilution assay was developed for the quantitative analysis of glycerol by GC/MS of triacetin with simultaneous determination of aliphatic monomers. To provide clues about the role of glycerol in dicarboxylic acid (DCA)-rich cutins, this methodology was applied to compare wild-type (WT) Arabidopsis cutin with a series of mutants that are defective in cutin synthesis. The molar ratio of glycerol to total DCAs in WT cutins was 2:1. Even when allowing for a small additional contribution from hydroxy fatty acids, this is a substantially higher glycerol to aliphatic monomer ratio than previously reported for any cutin. Glycerol content was strongly reduced in both stem and leaf cutin from all Arabidopsis mutants analyzed (gpat4/gpat8, att1-2 and lacs2-3). In addition, the molar reduction of glycerol was proportional to the molar reduction of total DCAs. These results suggest "glycerol-DCA-glycerol" may be the dominant motif in DCA-rich cutins. The ramifications and caveats for this hypothesis are presented.

  14. Retrieving Backbone String Neighbors Provides Insights Into Structural Modeling of Membrane Proteins*

    PubMed Central

    Sun, Jiang-Ming; Li, Tong-Hua; Cong, Pei-Sheng; Tang, Sheng-Nan; Xiong, Wen-Wei

    2012-01-01

    Identification of protein structural neighbors to a query is fundamental in structure and function prediction. Here we present BS-align, a systematic method to retrieve backbone string neighbors from primary sequences as templates for protein modeling. The backbone conformation of a protein is represented by the backbone string, as defined in Ramachandran space. The backbone string of a query can be accurately predicted by two innovative technologies: a knowledge-driven sequence alignment and encoding of a backbone string element profile. Then, the predicted backbone string is employed to align against a backbone string database and retrieve a set of backbone string neighbors. The backbone string neighbors were shown to be close to native structures of query proteins. BS-align was successfully employed to predict models of 10 membrane proteins with lengths ranging between 229 and 595 residues, and whose high-resolution structural determinations were difficult to elucidate both by experiment and prediction. The obtained TM-scores and root mean square deviations of the models confirmed that the models based on the backbone string neighbors retrieved by the BS-align were very close to the native membrane structures although the query and the neighbor shared a very low sequence identity. The backbone string system represents a new road for the prediction of protein structure from sequence, and suggests that the similarity of the backbone string would be more informative than describing a protein as belonging to a fold. PMID:22415040

  15. Structural studies of Pseudomonas and Chromobacterium ω-aminotransferases provide insights into their differing substrate specificity.

    PubMed

    Sayer, Christopher; Isupov, Michail N; Westlake, Aaron; Littlechild, Jennifer A

    2013-04-01

    The crystal structures and inhibitor complexes of two industrially important ω-aminotransferase enzymes from Pseudomonas aeruginosa and Chromobacterium violaceum have been determined in order to understand the differences in their substrate specificity. The two enzymes share 30% sequence identity and use the same amino acceptor, pyruvate; however, the Pseudomonas enzyme shows activity towards the amino donor β-alanine, whilst the Chromobacterium enzyme does not. Both enzymes show activity towards S-α-methylbenzylamine (MBA), with the Chromobacterium enzyme having a broader substrate range. The crystal structure of the P. aeruginosa enzyme has been solved in the holo form and with the inhibitor gabaculine bound. The C. violaceum enzyme has been solved in the apo and holo forms and with gabaculine bound. The structures of the holo forms of both enzymes are quite similar. There is little conformational difference observed between the inhibitor complex and the holoenzyme for the P. aeruginosa aminotransferase. In comparison, the crystal structure of the C. violaceum gabaculine complex shows significant structural rearrangements from the structures of both the apo and holo forms of the enzyme. It appears that the different rigidity of the protein scaffold contributes to the substrate specificity observed for the two ω-aminotransferases.

  16. Structural studies of Pseudomonas and Chromobacterium ω-aminotransferases provide insights into their differing substrate specificity

    PubMed Central

    Sayer, Christopher; Isupov, Michail N.; Westlake, Aaron; Littlechild, Jennifer A.

    2013-01-01

    The crystal structures and inhibitor complexes of two industrially important ω-aminotransferase enzymes from Pseudomonas aeruginosa and Chromobacterium violaceum have been determined in order to understand the differences in their substrate specificity. The two enzymes share 30% sequence identity and use the same amino acceptor, pyruvate; however, the Pseudomonas enzyme shows activity towards the amino donor β-alanine, whilst the Chromobacterium enzyme does not. Both enzymes show activity towards S-α-methylbenzylamine (MBA), with the Chromobacterium enzyme having a broader substrate range. The crystal structure of the P. aeruginosa enzyme has been solved in the holo form and with the inhibitor gabaculine bound. The C. violaceum enzyme has been solved in the apo and holo forms and with gabaculine bound. The structures of the holo forms of both enzymes are quite similar. There is little conformational difference observed between the inhibitor complex and the holoenzyme for the P. aeruginosa aminotransferase. In comparison, the crystal structure of the C. violaceum gabaculine complex shows significant structural rearrangements from the structures of both the apo and holo forms of the enzyme. It appears that the different rigidity of the protein scaffold contributes to the substrate specificity observed for the two ω-­aminotransferases. PMID:23519665

  17. Studies on cattle genomic structural variation provide insights into ruminant speciation and adaptation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genomic structural variations, including segmental duplications (SD) and copy number variations (CNV), contribute significantly to individual health and disease in primates and rodents. As a part of the bovine genome annotation effort, we performed the first genome-wide analysis of SD in cattle usin...

  18. Structure of a bimodular botulinum neurotoxin complex provides insights into its oral toxicity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Botulinum neurotoxins (BoNTs) are highly potent oral poisons produced by Clostridium botulinum. BoNTs are secreted along with several auxiliary proteins forming progenitor toxin complexes (PTC). Here, we report the structure of a ~760 kDa 14-subunit PTC using a combination of X-ray crystallography a...

  19. Structure of an asymmetric ternary protein complex provides insight for membrane interaction.

    PubMed

    Dempsey, Brian R; Rezvanpour, Atoosa; Lee, Ting-Wai; Barber, Kathryn R; Junop, Murray S; Shaw, Gary S

    2012-10-10

    Plasma membrane repair involves the coordinated effort of proteins and the inner phospholipid surface to mend the rupture and return the cell back to homeostasis. Here, we present the three-dimensional structure of a multiprotein complex that includes S100A10, annexin A2, and AHNAK, which along with dysferlin, functions in muscle and cardiac tissue repair. The 3.5 Å resolution X-ray structure shows that a single region from the AHNAK C terminus is recruited by an S100A10-annexin A2 heterotetramer, forming an asymmetric ternary complex. The AHNAK peptide adopts a coil conformation that arches across the heterotetramer contacting both annexin A2 and S100A10 protomers with tight affinity (∼30 nM) and establishing a structural rationale whereby both S100A10 and annexin proteins are needed in AHNAK recruitment. The structure evokes a model whereby AHNAK is targeted to the membrane surface through sandwiching of the binding region between the S100A10/annexin A2 complex and the phospholipid membrane.

  20. A high-resolution structure that provides insight into coiled-coil thiodepsipeptide dynamic chemistry.

    PubMed

    Dadon, Zehavit; Samiappan, Manickasundaram; Shahar, Anat; Zarivach, Raz; Ashkenasy, Gonen

    2013-09-16

    Stable and reactive: A crystal structure at 1.35 Å of a thioester coiled-coil protein reveals high similarity to all-peptide-bond proteins. In these assemblies, the thioester bonds are kept reactive towards thiol molecules in the mixture. This enables efficient domain exchange between proteins in response to changes in folding conditions or introduction of external templates.

  1. Huygens provides insights about Titan

    NASA Astrophysics Data System (ADS)

    Showstack, Randy

    2005-01-01

    Huygens provides insights about Titan Following the Huygens probe's successful 14 January soft landing on Titan, Saturn's largest moon, scientists at a 21 January European Space Agency (ESA) news briefing announced that the moon has Earth-like meteorology and geology, and that there is evidence for liquid methane. Martin Tomasko, principal investigator for the Huygens Descent Imager-Spectral Radiometer, said, ``Geological evidence for precipitation, erosion, mechanical abrasion and other fluvial activity says that the physical processes shaping Titan are much the same as those shaping Earth.''

  2. Structure of Ljungan virus provides insight into genome packaging of this picornavirus

    NASA Astrophysics Data System (ADS)

    Zhu, Ling; Wang, Xiangxi; Ren, Jingshan; Porta, Claudine; Wenham, Hannah; Ekström, Jens-Ola; Panjwani, Anusha; Knowles, Nick J.; Kotecha, Abhay; Siebert, C. Alistair; Lindberg, A. Michael; Fry, Elizabeth E.; Rao, Zihe; Tuthill, Tobias J.; Stuart, David I.

    2015-10-01

    Picornaviruses are responsible for a range of human and animal diseases, but how their RNA genome is packaged remains poorly understood. A particularly poorly studied group within this family are those that lack the internal coat protein, VP4. Here we report the atomic structure of one such virus, Ljungan virus, the type member of the genus Parechovirus B, which has been linked to diabetes and myocarditis in humans. The 3.78-Å resolution cryo-electron microscopy structure shows remarkable features, including an extended VP1 C terminus, forming a major protuberance on the outer surface of the virus, and a basic motif at the N terminus of VP3, binding to which orders some 12% of the viral genome. This apparently charge-driven RNA attachment suggests that this branch of the picornaviruses uses a different mechanism of genome encapsidation, perhaps explored early in the evolution of picornaviruses.

  3. Crystal structures of highly simplified BPTIs provide insights into hydration-driven increase of unfolding enthalpy.

    PubMed

    Islam, Mohammad Monirul; Yohda, Masafumi; Kidokoro, Shun-Ichi; Kuroda, Yutaka

    2017-03-07

    We report a thermodynamic and structural analysis of six extensively simplified bovine pancreatic trypsin inhibitor (BPTI) variants containing 19-24 alanines out of 58 residues. Differential scanning calorimetry indicated a two-state thermal unfolding, typical of a native protein with densely packed interior. Surprisingly, increasing the number of alanines induced enthalpy stabilization, which was however over-compensated by entropy destabilization. X-ray crystallography indicated that the alanine substitutions caused the recruitment of novel water molecules facilitating the formation of protein-water hydrogen bonds and improving the hydration shells around the alanine's methyl groups, both of which presumably contributed to enthalpy stabilization. There was a strong correlation between the number of water molecules and the thermodynamic parameters. Overall, our results demonstrate that, in contrast to our initial expectation, a protein sequence in which over 40% of the residues are alanines can retain a densely packed structure and undergo thermal denaturation with a large enthalpy change, mainly contributed by hydration.

  4. A Structural Model for a Self-Assembled Nanotube Provides Insight into Its Exciton Dynamics

    PubMed Central

    2016-01-01

    The design and synthesis of functional self-assembled nanostructures is frequently an empirical process fraught with critical knowledge gaps about atomic-level structure in these noncovalent systems. Here, we report a structural model for a semiconductor nanotube formed via the self-assembly of naphthalenediimide-lysine (NDI-Lys) building blocks determined using experimental 13C–13C and 13C–15N distance restraints from solid-state nuclear magnetic resonance supplemented by electron microscopy and X-ray powder diffraction data. The structural model reveals a two-dimensional-crystal-like architecture of stacked monolayer rings each containing ∼50 NDI-Lys molecules, with significant π-stacking interactions occurring both within the confines of the ring and along the long axis of the tube. Excited-state delocalization and energy transfer are simulated for the nanotube based on time-dependent density functional theory and an incoherent hopping model. Remarkably, these calculations reveal efficient energy migration from the excitonic bright state, which is in agreement with the rapid energy transfer within NDI-Lys nanotubes observed previously using fluorescence spectroscopy. PMID:26120375

  5. Apollo 17 Lunar Sounder Data provide Insight into Aitken Crater's Subsurface Structure

    NASA Technical Reports Server (NTRS)

    Cooper, Bonnie L.

    2007-01-01

    In preparation for the forthcoming avalanche of data from Lunar Reconnaissance Orbiter (LRO), we conducted a pilot study to demonstrate integration of multiple geophysical data sets. We applied methods of data integration that are used by the commercial mineral exploration industry to enhance the value of historical data sets and to provide a roadmap for future efforts.

  6. Crystal structure of a maltogenic amylase provides insights into a catalytic versatility.

    PubMed

    Kim, J S; Cha, S S; Kim, H J; Kim, T J; Ha, N C; Oh, S T; Cho, H S; Cho, M J; Kim, M J; Lee, H S; Kim, J W; Choi, K Y; Park, K H; Oh, B H

    1999-09-10

    Amylases catalyze the hydrolysis of starch material and play central roles in carbohydrate metabolism. Compared with many different amylases that are able to hydrolyze only alpha-D-(1,4)-glycosidic bonds, maltogenic amylases exhibit catalytic versatility: hydrolysis of alpha-D-(1,4)- and alpha-D-(1,6)-glycosidic bonds and transglycosylation of oligosaccharides to C3-, C4-, or C6-hydroxyl groups of various acceptor mono- or disaccharides. It has been speculated that the catalytic property of the enzymes is linked to the additional approximately 130 residues at the N terminus that are absent in other typical alpha-amylases. The crystal structure of a maltogenic amylase from a Thermus strain was determined at 2.8 A. The structure, an analytical centrifugation, and a size exclusion column chromatography proved that the enzyme is a dimer in solution. The N-terminal segment of the enzyme folds into a distinct domain and comprises the enzyme active site together with the central (alpha/beta)(8) barrel of the adjacent subunit. The active site is a narrow and deep cleft suitable for binding cyclodextrins, which are the preferred substrates to other starch materials. At the bottom of the active site cleft, an extra space, absent in the other typical alpha-amylases, is present whose size is comparable with that of a disaccharide. The space is most likely to host an acceptor molecule for the transglycosylation and to allow binding of a branched oligosaccharide for hydrolysis of alpha-D-(1,4)-glycosidic or alpha-D-(1,6)-glycosidic bond. The (alpha/beta)(8) barrel of the enzyme is the preserved scaffold in all the known amylases. The structure represents a novel example of how an enzyme acquires a different substrate profile and a catalytic versatility from a common active site and represents a framework for explaining the catalytic activities of transglycosylation and hydrolysis of alpha-D-(1,6)-glycosidic bond.

  7. Structures of human SRP72 complexes provide insights into SRP RNA remodeling and ribosome interaction

    PubMed Central

    Becker, Matthias M. M.; Lapouge, Karine; Segnitz, Bernd; Wild, Klemens; Sinning, Irmgard

    2017-01-01

    Co-translational protein targeting and membrane protein insertion is a fundamental process and depends on the signal recognition particle (SRP). In mammals, SRP is composed of the SRP RNA crucial for SRP assembly and function and six proteins. The two largest proteins SRP68 and SRP72 form a heterodimer and bind to a regulatory site of the SRP RNA. Despite their essential roles in the SRP pathway, structural information has been available only for the SRP68 RNA-binding domain (RBD). Here we present the crystal structures of the SRP68 protein-binding domain (PBD) in complex with SRP72-PBD and of the SRP72-RBD bound to the SRP S domain (SRP RNA, SRP19 and SRP68) detailing all interactions of SRP72 within SRP. The SRP72-PBD is a tetratricopeptide repeat, which binds an extended linear motif of SRP68 with high affinity. The SRP72-RBD is a flexible peptide crawling along the 5e- and 5f-loops of SRP RNA. A conserved tryptophan inserts into the 5e-loop forming a novel type of RNA kink-turn stabilized by a potassium ion, which we define as K+-turn. In addition, SRP72-RBD remodels the 5f-loop involved in ribosome binding and visualizes SRP RNA plasticity. Docking of the S domain structure into cryo-electron microscopy density maps reveals multiple contact sites between SRP68/72 and the ribosome, and explains the role of SRP72 in the SRP pathway. PMID:27899666

  8. The shades of gray of the chromatin fiber: recent literature provides new insights into the structure of chromatin.

    PubMed

    Ausió, Juan

    2015-01-01

    The chromatin fiber consists of a string of nucleosomes connected by linker DNA regions. The hierarchy of folding of this fiber within the cell has long been controversial, and the existence of an originally described 30 nm fiber has been debated and reviewed extensively. This review contextualizes two recent papers on this topic that suggest the 30 nm fiber to be an over-simplification. The idealized model from the first study provides good insight into the constraints and histone participation in the maintenance of the fiber structure. The second paper provides a theoretical description of a more realistic view of the highly heterogeneous and dynamic chromatin organization in the in vivo setting. It is now time to abandon the highly regular "one start" solenoidal 30 nm structure and replace it with a more realistic highly dynamic, polymorphic fiber.

  9. Near-planar Solution Structures of Mannose-binding Lectin Oligomers Provide Insight on Activation of Lectin Pathway of Complement

    PubMed Central

    Miller, Ami; Phillips, Anna; Gor, Jayesh; Wallis, Russell; Perkins, Stephen J.

    2012-01-01

    The complement system is a fundamental component of innate immunity that orchestrates complex immunological and inflammatory processes. Complement comprises over 30 proteins that eliminate invading microorganisms while maintaining host cell integrity. Protein-carbohydrate interactions play critical roles in both the activation and regulation of complement. Mannose-binding lectin (MBL) activates the lectin pathway of complement via the recognition of sugar arrays on pathogenic surfaces. To determine the solution structure of MBL, synchrotron x-ray scattering and analytical ultracentrifugation experiments showed that the carbohydrate-recognition domains in the MBL dimer, trimer, and tetramer are positioned close to each other in near-planar fan-like structures. These data were subjected to constrained modeling fits. A bent structure for the MBL monomer was identified starting from two crystal structures for its carbohydrate-recognition domain and its triple helical region. The MBL monomer structure was used to identify 10–12 near-planar solution structures for each of the MBL dimers, trimers, and tetramers starting from 900 to 6,859 randomized structures for each. These near-planar fan-like solution structures joined at an N-terminal hub clarified how the carbohydrate-recognition domain of MBL binds to pathogenic surfaces. They also provided insight on how MBL presents a structural template for the binding and auto-activation of the MBL-associated serine proteases to initiate the lectin pathway of complement activation. PMID:22167201

  10. Fish species introductions provide novel insights into the patterns and drivers of phylogenetic structure in freshwaters

    PubMed Central

    Strecker, Angela L.; Olden, Julian D.

    2014-01-01

    Despite long-standing interest of terrestrial ecologists, freshwater ecosystems are a fertile, yet unappreciated, testing ground for applying community phylogenetics to uncover mechanisms of species assembly. We quantify phylogenetic clustering and overdispersion of native and non-native fishes of a large river basin in the American Southwest to test for the mechanisms (environmental filtering versus competitive exclusion) and spatial scales influencing community structure. Contrary to expectations, non-native species were phylogenetically clustered and related to natural environmental conditions, whereas native species were not phylogenetically structured, likely reflecting human-related changes to the basin. The species that are most invasive (in terms of ecological impacts) tended to be the most phylogenetically divergent from natives across watersheds, but not within watersheds, supporting the hypothesis that Darwin's naturalization conundrum is driven by the spatial scale. Phylogenetic distinctiveness may facilitate non-native establishment at regional scales, but environmental filtering restricts local membership to closely related species with physiological tolerances for current environments. By contrast, native species may have been phylogenetically clustered in historical times, but species loss from contemporary populations by anthropogenic activities has likely shaped the phylogenetic signal. Our study implies that fundamental mechanisms of community assembly have changed, with fundamental consequences for the biogeography of both native and non-native species. PMID:24452027

  11. Crystal structures of highly simplified BPTIs provide insights into hydration-driven increase of unfolding enthalpy

    PubMed Central

    Islam, Mohammad Monirul; Yohda, Masafumi; Kidokoro, Shun-ichi; Kuroda, Yutaka

    2017-01-01

    We report a thermodynamic and structural analysis of six extensively simplified bovine pancreatic trypsin inhibitor (BPTI) variants containing 19–24 alanines out of 58 residues. Differential scanning calorimetry indicated a two-state thermal unfolding, typical of a native protein with densely packed interior. Surprisingly, increasing the number of alanines induced enthalpy stabilization, which was however over-compensated by entropy destabilization. X-ray crystallography indicated that the alanine substitutions caused the recruitment of novel water molecules facilitating the formation of protein–water hydrogen bonds and improving the hydration shells around the alanine’s methyl groups, both of which presumably contributed to enthalpy stabilization. There was a strong correlation between the number of water molecules and the thermodynamic parameters. Overall, our results demonstrate that, in contrast to our initial expectation, a protein sequence in which over 40% of the residues are alanines can retain a densely packed structure and undergo thermal denaturation with a large enthalpy change, mainly contributed by hydration. PMID:28266637

  12. Der p 5 Crystal Structure Provides Insight into the Group 5 Dust Mite Allergens

    SciTech Connect

    Mueller, G.; Gosavi, R; Krahn, J; Edwards, L; Cuneo, M; Glesner, J; Pomes, A; Chapman, M; London, R; Pedersen, L

    2010-01-01

    Group 5 allergens from house dust mites elicit strong IgE antibody binding in mite-allergic patients. The structure of Der p 5 was determined by x-ray crystallography to better understand the IgE epitopes, to investigate the biologic function in mites, and to compare with the conflicting published Blo t 5 structures, designated 2JMH and 2JRK in the Protein Data Bank. Der p 5 is a three-helical bundle similar to Blo t 5, but the interactions of the helices are more similar to 2JMH than 2JRK. The crystallographic asymmetric unit contains three dimers of Der p 5 that are not exactly alike. Solution scattering techniques were used to assess the multimeric state of Der p 5 in vitro and showed that the predominant state was monomeric, similar to Blo t 5, but larger multimeric species are also present. In the crystal, the formation of the Der p 5 dimer creates a large hydrophobic cavity of {approx}3000 {angstrom}{sup 3} that could be a ligand-binding site. Many allergens are known to bind hydrophobic ligands, which are thought to stimulate the innate immune system and have adjuvant-like effects on IgE-mediated inflammatory responses.

  13. Structure of ubiquitylated-Rpn10 provides insight into its autoregulation mechanism

    PubMed Central

    Keren-Kaplan, Tal; Zeev Peters, Lee; Levin-Kravets, Olga; Attali, Ilan; Kleifeld, Oded; Shohat, Noa; Artzi, Shay; Zucker, Ori; Pilzer, Inbar; Reis, Noa; Glickman, Michael H.; Ben-Aroya, Shay; Prag, Gali

    2016-01-01

    Ubiquitin receptors decode ubiquitin signals into many cellular responses. Ubiquitin receptors also undergo coupled monoubiquitylation, and rapid deubiquitylation has hampered the characterization of the ubiquitylated state. Using bacteria that express a ubiquitylation apparatus, we purified and determined the crystal structure of the proteasomal ubiquitin-receptor Rpn10 in its ubiquitylated state. The structure shows a novel ubiquitin-binding patch that directs K84 ubiquitylation. Superimposition of ubiquitylated-Rpn10 onto electron-microscopy models of proteasomes indicates that the Rpn10-conjugated ubiquitin clashes with Rpn9, suggesting that ubiquitylation might be involved in releasing Rpn10 from the proteasome. Indeed, ubiquitylation on immobilized proteasomes dissociates the modified Rpn10 from the complex, while unmodified Rpn10 mainly remains associated. In vivo experiments indicate that contrary to wild type, Rpn10-K84R is stably associated with the proteasomal subunit Rpn9. Similarly Rpn10, but not ubiquitylated-Rpn10, binds Rpn9 in vitro. Thus we suggest that ubiquitylation functions to dissociate modified ubiquitin receptors from their targets, a function that promotes cyclic activity of ubiquitin receptors. PMID:27698474

  14. Structure of ubiquitylated-Rpn10 provides insight into its autoregulation mechanism.

    PubMed

    Keren-Kaplan, Tal; Zeev Peters, Lee; Levin-Kravets, Olga; Attali, Ilan; Kleifeld, Oded; Shohat, Noa; Artzi, Shay; Zucker, Ori; Pilzer, Inbar; Reis, Noa; Glickman, Michael H; Ben-Aroya, Shay; Prag, Gali

    2016-10-04

    Ubiquitin receptors decode ubiquitin signals into many cellular responses. Ubiquitin receptors also undergo coupled monoubiquitylation, and rapid deubiquitylation has hampered the characterization of the ubiquitylated state. Using bacteria that express a ubiquitylation apparatus, we purified and determined the crystal structure of the proteasomal ubiquitin-receptor Rpn10 in its ubiquitylated state. The structure shows a novel ubiquitin-binding patch that directs K84 ubiquitylation. Superimposition of ubiquitylated-Rpn10 onto electron-microscopy models of proteasomes indicates that the Rpn10-conjugated ubiquitin clashes with Rpn9, suggesting that ubiquitylation might be involved in releasing Rpn10 from the proteasome. Indeed, ubiquitylation on immobilized proteasomes dissociates the modified Rpn10 from the complex, while unmodified Rpn10 mainly remains associated. In vivo experiments indicate that contrary to wild type, Rpn10-K84R is stably associated with the proteasomal subunit Rpn9. Similarly Rpn10, but not ubiquitylated-Rpn10, binds Rpn9 in vitro. Thus we suggest that ubiquitylation functions to dissociate modified ubiquitin receptors from their targets, a function that promotes cyclic activity of ubiquitin receptors.

  15. Structural characterization of encapsulated ferritin provides insight into iron storage in bacterial nanocompartments

    PubMed Central

    He, Didi; Hughes, Sam; Vanden-Hehir, Sally; Georgiev, Atanas; Altenbach, Kirsten; Tarrant, Emma; Mackay, C Logan; Waldron, Kevin J; Clarke, David J; Marles-Wright, Jon

    2016-01-01

    Ferritins are ubiquitous proteins that oxidise and store iron within a protein shell to protect cells from oxidative damage. We have characterized the structure and function of a new member of the ferritin superfamily that is sequestered within an encapsulin capsid. We show that this encapsulated ferritin (EncFtn) has two main alpha helices, which assemble in a metal dependent manner to form a ferroxidase center at a dimer interface. EncFtn adopts an open decameric structure that is topologically distinct from other ferritins. While EncFtn acts as a ferroxidase, it cannot mineralize iron. Conversely, the encapsulin shell associates with iron, but is not enzymatically active, and we demonstrate that EncFtn must be housed within the encapsulin for iron storage. This encapsulin nanocompartment is widely distributed in bacteria and archaea and represents a distinct class of iron storage system, where the oxidation and mineralization of iron are distributed between two proteins. DOI: http://dx.doi.org/10.7554/eLife.18972.001 PMID:27529188

  16. The picobirnavirus crystal structure provides functional insights into virion assembly and cell entry

    PubMed Central

    Duquerroy, Stéphane; Da Costa, Bruno; Henry, Céline; Vigouroux, Armelle; Libersou, Sonia; Lepault, Jean; Navaza, Jorge; Delmas, Bernard; Rey, Félix A

    2009-01-01

    Double-stranded (ds) RNA virus particles are organized around a central icosahedral core capsid made of 120 identical subunits. This core capsid is unable to invade cells from outside, and animal dsRNA viruses have acquired surrounding capsid layers that are used to deliver a transcriptionally active core particle across the membrane during cell entry. In contrast, dsRNA viruses infecting primitive eukaryotes have only a simple core capsid, and as a consequence are transmitted only vertically. Here, we report the 3.4 Å X-ray structure of a picobirnavirus—an animal dsRNA virus associated with diarrhoea and gastroenteritis in humans. The structure shows a simple core capsid with a distinctive icosahedral arrangement, displaying 60 two-fold symmetric dimers of a coat protein (CP) with a new 3D-fold. We show that, as many non-enveloped animal viruses, CP undergoes an autoproteolytic cleavage, releasing a post-translationally modified peptide that remains associated with nucleic acid within the capsid. Our data also show that picobirnavirus particles are capable of disrupting biological membranes in vitro, indicating that its simple 120-subunits capsid has evolved animal cell invasion properties. PMID:19407816

  17. Crystal structure of human thimet oligopeptidase provides insight into substrate recognition, regulation, and localization.

    PubMed

    Ray, Kallol; Hines, Christina S; Coll-Rodriguez, Jerry; Rodgers, David W

    2004-05-07

    Thimet oligopeptidase (TOP) is a zinc metallopeptidase that metabolizes a number of bioactive peptides and degrades peptides released by the proteasome, limiting antigenic presentation by MHC class I molecules. We present the crystal structure of human TOP at 2.0-A resolution. The active site is located at the base of a deep channel that runs the length of the elongated molecule, an overall fold first seen in the closely related metallopeptidase neurolysin. Comparison of the two related structures indicates hinge-like flexibility and identifies elements near one end of the channel that adopt different conformations. Relatively few of the sequence differences between TOP and neurolysin map to the proposed substrate-binding site, and four of these variable residues may account for differences in substrate specificity. In addition, a loop segment (residues 599-611) in TOP differs in conformation and degree of order from the corresponding neurolysin loop, suggesting it may also play a role in activity differences. Cysteines thought to mediate covalent oligomerization of rat TOP, which can inactivate the enzyme, are found to be surface-accessible in the human enzyme, and additional cysteines (residues 321,350, and 644) may also mediate multimerization in the human homolog. Disorder in the N terminus of TOP indicates it may be involved in subcellular localization, but a potential nuclear import element is found to be part of a helix and, therefore, unlikely to be involved in transport. A large acidic patch on the surface could potentially mediate a protein-protein interaction, possibly through formation of a covalent linkage.

  18. Catalysis and Structure of Zebrafish Urate Oxidase Provide Insights into the Origin of Hyperuricemia in Hominoids

    PubMed Central

    Marchetti, Marialaura; Liuzzi, Anastasia; Fermi, Beatrice; Corsini, Romina; Folli, Claudia; Speranzini, Valentina; Gandolfi, Francesco; Bettati, Stefano; Ronda, Luca; Cendron, Laura; Berni, Rodolfo; Zanotti, Giuseppe; Percudani, Riccardo

    2016-01-01

    Urate oxidase (Uox) catalyses the first reaction of oxidative uricolysis, a three-step enzymatic pathway that allows some animals to eliminate purine nitrogen through a water-soluble compound. Inactivation of the pathway in hominoids leads to elevated levels of sparingly soluble urate and puts humans at risk of hyperuricemia and gout. The uricolytic activities lost during evolution can be replaced by enzyme therapy. Here we report on the functional and structural characterization of Uox from zebrafish and the effects on the enzyme of the missense mutation (F216S) that preceded Uox pseudogenization in hominoids. Using a kinetic assay based on the enzymatic suppression of the spectroscopic interference of the Uox reaction product, we found that the F216S mutant has the same turnover number of the wild-type enzyme but a much-reduced affinity for the urate substrate and xanthine inhibitor. Our results indicate that the last functioning Uox in hominoid evolution had an increased Michaelis constant, possibly near to upper end of the normal range of urate in the human serum (~300 μM). Changes in the renal handling of urate during primate evolution can explain the genetic modification of uricolytic activities in the hominoid lineage without the need of assuming fixation of deleterious mutations. PMID:27922051

  19. Inter-basin movements of Mediterranean sperm whales provide insight into their population structure and conservation

    NASA Astrophysics Data System (ADS)

    Frantzis, A.; Airoldi, S.; Notarbartolo-di-Sciara, G.; Johnson, C.; Mazzariol, S.

    2011-04-01

    The sperm whale is one of the very few deep diving mammal species in the Mediterranean Sea. Following a rare mass stranding of male sperm whales in the Adriatic Sea in December 2009, photo-identification methods were used in order to investigate previous sightings of the stranded whales in the region. Fluke photos of the stranded whales were compared with those of 153 and 128 free-ranging individuals photographed in the western and eastern Mediterranean basins, respectively. Three out of the seven stranded whales had been previously photo-identified and some of them more than once. To reach the stranding place, two of these re-identified whales performed long-range inter-basin movements of about 1600-2100 km (in a straight line) either through the Strait of Sicily or the Strait of Messina. In addition, comparisons among all whales photographed in the two Mediterranean basins revealed that one more individual first photographed in the western basin (1991) was re-identified 13 years later in the eastern basin (2004). These three cases provide the first conclusive evidence of inter-basin movement of sperm whales in the Mediterranean Sea. Inter-basin gene flow is important for the survival of the small and endangered Mediterranean sperm whale population. Mitigating the disturbance created by human activities in the straits area is crucial for its conservation.

  20. The Structure of Allophanate Hydrolase from Granulibacter bethesdensis Provides Insights into Substrate Specificity in the Amidase Signature Family

    SciTech Connect

    Lin, Yi; Maurice, Martin

    2013-01-02

    Allophanate hydrolase (AH) catalyzes the hydrolysis of allophanate, an intermediate in atrazine degradation and urea catabolism pathways, to NH3 and CO2. AH belongs to the amidase signature family, which is characterized by a conserved block of 130 amino acids rich in Gly and Ser and a Ser-cis-Ser-Lys catalytic triad. In this study, the first structures of AH fromGranulibacter bethesdensis were determined, with and without the substrate analogue malonate, to 2.2 and 2.8 Å, respectively. The structures confirm the identity of the catalytic triad residues and reveal an altered dimerization interface that is not conserved in the amidase signature family. The structures also provide insights into previously unrecognized substrate specificity determinants in AH. Two residues, Tyr299 and Arg307, are within hydrogen bonding distance of a carboxylate moiety of malonate. Both Tyr299 and Arg307 were mutated, and the resulting modified enzymes revealed >3 order of magnitude reductions in both catalytic efficiency and substrate stringency. It is proposed that Tyr299 and Arg307 serve to anchor and orient the substrate for attack by the catalytic nucleophile, Ser172. The structure further suggests the presence of a unique C-terminal domain in AH. While this domain is conserved, it does not contribute to catalysis or to the structural integrity of the core domain, suggesting that it may play a role in mediating transient and specific interactions with the urea carboxylase component of urea amidolyase. Analysis of the AH active site architecture offers new insights into common determinants of catalysis and specificity among divergent members of the amidase signature family.

  1. Structural Analysis of the Myo1c and Neph1 Complex Provides Insight into the Intracellular Movement of Neph1

    PubMed Central

    Arif, Ehtesham; Sharma, Pankaj; Solanki, Ashish; Mallik, Leena; Rathore, Yogendra S.; Twal, Waleed O.; Nath, Samir K.; Gandhi, Darpan; Holzman, Lawrence B.; Ostap, E. Michael

    2016-01-01

    The Myo1c motor functions as a cargo transporter supporting various cellular events, including vesicular trafficking, cell migration, and stereociliary movements of hair cells. Although its partial crystal structures were recently described, the structural details of its interaction with cargo proteins remain unknown. This study presents the first structural demonstration of a cargo protein, Neph1, attached to Myo1c, providing novel insights into the role of Myo1c in intracellular movements of this critical slit diaphragm protein. Using small angle X-ray scattering studies, models of predominant solution conformation of unliganded full-length Myo1c and Myo1c bound to Neph1 were constructed. The resulting structures show an extended S-shaped Myo1c with Neph1 attached to its C-terminal tail. Importantly, binding of Neph1 did not induce a significant shape change in Myo1c, indicating this as a spontaneous process or event. Analysis of interaction surfaces led to the identification of a critical residue in Neph1 involved in binding to Myo1c. Indeed, a point mutant from this site abolished interaction between Neph1 and Myo1c when tested in the in vitro and in live-cell binding assays. Live-cell imaging, including fluorescence recovery after photobleaching, provided further support for the role of Myo1c in intracellular vesicular movement of Neph1 and its turnover at the membrane. PMID:27044863

  2. Crystal structure of a chimera of human and Plasmodium falciparum hypoxanthine guanine phosphoribosyltransferases provides insights into oligomerization.

    PubMed

    Gayathri, P; Sujay Subbayya, I N; Ashok, Chethan S; Selvi, T Senthamizh; Balaram, Hemalatha; Murthy, M R N

    2008-12-01

    The crystal structure of a chimera of Plasmodium falciparum (Pf) and human hypoxanthine guanine phosphoribosyltransferases (HGPRT), which consists of the core of the protein from the human enzyme and the hood region from the Pf enzyme, has been determined as a complex with the product guanosine monophosphate (GMP). The chimera can utilize hypoxanthine, guanine, and xanthine as substrates, similar to the Pf enzyme. It exists as a monomer-dimer mixture in solution, but shifts to a tetramer on addition of phosphoribosyl pyrophosphate (PRPP). The structural studies reveal that the asymmetric unit of the crystal consists of two monomers of the chimeric HGPRT. Surprisingly, the dimer interface of the chimera is the less extensive AC interface of the parent HGPRTs. An analysis of the crystal structures of the various human HGPRTs provides an explanation for the oligomeric characteristics of the chimera. Pro93 and Tyr197 form part of crucial interactions holding together the AB interface in the unliganded or GMP-bound forms of HGPRT, while Pro93 and His26 interact at the interface after binding of PRPP. Replacement of Tyr197 of human HGPRT by Ile207 in the chimera disrupts the interaction at the AB interface in the absence of PRPP. In the presence of PRPP, the interaction between Pro93 and His26 could restore the AB interface, shifting the chimeric enzyme to a tetrameric state. The structure provides valuable insights into the differences in the AB interface between Pf and human HGPRTs, which may be useful for designing selective inhibitors against the parasite enzyme.

  3. Insights into remodeling events during eukaryotic large ribosomal subunit assembly provided by high resolution cryo-EM structures.

    PubMed

    Biedka, Stephanie; Wu, Shan; LaPeruta, Amber J; Gao, Ning; Woolford, John L

    2017-03-07

    Ribosomes are responsible for translating the genome, in the form of mRNA, into the proteome in all organisms. Biogenesis of ribosomes in eukaryotes is a complex process involving numerous remodeling events driven in part by the concerted actions of hundreds of protein assembly factors. A major challenge in studying eukaryotic ribosome assembly has, until recently, been a lack of structural data to facilitate understanding of the conformational and compositional changes the pre-ribosome undergoes during its construction. Cryo-electron microscopy (cryo-EM) has begun filling these gaps; recent advances in cryo-EM have enabled the determination of several high resolution pre-ribosome structures. This review focuses mainly on lessons learned from the study of pre-60S particles purified from yeast using the assembly factor Nog2 as bait. These Nog2 particles provide insight into many aspects of nuclear stages of 60S subunit assembly, including construction of major 60S subunit functional centers and processing of the ITS2 spacer RNA.

  4. Novel UDP-GalNAc Derivative Structures Provide Insight into the Donor Specificity of Human Blood Group Glycosyltransferase.

    PubMed

    Wagner, Gerd K; Pesnot, Thomas; Palcic, Monica M; Jørgensen, Rene

    2015-12-25

    Two closely related glycosyltransferases are responsible for the final step of the biosynthesis of ABO(H) human blood group A and B antigens. The two enzymes differ by only four amino acid residues, which determine whether the enzymes transfer GalNAc from UDP-GalNAc or Gal from UDP-Gal to the H-antigen acceptor. The enzymes belong to the class of GT-A folded enzymes, grouped as GT6 in the CAZy database, and are characterized by a single domain with a metal dependent retaining reaction mechanism. However, the exact role of the four amino acid residues in the specificity of the enzymes is still unresolved. In this study, we report the first structural information of a dual specificity cis-AB blood group glycosyltransferase in complex with a synthetic UDP-GalNAc derivative. Interestingly, the GalNAc moiety adopts an unusual yet catalytically productive conformation in the binding pocket, which is different from the "tucked under" conformation previously observed for the UDP-Gal donor. In addition, we show that this UDP-GalNAc derivative in complex with the H-antigen acceptor provokes the same unusual binding pocket closure as seen for the corresponding UDP-Gal derivative. Despite this, the two derivatives show vastly different kinetic properties. Our results provide a important structural insight into the donor substrate specificity and utilization in blood group biosynthesis, which can very likely be exploited for the development of new glycosyltransferase inhibitors and probes.

  5. Crystal structure of peroxide stress regulator from Streptococcus pyogenes provides functional insights into the mechanism of oxidative stress sensing.

    PubMed

    Makthal, Nishanth; Rastegari, Sheila; Sanson, Misu; Ma, Zhen; Olsen, Randall J; Helmann, John D; Musser, James M; Kumaraswami, Muthiah

    2013-06-21

    Regulation of oxidative stress responses by the peroxide stress regulator (PerR) is critical for the in vivo fitness and virulence of group A Streptococcus. To elucidate the molecular mechanism of DNA binding, peroxide sensing, and gene regulation by PerR, we performed biochemical and structural characterization of PerR. Sequence-specific DNA binding by PerR does not require regulatory metal occupancy. However, metal binding promotes higher affinity PerR-DNA interactions. PerR metallated with iron directly senses peroxide stress and dissociates from operator sequences. The crystal structure revealed that PerR exists as a homodimer with two metal-binding sites per subunit as follows: a structural zinc site and a regulatory metal site that is occupied in the crystals by nickel. The regulatory metal-binding site in PerR involves a previously unobserved HXH motif located in its unique N-terminal extension. Mutational analysis of the regulatory site showed that the PerR metal ligands are involved in regulatory metal binding, and integrity of this site is critical for group A Streptococcus virulence. Interestingly, the metal-binding HXH motif is not present in the structurally characterized members of ferric uptake regulator (Fur) family but is fully conserved among PerR from the genus Streptococcus. Thus, it is likely that the PerR orthologs from streptococci share a common mechanism of metal binding, peroxide sensing, and gene regulation that is different from that of well characterized PerR from Bacillus subtilis. Together, our findings provide key insights into the peroxide sensing and regulation of the oxidative stress-adaptive responses by the streptococcal subfamily of PerR.

  6. Crystal Structure of Peroxide Stress Regulator from Streptococcus pyogenes Provides Functional Insights into the Mechanism of Oxidative Stress Sensing*

    PubMed Central

    Makthal, Nishanth; Rastegari, Sheila; Sanson, Misu; Ma, Zhen; Olsen, Randall J.; Helmann, John D.; Musser, James M.; Kumaraswami, Muthiah

    2013-01-01

    Regulation of oxidative stress responses by the peroxide stress regulator (PerR) is critical for the in vivo fitness and virulence of group A Streptococcus. To elucidate the molecular mechanism of DNA binding, peroxide sensing, and gene regulation by PerR, we performed biochemical and structural characterization of PerR. Sequence-specific DNA binding by PerR does not require regulatory metal occupancy. However, metal binding promotes higher affinity PerR-DNA interactions. PerR metallated with iron directly senses peroxide stress and dissociates from operator sequences. The crystal structure revealed that PerR exists as a homodimer with two metal-binding sites per subunit as follows: a structural zinc site and a regulatory metal site that is occupied in the crystals by nickel. The regulatory metal-binding site in PerR involves a previously unobserved HXH motif located in its unique N-terminal extension. Mutational analysis of the regulatory site showed that the PerR metal ligands are involved in regulatory metal binding, and integrity of this site is critical for group A Streptococcus virulence. Interestingly, the metal-binding HXH motif is not present in the structurally characterized members of ferric uptake regulator (Fur) family but is fully conserved among PerR from the genus Streptococcus. Thus, it is likely that the PerR orthologs from streptococci share a common mechanism of metal binding, peroxide sensing, and gene regulation that is different from that of well characterized PerR from Bacillus subtilis. Together, our findings provide key insights into the peroxide sensing and regulation of the oxidative stress-adaptive responses by the streptococcal subfamily of PerR. PMID:23645680

  7. The structure of C290A:C393A Aurora A provides structural insights into kinase regulation

    PubMed Central

    Burgess, Selena G.; Bayliss, Richard

    2015-01-01

    Aurora A is a Ser/Thr protein kinase that functions in cell-cycle regulation and is implicated in cancer development. During mitosis, Aurora A is activated by autophosphorylation on its activation loop at Thr288. The Aurora A catalytic domain (amino acids 122–403) expressed in Escherichia coli autophosphorylates on two activation-loop threonine residues (Thr288 and Thr287), whereas a C290A,C393A double point mutant of the Aurora A catalytic domain autophosphorylates only on Thr288. The structure of the complex of this mutant with ADP and magnesium was determined to 2.1 Å resolution using molecular replacement. This is an improvement on the existing 2.75 Å resolution structure of the equivalent wild-type complex. The structure confirms that single phosphorylation of the activation loop on Thr288 is insufficient to stabilize a ‘fully active’ conformation of the activation loop in the absence of binding to TPX2. PMID:25760707

  8. Crystal structure of viral serpin crmA provides insights into its mechanism of cysteine proteinase inhibition.

    PubMed Central

    Simonovic, M.; Gettins PGW; Volz, K.

    2000-01-01

    CrmA is an unusual viral serpin that inhibits both cysteine and serine proteinases involved in the regulation of host inflammatory and apoptosis processes. It differs from other members of the serpin superfamily by having a reactive center loop that is one residue shorter, and by its apparent inability to form SDS-stable covalent complexes with cysteine proteinases. To obtain insight into the inhibitory mechanism of crmA, we determined the crystal structure of reactive center loop-cleaved crmA to 2.9 A resolution. The structure, which is the first of a viral serpin, suggests that crmA can inhibit cysteine proteinases by a mechanism analogous to that used by other serpins against serine proteinases. However, one striking difference from other serpins, which may be significant for in vivo function, is an additional highly charged antiparallel strand for b sheet A, whose sequence and length are unique to crmA. PMID:10975564

  9. Structure of putrescine aminotransferase from Escherichia coli provides insights into the substrate specificity among class III aminotransferases.

    PubMed

    Cha, Hyung Jin; Jeong, Jae-Hee; Rojviriya, Catleya; Kim, Yeon-Gil

    2014-01-01

    YgjG is a putrescine aminotransferase enzyme that transfers amino groups from compounds with terminal primary amines to compounds with an aldehyde group using pyridoxal-5'-phosphate (PLP) as a cofactor. Previous biochemical data show that the enzyme prefers primary diamines, such as putrescine, over ornithine as a substrate. To better understand the enzyme's substrate specificity, crystal structures of YgjG from Escherichia coli were determined at 2.3 and 2.1 Å resolutions for the free and putrescine-bound enzymes, respectively. Sequence and structural analyses revealed that YgjG forms a dimer that adopts a class III PLP-dependent aminotransferase fold. A structural comparison between YgjG and other class III aminotransferases revealed that their structures are similar. However, YgjG has an additional N-terminal helical structure that partially contributes to a dimeric interaction with the other subunit via a helix-helix interaction. Interestingly, the YgjG substrate-binding site entrance size and charge distribution are smaller and more hydrophobic than other class III aminotransferases, which suggest that YgjG has a unique substrate binding site that could accommodate primary aliphatic diamine substrates, including putrescine. The YgjG crystal structures provide structural clues to putrescine aminotransferase substrate specificity and binding.

  10. Crystal structures of an archaeal oligosaccharyltransferase provide insights into the catalytic cycle of N-linked protein glycosylation.

    PubMed

    Matsumoto, Shunsuke; Shimada, Atsushi; Nyirenda, James; Igura, Mayumi; Kawano, Yoshiaki; Kohda, Daisuke

    2013-10-29

    Oligosaccharyltransferase transfers an oligosaccharide chain to the asparagine residues in proteins. The archaeal and eubacterial oligosaccharyltransferases are single subunit membrane enzymes, referred to as "AglB" (archaeal glycosylation B) and "PglB" (protein glycosylation B), respectively. Only one crystal structure of a full-length PglB has been solved. Here we report the crystal structures of the full-length AglB from a hyperthermophilic archaeon, Archaeoglobus fulgidus. The AglB and PglB proteins share the common overall topology of the 13 transmembrane helices, and a characteristic long plastic loop in the transmembrane region. This is the structural basis for the formation of the catalytic center, consisting of conserved acidic residues coordinating a divalent metal ion. In one crystal form, a sulfate ion was bound next to the metal ion. This structure appears to represent a dolichol-phosphate binding state, and suggests the release mechanism for the glycosylated product. The structure in the other crystal form corresponds to the resting state conformation with the well-ordered plastic loop in the transmembrane region. The overall structural similarity between the distantly related AglB and PglB proteins strongly indicates the conserved catalytic mechanism in the eukaryotic counterpart, the STT3 (stauroporine and temperature sensitivity 3) protein. The detailed structural comparison provided the dynamic view of the N-glycosylation reaction, involving the conversion between the structured and unstructured states of the plastic loop in the transmembrane region and the formation and collapse of the Ser/Thr-binding pocket in the C-terminal globular domain.

  11. Crystal Structures of Malonyl-Coenzyme A Decarboxylase Provide Insights into Its Catalytic Mechanism and Disease-Causing Mutations

    PubMed Central

    Froese, D. Sean; Forouhar, Farhad; Tran, Timothy H.; Vollmar, Melanie; Kim, Yi Seul; Lew, Scott; Neely, Helen; Seetharaman, Jayaraman; Shen, Yang; Xiao, Rong; Acton, Thomas B.; Everett, John K.; Cannone, Giuseppe; Puranik, Sriharsha; Savitsky, Pavel; Krojer, Tobias; Pilka, Ewa S.; Kiyani, Wasim; Lee, Wen Hwa; Marsden, Brian D.; von Delft, Frank; Allerston, Charles K.; Spagnolo, Laura; Gileadi, Opher; Montelione, Gaetano T.; Oppermann, Udo; Yue, Wyatt W.; Tong, Liang

    2013-01-01

    Summary Malonyl-coenzyme A decarboxylase (MCD) is found from bacteria to humans, has important roles in regulating fatty acid metabolism and food intake, and is an attractive target for drug discovery. We report here four crystal structures of MCD from human, Rhodopseudomonas palustris, Agrobacterium vitis, and Cupriavidus metallidurans at up to 2.3 Å resolution. The MCD monomer contains an N-terminal helical domain involved in oligomerization and a C-terminal catalytic domain. The four structures exhibit substantial differences in the organization of the helical domains and, consequently, the oligomeric states and intersubunit interfaces. Unexpectedly, the MCD catalytic domain is structurally homologous to those of the GCN5-related N-acetyltransferase superfamily, especially the curacin A polyketide synthase catalytic module, with a conserved His-Ser/Thr dyad important for catalysis. Our structures, along with mutagenesis and kinetic studies, provide a molecular basis for understanding pathogenic mutations and catalysis, as well as a template for structure-based drug design. PMID:23791943

  12. Insertion sequence element single nucleotide polymorphism typing provides insights into the population structure and evolution of Mycobacterium ulcerans across Africa.

    PubMed

    Vandelannoote, Koen; Jordaens, Kurt; Bomans, Pieter; Leirs, Herwig; Durnez, Lies; Affolabi, Dissou; Sopoh, Ghislain; Aguiar, Julia; Phanzu, Delphin Mavinga; Kibadi, Kapay; Eyangoh, Sara; Manou, Louis Bayonne; Phillips, Richard Odame; Adjei, Ohene; Ablordey, Anthony; Rigouts, Leen; Portaels, Françoise; Eddyani, Miriam; de Jong, Bouke C

    2014-02-01

    Buruli ulcer is an indolent, slowly progressing necrotizing disease of the skin caused by infection with Mycobacterium ulcerans. In the present study, we applied a redesigned technique to a vast panel of M. ulcerans disease isolates and clinical samples originating from multiple African disease foci in order to (i) gain fundamental insights into the population structure and evolutionary history of the pathogen and (ii) disentangle the phylogeographic relationships within the genetically conserved cluster of African M. ulcerans. Our analyses identified 23 different African insertion sequence element single nucleotide polymorphism (ISE-SNP) types that dominate in different areas where Buruli ulcer is endemic. These ISE-SNP types appear to be the initial stages of clonal diversification from a common, possibly ancestral ISE-SNP type. ISE-SNP types were found unevenly distributed over the greater West African hydrological drainage basins. Our findings suggest that geographical barriers bordering the basins to some extent prevented bacterial gene flow between basins and that this resulted in independent focal transmission clusters associated with the hydrological drainage areas. Different phylogenetic methods yielded two well-supported sister clades within the African ISE-SNP types. The ISE-SNP types from the "pan-African clade" were found to be widespread throughout Africa, while the ISE-SNP types of the "Gabonese/Cameroonian clade" were much rarer and found in a more restricted area, which suggested that the latter clade evolved more recently. Additionally, the Gabonese/Cameroonian clade was found to form a strongly supported monophyletic group with Papua New Guinean ISE-SNP type 8, which is unrelated to other Southeast Asian ISE-SNP types.

  13. Insertion Sequence Element Single Nucleotide Polymorphism Typing Provides Insights into the Population Structure and Evolution of Mycobacterium ulcerans across Africa

    PubMed Central

    Jordaens, Kurt; Bomans, Pieter; Leirs, Herwig; Durnez, Lies; Affolabi, Dissou; Sopoh, Ghislain; Aguiar, Julia; Phanzu, Delphin Mavinga; Kibadi, Kapay; Eyangoh, Sara; Manou, Louis Bayonne; Phillips, Richard Odame; Adjei, Ohene; Ablordey, Anthony; Rigouts, Leen; Portaels, Françoise; Eddyani, Miriam; de Jong, Bouke C.

    2014-01-01

    Buruli ulcer is an indolent, slowly progressing necrotizing disease of the skin caused by infection with Mycobacterium ulcerans. In the present study, we applied a redesigned technique to a vast panel of M. ulcerans disease isolates and clinical samples originating from multiple African disease foci in order to (i) gain fundamental insights into the population structure and evolutionary history of the pathogen and (ii) disentangle the phylogeographic relationships within the genetically conserved cluster of African M. ulcerans. Our analyses identified 23 different African insertion sequence element single nucleotide polymorphism (ISE-SNP) types that dominate in different areas where Buruli ulcer is endemic. These ISE-SNP types appear to be the initial stages of clonal diversification from a common, possibly ancestral ISE-SNP type. ISE-SNP types were found unevenly distributed over the greater West African hydrological drainage basins. Our findings suggest that geographical barriers bordering the basins to some extent prevented bacterial gene flow between basins and that this resulted in independent focal transmission clusters associated with the hydrological drainage areas. Different phylogenetic methods yielded two well-supported sister clades within the African ISE-SNP types. The ISE-SNP types from the “pan-African clade” were found to be widespread throughout Africa, while the ISE-SNP types of the “Gabonese/Cameroonian clade” were much rarer and found in a more restricted area, which suggested that the latter clade evolved more recently. Additionally, the Gabonese/Cameroonian clade was found to form a strongly supported monophyletic group with Papua New Guinean ISE-SNP type 8, which is unrelated to other Southeast Asian ISE-SNP types. PMID:24296504

  14. The Structure of a Sugar Transporter of the Glucose EIIC Superfamily Provides Insight into the Elevator Mechanism of Membrane Transport.

    PubMed

    McCoy, Jason G; Ren, Zhenning; Stanevich, Vitali; Lee, Jumin; Mitra, Sharmistha; Levin, Elena J; Poget, Sebastien; Quick, Matthias; Im, Wonpil; Zhou, Ming

    2016-06-07

    The phosphoenolpyruvate:carbohydrate phosphotransferase systems are found in bacteria, where they play central roles in sugar uptake and regulation of cellular uptake processes. Little is known about how the membrane-embedded components (EIICs) selectively mediate the passage of carbohydrates across the membrane. Here we report the functional characterization and 2.55-Å resolution structure of a maltose transporter, bcMalT, belonging to the glucose superfamily of EIIC transporters. bcMalT crystallized in an outward-facing occluded conformation, in contrast to the structure of another glucose superfamily EIIC, bcChbC, which crystallized in an inward-facing occluded conformation. The structures differ in the position of a structurally conserved substrate-binding domain that is suggested to play a central role in sugar transport. In addition, molecular dynamics simulations suggest a potential pathway for substrate entry from the periplasm into the bcMalT substrate-binding site. These results provide a mechanistic framework for understanding substrate recognition and translocation for the glucose superfamily EIIC transporters.

  15. Exploration of insights, opportunities and caveats provided by the X-ray structures of hSERT.

    PubMed

    Topiol, Sid; Bang-Andersen, Benny; Sanchez, Connie; Bøgesø, Klaus P

    2016-10-15

    The recently reported X-ray structures of the human serotonin (5-HT) transporter SERT with bound inhibitors open new opportunities for drug discovery at SERT, selectivity design with respect to other neurotransmitter sodium transporters, and enhanced understanding of the molecular events involved in SERT action. Through computational and structural analyses, we explore the binding and migration of 5-HT at SERT. Consistent with earlier studies of leucine migration at the bacterial homolog of SERT, LeuT, we find multiple potential 'stopover' sites for 5-HT binding at SERT including the two (transmembrane S1 and extracellular vestibule S2) seen in the binding of the SSRI (S)-citalopram (S-Cit) to SERT, as well as other sites. Docking studies reveal the possibility of both hetero- (S-Cit+5-HT) and homo-dimeric (5-HT+5-HT) co-binding at both these sites which may explain earlier published allosteric activity observations and provide novel design strategies. Comparisons with substrate bound X-ray structures of the dopamine transporter reveal a number of potential sources of selectivity, some of which may be 'artificial' including target based, species related, experimental design related, and ligand dependent examples including substrate versus inhibitor related features.

  16. Structure of the extracellular portion of CD46 provides insights into its interactions with complement proteins and pathogens.

    PubMed

    Persson, B David; Schmitz, Nikolaus B; Santiago, César; Zocher, Georg; Larvie, Mykol; Scheu, Ulrike; Casasnovas, José M; Stehle, Thilo

    2010-09-30

    The human membrane cofactor protein (MCP, CD46) is a central component of the innate immune system. CD46 protects autologous cells from complement attack by binding to complement proteins C3b and C4b and serving as a cofactor for their cleavage. Recent data show that CD46 also plays a role in mediating acquired immune responses, and in triggering autophagy. In addition to these physiologic functions, a significant number of pathogens, including select adenoviruses, measles virus, human herpes virus 6 (HHV-6), Streptococci, and Neisseria, use CD46 as a cell attachment receptor. We have determined the crystal structure of the extracellular region of CD46 in complex with the human adenovirus type 11 fiber knob. Extracellular CD46 comprises four short consensus repeats (SCR1-SCR4) that form an elongated structure resembling a hockey stick, with a long shaft and a short blade. Domains SCR1, SCR2 and SCR3 are arranged in a nearly linear fashion. Unexpectedly, however, the structure reveals a profound bend between domains SCR3 and SCR4, which has implications for the interactions with ligands as well as the orientation of the protein at the cell surface. This bend can be attributed to an insertion of five hydrophobic residues in a SCR3 surface loop. Residues in this loop have been implicated in interactions with complement, indicating that the bend participates in binding to C3b and C4b. The structure provides an accurate framework for mapping all known ligand binding sites onto the surface of CD46, thereby advancing an understanding of how CD46 acts as a receptor for pathogens and physiologic ligands of the immune system.

  17. The structure of Arabidopsis thaliana OST1 provides insights into the kinase regulation mechanism in response to osmotic stress.

    PubMed

    Yunta, Cristina; Martínez-Ripoll, Martín; Zhu, Jian-Kang; Albert, Armando

    2011-11-18

    SnRK [SNF1 (sucrose non-fermenting-1)-related protein kinase] 2.6 [open stomata 1 (OST1)] is well characterized at molecular and physiological levels to control stomata closure in response to water-deficit stress. OST1 is a member of a family of 10 protein kinases from Arabidopsis thaliana (SnRK2) that integrates abscisic acid (ABA)-dependent and ABA-independent signals to coordinate the cell response to osmotic stress. A subgroup of protein phosphatases type 2C binds OST1 and keeps the kinase dephosphorylated and inactive. Activation of OST1 relies on the ABA-dependent inhibition of the protein phosphatases type 2C and the subsequent self-phosphorylation of the kinase. The OST1 ABA-independent activation depends on a short sequence motif that is conserved among all the members of the SnRK2 family. However, little is known about the molecular mechanism underlying this regulation. The crystallographic structure of OST1 shows that ABA-independent regulation motif stabilizes the conformation of the kinase catalytically essential α C helix, and it provides the basis of the ABA-independent regulation mechanism for the SnRK2 family of protein kinases.

  18. The Structure of Arabidopsis thaliana OST1 Provides Insights into the Kinase Regulation Mechanism in Response to Osmotic Stress

    PubMed Central

    Yunta, Cristina; Martínez-Ripoll, Martín; Zhu, Jian-Kang; Albert, Armando

    2013-01-01

    SnRK [SNF1 (sucrose non-fermenting-1)-related protein kinase] 2.6 [open stomata 1 (OST1)] is well characterized at molecular and physiological levels to control stomata closure in response to water-deficit stress. OST1 is a member of a family of 10 protein kinases from Arabidopsis thaliana (SnRK2) that integrates abscisic acid (ABA)-dependent and ABA-independent signals to coordinate the cell response to osmotic stress. A subgroup of protein phosphatases type 2C binds OST1 and keeps the kinase dephosphorylated and inactive. Activation of OST1 relies on the ABA-dependent inhibition of the protein phosphatases type 2C and the subsequent self-phosphorylation of the kinase. The OST1 ABA-independent activation depends on a short sequence motif that is conserved among all the members of the SnRK2 family. However, little is known about the molecular mechanism underlying this regulation. The crystallographic structure of OST1 shows that ABA-independent regulation motif stabilizes the conformation of the kinase catalytically essential α C helix, and it provides the basis of the ABA-independent regulation mechanism for the SnRK2 family of protein kinases. PMID:21983340

  19. Spatially extensive microbial biogeography of the Indian Ocean provides insights into the unique community structure of a pristine coral atoll.

    PubMed

    Jeffries, Thomas C; Ostrowski, Martin; Williams, Rohan B; Xie, Chao; Jensen, Rachelle M; Grzymski, Joseph J; Senstius, Svend Jacob; Givskov, Michael; Hoeke, Ron; Philip, Gayle K; Neches, Russell Y; Drautz-Moses, Daniela I; Chénard, Caroline; Paulsen, Ian T; Lauro, Federico M

    2015-10-20

    Microorganisms act both as drivers and indicators of perturbations in the marine environment. In an effort to establish baselines to predict the response of marine habitats to environmental change, here we report a broad survey of microbial diversity across the Indian Ocean, including the first microbial samples collected in the pristine lagoon of Salomon Islands, Chagos Archipelago. This was the first large-scale ecogenomic survey aboard a private yacht employing a 'citizen oceanography' approach and tools and protocols easily adapted to ocean going sailboats. Our data highlighted biogeographic patterns in microbial community composition across the Indian Ocean. Samples from within the Salomon Islands lagoon contained a community which was different even from adjacent samples despite constant water exchange, driven by the dominance of the photosynthetic cyanobacterium Synechococcus. In the lagoon, Synechococcus was also responsible for driving shifts in the metatranscriptional profiles. Enrichment of transcripts related to photosynthesis and nutrient cycling indicated bottom-up controls of community structure. However a five-fold increase in viral transcripts within the lagoon during the day, suggested a concomitant top-down control by bacteriophages. Indeed, genome recruitment against Synechococcus reference genomes suggested a role of viruses in providing the ecological filter for determining the β-diversity patterns in this system.

  20. Spatially extensive microbial biogeography of the Indian Ocean provides insights into the unique community structure of a pristine coral atoll

    NASA Astrophysics Data System (ADS)

    Jeffries, Thomas C.; Ostrowski, Martin; Williams, Rohan B.; Xie, Chao; Jensen, Rachelle M.; Grzymski, Joseph J.; Senstius, Svend Jacob; Givskov, Michael; Hoeke, Ron; Philip, Gayle K.; Neches, Russell Y.; Drautz-Moses, Daniela I.; Chénard, Caroline; Paulsen, Ian T.; Lauro, Federico M.

    2015-10-01

    Microorganisms act both as drivers and indicators of perturbations in the marine environment. In an effort to establish baselines to predict the response of marine habitats to environmental change, here we report a broad survey of microbial diversity across the Indian Ocean, including the first microbial samples collected in the pristine lagoon of Salomon Islands, Chagos Archipelago. This was the first large-scale ecogenomic survey aboard a private yacht employing a ‘citizen oceanography’ approach and tools and protocols easily adapted to ocean going sailboats. Our data highlighted biogeographic patterns in microbial community composition across the Indian Ocean. Samples from within the Salomon Islands lagoon contained a community which was different even from adjacent samples despite constant water exchange, driven by the dominance of the photosynthetic cyanobacterium Synechococcus. In the lagoon, Synechococcus was also responsible for driving shifts in the metatranscriptional profiles. Enrichment of transcripts related to photosynthesis and nutrient cycling indicated bottom-up controls of community structure. However a five-fold increase in viral transcripts within the lagoon during the day, suggested a concomitant top-down control by bacteriophages. Indeed, genome recruitment against Synechococcus reference genomes suggested a role of viruses in providing the ecological filter for determining the β-diversity patterns in this system.

  1. Spatially extensive microbial biogeography of the Indian Ocean provides insights into the unique community structure of a pristine coral atoll

    PubMed Central

    Jeffries, Thomas C.; Ostrowski, Martin; Williams, Rohan B.; Xie, Chao; Jensen, Rachelle M.; Grzymski, Joseph J.; Senstius, Svend Jacob; Givskov, Michael; Hoeke, Ron; Philip, Gayle K.; Neches, Russell Y.; Drautz-Moses, Daniela I.; Chénard, Caroline; Paulsen, Ian T.; Lauro, Federico M.

    2015-01-01

    Microorganisms act both as drivers and indicators of perturbations in the marine environment. In an effort to establish baselines to predict the response of marine habitats to environmental change, here we report a broad survey of microbial diversity across the Indian Ocean, including the first microbial samples collected in the pristine lagoon of Salomon Islands, Chagos Archipelago. This was the first large-scale ecogenomic survey aboard a private yacht employing a ‘citizen oceanography’ approach and tools and protocols easily adapted to ocean going sailboats. Our data highlighted biogeographic patterns in microbial community composition across the Indian Ocean. Samples from within the Salomon Islands lagoon contained a community which was different even from adjacent samples despite constant water exchange, driven by the dominance of the photosynthetic cyanobacterium Synechococcus. In the lagoon, Synechococcus was also responsible for driving shifts in the metatranscriptional profiles. Enrichment of transcripts related to photosynthesis and nutrient cycling indicated bottom-up controls of community structure. However a five-fold increase in viral transcripts within the lagoon during the day, suggested a concomitant top-down control by bacteriophages. Indeed, genome recruitment against Synechococcus reference genomes suggested a role of viruses in providing the ecological filter for determining the β-diversity patterns in this system. PMID:26481089

  2. An exo-β-(1→3)-D-galactanase from Streptomyces sp. provides insights into type II arabinogalactan structure

    PubMed Central

    Ling, Naomi X.-Y.; Lee, Joanne; Ellis, Miriam; Liao, Ming-Long; Mau, Shaio-Lim; Guest, David; Janssen, Peter H.; Kováč, Pavol; Bacic, Antony; Pettolino, Filomena A.

    2012-01-01

    An exo-β-(1→3)-D-galactanase (SGalase1) that specifically cleaves the β-(1→3)-D-galactan backbone of arabinogalactan-proteins (AGPs) was isolated from culture filtrates of a soil Streptomyces sp. Internal peptide sequence information was used to clone and recombinantly express the gene in E. coli. The molecular mass of the isolated enzyme was ~45 kDa, similar to the 48.2 kDa mass predicted from the amino acid sequence. The pI, pH and temperature optima for the enzyme were ~7.45, 3.8 and 48 °C, respectively. The native and recombinant enzymes specifically hydrolysed β-(1→3)-D-galacto-oligo- or poly-saccharides from the upstream (non-reducing) end, typical of an exo-acting enzyme. A second homologous Streptomyces gene (SGalase2) was also cloned and expressed. SGalase2 was similar in size (47.9 kDa) and enzyme activity to SGalase1 but differed in its pH optimum (pH 5). Both SGalase1 and SGalase2 are predicted to belong to the CAZy glycosyl hydrolase family GH 43 based on activity, sequence homology and phylogenetic analysis. The Km and Vmax of the native exo-β-(1→3)-D-galactanase for de-arabinosylated gum arabic (dGA) were 19 mg/ml and 9.7 μmol D-Gal/min/mg protein, respectively. The activity of these enzymes is well suited for the study of type II galactan structures and provides an important tool for the investigation of the biological role of AGPs in plants. De-arabinosylated gum arabic (dGA) was used as a model to investigate the use of these enzymes in defining type II galactan structure. Exhaustive hydrolysis of dGA resulted in a limited number of oligosaccharide products with a trisaccharide of Gal2GlcA1 predominating. PMID:22464224

  3. Structural and Functional Analysis of the Globular Head Domain of p115 Provides Insight into Membrane Tethering

    PubMed Central

    An, Yu; Chen, Christine Y.; Moyer, Bryan; Rotkiewicz, Piotr; Elsliger, Marc-André; Godzik, Adam; Wilson, Ian A.; Balch, William E.

    2009-01-01

    Molecular tethers play a central role in the organization of the complex membrane architecture of eukaryotic cells. p115 is a ubiquitous, essential tether involved in vesicle transport and the structural organization of the exocytic pathway. We describe two crystal structures of the N-terminal domain of p115 at 2.0 Å resolution. The p115 structures show a novel α-solenoid architecture constructed of 12 armadillo-like, tether-repeat (TR), α-helical tripod motifs. We find that the H1 TR binds the Rab1 GTPase involved in ER to Golgi transport. Mutation of the H1 motif results in the dominant negative inhibition of ER to Golgi trafficking. We propose that the H1 helical tripod contributes to the assembly of Rab-dependent complexes responsible for the tether and SNARE-dependent fusion of membranes. PMID:19414022

  4. Structures of reduced and ligand-bound nitric oxide reductase provide insights into functional differences in respiratory enzymes.

    PubMed

    Sato, Nozomi; Ishii, Shoko; Sugimoto, Hiroshi; Hino, Tomoya; Fukumori, Yoshihiro; Sako, Yoshihiko; Shiro, Yoshitsugu; Tosha, Takehiko

    2014-07-01

    Nitric oxide reductase (NOR) catalyzes the generation of nitrous oxide (N2O) via the reductive coupling of two nitric oxide (NO) molecules at a heme/non-heme Fe center. We report herein on the structures of the reduced and ligand-bound forms of cytochrome c-dependent NOR (cNOR) from Pseudomonas aeruginosa at a resolution of 2.3-2.7 Å, to elucidate structure-function relationships in NOR, and compare them to those of cytochrome c oxidase (CCO) that is evolutionarily related to NOR. Comprehensive crystallographic refinement of the CO-bound form of cNOR suggested that a total of four atoms can be accommodated at the binuclear center. Consistent with this, binding of bulky acetaldoxime (CH3-CH=N-OH) to the binuclear center of cNOR was confirmed by the structural analysis. Active site reduction and ligand binding in cNOR induced only ∼0.5 Å increase in the heme/non-heme Fe distance, but no significant structural change in the protein. The highly localized structural change is consistent with the lack of proton-pumping activity in cNOR, because redox-coupled conformational changes are thought to be crucial for proton pumping in CCO. It also permits the rapid decomposition of cytotoxic NO in denitrification. In addition, the shorter heme/non-heme Fe distance even in the bulky ligand-bound form of cNOR (∼4.5 Å) than the heme/Cu distance in CCO (∼5 Å) suggests the ability of NOR to maintain two NO molecules within a short distance in the confined space of the active site, thereby facilitating N-N coupling to produce a hyponitrite intermediate for the generation of N2O.

  5. The human otubain2-ubiquitin structure provides insights into the cleavage specificity of poly-ubiquitin-linkages.

    PubMed

    Altun, Mikael; Walter, Thomas S; Kramer, Holger B; Herr, Patrick; Iphöfer, Alexander; Boström, Johan; David, Yael; Komsany, Alia; Ternette, Nicola; Navon, Ami; Stuart, David I; Ren, Jingshan; Kessler, Benedikt M

    2015-01-01

    Ovarian tumor domain containing proteases cleave ubiquitin (Ub) and ubiquitin-like polypeptides from proteins. Here we report the crystal structure of human otubain 2 (OTUB2) in complex with a ubiquitin-based covalent inhibitor, Ub-Br2. The ubiquitin binding mode is oriented differently to how viral otubains (vOTUs) bind ubiquitin/ISG15, and more similar to yeast and mammalian OTUs. In contrast to OTUB1 which has exclusive specificity towards Lys48 poly-ubiquitin chains, OTUB2 cleaves different poly-Ub linked chains. N-terminal tail swapping experiments between OTUB1 and OTUB2 revealed how the N-terminal structural motifs in OTUB1 contribute to modulating enzyme activity and Ub-chain selectivity, a trait not observed in OTUB2, supporting the notion that OTUB2 may affect a different spectrum of substrates in Ub-dependent pathways.

  6. The structure of the core NuRD repression complex provides insights into its interaction with chromatin

    PubMed Central

    Millard, Christopher J; Varma, Niranjan; Saleh, Almutasem; Morris, Kyle; Watson, Peter J; Bottrill, Andrew R; Fairall, Louise; Smith, Corinne J; Schwabe, John WR

    2016-01-01

    The NuRD complex is a multi-protein transcriptional corepressor that couples histone deacetylase and ATP-dependent chromatin remodelling activities. The complex regulates the higher-order structure of chromatin, and has important roles in the regulation of gene expression, DNA damage repair and cell differentiation. HDACs 1 and 2 are recruited by the MTA1 corepressor to form the catalytic core of the complex. The histone chaperone protein RBBP4, has previously been shown to bind to the carboxy-terminal tail of MTA1. We show that MTA1 recruits a second copy of RBBP4. The crystal structure reveals an extensive interface between MTA1 and RBBP4. An EM structure, supported by SAXS and crosslinking, reveals the architecture of the dimeric HDAC1:MTA1:RBBP4 assembly which forms the core of the NuRD complex. We find evidence that in this complex RBBP4 mediates interaction with histone H3 tails, but not histone H4, suggesting a mechanism for recruitment of the NuRD complex to chromatin. DOI: http://dx.doi.org/10.7554/eLife.13941.001 PMID:27098840

  7. The structure of the core NuRD repression complex provides insights into its interaction with chromatin.

    PubMed

    Millard, Christopher J; Varma, Niranjan; Saleh, Almutasem; Morris, Kyle; Watson, Peter J; Bottrill, Andrew R; Fairall, Louise; Smith, Corinne J; Schwabe, John W R

    2016-04-21

    The NuRD complex is a multi-protein transcriptional corepressor that couples histone deacetylase and ATP-dependent chromatin remodelling activities. The complex regulates the higher-order structure of chromatin, and has important roles in the regulation of gene expression, DNA damage repair and cell differentiation. HDACs 1 and 2 are recruited by the MTA1 corepressor to form the catalytic core of the complex. The histone chaperone protein RBBP4, has previously been shown to bind to the carboxy-terminal tail of MTA1. We show that MTA1 recruits a second copy of RBBP4. The crystal structure reveals an extensive interface between MTA1 and RBBP4. An EM structure, supported by SAXS and crosslinking, reveals the architecture of the dimeric HDAC1:MTA1:RBBP4 assembly which forms the core of the NuRD complex. We find evidence that in this complex RBBP4 mediates interaction with histone H3 tails, but not histone H4, suggesting a mechanism for recruitment of the NuRD complex to chromatin.

  8. The structural comparison between membrane-associated human carbonic anhydrases provides insights into drug design of selective inhibitors.

    PubMed

    Alterio, Vincenzo; Pan, Peiwen; Parkkila, Seppo; Buonanno, Martina; Supuran, Claudiu T; Monti, Simona M; De Simone, Giuseppina

    2014-07-01

    Carbonic anhydrase isoform XIV (CA XIV) is the last member of the human (h) CA family discovered so far, being localized in brain, kidneys, colon, small intestine, urinary bladder, liver, and spinal cord. It has recently been described as a possible drug target for treatment of epilepsy, some retinopathies as well as some skin tumors. Human carbonic anhydrase (hCA) XIV is a membrane-associated protein consisting of an N-terminal extracellular domain, a putative transmembrane region, and a small cytoplasmic tail. In this article, we report the expression, purification, and the crystallographic structure of the entire extracellular domain of this enzyme. The analysis of the structure revealed the typical α-CA fold, in which a 10-stranded β-sheet forms the core of the molecule, while the comparison with all the other membrane associated isoforms (hCAs IV, IX, and XII) allowed to identify the diverse oligomeric arrangement and the sequence and structural differences observed in the region 127-136 as the main factors to consider in the design of selective inhibitors for each one of the membrane associated α-CAs.

  9. The Crystal Structure of Peroxiredoxin Asp f3 Provides Mechanistic Insight into Oxidative Stress Resistance and Virulence of Aspergillus fumigatus.

    PubMed

    Hillmann, Falk; Bagramyan, Karine; Straßburger, Maria; Heinekamp, Thorsten; Hong, Teresa B; Bzymek, Krzysztof P; Williams, John C; Brakhage, Axel A; Kalkum, Markus

    2016-09-14

    Invasive aspergillosis and other fungal infections occur in immunocompromised individuals, including patients who received blood-building stem cell transplants, patients with chronic granulomatous disease (CGD), and others. Production of reactive oxygen species (ROS) by immune cells, which incidentally is defective in CGD patients, is considered to be a fundamental process in inflammation and antifungal immune response. Here we show that the peroxiredoxin Asp f3 of Aspergillus fumigatus inactivates ROS. We report the crystal structure and the catalytic mechanism of Asp f3, a two-cysteine type peroxiredoxin. The latter exhibits a thioredoxin fold and a homodimeric structure with two intermolecular disulfide bonds in its oxidized state. Replacement of the Asp f3 cysteines with serine residues retained its dimeric structure, but diminished Asp f3's peroxidase activity, and extended the alpha-helix with the former peroxidatic cysteine residue C61 by six residues. The asp f3 deletion mutant was sensitive to ROS, and this phenotype was rescued by ectopic expression of Asp f3. Furthermore, we showed that deletion of asp f3 rendered A. fumigatus avirulent in a mouse model of pulmonary aspergillosis. The conserved expression of Asp f3 homologs in medically relevant molds and yeasts prompts future evaluation of Asp f3 as a potential therapeutic target.

  10. The Crystal Structure of Peroxiredoxin Asp f3 Provides Mechanistic Insight into Oxidative Stress Resistance and Virulence of Aspergillus fumigatus

    PubMed Central

    Hillmann, Falk; Bagramyan, Karine; Straßburger, Maria; Heinekamp, Thorsten; Hong, Teresa B.; Bzymek, Krzysztof P.; Williams, John C.; Brakhage, Axel A.; Kalkum, Markus

    2016-01-01

    Invasive aspergillosis and other fungal infections occur in immunocompromised individuals, including patients who received blood-building stem cell transplants, patients with chronic granulomatous disease (CGD), and others. Production of reactive oxygen species (ROS) by immune cells, which incidentally is defective in CGD patients, is considered to be a fundamental process in inflammation and antifungal immune response. Here we show that the peroxiredoxin Asp f3 of Aspergillus fumigatus inactivates ROS. We report the crystal structure and the catalytic mechanism of Asp f3, a two-cysteine type peroxiredoxin. The latter exhibits a thioredoxin fold and a homodimeric structure with two intermolecular disulfide bonds in its oxidized state. Replacement of the Asp f3 cysteines with serine residues retained its dimeric structure, but diminished Asp f3’s peroxidase activity, and extended the alpha-helix with the former peroxidatic cysteine residue C61 by six residues. The asp f3 deletion mutant was sensitive to ROS, and this phenotype was rescued by ectopic expression of Asp f3. Furthermore, we showed that deletion of asp f3 rendered A. fumigatus avirulent in a mouse model of pulmonary aspergillosis. The conserved expression of Asp f3 homologs in medically relevant molds and yeasts prompts future evaluation of Asp f3 as a potential therapeutic target. PMID:27624005

  11. Structure of FliM Provides Insight into Assembly of the Switch Complex in the Bacterial Flagella Motor

    SciTech Connect

    Park,S.; Lowder, B.; Bilwes, A.; Blair, D.; Crane, B.

    2006-01-01

    Bacteria switch the direction their flagella rotate to control movement. FliM, along with FliN and FliG, compose a complex in the motor that, upon binding phosphorylated CheY, reverses the sense of flagellar rotation. The 2.0- Angstroms resolution structure of the FliM middle domain (FliMM) from Thermotoga maritima reveals a pseudo-2-fold symmetric topology similar to the CheY phosphatases CheC and CheX. A variable structural element, which, in CheC, mediates binding to CheD ({alpha}2') and, in CheX, mediates dimerization ({beta}x), has a truncated structure unique to FliM ({alpha}2'). An exposed helix of FliMM ({alpha}1) does not contain the catalytic residues of CheC and CheX but does include positions conserved in FliM sequences. Cross-linking experiments with site-directed cysteine mutants show that FliM self-associates through residues on {alpha}1 and {alpha}2'. CheY activated by BeF3- binds to FliM with {approx}40-fold higher affinity than CheY (Kd = 0.04 {micro}M vs. 2 {micro}M). Mapping residue conservation, suppressor mutation sites, binding data, and deletion analysis onto the FliMM surface defines regions important for contacts with the stator-interacting protein FliG and for either counterclockwise or clockwise rotation. Association of 33-35 FliM subunits would generate a 44- to 45-nm-diameter disk, consistent with the known dimensions of the C-ring. The localization of counterclockwise- and clockwise-biasing mutations to distinct surfaces suggests that the binding of phosphorylated CheY cooperatively realigns FliM around the ring.

  12. Structure of an Essential Type IV Pilus Biogenesis Protein Provides Insights into Pilus and Type II Secretion Systems

    PubMed Central

    Yamagata, Atsushi; Milgotina, Ekaterina; Scanlon, Karen; Craig, Lisa; Tainer, John A.; Donnenberg, Michael S.

    2012-01-01

    Type IV pili (T4Ps) are long cell surface filaments, essential for microcolony formation, tissue adherence, motility, transformation, and virulence by human pathogens. The enteropathogenic E. colibundle-forming pilus (BFP) is a prototypic T4P assembled and powered by BfpD, a conserved GspE secretion superfamily ATPase held by inner membrane proteins BfpC andBfpE, a GspF-family membrane protein. Although the T4P assembly machinery shares similarity with type II secretion (T2S) systems, the structural biochemistry of the T4P machine has been obscure. Here, we report the crystal structure of the two-domain BfpC cytoplasmic region (N-BfpC), responsible for binding to ATPase BfpD and membrane protein BfpE. The N-BfpC structure reveals a prominent central cleft between two α/β domains. Despite negligible sequence similarity, N-BfpC resembles PilM, a cytoplasmic T4P biogenesis protein.Yet surprisingly, N-BfpC has far greaterstructural similarity to T2S component EpsL, with which it also shares virtually no sequence identity. The C-terminus of the cytoplasmic domain, which leads to the transmembrane segment not present in the crystal structure, exits N-BfpC at a positively-charged surface that most likely interacts with the inner membrane, positioning its central cleft for interactions with other Bfp components.Point mutations in surface-exposed N-BfpC residues predicted to be critical for interactions among BfpC, BfpE and BfpD disrupt pilus biogenesis without precluding interactions with BfpE and BfpD and without affecting BfpD ATPase activity. These results illuminate the relationships between T4P biogenesis and T2S systems,imply that subtle changes in component residue interactions can have profound effects on function and pathogenesis, and suggest that T4P systems may be disrupted by inhibitors that donot preclude component assembly. PMID:22387466

  13. Structures of the yeast dynamin-like GTPase Sey1p provide insight into homotypic ER fusion

    PubMed Central

    Yan, Liming; Sun, Sha; Wang, Wei; Shi, Juanming; Hu, Xiaoyu; Wang, Shiyan; Su, Dan; Lou, Zhiyong

    2015-01-01

    Homotypic membrane fusion of the endoplasmic reticulum is mediated by dynamin-like guanosine triphosphatases (GTPases), which include atlastin (ATL) in metazoans and Sey1p in yeast. In this paper, we determined the crystal structures of the cytosolic domain of Sey1p derived from Candida albicans. The structures reveal a stalk-like, helical bundle domain following the GTPase, which represents a previously unidentified configuration of the dynamin superfamily. This domain is significantly longer than that of ATL and critical for fusion. Sey1p forms a side-by-side dimer in complex with GMP-PNP or GDP/AlF4− but is monomeric with GDP. Surprisingly, Sey1p could mediate fusion without GTP hydrolysis, even though fusion was much more efficient with GTP. Sey1p was able to replace ATL in mammalian cells, and the punctate localization of Sey1p was dependent on its GTPase activity. Despite the common function of fusogenic GTPases, our results reveal unique features of Sey1p. PMID:26370501

  14. The crystal structure of ribonuclease A in complex with thymidine-3'-monophosphate provides further insight into ligand binding.

    PubMed

    Doucet, Nicolas; Jayasundera, Thusitha B; Simonović, Miljan; Loria, J Patrick

    2010-08-15

    Thymidine-3'-monophosphate (3'-TMP) is a competitive inhibitor analogue of the 3'-CMP and 3'-UMP natural product inhibitors of bovine pancreatic ribonuclease A (RNase A). Isothermal titration calorimetry experiments show that 3'-TMP binds the enzyme with a dissociation constant (K(d)) of 15 microM making it one of the strongest binding members of the five natural bases found in nucleic acids (A, C, G, T, and U). To further investigate the molecular properties of this potent natural affinity, we have determined the crystal structure of bovine pancreatic RNase A in complex with 3'-TMP at 1.55 A resolution and we have performed NMR binding experiments with 3'-CMP and 3'-TMP. Our results show that binding of 3'-TMP is very similar to other natural and non-natural pyrimidine ligands, demonstrating that single nucleotide affinity is independent of the presence or absence of a 2'-hydroxyl on the ribose moiety of pyrimidines and suggesting that the pyrimidine binding subsite of RNase A is not a significant contributor of inhibitor discrimination. Accumulating evidence suggests that very subtle structural, chemical, and potentially motional variations contribute to ligand discrimination in this enzyme.

  15. The structure of the pleiotropic transcription regulator CodY provides insight into its GTP-sensing mechanism

    PubMed Central

    Han, Ah-reum; Kang, Hye-Ri; Son, Jonghyeon; Kwon, Do Hoon; Kim, Sulhee; Lee, Woo Cheol; Song, Hyun Kyu; Song, Moon Jung; Hwang, Kwang Yeon

    2016-01-01

    GTP and branched-chain amino acids (BCAAs) are metabolic sensors that are indispensable for the determination of the metabolic status of cells. However, their molecular sensing mechanism remains unclear. CodY is a unique global transcription regulator that recognizes GTP and BCAAs as specific signals and affects expression of more than 100 genes associated with metabolism. Herein, we report the first crystal structures of the full-length CodY complex with sensing molecules and describe their functional states. We observed two different oligomeric states of CodY: a dimeric complex of CodY from Staphylococcus aureus with the two metabolites GTP and isoleucine, and a tetrameric form (apo) of CodY from Bacillus cereus. Notably, the tetrameric state shows in an auto-inhibitory manner by blocking the GTP-binding site, whereas the binding sites of GTP and isoleucine are clearly visible in the dimeric state. The GTP is located at a hinge site between the long helical region and the metabolite-binding site. Together, data from structural and electrophoretic mobility shift assay analyses improve understanding of how CodY senses GTP and operates as a DNA-binding protein and a pleiotropic transcription regulator. PMID:27596595

  16. Structure and mutational analysis of the PhoN protein of Salmonella typhimurium provide insight into mechanistic details.

    PubMed

    Makde, Ravindra D; Mahajan, Suresh K; Kumar, Vinay

    2007-02-27

    The Salmonella typhimurium PhoN protein is a nonspecific acid phosphatase and belongs to the phosphatidic acid phosphatase type 2 (PAP2) superfamily. We report here the crystal structures of phosphate-bound PhoN, the PhoN-tungstate complex, and the T159D mutant of PhoN along with functional characterization of three mutants: L39T, T159D, and D201N. Invariant active site residues, Lys-123, Arg-130, Ser-156, Gly-157, His-158, and Arg-191, interact with phosphate and tungstate oxyanions. Ser-156 also accepts a hydrogen bond from Thr-159. The T159D mutation, surprisingly, severely diminishes phosphatase activity, apparently by disturbing the active site scaffold: Arg-191 is swung out of the active site resulting in conformational changes in His-158 and His-197 residues. Our results reveal a hitherto unknown functional role of Arg-191, namely, restricting the active conformation of catalytic His-158 and His-197 residues. Consistent with the conserved nature of Asp-201 in the PAP2 superfamily, the D201N mutation completely abolished phosphatase activity. On the basis of this observation and in silico analysis we suggest that the crucial mechanistic role of Asp-201 is to stabilize the positive charge on the phosphohistidine intermediate generated by the transfer of phosphoryl to the nucleophile, His-197, located within hydrogen bond distance to the invariant Asp-201. This is in contrast to earlier suggestions that Asp-201 stabilizes His-197 and the His197-Asp201 dyad facilitates formation of the phosphoenzyme intermediate through a charge-relay system. Finally, the L39T mutation in the conserved polyproline motif (39LPPPP43) of dimeric PhoN leads to a marginal reduction in activity, in contrast to the nearly 50-fold reduction observed for monomeric Prevotella intermedia acid phosphatase, suggesting that the varying quaternary structure of PhoN orthologues may have functional significance.

  17. Genome-Wide Analyses of Individual Strongyloides stercoralis (Nematoda: Rhabditoidea) Provide Insights into Population Structure and Reproductive Life Cycles

    PubMed Central

    Aung, Myo Pa Pa Thet Hnin Htwe; Afrin, Tanzila; Nagayasu, Eiji; Tanaka, Ryusei; Higashiarakawa, Miwa; Win, Kyu Kyu; Hirata, Tetsuo; Htike, Wah Win; Fujita, Jiro; Maruyama, Haruhiko

    2016-01-01

    The helminth Strongyloides stercoralis, which is transmitted through soil, infects 30–100 million people worldwide. S. stercoralis reproduces sexually outside the host as well as asexually within the host, which causes a life-long infection. To understand the population structure and transmission patterns of this parasite, we re-sequenced the genomes of 33 individual S. stercoralis nematodes collected in Myanmar (prevalent region) and Japan (non-prevalent region). We utilised a method combining whole genome amplification and next-generation sequencing techniques to detect 298,202 variant positions (0.6% of the genome) compared with the reference genome. Phylogenetic analyses of SNP data revealed an unambiguous geographical separation and sub-populations that correlated with the host geographical origin, particularly for the Myanmar samples. The relatively higher heterozygosity in the genomes of the Japanese samples can possibly be explained by the independent evolution of two haplotypes of diploid genomes through asexual reproduction during the auto-infection cycle, suggesting that analysing heterozygosity is useful and necessary to infer infection history and geographical prevalence. PMID:28033376

  18. Genetic structure of Populus hybrid zone along the Irtysh River provides insight into plastid-nuclear incompatibility

    PubMed Central

    Zeng, Yan-Fei; Zhang, Jian-Guo; Duan, Ai-Guo; Abuduhamiti, Bawerjan

    2016-01-01

    In plants, the maintenance of species integrity despite hybridization has often been explained by the co-adaption of nuclear gene complexes. However, the interaction between plastid and nuclear sub-genomes has been underestimated. Here, we analyzed the genetic structure of a Populus alba and P. tremula hybrid zone along the Irtysh River system in the Altai region, northwest China, using both nuclear microsatellites and plastid DNA sequences. We found high interspecific differentiation, although the hybrid P. × canescens was prevalent. Bayesian inference classified most hybrids into F1, followed by a few back-crosses to P. alba, and fewer F2 hybrids and back-crosses to P. tremula, indicating a few introgressions but preference toward P. alba. When plastid haplotypes in parental species were distinct, P. × canescens carried the haplotypes of both parents, but showed significant linkage between intraspecific haplotype and nuclear genotypes at several microsatellite loci. Selection, rather than migration and assortative mating, might have contributed to such plastid-nuclear disequilibria. By removing later-generated hybrids carrying interspecific combinations of haplotype and nuclear genotypes, plastid-nuclear incompatibility has greatly limited the gene exchange between P. alba and P. tremula via backcrossing with hybrids, demonstrating a significant association between plastid haplotype and the proportion of nuclear admixture. PMID:27306416

  19. Genetic structure of Populus hybrid zone along the Irtysh River provides insight into plastid-nuclear incompatibility.

    PubMed

    Zeng, Yan-Fei; Zhang, Jian-Guo; Duan, Ai-Guo; Abuduhamiti, Bawerjan

    2016-06-16

    In plants, the maintenance of species integrity despite hybridization has often been explained by the co-adaption of nuclear gene complexes. However, the interaction between plastid and nuclear sub-genomes has been underestimated. Here, we analyzed the genetic structure of a Populus alba and P. tremula hybrid zone along the Irtysh River system in the Altai region, northwest China, using both nuclear microsatellites and plastid DNA sequences. We found high interspecific differentiation, although the hybrid P. × canescens was prevalent. Bayesian inference classified most hybrids into F1, followed by a few back-crosses to P. alba, and fewer F2 hybrids and back-crosses to P. tremula, indicating a few introgressions but preference toward P. alba. When plastid haplotypes in parental species were distinct, P. × canescens carried the haplotypes of both parents, but showed significant linkage between intraspecific haplotype and nuclear genotypes at several microsatellite loci. Selection, rather than migration and assortative mating, might have contributed to such plastid-nuclear disequilibria. By removing later-generated hybrids carrying interspecific combinations of haplotype and nuclear genotypes, plastid-nuclear incompatibility has greatly limited the gene exchange between P. alba and P. tremula via backcrossing with hybrids, demonstrating a significant association between plastid haplotype and the proportion of nuclear admixture.

  20. Kinetic analyses of the magnesium chelatase provide insights into the mechanism, structure, and formation of the complex.

    PubMed

    Sawicki, Artur; Willows, Robert D

    2008-11-14

    The metabolic pathway known as (bacterio)chlorophyll biosynthesis is initiated by magnesium chelatase (BchI, BchD, BchH). This first step involves insertion of magnesium into protoporphyrin IX (proto), a process requiring ATP hydrolysis. Structural information shows that the BchI and BchD subunits form a double hexameric enzyme complex, whereas BchH binds proto and can be purified as BchH-proto. We utilized the Rhodobacter capsulatus magnesium chelatase subunits using continuous magnesium chelatase assays and treated the BchD subunit as the enzyme with both BchI and BchH-proto as substrates. Michaelis-Menten kinetics was observed with the BchI subunit, whereas the BchH subunit exhibited sigmoidal kinetics (Hill coefficient of 1.85). The BchI.BchD complex had intrinsic ATPase activity, and addition of BchH greatly increased ATPase activity. This was concentration-dependent and gave sigmoidal kinetics, indicating there is more than one binding site for the BchH subunit on the BchI.BchD complex. ATPase activity was approximately 40-fold higher than magnesium chelatase activity and continued despite cessation of magnesium chelation, implying one or more secondary roles for ATP hydrolysis and possibly an as yet unknown switch required to terminate ATPase activity. One of the secondary roles for BchH-stimulated ATP hydrolysis by a BchI.BchD complex is priming of BchH to facilitate correct binding of proto to BchH in a form capable of participating in magnesium chelation. This porphyrin binding is the rate-limiting step in catalysis. These data suggest that ATP hydrolysis by the BchI.BchD complex causes a series of conformational changes in BchH to effect substrate binding, magnesium chelation, and product release.

  1. THE STRUCTURE OF THE CRISPR-ASSOCIATED PROTEIN CSA3 PROVIDES INSIGHT INTO REGULATION OF THE CRISPR/CAS SYSTEM

    PubMed Central

    Lintner, Nathanael G.; Frankel, Kenneth A.; Tsutakawa, Susan E.; Alsbury, Donald L.; Copié, Valérie; Young, Mark J.; Tainer, John A.; Lawrence, C. Martin

    2015-01-01

    Adaptive immune systems have recently been recognized in prokaryotic organisms where, in response to viral infection, they incorporate short fragments of invader-derived DNA into loci called Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs). In subsequent infections, the CRISPR loci are transcribed and processed into guide sequences for the neutralization of the invading RNA or DNA. The CRISPR-associated protein machinery (Cas) lies at the heart of this process, yet many of the molecular details of the CRISPR/Cas system remain to be elucidated. Here we report the first structure of Csa3, a CRISPR-associated protein from Sulfolobus solfataricus (Sso1445), which reveals a dimeric two-domain protein. The N-terminal domain is a unique variation on the di-nucleotide binding-domain that orchestrates dimer formation. In addition, it utilizes two conserved sequence motifs (Thr-h-Gly-Phe-(Asn/Asp)-Glu-X4-Arg and Leu-X2-Gly-h-Arg) to construct a 2-fold symmetric pocket on the dimer axis. This pocket is likely to represent a regulatory ligand-binding site. The N-terminal domain is fused to a C-terminal MarR-like winged helix-turn-helix domain that is expected to be involved in DNA recognition. Overall, the unique domain architecture of Csa3 suggests a transcriptional regulator under allosteric control of the N-terminal domain. Alternatively, Csa3 may function in a larger complex, with the conserved cleft participating in protein-protein or protein-nucleic acid interactions. A similar N-terminal domain is also identified in Csx1, a second CRISPR associated protein family of unknown function. PMID:21093452

  2. Crystal Structure and Comparative Sequence Analysis of GmhA from Colwellia psychrerythraea Strain 34H Provides Insight into Functional Similarity with DiaA

    PubMed Central

    Do, Hackwon; Yun, Ji-Sook; Lee, Chang Woo; Choi, Young Jun; Kim, Hye-Yeon; Kim, Youn-Jung; Park, Hyun; Chang, Jeong Ho; Lee, Jun Hyuck

    2015-01-01

    The psychrophilic organism Colwellia psychrerythraea strain 34H produces extracellular polysaccharide substances to tolerate cold environments. Sedoheptulose 7-phosphate isomerase (GmhA) is essential for producing d-glycero-d-mannoheptose 7-phosphate, a key mediator in the lipopolysaccharide biosynthetic pathway. We determined the crystal structure of GmhA from C. psychrerythraea strain 34H (CpsGmhA, UniProtKB code: Q47VU0) at a resolution of 2.8 Å. The tetrameric structure is similar to that of homologous GmhA structures. Interestingly, one of the catalytic residues, glutamate, which has been reported to be critical for the activity of other homologous GmhA enzymes, is replaced by a glutamine residue in the CpsGmhA protein. We also found differences in the conformations of several other catalytic residues. Extensive structural and sequence analyses reveal that CpsGmhA shows high similarity to Escherichia coli DnaA initiator-associating protein A (DiaA). Therefore, the CpsGmhA structure reported here may provide insight into the structural and functional correlations between GmhA and DiaA among specific microorganisms. PMID:26612680

  3. Reannotation and extended community resources for the genome of the non-seed plant Physcomitrella patens provide insights into the evolution of plant gene structures and functions

    PubMed Central

    2013-01-01

    Background The moss Physcomitrella patens as a model species provides an important reference for early-diverging lineages of plants and the release of the genome in 2008 opened the doors to genome-wide studies. The usability of a reference genome greatly depends on the quality of the annotation and the availability of centralized community resources. Therefore, in the light of accumulating evidence for missing genes, fragmentary gene structures, false annotations and a low rate of functional annotations on the original release, we decided to improve the moss genome annotation. Results Here, we report the complete moss genome re-annotation (designated V1.6) incorporating the increased transcript availability from a multitude of developmental stages and tissue types. We demonstrate the utility of the improved P. patens genome annotation for comparative genomics and new extensions to the cosmoss.org resource as a central repository for this plant “flagship” genome. The structural annotation of 32,275 protein-coding genes results in 8387 additional loci including 1456 loci with known protein domains or homologs in Plantae. This is the first release to include information on transcript isoforms, suggesting alternative splicing events for at least 10.8% of the loci. Furthermore, this release now also provides information on non-protein-coding loci. Functional annotations were improved regarding quality and coverage, resulting in 58% annotated loci (previously: 41%) that comprise also 7200 additional loci with GO annotations. Access and manual curation of the functional and structural genome annotation is provided via the http://www.cosmoss.org model organism database. Conclusions Comparative analysis of gene structure evolution along the green plant lineage provides novel insights, such as a comparatively high number of loci with 5’-UTR introns in the moss. Comparative analysis of functional annotations reveals expansions of moss house-keeping and metabolic genes

  4. Crystal Structures of the Extracellular Domain from PepT1 and PepT2 Provide Novel Insights into Mammalian Peptide Transport.

    PubMed

    Beale, John H; Parker, Joanne L; Samsudin, Firdaus; Barrett, Anne L; Senan, Anish; Bird, Louise E; Scott, David; Owens, Raymond J; Sansom, Mark S P; Tucker, Stephen J; Meredith, David; Fowler, Philip W; Newstead, Simon

    2015-10-06

    Mammals obtain nitrogen via the uptake of di- and tri-peptides in the gastrointestinal tract through the action of PepT1 and PepT2, which are members of the POT family of proton-coupled oligopeptide transporters. PepT1 and PepT2 also play an important role in drug transport in the human body. Recent crystal structures of bacterial homologs revealed a conserved peptide-binding site and mechanism of transport. However, a key structural difference exists between bacterial and mammalian homologs with only the latter containing a large extracellular domain, the function of which is currently unknown. Here, we present the crystal structure of the extracellular domain from both PepT1 and PepT2 that reveal two immunoglobulin-like folds connected in tandem, providing structural insight into mammalian peptide transport. Functional and biophysical studies demonstrate that these domains interact with the intestinal protease trypsin, suggesting a role in clustering proteolytic activity to the site of peptide transport in eukaryotic cells.

  5. Can Economics Provide Insights into Trust Infrastructure?

    NASA Astrophysics Data System (ADS)

    Vishik, Claire

    Many security technologies require infrastructure for authentication, verification, and other processes. In many cases, viable and innovative security technologies are never adopted on a large scale because the necessary infrastructure is slow to emerge. Analyses of such technologies typically focus on their technical flaws, and research emphasizes innovative approaches to stronger implementation of the core features. However, an observation can be made that in many cases the success of adoption pattern depends on non-technical issues rather than technology-lack of economic incentives, difficulties in finding initial investment, inadequate government support. While a growing body of research is dedicated to economics of security and privacy in general, few theoretical studies in this area have been completed, and even fewer that look at the economics of “trust infrastructure” beyond simple “cost of ownership” models. This exploratory paper takes a look at some approaches in theoretical economics to determine if they can provide useful insights into security infrastructure technologies and architectures that have the best chance to be adopted. We attempt to discover if models used in theoretical economics can help inform technology developers of the optimal business models that offer a better chance for quick infrastructure deployment.

  6. Crystal structure of vaccinia virus mRNA capping enzyme provides insights into the mechanism and evolution of the capping apparatus

    PubMed Central

    Kyrieleis, Otto J.P.; Chang, Jonathan; de la Peña, Marcos; Shuman, Stewart; Cusack, Stephen

    2014-01-01

    Summary Vaccinia virus capping enzyme is a heterodimer of D1 (844-aa) and D12 (287-aa) polypeptides that executes all three steps in m7GpppRNA synthesis. The D1 subunit comprises an N-terminal RNA triphosphatase (TPase)–guanylyltransferase (GTase) module and a C-terminal guanine-N7-methyltransferase (MTase) module. The D12 subunit binds and allosterically stimulates the MTase module. Crystal structures of the complete D1•D12 heterodimer disclose the TPase and GTase as members of the triphosphate tunnel metalloenzyme and covalent nucleotidyltransferase superfamilies, respectively, albeit with distinctive active site features. An extensive TPase-GTase interface clamps the GTase nucleotidyltransferase and OB domains in a closed conformation around GTP. Mutagenesis confirms the importance of the TPase-GTase interface for GTase activity. The D1•D12 structure complements and rationalizes four decades of biochemical studies of this enzyme (the first capping enzyme to be purified and characterized) and provides new insights to the origins of the capping systems of other large DNA viruses. PMID:24607143

  7. Random and site-specific mutagenesis of the Helicobacter pylori ferric uptake regulator provides insight into Fur structure-function relationships.

    PubMed

    Gilbreath, Jeremy J; Pich, Oscar Q; Benoit, Stéphane L; Besold, Angelique N; Cha, Jeong-Heon; Maier, Robert J; Michel, Sarah L J; Maynard, Ernest L; Merrell, D Scott

    2013-07-01

    The ferric uptake regulator (Fur) of Helicobacter pylori is a global regulator that is important for colonization and survival within the gastric mucosa. H. pylori Fur is unique in its ability to activate and repress gene expression in both the iron-bound (Fe-Fur) and apo forms (apo-Fur). In the current study we combined random and site-specific mutagenesis to identify amino acid residues important for both Fe-Fur and apo-Fur function. We identified 25 mutations that affected Fe-Fur repression and 23 mutations that affected apo-Fur repression, as determined by transcriptional analyses of the Fe-Fur target gene amiE, and the apo-Fur target gene, pfr. In addition, eight of these mutations also significantly affected levels of Fur in the cell. Based on regulatory phenotypes, we selected several representative mutations to characterize further. Of those selected, we purified the wild-type (HpFurWT) and three mutant Fur proteins (HpFurE5A, HpFurA92T and HpFurH134Y), which represent mutations in the N-terminal extension, the regulatory metal binding site (S2) and the structural metal binding site (S3) respectively. Purified proteins were evaluated for secondary structure by circular dichroism spectroscopy, iron-binding by atomic absorption spectrophotometry, oligomerization in manganese-substituted and apo conditions by in vitro cross-linking assays, and DNA binding to Fe-Fur and apo-Fur target sequences by fluorescence anisotropy. The results showed that the N-terminal, S2 and S3 regions play distinct roles in terms of Fur structure-function relationships. Overall, these studies provide novel information regarding the role of these residues in Fur function, and provide mechanistic insight into how H. pylori Fur regulates gene expression in both the iron-bound and apo forms of the protein.

  8. Structural Characterization and Ligand/Inhibitor Identification Provide Functional Insights into the Mycobacterium tuberculosis Cytochrome P450 CYP126A1*

    PubMed Central

    Chenge, Jude T.; Duyet, Le Van; Swami, Shalini; McLean, Kirsty J.; Kavanagh, Madeline E.; Coyne, Anthony G.; Rigby, Stephen E. J.; Cheesman, Myles R.; Girvan, Hazel M.; Levy, Colin W.; Rupp, Bernd; von Kries, Jens P.; Abell, Chris; Leys, David; Munro, Andrew W.

    2017-01-01

    The Mycobacterium tuberculosis H37Rv genome encodes 20 cytochromes P450, including P450s crucial to infection and bacterial viability. Many M. tuberculosis P450s remain uncharacterized, suggesting that their further analysis may provide new insights into M. tuberculosis metabolic processes and new targets for drug discovery. CYP126A1 is representative of a P450 family widely distributed in mycobacteria and other bacteria. Here we explore the biochemical and structural properties of CYP126A1, including its interactions with new chemical ligands. A survey of azole antifungal drugs showed that CYP126A1 is inhibited strongly by azoles containing an imidazole ring but not by those tested containing a triazole ring. To further explore the molecular preferences of CYP126A1 and search for probes of enzyme function, we conducted a high throughput screen. Compounds containing three or more ring structures dominated the screening hits, including nitroaromatic compounds that induce substrate-like shifts in the heme spectrum of CYP126A1. Spectroelectrochemical measurements revealed a 155-mV increase in heme iron potential when bound to one of the newly identified nitroaromatic drugs. CYP126A1 dimers were observed in crystal structures of ligand-free CYP126A1 and for CYP126A1 bound to compounds discovered in the screen. However, ketoconazole binds in an orientation that disrupts the BC-loop regions at the P450 dimer interface and results in a CYP126A1 monomeric crystal form. Structural data also reveal that nitroaromatic ligands “moonlight” as substrates by displacing the CYP126A1 distal water but inhibit enzyme activity. The relatively polar active site of CYP126A1 distinguishes it from its most closely related sterol-binding P450s in M. tuberculosis, suggesting that further investigations will reveal its diverse substrate selectivity. PMID:27932461

  9. LiDAR: Providing structure

    USGS Publications Warehouse

    Vierling, Lee A.; Martinuzzi, Sebastián; Asner, Gregory P.; Stoker, Jason M.; Johnson, Brian R.

    2011-01-01

    Since the days of MacArthur, three-dimensional (3-D) structural information on the environment has fundamentally transformed scientific understanding of ecological phenomena (MacArthur and MacArthur 1961). Early data on ecosystem structure were painstakingly laborious to collect. However, as reviewed and reported in recent volumes of Frontiers(eg Vierling et al. 2008; Asner et al.2011), advances in light detection and ranging (LiDAR) remote-sensing technology provide quantitative and repeatable measurements of 3-D ecosystem structure that enable novel ecological insights at scales ranging from the plot, to the landscape, to the globe. Indeed, annual publication of studies using LiDAR to interpret ecological phenomena increased 17-fold during the past decade, with over 180 new studies appearing in 2010 (ISI Web of Science search conducted on 23 Mar 2011: [{lidar AND ecol*} OR {lidar AND fores*} OR {lidar AND plant*}]).

  10. Structure of a Single-Chain Fv Bound to the 17 N-Terminal Residues of Huntingtin Provides Insights into Pathogenic Amyloid Formation and Suppression

    PubMed Central

    De Genst, Erwin; Chirgadze, Dimitri Y.; Klein, Fabrice A.C.; Butler, David C.; Matak-Vinković, Dijana; Trottier, Yvon; Huston, James S.; Messer, Anne; Dobson, Christopher M.

    2015-01-01

    Huntington's disease is triggered by misfolding of fragments of mutant forms of the huntingtin protein (mHTT) with aberrant polyglutamine expansions. The C4 single-chain Fv antibody (scFv) binds to the first 17 residues of huntingtin [HTT(1-17)] and generates substantial protection against multiple phenotypic pathologies in situ and in vivo. We show in this paper that C4 scFv inhibits amyloid formation by exon1 fragments of huntingtin in vitro and elucidate the structural basis for this inhibition and protection by determining the crystal structure of the complex of C4 scFv and HTT(1-17). The peptide binds with residues 3–11 forming an amphipathic helix that makes contact with the antibody fragment in such a way that the hydrophobic face of this helix is shielded from the solvent. Residues 12–17 of the peptide are in an extended conformation and interact with the same region of another C4 scFv:HTT(1-17) complex in the asymmetric unit, resulting in a β-sheet interface within a dimeric C4 scFv:HTT(1-17) complex. The nature of this scFv–peptide complex was further explored in solution by high-resolution NMR and physicochemical analysis of species in solution. The results provide insights into the manner in which C4 scFv inhibits the aggregation of HTT, and hence into its therapeutic potential, and suggests a structural basis for the initial interactions that underlie the formation of disease-associated amyloid fibrils by HTT. PMID:25861763

  11. Crystal Structure of the Streptomyces coelicolor Sortase E1 Transpeptidase Provides Insight into the Binding Mode of the Novel Class E Sorting Signal

    PubMed Central

    Kattke, Michele D.; Chan, Albert H.; Duong, Andrew; Sexton, Danielle L.; Sawaya, Michael R.; Cascio, Duilio; Elliot, Marie A.; Clubb, Robert T.

    2016-01-01

    Many species of Gram-positive bacteria use sortase transpeptidases to covalently affix proteins to their cell wall or to assemble pili. Sortase-displayed proteins perform critical and diverse functions for cell survival, including cell adhesion, nutrient acquisition, and morphological development, among others. Based on their amino acid sequences, there are at least six types of sortases (class A to F enzymes); however, class E enzymes have not been extensively studied. Class E sortases are used by soil and freshwater-dwelling Actinobacteria to display proteins that contain a non-canonical LAXTG sorting signal, which differs from 90% of known sorting signals by substitution of alanine for proline. Here we report the first crystal structure of a class E sortase, the 1.93 Å resolution structure of the SrtE1 enzyme from Streptomyces coelicolor. The active site is bound to a tripeptide, providing insight into the mechanism of substrate binding. SrtE1 possesses β3/β4 and β6/β7 active site loops that contact the LAXTG substrate and are structurally distinct from other classes. We propose that SrtE1 and other class E sortases employ a conserved tyrosine residue within their β3/β4 loop to recognize the amide nitrogen of alanine at position P3 of the sorting signal through a hydrogen bond, as seen here. Incapability of hydrogen-bonding with canonical proline-containing sorting signals likely contributes to class E substrate specificity. Furthermore, we demonstrate that surface anchoring of proteins involved in aerial hyphae formation requires an N-terminal segment in SrtE1 that is presumably positioned within the cytoplasm. Combined, our results reveal unique features within class E enzymes that enable them to recognize distinct sorting signals, and could facilitate the development of substrate-based inhibitors of this important enzyme family. PMID:27936128

  12. Structure and specificity of a new class of Ca(2+) independent housekeeping sortase from Streptomyces avermitilis provides insights into its non-canonical substrate preference.

    PubMed

    Das, Sreetama; Pawale, Vijaykumar S; Dadireddy, Venkatareddy; Singh, Avinash Kumar; Ramakumar, Suryanarayanarao; Roy, Rajendra P

    2017-03-07

    Surface proteins in Gram-positive bacteria are incorporated into the cell wall through a peptide ligation reaction catalyzed by transpeptidase sortase. Six main classes (A-F) of sortase have been identified of which class A sortase is meant for housekeeping functions. The prototypic housekeeping sortase A (SaSrtA) from Staphylococcus aureus cleaves LPXTG-containing proteins at the scissile T-G peptide bond and ligates Protein-LPXT to the terminal Gly residue of the nascent cross-bridge of peptidoglycan Lipid II precursor. Sortase-mediated ligation ('sortagging') of LPXTG-containing substrates and Gly-terminated nucleophiles occurs in vitro as well as in cellulo in the presence of Ca(2+) and has been applied extensively for protein conjugations. Although majority of applications emanate from SaSrtA, low catalytic efficiency, LPXTG specificity restriction, and Ca(2+) requirement (particularly for in cellulo applications) remains a drawback. Given that Gram-positive bacterial genomes encode a variety of sortases, natural sortase mining can be a viable complementary approach akin to engineering of wild type SaSrtA. Here we describe the structure and specificity of a new class E sortase (SavSrtE) annotated to perform housekeeping roles in Streptomyces avermitilis Biochemical experiments define the attributes of an optimum peptide substrate, demonstrate Ca(2+)-independent activity and provide insights about contrasting functional characteristics of SavSrtE and SaSrtA. Crystal structure, substrate docking and mutagenesis experiments have identified a critical residue that dictates the preference for a non-canonical LAXTG recognition motif over LPXTG. These results have implications for rational tailoring of substrate tolerance in sortases. Besides, Ca(2+) independent orthogonal specificity of SavSrtE is likely to expand the sortagging toolkit.

  13. The Crystal Structure of N-Acetyl-L-glutamate Synthase from Neisseria gonorrhoeae Provides Insights into Mechanisms of Catalysis and Regulation

    SciTech Connect

    Shi, Dashuang; Sagar, Vatsala; Jin, Zhongmin; Yu, Xiaolin; Caldovic, Ljubica; Morizono, Hiroki; Allewell, Norma M.; Tuchman, Mendel

    2010-01-07

    The crystal structures of N-acetylglutamate synthase (NAGS) in the arginine biosynthetic pathway of Neisseria gonorrhoeae complexed with acetyl-CoA and with CoA plus N-acetylglutamate have been determined at 2.5- and 2.6-A resolution, respectively. The monomer consists of two separately folded domains, an amino acid kinase (AAK) domain and an N-acetyltransferase (NAT) domain connected through a 10-A linker. The monomers assemble into a hexameric ring that consists of a trimer of dimers with 32-point symmetry, inner and outer ring diameters of 20 and 100A, respectively, and a height of 110A(.) Each AAK domain interacts with the cognate domains of two adjacent monomers across two 2-fold symmetry axes and with the NAT domain from a second monomer of the adjacent dimer in the ring. The catalytic sites are located within the NAT domains. Three active site residues, Arg316, Arg425, and Ser427, anchor N-acetylglutamate in a position at the active site to form hydrogen bond interactions to the main chain nitrogen atoms of Cys356 and Leu314, and hydrophobic interactions to the side chains of Leu313 and Leu314. The mode of binding of acetyl-CoA and CoA is similar to other NAT family proteins. The AAK domain, although catalytically inactive, appears to bind arginine. This is the first reported crystal structure of any NAGS, and it provides insights into the catalytic function and arginine regulation of NAGS enzymes.

  14. Soil chemical insights provided through vibrational spectroscopy

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Vibrational spectroscopy techniques provide a powerful approach to study environmental materials and processes. These multifunctional analysis tools can be used to probe molecular vibrations of solid, liquid, and gaseous samples for characterizing materials, elucidating reaction mechanisms, and exam...

  15. The 2.0 Å X-ray structure for yeast acetohydroxyacid synthase provides new insights into its cofactor and quaternary structure requirements

    PubMed Central

    Lonhienne, Thierry; Garcia, Mario D.; Fraser, James A.; Williams, Craig M.; Guddat, Luke W.

    2017-01-01

    Acetohydroxyacid synthase (AHAS) catalyzes the first step of branched-chain amino acid biosynthesis, a pathway essential to the life-cycle of plants and micro-organisms. The catalytic subunit has thiamin diphosphate (ThDP) and flavin adenine dinucleotide (FAD) as indispensable co-factors. A new, high resolution, 2.0 Å crystal structure of Saccharomyces cerevisiae AHAS reveals that the dimer is asymmetric, with the catalytic centres having distinct structures where FAD is trapped in two different conformations indicative of different redox states. Two molecules of oxygen (O2) are bound on the surface of each active site and a tunnel in the polypeptide appears to passage O2 to the active site independently of the substrate. Thus, O2 appears to play a novel “co-factor” role in this enzyme. We discuss the functional implications of these features of the enzyme that have not previously been described. PMID:28178302

  16. Crystal structure and function of an unusual dimeric Hsp20.1 provide insight into the thermal protection mechanism of small heat shock proteins.

    PubMed

    Liu, Liang; Chen, Jiyun; Yang, Bo; Wang, Yonghua

    2015-03-06

    Small heat shock proteins (sHSPs) are ubiquitous chaperones that play a vital role in protein homeostasis. sHSPs are characterized by oligomeric architectures and dynamic exchange of subunits. The flexible oligomeric assembling associating with function remains poorly understood. Based on the structural data, it is certainly agreed that two dimerization models depend on the presence or absence of a β6 strand to differentiate nonmetazoan sHSPs from metazoan sHSPs. Here, we report the Sulfolobus solfataricus Hsp20.1 ACD dimer structure, which shows a distinct dimeric interface. We observed that, in the absence of β6, Hsp20.1 dimer does not depend on β7 strand for forming dimer interface as metazoan sHSPs, nor dissociates to monomers. This is in contrast to other published sHSPs. Our structure reveals a variable, highly polar dimer interface that has advantages for rapid subunits exchange and substrate binding. Remarkably, we find that the C-terminal truncation variant has chaperone activity comparable to that of wild-type despite lack of the oligomer structure. Our further study indicates that the N-terminal region is essential for the oligomer and dimer binding to the target protein. Together, the structure and function of Hsp20.1 give more insight into the thermal protection mechanism of sHSPs.

  17. COOH-Terminal Clustering of Autoantibody and T-Cell Determinants on the Structure of GAD65 Provide Insights Into the Molecular Basis of Autoreactivity

    SciTech Connect

    Fenalti, Gustavo; Hampe, Christiane S.; Arafat, Yasir; Law, Ruby H.P.; Banga, J. Paul; Mackay, Ian R.; Whisstock, James C.; Buckle, Ashley M.; Rowley, Merrill J.

    2008-11-19

    To gain structural insights into the autoantigenic properties of GAD65 in type 1 diabetes, we analyzed experimental epitope mapping data in the context of the recently determined crystal structures of GAD65 and GAD67, to allow 'molecular positioning' of epitope sites for B- and T-cell reactivity. Data were assembled from analysis of reported effects of mutagenesis of GAD65 on its reactivity with a panel of 11 human monoclonal antibodies (mAbs), supplemented by use of recombinant Fab to cross-inhibit reactivity with GAD65 by radioimmunoprecipitation of the same mAbs. COOH-terminal region on GAD65 was the major autoantigenic site. B-cell epitopes were distributed within two separate clusters around different faces of the COOH-terminal domain. Inclusion of epitope sites in the pyridoxal phosphate- and NH{sub 2}-terminal domains was attributed to the juxtaposition of all three domains in the crystal structure. Epitope preferences of different mAbs to GAD65 aligned with different clinical expressions of type 1 diabetes. Epitopes for four of five known reactive T-cell sequences restricted by HLA DRB1*0401 were aligned to solvent-exposed regions of the GAD65 structure and colocalized within the two B-cell epitope clusters. The continuous COOH-terminal epitope region of GAD65 was structurally highly flexible and therefore differed markedly from the equivalent region of GAD67. Structural features could explain the differing antigenicity, and perhaps immunogenicity, of GAD65 versus GAD67. The proximity of B- and T-cell epitopes within the GAD65 structure suggests that antigen-antibody complexes may influence antigen processing by accessory cells and thereby T-cell reactivity.

  18. High-Resolution Crystal Structures of Streptococcus pneumoniae Nicotinamidase with Trapped Intermediates Provide Insights into the Catalytic Mechanism and Inhibition by Aldehydes

    SciTech Connect

    French, Jarrod B.; Cen, Yana; Sauve, Anthony A.; Ealick, Steven E.

    2010-11-11

    Nicotinamidases are salvage enzymes that convert nicotinamide to nicotinic acid. These enzymes are essential for the recycling of nicotinamide into NAD{sup +} in most prokaryotes and most single-cell and multicellular eukaryotes, but not in mammals. The significance of these enzymes for nicotinamide salvage and for NAD{sup +} homeostasis has stimulated interest in nicotinamidases as possible antibiotic targets. Nicotinamidases are also regulators of intracellular nicotinamide concentrations, thereby regulating signaling of downstream NAD{sup +}-consuming enzymes, such as the NAD{sup +}-dependent deacetylases (sirtuins). Here, we report several high-resolution crystal structures of the nicotinamidase from Streptococcus pneumoniae (SpNic) in unliganded and ligand-bound forms. The structure of the C136S mutant in complex with nicotinamide provides details about substrate binding, while a trapped nicotinoyl thioester in a complex with SpNic reveals the structure of the proposed thioester reaction intermediate. Examination of the active site of SpNic reveals several important features, including a metal ion that coordinates the substrate and the catalytically relevant water molecule and an oxyanion hole that both orients the substrate and offsets the negative charge that builds up during catalysis. Structures of this enzyme with bound nicotinaldehyde inhibitors elucidate the mechanism of inhibition and provide further details about the catalytic mechanism. In addition, we provide a biochemical analysis of the identity and role of the metal ion that orients the ligand in the active site and activates the water molecule responsible for hydrolysis of the substrate. These data provide structural evidence of several proposed reaction intermediates and allow for a more complete understanding of the catalytic mechanism of this enzyme.

  19. The NMR structure of the II-III-VI three-way junction from the Neurospora VS ribozyme reveals a critical tertiary interaction and provides new insights into the global ribozyme structure.

    PubMed

    Bonneau, Eric; Girard, Nicolas; Lemieux, Sébastien; Legault, Pascale

    2015-09-01

    As part of an effort to structurally characterize the complete Neurospora VS ribozyme, NMR solution structures of several subdomains have been previously determined, including the internal loops of domains I and VI, the I/V kissing-loop interaction and the III-IV-V junction. Here, we expand this work by determining the NMR structure of a 62-nucleotide RNA (J236) that encompasses the VS ribozyme II-III-VI three-way junction and its adjoining stems. In addition, we localize Mg(2+)-binding sites within this structure using Mn(2+)-induced paramagnetic relaxation enhancement. The NMR structure of the J236 RNA displays a family C topology with a compact core stabilized by continuous stacking of stems II and III, a cis WC/WC G•A base pair, two base triples and two Mg(2+) ions. Moreover, it reveals a remote tertiary interaction between the adenine bulges of stems II and VI. Additional NMR studies demonstrate that both this bulge-bulge interaction and Mg(2+) ions are critical for the stable folding of the II-III-VI junction. The NMR structure of the J236 RNA is consistent with biochemical studies on the complete VS ribozyme, but not with biophysical studies performed with a minimal II-III-VI junction that does not contain the II-VI bulge-bulge interaction. Together with previous NMR studies, our findings provide important new insights into the three-dimensional architecture of this unique ribozyme.

  20. The NMR structure of the II–III–VI three-way junction from the Neurospora VS ribozyme reveals a critical tertiary interaction and provides new insights into the global ribozyme structure

    PubMed Central

    Bonneau, Eric; Girard, Nicolas; Lemieux, Sébastien; Legault, Pascale

    2015-01-01

    As part of an effort to structurally characterize the complete Neurospora VS ribozyme, NMR solution structures of several subdomains have been previously determined, including the internal loops of domains I and VI, the I/V kissing-loop interaction and the III–IV–V junction. Here, we expand this work by determining the NMR structure of a 62-nucleotide RNA (J236) that encompasses the VS ribozyme II–III–VI three-way junction and its adjoining stems. In addition, we localize Mg2+-binding sites within this structure using Mn2+-induced paramagnetic relaxation enhancement. The NMR structure of the J236 RNA displays a family C topology with a compact core stabilized by continuous stacking of stems II and III, a cis WC/WC G•A base pair, two base triples and two Mg2+ ions. Moreover, it reveals a remote tertiary interaction between the adenine bulges of stems II and VI. Additional NMR studies demonstrate that both this bulge–bulge interaction and Mg2+ ions are critical for the stable folding of the II–III–VI junction. The NMR structure of the J236 RNA is consistent with biochemical studies on the complete VS ribozyme, but not with biophysical studies performed with a minimal II–III–VI junction that does not contain the II–VI bulge–bulge interaction. Together with previous NMR studies, our findings provide important new insights into the three-dimensional architecture of this unique ribozyme. PMID:26124200

  1. Structure and Activity Analyses of Escherichia coli K-12 NagD Provide Insight into the Evolution of Biochemical Function in the Haloakanoic Acid Dehlogenase Superfamily

    SciTech Connect

    Tremblay,L.; Dunaway-Mariano, D.; Allen, K.

    2006-01-01

    The HAD superfamily is a large superfamily of proteins which share a conserved core domain that provides those active site residues responsible for the chemistry common to all family members. The superfamily is further divided into the four subfamilies I, IIA, IIB, and III, based on the topology and insertion site of a cap domain that provides substrate specificity. This structural and functional division implies that members of a given HAD structural subclass may target substrates that have similar structural characteristics. To understand the structure/function relationships in all of the subfamilies, a type IIA subfamily member, NagD from Escherichia coli K-12, was selected (type I, IIB, and III members have been more extensively studied). The structure of the NagD protein was solved to 1.80 Angstroms with R{sub work} = 19.8% and R{sub free} = 21.8%. Substrate screening and kinetic analysis showed NagD to have high specificity for nucleotide monophosphates with kcat/Km = 3.12 x 10{sup 4} and 1.28 x 10{sup 4} {micro}M{sup -1} s{sup -1} for UMP and GMP, respectively. This specificity is consistent with the presence of analogues of NagD that exist as fusion proteins with a nucleotide pyrophosphatase from the Nudix family. Docking of the nucleoside substrate in the active site brings it in contact with conserved residues from the cap domain that can act as a substrate specificity loop (NagD residues 144-149) in the type IIA subfamily. NagD and other subfamily IIA and IIB members show the common trait that substrate specificity and catalytic efficiencies (k{sub cat}/K{sub m}) are low (1 x 10{sup 4} M{sup -1} s{sup -1}) and the boundaries defining physiological substrates are somewhat overlapping. The ability to catabolize other related secondary metabolites indicates that there is regulation at the genetic level.

  2. Structure and activity analyses of Escherichia coli K-12 NagD provide insight into the evolution of biochemical function in the haloalkanoic acid dehalogenase superfamily.

    PubMed

    Tremblay, Lee W; Dunaway-Mariano, Debra; Allen, Karen N

    2006-01-31

    The HAD superfamily is a large superfamily of proteins which share a conserved core domain that provides those active site residues responsible for the chemistry common to all family members. The superfamily is further divided into the four subfamilies I, IIA, IIB, and III, based on the topology and insertion site of a cap domain that provides substrate specificity. This structural and functional division implies that members of a given HAD structural subclass may target substrates that have similar structural characteristics. To understand the structure/function relationships in all of the subfamilies, a type IIA subfamily member, NagD from Escherichia coli K-12, was selected (type I, IIB, and III members have been more extensively studied). The structure of the NagD protein was solved to 1.80 A with R(work) = 19.8% and R(free) = 21.8%. Substrate screening and kinetic analysis showed NagD to have high specificity for nucleotide monophosphates with k(cat)/K(m) = 3.12 x 10(4) and 1.28 x 10(4) microM(-)(1) s(-)(1) for UMP and GMP, respectively. This specificity is consistent with the presence of analogues of NagD that exist as fusion proteins with a nucleotide pyrophosphatase from the Nudix family. Docking of the nucleoside substrate in the active site brings it in contact with conserved residues from the cap domain that can act as a substrate specificity loop (NagD residues 144-149) in the type IIA subfamily. NagD and other subfamily IIA and IIB members show the common trait that substrate specificity and catalytic efficiencies (k(cat)/K(m)) are low (1 x 10(4) M(-)(1) s(-)(1)) and the boundaries defining physiological substrates are somewhat overlapping. The ability to catabolize other related secondary metabolites indicates that there is regulation at the genetic level.

  3. Population structure and historical demography of South American sea lions provide insights into the catastrophic decline of a marine mammal population

    PubMed Central

    Hoffman, J. I.; Kowalski, G. J.; Klimova, A.; Staniland, I. J.; Baylis, A. M. M.

    2016-01-01

    Understanding the causes of population decline is crucial for conservation management. We therefore used genetic analysis both to provide baseline data on population structure and to evaluate hypotheses for the catastrophic decline of the South American sea lion (Otaria flavescens) at the Falkland Islands (Malvinas) in the South Atlantic. We genotyped 259 animals from 23 colonies across the Falklands at 281 bp of the mitochondrial hypervariable region and 22 microsatellites. A weak signature of population structure was detected, genetic diversity was moderately high in comparison with other pinniped species, and no evidence was found for the decline being associated with a strong demographic bottleneck. By combining our mitochondrial data with published sequences from Argentina, Brazil, Chile and Peru, we also uncovered strong maternally directed population structure across the geographical range of the species. In particular, very few shared haplotypes were found between the Falklands and South America, and this was reflected in correspondingly low migration rate estimates. These findings do not support the prominent hypothesis that the decline was caused by migration to Argentina, where large-scale commercial harvesting operations claimed over half a million animals. Thus, our study not only provides baseline data for conservation management but also reveals the potential for genetic studies to shed light upon long-standing questions pertaining to the history and fate of natural populations. PMID:27493782

  4. Population structure and historical demography of South American sea lions provide insights into the catastrophic decline of a marine mammal population.

    PubMed

    Hoffman, J I; Kowalski, G J; Klimova, A; Eberhart-Phillips, L J; Staniland, I J; Baylis, A M M

    2016-07-01

    Understanding the causes of population decline is crucial for conservation management. We therefore used genetic analysis both to provide baseline data on population structure and to evaluate hypotheses for the catastrophic decline of the South American sea lion (Otaria flavescens) at the Falkland Islands (Malvinas) in the South Atlantic. We genotyped 259 animals from 23 colonies across the Falklands at 281 bp of the mitochondrial hypervariable region and 22 microsatellites. A weak signature of population structure was detected, genetic diversity was moderately high in comparison with other pinniped species, and no evidence was found for the decline being associated with a strong demographic bottleneck. By combining our mitochondrial data with published sequences from Argentina, Brazil, Chile and Peru, we also uncovered strong maternally directed population structure across the geographical range of the species. In particular, very few shared haplotypes were found between the Falklands and South America, and this was reflected in correspondingly low migration rate estimates. These findings do not support the prominent hypothesis that the decline was caused by migration to Argentina, where large-scale commercial harvesting operations claimed over half a million animals. Thus, our study not only provides baseline data for conservation management but also reveals the potential for genetic studies to shed light upon long-standing questions pertaining to the history and fate of natural populations.

  5. The 1.4 Å crystal structure of the ArsD arsenic metallochaperone provides insights into its interaction with the ArsA ATPase†

    PubMed Central

    Ye, Jun; Ajees, A. Abdul; Yang, Jianbo; Rosen, Barry P.

    2010-01-01

    Arsenic is a carcinogen that tops the Superfund list of hazardous chemicals. Bacterial resistance to arsenic is facilitated by ArsD, which delivers As(III) to the ArsA ATPase, the catalytic subunit of the ArsAB pump. Here we report the structure of the arsenic metallochaperone ArsD at 1.4 Å, and a model for its binding of metalloid. There are two ArsD molecules in the asymmetric unit. The overall structure of the ArsD monomer has a thioredoxin fold, with a core of four β-strands flanked by four α-helices. Based on data from structural homologues, ArsD was modeled with and without bound As(III). ArsD binds one arsenic per monomer coordinated with the three sulfur atoms of Cys12, Cys13 and Cys18. Using this structural model, an algorithm was used to dock ArsD and ArsA. The resulting docking model provides testable predictions of the contact points of the two proteins and forms the basis for future experiments. PMID:20507177

  6. The 1.4 Å Crystal Structure of the ArsD Arsenic Metallochaperone Provides Insights into Its Interaction with the ArsA ATPase

    SciTech Connect

    Ye, Jun; Ajees, A. Abdul; Yang, Jianbo; Rosen, Barry P.

    2010-12-07

    Arsenic is a carcinogen that tops the Superfund list of hazardous chemicals. Bacterial resistance to arsenic is facilitated by ArsD, which delivers As(III) to the ArsA ATPase, the catalytic subunit of the ArsAB pump. Here we report the structure of the arsenic metallochaperone ArsD at 1.4 {angstrom} and a model for its binding of metalloid. There are two ArsD molecules in the asymmetric unit. The overall structure of the ArsD monomer has a thioredoxin fold, with a core of four {beta}-strands flanked by four {alpha}-helices. Based on data from structural homologues, ArsD was modeled with and without bound As(III). ArsD binds one arsenic per monomer coordinated with the three sulfur atoms of Cys12, Cys13, and Cys18. Using this structural model, an algorithm was used to dock ArsD and ArsA. The resulting docking model provides testable predictions of the contact points of the two proteins and forms the basis for future experiments.

  7. Nuclear Protein-Only Ribonuclease P2 Structure and Biochemical Characterization Provide Insight into the Conserved Properties of tRNA 5' End Processing Enzymes.

    PubMed

    Karasik, Agnes; Shanmuganathan, Aranganathan; Howard, Michael J; Fierke, Carol A; Koutmos, Markos

    2016-01-16

    Protein-only RNase Ps (PRORPs) are a recently discovered class of RNA processing enzymes that catalyze maturation of the 5' end of precursor tRNAs in Eukaryotes. PRORPs are found in the nucleus and/or organelles of most eukaryotic organisms. Arabidopsis thaliana is a representative organism that contains PRORP enzymes (PRORP1, PRORP2 and PRORP3) in both its nucleus and its organelles; PRORP2 and PRORP3 localize to the nucleus and PRORP1 localizes to the chloroplast and the mitochondria. Apart from their identification, almost nothing is known about the structure and function of PRORPs that act in the nucleus. Here, we use a combination of biochemical assays and X-ray crystallography to characterize A. thaliana PRORP2. We solved the crystal structure of PRORP2 (3.2Å) revealing an overall V-shaped protein and conserved metallonuclease active-site structure. Our biochemical studies indicate that PRORP2 requires Mg(2+) for catalysis and catalyzes the maturation of nuclear encoded substrates up to 10-fold faster than mitochondrial encoded precursor nad6 t-element under single-turnover conditions. We also demonstrate that PRORP2 preferentially binds precursor tRNAs containing short 5' leaders and 3' trailers; however, leader and trailer lengths do not significantly alter the observed rate constants of PRORP2 in single-turnover cleavage assays. Our data provide a biochemical and structural framework to begin understanding how nuclear localized PRORPs recognize and cleave their substrates.

  8. The crystal structure of D-threonine aldolase from Alcaligenes xylosoxidans provides insight into a metal ion assisted PLP-dependent mechanism.

    PubMed

    Uhl, Michael K; Oberdorfer, Gustav; Steinkellner, Georg; Riegler-Berket, Lina; Mink, Daniel; van Assema, Friso; Schürmann, Martin; Gruber, Karl

    2015-01-01

    Threonine aldolases catalyze the pyridoxal phosphate (PLP) dependent cleavage of threonine into glycine and acetaldehyde and play a major role in the degradation of this amino acid. In nature, L- as well as D-specific enzymes have been identified, but the exact physiological function of D-threonine aldolases (DTAs) is still largely unknown. Both types of enantio-complementary enzymes have a considerable potential in biocatalysis for the stereospecific synthesis of various β-hydroxy amino acids, which are valuable building blocks for the production of pharmaceuticals. While several structures of L-threonine aldolases (LTAs) have already been determined, no structure of a DTA is available to date. Here, we report on the determination of the crystal structure of the DTA from Alcaligenes xylosoxidans (AxDTA) at 1.5 Å resolution. Our results underline the close relationship of DTAs and alanine racemases and allow the identification of a metal binding site close to the PLP-cofactor in the active site of the enzyme which is consistent with the previous observation that divalent cations are essential for DTA activity. Modeling of AxDTA substrate complexes provides a rationale for this metal dependence and indicates that binding of the β-hydroxy group of the substrate to the metal ion very likely activates this group and facilitates its deprotonation by His193. An equivalent involvement of a metal ion has been implicated in the mechanism of a serine dehydratase, which harbors a metal ion binding site in the vicinity of the PLP cofactor at the same position as in DTA. The structure of AxDTA is completely different to available structures of LTAs. The enantio-complementarity of DTAs and LTAs can be explained by an approximate mirror symmetry of crucial active site residues relative to the PLP-cofactor.

  9. The structural basis of chicken, swine and bovine CD8αα dimers provides insight into the co-evolution with MHC I in endotherm species.

    PubMed

    Liu, Yanjie; Li, Xin; Qi, Jianxun; Zhang, Nianzhi; Xia, Chun

    2016-04-28

    It is unclear how the pivotal molecules of the adaptive immune system (AIS) maintain their inherent characteristics and relationships with their co-receptors over the course of co-evolution. CD8α, a fundamental but simple AIS component with only one immunoglobulin variable (IgV) domain, is a good example with which to explore this question because it can fold correctly to form homodimers (CD8αα) and interact with peptide-MHC I (p/MHC I) with low sequence identities between different species. Hereby, we resolved the crystal structures of chicken, swine and bovine CD8αα. They are typical homodimers consisting of two symmetric IgV domains with distinct species specificities. The CD8αα structures indicated that a few highly conserved residues are important in CD8 dimerization and in interacting with p/MHC I. The dimerization of CD8αα mainly depends on the pivotal residues on the dimer interface; in particular, four aromatic residues provide many intermolecular forces and contact areas. Three residues on the surface of CD8α connecting cavities that formed most of the hydrogen bonds with p/MHC I were also completely conserved. Our data propose that a few key conserved residues are able to ensure the CD8α own structural characteristics despite the great sequence variation that occurs during evolution in endotherms.

  10. The structural basis of chicken, swine and bovine CD8αα dimers provides insight into the co-evolution with MHC I in endotherm species

    PubMed Central

    Liu, Yanjie; Li, Xin; Qi, Jianxun; Zhang, Nianzhi; Xia, Chun

    2016-01-01

    It is unclear how the pivotal molecules of the adaptive immune system (AIS) maintain their inherent characteristics and relationships with their co-receptors over the course of co-evolution. CD8α, a fundamental but simple AIS component with only one immunoglobulin variable (IgV) domain, is a good example with which to explore this question because it can fold correctly to form homodimers (CD8αα) and interact with peptide-MHC I (p/MHC I) with low sequence identities between different species. Hereby, we resolved the crystal structures of chicken, swine and bovine CD8αα. They are typical homodimers consisting of two symmetric IgV domains with distinct species specificities. The CD8αα structures indicated that a few highly conserved residues are important in CD8 dimerization and in interacting with p/MHC I. The dimerization of CD8αα mainly depends on the pivotal residues on the dimer interface; in particular, four aromatic residues provide many intermolecular forces and contact areas. Three residues on the surface of CD8α connecting cavities that formed most of the hydrogen bonds with p/MHC I were also completely conserved. Our data propose that a few key conserved residues are able to ensure the CD8α own structural characteristics despite the great sequence variation that occurs during evolution in endotherms. PMID:27122108

  11. The crystal structure of spermidine/spermine N1-acetyltransferase in complex with spermine provides insights into substrate binding and catalysis.

    PubMed

    Montemayor, Eric J; Hoffman, David W

    2008-09-02

    The enzyme spermidine/spermine N (1)-acetyltransferase (SSAT) catalyzes the transfer of acetyl groups from acetylcoenzyme A to spermidine and spermine, as part of a polyamine degradation pathway. This work describes the crystal structure of SSAT in complex with coenzyme A, with and without bound spermine. The complex with spermine provides a direct view of substrate binding by an SSAT and demonstrates structural plasticity near the active site of the enzyme. Associated water molecules bridge several of the intermolecular contacts between spermine and the enzyme and form a "proton wire" between the side chain of Glu92 and the N1 amine of spermine. A single water molecule can also be seen forming hydrogen bonds with the side chains of Glu92, Asp93, and the N4 amine of spermine. Site-directed mutation of Glu92 to glutamine had a detrimental effect on both substrate binding and catalysis and shifted the optimal pH for enzyme activity further into alkaline solution conditions, while mutation of Asp93 to asparagine affected both substrate binding and catalysis without changing the pH dependence of the enzyme. Considered together, the structural and kinetic data suggest that Glu92 functions as a catalytic base to drive an otherwise unfavorable deprotonation step at physiological pH.

  12. Molecular dynamics simulations and structure-guided mutagenesis provide insight into the architecture of the catalytic core of the ectoine hydroxylase.

    PubMed

    Widderich, Nils; Pittelkow, Marco; Höppner, Astrid; Mulnaes, Daniel; Buckel, Wolfgang; Gohlke, Holger; Smits, Sander H J; Bremer, Erhard

    2014-02-06

    Many bacteria amass compatible solutes to fend-off the detrimental effects of high osmolarity on cellular physiology and water content. These solutes also function as stabilizers of macromolecules, a property for which they are referred to as chemical chaperones. The tetrahydropyrimidine ectoine is such a compatible solute and is widely synthesized by members of the Bacteria. Many ectoine producers also synthesize the stress protectant 5-hydroxyectoine from the precursor ectoine, a process that is catalyzed by the ectoine hydroxylase (EctD). The EctD enzyme is a member of the non-heme-containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily. A crystal structure of the EctD protein from the moderate halophile Virgibacillus salexigens has previously been reported and revealed the coordination of the iron catalyst, but it lacked the substrate ectoine and the co-substrate 2-oxoglutarate. Here we used this crystal structure as a template to assess the likely positioning of the ectoine and 2-oxoglutarate ligands within the active site by structural comparison, molecular dynamics simulations, and site-directed mutagenesis. Collectively, these approaches suggest the positioning of the iron, ectoine, and 2-oxoglutarate ligands in close proximity to each other and with a spatial orientation that will allow the region-selective and stereo-specific hydroxylation of (4S)-ectoine to (4S,5S)-5-hydroxyectoine. Our study thus provides a view into the catalytic core of the ectoine hydroxylase and suggests an intricate network of interactions between the three ligands and evolutionarily highly conserved residues in members of the EctD protein family.

  13. Crystal structure of A. aeolicus argonaute, a site-specific DNA-guided endoribonuclease, provides insights into RISC-mediated mRNA cleavage

    SciTech Connect

    Yuan,Y.; Pei, Y.; Ma, J.; Kuryavyi, V.; Zhadina, M.; Meister, G.; Chen, H.; Dauter, Z.; Tuschi, T.; Patel, D.

    2005-01-01

    Argonaute (Ago) proteins constitute a key component of the RNA-induced silencing complex (RISC). We report the crystal structure of Aquifex aeolicus Ago (Aa-Ago) together with binding and cleavage studies, which establish this eubacterial Ago as a bona fide guide DNA strand-mediated site-specific RNA endonuclease. We have generated a stereochemically robust model of the complex, where the guide DNA-mRNA duplex is positioned within a basic channel spanning the bilobal interface, such that the 5' phosphate of the guide strand can be anchored in a basic pocket, and the mRNA can be positioned for site-specific cleavage by RNase H-type divalent cation-coordinated catalytic Asp residues of the PIWI domain. Domain swap experiments involving chimeras of human Ago (hAgo1) and cleavage-competent hAgo2 reinforce the role of the PIWI domain in 'slicer' activity. We propose a four-step Ago-mediated catalytic cleavage cycle model, which provides distinct perspectives into the mechanism of guide strand-mediated mRNA cleavage within the RISC.

  14. The crystal structure of maleylacetate reductase from Rhizobium sp. strain MTP-10005 provides insights into the reaction mechanism of enzymes in its original family.

    PubMed

    Fujii, Tomomi; Sato, Ai; Okamoto, Yuko; Yamauchi, Takae; Kato, Shiro; Yoshida, Masahiro; Oikawa, Tadao; Hata, Yasuo

    2016-08-01

    Maleylacetate reductase plays a crucial role in catabolism of resorcinol by catalyzing the NAD(P)H-dependent reduction of maleylacetate, at a carbon-carbon double bond, to 3-oxoadipate. The crystal structure of maleylacetate reductase from Rhizobium sp. strain MTP-10005, GraC, has been elucidated by the X-ray diffraction method at 1.5 Å resolution. GraC is a homodimer, and each subunit consists of two domains: an N-terminal NADH-binding domain adopting an α/β structure and a C-terminal functional domain adopting an α-helical structure. Such structural features show similarity to those of the two existing families of enzymes in dehydroquinate synthase-like superfamily. However, GraC is distinct in dimer formation and activity expression mechanism from the families of enzymes. Two subunits in GraC have different structures from each other in the present crystal. One subunit has several ligands mimicking NADH and the substrate in the cleft and adopts a closed domain arrangement. In contrast, the other subunit does not contain any ligand causing structural changes and adopts an open domain arrangement. The structure of GraC reveals those of maleylacetate reductase both in the coenzyme, substrate-binding state and in the ligand-free state. The comparison of both subunit structures reveals a conformational change of the Tyr326 loop for interaction with His243 on ligand binding. Structures of related enzymes suggest that His243 is likely a catalytic residue of GraC. Mutational analyses of His243 and Tyr326 support the catalytic roles proposed from structural information. The crystal structure of GraC characterizes the maleylacetate reductase family as a third family in the dehydroquinate synthase-like superfamily. Proteins 2016; 84:1029-1042. © 2016 Wiley Periodicals, Inc.

  15. Application of Structural-Dynamic Approaches Provide Novel Insights Into the Enzymatic Mechanism of the Tumor Necrosis Factor-Alpha Converting Enzyme (TACE)

    SciTech Connect

    Sagi, I.; Milla, M

    2008-01-01

    Zinc dependent metalloproteinases comprise a large family of structurally homologous enzymes with a wide variety of biological roles. Originally described as proteinases involved in extracellular matrix (ECM) catabolism, these enzymes were later found to serve major roles as initiators of signaling pathways in many aspects of biology, ranging from cell proliferation, differentiation and communication, to pathological states associated with tumor metastasis, inflammation, tissue degeneration and cell death. From these enzymes, the tumor necrosis factor-a converting enzyme (TACE) stands out as a central shedding activity mediating the regulated release of a host of cytokines, receptors and other cell surface molecules. Selective drugs targeted at blocking TACE for treatment of rheumatoid arthritis and other disease indications are highly sought. Yet, the structural and chemical knowledge underlying its enzymatic activity is very limited. This is in part due to the fact that the catalytic zinc atom of metalloproteinases is usually spectroscopically silent and hence difficult to study using conventional spectroscopic and analytical tools. Most structural and biochemical studies, as well as medicinal chemistry efforts carried out so far were limited to non-dynamic structure/function characterization. Thus, to date, our mechanistic knowledge comes from theoretical calculations derived from static crystal structures from family members that are highly similar in their amino acid sequence and three-dimensional structure.This review introduces the importance of real-time quantification of biophysical properties and structural kinetic behavior applied to the study of TACE and other zinc metalloproteinases to dissect their molecular mechanisms. The molecular details that link the catalytic chemistry to key kinetic, electronic and structural events have remained elusive because of the difficulties associated with probing time-dependent structure-function aspects of enzymatic

  16. Structure of an anti-DNA fab complexed with a non-DNA ligand provides insights into cross-reactivity and molecular mimicry.

    PubMed

    Schuermann, Jonathan P; Henzl, Michael T; Deutscher, Susan L; Tanner, John J

    2004-11-01

    Antibodies that recognize DNA (anti-DNA) are part of the autoimmune response underlying systemic lupus erythematosus. To better understand molecular recognition by anti-DNA antibodies, crystallographic studies have been performed using an anti-ssDNA antigen-binding fragment (Fab) known as DNA-1. The previously determined structure of a DNA-1/dT5 complex revealed that thymine bases insert into a narrow groove, and that ligand recognition primarily involves the bases of DNA. We now report the 1.75-A resolution structure of DNA-1 complexed with the biological buffer HEPES (4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid). All three light chain complementarity-determining regions (CDRs) and HCDR3 contribute to binding. The HEPES sulfonate hydrogen bonds to His L91, Asn L50, and to the backbone of Tyr H100 and Tyr H100A. The Tyr side-chains of L32, L92, H100, and H100A form nonpolar contacts with the HEPES ethylene and piperazine groups. Comparison to the DNA-1/dT5 structure reveals that the dual recognition of dT5 and HEPES requires a 13-A movement of HCDR3. This dramatic structural change converts the combining site from a narrow groove, appropriate for the edge-on insertion of thymine bases, to one sufficiently wide to accommodate the HEPES sulfonate and piperazine. Isothermal titration calorimetry verified the association of HEPES with DNA-1 under conditions similar those used for crystallization (2 M ammonium sulfate). Interestingly, the presence of 2 M ammonium sulfate increases the affinities of DNA-1 for both HEPES and dT5, suggesting that non-polar Fab-ligand interactions are important for molecular recognition in highly ionic solvent conditions. The structural and thermodynamic data suggest a molecular mimicry mechanism based on structural plasticity and hydrophobic interactions.

  17. Single-strand conformation polymorphism (SSCP) of oligodeoxyribonucleotides: an insight into solution structural dynamics of DNAs provided by gel electrophoresis and molecular dynamics simulations.

    PubMed

    Biyani, Manish; Nishigaki, Koichi

    2005-10-01

    Studies on the solution structure dynamics of RNA/DNA are becoming crucially important. The phenomena of SSCP (single-strand conformation polymorphism), small RNA dynamics in a cell, and others can be related to the conformational changes of single-stranded (ss) RNAs/DNAs in solution. However, little is known about those dynamics. Only the intra-structural transition of ssDNAs in solution has been reported based on Watson-Crick (W-C) base-pairing. Here, we found a general feature of the SSCP phenomenon by studying the simpler molecules of ss-oligodeoxyribonucleotides. A single base substitution or a positional exchange of nucleotide in a highly homologous series of ss-dodecanucleotides led to a change in the mobility-in-gel. This was unexpected, since most of these nucleotides [such as d(A(11)G) or d(A(11)C)] have no possibility of forming W-C base-pairing. MD (molecular dynamics) experiments revealed differences in shape and size between the dynamic structures of these molecules which could affect their mobility-in-gel. In addition, a high correlation was observed between the electrophoretic mobility and the size-related parameters such as end-to-end distance obtained from MD simulations. Because the simulation was considerably shorter (nanosecond) than the experimental time-scale (second), the result must be considered conservatively; but it is nevertheless encouraging for utilizing MD simulation for structural analysis of oligonucleotides.

  18. The structure of Medicago truncatula δ1-pyrroline-5-carboxylate reductase provides new insights into regulation of proline biosynthesis in plants

    PubMed Central

    Ruszkowski, Milosz; Nocek, Boguslaw; Forlani, Giuseppe; Dauter, Zbigniew

    2015-01-01

    The two pathways for proline biosynthesis in higher plants share the last step, the conversion of δ1-pyrroline-5-carboxylate (P5C) to L-proline, which is catalyzed by P5C reductase (P5CR, EC 1.5.1.2) with the use of NAD(P)H as a coenzyme. There is increasing amount of evidence to suggest a complex regulation of P5CR activity at the post-translational level, yet the molecular basis of these mechanisms is unknown. Here we report the three-dimensional structure of the P5CR enzyme from the model legume Medicago truncatula (Mt). The crystal structures of unliganded MtP5CR decamer, and its complexes with the products NAD+, NADP+, and L-proline were refined using x-ray diffraction data (at 1.7, 1.85, 1.95, and 2.1 Å resolution, respectively). Based on the presented structural data, the coenzyme preference for NADPH over NADH was explained, and NADPH is suggested to be the only coenzyme used by MtP5CR in vivo. Furthermore, the insensitivity of MtP5CR to feed-back inhibition by proline, revealed by enzymatic analysis, was correlated with structural features. Additionally, a mechanism for the modulation of enzyme activity by chloride anions is discussed, as well as the rationale for the possible development of effective enzyme inhibitors. PMID:26579138

  19. The structure of Medicago truncatula δ1-pyrroline-5-carboxylate reductase provides new insights into regulation of proline biosynthesis in plants

    DOE PAGES

    Ruszkowski, Milosz; Nocek, Boguslaw; Forlani, Giuseppe; ...

    2015-10-30

    The two pathways for proline biosynthesis in higher plants share the last step, the conversion of δ1-pyrroline-5-carboxylate (P5C) to L-proline, which is catalyzed by P5C reductase (P5CR, EC 1.5.1.2) with the use of NAD(P)H as a coenzyme. There is increasing amount of evidence to suggest a complex regulation of P5CR activity at the post-translational level, yet the molecular basis of these mechanisms is unknown. Here we report the three-dimensional structure of the P5CR enzyme from the model legume Medicago truncatula (Mt). The crystal structures of unliganded MtP5CR decamer, and its complexes with the products NAD+, NADP+, and L-proline were refinedmore » using x-ray diffraction data (at 1.7, 1.85, 1.95, and 2.1 Å resolution, respectively). Based on the presented structural data, the coenzyme preference for NADPH over NADH was explained, and NADPH is suggested to be the only coenzyme used by MtP5CR in vivo. Moreover, the insensitivity of MtP5CR to feed-back inhibition by proline, revealed by enzymatic analysis, was correlated with structural features. Additionally, a mechanism for the modulation of enzyme activity by chloride anions is discussed, as well as the rationale for the possible development of effective enzyme inhibitors.« less

  20. Structural Basis for Dual Nucleotide Selectivity of Aminoglycoside 2″-Phosphotransferase IVa Provides Insight on Determinants of Nucleotide Specificity of Aminoglycoside Kinases*♦

    PubMed Central

    Shi, Kun; Berghuis, Albert M.

    2012-01-01

    Enzymatic phosphorylation through a family of enzymes called aminoglycoside O-phosphotransferases (APHs) is a major mechanism by which bacteria confer resistance to aminoglycoside antibiotics. Members of the APH(2″) subfamily are of particular clinical interest because of their prevalence in pathogenic strains and their broad substrate spectra. APH(2″) enzymes display differential preferences between ATP or GTP as the phosphate donor, with aminoglycoside 2″-phosphotransferase IVa (APH(2″)-IVa) being a member that utilizes both nucleotides at comparable efficiencies. We report here four crystal structures of APH(2″)-IVa, two of the wild type enzyme and two of single amino acid mutants, each in complex with either adenosine or guanosine. Together, these structures afford a detailed look at the nucleoside-binding site architecture for this enzyme and reveal key elements that confer dual nucleotide specificity, including a solvent network in the interior of the nucleoside-binding pocket and the conformation of an interdomain linker loop. Steady state kinetic studies, as well as sequence and structural comparisons with members of the APH(2″) subfamily and other aminoglycoside kinases, rationalize the different substrate preferences for these enzymes. Finally, despite poor overall sequence similarity and structural homology, analysis of the nucleoside-binding pocket of APH(2″)-IVa shows a striking resemblance to that of eukaryotic casein kinase 2 (CK2), which also exhibits dual nucleotide specificity. These results, in complement with the multitude of existing inhibitors against CK2, can serve as a structural basis for the design of nucleotide-competitive inhibitors against clinically relevant APH enzymes. PMID:22371504

  1. Structural basis for dual nucleotide selectivity of aminoglycoside 2''-phosphotransferase IVa provides insight on determinants of nucleotide specificity of aminoglycoside kinases.

    PubMed

    Shi, Kun; Berghuis, Albert M

    2012-04-13

    Enzymatic phosphorylation through a family of enzymes called aminoglycoside O-phosphotransferases (APHs) is a major mechanism by which bacteria confer resistance to aminoglycoside antibiotics. Members of the APH(2″) subfamily are of particular clinical interest because of their prevalence in pathogenic strains and their broad substrate spectra. APH(2″) enzymes display differential preferences between ATP or GTP as the phosphate donor, with aminoglycoside 2″-phosphotransferase IVa (APH(2″)-IVa) being a member that utilizes both nucleotides at comparable efficiencies. We report here four crystal structures of APH(2″)-IVa, two of the wild type enzyme and two of single amino acid mutants, each in complex with either adenosine or guanosine. Together, these structures afford a detailed look at the nucleoside-binding site architecture for this enzyme and reveal key elements that confer dual nucleotide specificity, including a solvent network in the interior of the nucleoside-binding pocket and the conformation of an interdomain linker loop. Steady state kinetic studies, as well as sequence and structural comparisons with members of the APH(2″) subfamily and other aminoglycoside kinases, rationalize the different substrate preferences for these enzymes. Finally, despite poor overall sequence similarity and structural homology, analysis of the nucleoside-binding pocket of APH(2″)-IVa shows a striking resemblance to that of eukaryotic casein kinase 2 (CK2), which also exhibits dual nucleotide specificity. These results, in complement with the multitude of existing inhibitors against CK2, can serve as a structural basis for the design of nucleotide-competitive inhibitors against clinically relevant APH enzymes.

  2. High-resolution structures of Neotermes koshunensis β-glucosidase mutants provide insights into the catalytic mechanism and the synthesis of glucoconjugates.

    PubMed

    Jeng, Wen-Yih; Wang, Nai-Chen; Lin, Cheng-Tse; Chang, Wei-Jung; Liu, Chia-I; Wang, Andrew H-J

    2012-07-01

    NkBgl, a β-glucosidase from Neotermes koshunensis, is a β-retaining glycosyl hydrolase family 1 enzyme that cleaves β-glucosidic linkages in disaccharide or glucose-substituted molecules. β-Glucosidases have been widely used in several applications. For example, mutagenesis of the attacking nucleophile in β-glucosidase has been conducted to convert it into a glycosynthase for the synthesis of oligosaccharides. Here, several high-resolution structures of wild-type or mutated NkBgl in complex with different ligand molecules are reported. In the wild-type NkBgl structures it was found that glucose-like glucosidase inhibitors bind to the glycone-binding pocket, allowing the buffer molecule HEPES to remain in the aglycone-binding pocket. In the crystal structures of NkBgl E193A, E193S and E193D mutants Glu193 not only acts as the catalytic acid/base but also plays an important role in controlling substrate entry and product release. Furthermore, in crystal structures of the NkBgl E193D mutant it was found that new glucoconjugates were generated by the conjugation of glucose (hydrolyzed product) and HEPES/EPPS/opipramol (buffer components). Based on the wild-type and E193D-mutant structures of NkBgl, the glucosidic bond of cellobiose or salicin was hydrolyzed and a new bond was subsequently formed between glucose and HEPES/EPPS/opipramol to generate new glucopyranosidic products through the transglycosylation reaction in the NkBgl E193D mutant. This finding highlights an innovative way to further improve β-glucosidases for the enzymatic synthesis of oligosaccharides.

  3. Computational docking, molecular dynamics simulation and subsite structure analysis of a maltogenic amylase from Bacillus lehensis G1 provide insights into substrate and product specificity.

    PubMed

    Manas, Nor Hasmaliana Abdul; Bakar, Farah Diba Abu; Illias, Rosli Md

    2016-06-01

    Maltogenic amylase (MAG1) from Bacillus lehensis G1 displayed the highest hydrolysis activity on β-cyclodextrin (β-CD) to produce maltose as a main product and exhibited high transglycosylation activity on malto-oligosaccharides with polymerization degree of three and above. These substrate and product specificities of MAG1 were elucidated from structural point of view in this study. A three-dimensional structure of MAG1 was constructed using homology modeling. Docking of β-CD and malto-oligosaccharides was then performed in the MAG1 active site. An aromatic platform in the active site was identified which is responsible in substrate recognition especially in determining the enzyme's preference toward β-CD. Molecular dynamics (MD) simulation showed MAG1 structure is most stable when docked with β-CD and least stable when docked with maltose. The docking analysis and MD simulation showed that the main subsites for substrate stabilization in the active site are -2, -1, +1 and +2. A bulky residue, Trp359 at the +2 subsite was identified to cause steric interference to the bound linear malto-oligosaccharides thus prevented it to occupy subsite +3, which can only be reached by a highly bent glucose molecule such as β-CD. The resulted modes of binding from docking simulation show a good correlation with the experimentally determined hydrolysis pattern. The subsite structure generated from this study led to a possible mode of action that revealed how maltose was mainly produced during hydrolysis. Furthermore, maltose only occupies subsite +1 and +2, therefore could not be hydrolyzed or transglycosylated by the enzyme. This important knowledge has paved the way for a novel structure-based molecular design for modulation of its catalytic activities.

  4. Simultaneous visualization of two Citrus tristeza virus genotypes provides new insights into the structure of multi-component virus populations in a host.

    PubMed

    Bergua, María; Phelan, Dane M; Bak, Aurélie; Bloom, David C; Folimonova, Svetlana Y

    2016-04-01

    Complex Citrus tristeza virus (CTV) populations composed of mixtures of different strains of the virus are commonly found in citrus trees in the field. At present, little is known about how these populations are formed, maintained, and how they are structured within a host. Here we used a novel in situ hybridization approach allowing simultaneous visualization of two different RNA targets with high sensitivity and specificity to examine the distribution of two isolates, T36 and T68-1, representing phylogenetically distinct strains of CTV, in a citrus host in single and mixed infections. Remarkably, in doubly inoculated plants the two virus variants appeared to be well mixed within the infected tissue and showed no spatial segregation. In addition, both CTV variants were often found occupying the same cells. Possible mechanisms involved in shaping CTV populations and the biological significance of the observed lack of structural separation of the individual components are discussed.

  5. Crystal structures of the F88Y obelin mutant before and after bioluminescence provide molecular insight into spectral tuning among hydromedusan photoproteins.

    PubMed

    Natashin, Pavel V; Markova, Svetlana V; Lee, John; Vysotski, Eugene S; Liu, Zhi-Jie

    2014-03-01

    Ca(2+) -regulated photoproteins are responsible for the bioluminescence of a variety of marine coelenterates. All hydromedusan photoproteins are a single-chain polypeptide to which 2-hydroperoxycoelenterazine is tightly but non-covalently bound. Bioluminescence results from oxidative decarboxylation of 2-hydroperoxycoelenterazine, generating protein-bound coelenteramide in an excited state. The bioluminescence spectral maxima of recombinant photoproteins vary in the range 462-495 nm, despite a high degree of identity of amino acid sequences and spatial structures of these photoproteins. Based on studies of obelin and aequorin mutants with substitution of Phe to Tyr and Tyr to Phe, respectively [Stepanyuk GA et al. (2005) FEBS Lett 579, 1008-1014], it was suggested that the spectral differences may be accounted for by an additional hydrogen bond between the hydroxyl group of a Tyr residue and an oxygen atom of the 6-(p-hydroxyphenyl) substituent of coelenterazine. Here, we report the crystal structures of two conformation states of the F88Y obelin mutant that has bioluminescence and product fluorescence spectra resembling those of aequorin. Comparison of spatial structures of the F88Y obelin conformation states with those of wild-type obelin clearly shows that substitution of Phe to Tyr does not affect the overall structures of either F88Y obelin or its product following Ca(2+) discharge, compared to the conformation states of wild-type obelin. The hydrogen bond network in F88Y obelin being due to the Tyr substitution clearly supports the suggestion that different hydrogen bond patterns near the oxygen of the 6-(p-hydroxyphenyl) substituent are the basis for spectral modifications between hydromedusan photoproteins.

  6. Structure of turbulent flow at a river confluence with momentum and velocity ratios close to 1: Insight provided by an eddy-resolving numerical simulation

    NASA Astrophysics Data System (ADS)

    Constantinescu, George; Miyawaki, Shinjiro; Rhoads, Bruce; Sukhodolov, Alexander; Kirkil, Gokhan

    2011-05-01

    River confluences are complex hydrodynamic environments where convergence of incoming flows produces complicated patterns of fluid motion, including the development of large-scale turbulence structures. Accurately simulating confluence hydrodynamics represents a considerable challenge for numerical modeling of river flows. This study uses an eddy-resolving numerical model to simulate the mean flow and large-scale turbulence structure at an asymmetrical river confluence with a concordant bed when the momentum ratio between the two incoming streams is close to 1. Results of the simulation are compared with field data on mean flow and turbulence structure. The simulation shows that the mixing interface is populated by quasi-two-dimensional eddies. Successive eddies have opposing senses of rotation. The mixing layer structure resembles that of a wake behind a bluff body (wake mode). Strong streamwise-oriented vortical (SOV) cells form on both sides of the mixing layer, a finding consistent with patterns inferred from the field data. The predicted mean flow fields show that flow curvature has an important influence on streamwise variation of circulation within the cores of the two primary SOV cells. These SOV cells, along with vortices generated by flow over a submerged block of sediment at one of the banks, strongly influence distributions of the streamwise velocity and turbulent kinetic energy downstream of the junction. Comparison of the eddy-resolving simulation results with predictions from the steady Spalart-Allmaras RANS model shows that the latter fails to predict important features of the measured distributions of streamwise velocity and turbulent kinetic energy because the RANS model underpredicts the strength of the SOV cells. Analysis of instantaneous and mean bed shear stress distributions indicates that the SOV cells enhance bed shear stresses to a greater degree than the quasi-two-dimensional eddies in the mixing interface.

  7. Snowflake vitreoretinal degeneration (SVD) mutation R162W provides new insights into Kir7.1 ion channel structure and function.

    PubMed

    Pattnaik, Bikash R; Tokarz, Sara; Asuma, Matti P; Schroeder, Tyler; Sharma, Anil; Mitchell, Julie C; Edwards, Albert O; Pillers, De-Ann M

    2013-01-01

    Snowflake Vitreoretinal Degeneration (SVD) is associated with the R162W mutation of the Kir7.1 inwardly-rectifying potassium channel. Kir7.1 is found at the apical membrane of Retinal Pigment Epithelial (RPE) cells, adjacent to the photoreceptor neurons. The SVD phenotype ranges from RPE degeneration to an abnormal b-wave to a liquid vitreous. We sought to determine how this mutation alters the structure and function of the human Kir7.1 channel. In this study, we expressed a Kir7.1 construct with the R162W mutation in CHO cells to evaluate function of the ion channel. Compared to the wild-type protein, the mutant protein exhibited a non-functional Kir channel that resulted in depolarization of the resting membrane potential. Upon co-expression with wild-type Kir7.1, R162W mutant showed a reduction of IKir7.1 and positive shift in '0' current potential. Homology modeling based on the structure of a bacterial Kir channel protein suggested that the effect of R162W mutation is a result of loss of hydrogen bonding by the regulatory lipid binding domain of the cytoplasmic structure.

  8. Crystal structures of IFT70/52 and IFT52/46 provide insight into intraflagellar transport B core complex assembly

    PubMed Central

    Taschner, Michael; Kotsis, Fruzsina; Braeuer, Philipp; Kuehn, E. Wolfgang

    2014-01-01

    Cilia are microtubule-based organelles that assemble via intraflagellar transport (IFT) and function as signaling hubs on eukaryotic cells. IFT relies on molecular motors and IFT complexes that mediate the contacts with ciliary cargo. To elucidate the architecture of the IFT-B complex, we reconstituted and purified the nonameric IFT-B core from Chlamydomonas reinhardtii and determined the crystal structures of C. reinhardtii IFT70/52 and Tetrahymena IFT52/46 subcomplexes. The 2.5-Å resolution IFT70/52 structure shows that IFT52330–370 is buried deeply within the IFT70 tetratricopeptide repeat superhelix. Furthermore, the polycystic kidney disease protein IFT88 binds IFT52281–329 in a complex that interacts directly with IFT70/IFT52330–381 in trans. The structure of IFT52C/IFT46C was solved at 2.3 Å resolution, and we show that it is essential for IFT-B core integrity by mediating interaction between IFT88/70/52/46 and IFT81/74/27/25/22 subcomplexes. Consistent with this, overexpression of mammalian IFT52C in MDCK cells is dominant-negative and causes IFT protein mislocalization and disrupted ciliogenesis. These data further rationalize several ciliogenesis phenotypes of IFT mutant strains. PMID:25349261

  9. Insights into the conformations of three structurally diverse proteins: cytochrome c, p53, and MDM2, provided by variable-temperature ion mobility mass spectrometry.

    PubMed

    Dickinson, Eleanor R; Jurneczko, Ewa; Pacholarz, Kamila J; Clarke, David J; Reeves, Matthew; Ball, Kathryn L; Hupp, Ted; Campopiano, Dominic; Nikolova, Penka V; Barran, Perdita E

    2015-03-17

    Thermally induced conformational transitions of three proteins of increasing intrinsic disorder-cytochrome c, the tumor suppressor protein p53 DNA binding domain (p53 DBD), and the N-terminus of the oncoprotein murine double minute 2 (NT-MDM2)-have been studied by native mass spectrometry and variable-temperature drift time ion mobility mass spectrometry (VT-DT-IM-MS). Ion mobility measurements were carried out at temperatures ranging from 200 to 571 K. Multiple conformations are observable over several charge states for all three monomeric proteins, and for cytochrome c, dimers of significant intensity are also observed. Cytochrome c [M + 5H](5+) ions present in one conformer of CCS ∼1200 Å(2), undergoing compaction in line with the reported Tmelt = 360.15 K before slight unfolding at 571 K. The more extended [M + 7H](7+) cytochrome c monomer presents as two conformers undergoing similar compaction and structural rearrangements, prior to thermally induced unfolding. The [D + 11H](11+) dimer presents as two conformers, which undergo slight structural compaction or annealing before dissociation. p53 DBD follows a trend of structural collapse before an increase in the observed collision cross section (CCS), akin to that observed for cytochrome c but proceeding more smoothly. At 300 K, the monomeric charge states present in two conformational families, which compact to one conformer of CCS ∼1750 Å(2) at 365 K, in line with the low solution Tmelt = 315-317 K. The protein then extends to produce either a broad unresolved CCS distribution or, for z > 9, two conformers. NT-MDM2 exhibits a greater number of structural rearrangements, displaying charge-state-dependent unfolding pathways. DT-IM-MS experiments at 200 K resolve multiple conformers. Low charge state species of NT-MDM2 present as a single compact conformational family centered on CCS ∼1250 Å(2) at 300 K. This undergoes conformational tightening in line with the solution Tmelt = 348 K before unfolding at

  10. Structure of the AvrBs3–DNA complex provides new insights into the initial thymine-recognition mechanism

    SciTech Connect

    Stella, Stefano; Molina, Rafael; Yefimenko, Igor; Prieto, Jesús; Silva, George; Bertonati, Claudia; Juillerat, Alexandre; Duchateau, Phillippe; Montoya, Guillermo

    2013-09-01

    The crystal structure of the AvrBs3–DNA complex is reported. Transcription activator-like effectors contain a DNA-binding domain organized in tandem repeats. The repeats include two adjacent residues known as the repeat variable di-residue, which recognize a single base pair, establishing a direct code between the dipeptides and the target DNA. This feature suggests this scaffold as an excellent candidate to generate new protein–DNA specificities for biotechnological applications. Here, the crystal structure of AvrBs3 (residues 152–895, molecular mass 82 kDa) in complex with its target DNA sequence is presented, revealing a new mode of interaction with the initial thymine of the target sequence, together with an analysis of both the binding specificity and the thermodynamic properties of AvrBs3. This study quantifies the affinity and the specificity between AvrBs3 and its target DNA. Moreover, in vitro and in vivo analyses reveal that AvrBs3 does not show a strict nucleotide-binding preference for the nucleotide at the zero position of the DNA, widening the number of possible sequences that could be targeted by this scaffold.

  11. Fic domain-catalyzed adenylylation: Insight provided by the structural analysis of the type IV secretion system effector BepA

    PubMed Central

    Palanivelu, Dinesh V; Goepfert, Arnaud; Meury, Marcel; Guye, Patrick; Dehio, Christoph; Schirmer, Tilman

    2011-01-01

    Numerous bacterial pathogens subvert cellular functions of eukaryotic host cells by the injection of effector proteins via dedicated secretion systems. The type IV secretion system (T4SS) effector protein BepA from Bartonella henselae is composed of an N-terminal Fic domain and a C-terminal Bartonella intracellular delivery domain, the latter being responsible for T4SS-mediated translocation into host cells. A proteolysis resistant fragment (residues 10–302) that includes the Fic domain shows autoadenylylation activity and adenylyl transfer onto Hela cell extract proteins as demonstrated by autoradiography on incubation with α-[32P]-ATP. Its crystal structure, determined to 2.9-Å resolution by the SeMet-SAD method, exhibits the canonical Fic fold including the HPFxxGNGRxxR signature motif with several elaborations in loop regions and an additional β-rich domain at the C-terminus. On crystal soaking with ATP/Mg2+, additional electron density indicated the presence of a PPi/Mg2+ moiety, the side product of the adenylylation reaction, in the anion binding nest of the signature motif. On the basis of this information and that of the recent structure of IbpA(Fic2) in complex with the eukaryotic target protein Cdc42, we present a detailed model for the ternary complex of Fic with the two substrates, ATP/Mg2+ and target tyrosine. The model is consistent with an in-line nucleophilic attack of the deprotonated side-chain hydroxyl group onto the α-phosphorus of the nucleotide to accomplish AMP transfer. Furthermore, a general, sequence-independent mechanism of target positioning through antiparallel β-strand interactions between enzyme and target is suggested. PMID:21213248

  12. Structure and Properties of a Non-processive, Salt-requiring, and Acidophilic Pectin Methylesterase from Aspergillus niger Provide Insights into the Key Determinants of Processivity Control*

    PubMed Central

    Kent, Lisa M.; Loo, Trevor S.; Melton, Laurence D.; Mercadante, Davide; Williams, Martin A. K.; Jameson, Geoffrey B.

    2016-01-01

    Many pectin methylesterases (PMEs) are expressed in plants to modify plant cell-wall pectins for various physiological roles. These pectins are also attacked by PMEs from phytopathogens and phytophagous insects. The de-methylesterification by PMEs of the O6-methyl ester groups of the homogalacturonan component of pectin, exposing galacturonic acids, can occur processively or non-processively, respectively, describing sequential versus single de-methylesterification events occurring before enzyme-substrate dissociation. The high resolution x-ray structures of a PME from Aspergillus niger in deglycosylated and Asn-linked N-acetylglucosamine-stub forms reveal a 10⅔-turn parallel β-helix (similar to but with less extensive loops than bacterial, plant, and insect PMEs). Capillary electrophoresis shows that this PME is non-processive, halophilic, and acidophilic. Molecular dynamics simulations and electrostatic potential calculations reveal very different behavior and properties compared with processive PMEs. Specifically, uncorrelated rotations are observed about the glycosidic bonds of a partially de-methyl-esterified decasaccharide model substrate, in sharp contrast to the correlated rotations of processive PMEs, and the substrate-binding groove is negatively not positively charged. PMID:26567911

  13. Structure of the AvrBs3–DNA complex provides new insights into the initial thymine-recognition mechanism

    PubMed Central

    Stella, Stefano; Molina, Rafael; Yefimenko, Igor; Prieto, Jesús; Silva, George; Bertonati, Claudia; Juillerat, Alexandre; Duchateau, Phillippe; Montoya, Guillermo

    2013-01-01

    Transcription activator-like effectors contain a DNA-binding domain organized in tandem repeats. The repeats include two adjacent residues known as the repeat variable di-residue, which recognize a single base pair, establishing a direct code between the dipeptides and the target DNA. This feature suggests this scaffold as an excellent candidate to generate new protein–DNA specificities for biotechnological applications. Here, the crystal structure of AvrBs3 (residues 152–895, molecular mass 82 kDa) in complex with its target DNA sequence is presented, revealing a new mode of interaction with the initial thymine of the target sequence, together with an analysis of both the binding specificity and the thermodynamic properties of AvrBs3. This study quantifies the affinity and the specificity between AvrBs3 and its target DNA. Moreover, in vitro and in vivo analyses reveal that AvrBs3 does not show a strict nucleotide-binding preference for the nucleotide at the zero position of the DNA, widening the number of possible sequences that could be targeted by this scaffold. PMID:23999294

  14. Structure-based in silico identification of ubiquitin-binding domains provides insights into the ALIX-V:ubiquitin complex and retrovirus budding

    PubMed Central

    Keren-Kaplan, Tal; Attali, Ilan; Estrin, Michael; Kuo, Lillian S; Farkash, Efrat; Jerabek-Willemsen, Moran; Blutraich, Noa; Artzi, Shay; Peri, Aviyah; Freed, Eric O; Wolfson, Haim J; Prag, Gali

    2013-01-01

    The ubiquitylation signal promotes trafficking of endogenous and retroviral transmembrane proteins. The signal is decoded by a large set of ubiquitin (Ub) receptors that tether Ub-binding domains (UBDs) to the trafficking machinery. We developed a structure-based procedure to scan the protein data bank for hidden UBDs. The screen retrieved many of the known UBDs. Intriguingly, new potential UBDs were identified, including the ALIX-V domain. Pull-down, cross-linking and E3-independent ubiquitylation assays biochemically corroborated the in silico findings. Guided by the output model, we designed mutations at the postulated ALIX-V:Ub interface. Biophysical affinity measurements using microscale-thermophoresis of wild-type and mutant proteins revealed some of the interacting residues of the complex. ALIX-V binds mono-Ub with a Kd of 119 μM. We show that ALIX-V oligomerizes with a Hill coefficient of 5.4 and IC50 of 27.6 μM and that mono-Ub induces ALIX-V oligomerization. Moreover, we show that ALIX-V preferentially binds K63 di-Ub compared with mono-Ub and K48 di-Ub. Finally, an in vivo functionality assay demonstrates the significance of ALIX-V:Ub interaction in equine infectious anaemia virus budding. These results not only validate the new procedure, but also demonstrate that ALIX-V directly interacts with Ub in vivo and that this interaction can influence retroviral budding. PMID:23361315

  15. Structure-based in silico identification of ubiquitin-binding domains provides insights into the ALIX-V:ubiquitin complex and retrovirus budding.

    PubMed

    Keren-Kaplan, Tal; Attali, Ilan; Estrin, Michael; Kuo, Lillian S; Farkash, Efrat; Jerabek-Willemsen, Moran; Blutraich, Noa; Artzi, Shay; Peri, Aviyah; Freed, Eric O; Wolfson, Haim J; Prag, Gali

    2013-02-20

    The ubiquitylation signal promotes trafficking of endogenous and retroviral transmembrane proteins. The signal is decoded by a large set of ubiquitin (Ub) receptors that tether Ub-binding domains (UBDs) to the trafficking machinery. We developed a structure-based procedure to scan the protein data bank for hidden UBDs. The screen retrieved many of the known UBDs. Intriguingly, new potential UBDs were identified, including the ALIX-V domain. Pull-down, cross-linking and E3-independent ubiquitylation assays biochemically corroborated the in silico findings. Guided by the output model, we designed mutations at the postulated ALIX-V:Ub interface. Biophysical affinity measurements using microscale-thermophoresis of wild-type and mutant proteins revealed some of the interacting residues of the complex. ALIX-V binds mono-Ub with a K(d) of 119 μM. We show that ALIX-V oligomerizes with a Hill coefficient of 5.4 and IC(50) of 27.6 μM and that mono-Ub induces ALIX-V oligomerization. Moreover, we show that ALIX-V preferentially binds K63 di-Ub compared with mono-Ub and K48 di-Ub. Finally, an in vivo functionality assay demonstrates the significance of ALIX-V:Ub interaction in equine infectious anaemia virus budding. These results not only validate the new procedure, but also demonstrate that ALIX-V directly interacts with Ub in vivo and that this interaction can influence retroviral budding.

  16. Optimal fitting of Gaussian-apodized or under-resolved emission lines in Fourier transform spectra providing new insights on the velocity structure of NGC 6720

    NASA Astrophysics Data System (ADS)

    Martin, Thomas B.; Prunet, Simon; Drissen, Laurent

    2016-12-01

    An analysis of the kinematics of NGC 6720 is performed on the commissioning data obtained with SITELLE, the Canada-France-Hawaii Telescope's new imaging Fourier transform spectrometer. In order to measure carefully the small broadening effect of a shell expansion on an unresolved emission line, we have determined a computationally robust implementation of the convolution of a Gaussian with a sinc instrumental line shape which avoids arithmetic overflows. This model can be used to measure line broadening of typically a few km s-1 even at low spectral resolution (R < 5000). We have also designed the corresponding set of Gaussian apodizing functions that are now used by ORBS, the SITELLE's reduction pipeline. We have implemented this model in ORCS, a fitting engine for SITELLE's data, and used it to derive the [S II] density map of the central part of the nebula. The study of the broadening of the [N II] lines shows that the main ring and the central lobe are two different shells with different expansion velocities. We have also derived deep and spatially resolved velocity maps of the halo in [N II] and Hα and found that the brightest bubbles are originating from two bipolar structures with a velocity difference of more than 35 km s-1 lying at the poles of a possibly unique halo shell expanding at a velocity of more than 15 km s-1.

  17. Structure of a truncation mutant of the nuclear export factor CRM1 provides insights into the auto-inhibitory role of its C-terminal helix.

    PubMed

    Dian, Cyril; Bernaudat, Florent; Langer, Karla; Oliva, Mizar F; Fornerod, Maarten; Schoehn, Guy; Müller, Christoph W; Petosa, Carlo

    2013-08-06

    Chromosome region maintenance 1/exportin1/Xpo1 (CRM1) associates with the GTPase Ran to mediate the nuclear export of proteins bearing a leucine-rich nuclear export signal (NES). CRM1 consists of helical hairpin HEAT repeats and a C-terminal helical extension (C-extension) that inhibits the binding of NES-bearing cargos. We report the crystal structure and small-angle X-ray scattering analysis of a human CRM1 mutant with enhanced NES-binding activity due to deletion of the C-extension. We show that loss of the C-extension leads to a repositioning of CRM1's C-terminal repeats and to a more extended overall conformation. Normal mode analysis predicts reduced rigidity for the deletion mutant, consistent with an observed decrease in thermal stability. Point mutations that destabilize the C-extension shift CRM1 to the more extended conformation, reduce thermal stability, and enhance NES-binding activity. These findings suggest that an important mechanism by which the C-extension regulates CRM1's cargo-binding affinity is by modulating the conformation and flexibility of its HEAT repeats.

  18. Evolutionary and Structural Analyses of Mammalian Haloacid Dehalogenase-type Phosphatases AUM and Chronophin Provide Insight into the Basis of Their Different Substrate Specificities*

    PubMed Central

    Seifried, Annegrit; Knobloch, Gunnar; Duraphe, Prashant S.; Segerer, Gabriela; Manhard, Julia; Schindelin, Hermann; Schultz, Jörg; Gohla, Antje

    2014-01-01

    Mammalian haloacid dehalogenase (HAD)-type phosphatases are an emerging family of phosphatases with important functions in physiology and disease, yet little is known about the basis of their substrate specificity. Here, we characterize a previously unexplored HAD family member (gene annotation, phosphoglycolate phosphatase), which we termed AUM, for aspartate-based, ubiquitous, Mg2+-dependent phosphatase. AUM is a tyrosine-specific paralog of the serine/threonine-specific protein and pyridoxal 5′-phosphate-directed HAD phosphatase chronophin. Comparative evolutionary and biochemical analyses reveal that a single, differently conserved residue in the cap domain of either AUM or chronophin is crucial for phosphatase specificity. We have solved the x-ray crystal structure of the AUM cap fused to the catalytic core of chronophin to 2.65 Å resolution and present a detailed view of the catalytic clefts of AUM and chronophin that explains their substrate preferences. Our findings identify a small number of cap domain residues that encode the different substrate specificities of AUM and chronophin. PMID:24338473

  19. Wheat arabinoxylan structure provides insight into function

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recent attention to dietary fiber in wheat (Triticum aestivum L.) has invigorated research in the non-starch carbohydrate arabinoxylan. Arabinoxylan (AX) molecules are comprised of a linear xylose backbone with arabinose substitutions along the backbone. These arabinose substituents can also carry a...

  20. Eggshell Porosity Provides Insight on Evolution of Nesting in Dinosaurs.

    PubMed

    Tanaka, Kohei; Zelenitsky, Darla K; Therrien, François

    2015-01-01

    Knowledge about the types of nests built by dinosaurs can provide insight into the evolution of nesting and reproductive behaviors among archosaurs. However, the low preservation potential of their nesting materials and nesting structures means that most information can only be gleaned indirectly through comparison with extant archosaurs. Two general nest types are recognized among living archosaurs: 1) covered nests, in which eggs are incubated while fully covered by nesting material (as in crocodylians and megapodes), and 2) open nests, in which eggs are exposed in the nest and brooded (as in most birds). Previously, dinosaur nest types had been inferred by estimating the water vapor conductance (i.e., diffusive capacity) of their eggs, based on the premise that high conductance corresponds to covered nests and low conductance to open nests. However, a lack of statistical rigor and inconsistencies in this method render its application problematic and its validity questionable. As an alternative we propose a statistically rigorous approach to infer nest type based on large datasets of eggshell porosity and egg mass compiled for over 120 extant archosaur species and 29 archosaur extinct taxa/ootaxa. The presence of a strong correlation between eggshell porosity and nest type among extant archosaurs indicates that eggshell porosity can be used as a proxy for nest type, and thus discriminant analyses can help predict nest type in extinct taxa. Our results suggest that: 1) covered nests are likely the primitive condition for dinosaurs (and probably archosaurs), and 2) open nests first evolved among non-avian theropods more derived than Lourinhanosaurus and were likely widespread in non-avian maniraptorans, well before the appearance of birds. Although taphonomic evidence suggests that basal open nesters (i.e., oviraptorosaurs and troodontids) were potentially the first dinosaurs to brood their clutches, they still partially buried their eggs in sediment. Open nests

  1. Eggshell Porosity Provides Insight on Evolution of Nesting in Dinosaurs

    PubMed Central

    2015-01-01

    Knowledge about the types of nests built by dinosaurs can provide insight into the evolution of nesting and reproductive behaviors among archosaurs. However, the low preservation potential of their nesting materials and nesting structures means that most information can only be gleaned indirectly through comparison with extant archosaurs. Two general nest types are recognized among living archosaurs: 1) covered nests, in which eggs are incubated while fully covered by nesting material (as in crocodylians and megapodes), and 2) open nests, in which eggs are exposed in the nest and brooded (as in most birds). Previously, dinosaur nest types had been inferred by estimating the water vapor conductance (i.e., diffusive capacity) of their eggs, based on the premise that high conductance corresponds to covered nests and low conductance to open nests. However, a lack of statistical rigor and inconsistencies in this method render its application problematic and its validity questionable. As an alternative we propose a statistically rigorous approach to infer nest type based on large datasets of eggshell porosity and egg mass compiled for over 120 extant archosaur species and 29 archosaur extinct taxa/ootaxa. The presence of a strong correlation between eggshell porosity and nest type among extant archosaurs indicates that eggshell porosity can be used as a proxy for nest type, and thus discriminant analyses can help predict nest type in extinct taxa. Our results suggest that: 1) covered nests are likely the primitive condition for dinosaurs (and probably archosaurs), and 2) open nests first evolved among non-avian theropods more derived than Lourinhanosaurus and were likely widespread in non-avian maniraptorans, well before the appearance of birds. Although taphonomic evidence suggests that basal open nesters (i.e., oviraptorosaurs and troodontids) were potentially the first dinosaurs to brood their clutches, they still partially buried their eggs in sediment. Open nests

  2. Social network analysis provides insights into African swine fever epidemiology.

    PubMed

    Lichoti, Jacqueline Kasiiti; Davies, Jocelyn; Kitala, Philip M; Githigia, Samuel M; Okoth, Edward; Maru, Yiheyis; Bukachi, Salome A; Bishop, Richard P

    2016-04-01

    Pig movements play a significant role in the spread of economically important infectious diseases such as the African swine fever. Characterization of movement networks between pig farms and through other types of farm and household enterprises that are involved in pig value chains can provide useful information on the role that different participants in the networks play in pathogen transmission. Analysis of social networks that underpin these pig movements can reveal pathways that are important in the transmission of disease, trade in commodities, the dissemination of information and the influence of behavioural norms. We assessed pig movements among pig keeping households within West Kenya and East Uganda and across the shared Kenya-Uganda border in the study region, to gain insight into within-country and trans-boundary pig movements. Villages were sampled using a randomized cluster design. Data were collected through interviews in 2012 and 2013 from 683 smallholder pig-keeping households in 34 villages. NodeXL software was used to describe pig movement networks at village level. The pig movement and trade networks were localized and based on close social networks involving family ties, friendships and relationships with neighbours. Pig movement network modularity ranged from 0.2 to 0.5 and exhibited good community structure within the network implying an easy flow of knowledge and adoption of new attitudes and beliefs, but also promoting an enhanced rate of disease transmission. The average path length of 5 defined using NodeXL, indicated that disease could easily reach every node in a cluster. Cross-border boar service between Uganda and Kenya was also recorded. Unmonitored trade in both directions was prevalent. While most pig transactions in the absence of disease, were at a small scale (<5km) and characterized by regular agistment, most pig sales during ASF outbreaks were to traders or other farmers from outside the sellers' village at a range of >10km

  3. Sparse regularization techniques provide novel insights into outcome integration processes.

    PubMed

    Mohr, Holger; Wolfensteller, Uta; Frimmel, Steffi; Ruge, Hannes

    2015-01-01

    By exploiting information that is contained in the spatial arrangement of neural activations, multivariate pattern analysis (MVPA) can detect distributed brain activations which are not accessible by standard univariate analysis. Recent methodological advances in MVPA regularization techniques have made it feasible to produce sparse discriminative whole-brain maps with highly specific patterns. Furthermore, the most recent refinement, the Graph Net, explicitly takes the 3D-structure of fMRI data into account. Here, these advanced classification methods were applied to a large fMRI sample (N=70) in order to gain novel insights into the functional localization of outcome integration processes. While the beneficial effect of differential outcomes is well-studied in trial-and-error learning, outcome integration in the context of instruction-based learning has remained largely unexplored. In order to examine neural processes associated with outcome integration in the context of instruction-based learning, two groups of subjects underwent functional imaging while being presented with either differential or ambiguous outcomes following the execution of varying stimulus-response instructions. While no significant univariate group differences were found in the resulting fMRI dataset, L1-regularized (sparse) classifiers performed significantly above chance and also clearly outperformed the standard L2-regularized (dense) Support Vector Machine on this whole-brain between-subject classification task. Moreover, additional L2-regularization via the Elastic Net and spatial regularization by the Graph Net improved interpretability of discriminative weight maps but were accompanied by reduced classification accuracies. Most importantly, classification based on sparse regularization facilitated the identification of highly specific regions differentially engaged under ambiguous and differential outcome conditions, comprising several prefrontal regions previously associated with

  4. Structural analysis of APOB variants, p.(Arg3527Gln), p.(Arg1164Thr) and p.(Gln4494del), causing Familial Hypercholesterolaemia provides novel insights into variant pathogenicity.

    PubMed

    Fernández-Higuero, J A; Etxebarria, A; Benito-Vicente, A; Alves, A C; Arrondo, J L R; Ostolaza, H; Bourbon, M; Martin, C

    2015-12-08

    Familial hypercholesterolaemia (FH) is an inherited autosomal dominant disorder resulting from defects in the low-density lipoprotein receptor (LDLR), in the apolipoprotein B (APOB) or in the proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. In the majority of the cases FH is caused by mutations occurring within LDLR, while only few mutations in APOB and PCSK9 have been proved to cause disease. p.(Arg3527Gln) was the first mutation in APOB being identified and characterized. Recently two novel pathogenic APOB variants have been described: p.(Arg1164Thr) and p.(Gln4494del) showing impaired LDLR binding capacity, and diminished LDL uptake. The objective of this work was to analyse the structure of p.(Arg1164Thr) and p.(Gln4494del) variants to gain insight into their pathogenicity. Secondary structure of the human ApoB100 has been investigated by infrared spectroscopy (IR) and LDL particle size both by dynamic light scattering (DLS) and electron microscopy. The results show differences in secondary structure and/or in particle size of p.(Arg1164Thr) and p.(Gln4494del) variants compared with wild type. We conclude that these changes underlie the defective binding and uptake of p.(Arg1164Thr) and p.(Gln4494del) variants. Our study reveals that structural studies on pathogenic variants of APOB may provide very useful information to understand their role in FH disease.

  5. Structural analysis of APOB variants, p.(Arg3527Gln), p.(Arg1164Thr) and p.(Gln4494del), causing Familial Hypercholesterolaemia provides novel insights into variant pathogenicity

    PubMed Central

    Fernández-Higuero, J. A.; Etxebarria, A.; Benito-Vicente, A.; Alves, A. C.; Arrondo, J. L. R.; Ostolaza, H.; Bourbon, M.; Martin, C.

    2015-01-01

    Familial hypercholesterolaemia (FH) is an inherited autosomal dominant disorder resulting from defects in the low-density lipoprotein receptor (LDLR), in the apolipoprotein B (APOB) or in the proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. In the majority of the cases FH is caused by mutations occurring within LDLR, while only few mutations in APOB and PCSK9 have been proved to cause disease. p.(Arg3527Gln) was the first mutation in APOB being identified and characterized. Recently two novel pathogenic APOB variants have been described: p.(Arg1164Thr) and p.(Gln4494del) showing impaired LDLR binding capacity, and diminished LDL uptake. The objective of this work was to analyse the structure of p.(Arg1164Thr) and p.(Gln4494del) variants to gain insight into their pathogenicity. Secondary structure of the human ApoB100 has been investigated by infrared spectroscopy (IR) and LDL particle size both by dynamic light scattering (DLS) and electron microscopy. The results show differences in secondary structure and/or in particle size of p.(Arg1164Thr) and p.(Gln4494del) variants compared with wild type. We conclude that these changes underlie the defective binding and uptake of p.(Arg1164Thr) and p.(Gln4494del) variants. Our study reveals that structural studies on pathogenic variants of APOB may provide very useful information to understand their role in FH disease. PMID:26643808

  6. Saccharina genomes provide novel insight into kelp biology

    PubMed Central

    Ye, Naihao; Zhang, Xiaowen; Miao, Miao; Fan, Xiao; Zheng, Yi; Xu, Dong; Wang, Jinfeng; Zhou, Lin; Wang, Dongsheng; Gao, Yuan; Wang, Yitao; Shi, Wenyu; Ji, Peifeng; Li, Demao; Guan, Zheng; Shao, Changwei; Zhuang, Zhimeng; Gao, Zhengquan; Qi, Ji; Zhao, Fangqing

    2015-01-01

    Seaweeds are essential for marine ecosystems and have immense economic value. Here we present a comprehensive analysis of the draft genome of Saccharina japonica, one of the most economically important seaweeds. The 537-Mb assembled genomic sequence covered 98.5% of the estimated genome, and 18,733 protein-coding genes are predicted and annotated. Gene families related to cell wall synthesis, halogen concentration, development and defence systems were expanded. Functional diversification of the mannuronan C-5-epimerase and haloperoxidase gene families provides insight into the evolutionary adaptation of polysaccharide biosynthesis and iodine antioxidation. Additional sequencing of seven cultivars and nine wild individuals reveal that the genetic diversity within wild populations is greater than among cultivars. All of the cultivars are descendants of a wild S. japonica accession showing limited admixture with S. longissima. This study represents an important advance toward improving yields and economic traits in Saccharina and provides an invaluable resource for plant genome studies. PMID:25908475

  7. Saccharina genomes provide novel insight into kelp biology.

    PubMed

    Ye, Naihao; Zhang, Xiaowen; Miao, Miao; Fan, Xiao; Zheng, Yi; Xu, Dong; Wang, Jinfeng; Zhou, Lin; Wang, Dongsheng; Gao, Yuan; Wang, Yitao; Shi, Wenyu; Ji, Peifeng; Li, Demao; Guan, Zheng; Shao, Changwei; Zhuang, Zhimeng; Gao, Zhengquan; Qi, Ji; Zhao, Fangqing

    2015-04-24

    Seaweeds are essential for marine ecosystems and have immense economic value. Here we present a comprehensive analysis of the draft genome of Saccharina japonica, one of the most economically important seaweeds. The 537-Mb assembled genomic sequence covered 98.5% of the estimated genome, and 18,733 protein-coding genes are predicted and annotated. Gene families related to cell wall synthesis, halogen concentration, development and defence systems were expanded. Functional diversification of the mannuronan C-5-epimerase and haloperoxidase gene families provides insight into the evolutionary adaptation of polysaccharide biosynthesis and iodine antioxidation. Additional sequencing of seven cultivars and nine wild individuals reveal that the genetic diversity within wild populations is greater than among cultivars. All of the cultivars are descendants of a wild S. japonica accession showing limited admixture with S. longissima. This study represents an important advance toward improving yields and economic traits in Saccharina and provides an invaluable resource for plant genome studies.

  8. Prevalent Exon-Intron Structural Changes in the APETALA1/FRUITFULL, SEPALLATA, AGAMOUS-LIKE6, and FLOWERING LOCUS C MADS-Box Gene Subfamilies Provide New Insights into Their Evolution

    PubMed Central

    Yu, Xianxian; Duan, Xiaoshan; Zhang, Rui; Fu, Xuehao; Ye, Lingling; Kong, Hongzhi; Xu, Guixia; Shan, Hongyan

    2016-01-01

    AP1/FUL, SEP, AGL6, and FLC subfamily genes play important roles in flower development. The phylogenetic relationships among them, however, have been controversial, which impedes our understanding of the origin and functional divergence of these genes. One possible reason for the controversy may be the problems caused by changes in the exon-intron structure of genes, which, according to recent studies, may generate non-homologous sites and hamper the homology-based sequence alignment. In this study, we first performed exon-by-exon alignments of these and three outgroup subfamilies (SOC1, AG, and STK). Phylogenetic trees reconstructed based on these matrices show improved resolution and better congruence with species phylogeny. In the context of these phylogenies, we traced evolutionary changes of exon-intron structures in each subfamily. We found that structural changes have occurred frequently following gene duplication and speciation events. Notably, exons 7 and 8 (if present) suffered more structural changes than others. With the knowledge of exon-intron structural changes, we generated more reasonable alignments containing all the focal subfamilies. The resulting trees showed that the SEP subfamily is sister to the monophyletic group formed by AP1/FUL and FLC subfamily genes and that the AGL6 subfamily forms a sister group to the three abovementioned subfamilies. Based on this topology, we inferred the evolutionary history of exon-intron structural changes among different subfamilies. Particularly, we found that the eighth exon originated before the divergence of AP1/FUL, FLC, SEP, and AGL6 subfamilies and degenerated in the ancestral FLC-like gene. These results provide new insights into the origin and evolution of the AP1/FUL, FLC, SEP, and AGL6 subfamilies. PMID:27200066

  9. The Crystal Structures of Substrate and Nucleotide Complexes of Enterococcus faecium Aminoglycoside-2′′-Phosphotransferase-IIa [APH(2′′)-IIa] Provide Insights into Substrate Selectivity in the APH(2′′) Subfamily▿ ‡

    PubMed Central

    Young, Paul G.; Walanj, Rupa; Lakshmi, Vendula; Byrnes, Laura J.; Metcalf, Peter; Baker, Edward N.; Vakulenko, Sergei B.; Smith, Clyde A.

    2009-01-01

    Aminoglycoside-2′′-phosphotransferase-IIa [APH(2′′)-IIa] is one of a number of homologous bacterial enzymes responsible for the deactivation of the aminoglycoside family of antibiotics and is thus a major component in bacterial resistance to these compounds. APH(2′′)-IIa produces resistance to several clinically important aminoglycosides (including kanamycin and gentamicin) in both gram-positive and gram-negative bacteria, most notably in Enterococcus species. We have determined the structures of two complexes of APH(2′′)-IIa, the binary gentamicin complex and a ternary complex containing adenosine-5′-(β,γ-methylene)triphosphate (AMPPCP) and streptomycin. This is the first crystal structure of a member of the APH(2′′) family of aminoglycoside phosphotransferases. The structure of the gentamicin-APH(2′′)-IIa complex was solved by multiwavelength anomalous diffraction methods from a single selenomethionine-substituted crystal and was refined to a crystallographic R factor of 0.210 (Rfree, 0.271) at a resolution of 2.5 Å. The structure of the AMPPCP-streptomycin complex was solved by molecular replacement using the gentamicin-APH(2′′)-IIa complex as the starting model. The enzyme has a two-domain structure with the substrate binding site located in a cleft in the C-terminal domain. Gentamicin binding is facilitated by a number of conserved acidic residues lining the binding cleft, with the A and B rings of the substrate forming the majority of the interactions. The inhibitor streptomycin, although binding in the same pocket as gentamicin, is orientated such that no potential phosphorylation sites are adjacent to the catalytic aspartate residue. The binding of gentamicin and streptomycin provides structural insights into the substrate selectivity of the APH(2′′) subfamily of aminoglycoside phosphotransferases, specifically, the selectivity between the 4,6-disubstituted and the 4,5-disubstituted aminoglycosides. PMID:19429619

  10. Structural insights into microtubule doublet interactions inaxonemes

    SciTech Connect

    Downing, Kenneth H.; Sui, Haixin

    2007-06-06

    Coordinated sliding of microtubule doublets, driven by dynein motors, produces periodic beating of the axoneme. Recent structural studies of the axoneme have used cryo-electron tomography to reveal new details of the interactions among some of the multitude of proteins that form the axoneme and regulate its movement. Connections among the several sets of dyneins, in particular, suggest ways in which their actions may be coordinated. Study of the molecular architecture of isolated doublets has provided a structural basis for understanding the doublet's mechanical properties that are related to the bending of the axoneme, and has also offered insight into its potential role in the mechanism of dynein activity regulation.

  11. Structure of the LINGO-1-anti-LINGO-1 Li81 antibody complex provides insights into the biology of LINGO-1 and the mechanism of action of the antibody therapy.

    PubMed

    Pepinsky, R Blake; Arndt, Joseph W; Quan, Chao; Gao, Yan; Quintero-Monzon, Omar; Lee, Xinhua; Mi, Sha

    2014-07-01

    Multiple sclerosis (MS) is an autoimmune-inflammatory disease of the central nervous system (CNS) with prominent demyelination and axonal injury. While most MS therapies target the immunologic response, there is a large unmet need for treatments that can promote CNS repair. LINGO-1 (leucine-rich repeat and Ig-containing Nogo receptor interacting protein-1) is a membrane protein selectively expressed in the CNS that suppresses myelination, preventing the repair of damaged axons. We are investigating LINGO-1 antagonist antibodies that lead to remyelination as a new paradigm for treatment of individuals with MS. The anti-LINGO-1 Li81 antibody,BIIB033, is currently in clinical trials and is the first MS treatment targeting CNS repair. Here, to elucidate the mechanism of action of the antibody, we solved the crystal structure of the LINGO-1-Li81 Fab complex and used biochemical and functional studies to investigate structure-function relationships. Li81 binds to the convex surface of the leucine-rich repeat domain of LINGO-1 within repeats 4-8. Fab binding blocks contact points used in the oligomerization of LINGO-1 and produces a stable complex containing two copies each of LINGO-1 and Fab that results from a rearrangement of contacts stabilizing the quaternary structure of LINGO-1. The formation of the LINGO-1-Li81 Fab complex masks functional epitopes within the Ig domain of LINGO-1 that are important for its biologic activity in oligodendrocyte differentiation. These studies provide new insights into the structure and biology of LINGO-1 and how Li81 monoclonal antibody can block its function.

  12. CONSTRICTOR: Constraint Modification Provides Insight into Design of Biochemical Networks

    PubMed Central

    Erickson, Keesha E.; Gill, Ryan T.; Chatterjee, Anushree

    2014-01-01

    Advances in computational methods that allow for exploration of the combinatorial mutation space are needed to realize the potential of synthetic biology based strain engineering efforts. Here, we present Constrictor, a computational framework that uses flux balance analysis (FBA) to analyze inhibitory effects of genetic mutations on the performance of biochemical networks. Constrictor identifies engineering interventions by classifying the reactions in the metabolic model depending on the extent to which their flux must be decreased to achieve the overproduction target. The optimal inhibition of various reaction pathways is determined by restricting the flux through targeted reactions below the steady state levels of a baseline strain. Constrictor generates unique in silico strains, each representing an “expression state”, or a combination of gene expression levels required to achieve the overproduction target. The Constrictor framework is demonstrated by studying overproduction of ethylene in Escherichia coli network models iAF1260 and iJO1366 through the addition of the heterologous ethylene-forming enzyme from Pseudomonas syringae. Targeting individual reactions as well as combinations of reactions reveals in silico mutants that are predicted to have as high as 25% greater theoretical ethylene yields than the baseline strain during simulated exponential growth. Altering the degree of restriction reveals a large distribution of ethylene yields, while analysis of the expression states that return lower yields provides insight into system bottlenecks. Finally, we demonstrate the ability of Constrictor to scan networks and provide targets for a range of possible products. Constrictor is an adaptable technique that can be used to generate and analyze disparate populations of in silico mutants, select gene expression levels and provide non-intuitive strategies for metabolic engineering. PMID:25422896

  13. Small teleost fish provide new insights into human skeletal diseases.

    PubMed

    Witten, P E; Harris, M P; Huysseune, A; Winkler, C

    2017-01-01

    Small teleost fish such as zebrafish and medaka are increasingly studied as models for human skeletal diseases. Efficient new genome editing tools combined with advances in the analysis of skeletal phenotypes provide new insights into fundamental processes of skeletal development. The skeleton among vertebrates is a highly conserved organ system, but teleost fish and mammals have evolved unique traits or have lost particular skeletal elements in each lineage. Several unique features of the skeleton relate to the extremely small size of early fish embryos and the small size of adult fish used as models. A detailed analysis of the plethora of interesting skeletal phenotypes in zebrafish and medaka pushes available skeletal imaging techniques to their respective limits and promotes the development of new imaging techniques. Impressive numbers of zebrafish and medaka mutants with interesting skeletal phenotypes have been characterized, complemented by transgenic zebrafish and medaka lines. The advent of efficient genome editing tools, such as TALEN and CRISPR/Cas9, allows to introduce targeted deficiencies in genes of model teleosts to generate skeletal phenotypes that resemble human skeletal diseases. This review will also discuss other attractive aspects of the teleost skeleton. This includes the capacity for lifelong tooth replacement and for the regeneration of dermal skeletal elements, such as scales and fin rays, which further increases the value of zebrafish and medaka models for skeletal research.

  14. The complex jujube genome provides insights into fruit tree biology

    PubMed Central

    Liu, Meng-Jun; Zhao, Jin; Cai, Qing-Le; Liu, Guo-Cheng; Wang, Jiu-Rui; Zhao, Zhi-Hui; Liu, Ping; Dai, Li; Yan, Guijun; Wang, Wen-Jiang; Li, Xian-Song; Chen, Yan; Sun, Yu-Dong; Liu, Zhi-Guo; Lin, Min-Juan; Xiao, Jing; Chen, Ying-Ying; Li, Xiao-Feng; Wu, Bin; Ma, Yong; Jian, Jian-Bo; Yang, Wei; Yuan, Zan; Sun, Xue-Chao; Wei, Yan-Li; Yu, Li-Li; Zhang, Chi; Liao, Sheng-Guang; He, Rong-Jun; Guang, Xuan-Min; Wang, Zhuo; Zhang, Yue-Yang; Luo, Long-Hai

    2014-01-01

    The jujube (Ziziphus jujuba Mill.), a member of family Rhamnaceae, is a major dry fruit and a traditional herbal medicine for more than one billion people. Here we present a high-quality sequence for the complex jujube genome, the first genome sequence of Rhamnaceae, using an integrated strategy. The final assembly spans 437.65 Mb (98.6% of the estimated) with 321.45 Mb anchored to the 12 pseudo-chromosomes and contains 32,808 genes. The jujube genome has undergone frequent inter-chromosome fusions and segmental duplications, but no recent whole-genome duplication. Further analyses of the jujube-specific genes and transcriptome data from 15 tissues reveal the molecular mechanisms underlying some specific properties of the jujube. Its high vitamin C content can be attributed to a unique high level expression of genes involved in both biosynthesis and regeneration. Our study provides insights into jujube-specific biology and valuable genomic resources for the improvement of Rhamnaceae plants and other fruit trees. PMID:25350882

  15. The complex jujube genome provides insights into fruit tree biology.

    PubMed

    Liu, Meng-Jun; Zhao, Jin; Cai, Qing-Le; Liu, Guo-Cheng; Wang, Jiu-Rui; Zhao, Zhi-Hui; Liu, Ping; Dai, Li; Yan, Guijun; Wang, Wen-Jiang; Li, Xian-Song; Chen, Yan; Sun, Yu-Dong; Liu, Zhi-Guo; Lin, Min-Juan; Xiao, Jing; Chen, Ying-Ying; Li, Xiao-Feng; Wu, Bin; Ma, Yong; Jian, Jian-Bo; Yang, Wei; Yuan, Zan; Sun, Xue-Chao; Wei, Yan-Li; Yu, Li-Li; Zhang, Chi; Liao, Sheng-Guang; He, Rong-Jun; Guang, Xuan-Min; Wang, Zhuo; Zhang, Yue-Yang; Luo, Long-Hai

    2014-10-28

    The jujube (Ziziphus jujuba Mill.), a member of family Rhamnaceae, is a major dry fruit and a traditional herbal medicine for more than one billion people. Here we present a high-quality sequence for the complex jujube genome, the first genome sequence of Rhamnaceae, using an integrated strategy. The final assembly spans 437.65 Mb (98.6% of the estimated) with 321.45 Mb anchored to the 12 pseudo-chromosomes and contains 32,808 genes. The jujube genome has undergone frequent inter-chromosome fusions and segmental duplications, but no recent whole-genome duplication. Further analyses of the jujube-specific genes and transcriptome data from 15 tissues reveal the molecular mechanisms underlying some specific properties of the jujube. Its high vitamin C content can be attributed to a unique high level expression of genes involved in both biosynthesis and regeneration. Our study provides insights into jujube-specific biology and valuable genomic resources for the improvement of Rhamnaceae plants and other fruit trees.

  16. Marsupial Genome Sequences: Providing Insight into Evolution and Disease

    PubMed Central

    Deakin, Janine E.

    2012-01-01

    Marsupials (metatherians), with their position in vertebrate phylogeny and their unique biological features, have been studied for many years by a dedicated group of researchers, but it has only been since the sequencing of the first marsupial genome that their value has been more widely recognised. We now have genome sequences for three distantly related marsupial species (the grey short-tailed opossum, the tammar wallaby, and Tasmanian devil), with the promise of many more genomes to be sequenced in the near future, making this a particularly exciting time in marsupial genomics. The emergence of a transmissible cancer, which is obliterating the Tasmanian devil population, has increased the importance of obtaining and analysing marsupial genome sequence for understanding such diseases as well as for conservation efforts. In addition, these genome sequences have facilitated studies aimed at answering questions regarding gene and genome evolution and provided insight into the evolution of epigenetic mechanisms. Here I highlight the major advances in our understanding of evolution and disease, facilitated by marsupial genome projects, and speculate on the future contributions to be made by such sequences. PMID:24278712

  17. Comparative genomics provide insights into evolution of trichoderma nutrition style.

    PubMed

    Xie, Bin-Bin; Qin, Qi-Long; Shi, Mei; Chen, Lei-Lei; Shu, Yan-Li; Luo, Yan; Wang, Xiao-Wei; Rong, Jin-Cheng; Gong, Zhi-Ting; Li, Dan; Sun, Cai-Yun; Liu, Gui-Ming; Dong, Xiao-Wei; Pang, Xiu-Hua; Huang, Feng; Liu, Weifeng; Chen, Xiu-Lan; Zhou, Bai-Cheng; Zhang, Yu-Zhong; Song, Xiao-Yan

    2014-02-01

    Saprotrophy on plant biomass is a recently developed nutrition strategy for Trichoderma. However, the physiology and evolution of this new nutrition strategy is still elusive. We report the deep sequencing and analysis of the genome of Trichoderma longibrachiatum, an efficient cellulase producer. The 31.7-Mb genome, smallest among the sequenced Trichoderma species, encodes fewer nutrition-related genes than saprotrophic T. reesei (Tr), including glycoside hydrolases and nonribosomal peptide synthetase-polyketide synthase. Homology and phylogenetic analyses suggest that a large number of nutrition-related genes, including GH18 chitinases, β-1,3/1,6-glucanases, cellulolytic enzymes, and hemicellulolytic enzymes, were lost in the common ancestor of T. longibrachiatum (Tl) and Tr. dN/dS (ω) calculation indicates that all the nutrition-related genes analyzed are under purifying selection. Cellulolytic enzymes, the key enzymes for saprotrophy on plant biomass, are under stronger purifying selection pressure in Tl and Tr than in mycoparasitic species, suggesting that development of the nutrition strategy of saprotrophy on plant biomass has increased the selection pressure. In addition, aspartic proteases, serine proteases, and metalloproteases are subject to stronger purifying selection pressure in Tl and Tr, suggesting that these enzymes may also play important roles in the nutrition. This study provides insights into the physiology and evolution of the nutrition strategy of Trichoderma.

  18. Genetic Determinants of Epigenetic Patterns: Providing Insight into Disease.

    PubMed

    Cazaly, Emma; Charlesworth, Jac; Dickinson, Joanne L; Holloway, Adele F

    2015-03-26

    The field of epigenetics and our understanding of the mechanisms that regulate the establishment, maintenance and heritability of epigenetic patterns continue to grow at a remarkable rate. This information is providing increased understanding of the role of epigenetic changes in disease, insight into the underlying causes of these epigenetic changes and revealing new avenues for therapeutic intervention. Epigenetic modifiers are increasingly being pursued as therapeutic targets in a range of diseases, with a number of agents targeting epigenetic modifications already proving effective in diseases such as cancer. Although it is well established that DNA mutations and aberrant expression of epigenetic modifiers play a key role in disease, attention is now turning to the interplay between genetic and epigenetic factors in complex disease etiology. The role of genetic variability in determining epigenetic profiles, which can then be modified by environmental and stochastic factors, is becoming more apparent. Understanding the interplay between genetic and epigenetic factors is likely to aid in identifying individuals most likely to benefit from epigenetic therapies. This goal is coming closer to realization because of continual advances in laboratory and statistical tools enabling improvements in the integration of genomic, epigenomic and phenotypic data.

  19. Comparative Genomics Provide Insights into Evolution of Trichoderma Nutrition Style

    PubMed Central

    Xie, Bin-Bin; Qin, Qi-Long; Shi, Mei; Chen, Lei-Lei; Shu, Yan-Li; Luo, Yan; Wang, Xiao-Wei; Rong, Jin-Cheng; Gong, Zhi-Ting; Li, Dan; Sun, Cai-Yun; Liu, Gui-Ming; Dong, Xiao-Wei; Pang, Xiu-Hua; Huang, Feng; Liu, Weifeng; Chen, Xiu-Lan; Zhou, Bai-Cheng; Zhang, Yu-Zhong; Song, Xiao-Yan

    2014-01-01

    Saprotrophy on plant biomass is a recently developed nutrition strategy for Trichoderma. However, the physiology and evolution of this new nutrition strategy is still elusive. We report the deep sequencing and analysis of the genome of Trichoderma longibrachiatum, an efficient cellulase producer. The 31.7-Mb genome, smallest among the sequenced Trichoderma species, encodes fewer nutrition-related genes than saprotrophic T. reesei (Tr), including glycoside hydrolases and nonribosomal peptide synthetase–polyketide synthase. Homology and phylogenetic analyses suggest that a large number of nutrition-related genes, including GH18 chitinases, β-1,3/1,6-glucanases, cellulolytic enzymes, and hemicellulolytic enzymes, were lost in the common ancestor of T. longibrachiatum (Tl) and Tr. dN/dS (ω) calculation indicates that all the nutrition-related genes analyzed are under purifying selection. Cellulolytic enzymes, the key enzymes for saprotrophy on plant biomass, are under stronger purifying selection pressure in Tl and Tr than in mycoparasitic species, suggesting that development of the nutrition strategy of saprotrophy on plant biomass has increased the selection pressure. In addition, aspartic proteases, serine proteases, and metalloproteases are subject to stronger purifying selection pressure in Tl and Tr, suggesting that these enzymes may also play important roles in the nutrition. This study provides insights into the physiology and evolution of the nutrition strategy of Trichoderma. PMID:24482532

  20. Can tobacco dependence provide insights into other drug addictions?

    PubMed

    DiFranza, Joseph R

    2016-10-27

    Within the field of addiction research, individuals tend to operate within silos of knowledge focused on specific drug classes. The discovery that tobacco dependence develops in a progression of stages and that the latency to the onset of withdrawal symptoms after the last use of tobacco changes over time have provided insights into how tobacco dependence develops that might be applied to the study of other drugs.As physical dependence on tobacco develops, it progresses through previously unrecognized clinical stages of wanting, craving and needing. The latency to withdrawal is a measure of the asymptomatic phase of withdrawal, extending from the last use of tobacco to the emergence of withdrawal symptoms. Symptomatic withdrawal is characterized by a wanting phase, a craving phase, and a needing phase. The intensity of the desire to smoke that is triggered by withdrawal correlates with brain activity in addiction circuits. With repeated tobacco use, the latency to withdrawal shrinks from as long as several weeks to as short as several minutes. The shortening of the asymptomatic phase of withdrawal drives an escalation of smoking, first in terms of the number of smoking days/month until daily smoking commences, then in terms of cigarettes smoked/day.The discoveries of the stages of physical dependence and the latency to withdrawal raises the question, does physical dependence develop in stages with other drugs? Is the latency to withdrawal for other substances measured in weeks at the onset of dependence? Does it shorten over time? The research methods that uncovered how tobacco dependence emerges might be fruitfully applied to the investigation of other addictions.

  1. Latest insights on adenovirus structure and assembly.

    PubMed

    San Martín, Carmen

    2012-05-01

    Adenovirus (AdV) capsid organization is considerably complex, not only because of its large size (~950 Å) and triangulation number (pseudo T = 25), but also because it contains four types of minor proteins in specialized locations modulating the quasi-equivalent icosahedral interactions. Up until 2009, only its major components (hexon, penton, and fiber) had separately been described in atomic detail. Their relationships within the virion, and the location of minor coat proteins, were inferred from combining the known crystal structures with increasingly more detailed cryo-electron microscopy (cryoEM) maps. There was no structural information on assembly intermediates. Later on that year, two reports described the structural differences between the mature and immature adenoviral particle, starting to shed light on the different stages of viral assembly, and giving further insights into the roles of core and minor coat proteins during morphogenesis [1,2]. Finally, in 2010, two papers describing the atomic resolution structure of the complete virion appeared [3,4]. These reports represent a veritable tour de force for two structural biology techniques: X-ray crystallography and cryoEM, as this is the largest macromolecular complex solved at high resolution by either of them. In particular, the cryoEM analysis provided an unprecedented clear picture of the complex protein networks shaping the icosahedral shell. Here I review these latest developments in the field of AdV structural studies.

  2. Latest Insights on Adenovirus Structure and Assembly

    PubMed Central

    San Martín, Carmen

    2012-01-01

    Adenovirus (AdV) capsid organization is considerably complex, not only because of its large size (~950 Å) and triangulation number (pseudo T = 25), but also because it contains four types of minor proteins in specialized locations modulating the quasi-equivalent icosahedral interactions. Up until 2009, only its major components (hexon, penton, and fiber) had separately been described in atomic detail. Their relationships within the virion, and the location of minor coat proteins, were inferred from combining the known crystal structures with increasingly more detailed cryo-electron microscopy (cryoEM) maps. There was no structural information on assembly intermediates. Later on that year, two reports described the structural differences between the mature and immature adenoviral particle, starting to shed light on the different stages of viral assembly, and giving further insights into the roles of core and minor coat proteins during morphogenesis [1,2]. Finally, in 2010, two papers describing the atomic resolution structure of the complete virion appeared [3,4]. These reports represent a veritable tour de force for two structural biology techniques: X-ray crystallography and cryoEM, as this is the largest macromolecular complex solved at high resolution by either of them. In particular, the cryoEM analysis provided an unprecedented clear picture of the complex protein networks shaping the icosahedral shell. Here I review these latest developments in the field of AdV structural studies. PMID:22754652

  3. Fractal image perception provides novel insights into hierarchical cognition.

    PubMed

    Martins, M J; Fischmeister, F P; Puig-Waldmüller, E; Oh, J; Geissler, A; Robinson, S; Fitch, W T; Beisteiner, R

    2014-08-01

    Hierarchical structures play a central role in many aspects of human cognition, prominently including both language and music. In this study we addressed hierarchy in the visual domain, using a novel paradigm based on fractal images. Fractals are self-similar patterns generated by repeating the same simple rule at multiple hierarchical levels. Our hypothesis was that the brain uses different resources for processing hierarchies depending on whether it applies a "fractal" or a "non-fractal" cognitive strategy. We analyzed the neural circuits activated by these complex hierarchical patterns in an event-related fMRI study of 40 healthy subjects. Brain activation was compared across three different tasks: a similarity task, and two hierarchical tasks in which subjects were asked to recognize the repetition of a rule operating transformations either within an existing hierarchical level, or generating new hierarchical levels. Similar hierarchical images were generated by both rules and target images were identical. We found that when processing visual hierarchies, engagement in both hierarchical tasks activated the visual dorsal stream (occipito-parietal cortex, intraparietal sulcus and dorsolateral prefrontal cortex). In addition, the level-generating task specifically activated circuits related to the integration of spatial and categorical information, and with the integration of items in contexts (posterior cingulate cortex, retrosplenial cortex, and medial, ventral and anterior regions of temporal cortex). These findings provide interesting new clues about the cognitive mechanisms involved in the generation of new hierarchical levels as required for fractals.

  4. The relationship between consumer insight and provider-consumer agreement regarding consumer's quality of life.

    PubMed

    Hasson-Ohayon, Ilanit; Roe, David; Kravetz, Shlomo; Levy-Frank, Itamar; Meir, Taly

    2011-10-01

    This study examined the relationship between insight and mental health consumers and providers agreement regarding consumers rated quality of life (QoL). Seventy mental health consumers and their 23 care providers filled-out parallel questionnaires designed to measure consumer QoL. Consumers' insight was also assessed. For most QoL domains, agreement between consumers and providers was higher for persons with high insight. For the Psychological well being dimension a negative correlation was uncovered for persons with low insight indicating disagreement between consumer and provider. These findings are discussed within the context of the literature on insight and agreement between consumer and provider as related to the therapeutic alliance.

  5. Understanding cochleate formation: insights into structural development.

    PubMed

    Nagarsekar, Kalpa; Ashtikar, Mukul; Steiniger, Frank; Thamm, Jana; Schacher, Felix; Fahr, Alfred

    2016-04-20

    Understanding the structure and the self-assembly process of cochleates has become increasingly necessary considering the advances of this drug delivery system towards the pharmaceutical industry. It is well known that the addition of cations like calcium to a dispersion of anionic lipids such as phosphatidylserines results in stable, multilamellar cochleates through a spontaneous assembly. In the current investigation we have studied the intermediate structures generated during this self-assembly of cochleates. To achieve this, we have varied the process temperature for altering the rate of cochleate formation. Our findings from electron microscopy studies showed the formation of ribbonlike structures, which with proceeding interaction associate to form lipid stacks, networks and eventually cochleates. We also observed that the variation in lipid acyl chains did not make a remarkable difference to the type of structure evolved during the formation of cochleates. More generally, our observations provide a new insight into the self-assembly process of cochleates based on which we have proposed a pathway for cochleate formation from phosphatidylserine and calcium. This knowledge could be employed in using cochleates for a variety of possible biomedical applications in the future.

  6. Structural insights into RNA interference.

    PubMed

    Sashital, Dipali G; Doudna, Jennifer A

    2010-02-01

    Virtually all animals and plants utilize small RNA molecules to control protein expression during different developmental stages and in response to viral infection. Structural and mechanistic studies have begun to illuminate three fundamental aspects of these pathways: small RNA biogenesis, formation of RNA-induced silencing complexes (RISCs), and targeting of complementary mRNAs. Here we review exciting recent progress in understanding how regulatory RNAs are produced and how they trigger specific destruction of mRNAs during RNA interference (RNAi).

  7. Athena: Providing Insight into the History of the Universe

    NASA Technical Reports Server (NTRS)

    Murphy, Gloria A.

    2010-01-01

    The American Institute for Aeronautics and Astronautics has provided a Request for Proposal which calls for a manned mission to a Near-Earth Object. It is the goal of Team COLBERT to respond to their request by providing a reusable system that can be implemented as a solid stepping stone for future manned trips to Mars and beyond. Despite Team COLBERT consisting of only students in Aerospace Engineering, in order to achieve this feat, the team must employ the use of Systems Engineering. Tools and processes from Systems Engineering will provide quantitative and semi-quantitative tools for making design decisions and evaluating items such as budgets and schedules. This paper will provide an in-depth look at some of the Systems Engineering processes employed and will step through the design process of a Human Asteroid Exploration System.

  8. Structural insights into calicivirus attachment and uncoating.

    PubMed

    Bhella, David; Gatherer, Derek; Chaudhry, Yasmin; Pink, Rebecca; Goodfellow, Ian G

    2008-08-01

    The Caliciviridae family comprises positive-sense RNA viruses of medical and veterinary significance. In humans, caliciviruses are a major cause of acute gastroenteritis, while in animals respiratory illness, conjunctivitis, stomatitis, and hemorrhagic disease are documented. Investigation of virus-host interactions is limited by a lack of culture systems for many viruses in this family. Feline calicivirus (FCV), a member of the Vesivirus genus, provides a tractable model, since it may be propagated in cell culture. Feline junctional adhesion molecule 1 (fJAM-1) was recently identified as a functional receptor for FCV. We have analyzed the structure of this virus-receptor complex by cryo-electron microscopy and three-dimensional image reconstruction, combined with fitting of homology modeled high-resolution coordinates. We show that domain 1 of fJAM-1 binds to the outer face of the P2 domain of the FCV capsid protein VP1, inducing conformational changes in the viral capsid. This study provides the first structural view of a native calicivirus-protein receptor complex and insights into the mechanisms of virus attachment and uncoating.

  9. Structure of the Catalytic Domain of α-L-Arabinofuranosidase from Coprinopsis cinerea, CcAbf62A, Provides Insights into Structure-Function Relationships in Glycoside Hydrolase Family 62.

    PubMed

    Tonozuka, Takashi; Tanaka, Yutaro; Okuyama, Shunsaku; Miyazaki, Takatsugu; Nishikawa, Atsushi; Yoshida, Makoto

    2017-02-01

    α-L-Arabinofuranosidases, belonging to the glycoside hydrolase family (GH) 62, hydrolyze the α-1,2- or α-1,3-bond to liberate L-arabinofuranose from the xylan backbone. Here, we determined the structure of the C-terminal catalytic domain of CcAbf62A, a GH62 α-L-arabinofuranosidase from Coprinopsis cinerea. CcAbf62A is composed of a five-bladed β-propeller, as observed in other GH62 enzymes. The structure near the active site of CcAbf62A is also highly homologous to those of other GH62 enzymes. However, a calcium atom in the catalytic center interacts with an asparagine residue, Asn279, which is not found in other GH62 enzymes. In addition, some residues in subsites +3R, +2NR, +3NR, and +4NR of CcAbf62A are not conserved in other GH62 enzymes. In particular, a histidine residue, His221, is uniquely observed in subsite +2NR of CcAbf62A, which is likely to influence the substrate specificity. The results obtained here suggest that the amino acid residues that interact with the xylan backbone vary among the GH62 enzymes, despite the high similarity of their overall structures.

  10. Structural insights on complement activation.

    PubMed

    Alcorlo, Martín; López-Perrote, Andrés; Delgado, Sandra; Yébenes, Hugo; Subías, Marta; Rodríguez-Gallego, César; Rodríguez de Córdoba, Santiago; Llorca, Oscar

    2015-10-01

    The proteolytic cleavage of C3 to generate C3b is the central and most important step in the activation of complement, a major component of innate immunity. The comparison of the crystal structures of C3 and C3b illustrates large conformational changes during the transition from C3 to C3b. Exposure of a reactive thio-ester group allows C3b to bind covalently to surfaces such as pathogens or apoptotic cellular debris. The displacement of the thio-ester-containing domain (TED) exposes hidden surfaces that mediate the interaction with complement factor B to assemble the C3-convertase of the alternative pathway (AP). In addition, the displacement of the TED and its interaction with the macroglobulin 1 (MG1) domain generates an extended surface in C3b where the complement regulators factor H (FH), decay accelerating factor (DAF), membrane cofactor protein (MCP) and complement receptor 1 (CR1) can bind, mediating accelerated decay of the AP C3-convertase and proteolytic inactivation of C3b. In the last few years, evidence has accumulated revealing that the structure of C3b in solution is significantly more flexible than anticipated. We review our current knowledge on C3b structural flexibility to propose a general model where the TED can display a collection of conformations around the MG ring, as well as a few specialized positions where the TED is held in one of several fixed locations. Importantly, this conformational heterogeneity in C3b impacts complement regulation by affecting the interaction with regulators.

  11. The Atlantic salmon genome provides insights into rediploidization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The common ancestor of salmonids underwent an autotetraploid whole genome duplication event (Ss4R) approximately eighty million years ago, which provides unique opportunities to study the early evolutionary fate of a duplicated vertebrate genome in different extant lineages. Here, we present a high ...

  12. Structural insights into ribosome translocation

    PubMed Central

    Ling, Clarence

    2016-01-01

    During protein synthesis, tRNA and mRNA are translocated from the A to P to E sites of the ribosome thus enabling the ribosome to translate one codon of mRNA after the other. Ribosome translocation along mRNA is induced by the universally conserved ribosome GTPase, elongation factor G (EF‐G) in bacteria and elongation factor 2 (EF‐2) in eukaryotes. Recent structural and single‐molecule studies revealed that tRNA and mRNA translocation within the ribosome is accompanied by cyclic forward and reverse rotations between the large and small ribosomal subunits parallel to the plane of the intersubunit interface. In addition, during ribosome translocation, the ‘head’ domain of small ribosomal subunit undergoes forward‐ and back‐swiveling motions relative to the rest of the small ribosomal subunit around the axis that is orthogonal to the axis of intersubunit rotation. tRNA/mRNA translocation is also coupled to the docking of domain IV of EF‐G into the A site of the small ribosomal subunit that converts the thermally driven motions of the ribosome and tRNA into the forward translocation of tRNA/mRNA inside the ribosome. Despite recent and enormous progress made in the understanding of the molecular mechanism of ribosome translocation, the sequence of structural rearrangements of the ribosome, EF‐G and tRNA during translocation is still not fully established and awaits further investigation. WIREs RNA 2016, 7:620–636. doi: 10.1002/wrna.1354 For further resources related to this article, please visit the WIREs website. PMID:27117863

  13. Metaproteomics Provides Functional Insight into Activated Sludge Wastewater Treatment

    PubMed Central

    Wilmes, Paul; Wexler, Margaret; Bond, Philip L.

    2008-01-01

    Background Through identification of highly expressed proteins from a mixed culture activated sludge system this study provides functional evidence of microbial transformations important for enhanced biological phosphorus removal (EBPR). Methodology/Principal Findings A laboratory-scale sequencing batch reactor was successfully operated for different levels of EBPR, removing around 25, 40 and 55 mg/l P. The microbial communities were dominated by the uncultured polyphosphate-accumulating organism “Candidatus Accumulibacter phosphatis”. When EBPR failed, the sludge was dominated by tetrad-forming α-Proteobacteria. Representative and reproducible 2D gel protein separations were obtained for all sludge samples. 638 protein spots were matched across gels generated from the phosphate removing sludges. 111 of these were excised and 46 proteins were identified using recently available sludge metagenomic sequences. Many of these closely match proteins from “Candidatus Accumulibacter phosphatis” and could be directly linked to the EBPR process. They included enzymes involved in energy generation, polyhydroxyalkanoate synthesis, glycolysis, gluconeogenesis, glycogen synthesis, glyoxylate/TCA cycle, fatty acid β oxidation, fatty acid synthesis and phosphate transport. Several proteins involved in cellular stress response were detected. Conclusions/Significance Importantly, this study provides direct evidence linking the metabolic activities of “Accumulibacter” to the chemical transformations observed in EBPR. Finally, the results are discussed in relation to current EBPR metabolic models. PMID:18392150

  14. Isothermal microcalorimetry provides new insight into terrestrial carbon cycling.

    PubMed

    Herrmann, Anke M; Coucheney, Elsa; Nunan, Naoise

    2014-04-15

    Energy is continuously transformed in environmental systems through the metabolic activities of living organisms, but little is known about the relationship between the two. In this study, we tested the hypothesis that microbial energetics are controlled by microbial community composition in terrestrial ecosystems. We determined the functional diversity profiles of the soil biota (i.e., multiple substrate-induced respiration and microbial energetics) in soils from an arable ecosystem with contrasting long-term management regimes (54 years). These two functional profiling methods were then related to the soils' microbial community composition. Using isothermal microcalorimetry, we show that direct measures of energetics provide a functional link between energy flows and the composition of below-ground microbial communities at a high taxonomic level (Mantel R = 0.4602, P = 0.006). In contrast, this link was not apparent when carbon dioxide (CO2) was used as an aggregate measure of microbial metabolism (Mantel R = 0.2291, P = 0.11). Our work advocates that the microbial energetics approach provides complementary information to soil respiration for investigating the involvement of microbial communities in below-ground carbon dynamics. Empirical data of our proposed microbial energetics approach can feed into carbon-climate based ecosystem feedback modeling with the suggested conceptual ecological model as a base.

  15. Chromosomal Passports Provide New Insights into Diffusion of Emmer Wheat

    PubMed Central

    Badaeva, Ekaterina D.; Keilwagen, Jens; Knüpffer, Helmut; Waßermann, Louise; Dedkova, Olga S.; Mitrofanova, Olga P.; Kovaleva, Olga N.; Liapunova, Olga A.; Pukhalskiy, Vitaly A.; Özkan, Hakan; Graner, Andreas; Willcox, George; Kilian, Benjamin

    2015-01-01

    Emmer wheat, Triticum dicoccon schrank (syn. T. dicoccum (schrank) schÜbl.), is one of the earliest domesticated crops, harboring a wide range of genetic diversity and agronomically valuable traits. The crop, however, is currently largely neglected. We provide a wealth of karyotypic information from a comprehensive collection of emmer wheat and related taxa. In addition to C-banding polymorphisms, we identified 43 variants of chromosomal rearrangements in T. dicoccon; among them 26 (60.4%) were novel. The T7A:5B translocation was most abundant in Western Europe and the Mediterranean. The plant genetic resources investigated here might become important in the future for wheat improvement. Based on cluster analysis four major karyotypic groups were discriminated within the T. dicoccon genepool, each harboring characteristic C-banding patterns and translocation spectra: the balkan, asian, european and ethiopian groups. We postulate four major diffusion routes of the crop and discuss their migration out of the Fertile Crescent considering latest archaeobotanical findings. PMID:26024381

  16. Molecular insights provide the critical path to disease mitigation.

    PubMed

    Waldman, S A; Terzic, A

    2014-01-01

    The revolution in scientific innovation, driven by the engines of enabling technologies, is increasingly capable of deconstructing complex disease processes for the express purpose of reconstructing patient-specific solutions. These revelations in biological mechanisms provide the pressure points of opportunity for radical discovery and development to advance modern health care. Principles of mechanism-based discovery and their translation into therapeutic algorithms will, however, be challenged in the near term by emerging global public health crises that currently have no immediate solutions: chronic diseases, obesity, antibiotic-resistant infections, dementia, depression. The threat of these pandemics (multiplied in an increasingly aging population), the global burden of disease they represent, and their worldwide assault on human capital underscore the importance of continued and accelerated investments in science-propelled practice advancement, converting new knowledge into delivery of tangible health solutions. In that context, this annual issue of CPT on therapeutics innovations highlights remarkable recent successes in the discovery-development paradigm translating molecular innovations into diagnostic and therapeutic realities that transform the management of disease, impacting global health.

  17. Insights into early pig domestication provided by ancient DNA analysis

    PubMed Central

    Caliebe, Amke; Nebel, Almut; Makarewicz, Cheryl; Krawczak, Michael; Krause-Kyora, Ben

    2017-01-01

    Pigs (Sus scrofa) were first domesticated between 8,500 and 8,000 cal BC in the Near East, from where they were subsequently brought into Europe by agriculturalists. Soon after the arrival of the first domestic pigs in northern Europe (~4500 BC), farmers are thought to have started to incorporate local wild boars into their swine herds. This husbandry strategy ultimately resulted in the domestication of European wild boars. Here, we set out to provide a more precise geographic and temporal framework of the early management of suid populations in northern Europe, drawing upon mitochondrial DNA haplotype data from 116 Neolithic Sus specimens. We developed a quantitative mathematical model tracing the haplotypes of the domestic pigs back to their most likely geographic origin. Our modelling results suggest that, between 5000 and 4000 BC, almost all matrilines in the north originated from domesticated animals from the south of central Europe. In the following period (4000–3000 BC), an estimated 78–100% of domesticates in the north were of northern matrilineal origin, largely from local wild boars. These findings point towards a dramatic change in suid management strategies taking place throughout south-central and northern Europe after 4000 BC. PMID:28300151

  18. The Atlantic salmon genome provides insights into rediploidization.

    PubMed

    Lien, Sigbjørn; Koop, Ben F; Sandve, Simen R; Miller, Jason R; Kent, Matthew P; Nome, Torfinn; Hvidsten, Torgeir R; Leong, Jong S; Minkley, David R; Zimin, Aleksey; Grammes, Fabian; Grove, Harald; Gjuvsland, Arne; Walenz, Brian; Hermansen, Russell A; von Schalburg, Kris; Rondeau, Eric B; Di Genova, Alex; Samy, Jeevan K A; Olav Vik, Jon; Vigeland, Magnus D; Caler, Lis; Grimholt, Unni; Jentoft, Sissel; Våge, Dag Inge; de Jong, Pieter; Moen, Thomas; Baranski, Matthew; Palti, Yniv; Smith, Douglas R; Yorke, James A; Nederbragt, Alexander J; Tooming-Klunderud, Ave; Jakobsen, Kjetill S; Jiang, Xuanting; Fan, Dingding; Hu, Yan; Liberles, David A; Vidal, Rodrigo; Iturra, Patricia; Jones, Steven J M; Jonassen, Inge; Maass, Alejandro; Omholt, Stig W; Davidson, William S

    2016-05-12

    The whole-genome duplication 80 million years ago of the common ancestor of salmonids (salmonid-specific fourth vertebrate whole-genome duplication, Ss4R) provides unique opportunities to learn about the evolutionary fate of a duplicated vertebrate genome in 70 extant lineages. Here we present a high-quality genome assembly for Atlantic salmon (Salmo salar), and show that large genomic reorganizations, coinciding with bursts of transposon-mediated repeat expansions, were crucial for the post-Ss4R rediploidization process. Comparisons of duplicate gene expression patterns across a wide range of tissues with orthologous genes from a pre-Ss4R outgroup unexpectedly demonstrate far more instances of neofunctionalization than subfunctionalization. Surprisingly, we find that genes that were retained as duplicates after the teleost-specific whole-genome duplication 320 million years ago were not more likely to be retained after the Ss4R, and that the duplicate retention was not influenced to a great extent by the nature of the predicted protein interactions of the gene products. Finally, we demonstrate that the Atlantic salmon assembly can serve as a reference sequence for the study of other salmonids for a range of purposes.

  19. The African coelacanth genome provides insights into tetrapod evolution.

    PubMed

    Amemiya, Chris T; Alföldi, Jessica; Lee, Alison P; Fan, Shaohua; Philippe, Hervé; Maccallum, Iain; Braasch, Ingo; Manousaki, Tereza; Schneider, Igor; Rohner, Nicolas; Organ, Chris; Chalopin, Domitille; Smith, Jeramiah J; Robinson, Mark; Dorrington, Rosemary A; Gerdol, Marco; Aken, Bronwen; Biscotti, Maria Assunta; Barucca, Marco; Baurain, Denis; Berlin, Aaron M; Blatch, Gregory L; Buonocore, Francesco; Burmester, Thorsten; Campbell, Michael S; Canapa, Adriana; Cannon, John P; Christoffels, Alan; De Moro, Gianluca; Edkins, Adrienne L; Fan, Lin; Fausto, Anna Maria; Feiner, Nathalie; Forconi, Mariko; Gamieldien, Junaid; Gnerre, Sante; Gnirke, Andreas; Goldstone, Jared V; Haerty, Wilfried; Hahn, Mark E; Hesse, Uljana; Hoffmann, Steve; Johnson, Jeremy; Karchner, Sibel I; Kuraku, Shigehiro; Lara, Marcia; Levin, Joshua Z; Litman, Gary W; Mauceli, Evan; Miyake, Tsutomu; Mueller, M Gail; Nelson, David R; Nitsche, Anne; Olmo, Ettore; Ota, Tatsuya; Pallavicini, Alberto; Panji, Sumir; Picone, Barbara; Ponting, Chris P; Prohaska, Sonja J; Przybylski, Dariusz; Saha, Nil Ratan; Ravi, Vydianathan; Ribeiro, Filipe J; Sauka-Spengler, Tatjana; Scapigliati, Giuseppe; Searle, Stephen M J; Sharpe, Ted; Simakov, Oleg; Stadler, Peter F; Stegeman, John J; Sumiyama, Kenta; Tabbaa, Diana; Tafer, Hakim; Turner-Maier, Jason; van Heusden, Peter; White, Simon; Williams, Louise; Yandell, Mark; Brinkmann, Henner; Volff, Jean-Nicolas; Tabin, Clifford J; Shubin, Neil; Schartl, Manfred; Jaffe, David B; Postlethwait, John H; Venkatesh, Byrappa; Di Palma, Federica; Lander, Eric S; Meyer, Axel; Lindblad-Toh, Kerstin

    2013-04-18

    The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution.

  20. Elephant transcriptome provides insights into the evolution of eutherian placentation.

    PubMed

    Hou, Zhuo-Cheng; Sterner, Kirstin N; Romero, Roberto; Than, Nandor Gabor; Gonzalez, Juan M; Weckle, Amy; Xing, Jun; Benirschke, Kurt; Goodman, Morris; Wildman, Derek E

    2012-01-01

    The chorioallantoic placenta connects mother and fetus in eutherian pregnancies. In order to understand the evolution of the placenta and provide further understanding of placenta biology, we sequenced the transcriptome of a term placenta of an African elephant (Loxodonta africana) and compared these data with RNA sequence and microarray data from other eutherian placentas including human, mouse, and cow. We characterized the composition of 55,910 expressed sequence tag (i.e., cDNA) contigs using our custom annotation pipeline. A Markov algorithm was used to cluster orthologs of human, mouse, cow, and elephant placenta transcripts. We found 2,963 genes are commonly expressed in the placentas of these eutherian mammals. Gene ontology categories previously suggested to be important for placenta function (e.g., estrogen receptor signaling pathway, cell motion and migration, and adherens junctions) were significantly enriched in these eutherian placenta-expressed genes. Genes duplicated in different lineages and also specifically expressed in the placenta contribute to the great diversity observed in mammalian placenta anatomy. We identified 1,365 human lineage-specific, 1,235 mouse lineage-specific, 436 cow lineage-specific, and 904 elephant-specific placenta-expressed (PE) genes. The most enriched clusters of human-specific PE genes are signal/glycoprotein and immunoglobulin, and humans possess a deeply invasive human hemochorial placenta that comes into direct contact with maternal immune cells. Inference of phylogenetically conserved and derived transcripts demonstrates the power of comparative transcriptomics to trace placenta evolution and variation across mammals and identified candidate genes that may be important in the normal function of the human placenta, and their dysfunction may be related to human pregnancy complications.

  1. Fusion transcriptome profiling provides insights into alveolar rhabdomyosarcoma.

    PubMed

    Xie, Zhongqiu; Babiceanu, Mihaela; Kumar, Shailesh; Jia, Yuemeng; Qin, Fujun; Barr, Frederic G; Li, Hui

    2016-11-15

    Gene fusions and fusion products were thought to be unique features of neoplasia. However, more and more studies have identified fusion RNAs in normal physiology. Through RNA sequencing of 27 human noncancer tissues, a large number of fusion RNAs were found. By analyzing fusion transcriptome, we observed close clusterings between samples of same or similar tissues, supporting the feasibility of using fusion RNA profiling to reveal connections between biological samples. To put the concept into use, we selected alveolar rhabdomyosarcoma (ARMS), a myogenic pediatric cancer whose exact cell of origin is not clear. PAX3-FOXO1 (paired box gene 3 fused with forkhead box O1) fusion RNA, which is considered a hallmark of ARMS, was recently found during normal muscle cell differentiation. We performed and analyzed RNA sequencing from various time points during myogenesis and uncovered many chimeric fusion RNAs. Interestingly, we found that the fusion RNA profile of RH30, an ARMS cell line, is most similar to the myogenesis time point when PAX3-FOXO1 is expressed. In contrast, full transcriptome clustering analysis failed to uncover this connection. Strikingly, all of the 18 chimeric RNAs in RH30 cells could be detected at the same myogenic time point(s). In addition, the seven chimeric RNAs that follow the exact transient expression pattern as PAX3-FOXO1 are specific to rhabdomyosarcoma cells. Further testing with clinical samples also confirmed their specificity to rhabdomyosarcoma. These results provide further support for the link between at least some ARMSs and the PAX3-FOXO1-expressing myogenic cells and demonstrate that fusion RNA profiling can be used to investigate the etiology of fusion-gene-associated cancers.

  2. The genome of Laccaria bicolor provides insights into

    SciTech Connect

    Martin, F; Aerts, A.; Ahren, D; Brun, A; Danchin, E; Duchaussoy, F; Gibon, J; Kohler, A; Lindquist, E; Pereda, V; Salamov, A.; Shapiro, HJ; Wuyts, J; Blaudez, D.; Buee, M; Brokstein, P; Canbeck, B; Cohen, D; Courty, PE; Coutinho, PM; Delaruelle, C; Detter, J C; Deveau, A; DiFazio, Stephen P; Duplessis, S; Fraissinet-Tachet, L; Lucic, E; Frey-Klett, P; Fourrey, C; Feussner, I; Gay, G; Grimwood, Jane; Hoegger, P J; Jain, P; Kilaru, S; Labbe, J; Lin, Y C; Legue, V; Le Tacon, F; Marmeisse, R; Melayah, D; Montanini, B; Muratet, M; Nehls, U; Niculita-Hirzel, H; Oudot-Le Secq, M P; Peter, M; Quesneville, H; Rajashekar, B; Reich, M; Rouhler, N; Schmutz, Jeremy; Yin, Tongming; Tuskan, Gerald A; Chalot, M; Henrissat, B; Kues, U; Lucas, S; Van de Peer, Y; Podila, G; Polle, A; Pukkila, P J; Richardson, P M; Rouze, P; Sanders, I R; Stajich, J E; Tunlid, A; Grigoriev, I.

    2008-01-01

    Mycorrhizal symbioses the union of roots and soil fungi are universal in terrestrial ecosystems and may have been fundamental to land colonization by plants1,2. Boreal, temperate and montane forests all depend on ectomycorrhizae1. Identification of the primary factors that regulate symbiotic development and metabolic activity will therefore open the door to understanding the role of ectomycorrhizae in plant development and physiology, allowing the full ecological significance of this symbiosis to be explored. Here we report the genome sequence of the ectomycorrhizal basidiomycete Laccaria bicolor (Fig. 1) and highlight gene sets involved in rhizosphere colonization and symbiosis. This 65-megabase genome assembly contains 20,000 predicted protein-encoding genes and a very large number of transposons and repeated sequences. We detected unexpected genomic features, most notably a battery of effector-type small secreted proteins (SSPs) with unknown function, several of which are only expressed in symbiotic tissues. The most highly expressed SSP accumulates in the proliferating hyphae colonizing the host root. The ectomycorrhizae-specific SSPs probably have a decisive role in the establishment of the symbiosis. The unexpected observation that the genome of L. bicolor lacks carbohydrate-active enzymes involved in degradation of plant cell walls, but maintains the ability to degrade non-plant cell wall polysaccharides, reveals the dual saprotrophic and biotrophic lifestyle of the mycorrhizal fungus that enables it to grow within both soil and living plant roots. The predicted gene inventory of the L. bicolor genome, therefore, points to previously unknown mechanisms of symbiosis operating in biotrophic mycorrhizal fungi. The availability of this genome provides an unparalleled opportunity to develop a deeper understanding of the processes by which symbionts interact with plants within their ecosystem to perform vital functions in the carbon and nitrogen cycles that are

  3. The genome of Laccaria bicolor provides insights into mycorrhizal symbiosis

    SciTech Connect

    Martin, F.; Aerts, A.; Ahren, D.; Brun, A.; Danchin, E. G. J.; Duchaussoy, F.; Gibon, J.; Kohler, A.; Lindquist, E.; Peresa, V.; Salamov, A.; Shapiro, H. J.; Wuyts, J.; Blaudez, D.; Buee, M.; Brokstein, P.; Canback, B.; Cohen, D.; Courty, P. E.; Coutinho, P. M.; Delaruelle, C.; Detter, J. C.; Deveau, A.; DiFazio, S.; Duplessis, S.; Fraissinet-Tachet, L.; Lucic, E.; Frey-Klett, P.; Fourrey, C.; Feussner, I.; Gay, G.; Grimwood, J.; Hoegger, P. J.; Jain, P.; Kilaru, S.; Labbe, J.; Lin, Y. C.; Legue, V.; Le Tacon, F.; Marmeisse, R.; Melayah, D.; Montanini, B.; Muratet, M.; Nehls, U.; Niculita-Hirzel, H.; Secq, M. P. Oudot-Le; Peter, M.; Quesneville, H.; Rajashekar, B.; Reich, M.; Rouhier, N.; Schmutz, J.; Yin, T.; Chalot, M.; Henrissat, B.; Kues, U.; Lucas, S.; Van de Peer, Y.; Podila, G. K.; Polle, A.; Pukkila, P. J.; Richardson, P. M.; Rouze, P.; Sanders, I. R.; Stajich, J. E.; Tunlid, A.; Tuskan, G.; Grigoriev, I. V.

    2007-08-10

    Mycorrhizal symbioses the union of roots and soil fungi are universal in terrestrial ecosystems and may have been fundamental to land colonization by plants 1, 2. Boreal, temperate and montane forests all depend on ectomycorrhizae1. Identification of the primary factors that regulate symbiotic development and metabolic activity will therefore open the door to understanding the role of ectomycorrhizae in plant development and physiology, allowing the full ecological significance of this symbiosis to be explored. Here we report the genome sequence of the ectomycorrhizal basidiomycete Laccaria bicolor (Fig. 1) and highlight gene sets involved in rhizosphere colonization and symbiosis. This 65-megabase genome assembly contains 20,000 predicted protein-encoding genes and a very large number of transposons and repeated sequences. We detected unexpected genomic features, most notably a battery of effector-type small secreted proteins (SSPs) with unknown function, several of which are only expressed in symbiotic tissues. The most highly expressed SSP accumulates in the proliferating hyphae colonizing the host root. The ectomycorrhizae-specific SSPs probably have a decisive role in the establishment of the symbiosis. The unexpected observation that the genome of L. bicolor lacks carbohydrate-active enzymes involved in degradation of plant cell walls, but maintains the ability to degrade non-plant cell wall polysaccharides, reveals the dual saprotrophic and biotrophic lifestyle of the mycorrhizal fungus that enables it to grow within both soil and living plant roots. The predicted gene inventory of the L. bicolor genome, therefore, points to previously unknown mechanisms of symbiosis operating in biotrophic mycorrhizal fungi. The availability of this genome provides an unparalleled opportunity to develop a deeper understanding of the processes by which symbionts interact with plants within their ecosystem to perform vital functions in the carbon and nitrogen cycles that are

  4. Complexes of Thermotoga maritima S-adenosylmethionine decarboxylase provide insights into substrate specificity

    SciTech Connect

    Bale, Shridhar; Baba, Kavita; McCloskey, Diane E.; Pegg, Anthony E.; Ealick, Steven E.

    2010-06-25

    The polyamines putrescine, spermidine and spermine are ubiquitous aliphatic cations and are essential for cellular growth and differentiation. S-Adenosylmethionine decarboxylase (AdoMetDC) is a critical pyruvoyl-dependent enzyme in the polyamine-biosynthetic pathway. The crystal structures of AdoMetDC from humans and plants and of the AdoMetDC proenzyme from Thermotoga maritima have been obtained previously. Here, the crystal structures of activated T. maritima AdoMetDC (TmAdoMetDC) and of its complexes with S-adenosylmethionine methyl ester and 5{prime}-deoxy-5{prime}-dimethylthioadenosine are reported. The results demonstrate for the first time that TmAdoMetDC autoprocesses without the need for additional factors and that the enzyme contains two complete active sites, both of which use residues from both chains of the homodimer. The complexes provide insights into the substrate specificity and ligand binding of AdoMetDC in prokaryotes. The conservation of the ligand-binding mode and the active-site residues between human and T. maritima AdoMetDC provides insight into the evolution of AdoMetDC.

  5. Free energy calculation provides insight into the action mechanism of selective PARP-1 inhibitor.

    PubMed

    Cao, Ran

    2016-04-01

    Selective poly (ADP-ribose) polymerase (PARP)-1 inhibitor represents promising therapy against cancers with a good balance between efficacy and safety. Owing to the conserved structure between PARP-1 and PARP-2, most of the clinical and experimental drugs show equivalent inhibition against both targets. Most recently, it's disclosed a highly selective PARP-1 inhibitor (NMS-P118) with promising pharmacokinetic properties. Herein, we combined molecular simulation with free energy calculation to gain insights into the selective mechanism of NMS-P118. Our results suggest the reduction of binding affinity for PARP-2 is attributed to the unfavorable conformational change of protein, which is accompanied by a significant energy penalty. Alanine-scanning mutagenesis study further reveals the important role for a tyrosine residue of donor loop (Tyr889(PARP-1) and Tyr455(PARP-2)) in contributing to the ligand selectivity. Retrospective structural analysis indicates the ligand-induced movement of Tyr455(PARP-2) disrupts the intra-molecule hydrogen bonding network, which partially accounts for the "high-energy" protein conformation in the presence of NMS-P118. Interestingly, such effect isn't observed in other non-selective PARP inhibitors including BMN673 and A861695, which validates the computational prediction. Our work provides energetic insight into the subtle variations in the crystal structures and could facilitate rational design of new selective PARP inhibitor.

  6. Crystal Structure of the Human Cytomegalovirus pUL50-pUL53 Core Nuclear Egress Complex Provides Insight into a Unique Assembly Scaffold for Virus-Host Protein Interactions.

    PubMed

    Walzer, Sascha A; Egerer-Sieber, Claudia; Sticht, Heinrich; Sevvana, Madhumati; Hohl, Katharina; Milbradt, Jens; Muller, Yves A; Marschall, Manfred

    2015-11-13

    Nuclear replication of cytomegalovirus relies on elaborate mechanisms of nucleocytoplasmic egress of viral particles. Thus, the role of two essential and conserved viral nuclear egress proteins, pUL50 and pUL53, is pivotal. pUL50 and pUL53 heterodimerize and form a core nuclear egress complex (NEC), which is anchored to the inner nuclear membrane and provides a scaffold for the assembly of a multimeric viral-cellular NEC. Here, we report the crystal structure of the pUL50-pUL53 heterodimer (amino acids 1-175 and 50-292, respectively) at 2.44 Å resolution. Both proteins adopt a globular fold with mixed α and β secondary structure elements. pUL53-specific features include a zinc-binding site and a hook-like N-terminal extension, the latter representing a hallmark element of the pUL50-pUL53 interaction. The hook-like extension (amino acids 59-87) embraces pUL50 and contributes 1510 Å(2) to the total interface area (1880 Å(2)). The pUL50 structure overall resembles the recently published NMR structure of the murine cytomegalovirus homolog pM50 but reveals a considerable repositioning of the very C-terminal α-helix of pUL50 upon pUL53 binding. pUL53 shows structural resemblance with the GHKL domain of bacterial sensory histidine kinases. A close examination of the crystal structure indicates partial assembly of pUL50-pUL53 heterodimers to hexameric ring-like structures possibly providing additional scaffolding opportunities for NEC. In combination, the structural information on pUL50-pUL53 considerably improves our understanding of the mechanism of HCMV nuclear egress. It may also accelerate the validation of the NEC as a unique target for developing a novel type of antiviral drug and improved options of broad-spectrum antiherpesviral therapy.

  7. Feathered non-avian dinosaurs from North America provide insight into wing origins.

    PubMed

    Zelenitsky, Darla K; Therrien, François; Erickson, Gregory M; DeBuhr, Christopher L; Kobayashi, Yoshitsugu; Eberth, David A; Hadfield, Frank

    2012-10-26

    Previously described feathered dinosaurs reveal a fascinating record of feather evolution, although substantial phylogenetic gaps remain. Here we report the occurrence of feathers in ornithomimosaurs, a clade of non-maniraptoran theropods for which fossilized feathers were previously unknown. The Ornithomimus specimens, recovered from Upper Cretaceous deposits of Alberta, Canada, provide new insights into dinosaur plumage and the origin of the avian wing. Individuals from different growth stages reveal the presence of a filamentous feather covering throughout life and winglike structures on the forelimbs of adults. The appearance of winglike structures in older animals indicates that they may have evolved in association with reproductive behaviors. These specimens show that primordial wings originated earlier than previously thought, among non-maniraptoran theropods.

  8. NAP (davunetide) provides functional and structural neuroprotection.

    PubMed

    Gozes, Illana

    2011-01-01

    NAP (davunetide) is an eight amino acid peptide (NAPVSIPQ) that has been shown to provide potent neuroprotection, in vitro and in vivo. In human clinical trials, NAP has been shown to increase memory scores in patients suffering from amnestic mild cognitive impairment, a precursor to Alzheimer's disease and to enhance functional daily behaviors in schizophrenia patients. NAP is derived from activity-dependent neuroprotective protein (ADNP) a molecule that is essential for brain formation, interacting with chromatin associated protein alpha and the chromatin remodeling complex SWI/SNF and regulating >400 genes during embryonic development. Partial loss in ADNP results in cognitive deficits and pathology of the microtubule associated protein tau (tauopathy) that is ameliorated in part by NAP replacement therapy. Recent studies increased the scope of NAP neuroprotection and provided further insights into the NAP mechanisms of action. Thus, it has been hypothesized that the presence of tau on axonal microtubules renders them notably less sensitive to the microtubule-severing protein katanin, and NAP was shown to protect microtubules from katanin disruption in the face of reduced tau expression. Parallel studies showed that NAP reduced the number of apoptotic neurons through activation of PI-3K/Akt pathway in the cortical plate or both PI-3K/Akt and MAPK/MEK1 kinases in the white matter. The interaction of these disparate yet complementary pathways is the subject of future studies toward human brain neuroprotection in the clinical scenario.

  9. Orthobunyaviruses: recent genetic and structural insights.

    PubMed

    Elliott, Richard M

    2014-10-01

    Orthobunyaviruses, which have small, tripartite, negative-sense RNA genomes and structurally simple virions composed of just four proteins, can have devastating effects on human health and well-being, either by causing disease in humans or by causing disease in livestock and crops. In this Review, I describe the recent genetic and structural advances that have revealed important insights into the composition of orthobunyavirus virions, viral transcription and replication and viral interactions with the host innate immune response. Lastly, I highlight outstanding questions and areas of future research.

  10. Crystal structure of the external aldimine of Citrobacter freundii methionine γ-lyase with glycine provides insight in mechanisms of two stages of physiological reaction and isotope exchange of α- and β-protons of competitive inhibitors.

    PubMed

    Revtovich, Svetlana V; Faleev, Nicolai G; Morozova, Elena A; Anufrieva, Natalya V; Nikulin, Alexey D; Demidkina, Tatyana V

    2014-06-01

    The three-dimensional structure of the external aldimine of Citrobacter freundii methionine γ-lyase with competitive inhibitor glycine has been determined at 2.45 Å resolution. It revealed subtle conformational changes providing effective binding of the inhibitor and facilitating labilization of Cα-protons of the external aldimine. The structure shows that 1, 3-prototropic shift of Cα-proton to C4'-atom of the cofactor may proceed with participation of active site Lys210 residue whose location is favorable for performing this transformation by a concerted mechanism. The observed stereoselectivity of isotopic exchange of enantiotopic Cα-protons of glycine may be explained on the basis of external aldimine structure. The exchange of Cα-pro-(R)-proton of the external aldimine might proceed in the course of the concerted transfer of the proton from Cα-atom of glycine to C4'-atom of the cofactor. The exchange of Cα-pro-(S)-proton may be performed with participation of Tyr113 residue which should be present in its basic form. The isotopic exchange of β-protons, which is observed for amino acids bearing longer side groups, may be effected by two catalytic groups: Lys210 in its basic form, and Tyr113 acting as a general acid.

  11. Parameter sensitivity analysis of stochastic models provides insights into cardiac calcium sparks.

    PubMed

    Lee, Young-Seon; Liu, Ona Z; Hwang, Hyun Seok; Knollmann, Bjorn C; Sobie, Eric A

    2013-03-05

    We present a parameter sensitivity analysis method that is appropriate for stochastic models, and we demonstrate how this analysis generates experimentally testable predictions about the factors that influence local Ca(2+) release in heart cells. The method involves randomly varying all parameters, running a single simulation with each set of parameters, running simulations with hundreds of model variants, then statistically relating the parameters to the simulation results using regression methods. We tested this method on a stochastic model, containing 18 parameters, of the cardiac Ca(2+) spark. Results show that multivariable linear regression can successfully relate parameters to continuous model outputs such as Ca(2+) spark amplitude and duration, and multivariable logistic regression can provide insight into how parameters affect Ca(2+) spark triggering (a probabilistic process that is all-or-none in a single simulation). Benchmark studies demonstrate that this method is less computationally intensive than standard methods by a factor of 16. Importantly, predictions were tested experimentally by measuring Ca(2+) sparks in mice with knockout of the sarcoplasmic reticulum protein triadin. These mice exhibit multiple changes in Ca(2+) release unit structures, and the regression model both accurately predicts changes in Ca(2+) spark amplitude (30% decrease in model, 29% decrease in experiments) and provides an intuitive and quantitative understanding of how much each alteration contributes to the result. This approach is therefore an effective, efficient, and predictive method for analyzing stochastic mathematical models to gain biological insight.

  12. A new raptorial dinosaur with exceptionally long feathering provides insights into dromaeosaurid flight performance.

    PubMed

    Han, Gang; Chiappe, Luis M; Ji, Shu-An; Habib, Michael; Turner, Alan H; Chinsamy, Anusuya; Liu, Xueling; Han, Lizhuo

    2014-07-15

    Microraptorines are a group of predatory dromaeosaurid theropod dinosaurs with aerodynamic capacity. These close relatives of birds are essential for testing hypotheses explaining the origin and early evolution of avian flight. Here we describe a new 'four-winged' microraptorine, Changyuraptor yangi, from the Early Cretaceous Jehol Biota of China. With tail feathers that are nearly 30 cm long, roughly 30% the length of the skeleton, the new fossil possesses the longest known feathers for any non-avian dinosaur. Furthermore, it is the largest theropod with long, pennaceous feathers attached to the lower hind limbs (that is, 'hindwings'). The lengthy feathered tail of the new fossil provides insight into the flight performance of microraptorines and how they may have maintained aerial competency at larger body sizes. We demonstrate how the low-aspect-ratio tail of the new fossil would have acted as a pitch control structure reducing descent speed and thus playing a key role in landing.

  13. A Renaissance in Nepovirus Research Provides New Insights Into Their Molecular Interface With Hosts and Vectors.

    PubMed

    Fuchs, M; Schmitt-Keichinger, C; Sanfaçon, H

    2017-01-01

    Nepoviruses supplied seminal landmarks to the historical trail of plant virology. Among the first agriculturally relevant viruses recognized in the late 1920s and among the first plant viruses officially classified in the early 1970s, nepoviruses also comprise the first species for which a soil-borne ectoparasitic nematode vector was identified. Early research on nepoviruses shed light on the genome structure and expression, biological properties of the two genomic RNAs, and mode of transmission. In recent years, research on nepoviruses enjoyed an extraordinary renaissance. This resurgence provided new insights into the molecular interface between viruses and their plant hosts, and between viruses and dagger nematode vectors to advance our understanding of some of the major steps of the infectious cycle. Here we examine these recent findings, highlight ongoing work, and offer some perspectives for future research.

  14. Tools providing new insight into coastal anoxygenic purple bacterial mats: review and perspectives.

    PubMed

    Hubas, Cédric; Jesus, Bruno; Passarelli, Claire; Jeanthon, Christian

    2011-11-01

    Coastal photosynthetic microbial mats are highly structured microbial communities that populate a variety of shallow environments such as estuaries, sheltered sandy beaches, intertidal flats, salt marshes and hypersaline salterns. In soft sediments, most of these microbial mats are formed of vertically stratified, multicolored cohesive thin layers, of several functional groups of microorganisms, such as cyanobacteria, colorless sulfur bacteria, purple sulfur bacteria and sulfate-reducing bacteria, distributed along vertical microgradients of oxygen, sulfide and light. These microbial communities are highly productive and significant contributors to carbon, nitrogen and sulfur cycles and to sediment stability in shallow-water habitats. Many examples of these communities have been cited in the past, but comparatively few microbial mats have been presented for which mass developments of anoxygenic purple bacteria have been observed. Yet, application of molecular approaches has provided fresh insight into the ecology, diversity and evolution of microbial mats. In situ measurements using electrochemical and optical microprobes led to detailed characterization of their physical and chemical environment, whereas reflectance measurements revealed the spatial and temporal heterogeneity of microbial mat surfaces. We hereby report the main discoveries due to introduction of these powerful techniques and we point out the potential insight to be gained from the study of anoxygenic purple bacterial mats.

  15. Comparative and functional genomics provide insights into the pathogenicity of dermatophytic fungi

    PubMed Central

    2011-01-01

    Background Millions of humans and animals suffer from superficial infections caused by a group of highly specialized filamentous fungi, the dermatophytes, which exclusively infect keratinized host structures. To provide broad insights into the molecular basis of the pathogenicity-associated traits, we report the first genome sequences of two closely phylogenetically related dermatophytes, Arthroderma benhamiae and Trichophyton verrucosum, both of which induce highly inflammatory infections in humans. Results 97% of the 22.5 megabase genome sequences of A. benhamiae and T. verrucosum are unambiguously alignable and collinear. To unravel dermatophyte-specific virulence-associated traits, we compared sets of potentially pathogenicity-associated proteins, such as secreted proteases and enzymes involved in secondary metabolite production, with those of closely related onygenales (Coccidioides species) and the mould Aspergillus fumigatus. The comparisons revealed expansion of several gene families in dermatophytes and disclosed the peculiarities of the dermatophyte secondary metabolite gene sets. Secretion of proteases and other hydrolytic enzymes by A. benhamiae was proven experimentally by a global secretome analysis during keratin degradation. Molecular insights into the interaction of A. benhamiae with human keratinocytes were obtained for the first time by global transcriptome profiling. Given that A. benhamiae is able to undergo mating, a detailed comparison of the genomes further unraveled the genetic basis of sexual reproduction in this species. Conclusions Our results enlighten the genetic basis of fundamental and putatively virulence-related traits of dermatophytes, advancing future research on these medically important pathogens. PMID:21247460

  16. Crystal structure of a human cleavage factor CFI(m)25/CFI(m)68/RNA complex provides an insight into poly(A) site recognition and RNA looping.

    PubMed

    Yang, Qin; Coseno, Molly; Gilmartin, Gregory M; Doublié, Sylvie

    2011-03-09

    Cleavage factor I(m) (CFI(m)) is a highly conserved component of the eukaryotic mRNA 3' processing machinery that functions in sequence-specific poly(A) site recognition through the collaboration of a 25 kDa subunit containing a Nudix domain and a larger subunit of 59, 68, or 72 kDa containing an RNA recognition motif (RRM). Our previous work demonstrated that CFI(m)25 is both necessary and sufficient for sequence-specific binding of the poly(A) site upstream element UGUA. Here, we report the crystal structure of CFI(m)25 complexed with the RRM domain of CFI(m)68 and RNA. The CFI(m)25 dimer is clasped on opposite sides by two CFI(m)68 RRM domains. Each CFI(m)25 subunit binds one UGUA element specifically. Biochemical analysis indicates that the CFI(m)68 RRMs serve to enhance RNA binding and facilitate RNA looping. The intrinsic ability of CFI(m) to direct RNA looping may provide a mechanism for its function in the regulation of alternative poly(A) site selection.

  17. Structure of the Single-lobe Myosin Light Chain C in Complex with the Light Chain-binding Domains of Myosin-1C Provides Insights into Divergent IQ Motif Recognition.

    PubMed

    Langelaan, David N; Liburd, Janine; Yang, Yidai; Miller, Emily; Chitayat, Seth; Crawley, Scott W; Côté, Graham P; Smith, Steven P

    2016-09-09

    Myosin light chains are key regulators of class 1 myosins and typically comprise two domains, with calmodulin being the archetypal example. They bind IQ motifs within the myosin neck region and amplify conformational changes in the motor domain. A single lobe light chain, myosin light chain C (MlcC), was recently identified and shown to specifically bind to two sequentially divergent IQ motifs of the Dictyostelium myosin-1C. To provide a molecular basis of this interaction, the structures of apo-MlcC and a 2:1 MlcC·myosin-1C neck complex were determined. The two non-functional EF-hand motifs of MlcC pack together to form a globular four-helix bundle that opens up to expose a central hydrophobic groove, which interacts with the N-terminal portion of the divergent IQ1 and IQ2 motifs. The N- and C-terminal regions of MlcC make critical contacts that contribute to its specific interactions with the myosin-1C divergent IQ motifs, which are contacts that deviate from the traditional mode of calmodulin-IQ recognition.

  18. Forward dynamics simulations provide insight into muscle mechanical work during human locomotion.

    PubMed

    Neptune, Richard R; McGowan, Craig P; Kautz, Steven A

    2009-10-01

    Complex musculoskeletal models and computer simulations can provide critical insight into muscle mechanical work output during locomotion. Simulations provide both a consistent mechanical solution that can be interrogated at multiple levels (muscle fiber, musculotendon, net joint moment, and whole-body work) and an ideal framework to identify limitations with different estimates of muscle work and the resulting implications for metabolic cost and efficiency.

  19. Markov State Models Provide Insights into Dynamic Modulation of Protein Function

    PubMed Central

    2015-01-01

    Conspectus Protein function is inextricably linked to protein dynamics. As we move from a static structural picture to a dynamic ensemble view of protein structure and function, novel computational paradigms are required for observing and understanding conformational dynamics of proteins and its functional implications. In principle, molecular dynamics simulations can provide the time evolution of atomistic models of proteins, but the long time scales associated with functional dynamics make it difficult to observe rare dynamical transitions. The issue of extracting essential functional components of protein dynamics from noisy simulation data presents another set of challenges in obtaining an unbiased understanding of protein motions. Therefore, a methodology that provides a statistical framework for efficient sampling and a human-readable view of the key aspects of functional dynamics from data analysis is required. The Markov state model (MSM), which has recently become popular worldwide for studying protein dynamics, is an example of such a framework. In this Account, we review the use of Markov state models for efficient sampling of the hierarchy of time scales associated with protein dynamics, automatic identification of key conformational states, and the degrees of freedom associated with slow dynamical processes. Applications of MSMs for studying long time scale phenomena such as activation mechanisms of cellular signaling proteins has yielded novel insights into protein function. In particular, from MSMs built using large-scale simulations of GPCRs and kinases, we have shown that complex conformational changes in proteins can be described in terms of structural changes in key structural motifs or “molecular switches” within the protein, the transitions between functionally active and inactive states of proteins proceed via multiple pathways, and ligand or substrate binding modulates the flux through these pathways. Finally, MSMs also provide a

  20. Markov state models provide insights into dynamic modulation of protein function.

    PubMed

    Shukla, Diwakar; Hernández, Carlos X; Weber, Jeffrey K; Pande, Vijay S

    2015-02-17

    CONSPECTUS: Protein function is inextricably linked to protein dynamics. As we move from a static structural picture to a dynamic ensemble view of protein structure and function, novel computational paradigms are required for observing and understanding conformational dynamics of proteins and its functional implications. In principle, molecular dynamics simulations can provide the time evolution of atomistic models of proteins, but the long time scales associated with functional dynamics make it difficult to observe rare dynamical transitions. The issue of extracting essential functional components of protein dynamics from noisy simulation data presents another set of challenges in obtaining an unbiased understanding of protein motions. Therefore, a methodology that provides a statistical framework for efficient sampling and a human-readable view of the key aspects of functional dynamics from data analysis is required. The Markov state model (MSM), which has recently become popular worldwide for studying protein dynamics, is an example of such a framework. In this Account, we review the use of Markov state models for efficient sampling of the hierarchy of time scales associated with protein dynamics, automatic identification of key conformational states, and the degrees of freedom associated with slow dynamical processes. Applications of MSMs for studying long time scale phenomena such as activation mechanisms of cellular signaling proteins has yielded novel insights into protein function. In particular, from MSMs built using large-scale simulations of GPCRs and kinases, we have shown that complex conformational changes in proteins can be described in terms of structural changes in key structural motifs or "molecular switches" within the protein, the transitions between functionally active and inactive states of proteins proceed via multiple pathways, and ligand or substrate binding modulates the flux through these pathways. Finally, MSMs also provide a theoretical

  1. Mitochondrial DNA capture and divergence in Pinus provide new insights into the evolution of the genus.

    PubMed

    Wang, Baosheng; Wang, Xiao-Ru

    2014-11-01

    The evolution of the mitochondrial (mt) genome is far from being fully understood. Systematic investigations into the modes of inheritance, rates and patterns of recombination, nucleotide substitution, and structural changes in the mt genome are still lacking in many groups of plants. In this study, we sequenced >11kbp mtDNA segments from multiple accessions of 36 pine species to characterize the evolutionary patterns of mtDNA in the genus Pinus. We found extremely low substitution rates and complex repetitive sequences scattered across different genome regions, as well as chimeric structures that were probably generated by multiple intergenomic recombinations. The mtDNA-based phylogeny of the genus differed from that based on chloroplast and nuclear DNA in the placement of several groups of species. Such discordances suggest a series of mtDNA capture events during past range shifts of the pine species and that both vertical and horizontal inheritance are implicated in the evolution of mtDNA in Pinus. MtDNA dating revealed that most extant lineages of the genus originated during Oligocene-Miocene radiation and subgenus Strobus diversified earlier than subgenus Pinus. Our findings illustrate a reticular evolutionary pathway for the mt genome through capture and recombination in the genus Pinus, and provide new insights into the evolution of the genus.

  2. Structural insight into Slit-Robo signalling.

    PubMed

    Hohenester, Erhard

    2008-04-01

    Drosophila Slit and its vertebrate orthologues Slit1-Slit3 are secreted glycoproteins that play important roles in the development of the nervous system and other organs. Human Slits are also involved in a number of pathological situations, such as cancer and inflammation. Slits exert their effects by activating receptors of the Robo (Roundabout) family, which resemble cell adhesion molecules in their ectodomains and have large, mainly unstructured cytosolic domains. HS (heparan sulfate) is required for Slit-Robo signalling. The hallmark of Slit proteins is a tandem of four LRR (leucine-rich repeat) domains, which mediate binding to the IG (immunoglobulin-like) domains of Robos. A major question is how Slit binding is translated into the recruitment of effector molecules to the cytosolic domain of Robo. Detailed structure-function studies have shown that the second LRR domain of Slit (D2) binds to the first two IG domains of Robo, and that HS serves to stabilize the Slit-Robo interaction and is required for biological activity of Slit D2. Very recently, the crystal structure of a minimal Slit-Robo complex revealed that the IG1 domain of Robo is bound by the concave face of Slit D2, confirming earlier mutagenesis data. To define the mechanism of Robo transmembrane signalling, these structural insights will have to be complemented by new cell biology and microscopy approaches.

  3. The Active Site of Oligogalacturonate Lyase Provides Unique Insights into Cytoplasmic Oligogalacturonate β-Elimination*

    PubMed Central

    Abbott, D. Wade; Gilbert, Harry J.; Boraston, Alisdair B.

    2010-01-01

    Oligogalacturonate lyases (OGLs; now also classified as pectate lyase family 22) are cytoplasmic enzymes found in pectinolytic members of Enterobacteriaceae, such as the enteropathogen Yersinia enterocolitica. OGLs utilize a β-elimination mechanism to preferentially catalyze the conversion of saturated and unsaturated digalacturonate into monogalacturonate and the 4,5-unsaturated monogalacturonate-like molecule, 5-keto-4-deoxyuronate. To provide mechanistic insights into the specificity of this enzyme activity, we have characterized the OGL from Y. enterocolitica, YeOGL, on oligogalacturonides and determined its three-dimensional x-ray structure to 1.65 Å. The model contains a Mn2+ atom in the active site, which is coordinated by three histidines, one glutamine, and an acetate ion. The acetate mimics the binding of the uronate group of galactourono-configured substrates. These findings, in combination with enzyme kinetics and metal supplementation assays, provide a framework for modeling the active site architecture of OGL. This enzyme appears to contain a histidine for the abstraction of the α-proton in the −1 subsite, a residue that is highly conserved throughout the OGL family and represents a unique catalytic base among pectic active lyases. In addition, we present a hypothesis for an emerging relationship observed between the cellular distribution of pectate lyase folding and the distinct metal coordination chemistries of pectate lyases. PMID:20851883

  4. The charophycean green algae provide insights into the early origins of plant cell walls.

    PubMed

    Sørensen, Iben; Pettolino, Filomena A; Bacic, Antony; Ralph, John; Lu, Fachuang; O'Neill, Malcolm A; Fei, Zhangzhun; Rose, Jocelyn K C; Domozych, David S; Willats, William G T

    2011-10-01

    Numerous evolutionary innovations were required to enable freshwater green algae to colonize terrestrial habitats and thereby initiate the evolution of land plants (embryophytes). These adaptations probably included changes in cell-wall composition and architecture that were to become essential for embryophyte development and radiation. However, it is not known to what extent the polymers that are characteristic of embryophyte cell walls, including pectins, hemicelluloses, glycoproteins and lignin, evolved in response to the demands of the terrestrial environment or whether they pre-existed in their algal ancestors. Here we show that members of the advanced charophycean green algae (CGA), including the Charales, Coleochaetales and Zygnematales, but not basal CGA (Klebsormidiales and Chlorokybales), have cell walls that are comparable in several respects to the primary walls of embryophytes. Moreover, we provide both chemical and immunocytochemical evidence that selected Coleochaete species have cell walls that contain small amounts of lignin or lignin-like polymers derived from radical coupling of hydroxycinnamyl alcohols. Thus, the ability to synthesize many of the components that characterize extant embryophyte walls evolved during divergence within CGA. Our study provides new insight into the evolutionary window during which the structurally complex walls of embryophytes originated, and the significance of the advanced CGA during these events.

  5. The structure of Medicago truncatula δ1-pyrroline-5-carboxylate reductase provides new insights into regulation of proline biosynthesis in plants

    SciTech Connect

    Ruszkowski, Milosz; Nocek, Boguslaw; Forlani, Giuseppe; Dauter, Zbigniew

    2015-10-30

    The two pathways for proline biosynthesis in higher plants share the last step, the conversion of δ1-pyrroline-5-carboxylate (P5C) to L-proline, which is catalyzed by P5C reductase (P5CR, EC 1.5.1.2) with the use of NAD(P)H as a coenzyme. There is increasing amount of evidence to suggest a complex regulation of P5CR activity at the post-translational level, yet the molecular basis of these mechanisms is unknown. Here we report the three-dimensional structure of the P5CR enzyme from the model legume Medicago truncatula (Mt). The crystal structures of unliganded MtP5CR decamer, and its complexes with the products NAD+, NADP+, and L-proline were refined using x-ray diffraction data (at 1.7, 1.85, 1.95, and 2.1 Å resolution, respectively). Based on the presented structural data, the coenzyme preference for NADPH over NADH was explained, and NADPH is suggested to be the only coenzyme used by MtP5CR in vivo. Moreover, the insensitivity of MtP5CR to feed-back inhibition by proline, revealed by enzymatic analysis, was correlated with structural features. Additionally, a mechanism for the modulation of enzyme activity by chloride anions is discussed, as well as the rationale for the possible development of effective enzyme inhibitors.

  6. Visualization of elusive structures using intracardiac echocardiography: Insights from electrophysiology

    PubMed Central

    Szili-Torok, T; McFadden, EP; Jordaens, LJ; Roelandt, JRTC

    2004-01-01

    Electrophysiological mapping and ablation techniques are increasingly used to diagnose and treat many types of supraventricular and ventricular tachycardias. These procedures require an intimate knowledge of intracardiac anatomy and their use has led to a renewed interest in visualization of specific structures. This has required collaborative efforts from imaging as well as electrophysiology experts. Classical imaging techniques may be unable to visualize structures involved in arrhythmia mechanisms and therapy. Novel methods, such as intracardiac echocardiography and three-dimensional echocardiography, have been refined and these technological improvements have opened new perspectives for more effective and accurate imaging during electrophysiology procedures. Concurrently, visualization of these structures noticeably improved our ability to identify intracardiac structures. The aim of this review is to provide electrophysiologists with an overview of recent insights into the structure of the heart obtained with intracardiac echocardiography and to indicate to the echo-specialist which structures are potentially important for the electrophysiologist. PMID:15253772

  7. The structure- and metal-dependent activity of Escherichia coli PgaB provides insight into the partial de-N-acetylation of poly-β-1,6-N-acetyl-D-glucosamine.

    PubMed

    Little, Dustin J; Poloczek, Joanna; Whitney, John C; Robinson, Howard; Nitz, Mark; Howell, P Lynne

    2012-09-07

    Exopolysaccharides are required for the development and integrity of biofilms produced by a wide variety of bacteria. In Escherichia coli, partial de-N-acetylation of the exopolysaccharide poly-β-1,6-N-acetyl-D-glucosamine (PNAG) by the periplasmic protein PgaB is required for polysaccharide intercellular adhesin-dependent biofilm formation. To understand the molecular basis for PNAG de-N-acetylation, the structure of PgaB in complex with Ni(2+) and Fe(3+) have been determined to 1.9 and 2.1 Å resolution, respectively, and its activity on β-1,6-GlcNAc oligomers has been characterized. The structure of PgaB reveals two (β/α)(x) barrel domains: a metal-binding de-N-acetylase that is a member of the family 4 carbohydrate esterases (CE4s) and a domain structurally similar to glycoside hydrolases. PgaB displays de-N-acetylase activity on β-1,6-GlcNAc oligomers but not on the β-1,4-(GlcNAc)(4) oligomer chitotetraose and is the first CE4 member to exhibit this substrate specificity. De-N-acetylation occurs in a length-dependent manor, and specificity is observed for the position of de-N-acetylation. A key aspartic acid involved in de-N-acetylation, normally seen in other CE4s, is missing in PgaB, suggesting that the activity of PgaB is attenuated to maintain the low levels of de-N-acetylation of PNAG observed in vivo. The metal dependence of PgaB is different from most CE4s, because PgaB shows increased rates of de-N-acetylation with Co(2+) and Ni(2+) under aerobic conditions, and Co(2+), Ni(2+) and Fe(2+) under anaerobic conditions, but decreased activity with Zn(2+). The work presented herein will guide inhibitor design to combat biofilm formation by E. coli and potentially a wide range of medically relevant bacteria producing polysaccharide intercellular adhesin-dependent biofilms.

  8. The crystal structure of a ternary complex of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa Provides new insight into the reaction mechanism and shows a novel binding mode of the 2'-phosphate of NADP+ and a novel cation binding site.

    PubMed

    González-Segura, Lilian; Rudiño-Piñera, Enrique; Muñoz-Clares, Rosario A; Horjales, Eduardo

    2009-01-16

    In the human pathogen Pseudomonas aeruginosa, the NAD(P)(+)-dependent betaine aldehyde dehydrogenase (PaBADH) may play the dual role of assimilating carbon and nitrogen from choline or choline precursors--abundant at infection sites--and producing glycine betaine and NADPH, potentially protective against the high-osmolarity and oxidative stresses prevalent in the infected tissues. Disruption of the PaBADH gene negatively affects the growth of bacteria, suggesting that this enzyme could be a target for antibiotic design. PaBADH is one of the few ALDHs that efficiently use NADP(+) and one of the even fewer that require K(+) ions for stability. Crystals of PaBADH were obtained under aerobic conditions in the presence of 2-mercaptoethanol, glycerol, NADP(+) and K(+) ions. The three-dimensional structure was determined at 2.1-A resolution. The catalytic cysteine (C286, corresponding to C302 of ALDH2) is oxidized to sulfenic acid or forms a mixed disulfide with 2-mercaptoethanol. The glutamyl residue involved in the deacylation step (E252, corresponding to E268 of ALDH2) is in two conformations, suggesting a proton relay system formed by two well-conserved residues (E464 and K162, corresponding to E476 and K178, respectively, of ALDH2) that connects E252 with the bulk water. In some active sites, a bound glycerol molecule mimics the thiohemiacetal intermediate; its hydroxyl oxygen is hydrogen bonded to the nitrogen of the amide groups of the side chain of the conserved N153 (N169 of ALDH2) and those of the main chain of C286, which form the "oxyanion hole." The nicotinamide moiety of the nucleotide is not observed in the crystal, and the adenine moiety binds in the usual way. A salt bridge between E179 (E195 of ALDH2) and R40 (E53 of ALDH2) moves the carboxylate group of the former away from the 2'-phosphate of the NADP(+), thus avoiding steric clashes and/or electrostatic repulsion between the two groups. Finally, the crystal shows two K(+) binding sites per subunit

  9. The Crystal Structure of a Ternary Complex of Betaine Aldehyde Dehydrogenase from Pseudomonas aeruginosa Provides New Insight Into the Reaction Mechansim and Shows A Novel Binding Mode of the 2'-Phosphate of NADP+ and A Novel Cation Binding Site

    SciTech Connect

    Gonzalez-Segura, L.; Rudino-Pinera, E; Munoz-Clares, R; Horjales, E

    2009-01-01

    In the human pathogen Pseudomonas aeruginosa, the NAD(P)+-dependent betaine aldehyde dehydrogenase (PaBADH) may play the dual role of assimilating carbon and nitrogen from choline or choline precursors-abundant at infection sites-and producing glycine betaine and NADPH, potentially protective against the high-osmolarity and oxidative stresses prevalent in the infected tissues. Disruption of the PaBADH gene negatively affects the growth of bacteria, suggesting that this enzyme could be a target for antibiotic design. PaBADH is one of the few ALDHs that efficiently use NADP+ and one of the even fewer that require K+ ions for stability. Crystals of PaBADH were obtained under aerobic conditions in the presence of 2-mercaptoethanol, glycerol, NADP+ and K+ ions. The three-dimensional structure was determined at 2.1-A resolution. The catalytic cysteine (C286, corresponding to C302 of ALDH2) is oxidized to sulfenic acid or forms a mixed disulfide with 2-mercaptoethanol. The glutamyl residue involved in the deacylation step (E252, corresponding to E268 of ALDH2) is in two conformations, suggesting a proton relay system formed by two well-conserved residues (E464 and K162, corresponding to E476 and K178, respectively, of ALDH2) that connects E252 with the bulk water. In some active sites, a bound glycerol molecule mimics the thiohemiacetal intermediate; its hydroxyl oxygen is hydrogen bonded to the nitrogen of the amide groups of the side chain of the conserved N153 (N169 of ALDH2) and those of the main chain of C286, which form the 'oxyanion hole.' The nicotinamide moiety of the nucleotide is not observed in the crystal, and the adenine moiety binds in the usual way. A salt bridge between E179 (E195 of ALDH2) and R40 (E53 of ALDH2) moves the carboxylate group of the former away from the 2?-phosphate of the NADP+, thus avoiding steric clashes and/or electrostatic repulsion between the two groups. Finally, the crystal shows two K+ binding sites per subunit. One is in an

  10. Opioid receptors: Structural and mechanistic insights into pharmacology and signaling

    PubMed Central

    Shang, Yi; Filizola, Marta

    2015-01-01

    Opioid receptors are important drug targets for pain management, addiction, and mood disorders. Although substantial research on these important subtypes of G protein-coupled receptors has been conducted over the past two decades to discover ligands with higher specificity and diminished side effects, currently used opioid therapeutics remain suboptimal. Luckily, recent advances in structural biology of opioid receptors provide unprecedented insights into opioid receptor pharmacology and signaling. We review here a few recent studies that have used the crystal structures of opioid receptors as a basis for revealing mechanistic details of signal transduction mediated by these receptors, and for the purpose of drug discovery. PMID:25981301

  11. Opioid receptors: Structural and mechanistic insights into pharmacology and signaling.

    PubMed

    Shang, Yi; Filizola, Marta

    2015-09-15

    Opioid receptors are important drug targets for pain management, addiction, and mood disorders. Although substantial research on these important subtypes of G protein-coupled receptors has been conducted over the past two decades to discover ligands with higher specificity and diminished side effects, currently used opioid therapeutics remain suboptimal. Luckily, recent advances in structural biology of opioid receptors provide unprecedented insights into opioid receptor pharmacology and signaling. We review here a few recent studies that have used the crystal structures of opioid receptors as a basis for revealing mechanistic details of signal transduction mediated by these receptors, and for the purpose of drug discovery.

  12. New technologies provide insights into genetic basis of psychiatric disorders and explain their co-morbidity.

    PubMed

    Rudan, Igor

    2010-06-01

    The completion of Human Genome Project and the "HapMap" project was followed by translational activities from companies within the private sector. This led to the introduction of genome-wide scans based on hundreds of thousands of single nucleotide polymorphysms (SNP). These scans were based on common genetic variants in human populations. This new and powerful technology was then applied to the existing DNA-based datasets with information on psychiatric disorders. As a result, an unprecedented amount of novel scientific insights related to the underlying biology and genetics of psychiatric disorders was obtained. The dominant design of these studies, so called "genome-wide association studies" (GWAS), used statistical methods which minimized the risk of false positive reports and provided much greater power to detect genotype-phenotype associations. All findings were entirely data-driven rather than hypothesis-driven, which often made it difficult for researchers to understand or interpret the findings. Interestingly, this work in genetics is indicating how non-specific some genes are for psychiatric disorders, having associations in common for schizophrenia, bipolar disorder and autism. This suggests that the earlier stages of psychiatric disorders may be multi-valent and that early detection, coupled with a clearer understanding of the environmental factors, may allow prevention. At the present time, the rich "harvest" from GWAS still has very limited power to predict the variation in psychiatric disease status at individual level, typically explaining less than 5% of the total risk variance. The most recent studies of common genetic variation implicated the role of major histocompatibility complex in schizophrenia and other disorders. They also provided molecular evidence for a substantial polygenic component to the risk of psychiatric diseases, involving thousands of common alleles of very small effect. The studies of structural genetic variation, such as copy

  13. X-ray Structure Analysis of Indazolium trans-[Tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019) Bound to Human Serum Albumin Reveals Two Ruthenium Binding Sites and Provides Insights into the Drug Binding Mechanism

    PubMed Central

    2016-01-01

    Ruthenium(III) complexes are promising candidates for anticancer drugs, especially the clinically studied indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019) and its analogue sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (NKP-1339). Several studies have emphasized the likely role of human serum proteins in the transportation and accumulation of ruthenium(III) complexes in tumors. Therefore, the interaction between KP1019 and human serum albumin was investigated by means of X-ray crystallography and inductively coupled plasma mass spectrometry (ICP-MS). The structural data unambiguously reveal the binding of two ruthenium atoms to histidine residues 146 and 242, which are both located within well-known hydrophobic binding pockets of albumin. The ruthenium centers are octahedrally coordinated by solvent molecules revealing the dissociation of both indazole ligands from the ruthenium-based drug. However, a binding mechanism is proposed indicating the importance of the indazole ligands for binding site recognition and thus their indispensable role for the binding of KP1019. PMID:27196130

  14. Structural Determination and Tryptophan Fluorescence of Heterokaryon Incompatibility C2 Protein (HET-C2), a Fungal Glycolipid Transfer Protein (GLTP), Provide Novel Insights into Glycolipid Specificity and Membrane Interaction by the GLTP Fold

    SciTech Connect

    Kenoth, Roopa; Simanshu, Dhirendra K.; Kamlekar, Ravi Kanth; Pike, Helen M.; Molotkovsky, Julian G.; Benson, Linda M.; Bergen, III, H. Robert; Prendergast, Franklyn G.; Malinina, Lucy; Venyaminov, Sergei Y.; Patel, Dinshaw J.; Brown, Rhoderick E.

    2010-06-21

    HET-C2 is a fungal protein that transfers glycosphingolipids between membranes and has limited sequence homology with human glycolipid transfer protein (GLTP). The human GLTP fold is unique among lipid binding/transfer proteins, defining the GLTP superfamily. Herein, GLTP fold formation by HET-C2, its glycolipid transfer specificity, and the functional role(s) of its two Trp residues have been investigated. X-ray diffraction (1.9 {angstrom}) revealed a GLTP fold with all key sugar headgroup recognition residues (Asp{sup 66}, Asn{sup 70}, Lys{sup 73}, Trp{sup 109}, and His{sup 147}) conserved and properly oriented for glycolipid binding. Far-UV CD showed secondary structure dominated by {alpha}-helices and a cooperative thermal unfolding transition of 49 C, features consistent with a GLTP fold. Environmentally induced optical activity of Trp/Tyr/Phe (2:4:12) detected by near-UV CD was unaffected by membranes containing glycolipid but was slightly altered by membranes lacking glycolipid. Trp fluorescence was maximal at {approx}355 nm and accessible to aqueous quenchers, indicating free exposure to the aqueous milieu and consistent with surface localization of the two Trps. Interaction with membranes lacking glycolipid triggered significant decreases in Trp emission intensity but lesser than decreases induced by membranes containing glycolipid. Binding of glycolipid (confirmed by electrospray injection mass spectrometry) resulted in a blue-shifted emission wavelength maximum ({approx}6 nm) permitting determination of binding affinities. The unique positioning of Trp{sup 208} at the HET-C2 C terminus revealed membrane-induced conformational changes that precede glycolipid uptake, whereas key differences in residues of the sugar headgroup recognition center accounted for altered glycolipid specificity and suggested evolutionary adaptation for the simpler glycosphingolipid compositions of filamentous fungi.

  15. Plasmodium malariae and P. ovale genomes provide insights into malaria parasite evolution.

    PubMed

    Rutledge, Gavin G; Böhme, Ulrike; Sanders, Mandy; Reid, Adam J; Cotton, James A; Maiga-Ascofare, Oumou; Djimdé, Abdoulaye A; Apinjoh, Tobias O; Amenga-Etego, Lucas; Manske, Magnus; Barnwell, John W; Renaud, François; Ollomo, Benjamin; Prugnolle, Franck; Anstey, Nicholas M; Auburn, Sarah; Price, Ric N; McCarthy, James S; Kwiatkowski, Dominic P; Newbold, Chris I; Berriman, Matthew; Otto, Thomas D

    2017-02-02

    Elucidation of the evolutionary history and interrelatedness of Plasmodium species that infect humans has been hampered by a lack of genetic information for three human-infective species: P. malariae and two P. ovale species (P. o. curtisi and P. o. wallikeri). These species are prevalent across most regions in which malaria is endemic and are often undetectable by light microscopy, rendering their study in human populations difficult. The exact evolutionary relationship of these species to the other human-infective species has been contested. Using a new reference genome for P. malariae and a manually curated draft P. o. curtisi genome, we are now able to accurately place these species within the Plasmodium phylogeny. Sequencing of a P. malariae relative that infects chimpanzees reveals similar signatures of selection in the P. malariae lineage to another Plasmodium lineage shown to be capable of colonization of both human and chimpanzee hosts. Molecular dating suggests that these host adaptations occurred over similar evolutionary timescales. In addition to the core genome that is conserved between species, differences in gene content can be linked to their specific biology. The genome suggests that P. malariae expresses a family of heterodimeric proteins on its surface that have structural similarities to a protein crucial for invasion of red blood cells. The data presented here provide insight into the evolution of the Plasmodium genus as a whole.

  16. Analysis of the FGF gene family provides insights into aquatic adaptation in cetaceans

    PubMed Central

    Nam, Kiwoong; Lee, Kyeong Won; Chung, Oksung; Yim, Hyung-Soon; Cha, Sun-Shin; Lee, Sae-Won; Jun, JeHoon; Cho, Yun Sung; Bhak, Jong; Magalhães, João Pedro de; Lee, Jung-Hyun; Jeong, Jae-Yeon

    2017-01-01

    Cetacean body structure and physiology exhibit dramatic adaptations to their aquatic environment. Fibroblast growth factors (FGFs) are a family of essential factors that regulate animal development and physiology; however, their role in cetacean evolution is not clearly understood. Here, we sequenced the fin whale genome and analysed FGFs from 8 cetaceans. FGF22, a hair follicle-enriched gene, exhibited pseudogenization, indicating that the function of this gene is no longer necessary in cetaceans that have lost most of their body hair. An evolutionary analysis revealed signatures of positive selection for FGF3 and FGF11, genes related to ear and tooth development and hypoxia, respectively. We found a D203G substitution in cetacean FGF9, which was predicted to affect FGF9 homodimerization, suggesting that this gene plays a role in the acquisition of rigid flippers for efficient manoeuvring. Cetaceans utilize low bone density as a buoyancy control mechanism, but the underlying genes are not known. We found that the expression of FGF23, a gene associated with reduced bone density, is greatly increased in the cetacean liver under hypoxic conditions, thus implicating FGF23 in low bone density in cetaceans. Altogether, our results provide novel insights into the roles of FGFs in cetacean adaptation to the aquatic environment. PMID:28074842

  17. Obsessive Compulsive Disorder Networks: Positron Emission Tomography and Neuropsychology Provide New Insights

    PubMed Central

    Millet, Bruno; Dondaine, Thibaut; Reymann, Jean-Michel; Bourguignon, Aurélie; Naudet, Florian; Jaafari, Nematollah; Drapier, Dominique; Turmel, Valérie; Mesbah, Habiba; Vérin, Marc; Le Jeune, Florence

    2013-01-01

    Background Deep brain stimulation has shed new light on the central role of the prefrontal cortex (PFC) in obsessive compulsive disorder (OCD). We explored this structure from a functional perspective, synchronizing neuroimaging and cognitive measures. Methods and Findings This case-control cross-sectional study compared 15 OCD patients without comorbidities and not currently on serotonin reuptake inhibitors or cognitive behavioural therapy with 15 healthy controls (matched for age, sex and education level) on resting-state 18FDG-PET scans and a neuropsychological battery assessing executive functions. We looked for correlations between metabolic modifications and impaired neuropsychological scores. Modifications in glucose metabolism were found in frontal regions (orbitofrontal cortex and dorsolateral cortices), the cingulate gyrus, insula and parietal gyrus. Neuropsychological differences between patients and controls, which were subtle, were correlated with the metabolism of the prefrontal, parietal, and temporal cortices. Conclusion As expected, we confirmed previous reports of a PFC dysfunction in OCD patients, and established a correlation with cognitive deficits. Other regions outside the prefrontal cortex, including the dorsoparietal cortex and the insula, also appeared to be implicated in the pathophysiology of OCD, providing fresh insights on the complexity of OCD syndromes. PMID:23326403

  18. Plasmodium malariae and P. ovale genomes provide insights into malaria parasite evolution

    PubMed Central

    Rutledge, Gavin G.; Böhme, Ulrike; Sanders, Mandy; Reid, Adam J.; Cotton, James A.; Maiga-Ascofare, Oumou; Djimdé, Abdoulaye A.; Apinjoh, Tobias O.; Amenga-Etego, Lucas; Manske, Magnus; Barnwell, John W.; Renaud, François; Ollomo, Benjamin; Prugnolle, Franck; Anstey, Nicholas M.; Auburn, Sarah; Price, Ric N.; McCarthy, James S.; Kwiatkowski, Dominic P.; Newbold, Chris I.; Berriman, Matthew; Otto, Thomas D.

    2017-01-01

    Elucidation of the evolutionary history and interrelatedness of Plasmodium species that infect humans has been hampered by a lack of genetic information for three human-infective species: P. malariae and two P. ovale species (P. o. curtisi and P. o. wallikeri)1. These species are prevalent across most regions in which malaria is endemic2,3 and are often undetectable by light microscopy4, rendering their study in human populations difficult5. The exact evolutionary relationship of these species to the other human-infective species has been contested6,7. Using a new reference genome for P. malariae and a manually curated draft P. o. curtisi genome, we are now able to accurately place these species within the Plasmodium phylogeny. Sequencing of a P. malariae relative that infects chimpanzees reveals similar signatures of selection in the P. malariae lineage to another Plasmodium lineage shown to be capable of colonization of both human and chimpanzee hosts. Molecular dating suggests that these host adaptations occurred over similar evolutionary timescales. In addition to the core genome that is conserved between species, differences in gene content can be linked to their specific biology. The genome suggests that P. malariae expresses a family of heterodimeric proteins on its surface that have structural similarities to a protein crucial for invasion of red blood cells. The data presented here provide insight into the evolution of the Plasmodium genus as a whole. PMID:28117441

  19. Unusual mutation clusters provide insight into class I gene conversion mechanisms.

    PubMed Central

    Pease, L R; Horton, R M; Pullen, J K; Yun, T J

    1993-01-01

    Genetic diversity among the K and D alleles of the mouse major histocompatibility complex is generated by gene conversion among members of the class I multigene family. The majority of known class I mutants contain clusters of nucleotide changes that can be traced to linked family members. However, the details of the gene conversion mechanism are not known. The bm3 and bm23 mutations represent exceptions to the usual pattern and provide insight into intermediates generated during the gene conversion process. Both of these variants contain clusters of five nucleotide substitutions, but they differ from the classic conversion mutants in the important respect that no donor gene for either mutation could be identified in the parental genome. Nevertheless, both mutation clusters are composed of individual mutations that do exist within the parent. Therefore, they are not random and appear to be templated. Significantly, the bm3 and bm23 mutation clusters are divided into overlapping regions that match class I genes which have functioned as donor genes in other characterized gene conversion events. The unusual structure of the mutation clusters indicates an underlying gene conversion mechanism that can generate mutation clusters as a result of the interaction of three genes in a single genetic event. The unusual mutation clusters are consistent with a hypothetical gene conversion model involving extrachromosomal intermediates. Images PMID:8321237

  20. First fossil gravid turtle provides insight into the evolution of reproductive traits in turtles.

    PubMed

    Zelenitsky, Darla K; Therrien, Franc Ois; Joyce, Walter G; Brinkman, Donald B

    2008-12-23

    Here we report on the first discovery of shelled eggs inside the body cavity of a fossil turtle and on an isolated egg clutch, both referable to the Cretaceous turtle Adocus. These discoveries provide a unique opportunity to gain insight into the reproductive traits of an extinct turtle and to understand the evolution of such traits among living turtles. The gravid adult and egg clutch indicate that Adocus laid large clutches of rigid-shelled spherical eggs and established their nests near rivers, traits that are shared by its closest living relatives, the soft-shelled turtles. Adocus eggshell, however, was probably more rigid than that of living turtles, based on its great thickness and structure, features that may represent unique adaptations to intense predation or to arid nest environments. In light of the reproductive traits observed in Adocus, the distribution of reproductive traits among turtles reveals that large clutches of rigid-shelled eggs are primitive for hidden-necked turtles (cryptodirans) and that spherical eggs may have evolved independently within this group.

  1. Microstructure provides insights into evolutionary design and resilience of Coscinodiscus sp. frustule

    PubMed Central

    Aitken, Zachary H.; Luo, Shi; Reynolds, Stephanie N.; Thaulow, Christian; Greer, Julia R.

    2016-01-01

    We conducted in situ three-point bending experiments on beams with roughly square cross-sections, which we fabricated from the frustule of Coscinodiscus sp. We observe failure by brittle fracture at an average stress of 1.1 GPa. Analysis of crack propagation and shell morphology reveals a differentiation in the function of the frustule layers with the basal layer pores, which deflect crack propagation. We calculated the relative density of the frustule to be ∼30% and show that at this density the frustule has the highest strength-to-density ratio of 1,702 kN⋅m/kg, a significant departure from all reported biologic materials. We also performed nanoindentation on both the single basal layer of the frustule as well as the girdle band and show that these components display similar mechanical properties that also agree well with bending tests. Transmission electron microscopy analysis reveals that the frustule is made almost entirely of amorphous silica with a nanocrystalline proximal layer. No flaws are observed within the frustule material down to 2 nm. Finite element simulations of the three-point bending experiments show that the basal layer carries most of the applied load whereas stresses within the cribrum and areolae layer are an order of magnitude lower. These results demonstrate the natural development of architecture in live organisms to simultaneously achieve light weight, strength, and exceptional structural integrity and may provide insight into evolutionary design. PMID:26858446

  2. Peeping at TOMs-Diverse Entry Gates to Mitochondria Provide Insights into the Evolution of Eukaryotes.

    PubMed

    Mani, Jan; Meisinger, Chris; Schneider, André

    2016-02-01

    Mitochondria are essential for eukaryotic life and more than 95% of their proteins are imported as precursors from the cytosol. The targeting signals for this posttranslational import are conserved in all eukaryotes. However, this conservation does not hold true for the protein translocase of the mitochondrial outer membrane that serves as entry gate for essentially all precursor proteins. Only two of its subunits, Tom40 and Tom22, are conserved and thus likely were present in the last eukaryotic common ancestor. Tom7 is found in representatives of all supergroups except the Excavates. This suggests that it was added to the core of the translocase after the Excavates segregated from all other eukaryotes. A comparative analysis of the biochemically and functionally characterized outer membrane translocases of yeast, plants, and trypanosomes, which represent three eukaryotic supergroups, shows that the receptors that recognize the conserved import signals differ strongly between the different systems. They present a remarkable example of convergent evolution at the molecular level. The structural diversity of the functionally conserved import receptors therefore provides insight into the early evolutionary history of mitochondria.

  3. Segmentation studies provide insights to better understanding attitudes towards science and technology.

    PubMed

    Cormick, Craig; Romanach, Lygia Malzoni

    2014-03-01

    Values-based studies of people's attitudes towards science and technology not only provide great insights into what drives different attitudes to issues like climate change and genetically modified foods, but allow for segmenting the general public by homogeneous values. Such segmentations both provide better predictions of people's attitudes to new technologies or contentious science issues than age, sex, or other standard demographics, and allow a better matching of different messages with different community values.

  4. Canine CNGA3 Gene Mutations Provide Novel Insights into Human Achromatopsia-Associated Channelopathies and Treatment.

    PubMed

    Tanaka, Naoto; Dutrow, Emily V; Miyadera, Keiko; Delemotte, Lucie; MacDermaid, Christopher M; Reinstein, Shelby L; Crumley, William R; Dixon, Christopher J; Casal, Margret L; Klein, Michael L; Aguirre, Gustavo D; Tanaka, Jacqueline C; Guziewicz, Karina E

    2015-01-01

    Cyclic nucleotide-gated (CNG) ion channels are key mediators underlying signal transduction in retinal and olfactory receptors. Genetic defects in CNGA3 and CNGB3, encoding two structurally related subunits of cone CNG channels, lead to achromatopsia (ACHM). ACHM is a congenital, autosomal recessive retinal disorder that manifests by cone photoreceptor dysfunction, severely reduced visual acuity, impaired or complete color blindness and photophobia. Here, we report the first canine models for CNGA3-associated channelopathy caused by R424W or V644del mutations in the canine CNGA3 ortholog that accurately mimic the clinical and molecular features of human CNGA3-associated ACHM. These two spontaneous mutations exposed CNGA3 residues essential for the preservation of channel function and biogenesis. The CNGA3-R424W results in complete loss of cone function in vivo and channel activity confirmed by in vitro electrophysiology. Structural modeling and molecular dynamics (MD) simulations revealed R424-E306 salt bridge formation and its disruption with the R424W mutant. Reversal of charges in a CNGA3-R424E-E306R double mutant channel rescued cGMP-activated currents uncovering new insights into channel gating. The CNGA3-V644del affects the C-terminal leucine zipper (CLZ) domain destabilizing intersubunit interactions of the coiled-coil complex in the MD simulations; the in vitro experiments showed incompetent trimeric CNGA3 subunit assembly consistent with abnormal biogenesis of in vivo channels. These newly characterized large animal models not only provide a valuable system for studying cone-specific CNG channel function in health and disease, but also represent prime candidates for proof-of-concept studies of CNGA3 gene replacement therapy for ACHM patients.

  5. Canine CNGA3 Gene Mutations Provide Novel Insights into Human Achromatopsia-Associated Channelopathies and Treatment

    PubMed Central

    Miyadera, Keiko; Delemotte, Lucie; MacDermaid, Christopher M.; Reinstein, Shelby L.; Crumley, William R.; Dixon, Christopher J.; Casal, Margret L.; Klein, Michael L.; Aguirre, Gustavo D.; Tanaka, Jacqueline C.; Guziewicz, Karina E.

    2015-01-01

    Cyclic nucleotide-gated (CNG) ion channels are key mediators underlying signal transduction in retinal and olfactory receptors. Genetic defects in CNGA3 and CNGB3, encoding two structurally related subunits of cone CNG channels, lead to achromatopsia (ACHM). ACHM is a congenital, autosomal recessive retinal disorder that manifests by cone photoreceptor dysfunction, severely reduced visual acuity, impaired or complete color blindness and photophobia. Here, we report the first canine models for CNGA3-associated channelopathy caused by R424W or V644del mutations in the canine CNGA3 ortholog that accurately mimic the clinical and molecular features of human CNGA3-associated ACHM. These two spontaneous mutations exposed CNGA3 residues essential for the preservation of channel function and biogenesis. The CNGA3-R424W results in complete loss of cone function in vivo and channel activity confirmed by in vitro electrophysiology. Structural modeling and molecular dynamics (MD) simulations revealed R424-E306 salt bridge formation and its disruption with the R424W mutant. Reversal of charges in a CNGA3-R424E-E306R double mutant channel rescued cGMP-activated currents uncovering new insights into channel gating. The CNGA3-V644del affects the C-terminal leucine zipper (CLZ) domain destabilizing intersubunit interactions of the coiled-coil complex in the MD simulations; the in vitro experiments showed incompetent trimeric CNGA3 subunit assembly consistent with abnormal biogenesis of in vivo channels. These newly characterized large animal models not only provide a valuable system for studying cone-specific CNG channel function in health and disease, but also represent prime candidates for proof-of-concept studies of CNGA3 gene replacement therapy for ACHM patients. PMID:26407004

  6. Transcriptional profiling of a yeast colony provides new insight into the heterogeneity of multicellular fungal communities.

    PubMed

    Traven, Ana; Jänicke, Amrei; Harrison, Paul; Swaminathan, Angavai; Seemann, Torsten; Beilharz, Traude H

    2012-01-01

    Understanding multicellular fungal structures is important for designing better strategies against human fungal pathogens. For example, the ability to form multicellular biofilms is a key virulence property of the yeast Candida albicans. C. albicans biofilms form on indwelling medical devices and are drug resistant, causing serious infections in hospital settings. Multicellular fungal communities are heterogeneous, consisting of cells experiencing different environments. Heterogeneity is likely important for the phenotypic characteristics of communities, yet it is poorly understood. Here we used colonies of the yeast Saccharomyces cerevisiae as a model fungal multicellular structure. We fractionated the outside colony layers from the cells in the center by FACS, using a Cit1-GFP marker expressed exclusively on the outside. Transcriptomics analysis of the two subpopulations revealed that the outside colony layers are actively growing by fermentative metabolism, while the cells residing on the inside are in a resting state and experience changes to mitochondrial activity. Our data shows several parallels with C. albicans biofilms providing insight into the contributions of heterogeneity to biofilm phenotypes. Hallmarks of C. albicans biofilms - the expression of ribosome and translation functions and activation of glycolysis and ergosterol biosynthesis occur on the outside of colonies, while expression of genes associates with sulfur assimilation is observed in the colony center. Cell wall restructuring occurs in biofilms, and cell wall functions are enriched in both fractions: the outside cells display enrichment of cell wall biosynthesis enzymes and cell wall proteins, while the inside cells express cell wall degrading enzymes. Our study also suggests that noncoding transcription and posttranscriptional mRNA regulation play important roles during growth of yeast in colonies, setting the scene for investigating these pathways in the development of multicellular

  7. Transcriptional Profiling of a Yeast Colony Provides New Insight into the Heterogeneity of Multicellular Fungal Communities

    PubMed Central

    Traven, Ana; Jänicke, Amrei; Harrison, Paul; Swaminathan, Angavai; Seemann, Torsten; Beilharz, Traude H.

    2012-01-01

    Understanding multicellular fungal structures is important for designing better strategies against human fungal pathogens. For example, the ability to form multicellular biofilms is a key virulence property of the yeast Candida albicans. C. albicans biofilms form on indwelling medical devices and are drug resistant, causing serious infections in hospital settings. Multicellular fungal communities are heterogeneous, consisting of cells experiencing different environments. Heterogeneity is likely important for the phenotypic characteristics of communities, yet it is poorly understood. Here we used colonies of the yeast Saccharomyces cerevisiae as a model fungal multicellular structure. We fractionated the outside colony layers from the cells in the center by FACS, using a Cit1-GFP marker expressed exclusively on the outside. Transcriptomics analysis of the two subpopulations revealed that the outside colony layers are actively growing by fermentative metabolism, while the cells residing on the inside are in a resting state and experience changes to mitochondrial activity. Our data shows several parallels with C. albicans biofilms providing insight into the contributions of heterogeneity to biofilm phenotypes. Hallmarks of C. albicans biofilms – the expression of ribosome and translation functions and activation of glycolysis and ergosterol biosynthesis occur on the outside of colonies, while expression of genes associates with sulfur assimilation is observed in the colony center. Cell wall restructuring occurs in biofilms, and cell wall functions are enriched in both fractions: the outside cells display enrichment of cell wall biosynthesis enzymes and cell wall proteins, while the inside cells express cell wall degrading enzymes. Our study also suggests that noncoding transcription and posttranscriptional mRNA regulation play important roles during growth of yeast in colonies, setting the scene for investigating these pathways in the development of multicellular

  8. Emerging structural insights into the function of ionotropic glutamate receptors

    PubMed Central

    Karakas, Erkan; Regan, Michael C.; Furukawa, Hiro

    2015-01-01

    Summary Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate excitatory neurotransmission crucial for brain development and function including learning and memory formation. Recently a wealth of structural studies on iGluRs, including AMPA receptors (AMPARs), kainate receptors, and NMDA receptors (NMDARs) became available.. These studies showed structures of non-NMDARs including AMPAR and kainate receptor in various functional states, thereby providing the first visual sense of how non-NMDAR iGluRs may function in the context of homotetramers. Furthermore, they provided the first view of heterotetrameric NMDAR ion channels, which illuminated the similarities with and differences from non-NMDARs, thus raising a mechanistic distinction between the two groups of iGluRs. Here we review mechanistic insights into iGluR functions gained through structural studies of multiple groups. PMID:25941168

  9. Modeling fMRI signals can provide insights into neural processing in the cerebral cortex.

    PubMed

    Vanni, Simo; Sharifian, Fariba; Heikkinen, Hanna; Vigário, Ricardo

    2015-08-01

    Every stimulus or task activates multiple areas in the mammalian cortex. These distributed activations can be measured with functional magnetic resonance imaging (fMRI), which has the best spatial resolution among the noninvasive brain imaging methods. Unfortunately, the relationship between the fMRI activations and distributed cortical processing has remained unclear, both because the coupling between neural and fMRI activations has remained poorly understood and because fMRI voxels are too large to directly sense the local neural events. To get an idea of the local processing given the macroscopic data, we need models to simulate the neural activity and to provide output that can be compared with fMRI data. Such models can describe neural mechanisms as mathematical functions between input and output in a specific system, with little correspondence to physiological mechanisms. Alternatively, models can be biomimetic, including biological details with straightforward correspondence to experimental data. After careful balancing between complexity, computational efficiency, and realism, a biomimetic simulation should be able to provide insight into how biological structures or functions contribute to actual data processing as well as to promote theory-driven neuroscience experiments. This review analyzes the requirements for validating system-level computational models with fMRI. In particular, we study mesoscopic biomimetic models, which include a limited set of details from real-life networks and enable system-level simulations of neural mass action. In addition, we discuss how recent developments in neurophysiology and biophysics may significantly advance the modelling of fMRI signals.

  10. Modeling fMRI signals can provide insights into neural processing in the cerebral cortex

    PubMed Central

    Sharifian, Fariba; Heikkinen, Hanna; Vigário, Ricardo

    2015-01-01

    Every stimulus or task activates multiple areas in the mammalian cortex. These distributed activations can be measured with functional magnetic resonance imaging (fMRI), which has the best spatial resolution among the noninvasive brain imaging methods. Unfortunately, the relationship between the fMRI activations and distributed cortical processing has remained unclear, both because the coupling between neural and fMRI activations has remained poorly understood and because fMRI voxels are too large to directly sense the local neural events. To get an idea of the local processing given the macroscopic data, we need models to simulate the neural activity and to provide output that can be compared with fMRI data. Such models can describe neural mechanisms as mathematical functions between input and output in a specific system, with little correspondence to physiological mechanisms. Alternatively, models can be biomimetic, including biological details with straightforward correspondence to experimental data. After careful balancing between complexity, computational efficiency, and realism, a biomimetic simulation should be able to provide insight into how biological structures or functions contribute to actual data processing as well as to promote theory-driven neuroscience experiments. This review analyzes the requirements for validating system-level computational models with fMRI. In particular, we study mesoscopic biomimetic models, which include a limited set of details from real-life networks and enable system-level simulations of neural mass action. In addition, we discuss how recent developments in neurophysiology and biophysics may significantly advance the modelling of fMRI signals. PMID:25972586

  11. Structural insights into the transport of small molecules across membranes

    PubMed Central

    Noinaj, Nicholas; Buchanan, Susan K.

    2014-01-01

    While hydrophobic small molecules often can freely permeate a lipid bilayer, ions and other polar molecules cannot and require transporters to mediate their transport. Recently, a number of important structures have been reported which have advanced our understanding of how membrane protein transporters function to transport small molecules. Structures of TbpA/B and HmuUV provided new insight into iron uptake by pathogenic bacteria while the structures of NarK, ASBT, and VcINDY revealed molecular details about the transport of nitrate, bile acids and dicarboxylates, respectively. The structure of the folate ECF transporter indicated that the S component likely undergoes a large conformational shift to mediate folate transport, while the cellulose synthase/transporter contains an elongated translocation pore for passage through the inner membrane. PMID:24681594

  12. Structural and mechanistic insights into hepatitis C viral translation initiation.

    PubMed

    Fraser, Christopher S; Doudna, Jennifer A

    2007-01-01

    Hepatitis C virus uses an internal ribosome entry site (IRES) to control viral protein synthesis by directly recruiting ribosomes to the translation-start site in the viral mRNA. Structural insights coupled with biochemical studies have revealed that the IRES substitutes for the activities of translation-initiation factors by binding and inducing conformational changes in the 40S ribosomal subunit. Direct interactions of the IRES with initiation factor eIF3 are also crucial for efficient translation initiation, providing clues to the role of eIF3 in protein synthesis.

  13. Association genetics in Solanum tuberosum provides new insights into potato tuber bruising and enzymatic tissue discoloration

    PubMed Central

    2011-01-01

    Background Most agronomic plant traits result from complex molecular networks involving multiple genes and from environmental factors. One such trait is the enzymatic discoloration of fruit and tuber tissues initiated by mechanical impact (bruising). Tuber susceptibility to bruising is a complex trait of the cultivated potato (Solanum tuberosum) that is crucial for crop quality. As phenotypic evaluation of bruising is cumbersome, the application of diagnostic molecular markers would empower the selection of low bruising potato varieties. The genetic factors and molecular networks underlying enzymatic tissue discoloration are sparsely known. Hitherto there is no association study dealing with tuber bruising and diagnostic markers for enzymatic discoloration are rare. Results The natural genetic diversity for bruising susceptibility was evaluated in elite middle European potato germplasm in order to elucidate its molecular basis. Association genetics using a candidate gene approach identified allelic variants in genes that function in tuber bruising and enzymatic browning. Two hundred and five tetraploid potato varieties and breeding clones related by descent were evaluated for two years in six environments for tuber bruising susceptibility, specific gravity, yield, shape and plant maturity. Correlations were found between different traits. In total 362 polymorphic DNA fragments, derived from 33 candidate genes and 29 SSR loci, were scored in the population and tested for association with the traits using a mixed model approach, which takes into account population structure and kinship. Twenty one highly significant (p < 0.001) and robust marker-trait associations were identified. Conclusions The observed trait correlations and associated marker fragments provide new insight in the molecular basis of bruising susceptibility and its natural variation. The markers diagnostic for increased or decreased bruising susceptibility will facilitate the combination of superior

  14. Genome-wide analysis of Pax8 binding provides new insights into thyroid functions

    PubMed Central

    2012-01-01

    Background The transcription factor Pax8 is essential for the differentiation of thyroid cells. However, there are few data on genes transcriptionally regulated by Pax8 other than thyroid-related genes. To better understand the role of Pax8 in the biology of thyroid cells, we obtained transcriptional profiles of Pax8-silenced PCCl3 thyroid cells using whole genome expression arrays and integrated these signals with global cis-regulatory sequencing studies performed by ChIP-Seq analysis Results Exhaustive analysis of Pax8 immunoprecipitated peaks demonstrated preferential binding to intragenic regions and CpG-enriched islands, which suggests a role of Pax8 in transcriptional regulation of orphan CpG regions. In addition, ChIP-Seq allowed us to identify Pax8 partners, including proteins involved in tertiary DNA structure (CTCF) and chromatin remodeling (Sp1), and these direct transcriptional interactions were confirmed in vivo. Moreover, both factors modulate Pax8-dependent transcriptional activation of the sodium iodide symporter (Nis) gene promoter. We ultimately combined putative and novel Pax8 binding sites with actual target gene expression regulation to define Pax8-dependent genes. Functional classification suggests that Pax8-regulated genes may be directly involved in important processes of thyroid cell function such as cell proliferation and differentiation, apoptosis, cell polarity, motion and adhesion, and a plethora of DNA/protein-related processes. Conclusion Our study provides novel insights into the role of Pax8 in thyroid biology, exerted through transcriptional regulation of important genes involved in critical thyrocyte processes. In addition, we found new transcriptional partners of Pax8, which functionally cooperate with Pax8 in the regulation of thyroid gene transcription. Besides, our data demonstrate preferential location of Pax8 in non-promoter CpG regions. These data point to an orphan CpG island-mediated mechanism that represents a novel role

  15. Proteomic analysis of FUS interacting proteins provides insights into FUS function and its role in ALS.

    PubMed

    Kamelgarn, Marisa; Chen, Jing; Kuang, Lisha; Arenas, Alexandra; Zhai, Jianjun; Zhu, Haining; Gal, Jozsef

    2016-10-01

    Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease. Mutations in the Fused in Sarcoma/Translocated in Liposarcoma (FUS/TLS) gene cause a subset of familial ALS cases and are also implicated in sporadic ALS. FUS is typically localized to the nucleus. The ALS-related FUS mutations cause cytoplasmic mis-localization and the formation of stress granule-like structures. Abnormal cytoplasmic FUS localization was also found in a subset of frontotemporal dementia (FTLD) cases without FUS mutations. To better understand the function of FUS, we performed wild-type and mutant FUS pull-downs followed by proteomic identification of the interacting proteins. The FUS interacting partners we identified are involved in multiple pathways, including chromosomal organization, transcription, RNA splicing, RNA transport, localized translation, and stress response. FUS interacted with hnRNPA1 and Matrin-3, RNA binding proteins whose mutations were also reported to cause familial ALS, suggesting that hnRNPA1 and Matrin-3 may play common pathogenic roles with FUS. The FUS interactions displayed varied RNA dependence. Numerous FUS interacting partners that we identified are components of exosomes. We found that FUS itself was present in exosomes, suggesting that the secretion of FUS might contribute to the cell-to-cell spreading of FUS pathology. FUS interacting proteins were sequestered into the cytoplasmic mutant FUS inclusions that could lead to their mis-regulation or loss of function, contributing to ALS pathogenesis. Our results provide insights into the physiological functions of FUS as well as important pathways where mutant FUS can interfere with cellular processes and potentially contribute to the pathogenesis of ALS.

  16. BK channel activation: structural and functional insights

    PubMed Central

    Lee, Urvi S.; Cui, Jianmin

    2010-01-01

    The voltage and Ca2+ activated K+ (BK) channels are involved in the regulation of neurotransmitter release and neuronal excitability. Structurally, BK channels are homologous to voltage- and ligand-gated K+ channels, having a voltage sensor and pore as the membrane-spanning domain and a cytosolic domain containing metal binding sites. Recently published electron cryomicroscopy (cryo-EM) and X-ray crystallographic structures of the BK channel provided the first look into the assembly of these domains, corroborating the close interactions among these domains during channel gating that have been suggested by functional studies. This review discusses these latest findings and an emerging new understanding about BK channel gating and implications for diseases such as epilepsy, in which mutations in BK channel genes have been associated. PMID:20663573

  17. Structural and functional insights into asymmetric enzymatic dehydration of alkenols.

    PubMed

    Nestl, Bettina M; Geinitz, Christopher; Popa, Stephanie; Rizek, Sari; Haselbeck, Robert J; Stephen, Rosary; Noble, Michael A; Fischer, Max-Philipp; Ralph, Erik C; Hau, Hoi Ting; Man, Henry; Omar, Muhiadin; Turkenburg, Johan P; van Dien, Stephen; Culler, Stephanie J; Grogan, Gideon; Hauer, Bernhard

    2017-03-01

    The asymmetric dehydration of alcohols is an important process for the direct synthesis of alkenes. We report the structure and substrate specificity of the bifunctional linalool dehydratase isomerase (LinD) from the bacterium Castellaniella defragrans that catalyzes in nature the hydration of β-myrcene to linalool and the subsequent isomerization to geraniol. Enzymatic kinetic resolutions of truncated and elongated aromatic and aliphatic tertiary alcohols (C5-C15) that contain a specific signature motif demonstrate the broad substrate specificity of LinD. The three-dimensional structure of LinD from Castellaniella defragrans revealed a pentamer with active sites at the protomer interfaces. Furthermore, the structure of LinD in complex with the product geraniol provides initial mechanistic insights into this bifunctional enzyme. Site-directed mutagenesis confirmed active site amino acid residues essential for its dehydration and isomerization activity. These structural and mechanistic insights facilitate the development of hydrating catalysts, enriching the toolbox for novel bond-forming biocatalysis.

  18. VET Provider Market Structures: History, Growth and Change. Research Report

    ERIC Educational Resources Information Center

    Korbel, Patrick; Misko, Josie

    2016-01-01

    This paper tracks the development of the Australian vocational education and training (VET) provider market over the last two decades in the context of significant policy changes and generally increased competition. It provides an insight into how the sector has arrived at its current position, painting a present-day picture of great diversity.…

  19. Structural Insights into the Mechanism of PEPCK Catalysis

    SciTech Connect

    Holyoak,T.; Sullivan, S.; Nowak, T.

    2006-01-01

    Phosphoenolpyruvate carboxykinase catalyzes the reversible decarboxylation of oxaloacetic acid with the concomitant transfer of the {gamma}-phosphate of GTP to form PEP and GDP as the first committed step of gluconeogenesis and glyceroneogenesis. The three structures of the mitochondrial isoform of PEPCK reported are complexed with Mn{sup 2+}, Mn{sup 2+}-PEP, or Mn{sup 2+}-malonate-Mn{sup 2+}GDP and provide the first observations of the structure of the mitochondrial isoform and insight into the mechanism of catalysis mediated by this enzyme. The structures show the involvement of the hyper-reactive cysteine (C307) in the coordination of the active site Mn{sup 2+}. Upon formation of the PEPCK-Mn{sup 2+}-PEP or PEPCK-Mn{sup 2+}-malonate-Mn{sup 2+}GDP complexes, C307 coordination is lost as the P-loop in which it resides adopts a different conformation. The structures suggest that stabilization of the cysteine-coordinated metal geometry holds the enzyme as a catalytically incompetent metal complex and may represent a previously unappreciated mechanism of regulation. A third conformation of the mobile P-loop in the PEPCK-Mn{sup 2+}-malonate-Mn{sup 2+}GDP complex demonstrates the participation of a previously unrecognized, conserved serine residue (S305) in mediating phosphoryl transfer. The ordering of the mobile active site lid in the PEPCK-Mn{sup 2+}-malonate-Mn{sup 2+}GDP complex yields the first observation of this structural feature and provides additional insight into the mechanism of phosphoryl transfer.

  20. Structure-function insights into prokaryotic and eukaryotic translation initiation.

    PubMed

    Myasnikov, Alexander G; Simonetti, Angelita; Marzi, Stefano; Klaholz, Bruno P

    2009-06-01

    Translation initiation is the rate-limiting and most complexly regulated step of protein synthesis in prokaryotes and eukaryotes. In the last few years, cryo-electron microscopy has provided several novel insights into the universal process of translation initiation. Structures of prokaryotic 30S and 70S ribosomal initiation complexes with initiator transfer RNA (tRNA), messenger RNA (mRNA), and initiation factors have recently revealed the mechanism of initiator tRNA recruitment to the assembling ribosomal machinery, involving molecular rearrangements of the ribosome and associated factors. First three-dimensional pictures of the particularly complex eukaryotic translation initiation machinery have been obtained, revealing how initiation factors tune the ribosome for recruiting the mRNA. A comparison of the available prokaryotic and eukaryotic structures shows that--besides significant differences--some key ribosomal features are universally conserved.

  1. Structural insights into Bacillus thuringiensis Cry, Cyt and parasporin toxins.

    PubMed

    Xu, Chengchen; Wang, Bi-Cheng; Yu, Ziniu; Sun, Ming

    2014-09-16

    Since the first X-ray structure of Cry3Aa was revealed in 1991, numerous structures of B. thuringiensis toxins have been determined and published. In recent years, functional studies on the mode of action and resistance mechanism have been proposed, which notably promoted the developments of biological insecticides and insect-resistant transgenic crops. With the exploration of known pore-forming toxins (PFTs) structures, similarities between PFTs and B. thuringiensis toxins have provided great insights into receptor binding interactions and conformational changes from water-soluble to membrane pore-forming state of B. thuringiensis toxins. This review mainly focuses on the latest discoveries of the toxin working mechanism, with the emphasis on structural related progress. Based on the structural features, B. thuringiensis Cry, Cyt and parasporin toxins could be divided into three categories: three-domain type α-PFTs, Cyt toxin type β-PFTs and aerolysin type β-PFTs. Structures from each group are elucidated and discussed in relation to the latest data, respectively.

  2. Structural Insights into Bacillus thuringiensis Cry, Cyt and Parasporin Toxins

    PubMed Central

    Xu, Chengchen; Wang, Bi-Cheng; Yu, Ziniu; Sun, Ming

    2014-01-01

    Since the first X-ray structure of Cry3Aa was revealed in 1991, numerous structures of B. thuringiensis toxins have been determined and published. In recent years, functional studies on the mode of action and resistance mechanism have been proposed, which notably promoted the developments of biological insecticides and insect-resistant transgenic crops. With the exploration of known pore-forming toxins (PFTs) structures, similarities between PFTs and B. thuringiensis toxins have provided great insights into receptor binding interactions and conformational changes from water-soluble to membrane pore-forming state of B. thuringiensis toxins. This review mainly focuses on the latest discoveries of the toxin working mechanism, with the emphasis on structural related progress. Based on the structural features, B. thuringiensis Cry, Cyt and parasporin toxins could be divided into three categories: three-domain type α-PFTs, Cyt toxin type β-PFTs and aerolysin type β-PFTs. Structures from each group are elucidated and discussed in relation to the latest data, respectively. PMID:25229189

  3. Comparison of the Internal Dynamics of Metalloproteases Provides New Insights on Their Function and Evolution

    PubMed Central

    Carvalho, Henrique F.; Roque, Ana C. A.; Iranzo, Olga; Branco, Ricardo J. F.

    2015-01-01

    Metalloproteases have evolved in a vast number of biological systems, being one of the most diverse types of proteases and presenting a wide range of folds and catalytic metal ions. Given the increasing understanding of protein internal dynamics and its role in enzyme function, we are interested in assessing how the structural heterogeneity of metalloproteases translates into their dynamics. Therefore, the dynamical profile of the clan MA type protein thermolysin, derived from an Elastic Network Model of protein structure, was evaluated against those obtained from a set of experimental structures and molecular dynamics simulation trajectories. A close correspondence was obtained between modes derived from the coarse-grained model and the subspace of functionally-relevant motions observed experimentally, the later being shown to be encoded in the internal dynamics of the protein. This prompted the use of dynamics-based comparison methods that employ such coarse-grained models in a representative set of clan members, allowing for its quantitative description in terms of structural and dynamical variability. Although members show structural similarity, they nonetheless present distinct dynamical profiles, with no apparent correlation between structural and dynamical relatedness. However, previously unnoticed dynamical similarity was found between the relevant members Carboxypeptidase Pfu, Leishmanolysin, and Botulinum Neurotoxin Type A, despite sharing no structural similarity. Inspection of the respective alignments shows that dynamical similarity has a functional basis, namely the need for maintaining proper intermolecular interactions with the respective substrates. These results suggest that distinct selective pressure mechanisms act on metalloproteases at structural and dynamical levels through the course of their evolution. This work shows how new insights on metalloprotease function and evolution can be assessed with comparison schemes that incorporate

  4. The African turquoise killifish genome provides insights into evolution and genetic architecture of lifespan

    PubMed Central

    Valenzano, Dario Riccardo; Benayoun, Bérénice A.; Singh, Param Priya; Zhang, Elisa; Etter, Paul D.; Hu, Chi-Kuo; Clément-Ziza, Mathieu; Willemsen, David; Cui, Rongfeng; Harel, Itamar; Machado, Ben E.; Yee, Muh-Ching; Sharp, Sabrina C.; Bustamante, Carlos D.; Beyer, Andreas; Johnson, Eric A.; Brunet, Anne

    2015-01-01

    Summary Lifespan is a remarkably diverse trait ranging from a few days to several hundred years in nature, but the mechanisms underlying the evolution of lifespan differences remain elusive. Here we de novo assemble a reference genome for the naturally short-lived African turquoise killifish, providing a unique resource for comparative and experimental genomics. The identification of genes under positive selection in this fish reveals potential candidates to explain its compressed lifespan. Several aging genes are under positive selection in this short-lived fish and long-lived species, raising the intriguing possibility that the same gene could underlie evolution of both compressed and extended lifespans. Comparative genomics and linkage analysis identify candidate genes associated with lifespan differences between various turquoise killifish strains. Remarkably, these genes are clustered on the sex chromosome, suggesting that short lifespan might have co-evolved with sex determination. Our study provides insights into the evolutionary forces that shape lifespan in nature. PMID:26638078

  5. Mechanisms of bacterial morphogenesis: evolutionary cell biology approaches provide new insights.

    PubMed

    Jiang, Chao; Caccamo, Paul D; Brun, Yves V

    2015-04-01

    How Darwin's "endless forms most beautiful" have evolved remains one of the most exciting questions in biology. The significant variety of bacterial shapes is most likely due to the specific advantages they confer with respect to the diverse environments they occupy. While our understanding of the mechanisms generating relatively simple shapes has improved tremendously in the last few years, the molecular mechanisms underlying the generation of complex shapes and the evolution of shape diversity are largely unknown. The emerging field of bacterial evolutionary cell biology provides a novel strategy to answer this question in a comparative phylogenetic framework. This relatively novel approach provides hypotheses and insights into cell biological mechanisms, such as morphogenesis, and their evolution that would have been difficult to obtain by studying only model organisms. We discuss the necessary steps, challenges, and impact of integrating "evolutionary thinking" into bacterial cell biology in the genomic era.

  6. Mechanisms of bacterial morphogenesis: Evolutionary cell biology approaches provide new insights

    PubMed Central

    Jiang, Chao; Caccamo, Paul D.; Brun, Yves V.

    2015-01-01

    How Darwin’s “endless forms most beautiful” have evolved remains one of the most exciting questions in biology. The significant variety of bacterial shapes is most likely due to the specific advantages they confer with respect to the diverse environments they occupy. While our understanding of the mechanisms generating relatively simple shapes has improved tremendously in the last few years, the molecular mechanisms underlying the generation of complex shapes and the evolution of shape diversity are largely unknown. The emerging field of bacterial evolutionary cell biology provides a novel strategy to answer this question in a comparative phylogenetic framework. This relatively novel approach provides hypotheses and insights into cell biological mechanisms, such as morphogenesis, and their evolution that would have been difficult to obtain by studying only model organisms. We discuss the necessary steps, challenges, and impact of integrating “evolutionary thinking” into bacterial cell biology in the genomic era. PMID:25664446

  7. The African Turquoise Killifish Genome Provides Insights into Evolution and Genetic Architecture of Lifespan.

    PubMed

    Valenzano, Dario Riccardo; Benayoun, Bérénice A; Singh, Param Priya; Zhang, Elisa; Etter, Paul D; Hu, Chi-Kuo; Clément-Ziza, Mathieu; Willemsen, David; Cui, Rongfeng; Harel, Itamar; Machado, Ben E; Yee, Muh-Ching; Sharp, Sabrina C; Bustamante, Carlos D; Beyer, Andreas; Johnson, Eric A; Brunet, Anne

    2015-12-03

    Lifespan is a remarkably diverse trait ranging from a few days to several hundred years in nature, but the mechanisms underlying the evolution of lifespan differences remain elusive. Here we de novo assemble a reference genome for the naturally short-lived African turquoise killifish, providing a unique resource for comparative and experimental genomics. The identification of genes under positive selection in this fish reveals potential candidates to explain its compressed lifespan. Several aging genes are under positive selection in this short-lived fish and long-lived species, raising the intriguing possibility that the same gene could underlie evolution of both compressed and extended lifespans. Comparative genomics and linkage analysis identify candidate genes associated with lifespan differences between various turquoise killifish strains. Remarkably, these genes are clustered on the sex chromosome, suggesting that short lifespan might have co-evolved with sex determination. Our study provides insights into the evolutionary forces that shape lifespan in nature.

  8. Structure Prediction: New Insights into Decrypting Long Noncoding RNAs

    PubMed Central

    Yan, Kun; Arfat, Yasir; Li, Dijie; Zhao, Fan; Chen, Zhihao; Yin, Chong; Sun, Yulong; Hu, Lifang; Yang, Tuanmin; Qian, Airong

    2016-01-01

    Long noncoding RNAs (lncRNAs), which form a diverse class of RNAs, remain the least understood type of noncoding RNAs in terms of their nature and identification. Emerging evidence has revealed that a small number of newly discovered lncRNAs perform important and complex biological functions such as dosage compensation, chromatin regulation, genomic imprinting, and nuclear organization. However, understanding the wide range of functions of lncRNAs related to various processes of cellular networks remains a great experimental challenge. Structural versatility is critical for RNAs to perform various functions and provides new insights into probing the functions of lncRNAs. In recent years, the computational method of RNA structure prediction has been developed to analyze the structure of lncRNAs. This novel methodology has provided basic but indispensable information for the rapid, large-scale and in-depth research of lncRNAs. This review focuses on mainstream RNA structure prediction methods at the secondary and tertiary levels to offer an additional approach to investigating the functions of lncRNAs. PMID:26805815

  9. Quantitative measures of walking and strength provide insight into brain corticospinal tract pathology in multiple sclerosis.

    PubMed

    Fritz, Nora E; Keller, Jennifer; Calabresi, Peter A; Zackowski, Kathleen M

    2017-01-01

    At least 85% of individuals with multiple sclerosis report walking dysfunction as their primary complaint. Walking and strength measures are common clinical measures to mark increasing disability or improvement with rehabilitation. Previous studies have shown an association between strength or walking ability and spinal cord MRI measures, and strength measures with brainstem corticospinal tract magnetization transfer ratio. However, the relationship between walking performance and brain corticospinal tract magnetization transfer imaging measures and the contribution of clinical measurements of walking and strength to the underlying integrity of the corticospinal tract has not been explored in multiple sclerosis. The objectives of this study were explore the relationship of quantitative measures of walking and strength to whole-brain corticospinal tract-specific MRI measures and to determine the contribution of quantitative measures of function in addition to basic clinical measures (age, gender, symptom duration and Expanded Disability Status Scale) to structural imaging measures of the corticospinal tract. We hypothesized that quantitative walking and strength measures would be related to brain corticospinal tract-specific measures, and would provide insight into the heterogeneity of brain pathology. Twenty-nine individuals with relapsing-remitting multiple sclerosis (mean(SD) age 48.7 (11.5) years; symptom duration 11.9(8.7); 17 females; median[range] Expanded Disability Status Scale 4.0 [1.0-6.5]) and 29 age and gender-matched healthy controls (age 50.8(11.6) years; 20 females) participated in clinical tests of strength and walking (Timed Up and Go, Timed 25 Foot Walk, Two Minute Walk Test ) as well as 3 T imaging including diffusion tensor imaging and magnetization transfer imaging. Individuals with multiple sclerosis were weaker (p = 0.0024) and walked slower (p = 0.0013) compared to controls. Quantitative measures of walking and strength were

  10. Supramolecular polymerization of a prebiotic nucleoside provides insights into the creation of sequence-controlled polymers

    NASA Astrophysics Data System (ADS)

    Wang, Jun; Bonnesen, Peter V.; Rangel, E.; Vallejo, E.; Sanchez-Castillo, Ariadna; James Cleaves, H., II; Baddorf, Arthur P.; Sumpter, Bobby G.; Pan, Minghu; Maksymovych, Petro; Fuentes-Cabrera, Miguel

    2016-01-01

    Self-assembly of a nucleoside on Au(111) was studied to ascertain whether polymerization on well-defined substrates constitutes a promising approach for making sequence-controlled polymers. Scanning tunneling microscopy and density functional theory were used to investigate the self-assembly on Au(111) of (RS)-N9-(2,3-dihydroxypropyl)adenine (DHPA), a plausibly prebiotic nucleoside analog of adenosine. It is found that DHPA molecules self-assemble into a hydrogen-bonded polymer that grows almost exclusively along the herringbone reconstruction pattern, has a two component sequence that is repeated over hundreds of nanometers, and is erasable with electron-induced excitation. Although the sequence is simple, more complicated ones are envisioned if two or more nucleoside types are combined. Because polymerization occurs on a substrate in a dry environment, the success of each combination can be gauged with high-resolution imaging and accurate modeling techniques. These characteristics make nucleoside self-assembly on a substrate an attractive approach for designing sequence-controlled polymers. Further, by choosing plausibly prebiotic nucleosides, insights may be provided into how nature created the first sequence-controlled polymers capable of storing information. Such insights, in turn, can inspire new ways of synthesizing sequence-controlled polymers.

  11. Supramolecular polymerization of a prebiotic nucleoside provides insights into the creation of sequence-controlled polymers

    PubMed Central

    Wang, Jun; Bonnesen, Peter V.; Rangel, E.; Vallejo, E.; Sanchez-Castillo, Ariadna; James Cleaves II, H.; Baddorf, Arthur P.; Sumpter, Bobby G.; Pan, Minghu; Maksymovych, Petro; Fuentes-Cabrera, Miguel

    2016-01-01

    Self-assembly of a nucleoside on Au(111) was studied to ascertain whether polymerization on well-defined substrates constitutes a promising approach for making sequence-controlled polymers. Scanning tunneling microscopy and density functional theory were used to investigate the self-assembly on Au(111) of (RS)-N9-(2,3-dihydroxypropyl)adenine (DHPA), a plausibly prebiotic nucleoside analog of adenosine. It is found that DHPA molecules self-assemble into a hydrogen-bonded polymer that grows almost exclusively along the herringbone reconstruction pattern, has a two component sequence that is repeated over hundreds of nanometers, and is erasable with electron-induced excitation. Although the sequence is simple, more complicated ones are envisioned if two or more nucleoside types are combined. Because polymerization occurs on a substrate in a dry environment, the success of each combination can be gauged with high-resolution imaging and accurate modeling techniques. These characteristics make nucleoside self-assembly on a substrate an attractive approach for designing sequence-controlled polymers. Further, by choosing plausibly prebiotic nucleosides, insights may be provided into how nature created the first sequence-controlled polymers capable of storing information. Such insights, in turn, can inspire new ways of synthesizing sequence-controlled polymers. PMID:26725380

  12. Supramolecular polymerization of a prebiotic nucleoside provides insights into the creation of sequence-controlled polymers

    SciTech Connect

    Wang, Jun; Bonnesen, Peter V; Rangel, E.; Vallejo, E.; Sanchez-Castillo, Ariadna; Cleaves, II, H. James; Baddorf, Arthur P; Sumpter, Bobby G; Pan, Minghu; Maksymovych, Petro; Fuentes-Cabrera, Miguel A

    2016-01-04

    The self-assembly of a nucleoside on Au(111) was studied to ascertain whether polymerization on well-defined substrates constitutes a promising approach for making sequence-controlled polymers. Scanning tunneling microscopy and density functional theory were used to investigate the self-assembly on Au(111) of (RS)-N9-(2,3-dihydroxypropyl)adenine (DHPA), a plausibly prebiotic nucleoside analog of adenosine. It is found that DHPA molecules self-assemble into a hydrogen-bonded polymer that grows almost exclusively along the herringbone reconstruction pattern, has a two component sequence that is repeated over hundreds of nanometers, and is erasable with electron-induced excitation. Although the sequence is simple, more complicated ones are envisioned if two or more nucleoside types are combined. Because polymerization occurs on a substrate in a dry environment, the success of each combination can be gauged with high-resolution imaging and accurate modeling techniques. The resulting characteristics make nucleoside self-assembly on a substrate an attractive approach for designing sequence-controlled polymers. Moreover, by choosing plausibly prebiotic nucleosides, insights may be provided into how nature created the first sequence-controlled polymers capable of storing information. Such insights, in turn, can inspire new ways of synthesizing sequence-controlled polymers.

  13. Supramolecular polymerization of a prebiotic nucleoside provides insights into the creation of sequence-controlled polymers

    DOE PAGES

    Wang, Jun; Bonnesen, Peter V; Rangel, E.; ...

    2016-01-04

    The self-assembly of a nucleoside on Au(111) was studied to ascertain whether polymerization on well-defined substrates constitutes a promising approach for making sequence-controlled polymers. Scanning tunneling microscopy and density functional theory were used to investigate the self-assembly on Au(111) of (RS)-N9-(2,3-dihydroxypropyl)adenine (DHPA), a plausibly prebiotic nucleoside analog of adenosine. It is found that DHPA molecules self-assemble into a hydrogen-bonded polymer that grows almost exclusively along the herringbone reconstruction pattern, has a two component sequence that is repeated over hundreds of nanometers, and is erasable with electron-induced excitation. Although the sequence is simple, more complicated ones are envisioned if two ormore » more nucleoside types are combined. Because polymerization occurs on a substrate in a dry environment, the success of each combination can be gauged with high-resolution imaging and accurate modeling techniques. The resulting characteristics make nucleoside self-assembly on a substrate an attractive approach for designing sequence-controlled polymers. Moreover, by choosing plausibly prebiotic nucleosides, insights may be provided into how nature created the first sequence-controlled polymers capable of storing information. Such insights, in turn, can inspire new ways of synthesizing sequence-controlled polymers.« less

  14. Basic Science Simulations Provide New Insights to Aid Hydrogen Gas Turbine Development (Fact Sheet), NREL Highlights, Science

    SciTech Connect

    Not Available

    2011-11-01

    Massive first-principles simulation provides insight into flame anchoring in a hydrogen-rich jet in cross-flow. When gas turbine designers want to use gasified biomass for stationary power generation, they are faced with a challenge: bio-derived syngas typically contains significant amounts of hydrogen, which is far more reactive than the methane that is the traditional gas turbine fuel. This reactivity leads to a safety design issue, because with hydrogen-rich fuels a flame may anchor in the fuel injection section of the combustor instead of the downstream design point. In collaboration with Jacqueline Chen of Sandia National Laboratories and Andrea Gruber of SINTEF, a Norwegian energy think tank, the National Renewable Energy Laboratory (NREL) is carrying out fundamental simulations to provide new insight into the physics of flame anchoring in canonical 'jet in cross-flow' configurations using hydrogen-rich fuels. To deal with the large amount and complexity of the data, the combustion scientists also teamed up with computer scientists from across the U.S. Department of Energy's laboratories to develop novel ways to analyze the data. These simulations have shown that fine-scale turbulence structures formed at the jet boundary provide particularly intense mixing between the fuel and air, which then enters a quiescent region formed downstream of the jet in a separate, larger turbulent structure. This insight explains the effect that reducing the wall-normal velocity of the fuel jet causes the flame to blow off; with the aid of the simulation, we now understand this counterintuitive result because reducing the wall-normal velocity would reduce the intensity of the mixing as well as move the quiescent region farther downstream. NREL and its research partners are conducting simulations that provide new insight into the physics of flame anchoring in canonical 'jet in cross-flow' configurations using hydrogen-rich fuels. Simulation results explain the mechanism behind

  15. Structural Insights on the Mycobacterium tuberculosis Proteasomal ATPase Mpa

    SciTech Connect

    Wang, T.; Li, H; Lin, G; Tang, C; Li, D; Nathan, C; Heran Darwin, K

    2009-01-01

    Proteasome-mediated protein turnover in all domains of life is an energy-dependent process that requires ATPase activity. Mycobacterium tuberculosis (Mtb) was recently shown to possess a ubiquitin-like proteasome pathway that plays an essential role in Mtb resistance to killing by products of host macrophages. Here we report our structural and biochemical investigation of Mpa, the presumptive Mtb proteasomal ATPase. We demonstrate that Mpa binds to the Mtb proteasome in the presence of ATPS, providing the physical evidence that Mpa is the proteasomal ATPase. X-ray crystallographic determination of the conserved interdomain showed a five stranded double {beta} barrel structure containing a Greek key motif. Structure and mutational analysis indicate a major role of the interdomain for Mpa hexamerization. Our mutational and functional studies further suggest that the central channel in the Mpa hexamer is involved in protein substrate translocation and degradation. These studies provide insights into how a bacterial proteasomal ATPase interacts with and facilitates protein degradation by the proteasome.

  16. The Genome of Undifilum oxytropis Provides Insights into Swainsonine Biosynthesis and Locoism

    PubMed Central

    Lu, Hao; Quan, Haiyun; Ren, Zhenhui; Wang, Shuai; Xue, Ruixu; Zhao, Baoyu

    2016-01-01

    Undifilum oxytropis is a fungal endophyte of locoweeds. It produces swainsonine, which is the principal toxic ingredient of locoweeds. However, the genes, pathways and mechanisms of swainsonine biosynthesis are not known. In this study, the genome of U. oxytropis was firstly sequenced and assembled into a 70.05 megabases (Mb) draft genome, which encoded 11,057 protein-coding genes, and 54% of them were similar to current publicly available sequences. U. oxytropis genes were annotated and 164 putative genes were annotated into enzymes, such as Saccharopine dehydrogenase, Saccharopine oxidase, and Pyrroline-5-carboxylate reductase, hypothesized to be involved in the biosynthesis pathway of swainsonine. The genome sequence and gene annotation of U. oxytropis will provide new insights into functional analyses. The characterization of genes in swainsonine biosynthesis will greatly facilitate locoweed poisoning research and help direct locoism management. PMID:27477109

  17. The genome and transcriptome of Japanese flounder provide insights into flatfish asymmetry.

    PubMed

    Shao, Changwei; Bao, Baolong; Xie, Zhiyuan; Chen, Xinye; Li, Bo; Jia, Xiaodong; Yao, Qiulin; Ortí, Guillermo; Li, Wenhui; Li, Xihong; Hamre, Kristin; Xu, Juan; Wang, Lei; Chen, Fangyuan; Tian, Yongsheng; Schreiber, Alex M; Wang, Na; Wei, Fen; Zhang, Jilin; Dong, Zhongdian; Gao, Lei; Gai, Junwei; Sakamoto, Takashi; Mo, Sudong; Chen, Wenjun; Shi, Qiong; Li, Hui; Xiu, Yunji; Li, Yangzhen; Xu, Wenteng; Shi, Zhiyi; Zhang, Guojie; Power, Deborah M; Wang, Qingyin; Schartl, Manfred; Chen, Songlin

    2017-01-01

    Flatfish have the most extreme asymmetric body morphology of vertebrates. During metamorphosis, one eye migrates to the contralateral side of the skull, and this migration is accompanied by extensive craniofacial transformations and simultaneous development of lopsided body pigmentation. The evolution of this developmental and physiological innovation remains enigmatic. Comparative genomics of two flatfish and transcriptomic analyses during metamorphosis point to a role for thyroid hormone and retinoic acid signaling, as well as phototransduction pathways. We demonstrate that retinoic acid is critical in establishing asymmetric pigmentation and, via cross-talk with thyroid hormones, in modulating eye migration. The unexpected expression of the visual opsins from the phototransduction pathway in the skin translates illumination differences and generates retinoic acid gradients that underlie the generation of asymmetry. Identifying the genetic underpinning of this unique developmental process answers long-standing questions about the evolutionary origin of asymmetry, but it also provides insight into the mechanisms that control body shape in vertebrates.

  18. Genomic analyses of primitive, wild and cultivated citrus provide insights into asexual reproduction.

    PubMed

    Wang, Xia; Xu, Yuantao; Zhang, Siqi; Cao, Li; Huang, Yue; Cheng, Junfeng; Wu, Guizhi; Tian, Shilin; Chen, Chunli; Liu, Yan; Yu, Huiwen; Yang, Xiaoming; Lan, Hong; Wang, Nan; Wang, Lun; Xu, Jidi; Jiang, Xiaolin; Xie, Zongzhou; Tan, Meilian; Larkin, Robert M; Chen, Ling-Ling; Ma, Bin-Guang; Ruan, Yijun; Deng, Xiuxin; Xu, Qiang

    2017-04-10

    The emergence of apomixis-the transition from sexual to asexual reproduction-is a prominent feature of modern citrus. Here we de novo sequenced and comprehensively studied the genomes of four representative citrus species. Additionally, we sequenced 100 accessions of primitive, wild and cultivated citrus. Comparative population analysis suggested that genomic regions harboring energy- and reproduction-associated genes are probably under selection in cultivated citrus. We also narrowed the genetic locus responsible for citrus polyembryony, a form of apomixis, to an 80-kb region containing 11 candidate genes. One of these, CitRWP, is expressed at higher levels in ovules of polyembryonic cultivars. We found a miniature inverted-repeat transposable element insertion in the promoter region of CitRWP that cosegregated with polyembryony. This study provides new insights into citrus apomixis and constitutes a promising resource for the mining of agriculturally important genes.

  19. The Lingula genome provides insights into brachiopod evolution and the origin of phosphate biomineralization

    PubMed Central

    Luo, Yi-Jyun; Takeuchi, Takeshi; Koyanagi, Ryo; Yamada, Lixy; Kanda, Miyuki; Khalturina, Mariia; Fujie, Manabu; Yamasaki, Shin-ichi; Endo, Kazuyoshi; Satoh, Noriyuki

    2015-01-01

    The evolutionary origins of lingulid brachiopods and their calcium phosphate shells have been obscure. Here we decode the 425-Mb genome of Lingula anatina to gain insights into brachiopod evolution. Comprehensive phylogenomic analyses place Lingula close to molluscs, but distant from annelids. The Lingula gene number has increased to ∼34,000 by extensive expansion of gene families. Although Lingula and vertebrates have superficially similar hard tissue components, our genomic, transcriptomic and proteomic analyses show that Lingula lacks genes involved in bone formation, indicating an independent origin of their phosphate biominerals. Several genes involved in Lingula shell formation are shared by molluscs. However, Lingula has independently undergone domain combinations to produce shell matrix collagens with EGF domains and carries lineage-specific shell matrix proteins. Gene family expansion, domain shuffling and co-option of genes appear to be the genomic background of Lingula's unique biomineralization. This Lingula genome provides resources for further studies of lophotrochozoan evolution. PMID:26383154

  20. Food allergy: Insights into etiology, prevention and treatment provided by murine models

    PubMed Central

    Oyoshi, Michiko K.; Oettgen, Hans C.; Chatila, Talal A.; Geha, Raif S.; Bryce, Paul J.

    2014-01-01

    Food allergy is a rapidly growing public health concern due to its increasing prevalence and its life threatening potential. Animal models of food allergy have emerged as a tool for identifying mechanisms involved in the development of sensitization to normally harmless food allergens as well as delineating the critical immune components of the effector phase of allergic reactions to food. However, the role animal models might play in understanding human diseases remain contentious. This review summarizes how animal models have provided insights on the etiology of human food allergy, experimental corroboration for epidemiological findings that might facilitate prevention strategies, and validation for the utility of new therapies for food allergy. Improved understanding of food allergy from the study of animal models together with human studies are likely to contribute to the development of novel strategies to prevent and treat food allergy. PMID:24636470

  1. Whole Genome Analysis of Leptospira licerasiae Provides Insight into Leptospiral Evolution and Pathogenicity

    PubMed Central

    Selengut, Jeremy D.; Harkins, Derek M.; Patra, Kailash P.; Moreno, Angelo; Lehmann, Jason S.; Purushe, Janaki; Sanka, Ravi; Torres, Michael; Webster, Nicholas J.; Vinetz, Joseph M.; Matthias, Michael A.

    2012-01-01

    The whole genome analysis of two strains of the first intermediately pathogenic leptospiral species to be sequenced (Leptospira licerasiae strains VAR010 and MMD0835) provides insight into their pathogenic potential and deepens our understanding of leptospiral evolution. Comparative analysis of eight leptospiral genomes shows the existence of a core leptospiral genome comprising 1547 genes and 452 conserved genes restricted to infectious species (including L. licerasiae) that are likely to be pathogenicity-related. Comparisons of the functional content of the genomes suggests that L. licerasiae retains several proteins related to nitrogen, amino acid and carbohydrate metabolism which might help to explain why these Leptospira grow well in artificial media compared with pathogenic species. L. licerasiae strains VAR010T and MMD0835 possess two prophage elements. While one element is circular and shares homology with LE1 of L. biflexa, the second is cryptic and homologous to a previously identified but unnamed region in L. interrogans serovars Copenhageni and Lai. We also report a unique O-antigen locus in L. licerasiae comprised of a 6-gene cluster that is unexpectedly short compared with L. interrogans in which analogous regions may include >90 such genes. Sequence homology searches suggest that these genes were acquired by lateral gene transfer (LGT). Furthermore, seven putative genomic islands ranging in size from 5 to 36 kb are present also suggestive of antecedent LGT. How Leptospira become naturally competent remains to be determined, but considering the phylogenetic origins of the genes comprising the O-antigen cluster and other putative laterally transferred genes, L. licerasiae must be able to exchange genetic material with non-invasive environmental bacteria. The data presented here demonstrate that L. licerasiae is genetically more closely related to pathogenic than to saprophytic Leptospira and provide insight into the genomic bases for its infectiousness

  2. Hyper-dry conditions provide new insights into the cause of extreme floods after wildfire

    USGS Publications Warehouse

    Moody, John A.; Ebel, Brian A.

    2012-01-01

    A catastrophic wildfire in the foothills of the Rocky Mountains near Boulder, Colorado provided a unique opportunity to investigate soil conditions immediately after a wildfire and before alteration by rainfall. Measurements of near-surface (θ; and matric suction, ψ), rainfall, and wind velocity were started 8 days after the wildfire began. These measurements established that hyper-dryconditions (θ 3 cm-3; ψ > ~ 3 x 105 cm) existed and provided an in-situ retention curve for these conditions. These conditions exacerbate the effects of water repellency (natural and fire-induced) and limit the effectiveness of capillarity and gravity driven infiltration into fire-affected soils. The important consequence is that given hyper-dryconditions, the critical rewetting process before the first rain is restricted to the diffusion–adsorption of water-vapor. This process typically has a time scale of days to weeks (especially when the hydrologic effects of the ash layer are included) that is longer than the typical time scale (minutes to hours) of some rainstorms, such that under hyper-dryconditions essentially no rain infiltrates. The existence of hyper-dryconditions provides insight into why, frequently during the first rain storm after a wildfire, nearly all rainfall becomes runoff causing extremefloods and debris flows.

  3. Circulating Tumor Cells and Circulating Tumor DNA Provide New Insights into Pancreatic Cancer

    PubMed Central

    Gao, Yang; Zhu, Yayun; Yuan, Zhou

    2016-01-01

    Pancreatic cancer has a rather dismal prognosis mainly due to high malignance of tumor biology. Up to now, the relevant researches on pancreatic cancer lag behind seriously partly due to the obstacles for tissue biopsy, which handicaps the understanding of molecular and genetic features of pancreatic cancer. In the last two decades, liquid biopsy, including circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), is promising to provide new insights into the biological and clinical characteristics of malignant tumors. Both CTCs and ctDNA provide an opportunity for studying tumor heterogeneity, drug resistance, and metastatic mechanism for pancreatic cancer. Furthermore, they can also play important roles in detecting early-stage tumors, providing prognostic information, monitoring tumor progression and guiding treatment regimens. In this review, we will introduce the latest findings on biological features and clinical applications of both CTCs and ctDNA in pancreatic cancer. In a word, CTCs and ctDNA are promising to promote precision medicine in pancreatic cancer. PMID:27994495

  4. Structural insights into the translational infidelity mechanism

    NASA Astrophysics Data System (ADS)

    Rozov, Alexey; Demeshkina, Natalia; Westhof, Eric; Yusupov, Marat; Yusupova, Gulnara

    2015-06-01

    The decoding of mRNA on the ribosome is the least accurate process during genetic information transfer. Here we propose a unified decoding mechanism based on 11 high-resolution X-ray structures of the 70S ribosome that explains the occurrence of missense errors during translation. We determined ribosome structures in rare states where incorrect tRNAs were incorporated into the peptidyl-tRNA-binding site. These structures show that in the codon-anticodon duplex, a G.U mismatch adopts the Watson-Crick geometry, indicating a shift in the tautomeric equilibrium or ionization of the nucleobase. Additional structures with mismatches in the 70S decoding centre show that the binding of any tRNA induces identical rearrangements in the centre, which favours either isosteric or close to the Watson-Crick geometry codon-anticodon pairs. Overall, the results suggest that a mismatch escapes discrimination by preserving the shape of a Watson-Crick pair and indicate that geometric selection via tautomerism or ionization dominates the translational infidelity mechanism.

  5. Insight into Amyloid Structure Using Chemical Probes

    PubMed Central

    Reinke, Ashley A.; Gestwicki, Jason E.

    2011-01-01

    Alzheimer’s disease (AD) is a common neurodegenerative disorder characterized by the deposition of amyloids in the brain. One prominent form of amyloid is composed of repeating units of the amyloid-β (Aβ) peptide. Over the past decade, it has become clear that these Aβ amyloids are not homogeneous; rather, they are composed of a series of structures varying in their overall size and shape and the number of Aβ peptides they contain. Recent theories suggest that these different amyloid conformations may play distinct roles in disease, although their relative contributions are still being discovered. Here, we review how chemical probes, such as congo red, thioflavin T and their derivatives, have been powerful tools for better understanding amyloid structure and function. Moreover, we discuss how design and deployment of conformationally selective probes might be used to test emerging models of AD. PMID:21457473

  6. Six decades of vitiligo genetics: genome-wide studies provide insights into autoimmune pathogenesis.

    PubMed

    Spritz, Richard A

    2012-02-01

    Generalized vitiligo (GV) is a complex disease in which patchy depigmentation results from autoimmune loss of melanocytes from affected regions. Genetic analyses of GV span six decades, with the goal of understanding biological mechanisms and elucidating pathways that underlie the disease. The earliest studies attempted to describe the mode of inheritance and genetic epidemiology. Early genetic association studies of biological candidate genes resulted in some successes, principally HLA and PTPN22, but in hindsight many such reports now seem to be false-positives. Later, genome-wide linkage studies of multiplex GV families identified NLRP1 and XBP1, which appear to be valid GV susceptibility genes that control key aspects of immune regulation. Recently, the application of genome-wide association studies to analysis of GV has produced a rich yield of validated GV susceptibility genes that encode components of biological pathways reaching from immune cells to the melanocyte. These genes and pathways provide insights into underlying pathogenetic mechanisms and possible triggers of GV, establish relationships to other autoimmune diseases, and may provide clues to potential new approaches to GV treatment and perhaps even prevention. These results thus validate the hopes and efforts of the early investigators who first attempted to comprehend the genetic basis of vitiligo.

  7. The presence of bacteria within tissue provides insights into the pathogenesis of oral lichen planus

    PubMed Central

    Choi, Yun Sik; Kim, Yunji; Yoon, Hye-Jung; Baek, Keum Jin; Alam, Jehan; Park, Hee Kyung; Choi, Youngnim

    2016-01-01

    Oral lichen planus (OLP) is a chronic T cell-mediated mucocutaneous disease of unknown etiopathogenesis. Although various antigens have been considered, what actually triggers the inflammatory response of T cells is unknown. In the present study, we propose that intracellular bacteria present within tissues trigger T cell infiltration and provide target antigens. Sections of OLP (n = 36) and normal (n = 10) oral mucosal tissues were subjected to in situ hybridization using a universal probe targeting the bacterial 16S rRNA gene and immunohistochemistry with anti-CD3, anti-CD4, anti-CD8, and anti-macrophage-specific antibodies. Bacteria were abundant throughout the epithelium and the lamina propria of OLP tissues, which exhibited positive correlations with the levels of infiltrated CD3+, CD4+, and CD8+ cells. Furthermore, bacteria were detected within the infiltrated T cells. Pyrosequencing analysis of the mucosal microbiota from OLP patients (n = 13) and control subjects (n = 11) revealed a decrease in Streptococcus and increases in gingivitis/periodontitis-associated bacteria in OLP lesions. Using the selected bacterial species, we demonstrated that certain oral bacteria damage the epithelial physical barrier, are internalized into epithelial cells or T cells, and induce production of T cell chemokines CXCL10 and CCL5. Our findings provide insights into the pathogenesis of OLP. PMID:27383402

  8. Comprehensive population-based genome sequencing provides insight into hematopoietic regulatory mechanisms

    PubMed Central

    Guo, Michael H.; Nandakumar, Satish K.; Ulirsch, Jacob C.; Zekavat, Seyedeh M.; Buenrostro, Jason D.; Natarajan, Pradeep; Salem, Rany M.; Chiarle, Roberto; Mitt, Mario; Kals, Mart; Pärn, Kalle; Fischer, Krista; Milani, Lili; Mägi, Reedik; Palta, Priit; Gabriel, Stacey B.; Metspalu, Andres; Lander, Eric S.; Kathiresan, Sekar; Hirschhorn, Joel N.; Esko, Tõnu; Sankaran, Vijay G.

    2017-01-01

    Genetic variants affecting hematopoiesis can influence commonly measured blood cell traits. To identify factors that affect hematopoiesis, we performed association studies for blood cell traits in the population-based Estonian Biobank using high-coverage whole-genome sequencing (WGS) in 2,284 samples and SNP genotyping in an additional 14,904 samples. Using up to 7,134 samples with available phenotype data, our analyses identified 17 associations across 14 blood cell traits. Integration of WGS-based fine-mapping and complementary epigenomic datasets provided evidence for causal mechanisms at several loci, including at a previously undiscovered basophil count-associated locus near the master hematopoietic transcription factor CEBPA. The fine-mapped variant at this basophil count association near CEBPA overlapped an enhancer active in common myeloid progenitors and influenced its activity. In situ perturbation of this enhancer by CRISPR/Cas9 mutagenesis in hematopoietic stem and progenitor cells demonstrated that it is necessary for and specifically regulates CEBPA expression during basophil differentiation. We additionally identified basophil count-associated variation at another more pleiotropic myeloid enhancer near GATA2, highlighting regulatory mechanisms for ordered expression of master hematopoietic regulators during lineage specification. Our study illustrates how population-based genetic studies can provide key insights into poorly understood cell differentiation processes of considerable physiologic relevance. PMID:28031487

  9. Dynamic transcriptional profiling provides insights into tuberous root development in Rehmannia glutinosa

    PubMed Central

    Sun, Peng; Xiao, Xingguo; Duan, Liusheng; Guo, Yuhai; Qi, Jianjun; Liao, Dengqun; Zhao, Chunli; Liu, Yan; Zhou, Lili; Li, Xianen

    2015-01-01

    Rehmannia glutinosa, an herb of the Scrophulariaceae family, is widely cultivated in the Northern part of China. The tuberous root has well-known medicinal properties; however, yield and quality are threatened by abiotic and biotic stresses. Understanding the molecular process of tuberous root development may help identify novel targets for its control. In the present study, we used Illumina sequencing and de novo assembly strategies to obtain a reference transcriptome that is relevant to tuberous root development. We then conducted RNA-seq quantification analysis to determine gene expression profiles of the adventitious root (AR), thickening adventitious root (TAR), and the developing tuberous root (DTR). Expression profiling identified a total of 6794 differentially expressed unigenes during root development. Bioinformatics analysis and gene expression profiling revealed changes in phenylpropanoid biosynthesis, starch and sucrose metabolism, and plant hormone biosynthesis during root development. Moreover, we identified and allocated putative functions to the genes involved in tuberous root development, including genes related to major carbohydrate metabolism, hormone metabolism, and transcription regulation. The present study provides the initial description of gene expression profiles of AR, TAR, and DTR, which facilitates identification of genes of interest. Moreover, our work provides insights into the molecular mechanisms underlying tuberous root development and may assist in the design and development of improved breeding schemes for different R. glutinosa varieties through genetic manipulation. PMID:26113849

  10. Vertebral development of modern salamanders provides insights into a unique event of their evolutionary history.

    PubMed

    Boisvert, Catherine Anne

    2009-01-15

    The origin of salamanders and their interrelationships to the two other modern amphibian orders (frogs and caecilians) are problematic owing to an 80-100 million year gap in the fossil record between the Carboniferous to the Lower Jurassic. This is compounded by a scarcity of adult skeletal characters linking the early representatives of the modern orders to their stem-group in the Paleozoic. The use of ontogenetic characters can be of great use in the resolution of these questions. Growth series of all ten modern salamander families (a 120 cleared and stained larvae) were examined for pattern and timing of vertebral elements chondrification and ossification. The primitive pattern is that of the neural arches developing before the centra, while the reverse represents the derived condition. Both the primitive and derived conditions are observed within the family Hynobiidae, whereas only the derived condition is observed in all other salamanders. This provides support to the claims that Hynobiidae is both the most basal of modern families and potentially polyphyletic (with Ranodon and Hybobius forming the most basal clade and Salamandrella being a part of the most derived clade). This provides insight into a unique event in salamander evolutionary history and suggests that the developmental pattern switch occurred between the Triassic and the mid-Jurassic before the last major radiation.

  11. Transcriptome Analysis of Manganese-deficient Chlamydomonas reinhardtii Provides Insight on the Chlorophyll Biosynthesis Pathway

    SciTech Connect

    Lockhart, Ainsley; Zvenigorodsky, Natasha; Pedraza, Mary Ann; Lindquist, Erika

    2011-08-11

    The biosynthesis of chlorophyll and other tetrapyrroles is a vital but poorly understood process. Recent genomic advances with the unicellular green algae Chlamydomonas reinhardtii have created opportunity to more closely examine the mechanisms of the chlorophyll biosynthesis pathway via transcriptome analysis. Manganese is a nutrient of interest for complex reactions because of its multiple stable oxidation states and role in molecular oxygen coordination. C. reinhardtii was cultured in Manganese-deplete Tris-acetate-phosphate (TAP) media for 24 hours and used to create cDNA libraries for sequencing using Illumina TruSeq technology. Transcriptome analysis provided intriguing insight on possible regulatory mechanisms in the pathway. Evidence supports similarities of GTR (Glutamyl-tRNA synthase) to its Chlorella vulgaris homolog in terms of Mn requirements. Data was also suggestive of Mn-related compensatory up-regulation for pathway proteins CHLH1 (Manganese Chelatase), GUN4 (Magnesium chelatase activating protein), and POR1 (Light-dependent protochlorophyllide reductase). Intriguingly, data suggests possible reciprocal expression of oxygen dependent CPX1 (coproporphyrinogen III oxidase) and oxygen independent CPX2. Further analysis using RT-PCR could provide compelling evidence for several novel regulatory mechanisms in the chlorophyll biosynthesis pathway.

  12. Structural insights into sulfite oxidase deficiency.

    PubMed

    Karakas, Erkan; Wilson, Heather L; Graf, Tyler N; Xiang, Song; Jaramillo-Busquets, Sandra; Rajagopalan, K V; Kisker, Caroline

    2005-09-30

    Sulfite oxidase deficiency is a lethal genetic disease that results from defects either in the genes encoding proteins involved in molybdenum cofactor biosynthesis or in the sulfite oxidase gene itself. Several point mutations in the sulfite oxidase gene have been identified from patients suffering from this disease worldwide. Although detailed biochemical analyses have been carried out on these mutations, no structural data could be obtained because of problems in crystallizing recombinant human and rat sulfite oxidases and the failure to clone the chicken sulfite oxidase gene. We synthesized the gene for chicken sulfite oxidase de novo, working backward from the amino acid sequence of the native chicken liver enzyme by PCR amplification of a series of 72 overlapping primers. The recombinant protein displayed the characteristic absorption spectrum of sulfite oxidase and exhibited steady state and rapid kinetic parameters comparable with those of the tissue-derived enzyme. We solved the crystal structures of the wild type and the sulfite oxidase deficiency-causing R138Q (R160Q in humans) variant of recombinant chicken sulfite oxidase in the resting and sulfate-bound forms. Significant alterations in the substrate-binding pocket were detected in the structure of the mutant, and a comparison between the wild type and mutant protein revealed that the active site residue Arg-450 adopts different conformations in the presence and absence of bound sulfate. The size of the binding pocket is thereby considerably reduced, and its position relative to the cofactor is shifted, causing an increase in the distance of the sulfur atom of the bound sulfate to the molybdenum.

  13. New insights into prion structure and toxicity.

    PubMed

    Harris, David A; True, Heather L

    2006-05-04

    Prion diseases in humans and animals are due to conformational conversion of PrP(C), a cellular glycoprotein of unknown function, into PrP(Sc), an isoform that appears to be infectious in the absence of nucleic acids. Proteins that behave as prions are also found in yeast and filamentous fungi. Although there is now strong experimental support for the hypothesis that prions are infectious proteins, two subjects have remained poorly understood: the structure of prions, and the mechanisms by which they kill neurons. In this review, we will highlight recent studies that shed new light on these important issues.

  14. A neurophylogenetic approach provides new insight to the evolution of Scaphopoda.

    PubMed

    Sumner-Rooney, Lauren H; Schrödl, Michael; Lodde-Bensch, Eva; Lindberg, David R; Heß, Martin; Brennan, Gerard P; Sigwart, Julia D

    2015-01-01

    The position of scaphopods in molluscan phylogeny remains singularly contentious, with several sister relationships supported by morphological and phylogenomic data: Scaphopoda + Bivalvia (Diasoma), Scaphopoda + Cephalopoda (Variopoda), and Scaphopoda + Gastropoda. Nervous system architecture has contributed significant insights to reconstructing phylogeny in the Mollusca and other invertebrate groups, but a modern neurophylogenetic approach has not been applied to molluscs, hampered by a lack of clearly defined homologous characters that can be unequivocally compared across the radical body plan disparity among the living clades. We present the first three-dimensional reconstruction of the anterior nervous system of a scaphopod, Rhabdus rectius, using histological tomography. We also describe a new putative sensory organ, a paired and pigmented sensory mantle slit. This structure is restricted to our study species and not a general feature of scaphopods, but it forms an integral part of the description of the nervous system in R. rectius. It also highlights the potential utility of neuro-anatomical characters for multiple levels of phylogenetic inference beyond this study. This potential has not previously been exploited for the thorny problem of molluscan phylogeny. The neuroanatomy of scaphopods demonstrates a highly derived architecture that shares a number of key characters with the cephalopod nervous system, and supports a Scaphopoda + Cephalopoda grouping.

  15. Structural and functional analyses of the archaeal tRNA m2G/m22G10 methyltransferase aTrm11 provide mechanistic insights into site specificity of a tRNA methyltransferase that contains common RNA-binding modules

    PubMed Central

    Hirata, Akira; Nishiyama, Seiji; Tamura, Toshihiro; Yamauchi, Ayano; Hori, Hiroyuki

    2016-01-01

    N2-methylguanosine is one of the most universal modified nucleosides required for proper function in transfer RNA (tRNA) molecules. In archaeal tRNA species, a specific S-adenosyl-L-methionine (SAM)-dependent tRNA methyltransferase (MTase), aTrm11, catalyzes formation of N2-methylguanosine and N2,N2-dimethylguanosine at position 10. Here, we report the first X-ray crystal structures of aTrm11 from Thermococcus kodakarensis (Tko), of the apo-form, and of its complex with SAM. The structures show that TkoTrm11 consists of three domains: an N-terminal ferredoxinlike domain (NFLD), THUMP domain and Rossmann-fold MTase (RFM) domain. A linker region connects the THUMP-NFLD and RFM domains. One SAM molecule is bound in the pocket of the RFM domain, suggesting that TkoTrm11 uses a catalytic mechanism similar to that of other tRNA MTases containing an RFM domain. Furthermore, the conformation of NFLD and THUMP domains in TkoTrm11 resembles that of other tRNA-modifying enzymes specifically recognizing the tRNA acceptor stem. Our docking model of TkoTrm11-SAM in complex with tRNA, combined with biochemical analyses and pre-existing evidence, provides insights into the substrate tRNA recognition mechanism: The THUMP domain recognizes a 3′-ACCA end, and the linker region and RFM domain recognize the T-stem, acceptor stem and V-loop of tRNA, thereby causing TkoTrm11 to specifically identify its methylation site. PMID:27325738

  16. Iron-Sulfur Cluster Engineering Provides Insight into the Evolution of Substrate Specificity among Sulfonucleotide Reductases

    PubMed Central

    Bhave, Devayani P.; Hong, Jiyoung A.; Keller, Rebecca L.; Krebs, Carsten; Carroll, Kate S.

    2011-01-01

    Assimilatory sulfate reduction supplies prototrophic organisms with reduced sulfur that is required for the biosynthesis of all sulfur-containing metabolites, including cysteine and methionine. The reduction of sulfate requires its activation via an ATP-dependent activation to form adenosine-5′-phosphosulfate (APS). Depending on the species, APS can be reduced directly to sulfite by APS reductase (APR) or undergo a second phosphorylation to yield 3′-phosphoadenosine-5′-phosphosulfate (PAPS), the substrate for PAPS reductase (PAPR). These essential enzymes have no human homolog, rendering them attractive targets for the development of novel antibacterial drugs. APR and PAPR share sequence and structure homology as well as a common catalytic mechanism, but the enzymes are distinguished by two features, namely, the amino acid sequence of the phosphate-binding loop (P-loop) and an iron-sulfur cofactor in APRs. Based on the crystal structures of APR and PAPR, two P-loop residues are proposed to determine substrate specificity; however, this hypothesis has not been tested. In contrast to this prevailing view, we report here that the P-loop motif has a modest effect on substrate discrimination. Instead, by means of metalloprotein engineering, spectroscopic and kinetic analyses, we demonstrate that the iron-sulfur cluster cofactor enhances APS reduction by nearly 1000-fold, thereby playing a pivotal role in substrate specificity and catalysis. These findings offer new insights into the evolution of this enzyme family, and extend the known functions of protein-bound iron-sulfur clusters. PMID:22023093

  17. The Microbial Signature Provides Insight into the Mechanistic Basis of Coral Success across Reef Habitats

    PubMed Central

    Leggat, William; Bongaerts, Pim

    2016-01-01

    ABSTRACT For ecosystems vulnerable to environmental change, understanding the spatiotemporal stability of functionally crucial symbioses is fundamental to determining the mechanisms by which these ecosystems may persist. The coral Pachyseris speciosa is a successful environmental generalist that succeeds in diverse reef habitats. The generalist nature of this coral suggests it may have the capacity to form functionally significant microbial partnerships to facilitate access to a range of nutritional sources within different habitats. Here, we propose that coral is a metaorganism hosting three functionally distinct microbial interactions: a ubiquitous core microbiome of very few symbiotic host-selected bacteria, a microbiome of spatially and/or regionally explicit core microbes filling functional niches (<100 phylotypes), and a highly variable bacterial community that is responsive to biotic and abiotic processes across spatial and temporal scales (>100,000 phylotypes). We find that this coral hosts upwards of 170,000 distinct phylotypes and provide evidence for the persistence of a select group of bacteria in corals across environmental habitats of the Great Barrier Reef and Coral Sea. We further show that a higher number of bacteria are consistently associated with corals on mesophotic reefs than on shallow reefs. An increase in microbial diversity with depth suggests reliance by this coral on bacteria for nutrient acquisition on reefs exposed to nutrient upwelling. Understanding the complex microbial communities of host organisms across broad biotic and abiotic environments as functionally distinct microbiomes can provide insight into those interactions that are ubiquitous niche symbioses and those that provide competitive advantage within the hosts’ environment. PMID:27460792

  18. Functional Analysis of Environmental DNA-Derived Microviridins Provides New Insights into the Diversity of the Tricyclic Peptide Family

    PubMed Central

    Gatte-Picchi, Douglas; Weiz, Annika; Ishida, Keishi; Hertweck, Christian

    2014-01-01

    Microviridins represent a unique family of ribosomally synthesized cage-like depsipeptides from cyanobacteria with potent protease-inhibitory activities. The natural diversity of these peptides is largely unexplored. Here, we describe two methodologies that were developed to functionally characterize cryptic microviridin gene clusters from metagenomic DNA. Environmental samples were collected and enriched from cyanobacterial freshwater blooms of different geographical origins containing predominantly Microcystis sp. Microviridins were produced either directly from fosmid clones or after insertion of environmental DNA-derived gene cassettes into a minimal expression platform in Escherichia coli. Three novel microviridin variants were isolated and tested against different serine-type proteases. The comparison of the bioactivity profiles of the new congeners allows deduction of further structure-function relationships for microviridins. Moreover, this study provides new insights into microviridin processing and gene cluster organization. PMID:24334668

  19. The Jujube Genome Provides Insights into Genome Evolution and the Domestication of Sweetness/Acidity Taste in Fruit Trees

    PubMed Central

    Wan, KangKang; Zhang, Zhong; Pang, Xiaoming; Yin, Xiao; Bai, Yang; Sun, Xiaoqing; Gao, Lizhi; Li, Ruiqiang; Zhang, Jinbo

    2016-01-01

    Jujube (Ziziphus jujuba Mill.) belongs to the Rhamnaceae family and is a popular fruit tree species with immense economic and nutritional value. Here, we report a draft genome of the dry jujube cultivar ‘Junzao’ and the genome resequencing of 31 geographically diverse accessions of cultivated and wild jujubes (Ziziphus jujuba var. spinosa). Comparative analysis revealed that the genome of ‘Dongzao’, a fresh jujube, was ~86.5 Mb larger than that of the ‘Junzao’, partially due to the recent insertions of transposable elements in the ‘Dongzao’ genome. We constructed eight proto-chromosomes of the common ancestor of Rhamnaceae and Rosaceae, two sister families in the order Rosales, and elucidated the evolutionary processes that have shaped the genome structures of modern jujubes. Population structure analysis revealed the complex genetic background of jujubes resulting from extensive hybridizations between jujube and its wild relatives. Notably, several key genes that control fruit organic acid metabolism and sugar content were identified in the selective sweep regions. We also identified S-locus genes controlling gametophytic self-incompatibility and investigated haplotype patterns of the S locus in the jujube genomes, which would provide a guideline for parent selection for jujube crossbreeding. This study provides valuable genomic resources for jujube improvement, and offers insights into jujube genome evolution and its population structure and domestication. PMID:28005948

  20. Evolution of Digestive Enzymes and RNASE1 Provides Insights into Dietary Switch of Cetaceans

    PubMed Central

    Wang, Zhengfei; Xu, Shixia; Du, Kexing; Huang, Fang; Chen, Zhuo; Zhou, Kaiya; Ren, Wenhua; Yang, Guang

    2016-01-01

    Although cetaceans (whales, porpoises, and dolphins) have multi-chambered stomachs, feeding habits of modern cetaceans have dramatically changed from herbivorous to carnivorous. However, the genetic basis underlying this dietary switch remains unexplored. Here, we present the first systematic investigation of 10 digestive enzymes genes (i.e., CYP7A1, CTRC, LIPC, LIPF, PNLIP, PGC, PRSS1, SI, SLC5A1, and TMPRSS15) of representative cetaceans, and the evolutionary trajectory of RNASE1 in cetartiodactylans. Positive selections were detected with proteinases (i.e., CTRC, PRSS1, and TMPRSS15) and lipases (i.e., CYP7A1, LIPF, and PNLIP) suggesting that cetaceans have evolved an enhanced digestion capacity for proteins and lipids, the major nutritional components of their prey (fishes and invertebrates). In addition, it was found that RNASE1 gene duplicated after the cetartiodactylan speciation and two independent gene duplication events took place in Camelidae and Ruminantia. Positive selection was detected with RNASE1 of Camelidae and Bovidae, suggesting enhanced digestive efficiency in the ruminants. Remarkably, even though the ancestors of cetaceans were terrestrial artiodactyls that are herbivorous, modern cetaceans lost the pancreatic RNASE1 copy with digestive function, which is in accordance with the dietary change from herbivorous to carnivorous. In sum, this is the first study that provides new insights into the evolutionary mechanism of dietary switch in cetaceans. PMID:27651393

  1. Comparative transcriptome analysis of chemosensory genes in two sister leaf beetles provides insights into chemosensory speciation.

    PubMed

    Zhang, Bin; Zhang, Wei; Nie, Rui-E; Li, Wen-Zhu; Segraves, Kari A; Yang, Xing-Ke; Xue, Huai-Jun

    2016-12-01

    Divergence in chemosensory traits has been posited as an important component of chemosensory speciation in insects. In particular, chemosensory genes expressed in the peripheral sensory neurons are likely to influence insect behaviors such as preference for food, oviposition sites, and mates. Despite their key role in insect behavior and potentially speciation, the underlying genetic basis for divergence in chemosensory traits remains largely unexplored. One way to ascertain the role of chemosensory genes in speciation is to make comparisons of these genes across closely related species to detect the genetic signatures of divergence. Here, we used high throughput transcriptome analysis to compare chemosensory genes of the sister leaf beetles species Pyrrhalta maculicollis and P. aenescens, whose sexual isolation and host plant preference are mediated by divergent chemical signals. Although there was low overall divergence between transcriptome profiles, there were a number of genes that were differentially expressed between the species. Furthermore, we also detected two chemosensory genes under positive selection, one of which that was also differentially expressed between the species, suggesting a possible role for these genes in chemical-based premating reproductive isolation and host use. Combined with the available chemical and ecological work in this system, further studies of the divergent chemosensory genes presented here will provide insight into the process of chemosensory speciation among Pyrrhalta beetles.

  2. Metatranscriptome Analysis of Fig Flowers Provides Insights into Potential Mechanisms for Mutualism Stability and Gall Induction

    PubMed Central

    Martinson, Ellen O.; Hackett, Jeremiah D.; Machado, Carlos A.; Arnold, A. Elizabeth

    2015-01-01

    A striking property of the mutualism between figs and their pollinating wasps is that wasps consistently oviposit in the inner flowers of the fig syconium, which develop into galls that house developing larvae. Wasps typically do not use the outer ring of flowers, which develop into seeds. To better understand differences between gall and seed flowers, we used a metatranscriptomic approach to analyze eukaryotic gene expression within fig flowers at the time of oviposition choice and early gall development. Consistent with the unbeatable seed hypothesis, we found significant differences in gene expression between gall- and seed flowers in receptive syconia prior to oviposition. In particular, transcripts assigned to flavonoids and carbohydrate metabolism were significantly up-regulated in gall flowers relative to seed flowers. In response to oviposition, gall flowers significantly up-regulated the expression of chalcone synthase, which previously has been connected to gall formation in other plants. We propose several genes encoding proteins with signal peptides or associations with venom of other Hymenoptera as candidate genes for gall initiation or growth. This study simultaneously evaluates the gene expression profile of both mutualistic partners in a plant-insect mutualism and provides insight into a possible stability mechanism in the ancient fig-fig wasp association. PMID:26090817

  3. Biological responsiveness to pheromones provides fundamental and unique insight into olfactory function.

    PubMed

    Sorensen, P W

    1996-04-01

    When exposed to the odor of conspecifics, most organisms exhibit an adaptive behavioral response, particularly if the individuals are sexually mature. Evidence increasingly suggests that behavioral responsiveness to these odors, which are termed 'pheromones', reflects neuroethological mechanisms associated with olfactory function. Reproductive pheromones, which are the best understood, are commonly used by both invertebrates and vertebrates. In both instances they are generally comprised of mixtures of compounds and behavioral responsiveness to them is largely instinctual, sexually-dimorphic, and attributable to a specialized component(s) of the olfactory system. While pheromonal responsiveness in some systems (e.g. moths) appears highly stereotypic and symptomatic of a relatively simple 'labeled line', behavioral responsiveness of other animals (e.g. rodents) can be modified by experience, suggesting a more complex underlying central mechanism. In any case, our understanding of these fascinating systems is progressing only because of an active dialogue between behavioral and neurological investigations. This review briefly examines how behavioral studies have provided fundamental insight into the neuroethology of olfactory function by drawing comparisons between some of the better understood sex pheromone systems which have been described in heliothine moths, the goldfish, and the pig. Many similarities between invertebrate and vertebrate pheromone systems are noted.

  4. Placental Proteomics Provides Insights into Pathophysiology of Pre-Eclampsia and Predicts Possible Markers in Plasma.

    PubMed

    Mary, Sheon; Kulkarni, Mahesh J; Malakar, Dipankar; Joshi, Sadhana R; Mehendale, Savita S; Giri, Ashok P

    2017-02-03

    Pre-eclampsia is a hypertensive disorder characterized by the new onset of hypertension >140/90 mmHg and proteinuria after the 20th week of gestation. The disorder is multifactorial and originates with abnormal placentation. Comparison of the placental proteome of normotensive (n = 25) and pre-eclamptic (n = 25) patients by gel-free proteomic techniques identified a total of 2145 proteins in the placenta of which 180 were differentially expressed (>1.3 fold, p < 0.05). Gene ontology enrichment analysis of biological process suggested that the differentially expressed proteins belonged to various physiological processes such as angiogenesis, apoptosis, oxidative stress, hypoxia, and placental development, which are implicated in the pathophysiology of pre-eclampsia. Some of the differentially expressed proteins were monitored in the plasma by multiple reaction monitoring analysis, which showed an increase in apolipoproteins A-I and A-II in gestational weeks 26-30 (2-fold, p < 0.01), while haptoglobin and hemopexin decreased in gestational weeks 26-30 and week 40/at delivery (1.8 fold, p < 0.01) in pre-eclamptic patients. This study provides a proteomic insight into the pathophysiology of pre-eclampsia. Identified candidate proteins can be evaluated further for the development of potential biomarkers associated with pre-eclampsia pathogenesis.

  5. Systematic characterization of the peroxidase gene family provides new insights into fungal pathogenicity in Magnaporthe oryzae.

    PubMed

    Mir, Albely Afifa; Park, Sook-Young; Abu Sadat, Md; Kim, Seongbeom; Choi, Jaeyoung; Jeon, Junhyun; Lee, Yong-Hwan

    2015-07-02

    Fungal pathogens have evolved antioxidant defense against reactive oxygen species produced as a part of host innate immunity. Recent studies proposed peroxidases as components of antioxidant defense system. However, the role of fungal peroxidases during interaction with host plants has not been explored at the genomic level. Here, we systematically identified peroxidase genes and analyzed their impact on fungal pathogenesis in a model plant pathogenic fungus, Magnaporthe oryzae. Phylogeny reconstruction placed 27 putative peroxidase genes into 15 clades. Expression profiles showed that majority of them are responsive to in planta condition and in vitro H2O2. Our analysis of individual deletion mutants for seven selected genes including MoPRX1 revealed that these genes contribute to fungal development and/or pathogenesis. We identified significant and positive correlations among sensitivity to H2O2, peroxidase activity and fungal pathogenicity. In-depth analysis of MoPRX1 demonstrated that it is a functional ortholog of thioredoxin peroxidase in Saccharomyces cerevisiae and is required for detoxification of the oxidative burst within host cells. Transcriptional profiling of other peroxidases in ΔMoprx1 suggested interwoven nature of the peroxidase-mediated antioxidant defense system. The results from this study provide insight into the infection strategy built on evolutionarily conserved peroxidases in the rice blast fungus.

  6. Can transcriptomics provide insight into the chemopreventive mechanisms of complex mixtures of phytochemicals in humans?

    PubMed

    van Breda, Simone G J; Wilms, Lonneke C; Gaj, Stan; Jennen, Danyel G J; Briedé, Jacob J; Helsper, Johannes P; Kleinjans, Jos C S; de Kok, Theo M C M

    2014-05-10

    Blueberries contain relatively large amounts of different phytochemicals, which are suggested to have chemopreventive properties, but little information is available on the underlying molecular modes of action. This study investigates whole genome gene expression changes in lymphocytes of 143 humans after a 4-week blueberry-apple juice dietary intervention. Differentially expressed genes and genes correlating with the extent of antioxidant protection were identified in four subgroups. The magnitude of the preventive effect after the intervention differed between these four subgroups. Furthermore, subjects in two groups carried genetic polymorphisms that were previously found to influence the chemopreventive response. Pathway analysis of the identified genes showed strong but complex gene expression changes in pathways signaling for apoptosis, immune response, cell adhesion, and lipid metabolism. These pathways indicate increased apoptosis, upgraded growth control, induced immunity, reduced platelet aggregation and activation, blood glucose homeostasis, and regulation of fatty acid metabolism. Based on these observations, we hypothesize that combining transcriptomic data with phenotypic markers of oxidative stress may provide insight into the relevant cellular processes and genetic pathways, which contribute to the antioxidant response of complex mixtures of phytochemicals, such as found in blueberry-apple juice.

  7. Comparative proteomic analysis provides new insights into mulberry dwarf responses in mulberry (Morus alba L.).

    PubMed

    Ji, Xianling; Gai, Yingping; Zheng, Chengchao; Mu, Zhimei

    2009-12-01

    Mulberry dwarf (MD) is a serious infectious disease of mulberry caused by phytoplasma. Infection with MD phytoplasma results in stress phenotypes of yellowing, phyllody, stunting, proliferation, and witches' broom. Physiological and biochemical analysis has shown that infection with MD phytoplasma causes an increase in soluble carbohydrate and starch content, and a decrease in the net photosynthesis rate, carboxylation efficiency, and pigment content of leaves. Furthermore, damage to the chloroplast ultrastructure was detected in infected leaves. To better understand the pathogen-stress response of mulberry (Morus alba L.) to MD phytoplasma, we conducted a comparative proteomic analysis using 2-DE of infected and healthy leaves. Among 500 protein spots that were reproducibly detected, 20 were down-regulated and 17 were up-regulated. MS identified 16 differentially expressed proteins. The photosynthetic proteins rubisco large subunit, rubisco activase, and sedoheptulose-1,7-bisphosphatase showed enhanced degradation in infected leaves. Based these results, a model for the occurrence mechanism of MD is proposed. In conclusion, this study provides new insights into the mulberry response to MD phytoplasma infection.

  8. Metatranscriptome Analysis of Fig Flowers Provides Insights into Potential Mechanisms for Mutualism Stability and Gall Induction.

    PubMed

    Martinson, Ellen O; Hackett, Jeremiah D; Machado, Carlos A; Arnold, A Elizabeth

    2015-01-01

    A striking property of the mutualism between figs and their pollinating wasps is that wasps consistently oviposit in the inner flowers of the fig syconium, which develop into galls that house developing larvae. Wasps typically do not use the outer ring of flowers, which develop into seeds. To better understand differences between gall and seed flowers, we used a metatranscriptomic approach to analyze eukaryotic gene expression within fig flowers at the time of oviposition choice and early gall development. Consistent with the unbeatable seed hypothesis, we found significant differences in gene expression between gall- and seed flowers in receptive syconia prior to oviposition. In particular, transcripts assigned to flavonoids and carbohydrate metabolism were significantly up-regulated in gall flowers relative to seed flowers. In response to oviposition, gall flowers significantly up-regulated the expression of chalcone synthase, which previously has been connected to gall formation in other plants. We propose several genes encoding proteins with signal peptides or associations with venom of other Hymenoptera as candidate genes for gall initiation or growth. This study simultaneously evaluates the gene expression profile of both mutualistic partners in a plant-insect mutualism and provides insight into a possible stability mechanism in the ancient fig-fig wasp association.

  9. Ambient mass spectrometry imaging metabolomics method provides novel insights into the action mechanism of drug candidates.

    PubMed

    He, Jingjing; Luo, Zhigang; Huang, Lan; He, Jiuming; Chen, Yi; Rong, Xianfang; Jia, Shaobo; Tang, Fei; Wang, Xiaohao; Zhang, Ruiping; Zhang, Jianjun; Shi, Jiangong; Abliz, Zeper

    2015-01-01

    Elucidation of the mechanism of action for drug candidates is fundamental to drug development, and it is strongly facilitated by metabolomics. Herein, we developed an imaging metabolomics method based on air-flow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI) under ambient conditions. This method was subsequently applied to simultaneously profile a novel anti-insomnia drug candidate, N(6)-(4-hydroxybenzyl)-adenosine (NHBA), and various endogenous metabolites in rat whole-body tissue sections after the administration of NHBA. The principal component analysis (PCA) represented by an intuitive color-coding scheme based on hyperspectral imaging revealed in situ molecular profiling alterations in response to stimulation of NHBA, which are in a very low intensity and hidden in massive interferential peaks. We found that the abundance of six endogenous metabolites changed after drug administration. The spatiotemporal distribution indicated that five altered molecules—including neurotransmitter γ-aminobutyric acid, neurotransmitter precursors choline and glycerophosphocholine, energy metabolism-related molecules adenosine (an endogenous sleep factor), and creatine—are closely associated with insomnia or other neurological disorders. These findings not only provide insights into a deep understanding on the mechanism of action of NHBA, but also demonstrate that the AFADESI-MSI-based imaging metabolomics is a powerful technique to investigate the molecular mechanism of drug action, especially for drug candidates with multitarget or undefined target in the preclinical study stage.

  10. The sacred lotus genome provides insights into the evolution of flowering plants.

    PubMed

    Wang, Yun; Fan, Guangyi; Liu, Yiman; Sun, Fengming; Shi, Chengcheng; Liu, Xin; Peng, Jing; Chen, Wenbin; Huang, Xinfang; Cheng, Shifeng; Liu, Yuping; Liang, Xinming; Zhu, Honglian; Bian, Chao; Zhong, Lan; Lv, Tian; Dong, Hongxia; Liu, Weiqing; Zhong, Xiao; Chen, Jing; Quan, Zhiwu; Wang, Zhihong; Tan, Benzhong; Lin, Chufa; Mu, Feng; Xu, Xun; Ding, Yi; Guo, An-Yuan; Wang, Jun; Ke, Weidong

    2013-11-01

    Sacred lotus (Nelumbo nucifera) is an ornamental plant that is also used for food and medicine. This basal eudicot species is especially important from an evolutionary perspective, as it occupies a critical phylogenetic position in flowering plants. Here we report the draft genome of a wild strain of sacred lotus. The assembled genome is 792 Mb, which is approximately 85-90% of genome size estimates. We annotated 392 Mb of repeat sequences and 36,385 protein-coding genes within the genome. Using these sequence data, we constructed a phylogenetic tree and confirmed the basal location of sacred lotus within eudicots. Importantly, we found evidence for a relatively recent whole-genome duplication event; any indication of the ancient paleo-hexaploid event was, however, absent. Genomic analysis revealed evidence of positive selection within 28 embryo-defective genes and one annexin gene that may be related to the long-term viability of sacred lotus seed. We also identified a significant expansion of starch synthase genes, which probably elevated starch levels within the rhizome of sacred lotus. Sequencing this strain of sacred lotus thus provided important insights into the evolution of flowering plant and revealed genetic mechanisms that influence seed dormancy and starch synthesis.

  11. Digital expression profiling of novel diatom transcripts provides insight into their biological functions

    PubMed Central

    2010-01-01

    Background Diatoms represent the predominant group of eukaryotic phytoplankton in the oceans and are responsible for around 20% of global photosynthesis. Two whole genome sequences are now available. Notwithstanding, our knowledge of diatom biology remains limited because only around half of their genes can be ascribed a function based onhomology-based methods. High throughput tools are needed, therefore, to associate functions with diatom-specific genes. Results We have performed a systematic analysis of 130,000 ESTs derived from Phaeodactylum tricornutum cells grown in 16 different conditions. These include different sources of nitrogen, different concentrations of carbon dioxide, silicate and iron, and abiotic stresses such as low temperature and low salinity. Based on unbiased statistical methods, we have catalogued transcripts with similar expression profiles and identified transcripts differentially expressed in response to specific treatments. Functional annotation of these transcripts provides insights into expression patterns of genes involved in various metabolic and regulatory pathways and into the roles of novel genes with unknown functions. Specific growth conditions could be associated with enhanced gene diversity, known gene product functions, and over-representation of novel transcripts. Comparative analysis of data from the other sequenced diatom, Thalassiosira pseudonana, helped identify several unique diatom genes that are specifically regulated under particular conditions, thus facilitating studies of gene function, genome annotation and the molecular basis of species diversity. Conclusions The digital gene expression database represents a new resource for identifying candidate diatom-specific genes involved in processes of major ecological relevance. PMID:20738856

  12. Transcriptome analyses provide insights into the phylogeny and adaptive evolution of the mangrove fern genus Acrostichum

    PubMed Central

    Zhang, Zhang; He, Ziwen; Xu, Shaohua; Li, Xinnian; Guo, Wuxia; Yang, Yuchen; Zhong, Cairong; Zhou, Renchao; Shi, Suhua

    2016-01-01

    The mangrove fern genus Acrostichum grows in the extremely unstable marine intertidal zone under harsh conditions, such as high salt concentrations, tidal rhythms and long-term climate changes. To explore the phylogenetic relationships and molecular mechanisms underlying adaptations in this genus, we sequenced the transcriptomes of two species of Acrostichum, A. aureum and A. speciosum, as well as a species in the sister genus, Ceratopteris thalictroides. We obtained 47,517, 36,420 and 60,823 unigenes for the three ferns, of which 24.39–45.63% were annotated using public databases. The estimated divergence time revealed that Acrostichum adapted to the coastal region during the late Cretaceous, whereas the two mangrove ferns from the Indo West-Pacific (IWP) area diverged more recently. Two methods (the modified branch-site model and the Kh method) were used to identify several positively selected genes, which may contribute to differential adaptation of the two Acrostichum species to different light and salt conditions. Our study provides abundant transcriptome data and new insights into the evolution and adaptations of mangrove ferns in the inhospitable intertidal zone. PMID:27782130

  13. Network component analysis provides quantitative insights on an Arabidopsis transcription factor-gene regulatory network

    PubMed Central

    2013-01-01

    activity. However, since NCA relies on documented connectivity information about the underlying TF-GRN, it is currently limited in its application to larger plant networks because of the lack of documented connectivities. In the future, the identification of interactions between plant TFs and their target genes on a genome scale would allow the use of NCA to provide quantitative regulatory information about plant TF-GRNs, leading to improved insights on cellular regulatory programs. PMID:24228871

  14. Phylogenetic analyses provide the first insights into the evolution of OVATE family proteins in land plants

    PubMed Central

    Liu, Di; Sun, Wei; Yuan, Yaowu; Zhang, Ning; Hayward, Alice; Liu, Yongliang; Wang, Ying

    2014-01-01

    Background and Aims The OVATE gene encodes a nuclear-localized regulatory protein belonging to a distinct family of plant-specific proteins known as the OVATE family proteins (OFPs). OVATE was first identified as a key regulator of fruit shape in tomato, with nonsense mutants displaying pear-shaped fruits. However, the role of OFPs in plant development has been poorly characterized. Methods Public databases were searched and a total of 265 putative OVATE protein sequences were identified from 13 sequenced plant genomes that represent the major evolutionary lineages of land plants. A phylogenetic analysis was conducted based on the alignment of the conserved OVATE domain from these 13 selected plant genomes. The expression patterns of tomato SlOFP genes were analysed via quantitative real-time PCR. The pattern of OVATE gene duplication resulting in the expansion of the gene family was determined in arabidopsis, rice and tomato. Key Results Genes for OFPs were found to be present in all the sampled land plant genomes, including the early-diverged lineages, mosses and lycophytes. Phylogenetic analysis based on the amino acid sequences of the conserved OVATE domain defined 11 sub-groups of OFPs in angiosperms. Different evolutionary mechanisms are proposed for OVATE family evolution, namely conserved evolution and divergent expansion. Characterization of the AtOFP family in arabidopsis, the OsOFP family in rice and the SlOFP family in tomato provided further details regarding the evolutionary framework and revealed a major contribution of tandem and segmental duplications towards expansion of the OVATE gene family. Conclusions This first genome-wide survey on OFPs provides new insights into the evolution of the OVATE protein family and establishes a solid base for future functional genomics studies on this important but poorly characterized regulatory protein family in plants. PMID:24812252

  15. Insights from the structural analysis of protein heterodimer interfaces.

    PubMed

    Sowmya, Gopichandran; Anita, Sathyanarayanan; Kangueane, Pandjassarame

    2011-05-07

    Protein heterodimer complexes are often involved in catalysis, regulation, assembly, immunity and inhibition. This involves the formation of stable interfaces between the interacting partners. Hence, it is of interest to describe heterodimer interfaces using known structural complexes. We use a non-redundant dataset of 192 heterodimer complex structures from the protein databank (PDB) to identify interface residues and describe their interfaces using amino-acids residue property preference. Analysis of the dataset shows that the heterodimer interfaces are often abundant in polar residues. The analysis also shows the presence of two classes of interfaces in heterodimer complexes. The first class of interfaces (class A) with more polar residues than core but less than surface is known. These interfaces are more hydrophobic than surfaces, where protein-protein binding is largely hydrophobic. The second class of interfaces (class B) with more polar residues than core and surface is shown. These interfaces are more polar than surfaces, where binding is mainly polar. Thus, these findings provide insights to the understanding of protein-protein interactions.

  16. Amyloid-beta fibrillogenesis: structural insight and therapeutic intervention.

    PubMed

    Dasilva, Kevin A; Shaw, James E; McLaurin, Joanne

    2010-06-01

    Structural insight into the conformational changes associated with aggregation and assembly of fibrils has provided a number of targets for therapeutic intervention. Solid-state NMR, hydrogen/deuterium exchange and mutagenesis strategies have been used to probe the secondary and tertiary structure of amyloid fibrils and key intermediates. Rational design of peptide inhibitors directed against key residues important for aggregation and stabilization of fibrils has demonstrated effectiveness at inhibiting fibrillogenesis. Studies on the interaction between Abeta and cell membranes led to the discovery that inositol, the head group of phosphatidylinositol, inhibits fibrillogenesis. As a result, scyllo-inositol is currently in clinical trials for the treatment of AD. Additional small-molecule inhibitors, including polyphenolic compounds such as curcumin, (-)-epigallocatechin gallate (EGCG), and grape seed extract have been shown to attenuate Abeta aggregation through distinct mechanisms, and have shown effectiveness at reducing amyloid levels when administered to transgenic mouse models of AD. Although the results of ongoing clinical trials remain to be seen, these compounds represent the first generation of amyloid-based therapeutics, with the potential to alter the progression of AD and, when used prophylactically, alleviate the deposition of Abeta.

  17. Improved prediction of RNA tertiary structure with insights into native state dynamics.

    PubMed

    Bida, John Paul; Maher, L James

    2012-03-01

    The importance of RNA tertiary structure is evident from the growing number of published high resolution NMR and X-ray crystallographic structures of RNA molecules. These structures provide insights into function and create a knowledge base that is leveraged by programs such as Assemble, ModeRNA, RNABuilder, NAST, FARNA, Mc-Sym, RNA2D3D, and iFoldRNA for tertiary structure prediction and design. While these methods sample native-like RNA structures during simulations, all struggle to capture the native RNA conformation after scoring. We propose RSIM, an improved RNA fragment assembly method that preserves RNA global secondary structure while sampling conformations. This approach enhances the quality of predicted RNA tertiary structure, provides insights into the native state dynamics, and generates a powerful visualization of the RNA conformational space. RSIM is available for download from http://www.github.com/jpbida/rsim.

  18. Whole-Genome Sequencing of Native Sheep Provides Insights into Rapid Adaptations to Extreme Environments

    PubMed Central

    Yang, Ji; Li, Wen-Rong; Lv, Feng-Hua; He, San-Gang; Tian, Shi-Lin; Peng, Wei-Feng; Sun, Ya-Wei; Zhao, Yong-Xin; Tu, Xiao-Long; Zhang, Min; Xie, Xing-Long; Wang, Yu-Tao; Li, Jin-Quan; Liu, Yong-Gang; Shen, Zhi-Qiang; Wang, Feng; Liu, Guang-Jian; Lu, Hong-Feng; Kantanen, Juha; Han, Jian-Lin; Li, Meng-Hua; Liu, Ming-Jun

    2016-01-01

    Global climate change has a significant effect on extreme environments and a profound influence on species survival. However, little is known of the genome-wide pattern of livestock adaptations to extreme environments over a short time frame following domestication. Sheep (Ovis aries) have become well adapted to a diverse range of agroecological zones, including certain extreme environments (e.g., plateaus and deserts), during their post-domestication (approximately 8–9 kya) migration and differentiation. Here, we generated whole-genome sequences from 77 native sheep, with an average effective sequencing depth of ∼5× for 75 samples and ∼42× for 2 samples. Comparative genomic analyses among sheep in contrasting environments, that is, plateau (>4,000 m above sea level) versus lowland (<100 m), high-altitude region (>1500 m) versus low-altitude region (<1300 m), desert (<10 mm average annual precipitation) versus highly humid region (>600 mm), and arid zone (<400 mm) versus humid zone (>400 mm), detected a novel set of candidate genes as well as pathways and GO categories that are putatively associated with hypoxia responses at high altitudes and water reabsorption in arid environments. In addition, candidate genes and GO terms functionally related to energy metabolism and body size variations were identified. This study offers novel insights into rapid genomic adaptations to extreme environments in sheep and other animals, and provides a valuable resource for future research on livestock breeding in response to climate change. PMID:27401233

  19. Perigone Lobe Transcriptome Analysis Provides Insights into Rafflesia cantleyi Flower Development.

    PubMed

    Lee, Xin-Wei; Mat-Isa, Mohd-Noor; Mohd-Elias, Nur-Atiqah; Aizat-Juhari, Mohd Afiq; Goh, Hoe-Han; Dear, Paul H; Chow, Keng-See; Haji Adam, Jumaat; Mohamed, Rahmah; Firdaus-Raih, Mohd; Wan, Kiew-Lian

    2016-01-01

    Rafflesia is a biologically enigmatic species that is very rare in occurrence and possesses an extraordinary morphology. This parasitic plant produces a gigantic flower up to one metre in diameter with no leaves, stem or roots. However, little is known about the floral biology of this species especially at the molecular level. In an effort to address this issue, we have generated and characterised the transcriptome of the Rafflesia cantleyi flower, and performed a comparison with the transcriptome of its floral bud to predict genes that are expressed and regulated during flower development. Approximately 40 million sequencing reads were generated and assembled de novo into 18,053 transcripts with an average length of 641 bp. Of these, more than 79% of the transcripts had significant matches to annotated sequences in the public protein database. A total of 11,756 and 7,891 transcripts were assigned to Gene Ontology categories and clusters of orthologous groups respectively. In addition, 6,019 transcripts could be mapped to 129 pathways in Kyoto Encyclopaedia of Genes and Genomes Pathway database. Digital abundance analysis identified 52 transcripts with very high expression in the flower transcriptome of R. cantleyi. Subsequently, analysis of differential expression between developing flower and the floral bud revealed a set of 105 transcripts with potential role in flower development. Our work presents a deep transcriptome resource analysis for the developing flower of R. cantleyi. Genes potentially involved in the growth and development of the R. cantleyi flower were identified and provide insights into biological processes that occur during flower development.

  20. Perigone Lobe Transcriptome Analysis Provides Insights into Rafflesia cantleyi Flower Development

    PubMed Central

    Lee, Xin-Wei; Mat-Isa, Mohd-Noor; Mohd-Elias, Nur-Atiqah; Aizat-Juhari, Mohd Afiq; Goh, Hoe-Han; Dear, Paul H.; Chow, Keng-See; Haji Adam, Jumaat; Mohamed, Rahmah; Firdaus-Raih, Mohd; Wan, Kiew-Lian

    2016-01-01

    Rafflesia is a biologically enigmatic species that is very rare in occurrence and possesses an extraordinary morphology. This parasitic plant produces a gigantic flower up to one metre in diameter with no leaves, stem or roots. However, little is known about the floral biology of this species especially at the molecular level. In an effort to address this issue, we have generated and characterised the transcriptome of the Rafflesia cantleyi flower, and performed a comparison with the transcriptome of its floral bud to predict genes that are expressed and regulated during flower development. Approximately 40 million sequencing reads were generated and assembled de novo into 18,053 transcripts with an average length of 641 bp. Of these, more than 79% of the transcripts had significant matches to annotated sequences in the public protein database. A total of 11,756 and 7,891 transcripts were assigned to Gene Ontology categories and clusters of orthologous groups respectively. In addition, 6,019 transcripts could be mapped to 129 pathways in Kyoto Encyclopaedia of Genes and Genomes Pathway database. Digital abundance analysis identified 52 transcripts with very high expression in the flower transcriptome of R. cantleyi. Subsequently, analysis of differential expression between developing flower and the floral bud revealed a set of 105 transcripts with potential role in flower development. Our work presents a deep transcriptome resource analysis for the developing flower of R. cantleyi. Genes potentially involved in the growth and development of the R. cantleyi flower were identified and provide insights into biological processes that occur during flower development. PMID:27977777

  1. Gene expression profiling provides insights into pathways of oxaliplatin-related sinusoidal obstruction syndrome in humans.

    PubMed

    Rubbia-Brandt, Laura; Tauzin, Sébastien; Brezault, Catherine; Delucinge-Vivier, Céline; Descombes, Patrick; Dousset, Bertand; Majno, Pietro E; Mentha, Gilles; Terris, Benoit

    2011-04-01

    Sinusoidal obstruction syndrome (SOS; formerly veno-occlusive disease) is a well-established complication of hematopoietic stem cell transplantation, pyrrolizidine alkaloid intoxication, and widely used chemotherapeutic agents such as oxaliplatin. It is associated with substantial morbidity and mortality. Pathogenesis of SOS in humans is poorly understood. To explore its molecular mechanisms, we used Affymetrix U133 Plus 2.0 microarrays to investigate the gene expression profile of 11 human livers with oxaliplatin-related SOS and compared it to 12 matched controls. Hierarchical clustering analysis showed that profiles from SOS and controls formed distinct clusters. To identify functional networks and gene ontologies, data were analyzed by the Ingenuity Pathway Analysis Tool. A total of 913 genes were differentially expressed in SOS: 613 being upregulated and 300 downregulated. Reverse transcriptase-PCR results showed excellent concordance with microarray data. Pathway analysis showed major gene upregulation in six pathways in SOS compared with controls: acute phase response (notably interleukin 6), coagulation system (Serpine1, THBD, and VWF), hepatic fibrosis/hepatic stellate cell activation (COL3a1, COL3a2, PDGF-A, TIMP1, and MMP2), and oxidative stress. Angiogenic factors (VEGF-C) and hypoxic factors (HIF1A) were upregulated. The most significant increase was seen in CCL20 mRNA. In conclusion, oxaliplatin-related SOS can be readily distinguished according to morphologic characteristics but also by a molecular signature. Global gene analysis provides new insights into mechanisms underlying chemotherapy-related hepatotoxicity in humans and potential targets relating to its diagnosis, prevention, and treatment. Activation of VEGF and coagulation (vWF) pathways could partially explain at a molecular level the clinical observations that bevacizumab and aspirin have a preventive effect in SOS.

  2. Quantitative Proteomic Analysis Provides Novel Insights into Cold Stress Responses in Petunia Seedlings.

    PubMed

    Zhang, Wei; Zhang, Huilin; Ning, Luyun; Li, Bei; Bao, Manzhu

    2016-01-01

    Low temperature is a major adverse environmental factor that impairs petunia growth and development. To better understand the molecular mechanisms of cold stress adaptation of petunia plants, a quantitative proteomic analysis using iTRAQ technology was performed to detect the effects of cold stress on protein expression profiles in petunia seedlings which had been subjected to 2°C for 5 days. Of the 2430 proteins whose levels were quantitated, a total of 117 proteins were discovered to be differentially expressed under low temperature stress in comparison to unstressed controls. As an initial study, 44 proteins including well known and novel cold-responsive proteins were successfully annotated. By integrating the results of two independent Gene Ontology (GO) enrichment analyses, seven common GO terms were found of which "oxidation-reduction process" was the most notable for the cold-responsive proteins. By using the subcellular localization tool Plant-mPLoc predictor, as much as 40.2% of the cold-responsive protein group was found to be located within chloroplasts, suggesting that the chloroplast proteome is particularly affected by cold stress. Gene expression analyses of 11 cold-responsive proteins by real time PCR demonstrated that the mRNA levels were not strongly correlated with the respective protein levels. Further activity assay of anti-oxidative enzymes showed different alterations in cold treated petunia seedlings. Our investigation has highlighted the role of antioxidation mechanisms and also epigenetic factors in the regulation of cold stress responses. Our work has provided novel insights into the plant response to cold stress and should facilitate further studies regarding the molecular mechanisms which determine how plant cells cope with environmental perturbation. The data have been deposited to the ProteomeXchange with identifier PXD002189.

  3. Porphyra (Bangiophyceae) Transcriptomes Provide Insights Into Red Algal Development And Metabolism.

    PubMed

    Chan, Cheong Xin; Blouin, Nicolas A; Zhuang, Yunyun; Zäuner, Simone; Prochnik, Simon E; Lindquist, Erika; Lin, Senjie; Benning, Christoph; Lohr, Martin; Yarish, Charles; Gantt, Elisabeth; Grossman, Arthur R; Lu, Shan; Müller, Kirsten; W Stiller, John; Brawley, Susan H; Bhattacharya, Debashish

    2012-12-01

    The red seaweed Porphyra (Bangiophyceae) and related Bangiales have global economic importance. Here, we report the analysis of a comprehensive transcriptome comprising ca. 4.7 million expressed sequence tag (EST) reads from P. umbilicalis (L.) J. Agardh and P. purpurea (Roth) C. Agardh (ca. 980 Mbp of data generated using 454 FLX pyrosequencing). These ESTs were isolated from the haploid gametophyte (blades from both species) and diploid conchocelis stage (from P. purpurea). In a bioinformatic analysis, only 20% of the contigs were found to encode proteins of known biological function. Comparative analysis of predicted protein functions in mesophilic (including Porphyra) and extremophilic red algae suggest that the former has more putative functions related to signaling, membrane transport processes, and establishment of protein complexes. These enhanced functions may reflect general mesophilic adaptations. A near-complete repertoire of genes encoding histones and ribosomal proteins was identified, with some differentially regulated between the blade and conchocelis stage in P. purpurea. This finding may reflect specific regulatory processes associated with these distinct phases of the life history. Fatty acid desaturation patterns, in combination with gene expression profiles, demonstrate differences from seed plants with respect to the transport of fatty acid/lipid among subcellular compartments and the molecular machinery of lipid assembly. We also recovered a near-complete gene repertoire for enzymes involved in the formation of sterols and carotenoids, including candidate genes for the biosynthesis of lutein. Our findings provide key insights into the evolution, development, and biology of Porphyra, an important lineage of red algae.

  4. Characterization of the Arginolytic Microflora Provides Insights into pH Homeostasis in Human Oral Biofilms

    PubMed Central

    Huang, Xuelian; Schulte, Renee M.; Burne, Robert A.; Nascimento, Marcelle M.

    2014-01-01

    A selected group of oral bacteria commonly associated with dental health is capable of producing alkali via the arginine deiminase system (ADS), which has a profound impact on the pH of human oral biofilms. An increased risk for dental caries has been associated with reduced ADS activity of the bacteria in oral biofilms. Arginolytic bacterial strains from dental plaque samples of caries-free (CF) and caries-active (CA) adults were isolated and characterized to investigate the basis for differences in plaque ADS activity between individuals. Fifty-six ADS-positive bacterial strains were identified by 16S rRNA gene sequencing and their ADS activity levels were compared under standard growth conditions. The spectrum of bacterial ADS activity ranged from 45.2 to 688.0 units (mg protein)−1. Although Streptococcus sanguinis was the most prevalent species, other Streptococcus were also represented. Biochemical assays carried out using twenty-seven ADS-positive strains under conditions known to induce or repress ADS gene expression, showed substantial variation in arginolytic activity in response to pH, oxygen, and the availability of carbohydrate or arginine. This study reveals that the basis for the wide spectrum of arginolytic expression observed among clinical strains is, at least in part, attributable to differences in the regulation of the ADS within and between species. The results provide insights into the microbiological basis for inter-subject differences in ADS activity in oral biofilms and enhance our understanding of dental caries as an ecologically-driven disease in which arginine metabolism moderates plaque pH and promotes dental health. PMID:25634570

  5. Comparative Genome Analysis Provides Insights into the Pathogenicity of Flavobacterium psychrophilum

    PubMed Central

    Castillo, Daniel; Christiansen, Rói Hammershaimb; Dalsgaard, Inger; Madsen, Lone; Espejo, Romilio

    2016-01-01

    phenotypic properties may provide new insights to the mechanisms of pathogenicity in F. psychrophilum. PMID:27071075

  6. Assessing stability in mild and moderate Parkinson's disease: Can clinical measures provide insight?

    PubMed

    Hubble, Ryan P; Silburn, Peter A; Naughton, Geraldine A; Cole, Michael H

    2016-09-01

    This cross-sectional study aimed to investigate the relationship between accelerometer-derived measures of movement rhythmicity and clinical measures of mobility, balance confidence and gait difficulty in people with Parkinson's disease (PD). Twenty-nine independently-living PD patients (Hoehn & Yahr Stages 1-3) with no history of significant injury or orthopaedic/deep brain stimulation surgery were recruited from a database of patients who had expressed an interest to participate in research. Participants completed clinical assessments of mobility, postural stability, balance confidence and symptom severity, while head and trunk rhythmicity was evaluated during gait using accelerometers. Following data collection, patients were stratified based on disease stage into either a Mild (Hoehn & Yahr Stage 1) or Moderate (Hoehn & Yahr Stages 2-3) PD group. The results highlighted that the Moderate PD group had poorer quality of life, reduced balance confidence and increased gait and falls difficulty. Furthermore, for these patients, gait disability and the number of previous falls were both negatively correlated with multiple components of head and trunk rhythmicity. For the Mild PD group, six-meter walk time was positively correlated with ML head rhythmicity and linear regression highlighted a significant predictive relationship between these outcomes. For the Mild and Moderate PD groups, balance confidence respectively predicted anterior-posterior trunk rhythmicity and vertical head rhythmicity. While these findings demonstrate that falls history and the Gait and Falls questionnaire provide moderate insight into head and trunk rhythmicity in Moderate PD patients, objective and clinically-feasible measures of postural instability would assist with the management of these symptoms.

  7. DNA Methylation Profiling in Inflammatory Bowel Disease Provides New Insights into Disease Pathogenesis

    PubMed Central

    McDermott, Edel; Ryan, Elizabeth J.; Tosetto, Miriam; Gibson, David; Burrage, Joe; Keegan, Denise; Byrne, Kathryn; Crowe, Eimear; Sexton, Gillian; Malone, Kevin; Harris, R. Alan; Kellermayer, Richard; Mill, Jonathan; Cullen, Garret; Doherty, Glen A.; Mulcahy, Hugh; Murphy, Therese M.

    2016-01-01

    Background and Aims: Inflammatory bowel diseases (IBDs) are heterogeneous disorders with complex aetiology. Quantitative genetic studies suggest that only a small proportion of the disease variance observed in IBD is accounted for by genetic variation, indicating a potential role for differential epigenetic regulation in disease aetiology. The aim of this study was to assess genome-wide DNA methylation changes specifically associated with ulcerative colitis (UC), Crohn’s disease (CD) and IBD activity. Methods: DNA methylation was quantified in peripheral blood mononuclear cells (PBMCs) from 149 IBD cases (61 UC, 88 CD) and 39 controls using the Infinium HumanMethylation450 BeadChip. Technical and functional validation was performed using pyrosequencing and the real-time polymerase chain reaction. Cross-tissue replication of the top differentially methylated positions (DMPs) was tested in colonic mucosa tissue samples obtained from paediatric IBD cases and controls. Results: A total of 3196 probes were differentially methylated between CD cases and controls, while 1481 probes were differentially methylated between UC cases and controls. There was considerable (45%) overlap between UC and CD DMPs. The top-ranked IBD-associated PBMC differentially methylated region (promoter region of TRIM39-RPP2) was also significantly hypomethylated in colonic mucosa from paediatric UC patients. In addition, we confirmed TRAF6 hypermethylation using pyrosequencing and found reduced TRAF6 gene expression in PBMCs of IBD patients. Conclusions: Our data provide new insights into differential epigenetic regulation of genes and molecular pathways, which may contribute to the pathogenesis and activity of IBD. PMID:26419460

  8. Quantitative Proteomic Analysis Provides Novel Insights into Cold Stress Responses in Petunia Seedlings

    PubMed Central

    Zhang, Wei; Zhang, Huilin; Ning, Luyun; Li, Bei; Bao, Manzhu

    2016-01-01

    Low temperature is a major adverse environmental factor that impairs petunia growth and development. To better understand the molecular mechanisms of cold stress adaptation of petunia plants, a quantitative proteomic analysis using iTRAQ technology was performed to detect the effects of cold stress on protein expression profiles in petunia seedlings which had been subjected to 2°C for 5 days. Of the 2430 proteins whose levels were quantitated, a total of 117 proteins were discovered to be differentially expressed under low temperature stress in comparison to unstressed controls. As an initial study, 44 proteins including well known and novel cold-responsive proteins were successfully annotated. By integrating the results of two independent Gene Ontology (GO) enrichment analyses, seven common GO terms were found of which “oxidation-reduction process” was the most notable for the cold-responsive proteins. By using the subcellular localization tool Plant-mPLoc predictor, as much as 40.2% of the cold-responsive protein group was found to be located within chloroplasts, suggesting that the chloroplast proteome is particularly affected by cold stress. Gene expression analyses of 11 cold-responsive proteins by real time PCR demonstrated that the mRNA levels were not strongly correlated with the respective protein levels. Further activity assay of anti-oxidative enzymes showed different alterations in cold treated petunia seedlings. Our investigation has highlighted the role of antioxidation mechanisms and also epigenetic factors in the regulation of cold stress responses. Our work has provided novel insights into the plant response to cold stress and should facilitate further studies regarding the molecular mechanisms which determine how plant cells cope with environmental perturbation. The data have been deposited to the ProteomeXchange with identifier PXD002189. PMID:26941746

  9. A semisynthetic Eph receptor tyrosine kinase provides insight into ligand-induced kinase activation

    PubMed Central

    Singla, Nikhil; Erdjument-Bromage, Hediye; Himanen, Juha P.; Muir, Tom W.; Nikolov, Dimitar B.

    2011-01-01

    SUMMARY We have developed a methodology for generating milligram amounts of functional Eph tyrosine kinase receptor using the protein engineering approach of expressed protein ligation. Stimulation with ligand induces efficient autophosphorylation of the semisynthetic Eph construct. The in vitro phosphorylation of key Eph tyrosine residues upon ligand-induced activation was monitored via time-resolved, quantitative phosphoproteomics, suggesting a precise and unique order of phosphorylation of the Eph tyrosines in the kinase activation process. To our knowledge, this work represents the first reported semisynthesis of a receptor tyrosine kinase and provides a potentially general method for producing single-pass membrane proteins for structural and biochemical characterization. PMID:21439481

  10. Field Studies Provide Insight into Tritium Migration in an Arid Environment

    NASA Astrophysics Data System (ADS)

    Andraski, B. J.; Garcia, C.; Michel, R. L.; Stonestrom, D. A.; Mayers, C.; Johnson, M.

    2009-12-01

    directly beneath the root zone. On the west site of the facility, a drainage-diversion ditch cuts through this near-surface gravel layer, but the gravel-layer discontinuity created by the ditch is not reflected in the plant and soil water-vapor tritium distributions. Thus, an alternative conceptual model is needed for the western plume. Recognizing the importance of upward flow processes in arid unsaturated zones, it is likely that the western plume represents tritium moving laterally through a deeper gravel layer and then upward into near-surface soils with subsequent release to the atmosphere. This evaluation of tritium distributions in relation to site features provides insight into field-scale transport in an arid environment.

  11. Substrate and Product Complexes of Escherichia Coli Adenylosuccinate Lyase Provide New Insights into the Enzymatic Mechanism

    SciTech Connect

    Tsai,M.; Koo, J.; Yip, P.; Colman, R.; Segall, M.; Howell, P.

    2007-01-01

    Adenylosuccinate lyase (ADL) catalyzes the breakdown of 5-aminoimidazole- (N-succinylocarboxamide) ribotide (SAICAR) to 5-aminoimidazole-4-carboxamide ribotide (AICAR) and fumarate, and of adenylosuccinate (ADS) to adenosine monophosphate (AMP) and fumarate in the de novo purine biosynthetic pathway. ADL belongs to the argininosuccinate lyase (ASL)/fumarase C superfamily of enzymes. Members of this family share several common features including: a mainly {alpha}-helical, homotetrameric structure; three regions of highly conserved amino acid residues; and a general acid-base catalytic mechanism with the overall {beta}-elimination of fumarate as a product. The crystal structures of wild-type Escherichia coli ADL (ec-ADL), and mutant-substrate (H171A-ADS) and -product (H171N-AMP{center_dot}FUM) complexes have been determined to 2.0, 1.85, and 2.0 {angstrom} resolution, respectively. The H171A-ADS and H171N-AMP{center_dot}FUM structures provide the first detailed picture of the ADL active site, and have enabled the precise identification of substrate binding and putative catalytic residues. Contrary to previous suggestions, the ec-ADL structures implicate S295 and H171 in base and acid catalysis, respectively. Furthermore, structural alignments of ec-ADL with other superfamily members suggest for the first time a large conformational movement of the flexible C3 loop (residues 287-303) in ec-ADL upon substrate binding and catalysis, resulting in its closure over the active site. This loop movement has been observed in other superfamily enzymes, and has been proposed to be essential for catalysis. The ADL catalytic mechanism is re-examined in light of the results presented here.

  12. Support Needs for Canadian Health Providers Responding to Disaster: New Insights from a Grounded Theory Approach

    PubMed Central

    Fahim, Christine; O'Sullivan, Tracey L.; Lane, Dan

    2015-01-01

    Introduction: An earlier descriptive study exploring the various supports available to Canadian health and social service providers who deployed to the 2010 earthquake disaster in Haiti, indicated that when systems are compromised, professionals are at physical, emotional and mental risk during overseas deployment. While these risks are generally well-identified, there is little literature that explores the effectiveness of the supports in place to mitigate this risk. This study provides evidence to inform policy development regarding future disaster relief, and the effectiveness of supports available to responders assisting with international disaster response. Methods: This study follows Strauss and Corbin’s 1990 structured approach to grounded theory to develop a framework for effective disaster support systems. N=21 interviews with Canadian health and social service providers, who deployed to Haiti in response to the 2010 earthquake, were conducted and analyzed. Resulting data were transcribed, coded and analysed for emergent themes. Results and Discussion: Three themes were identified in the data and were used to develop the evolving theory. The interview data indicate that the experiences of responders are determined based on an interaction between the individual’s ‘lens’ or personal expectations, as well as the supports that an organization is able to provide. Therefore, organizations should consider the following factors: experience, expectations, and supports, to tailor a successful support initiative that caters to the needs of the volunteer workforce. PMID:26203399

  13. Resiniferatoxin and its analogs provide novel insights into the pharmacology of the vanilloid (capsaicin) receptor

    SciTech Connect

    Szallasi, A.; Blumberg, P.M. )

    1990-01-01

    Capsaicin, the pungent constituent of chili peppers, represents the paradigm for the capsaicinoids or vanilloids, a family of compounds shown to stimulate and then desensitize specific subpopulations of sensory receptors, including C-polymodal nociceptors, A-delta mechanoheat nociceptors and warm receptors of the skin, as well as enteroceptors of thin afferent fibers. An exciting recent advance in the field has been the finding that resiniferatoxin (RTX), a naturally occurring diterpene containing a homovanillic acid ester, a key structural motif of capsaicin, functions as an ultrapotent capsaicin analog. For most of the responses characteristic of capsaicin, RTX is 100-10,000 fold more potent. Structure/activity analysis indicates, however, that RTX and related homovanillyl-diterpene esters display distinct spectra of activity. Specific ({sup 3}H)RTX binding provides the first direct proof for the existence of vanilloid receptors. We expect that the RTX class of vanilloids will promote rapid progress in understanding of vanilloid structure/activity requirements and mechanism.

  14. Multiple Problem-Solving Strategies Provide Insight into Students' Understanding of Open-Ended Linear Programming Problems

    ERIC Educational Resources Information Center

    Sole, Marla A.

    2016-01-01

    Open-ended questions that can be solved using different strategies help students learn and integrate content, and provide teachers with greater insights into students' unique capabilities and levels of understanding. This article provides a problem that was modified to allow for multiple approaches. Students tended to employ high-powered, complex,…

  15. An integrated Biophysical CGE model to provide Sustainable Development Goal insights

    NASA Astrophysics Data System (ADS)

    Sanchez, Marko; Cicowiez, Martin; Howells, Mark; Zepeda, Eduardo

    2016-04-01

    Future projected changes in the energy system will inevitably result in changes to the level of appropriation of environmental resources, particularly land and water, and this will have wider implications for environmental sustainability, and may affect other sectors of the economy. An integrated climate, land, energy and water (CLEW) system will provide useful insights, particularly with regard to the environmental sustainability. However, it will require adequate integration with other tools to detect economic impacts and broaden the scope for policy analysis. A computable general equilibrium (CGE) model is a well suited tool to channel impacts, as detected in a CLEW analysis, onto all sectors of the economy, and evaluate trade-offs and synergies, including those of possible policy responses. This paper will show an application of such integration in a single-country CGE model with the following key characteristics. Climate is partly exogenous (as proxied by temperature and rainfall) and partly endogenous (as proxied by emissions generated by different sectors) and has an impact on endogenous variables such as land productivity and labor productivity. Land is a factor of production used in agricultural and forestry activities which can be of various types if land use alternatives (e.g., deforestation) are to be considered. Energy is an input to the production process of all economic sectors and a consumption good for households. Because it is possible to allow for substitution among different energy sources (e.g. renewable vs non-renewable) in the generation of electricity, the production process of energy products can consider the use of natural resources such as oil and water. Water, data permitting, can be considered as an input into the production process of agricultural sectors, which is particularly relevant in case of irrigation. It can also be considered as a determinant of total factor productivity in hydro-power generation. The integration of a CLEW

  16. Scrutiny of Mycobacterium tuberculosis 19 kDa antigen proteoforms provides new insights in the lipoglycoprotein biogenesis paradigm

    PubMed Central

    Parra, Julien; Marcoux, Julien; Poncin, Isabelle; Canaan, Stéphane; Herrmann, Jean Louis; Nigou, Jérôme; Burlet-Schiltz, Odile; Rivière, Michel

    2017-01-01

    Post-translational modifications (PTMs) are essential processes conditioning the biophysical properties and biological activities of the vast majority of mature proteins. However, occurrence of several distinct PTMs on a same protein dramatically increases its molecular diversity. The comprehensive understanding of the functionalities resulting from any particular PTM association requires a highly challenging full structural description of the PTM combinations. Here, we report the in-depth exploration of the natural structural diversity of the M. tuberculosis (Mtb) virulence associated 19 kDa lipoglycoprotein antigen (LpqH) using intact protein high-resolution mass spectrometry (HR-MS) coupled to liquid chromatography. Combined top-down and bottom-up HR-MS analyses of the purified Mtb LpqH protein allow, for the first time, to uncover a complex repertoire of about 130 molecular species resulting from the intrinsically heterogeneous combination of lipidation and glycosylation together with some truncations. Direct view on the co-occurring PTMs stoichiometry reveals the presence of functionally distinct LpqH lipidation states and indicates that glycosylation is independent from lipidation. This work allowed the identification of a novel unsuspected phosphorylated form of the unprocessed preprolipoglycoprotein totally absent from the current lipoglycoprotein biogenesis pathway and providing new insights into the biogenesis and functional determinants of the mycobacterial lipoglycoprotein interacting with the host immune PRRs. PMID:28272507

  17. Information theory provides a comprehensive framework for the evaluation of protein structure predictions

    PubMed Central

    Swanson, Rosemarie; Vannucci, Marina; Tsai, Jerry W.

    2008-01-01

    Protein structure prediction has a number of important ad hoc similarity measures for evaluating predictions, but would benefit from a measure that is able to provide a common framework for a broad range of comparisons. Here we show that a mutual information-like measure can provide a comprehensive framework for evaluating protein structure prediction of all types. We discuss the concept of information, its application to secondary structure, and the obstacle to applying it to 3D structure. Based on insights from the secondary structure case, we present an approach to work around the 3D difficulties, and develop a method to measure the mutual information provided by a 3D structure prediction. We integrate the evaluation of all types of protein structure prediction into a single frame work, and compare the amount of information provided by various prediction methods, including secondary structure prediction. Within this broadened framework, the idea that structure is better preserved than sequence during evolution is evaluated quantitatively for the globin family. A nearly perfect sequence match in the globin family corresponds to about 300 bits of information, whereas a nearly perfect structural match for the same two proteins corresponds to about 2500 bits of information, where bits of information describes the probability of obtaining a match of similar closeness by chance. Mutual information provides both a theoretical basis for evaluating structure similarity and an explanatory surround for existing similarity measures. PMID:18704942

  18. Proteomic analysis of scallop hepatopancreatic extract provides insights into marine polysaccharide digestion

    PubMed Central

    Lyu, Qianqian; Jiao, Wenqian; Zhang, Keke; Bao, Zhenmin; Wang, Shi; Liu, Weizhi

    2016-01-01

    Marine polysaccharides are used in a variety of applications, and the enzymes that degrade these polysaccharides are of increasing interest. The main food source of herbivorous marine mollusks is seaweed, and several polysaccharide-degrading enzymes have been extracted from mollusk digestive glands (hepatopancreases). Here, we used a comprehensive proteomic approach to examine the hepatopancreatic proteins of the Zhikong scallop (Chlamys farreri). We identified 435 proteins, the majority of which were lysosomal enzymes and carbohydrate and protein metabolism enzymes. However, several new enzymes related to polysaccharide metabolism were also identified. Phylogenetic and structural analyses of these enzymes suggest that these polysaccharide-degrading enzymes may have a variety of potential substrate specificities. Taken together, our study characterizes several novel polysaccharide-degrading enzymes in the scallop hepatopancreas and provides an enhanced view of these enzymes and a greater understanding of marine polysaccharide digestion. PMID:27982037

  19. Electron density deformations provide new insights into the spectral shift of rhodopsins.

    PubMed

    Hernández-Rodríguez, Erix Wiliam; Montero-Alejo, Ana Lilian; López, Rafael; Sánchez-García, Elsa; Montero-Cabrera, Luis Alberto; de la Vega, José Manuel García

    2013-10-30

    Spectral shifts of rhodopsin, which are related to variations of the electron distribution in 11-cis-retinal, are investigated here using the method of deformed atoms in molecules. We found that systems carrying the M207R and S186W mutations display large perturbations of the π-conjugated system with respect to wild-type rhodopsins. These changes agree with the predicted behavior of the bond length alternation (BLA) and the blue shifts of vertical excitation energies of these systems. The effect of the planarity of the central and Schiff-base regions of retinal chain on the electronic structure of the chromophore is also investigated. By establishing nonlinear polynomial relations between BLA, chain distortions, and vertical excitation energies, we are also able to provide a semiquantitative approach for the understanding of the mechanisms regulating spectral shifts in rhodopsin and its mutants.

  20. Variation in breeding phenology provides insights into drivers of long-term population change in harbour seals

    PubMed Central

    Cordes, Line S.; Thompson, Paul M.

    2013-01-01

    Phenological trends provide important indicators of environmental change and population dynamics. However, the use of untested population-level measures can lead to incorrect conclusions about phenological trends, particularly when changes in population structure or density are ignored. We used individual-based estimates of birth date and lactation duration of harbour seals (Phoca vitulina) to investigate energetic consequences of changes in pupping phenology. Using generalized linear mixed models, we first demonstrate annual variation in pupping phenology. Second, we show a negative relationship between lactation duration and the timing of pupping, indicating that females who pup early nurse their pups longer, thereby highlighting lactation duration as a useful proxy of female condition and resource availability. Third, individual-based data were used to derive a population-level proxy that demonstrated an advance in pupping date over the last 25 years, co-incident with a reduction in population abundance that resulted from fisheries-related shootings. These findings demonstrate that phenological studies examining the impacts of climate change on mammal populations must carefully control for changes in population density and highlight how joint investigations of phenological and demographic change provide insights into the drivers of population declines. PMID:23782881

  1. Variation in breeding phenology provides insights into drivers of long-term population change in harbour seals.

    PubMed

    Cordes, Line S; Thompson, Paul M

    2013-08-07

    Phenological trends provide important indicators of environmental change and population dynamics. However, the use of untested population-level measures can lead to incorrect conclusions about phenological trends, particularly when changes in population structure or density are ignored. We used individual-based estimates of birth date and lactation duration of harbour seals (Phoca vitulina) to investigate energetic consequences of changes in pupping phenology. Using generalized linear mixed models, we first demonstrate annual variation in pupping phenology. Second, we show a negative relationship between lactation duration and the timing of pupping, indicating that females who pup early nurse their pups longer, thereby highlighting lactation duration as a useful proxy of female condition and resource availability. Third, individual-based data were used to derive a population-level proxy that demonstrated an advance in pupping date over the last 25 years, co-incident with a reduction in population abundance that resulted from fisheries-related shootings. These findings demonstrate that phenological studies examining the impacts of climate change on mammal populations must carefully control for changes in population density and highlight how joint investigations of phenological and demographic change provide insights into the drivers of population declines.

  2. The molecular analysis of Trypanosoma cruzi metallocarboxypeptidase 1 provides insight into fold and substrate specificity.

    PubMed

    Niemirowicz, Gabriela; Fernández, Daniel; Solà, Maria; Cazzulo, Juan J; Avilés, Francesc X; Gomis-Rüth, F Xavier

    2008-11-01

    Trypanosoma cruzi is the aetiological agent of Chagas' disease, a chronic infection that affects millions in Central and South America. Proteolytic enzymes are involved in the development and progression of this disease and two metallocarboxypeptidases, isolated from T. cruzi CL Brener clone, have recently been characterized: TcMCP-1 and TcMCP-2. Although both are cytosolic and closely related in sequence, they display different temporary expression patterns and substrate preferences. TcMCP-1 removes basic C-terminal residues, whereas TcMCP-2 prefers hydrophobic/aromatic residues. Here we report the three-dimensional structure of TcMCP-1. It resembles an elongated cowry, with a long, deep, narrow active-site cleft mimicking the aperture. It has an N-terminal dimerization subdomain, involved in a homodimeric catalytically active quaternary structure arrangement, and a proteolytic subdomain partitioned by the cleft into an upper and a lower moiety. The cleft accommodates a catalytic metal ion, most likely a cobalt, which is co-ordinated by residues included in a characteristic zinc-binding sequence, HEXXH and a downstream glutamate. The structure of TcMCP-1 shows strong topological similarity with archaeal, bacterial and mammalian metallopeptidases including angiotensin-converting enzyme, neurolysin and thimet oligopeptidase. A crucial residue for shaping the S(1') pocket in TcMCP-1, Met-304, was mutated to the respective residue in TcMCP-2, an arginine, leading to a TcMCP-1 variant with TcMCP-2 specificity. The present studies pave the way for a better understanding of a potential target in Chagas' disease at the molecular level and provide a template for the design of novel therapeutic approaches.

  3. Directed Evolution of a Model Primordial Enzyme Provides Insights into the Development of the Genetic Code

    PubMed Central

    Müller, Manuel M.; Allison, Jane R.; Hongdilokkul, Narupat; Gaillon, Laurent; Kast, Peter; van Gunsteren, Wilfred F.; Marlière, Philippe; Hilvert, Donald

    2013-01-01

    The contemporary proteinogenic repertoire contains 20 amino acids with diverse functional groups and side chain geometries. Primordial proteins, in contrast, were presumably constructed from a subset of these building blocks. Subsequent expansion of the proteinogenic alphabet would have enhanced their capabilities, fostering the metabolic prowess and organismal fitness of early living systems. While the addition of amino acids bearing innovative functional groups directly enhances the chemical repertoire of proteomes, the inclusion of chemically redundant monomers is difficult to rationalize. Here, we studied how a simplified chorismate mutase evolves upon expanding its amino acid alphabet from nine to potentially 20 letters. Continuous evolution provided an enhanced enzyme variant that has only two point mutations, both of which extend the alphabet and jointly improve protein stability by >4 kcal/mol and catalytic activity tenfold. The same, seemingly innocuous substitutions (Ile→Thr, Leu→Val) occurred in several independent evolutionary trajectories. The increase in fitness they confer indicates that building blocks with very similar side chain structures are highly beneficial for fine-tuning protein structure and function. PMID:23300488

  4. The Manifest Association Structure of the Single-Factor Model: Insights from Partial Correlations

    ERIC Educational Resources Information Center

    Salgueiro, Maria de Fatima; Smith, Peter W. F.; McDonald, John W.

    2008-01-01

    The association structure between manifest variables arising from the single-factor model is investigated using partial correlations. The additional insights to the practitioner provided by partial correlations for detecting a single-factor model are discussed. The parameter space for the partial correlations is presented, as are the patterns of…

  5. The channel catfish genome sequence provides insights into the evolution of scale formation in teleosts

    PubMed Central

    Liu, Zhanjiang; Liu, Shikai; Yao, Jun; Bao, Lisui; Zhang, Jiaren; Li, Yun; Jiang, Chen; Sun, Luyang; Wang, Ruijia; Zhang, Yu; Zhou, Tao; Zeng, Qifan; Fu, Qiang; Gao, Sen; Li, Ning; Koren, Sergey; Jiang, Yanliang; Zimin, Aleksey; Xu, Peng; Phillippy, Adam M.; Geng, Xin; Song, Lin; Sun, Fanyue; Li, Chao; Wang, Xiaozhu; Chen, Ailu; Jin, Yulin; Yuan, Zihao; Yang, Yujia; Tan, Suxu; Peatman, Eric; Lu, Jianguo; Qin, Zhenkui; Dunham, Rex; Li, Zhaoxia; Sonstegard, Tad; Feng, Jianbin; Danzmann, Roy G.; Schroeder, Steven; Scheffler, Brian; Duke, Mary V.; Ballard, Linda; Kucuktas, Huseyin; Kaltenboeck, Ludmilla; Liu, Haixia; Armbruster, Jonathan; Xie, Yangjie; Kirby, Mona L.; Tian, Yi; Flanagan, Mary Elizabeth; Mu, Weijie; Waldbieser, Geoffrey C.

    2016-01-01

    Catfish represent 12% of teleost or 6.3% of all vertebrate species, and are of enormous economic value. Here we report a high-quality reference genome sequence of channel catfish (Ictalurus punctatus), the major aquaculture species in the US. The reference genome sequence was validated by genetic mapping of 54,000 SNPs, and annotated with 26,661 predicted protein-coding genes. Through comparative analysis of genomes and transcriptomes of scaled and scaleless fish and scale regeneration experiments, we address the genomic basis for the most striking physical characteristic of catfish, the evolutionary loss of scales and provide evidence that lack of secretory calcium-binding phosphoproteins accounts for the evolutionary loss of scales in catfish. The channel catfish reference genome sequence, along with two additional genome sequences and transcriptomes of scaled catfishes, provide crucial resources for evolutionary and biological studies. This work also demonstrates the power of comparative subtraction of candidate genes for traits of structural significance. PMID:27249958

  6. The Epigenome of Schistosoma mansoni Provides Insight about How Cercariae Poise Transcription until Infection

    PubMed Central

    Freitag, Michael; Parrinello, Hugues; Groth, Marco; Emans, Rémi; Cosseau, Céline; Grunau, Christoph

    2015-01-01

    Background Chromatin structure can control gene expression and can define specific transcription states. For example, bivalent methylation of histone H3K4 and H3K27 is linked to poised transcription in vertebrate embryonic stem cells (ESC). It allows them to rapidly engage specific developmental pathways. We reasoned that non-vertebrate metazoans that encounter a similar developmental constraint (i.e. to quickly start development into a new phenotype) might use a similar system. Schistosomes are parasitic platyhelminthes that are characterized by passage through two hosts: a mollusk as intermediate host and humans or rodents as definitive host. During its development, the parasite undergoes drastic changes, most notable immediately after infection of the definitive host, i.e. during the transition from the free-swimming cercariae into adult worms. Methodology/Principal Findings We used Chromatin Immunoprecipitation followed by massive parallel sequencing (ChIP-Seq) to analyze genome-wide chromatin structure of S. mansoni on the level of histone modifications (H3K4me3, H3K27me3, H3K9me3, and H3K9ac) in cercariae, schistosomula and adults (available at http://genome.univ-perp.fr). We saw striking differences in chromatin structure between the developmental stages, but most importantly we found that cercariae possess a specific combination of marks at the transcription start sites (TSS) that has similarities to a structure found in ESC. We demonstrate that in cercariae no transcription occurs, and we provide evidences that cercariae do not possess large numbers of canonical stem cells. Conclusions/Significance We describe here a broad view on the epigenome of a metazoan parasite. Most notably, we find bivalent histone H3 methylation in cercariae. Methylation of H3K27 is removed during transformation into schistosomula (and stays absent in adults) and transcription is activated. In addition, shifts of H3K9 methylation and acetylation occur towards upstream and

  7. Signal Transduction in Histidine Kinases: Insights from New Structures

    PubMed Central

    Bhate, Manasi P.; Molnar, Kathleen S.; Goulian, Mark; DeGrado, William F.

    2015-01-01

    Histidine kinases (HKs) are major players in bacterial signaling. There has been an explosion of new HK crystal structures in the last five years. We globally analyze the structures of HKs to yield insights into the mechanisms by which signals are transmitted to and across protein structures in this family. We interpret known enzymological data in the context of new structural data to show how asymmetry across the dimer interface is a key feature of signal transduction in HKs, and discuss how different HK domains undergo asymmetric-to-symmetric transitions during signal transduction and catalysis. A thermodynamic framework for signaling that encompasses these various properties is presented and the consequences of weak thermodynamic coupling are discussed. The synthesis of observations from enzymology, structural biology, protein engineering and thermodynamics paves the way for a deeper molecular understanding of histidine kinase signal transduction. PMID:25982528

  8. Discovery of cyclotides in the fabaceae plant family provides new insights into the cyclization, evolution, and distribution of circular proteins.

    PubMed

    Poth, Aaron G; Colgrave, Michelle L; Philip, Reynold; Kerenga, Bomai; Daly, Norelle L; Anderson, Marilyn A; Craik, David J

    2011-04-15

    Cyclotides are plant proteins whose defining structural features are a head-to-tail cyclized backbone and three interlocking disulfide bonds, which in combination are known as a cyclic cystine knot. This unique structural motif confers cyclotides with exceptional resistance to proteolysis. Their endogenous function is thought to be as plant defense agents, associated with their insecticidal and larval growth-inhibitory properties. However, in addition, an array of pharmaceutically relevant biological activities has been ascribed to cyclotides, including anti-HIV, anthelmintic, uterotonic, and antimicrobial effects. So far, >150 cyclotides have been elucidated from members of the Rubiaceae, Violaceae, and Cucurbitaceae plant families, but their wider distribution among other plant families remains unclear. Clitoria ternatea (Butterfly pea) is a member of plant family Fabaceae and through its usage in traditional medicine to aid childbirth bears similarity to Oldenlandia affinis, from which many cyclotides have been isolated. Using a combination of nanospray and matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) analyses, we examined seed extracts of C. ternatea and discovered cyclotides in the Fabaceae, the third-largest family of flowering plants. We characterized 12 novel cyclotides, thus expanding knowledge of cyclotide distribution and evolution within the plant kingdom. The discovery of cyclotides containing novel sequence motifs near the in planta cyclization site has provided new insights into cyclotide biosynthesis. In particular, MS analyses of the novel cyclotides from C. ternatea suggest that Asn to Asp variants at the cyclization site are more common than previously recognized. Moreover, this study provides impetus for the examination of other economically and agriculturally significant species within Fabaceae, now the largest plant family from which cyclotides have been described.

  9. Battling Carpal Tunnel Syndrome through Ergonomics: A Case Study of Texas A&M's Library Provides Insights and Answers.

    ERIC Educational Resources Information Center

    Thornton, Joyce K.

    1995-01-01

    Current library automation practices and new technologies have forced library managers to seek some means of reducing carpal tunnel syndrome, and a case study of Texas A&M's library provides insights. Highlights include identifying and assessing the injuries, adjusting work surfaces, testing and selecting new keyboards, and developing…

  10. The Medicago Genome Provides Insight into the Evolution of Rhizobial Symbioses

    PubMed Central

    Young, Nevin D.; Debellé, Frédéric; Oldroyd, Giles E. D.; Geurts, Rene; Cannon, Steven B.; Udvardi, Michael K.; Benedito, Vagner A.; Mayer, Klaus F. X.; Gouzy, Jérôme; Schoof, Heiko; Van de Peer, Yves; Proost, Sebastian; Cook, Douglas R.; Meyers, Blake C.; Spannagl, Manuel; Cheung, Foo; De Mita, Stéphane; Krishnakumar, Vivek; Gundlach, Heidrun; Zhou, Shiguo; Mudge, Joann; Bharti, Arvind K.; Murray, Jeremy D.; Naoumkina, Marina A.; Rosen, Benjamin; Silverstein, Kevin A. T.; Tang, Haibao; Rombauts, Stephane; Zhao, Patrick X.; Zhou, Peng; Barbe, Valérie; Bardou, Philippe; Bechner, Michael; Bellec, Arnaud; Berger, Anne; Bergès, Hélène; Bidwell, Shelby; Bisseling, Ton; Choisne, Nathalie; Couloux, Arnaud; Denny, Roxanne; Deshpande, Shweta; Dai, Xinbin; Doyle, Jeff; Dudez, Anne-Marie; Farmer, Andrew D.; Fouteau, Stéphanie; Franken, Carolien; Gibelin, Chrystel; Gish, John; Goldstein, Steven; González, Alvaro J.; Green, Pamela J.; Hallab, Asis; Hartog, Marijke; Hua, Axin; Humphray, Sean; Jeong, Dong-Hoon; Jing, Yi; Jöcker, Anika; Kenton, Steve M.; Kim, Dong-Jin; Klee, Kathrin; Lai, Hongshing; Lang, Chunting; Lin, Shaoping; Macmil, Simone L; Magdelenat, Ghislaine; Matthews, Lucy; McCorrison, Jamison; Monaghan, Erin L.; Mun, Jeong-Hwan; Najar, Fares Z.; Nicholson, Christine; Noirot, Céline; O’Bleness, Majesta; Paule, Charles R.; Poulain, Julie; Prion, Florent; Qin, Baifang; Qu, Chunmei; Retzel, Ernest F.; Riddle, Claire; Sallet, Erika; Samain, Sylvie; Samson, Nicolas; Sanders, Iryna; Saurat, Olivier; Scarpelli, Claude; Schiex, Thomas; Segurens, Béatrice; Severin, Andrew J.; Sherrier, D. Janine; Shi, Ruihua; Sims, Sarah; Singer, Susan R.; Sinharoy, Senjuti; Sterck, Lieven; Viollet, Agnès; Wang, Bing-Bing; Wang, Keqin; Wang, Mingyi; Wang, Xiaohong; Warfsmann, Jens; Weissenbach, Jean; White, Doug D.; White, Jim D.; Wiley, Graham B.; Wincker, Patrick; Xing, Yanbo; Yang, Limei; Yao, Ziyun; Ying, Fu; Zhai, Jixian; Zhou, Liping; Zuber, Antoine; Dénarié, Jean; Dixon, Richard A.; May, Gregory D.; Schwartz, David C.; Rogers, Jane; Quétier, Francis; Town, Christopher D.; Roe, Bruce A.

    2011-01-01

    Legumes (Fabaceae or Leguminosae) are unique among cultivated plants for their ability to carry out endosymbiotic nitrogen fixation with rhizobial bacteria, a process that takes place in a specialized structure known as the nodule. Legumes belong to one of the two main groups of eurosids, the Fabidae, which includes most species capable of endosymbiotic nitrogen fixation 1. Legumes comprise several evolutionary lineages derived from a common ancestor 60 million years ago (Mya). Papilionoids are the largest clade, dating nearly to the origin of legumes and containing most cultivated species 2. Medicago truncatula (Mt) is a long-established model for the study of legume biology. Here we describe the draft sequence of the Mt euchromatin based on a recently completed BAC-assembly supplemented with Illumina-shotgun sequence, together capturing ~94% of all Mt genes. A whole-genome duplication (WGD) approximately 58 Mya played a major role in shaping the Mt genome and thereby contributed to the evolution of endosymbiotic nitrogen fixation. Subsequent to the WGD, the Mt genome experienced higher levels of rearrangement than two other sequenced legumes, Glycine max (Gm) and Lotus japonicus (Lj). Mt is a close relative of alfalfa (M. sativa), a widely cultivated crop with limited genomics tools and complex autotetraploid genetics. As such, the Mt genome sequence provides significant opportunities to expand alfalfa’s genomic toolbox. PMID:22089132

  11. Ancient DNA from Nubian and Somali wild ass provides insights into donkey ancestry and domestication.

    PubMed

    Kimura, Birgitta; Marshall, Fiona B; Chen, Shanyuan; Rosenbom, Sónia; Moehlman, Patricia D; Tuross, Noreen; Sabin, Richard C; Peters, Joris; Barich, Barbara; Yohannes, Hagos; Kebede, Fanuel; Teclai, Redae; Beja-Pereira, Albano; Mulligan, Connie J

    2011-01-07

    Genetic data from extant donkeys (Equus asinus) have revealed two distinct mitochondrial DNA haplogroups, suggestive of two separate domestication events in northeast Africa about 5000 years ago. Without distinct phylogeographic structure in domestic donkey haplogroups and with little information on the genetic makeup of the ancestral African wild ass, however, it has been difficult to identify wild ancestors and geographical origins for the domestic mitochondrial clades. Our analysis of ancient archaeological and historic museum samples provides the first genetic information on the historic Nubian wild ass (Equus africanus africanus), Somali wild ass (Equus africanus somaliensis) and ancient donkey. The results demonstrate that the Nubian wild ass was an ancestor of the first donkey haplogroup. In contrast, the Somali wild ass has considerable mitochondrial divergence from the Nubian wild ass and domestic donkeys. These findings resolve the long-standing issue of the role of the Nubian wild ass in the domestication of the donkey, but raise new questions regarding the second ancestor for the donkey. Our results illustrate the complexity of animal domestication, and have conservation implications for critically endangered Nubian and Somali wild ass.

  12. Structural and Mechanistic Insights into C-P Bond Hydrolysis by Phosphonoacetate Hydrolase

    SciTech Connect

    Agarwal, Vinayak; Borisova, Svetlana A.; Metcalf, William W.; van der Donk, Wilfred A.; Nair, Satish K.

    2011-12-22

    Bacteria have evolved pathways to metabolize phosphonates as a nutrient source for phosphorus. In Sinorhizobium meliloti 1021, 2-aminoethylphosphonate is catabolized to phosphonoacetate, which is converted to acetate and inorganic phosphate by phosphonoacetate hydrolase (PhnA). Here we present detailed biochemical and structural characterization of PhnA that provides insights into the mechanism of C-P bond cleavage. The 1.35 {angstrom} resolution crystal structure reveals a catalytic core similar to those of alkaline phosphatases and nucleotide pyrophosphatases but with notable differences, such as a longer metal-metal distance. Detailed structure-guided analysis of active site residues and four additional cocrystal structures with phosphonoacetate substrate, acetate, phosphonoformate inhibitor, and a covalently bound transition state mimic provide insight into active site features that may facilitate cleavage of the C-P bond. These studies expand upon the array of reactions that can be catalyzed by enzymes of the alkaline phosphatase superfamily.

  13. Structural and Mechanistic Insights into C-P Bond Hydrolysis by Phosphonoacetate Hydrolase

    PubMed Central

    Agarwal, Vinayak; Borisova, Svetlana A.; Metcalf, William W.; van der Donk, Wilfred A.; Nair, Satish K.

    2015-01-01

    SUMMARY Bacteria have evolved pathways to metabolize phosphonates as a nutrient source for phosphorus. In Sinorhizobium meliloti 1021, 2-aminoethylphosphonate is catabolized to phosphonoacetate, which is converted to acetate and inorganic phosphate by phosphonoacetate hydrolase (PhnA). Here we present detailed biochemical and structural characterization of PhnA that provides insights into the mechanism of C-P bond cleavage. The 1.35 Å resolution crystal structure reveals a catalytic core similar to those of alkaline phosphatases and nucleotide pyrophosphatases, but with notable differences such as a longer metal-metal distance. Detailed structure-guided analysis of active site residues and four additional co-crystal structures with phosphonoacetate substrate, acetate, phosphonoformate inhibitor, and a covalently-bound transition state mimic, provide insight into active site features that may facilitate cleavage of the C-P bond. These studies expand upon the array of reactions that can be catalyzed by enzymes of the alkaline phosphatase superfamily. PMID:22035792

  14. Analysis of the Phlebiopsis gigantea Genome, Transcriptome and Secretome Provides Insight into Its Pioneer Colonization Strategies of Wood

    DOE PAGES

    Hori, Chiaki; Ishida, Takuya; Igarashi, Kiyohiko; ...

    2014-12-04

    Collectively classified as white-rot fungi, certain basidiomycetes efficiently degrade the major structural polymers of wood cell walls. A small subset of these Agaricomycetes, exemplified by Phlebiopsis gigantea, is capable of colonizing freshly exposed conifer sapwood despite its high content of extractives, which retards the establishment of other fungal species. The mechanism(s) by which P. gigantea tolerates and metabolizes resinous compounds have not been explored. Here, we report the annotated P. gigantea genome and compare profiles of its transcriptome and secretome when cultured on freshcut versus solvent-extracted loblolly pine wood. The P. gigantea genome contains a conventional repertoire of hydrolase genesmore » involved in cellulose/hemicellulose degradation, whose patterns of expression were relatively unperturbed by the absence of extractives. The expression of genes typically ascribed to lignin degradation was also largely unaffected. In contrast, genes likely involved in the transformation and detoxification of wood extractives were highly induced in its presence. Their products included an ABC transporter, lipases, cytochrome P450s, glutathione S-transferase and aldehyde dehydrogenase. Other regulated genes of unknown function and several constitutively expressed genes are also likely involved in P. gigantea’s extractives metabolism. These results contribute to our fundamental understanding of pioneer colonization of conifer wood and provide insight into the diverse chemistries employed by fungi in carbon cycling processes.« less

  15. Analysis of the Phlebiopsis gigantea Genome, Transcriptome and Secretome Provides Insight into Its Pioneer Colonization Strategies of Wood

    SciTech Connect

    Hori, Chiaki; Ishida, Takuya; Igarashi, Kiyohiko; Samejima, Masahiro; Suzuki, Hitoshi; Master, Emma; Ferreira, Patricia; Ruiz-Dueñas, Francisco J.; Held, Benjamin; Canessa, Paulo; Larrondo, Luis F.; Schmoll, Monika; Druzhinina, Irina S.; Kubicek, Christian P.; Gaskell, Jill A.; Kersten, Phil; St. John, Franz; Glasner, Jeremy; Sabat, Grzegorz; Splinter BonDurant, Sandra; Syed, Khajamohiddin; Yadav, Jagjit; Mgbeahuruike, Anthony C.; Kovalchuk, Andriy; Asiegbu, Fred O.; Lackner, Gerald; Hoffmeister, Dirk; Rencoret, Jorge; Gutiérrez, Ana; Sun, Hui; Lindquist, Erika; Barry, Kerrie; Riley, Robert; Grigoriev, Igor V.; Henrissat, Bernard; Berka, Randy M.; Martínez, Angel T.; Covert, Sarah F.; Blanchette, Robert A.; Cullen, Daniel; Copenhaver, Gregory P.

    2014-12-04

    Collectively classified as white-rot fungi, certain basidiomycetes efficiently degrade the major structural polymers of wood cell walls. A small subset of these Agaricomycetes, exemplified by Phlebiopsis gigantea, is capable of colonizing freshly exposed conifer sapwood despite its high content of extractives, which retards the establishment of other fungal species. The mechanism(s) by which P. gigantea tolerates and metabolizes resinous compounds have not been explored. Here, we report the annotated P. gigantea genome and compare profiles of its transcriptome and secretome when cultured on freshcut versus solvent-extracted loblolly pine wood. The P. gigantea genome contains a conventional repertoire of hydrolase genes involved in cellulose/hemicellulose degradation, whose patterns of expression were relatively unperturbed by the absence of extractives. The expression of genes typically ascribed to lignin degradation was also largely unaffected. In contrast, genes likely involved in the transformation and detoxification of wood extractives were highly induced in its presence. Their products included an ABC transporter, lipases, cytochrome P450s, glutathione S-transferase and aldehyde dehydrogenase. Other regulated genes of unknown function and several constitutively expressed genes are also likely involved in P. gigantea’s extractives metabolism. These results contribute to our fundamental understanding of pioneer colonization of conifer wood and provide insight into the diverse chemistries employed by fungi in carbon cycling processes.

  16. Proteomic Analysis of the Eyespot of Chlamydomonas reinhardtii Provides Novel Insights into Its Components and Tactic Movements[W

    PubMed Central

    Schmidt, Melanie; Geßner, Gunther; Luff, Matthias; Heiland, Ines; Wagner, Volker; Kaminski, Marc; Geimer, Stefan; Eitzinger, Nicole; Reißenweber, Tobias; Voytsekh, Olga; Fiedler, Monika; Mittag, Maria; Kreimer, Georg

    2006-01-01

    Flagellate green algae have developed a visual system, the eyespot apparatus, which allows the cell to phototax. To further understand the molecular organization of the eyespot apparatus and the phototactic movement that is controlled by light and the circadian clock, a detailed understanding of all components of the eyespot apparatus is needed. We developed a procedure to purify the eyespot apparatus from the green model alga Chlamydomonas reinhardtii. Its proteomic analysis resulted in the identification of 202 different proteins with at least two different peptides (984 in total). These data provide new insights into structural components of the eyespot apparatus, photoreceptors, retina(l)-related proteins, members of putative signaling pathways for phototaxis and chemotaxis, and metabolic pathways within an algal visual system. In addition, we have performed a functional analysis of one of the identified putative components of the phototactic signaling pathway, casein kinase 1 (CK1). CK1 is also present in the flagella and thus is a promising candidate for controlling behavioral responses to light. We demonstrate that silencing CK1 by RNA interference reduces its level in both flagella and eyespot. In addition, we show that silencing of CK1 results in severe disturbances in hatching, flagellum formation, and circadian control of phototaxis. PMID:16798888

  17. Analysis of the Phlebiopsis gigantea Genome, Transcriptome and Secretome Provides Insight into Its Pioneer Colonization Strategies of Wood

    PubMed Central

    Hori, Chiaki; Ishida, Takuya; Igarashi, Kiyohiko; Samejima, Masahiro; Suzuki, Hitoshi; Master, Emma; Ferreira, Patricia; Ruiz-Dueñas, Francisco J.; Held, Benjamin; Canessa, Paulo; Larrondo, Luis F.; Schmoll, Monika; Druzhinina, Irina S.; Kubicek, Christian P.; Gaskell, Jill A.; Kersten, Phil; St. John, Franz; Glasner, Jeremy; Sabat, Grzegorz; Splinter BonDurant, Sandra; Syed, Khajamohiddin; Yadav, Jagjit; Mgbeahuruike, Anthony C.; Kovalchuk, Andriy; Asiegbu, Fred O.; Lackner, Gerald; Hoffmeister, Dirk; Rencoret, Jorge; Gutiérrez, Ana; Sun, Hui; Lindquist, Erika; Barry, Kerrie; Riley, Robert; Grigoriev, Igor V.; Henrissat, Bernard; Kües, Ursula; Berka, Randy M.; Martínez, Angel T.; Covert, Sarah F.; Blanchette, Robert A.; Cullen, Daniel

    2014-01-01

    Collectively classified as white-rot fungi, certain basidiomycetes efficiently degrade the major structural polymers of wood cell walls. A small subset of these Agaricomycetes, exemplified by Phlebiopsis gigantea, is capable of colonizing freshly exposed conifer sapwood despite its high content of extractives, which retards the establishment of other fungal species. The mechanism(s) by which P. gigantea tolerates and metabolizes resinous compounds have not been explored. Here, we report the annotated P. gigantea genome and compare profiles of its transcriptome and secretome when cultured on fresh-cut versus solvent-extracted loblolly pine wood. The P. gigantea genome contains a conventional repertoire of hydrolase genes involved in cellulose/hemicellulose degradation, whose patterns of expression were relatively unperturbed by the absence of extractives. The expression of genes typically ascribed to lignin degradation was also largely unaffected. In contrast, genes likely involved in the transformation and detoxification of wood extractives were highly induced in its presence. Their products included an ABC transporter, lipases, cytochrome P450s, glutathione S-transferase and aldehyde dehydrogenase. Other regulated genes of unknown function and several constitutively expressed genes are also likely involved in P. gigantea's extractives metabolism. These results contribute to our fundamental understanding of pioneer colonization of conifer wood and provide insight into the diverse chemistries employed by fungi in carbon cycling processes. PMID:25474575

  18. Chimeric DCL1-Partnering Proteins Provide Insights into the MicroRNA Pathway

    PubMed Central

    Reis, Rodrigo S.; Eamens, Andrew L.; Roberts, Thomas H.; Waterhouse, Peter M.

    2016-01-01

    In Arabidopsis thaliana, efficient microRNA (miRNA) production requires DICER-LIKE1 (DCL1) with the assistance of a partnering protein, DOUBLE-STRANDED RNA BINDING1 (DRB1) or DRB2. The presence of either of these DRB proteins is crucial to determine the mode of action of a miRNA; i.e., cleavage or translation inhibition. Here we studied the structural determinants for the role of DRB1 and DRB2 in the miRNA pathway. We developed a series of chimeric vectors encoding different functional domains of DRB1 and DRB2, and expressed these in the drb1 mutant background in Arabidopsis under the control of the native DRB1 promoter. Complementation of the drb1 developmental phenotype was used to assess the biological role that each functional domain of DRB1 and DRB2 mediates in the miRNA-guided transcript cleavage pathway. The DRB1 amino acid sequence differs considerably to that of DRB2, and analysis of drb1 transgenic lines revealed that the first dsRNA-binding domains of DRB1 and DRB2 are functionally similar; in contrast, the dsRBD2 of DRB1 and DRB2 appear functionally distinct. Our bioinformatic analysis further suggests that the C-terminal domain of DRB2 mediates a functional role in the miRNA pathway, whereas its counterpart in DRB1 is known to be dispensable. Our results provide evidence for the differences between DRB1 and DRB2 proteins in vivo, which may be essential for the selection of the miRNA regulatory mechanisms, and suggest that these features are conserved among land plants. PMID:26779232

  19. Spider genomes provide insight into composition and evolution of venom and silk

    PubMed Central

    Sanggaard, Kristian W.; Bechsgaard, Jesper S.; Fang, Xiaodong; Duan, Jinjie; Dyrlund, Thomas F.; Gupta, Vikas; Jiang, Xuanting; Cheng, Ling; Fan, Dingding; Feng, Yue; Han, Lijuan; Huang, Zhiyong; Wu, Zongze; Liao, Li; Settepani, Virginia; Thøgersen, Ida B.; Vanthournout, Bram; Wang, Tobias; Zhu, Yabing; Funch, Peter; Enghild, Jan J.; Schauser, Leif; Andersen, Stig U.; Villesen, Palle; Schierup, Mikkel H; Bilde, Trine; Wang, Jun

    2014-01-01

    Spiders are ecologically important predators with complex venom and extraordinarily tough silk that enables capture of large prey. Here we present the assembled genome of the social velvet spider and a draft assembly of the tarantula genome that represent two major taxonomic groups of spiders. The spider genomes are large with short exons and long introns, reminiscent of mammalian genomes. Phylogenetic analyses place spiders and ticks as sister groups supporting polyphyly of the Acari. Complex sets of venom and silk genes/proteins are identified. We find that venom genes evolved by sequential duplication, and that the toxic effect of venom is most likely activated by proteases present in the venom. The set of silk genes reveals a highly dynamic gene evolution, new types of silk genes and proteins, and a novel use of aciniform silk. These insights create new opportunities for pharmacological applications of venom and biomaterial applications of silk. PMID:24801114

  20. The elite cross-country skier provides unique insights into human exercise physiology.

    PubMed

    Holmberg, H-C

    2015-12-01

    Successful cross-country skiing, one of the most demanding of endurance sports, involves considerable physiological challenges posed by the combined upper- and lower-body effort of varying intensity and duration, on hilly terrain, often at moderate altitude and in a cold environment. Over the years, this unique sport has helped physiologists gain novel insights into the limits of human performance and regulatory capacity. There is a long-standing tradition of researchers in this field working together with coaches and athletes to improve training routines, monitor progress, and refine skiing techniques. This review summarizes research on elite cross-country skiers, with special emphasis on the studies initiated by Professor Bengt Saltin. He often employed exercise as a means to learn more about the human body, successfully engaging elite endurance athletes to improve our understanding of the demands, characteristics, and specific effects associated with different types of exercise.

  1. Whole Genome Sequencing of Mycobacterium africanum Strains from Mali Provides Insights into the Mechanisms of Geographic Restriction

    PubMed Central

    Maiga, Mamoudou; Abeel, Thomas; Shea, Terrance; Desjardins, Christopher A.; Diarra, Bassirou; Baya, Bocar; Sanogo, Moumine; Diallo, Souleymane; Earl, Ashlee M.; Bishai, William R.

    2016-01-01

    Background Mycobacterium africanum, made up of lineages 5 and 6 within the Mycobacterium tuberculosis complex (MTC), causes up to half of all tuberculosis cases in West Africa, but is rarely found outside of this region. The reasons for this geographical restriction remain unknown. Possible reasons include a geographically restricted animal reservoir, a unique preference for hosts of West African ethnicity, and an inability to compete with other lineages outside of West Africa. These latter two hypotheses could be caused by loss of fitness or altered interactions with the host immune system. Methodology/Principal Findings We sequenced 92 MTC clinical isolates from Mali, including two lineage 5 and 24 lineage 6 strains. Our genome sequencing assembly, alignment, phylogeny and average nucleotide identity analyses enabled us to identify features that typify lineages 5 and 6 and made clear that these lineages do not constitute a distinct species within the MTC. We found that in Mali, lineage 6 and lineage 4 strains have similar levels of diversity and evolve drug resistance through similar mechanisms. In the process, we identified a putative novel streptomycin resistance mutation. In addition, we found evidence of person-to-person transmission of lineage 6 isolates and showed that lineage 6 is not enriched for mutations in virulence-associated genes. Conclusions This is the largest collection of lineage 5 and 6 whole genome sequences to date, and our assembly and alignment data provide valuable insights into what distinguishes these lineages from other MTC lineages. Lineages 5 and 6 do not appear to be geographically restricted due to an inability to transmit between West African hosts or to an elevated number of mutations in virulence-associated genes. However, lineage-specific mutations, such as mutations in cell wall structure, secretion systems and cofactor biosynthesis, provide alternative mechanisms that may lead to host specificity. PMID:26751217

  2. Insights into RNA structure and function from genome-wide studies.

    PubMed

    Mortimer, Stefanie A; Kidwell, Mary Anne; Doudna, Jennifer A

    2014-07-01

    A comprehensive understanding of RNA structure will provide fundamental insights into the cellular function of both coding and non-coding RNAs. Although many RNA structures have been analysed by traditional biophysical and biochemical methods, the low-throughput nature of these approaches has prevented investigation of the vast majority of cellular transcripts. Triggered by advances in sequencing technology, genome-wide approaches for probing the transcriptome are beginning to reveal how RNA structure affects each step of protein expression and RNA stability. In this Review, we discuss the emerging relationships between RNA structure and the regulation of gene expression.

  3. Neutral nuclear variation in Baboons (genus Papio) provides insights into their evolutionary and demographic histories.

    PubMed

    Boissinot, Stéphane; Alvarez, Lauren; Giraldo-Ramirez, Juliana; Tollis, Marc

    2014-12-01

    Baboons (genus Papio) are distributed over most of sub-Saharan Africa and in the southern portion of the Arabian Peninsula. Six distinct morphotypes, with clearly defined geographic distributions, are recognized (the olive, chacma, yellow, Guinea, Kinda, and hamadryas baboons). The evolutionary relationships among baboon forms have long been a controversial issue. Phylogenetic analyses based on mitochondrial DNA sequences revealed that the modern baboon morphotypes are mitochondrially paraphyletic or polyphyletic. The discordance between mitochondrial lineages and morphology is indicative of extensive introgressive hybridization between ancestral baboon populations. To gain insights into the evolutionary relationships among morphotypes and their demographic history, we performed an analysis of nuclear variation in baboons. We sequenced 13 noncoding, putatively neutral, nuclear regions, and scored the presence/absence of 18 polymorphic transposable elements in a sample of 45 baboons belonging to five of the six recognized baboon forms. We found that the chacma baboon is the sister-taxon to all other baboons and the yellow baboon is the sister-taxon to an unresolved northern clade containing the olive, Guinea, and hamadryas baboons. We estimated that the diversification of baboons occurred entirely in the Pleistocene, the earliest split dating ∼1.5 million years ago, and that baboons have experienced relatively large and constant effective population sizes for most of their evolutionary history (∼30,000 to 95,000 individuals).

  4. Kynurenine pathway metabolomics predicts and provides mechanistic insight into multiple sclerosis progression

    PubMed Central

    Lim, Chai K.; Bilgin, Ayse; Lovejoy, David B.; Tan, Vanessa; Bustamante, Sonia; Taylor, Bruce V.; Bessede, Alban; Brew, Bruce J.; Guillemin, Gilles J.

    2017-01-01

    Activation of the kynurenine pathway (KP) of tryptophan metabolism results from chronic inflammation and is known to exacerbate progression of neurodegenerative disease. To gain insights into the links between inflammation, the KP and multiple sclerosis (MS) pathogenesis, we investigated the KP metabolomics profile of MS patients. Most significantly, we found aberrant levels of two key KP metabolites, kynurenic acid (KA) and quinolinic acid (QA). The balance between these metabolites is important as it determines overall excitotoxic activity at the N-methyl-D-Aspartate (NMDA) receptor. We also identified that serum KP metabolic signatures in patients can discriminate clinical MS subtypes with high sensitivity and specificity. A C5.0 Decision Tree classification model discriminated the clinical subtypes of MS with a sensitivity of 91%. After validation in another independent cohort, sensitivity was maintained at 85%. Collectively, our studies suggest that abnormalities in the KP may be associated with the switch from early-mild stage MS to debilitating progressive forms of MS and that analysis of KP metabolites in MS patient serum may have application as MS disease biomarkers. PMID:28155867

  5. Neutral Nuclear Variation in Baboons (genus Papio) Provides Insights into their Evolutionary and Demographic Histories

    PubMed Central

    Boissinot, Stéphane; Alvarez, Lauren; Giraldo-Ramirez, Juliana; Tollis, Marc

    2015-01-01

    Baboons (genus Papio) are distributed over most of sub-Saharan Africa and in the southern portion of the Arabian Peninsula. Six distinct morphotypes, with clearly defined geographic distributions, are recognized (the olive, chacma, yellow, Guinea, Kinda and hamadryas baboons). The evolutionary relationships among baboon forms have long been a controversial issue. Phylogenetic analyses based on mitochondrial DNA sequences revealed that the modern baboon morphotypes are mitochondrially paraphyletic or polyphyletic. The discordance between mitochondrial lineages and morphology is indicative of extensive introgressive hybridization between ancestral baboon populations. To gain insights into the evolutionary relationships among morphotypes and their demographic history, we performed an analysis of nuclear variation in baboons. We sequenced 13 non-coding, putatively neutral, nuclear regions and scored the presence/absence of 18 polymorphic transposable elements in a sample of 45 baboons belonging to five of the six recognized baboon forms. We found that the chacma baboon is the sister-taxon to all other baboons and the yellow baboon is the sister-taxon to an unresolved northern clade containing the olive, Guinea and hamadryas baboons. We estimated that the diversification of baboons occurred entirely in the Pleistocene, the earliest split dating ~1.5 million years ago, and that baboons have experienced relatively large and constant population sizes for most of their evolutionary history (~30,000 to 95,000 individuals). PMID:25234435

  6. Strain-specific estimation of epidemic success provides insights into the transmission dynamics of tuberculosis

    PubMed Central

    Rasigade, Jean-Philippe; Barbier, Maxime; Dumitrescu, Oana; Pichat, Catherine; Carret, Gérard; Ronnaux-Baron, Anne-Sophie; Blasquez, Ghislaine; Godin-Benhaim, Christine; Boisset, Sandrine; Carricajo, Anne; Jacomo, Véronique; Fredenucci, Isabelle; Pérouse de Montclos, Michèle; Flandrois, Jean-Pierre; Ader, Florence; Supply, Philip; Lina, Gérard; Wirth, Thierry

    2017-01-01

    The transmission dynamics of tuberculosis involves complex interactions of socio-economic and, possibly, microbiological factors. We describe an analytical framework to infer factors of epidemic success based on the joint analysis of epidemiological, clinical and pathogen genetic data. We derive isolate-specific, genetic distance-based estimates of epidemic success, and we represent success-related time-dependent concepts, namely epidemicity and endemicity, by restricting analysis to specific time scales. The method is applied to analyze a surveillance-based cohort of 1,641 tuberculosis patients with minisatellite-based isolate genotypes. Known predictors of isolate endemicity (older age, native status) and epidemicity (younger age, sputum smear positivity) were identified with high confidence (P < 0.001). Long-term epidemic success also correlated with the ability of Euro-American and Beijing MTBC lineages to cause active pulmonary infection, independent of patient age and country of origin. Our results demonstrate how important insights into the transmission dynamics of tuberculosis can be gained from active surveillance data. PMID:28349973

  7. Phenotypical, physiological and biochemical analyses provide insight into selenium-induced phytotoxicity in rice plants.

    PubMed

    Mostofa, Mohammad Golam; Hossain, Mohammad Anwar; Siddiqui, Md Nurealam; Fujita, Masayuki; Tran, Lam-Son Phan

    2017-03-11

    The present study investigated the phenotypical, physiological and biochemical changes of rice plants exposed to high selenium (Se) concentrations to gain an insight into Se-induced phytotoxicity. Results showed that exposure of rice plants to excessive Se resulted in growth retardation and biomass reduction in connection with the decreased levels of chlorophyll, carotenoids and soluble proteins. The reduced water status and an associated increase in sugar and proline levels indicated Se-induced osmotic stress in rice plants. Measurements of Se contents in roots, leaf sheaths and leaves revealed that Se was highly accumulated in leaves followed by leaf sheaths and roots. Se also potentiated its toxicity by impairing oxidative metabolism, as evidenced by enhanced accumulation of hydrogen peroxide, superoxide and lipid peroxidation product. Se toxicity also displayed a desynchronized antioxidant system by elevating the level of glutathione and the activities of superoxide dismutase, glutathione-S-transferase and glutathione peroxidase, whereas decreasing the level of ascorbic acid and the activities of catalase, glutathione reductase and dehydroascorbate reductase. Furthermore, Se triggered methylglyoxal toxicity by inhibiting the activities of glyoxalases I and II, particularly at higher concentrations of Se. Collectively, our results suggest that excessive Se caused phytotoxic effects on rice plants by inducing chlorosis, reducing sugar, protein and antioxidant contents, and exacerbating oxidative stress and methylglyoxal toxicity. Accumulation levels of Se, proline and glutathione could be considered as efficient biomarkers to indicate degrees of Se-induced phytotoxicity in rice, and perhaps in other crops.

  8. Construction patterns of birds’ nests provide insight into nest-building behaviours

    PubMed Central

    Goodman, Adrian M.

    2017-01-01

    Previous studies have suggested that birds and mammals select materials needed for nest building based on their thermal or structural properties, although the amounts or properties of the materials used have been recorded for only a very small number of species. Some of the behaviours underlying the construction of nests can be indirectly determined by careful deconstruction of the structure and measurement of the biomechanical properties of the materials used. Here we examined this idea in an investigation of Bullfinch (Pyrrhula pyrrhula) nests as a model for open-nesting songbird species that construct a “twig” nest, and tested the hypothesis that materials in different parts of nests serve different functions. The quantities of materials present in the nest base, sides and cup were recorded before structural analysis. Structural analysis showed that the base of the outer nests were composed of significantly thicker, stronger and more rigid materials compared to the side walls, which in turn were significantly thicker, stronger and more rigid than materials used in the cup. These results suggest that the placement of particular materials in nests may not be random, but further work is required to determine if the final structure of a nest accurately reflects the construction process. PMID:28265501

  9. Construction patterns of birds' nests provide insight into nest-building behaviours.

    PubMed

    Biddle, Lucia; Goodman, Adrian M; Deeming, D Charles

    2017-01-01

    Previous studies have suggested that birds and mammals select materials needed for nest building based on their thermal or structural properties, although the amounts or properties of the materials used have been recorded for only a very small number of species. Some of the behaviours underlying the construction of nests can be indirectly determined by careful deconstruction of the structure and measurement of the biomechanical properties of the materials used. Here we examined this idea in an investigation of Bullfinch (Pyrrhula pyrrhula) nests as a model for open-nesting songbird species that construct a "twig" nest, and tested the hypothesis that materials in different parts of nests serve different functions. The quantities of materials present in the nest base, sides and cup were recorded before structural analysis. Structural analysis showed that the base of the outer nests were composed of significantly thicker, stronger and more rigid materials compared to the side walls, which in turn were significantly thicker, stronger and more rigid than materials used in the cup. These results suggest that the placement of particular materials in nests may not be random, but further work is required to determine if the final structure of a nest accurately reflects the construction process.

  10. Proline and lysine residues provide modulatory switches in amyloid formation: Insights from prion protein.

    PubMed

    Kraus, Allison

    2016-01-01

    Amyloidogenic proteins have an increased propensity to reorganize into the highly structured, β sheet rich structures that characterize amyloid. The probability of attaining these highly structured assemblies is influenced by multiple factors, including amino acid composition and environmental conditions. Evolutionary selection for amino acid sequences that prevent amyloid formation could further modulate amyloid-forming propensity. Indeed, we have recently identified specific proline and lysine residues, contained within a highly conserved central region of prion protein (PrP), that impede PrP amyloid formation in vitro. These prolines are mutated in certain forms of the human familial genetic disease, Gerstmann-Straüssler-Schneiker (GSS) syndrome. Here, I discuss the influence of these proline and lysine residues on PrP amyloid formation and how such anti-amyloidogenic primary amino acid sequences might be modulated to influence protein amyloidogenicity.

  11. Comparative molecular epidemiology provides new insights into Zucchini yellow mosaic virus occurrence in France.

    PubMed

    Lecoq, H; Wipf-Scheibel, C; Nozeran, K; Millot, P; Desbiez, C

    2014-06-24

    Zucchini yellow mosaic virus (ZYMV, genus Potyvirus) causes important crop losses in cucurbits worldwide. In France, ZYMV epidemics are sporadic but occasionally very severe. This contrasts with Watermelon mosaic virus (WMV, genus Potyvirus) which causes regular and early epidemics. Factors influencing ZYMV epidemiology are still poorly understood. In order to gain new insights on the ecology and epidemiology of this virus, a 5-year multilocation trial was conducted in which ZYMV spread and populations were studied in each of the 20 plot/year combinations and compared with WMV. Search for ZYMV alternative hosts was conducted by testing weeds growing naturally around one plot and also by checking ZYMV natural infections in selected ornamental species. Although similar ZYMV populations were observed occasionally in the same plot in two successive years suggesting the occurrence of overwintering hosts nearby, only two Lamium amplexicaule plants were found to be infected by ZYMV of 3459 weed samples that were tested. The scarcity of ZYMV reservoirs contrasts with the frequent detection of WMV in the same samples. Since ZYMV and WMV have many aphid vectors in common and are transmitted with similar efficiencies, the differences observed in ZYMV and WMV reservoir abundances could be a major explanatory factor for the differences observed in the typology of ZYMV and WMV epidemics in France. Other potential ZYMV alternative hosts have been identified in ornamental species including begonia. Although possible in a few cases, exchanges of populations between different plots located from 500 m to 4 km apart seem uncommon. Therefore, the potential dissemination range of ZYMV by its aphid vectors seems to be rather limited in a fragmented landscape.

  12. Phylogenetic analyses provide insights into the historical biogeography and evolution of Brachyrhaphis fishes.

    PubMed

    Ingley, Spencer J; Reina, Ruth G; Bermingham, Eldredge; Johnson, Jerald B

    2015-08-01

    The livebearing fish genus Brachyrhaphis (Poeciliidae) has become an increasingly important model in evolution and ecology research, yet the phylogeny of this group is not well understood, nor has it been examined thoroughly using modern phylogenetic methods. Here, we present the first comprehensive phylogenetic analysis of Brachyrhaphis by using four molecular markers (3mtDNA, 1nucDNA) to infer relationships among species in this genus. We tested the validity of this genus as a monophyletic group using extensive outgroup sampling based on recent phylogenetic hypotheses of Poeciliidae. We also tested the validity of recently described species of Brachyrhaphis that are part of the B. episcopi complex in Panama. Finally, we examined the impact of historical events on diversification of Brachyrhaphis, and made predictions regarding the role of different ecological environments on evolutionary diversification where known historical events apparently fail to explain speciation. Based on our results, we reject the monophyly of Brachyrhaphis, and question the validity of two recently described species (B. hessfeldi and B. roswithae). Historical biogeography of Brachyrhaphis generally agrees with patterns found in other freshwater taxa in Lower Central America, which show that geological barriers frequently predict speciation. Specifically, we find evidence in support of an 'island' model of Lower Central American formation, which posits that the nascent isthmus was partitioned by several marine connections before linking North and South America. In some cases where historic events (e.g., vicariance) fail to explain allopatric species breaks in Brachyrhaphis, ecological processes (e.g., divergent predation environments) offer additional insight into our understanding of phylogenetic diversification in this group.

  13. Insights into structural mechanisms of gating induced regulation of aquaporins.

    PubMed

    Sachdeva, Ruchi; Singh, Balvinder

    2014-04-01

    Aquaporin family comprises of transmembrane channels that are specialized in conducting water and certain small, uncharged molecules across cell membranes. Essential roles of aquaporins in various physiological and pathophysiological conditions have attracted great scientific interest. Pioneering structural studies on aquaporins have almost solved the basic question of mechanism of selective water transport through these channels. Another important structural aspect of aquaporins which seeks attention is that how the flow of water through the channel is regulated by the mechanism of gating. Aquaporins are also regulated at the protein level, i.e. by trafficking which includes changes in their expression levels in the membrane. Availability of high resolution structures along with numerous molecular dynamics simulation studies have helped to gain an understanding of the structural mechanisms by which water flux through aquaporins is controlled. This review will summarize the highlights regarding structural features of aquaporins, mechanisms governing water permeation, proton exclusion and substrate specificity, and describe the structural insights into the mechanisms of aquaporin gating whereby water conduction is regulated by post translational modifications, such as phosphorylation.

  14. Targeted protein engineering provides insights into binding mechanism and affinities of bacterial collagen adhesins.

    PubMed

    Ross, Caná L; Liang, Xiaowen; Liu, Qing; Murray, Barbara E; Höök, Magnus; Ganesh, Vannakambadi K

    2012-10-05

    The collagen-binding bacterial proteins, Ace and Cna, are well characterized on the biochemical and structural level. Despite overall structural similarity, recombinant forms of the Ace and Cna ligand-binding domains exhibit significantly different affinities and binding kinetics for collagen type I (CI) in vitro. In this study, we sought to understand, in submolecular detail, the bases for these differences. Using a structure-based approach, we engineered Cna and Ace variants by altering specific structural elements within the ligand-binding domains. Surface plasmon resonance-based binding analysis demonstrated that mutations that are predicted to alter the orientation of the Ace and Cna N(1) and N(2) subdomains significantly affect the interaction between the MSCRAMM (microbial surface components recognizing adhesive matrix molecule) and CI in vitro, including affinity, association/dissociation rates and binding ratio. Moreover, we utilized this information to engineer an Ace variant with an 11,000-fold higher CI affinity than the parent protein. Finally, we noted that several engineered proteins that exhibited a weak interaction with CI recognized more sites on CI, suggesting an inverse correlation between affinity and specificity.

  15. Structural insight into CIDE domains: the Janus face of CIDEs.

    PubMed

    Park, Hyun Ho

    2015-02-01

    Cell-death inducing DFF45-like effect domain (CIDE domain) is a protein interaction module that was initially found in DNA fragmentation factor (DFF) proteins DFF40 and DFF45. Several other CIDE-containing proteins, CIDE-A, CIDE-B, and CIDE-3, have since been identified in humans. Although the main function of these proteins is associated with apoptosis, recent studies have identified roles of CIDE-containing proteins in energy metabolism, especially involvement in control of the size of lipid droplets. Because CIDE-containing proteins perform critical tasks in apoptosis and energy metabolism and have been linked to many human diseases including cancer and obesity, studies of CIDE domains and CIDE-containing proteins are of great biological importance. This review summarizes the structural insight into CIDE and the CIDE-CIDE complex and speculates on a generalized strategy for the CIDE-CIDE interaction based on the available CIDE structures and molecular modelling.

  16. Barley Brassinosteroid Mutants Provide an Insight into Phytohormonal Homeostasis in Plant Reaction to Drought Stress

    PubMed Central

    Gruszka, Damian; Janeczko, Anna; Dziurka, Michal; Pociecha, Ewa; Oklestkova, Jana; Szarejko, Iwona

    2016-01-01

    Brassinosteroids (BRs) are a class of steroid phytohormones, which regulate various processes of morphogenesis and physiology—from seed development to regulation of flowering and senescence. An accumulating body of evidence indicates that BRs take part in regulation of physiological reactions to various stress conditions, including drought. Many of the physiological functions of BRs are regulated by a complicated, and not fully elucidated network of interactions with metabolic pathways of other phytohormones. Therefore, the aim of this study was to characterize phytohormonal homeostasis in barley (Hordeum vulgare) in reaction to drought and validate role of BRs in regulation of this process. Material of this study included the barley cultivar “Bowman” and five Near-Isogenic Lines (NILs) representing characterized semi-dwarf mutants of several genes encoding enzymes participating in BR biosynthesis and signaling. Analysis of endogenous BRs concentrations in these NILs confirmed that their phenotypes result from abnormalities in BR metabolism. In general, concentrations of 18 compounds, representing various classes of phytohormones, including brassinosteroids, auxins, cytokinins, gibberellins, abscisic acid, salicylic acid and jasmonic acid were analyzed under control and drought conditions in the “Bowman” cultivar and the BR-deficient NILs. Drought induced a significant increase in accumulation of the biologically active form of BRs—castasterone in all analyzed genotypes. Another biologically active form of BRs—24-epi-brassinolide—was identified in one, BR-insensitive NIL under normal condition, but its accumulation was drought-induced in all analyzed genotypes. Analysis of concentration profiles of several compounds representing gibberellins allowed an insight into the BR-dependent regulation of gibberellin biosynthesis. The concentration of the gibberellic acid GA7 was significantly lower in all NILs when compared with the “Bowman” cultivar

  17. Aurone synthase is a catechol oxidase with hydroxylase activity and provides insights into the mechanism of plant polyphenol oxidases.

    PubMed

    Molitor, Christian; Mauracher, Stephan Gerhard; Rompel, Annette

    2016-03-29

    Tyrosinases and catechol oxidases belong to the family of polyphenol oxidases (PPOs). Tyrosinases catalyze theo-hydroxylation and oxidation of phenolic compounds, whereas catechol oxidases were so far defined to lack the hydroxylation activity and catalyze solely the oxidation of o-diphenolic compounds. Aurone synthase from Coreopsis grandiflora (AUS1) is a specialized plant PPO involved in the anabolic pathway of aurones. We present, to our knowledge, the first crystal structures of a latent plant PPO, its mature active and inactive form, caused by a sulfation of a copper binding histidine. Analysis of the latent proenzyme's interface between the shielding C-terminal domain and the main core provides insights into its activation mechanisms. As AUS1 did not accept common tyrosinase substrates (tyrosine and tyramine), the enzyme is classified as a catechol oxidase. However, AUS1 showed hydroxylase activity toward its natural substrate (isoliquiritigenin), revealing that the hydroxylase activity is not correlated with the acceptance of common tyrosinase substrates. Therefore, we propose that the hydroxylase reaction is a general functionality of PPOs. Molecular dynamics simulations of docked substrate-enzyme complexes were performed, and a key residue was identified that influences the plant PPO's acceptance or rejection of tyramine. Based on the evidenced hydroxylase activity and the interactions of specific residues with the substrates during the molecular dynamics simulations, a novel catalytic reaction mechanism for plant PPOs is proposed. The presented results strongly suggest that the physiological role of plant catechol oxidases were previously underestimated, as they might hydroxylate their--so far unknown--natural substrates in vivo.

  18. The teeth and faces of twins: providing insights into dentofacial development and oral health for practising oral health professionals.

    PubMed

    Hughes, T E; Townsend, G C; Pinkerton, S K; Bockmann, M R; Seow, W K; Brook, A H; Richards, L C; Mihailidis, S; Ranjitkar, S; Lekkas, D

    2014-06-01

    The continuing studies of the teeth and faces of Australian twins and their families in the Craniofacial Biology Research Group in the School of Dentistry at the University of Adelaide began 30 years ago. Three main cohorts of twins have been recruited, enabling various objectives and specific hypotheses to be addressed about the roles of genetic, epigenetic and environmental influences on human dentofacial growth and development, as well as oral health. This paper highlights some key findings arising from these studies, emphasizing those of direct relevance to practising oral health professionals. We also draw on published literature to review the significant developments in relation to the use of precision 2D and 3D imaging equipment, the application of modern molecular techniques, and the development of sophisticated computer software for analysing genetic relationships and comparing complex shapes. Such developments are valuable for current and future work. Apart from the classical or traditional twin model, there are several other twin models that can be used in research to clarify the relative contributions of genetic, epigenetic and environmental contributions to phenotypic variation. The monozygotic (MZ) co-twin model is one particularly valuable method, given that examination of only one pair of MZ twins can provide considerable insights into underlying causes of observed variation. This model can be used in a dental practice environment, with oral health professionals having the opportunity to explore differences in orofacial structures between MZ co-twins who are attending as patients. As researchers have become more aware of the complexities of the interactions between the genome, the epigenome and the environment during development, there is the need to collect more phenotypic data and define new phenotypes that will better characterize variations in growth processes and health status. When coupled with powerful new genetic approaches, including genome

  19. A Flexible Binding Site Architecture Provides New Insights into CcpA Global Regulation in Gram-Positive Bacteria

    PubMed Central

    Yang, Yunpeng; Zhang, Lu; Huang, He; Yang, Chen; Yang, Sheng

    2017-01-01

    ABSTRACT Catabolite control protein A (CcpA) is the master regulator in Gram-positive bacteria that mediates carbon catabolite repression (CCR) and carbon catabolite activation (CCA), two fundamental regulatory mechanisms that enable competitive advantages in carbon catabolism. It is generally regarded that CcpA exerts its regulatory role by binding to a typical 14- to 16-nucleotide (nt) consensus site that is called a catabolite response element (cre) within the target regions. However, here we report a previously unknown noncanonical flexible architecture of the CcpA-binding site in solventogenic clostridia, providing new mechanistic insights into catabolite regulation. This novel CcpA-binding site, named crevar, has a unique architecture that consists of two inverted repeats and an intervening spacer, all of which are variable in nucleotide composition and length, except for a 6-bp core palindromic sequence (TGTAAA/TTTACA). It was found that the length of the intervening spacer of crevar can affect CcpA binding affinity, and moreover, the core palindromic sequence of crevar is the key structure for regulation. Such a variable architecture of crevar shows potential importance for CcpA’s diverse and fine regulation. A total of 103 potential crevar sites were discovered in solventogenic Clostridium acetobutylicum, of which 42 sites were picked out for electrophoretic mobility shift assays (EMSAs), and 30 sites were confirmed to be bound by CcpA. These 30 crevar sites are associated with 27 genes involved in many important pathways. Also of significance, the crevar sites are found to be widespread and function in a great number of taxonomically different Gram-positive bacteria, including pathogens, suggesting their global role in Gram-positive bacteria. PMID:28119470

  20. Aurone synthase is a catechol oxidase with hydroxylase activity and provides insights into the mechanism of plant polyphenol oxidases

    PubMed Central

    Molitor, Christian; Mauracher, Stephan Gerhard

    2016-01-01

    Tyrosinases and catechol oxidases belong to the family of polyphenol oxidases (PPOs). Tyrosinases catalyze the o-hydroxylation and oxidation of phenolic compounds, whereas catechol oxidases were so far defined to lack the hydroxylation activity and catalyze solely the oxidation of o-diphenolic compounds. Aurone synthase from Coreopsis grandiflora (AUS1) is a specialized plant PPO involved in the anabolic pathway of aurones. We present, to our knowledge, the first crystal structures of a latent plant PPO, its mature active and inactive form, caused by a sulfation of a copper binding histidine. Analysis of the latent proenzyme’s interface between the shielding C-terminal domain and the main core provides insights into its activation mechanisms. As AUS1 did not accept common tyrosinase substrates (tyrosine and tyramine), the enzyme is classified as a catechol oxidase. However, AUS1 showed hydroxylase activity toward its natural substrate (isoliquiritigenin), revealing that the hydroxylase activity is not correlated with the acceptance of common tyrosinase substrates. Therefore, we propose that the hydroxylase reaction is a general functionality of PPOs. Molecular dynamics simulations of docked substrate–enzyme complexes were performed, and a key residue was identified that influences the plant PPO’s acceptance or rejection of tyramine. Based on the evidenced hydroxylase activity and the interactions of specific residues with the substrates during the molecular dynamics simulations, a novel catalytic reaction mechanism for plant PPOs is proposed. The presented results strongly suggest that the physiological role of plant catechol oxidases were previously underestimated, as they might hydroxylate their—so far unknown—natural substrates in vivo. PMID:26976571

  1. Reconstitution of the augmin complex provides insights into its architecture and function

    PubMed Central

    Hsia, Kuo-Chiang; Wilson-Kubalek, Elizabeth M.; Dottore, Alejandro; Hao, Qi; Tsai, Kuang-Lei; Forth, Scott; Shimamoto, Yuta; Milligan, Ronald A.; Kapoor, Tarun M.

    2014-01-01

    Proper microtubule nucleation during cell division requires augmin, a microtubule-associated hetero-octameric protein complex. In current models, augmin recruits γ-tubulin, via its hDgt6 subunit’s C-terminus, to nucleate microtubules within spindles. However, augmin’s biochemical complexity has restricted analysis of its structural organization and function. Here, we reconstitute human augmin and show it is a Y-shaped complex that can adopt multiple conformations. Further, we find that a dimeric sub-complex retains in vitro microtubule-binding properties of octameric complexes, but not proper metaphase spindle localization. Addition of octameric augmin complexes to Xenopus egg extracts promotes microtubule aster formation, an activity enhanced by Ran-GTP. This activity requires microtubule binding, but not the characterized hDgt6 γ-tubulin-recruitment domain. Tetrameric sub-complexes induce asters, but activity and microtubule bundling within asters are reduced compared to octameric complexes. Together, our findings shed light on augmin’s structural organization, microtubule binding properties and define subunits required for its function in organizing microtubule-based structures. PMID:25173975

  2. The carrot genome provides insights into crop origins and a foundation for future crop improvement

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The sequencing of the carrot genome was an effort that formally began in 2012 and culminated with the publication and release of the genome in 2016. A full genome sequence provides the ultimate foundation to study genetics, gene function, and evolution of a species. The primary goal of the carrot ge...

  3. Variation revealed by SNP genotyping and morphology provides insight into the origin of the tomato.

    PubMed

    Blanca, Jose; Cañizares, Joaquín; Cordero, Laura; Pascual, Laura; Diez, María José; Nuez, Fernando

    2012-01-01

    Tomato, Solanum lycopersicum, is divided into two widely distributed varieties: the cultivated S. lycopersicum var. lycopersicum, and the weedy S. lycopersicum var. cerasiforme. Solanum pimpinellifolium is the most closely related wild species of tomato.The roles of S. pimpinellifolium and S. l. cerasiforme during the domestication of tomato are still under debate. Some authors consider S. l. cerasiforme to be the ancestor, whereas others think that S. l. cerasiforme is an admixture of S. pimpinellifolium and the cultivated S. l. lycopersicum. It is also not clear whether the domestication occurred in the Andean region or in Mesoamerica. We characterized 272 accessions (63 S. pimpinellifolium, 106 S. l. cerasiforme, 95 S. l. lycopersicum and 8 derived from hybridization processes) were morphologically and genetically using the SolCap platform (7,414 SNPs). The two species were distinguished in a PCA analysis and displayed a rich geographic structure. Solanum lycopersicum var. cerasiforme and S. l. lycopersicum were also differentiated in the PCA and Structure analyses, which supports maintaining them as different varieties. Solanum pimpinellifolium and the Andean S. l. cerasiforme were more diverse than the non-Andean S. lycopersicum. Solanum lycopersicum var. cerasiforme was morphologically and molecularly intermediate between S. pimpinellifolium and tomato. Solanum lycopersicum var. cerasiforme, with the exception of several Ecuadorian and Mexican accessions, is composed of the products of admixture processes according to the Structure analysis. The non-admixtured S. l. cerasiforme might be similar to the ancestral cultivars from which the cultivated tomato originated, and presents remarkable morphological diversity, including fruits of up to 6 cm in diameter. The data obtained would fit a model in which a pre-domestication took place in the Andean region, with the domestication being completed in Mesoamerica. Subsequently, the Spaniards took plants from

  4. Variation Revealed by SNP Genotyping and Morphology Provides Insight into the Origin of the Tomato

    PubMed Central

    Cordero, Laura; Pascual, Laura; Diez, María José; Nuez, Fernando

    2012-01-01

    Tomato, Solanum lycopersicum, is divided into two widely distributed varieties: the cultivated S. lycopersicum var. lycopersicum, and the weedy S. lycopersicum var. cerasiforme. Solanum pimpinellifolium is the most closely related wild species of tomato. The roles of S. pimpinellifolium and S. l. cerasiforme during the domestication of tomato are still under debate. Some authors consider S. l. cerasiforme to be the ancestor, whereas others think that S. l. cerasiforme is an admixture of S. pimpinellifolium and the cultivated S. l. lycopersicum. It is also not clear whether the domestication occurred in the Andean region or in Mesoamerica. We characterized 272 accessions (63 S. pimpinellifolium, 106 S. l. cerasiforme, 95 S. l. lycopersicum and 8 derived from hybridization processes) were morphologically and genetically using the SolCap platform (7,414 SNPs). The two species were distinguished in a PCA analysis and displayed a rich geographic structure. Solanum lycopersicum var. cerasiforme and S. l. lycopersicum were also differentiated in the PCA and Structure analyses, which supports maintaining them as different varieties. Solanum pimpinellifolium and the Andean S. l. cerasiforme were more diverse than the non-Andean S. lycopersicum. Solanum lycopersicum var. cerasiforme was morphologically and molecularly intermediate between S. pimpinellifolium and tomato. Solanum lycopersicum var. cerasiforme, with the exception of several Ecuadorian and Mexican accessions, is composed of the products of admixture processes according to the Structure analysis. The non-admixtured S. l. cerasiforme might be similar to the ancestral cultivars from which the cultivated tomato originated, and presents remarkable morphological diversity, including fruits of up to 6 cm in diameter. The data obtained would fit a model in which a pre-domestication took place in the Andean region, with the domestication being completed in Mesoamerica. Subsequently, the Spaniards took plants from

  5. Time-resolved neutron scattering provides new insight into protein substrate processing by a AAA+ unfoldase

    PubMed Central

    Ibrahim, Ziad; Martel, Anne; Moulin, Martine; Kim, Henry S.; Härtlein, Michael; Franzetti, Bruno; Gabel, Frank

    2017-01-01

    We present a combination of small-angle neutron scattering, deuterium labelling and contrast variation, temperature activation and fluorescence spectroscopy as a novel approach to obtain time-resolved, structural data individually from macromolecular complexes and their substrates during active biochemical reactions. The approach allowed us to monitor the mechanical unfolding of a green fluorescent protein model substrate by the archaeal AAA+ PAN unfoldase on the sub-minute time scale. Concomitant with the unfolding of its substrate, the PAN complex underwent an energy-dependent transition from a relaxed to a contracted conformation, followed by a slower expansion to its initial state at the end of the reaction. The results support a model in which AAA ATPases unfold their substrates in a reversible power stroke mechanism involving several subunits and demonstrate the general utility of this time-resolved approach for studying the structural molecular kinetics of multiple protein remodelling complexes and their substrates on the sub-minute time scale. PMID:28102317

  6. Inhalation dosimetry modeling provides insights into regional respiratory tract toxicity of inhaled diacetyl.

    PubMed

    Cichocki, Joseph A; Morris, John B

    2016-11-13

    Vapor dosimetry models provide a means of assessing the role of delivered dose in determining the regional airway response to inspired vapors. A validated hybrid computational fluid dynamics physiologically based pharmacokinetic model for inhaled diacetyl has been developed to describe inhaled diacetyl dosimetry in both the rat and human respiratory tracts. Comparison of the distribution of respiratory tract injury with dosimetry estimates provides strong evidence that regional delivered dose rather than regional airway tissue sensitivity to diacetyl-induced injury is the critical determinant of the regional respiratory tract response to this water soluble reactive vapor. In the rat, inhalation exposure to diacetyl causes much lesser injury in the distal bronchiolar airways compared to nose and large tracheobronchial airways. The degree of injury correlates very strongly to model based estimates of local airway diacetyl concentrations. According to the model, regional dosimetry patterns of diacetyl in the human differ greatly from those in the rat with much greater penetration of diacetyl to the bronchiolar airways in the lightly exercising mouth breathing human compared to the rat, providing evidence that rat inhalation toxicity studies underpredict the risk of bronchiolar injury in the human. For example, repeated exposure of the rat to 200ppm diacetyl results in bronchiolar injury; the estimated bronchiolar tissue concentration in rats exposed to 200ppm diacetyl would occur in lightly exercising mouth breathing humans exposed to 12ppm. Consideration of airway dosimetry patterns of inspired diacetyl is critical to the proper evaluation of rodent toxicity data and its relevance for predicting human risk.

  7. New Insights about Enzyme Evolution from Large Scale Studies of Sequence and Structure Relationships*

    PubMed Central

    Brown, Shoshana D.; Babbitt, Patricia C.

    2014-01-01

    Understanding how enzymes have evolved offers clues about their structure-function relationships and mechanisms. Here, we describe evolution of functionally diverse enzyme superfamilies, each representing a large set of sequences that evolved from a common ancestor and that retain conserved features of their structures and active sites. Using several examples, we describe the different structural strategies nature has used to evolve new reaction and substrate specificities in each unique superfamily. The results provide insight about enzyme evolution that is not easily obtained from studies of one or only a few enzymes. PMID:25210038

  8. New insights about enzyme evolution from large scale studies of sequence and structure relationships.

    PubMed

    Brown, Shoshana D; Babbitt, Patricia C

    2014-10-31

    Understanding how enzymes have evolved offers clues about their structure-function relationships and mechanisms. Here, we describe evolution of functionally diverse enzyme superfamilies, each representing a large set of sequences that evolved from a common ancestor and that retain conserved features of their structures and active sites. Using several examples, we describe the different structural strategies nature has used to evolve new reaction and substrate specificities in each unique superfamily. The results provide insight about enzyme evolution that is not easily obtained from studies of one or only a few enzymes.

  9. In situ neutron diffraction under high pressure—Providing an insight into working catalysts

    NASA Astrophysics Data System (ADS)

    Kandemir, Timur; Wallacher, Dirk; Hansen, Thomas; Liss, Klaus-Dieter; Naumann d'Alnoncourt, Raoul; Schlögl, Robert; Behrens, Malte

    2012-05-01

    In the present work the construction and application of a continuous flow cell is presented, from which neutron diffraction data could be obtained during catalytic reactions at high pressure. By coupling an online gas detection system, parallel structure and activity investigations of working catalysts under industrial relevant conditions are possible. The flow cell can be operated with different feed gases in a wide range from room temperature to 603 K. Pressures from ambient up to 6 MPa are applicable. An exchangeable sample positioning system makes the flow cell suitable for several different goniomter types on a variety of instrument beam lines. Complementary operational test measurements were carried out monitoring reduction of and methanol synthesis over a Cu/ZnO/Al2O3 catalyst at the high-flux powder diffraction beamline D1B at ILL and high-resolution diffraction beamline Echidna at ANSTO.

  10. A Devonian predatory fish provides insights into the early evolution of modern sarcopterygians

    PubMed Central

    Lu, Jing; Zhu, Min; Ahlberg, Per Erik; Qiao, Tuo; Zhu, You’an; Zhao, Wenjin; Jia, Liantao

    2016-01-01

    Crown or modern sarcopterygians (coelacanths, lungfishes, and tetrapods) differ substantially from stem sarcopterygians, such as Guiyu and Psarolepis, and a lack of transitional fossil taxa limits our understanding of the origin of the crown group. The Onychodontiformes, an enigmatic Devonian predatory fish group, seems to have characteristics of both stem and crown sarcopterygians but is difficult to place because of insufficient anatomical information. We describe the new skull material of Qingmenodus, a Pragian (~409-million-year-old) onychodont from China, using high-resolution computed tomography to image internal structures of the braincase. In addition to its remarkable similarities with stem sarcopterygians in the ethmosphenoid portion, Qingmenodus exhibits coelacanth-like neurocranial features in the otic region. A phylogenetic analysis based on a revised data set unambiguously assigns onychodonts to crown sarcopterygians as stem coelacanths. Qingmenodus thus bridges the morphological gap between stem sarcopterygians and coelacanths and helps to illuminate the early evolution and diversification of crown sarcopterygians. PMID:27386576

  11. Glossina fuscipes populations provide insights for Human African Trypanosomiasis transmission in Uganda

    PubMed Central

    Aksoy, Serap; Caccone, Adalgisa; Galvani, Alison P.; Okedi, Loyce M.

    2013-01-01

    Uganda has both forms of human African trypanosomiasis (HAT): the chronic gambiense disease in the northwest and the acute rhodesiense disease in the south. The recent spread of rhodesiense into central Uganda has raised concerns given the different control strategies the two diseases require. We present knowledge on the population genetics of the major vector species Glossina fuscipes fuscipes in Uganda with a focus on population structure, measures of gene flow between populations, and the occurrence of polyandry. The microbiome composition and diversity is discussed, focusing on their potential role on trypanosome infection outcomes. We discuss the implications of these findings for large-scale tsetse control programs, including suppression or eradication, being undertaken in Uganda and potential future genetic applications. PMID:23845311

  12. Glossina fuscipes populations provide insights for human African trypanosomiasis transmission in Uganda.

    PubMed

    Aksoy, Serap; Caccone, Adalgisa; Galvani, Alison P; Okedi, Loyce M

    2013-08-01

    Uganda has both forms of human African trypanosomiasis (HAT): the chronic gambiense disease in the northwest and the acute rhodesiense disease in the south. The recent spread of rhodesiense into central Uganda has raised concerns given the different control strategies the two diseases require. We present knowledge on the population genetics of the major vector species Glossina fuscipes fuscipes in Uganda with a focus on population structure, measures of gene flow between populations, and the occurrence of polyandry. The microbiome composition and diversity is discussed, focusing on their potential role on trypanosome infection outcomes. We discuss the implications of these findings for large-scale tsetse control programs, including suppression or eradication, being undertaken in Uganda, and potential future genetic applications.

  13. DNA Barcode Libraries Provide Insight into Continental Patterns of Avian Diversification

    PubMed Central

    Lijtmaer, Darío A.; Kerr, Kevin C. R.; Barreira, Ana S.; Hebert, Paul D. N.; Tubaro, Pablo L.

    2011-01-01

    Background The causes for the higher biodiversity in the Neotropics as compared to the Nearctic and the factors promoting species diversification in each region have been much debated. The refuge hypothesis posits that high tropical diversity reflects high speciation rates during the Pleistocene, but this conclusion has been challenged. The present study investigates this matter by examining continental patterns of avian diversification through the analysis of large-scale DNA barcode libraries. Methodology and Principal Findings Standardized COI datasets from the avifaunas of Argentina, the Nearctic, and the Palearctic were analyzed. Average genetic distances between closest congeners and sister species were higher in Argentina than in North America reflecting a much higher percentage of recently diverged species in the latter region. In the Palearctic genetic distances between closely related species appeared to be more similar to those of the southern Neotropics. Average intraspecific variation was similar in Argentina and North America, while the Palearctic fauna had a higher value due to a higher percentage of variable species. Geographic patterning of intraspecific structure was more complex in the southern Neotropics than in the Nearctic, while the Palearctic showed an intermediate level of complexity. Conclusions and Significance DNA barcodes can reveal continental patterns of diversification. Our analysis suggests that avian species are older in Argentina than in the Nearctic, supporting the idea that the greater diversity of the Neotropical avifauna is not caused by higher recent speciation rates. Species in the Palearctic also appear to be older than those in the Nearctic. These results, combined with the patterns of geographic structuring found in each region, suggest a major impact of Pleistocene glaciations in the Nearctic, a lesser effect in the Palearctic and a mild effect in the southern Neotropics. PMID:21818252

  14. Comparative genomics of Ceriporiopsis subvermispora and Phanerochaete chrysosporium provide insight into selective ligninolysis

    SciTech Connect

    Fernandez-Fueyo, Elena; Ruiz-Duenas, Francisco J.; Ferreira, Patrica; Floudas, Dimitrios; HIbbett, David S.; Canessa, Paulo; Larrondo, Luis F.; James, Tim Y.; Seelenfreund, Daniela; Lobos, Sergio; Polanco, Ruben; Tello, Mario; Honda, Yoichi; Watanabe, Takahito; Watanabe, Takashi; Ryu, Jae San; Kubicek, Christian P.; Schmoll, Monika; Gaskell, Jill; Hammel, Kenneth E.; John, Franz J.; Vanden Wymelenberg, Amber; Sabat, Grzegorz; Splinter BonDurant, Sandra; Syed, Khajamohiddin; Yadav, Jagjit S.; Doddapaneni, Harshavardhan; Subramanian, Venkataramanan; Lavin, Jose L.; Oguiza, Jose A.; Perez, Gumer; Pisabarro, Antonio G.; Ramirez, Lucia; Santoyo, Francisco; Master, Emma; Coutinho, Pedro M.; Henrissat, Bernard; Lombard, Vincent; Magnuson, Jon Karl; Kues, Ursula; Hori, Chiaki; Igarashi, Kiyohiko; Samejima, Masahiro; Held, Benjamin W.; Barry, Kerrie W.; LaButti, Kurt M.; Lapidus, Alla; Lindquist, Erika A.; Lucas, Susan M.; Riley, Robert; Salamov, Asaf A.; Hoffmeister, Dirk; Schwenk, Daniel; Hadar, Yitzhak; Yarden, Oded; de Vries, Ronald P.; Wiebenga, Ad; Stenlid, Jan; Eastwood, Daniel; Grigoriev, Igor V.; Berka, Randy M.; Blanchette, Robert A.; Kersten, Phil; Martinez, Angel T.; Vicuna, Rafael; Cullen, Dan

    2011-12-06

    Efficient lignin depolymerization is unique to the wood decay basidiomycetes, collectively referred to as white rot fungi. Phanerochaete chrysosporium simultaneously degrades lignin and cellulose, whereas the closely related species, Ceriporiopsis subvermispora, also depolymerizes lignin but may do so with relatively little cellulose degradation. To investigate the basis for selective ligninolysis, we conducted comparative genome analysis of C. subvermispora and P. chrysosporium. Genes encoding manganese peroxidase numbered 13 and five in C. subvermispora and P. chrysosporium, respectively. In addition, the C. subvermispora genome contains at least seven genes predicted to encode laccases, whereas the P. chrysosporium genome contains none. We also observed expansion of the number of C. subvermispora desaturase-encoding genes putatively involved in lipid metabolism. Microarray-based transcriptome analysis showed substantial up-regulation of several desaturase and MnP genes in wood-containing medium. MS identified MnP proteins in C. subvermispora culture filtrates, but none in P. chrysosporium cultures. These results support the importance of MnP and a lignin degradation mechanism whereby cleavage of the dominant nonphenolic structures is mediated by lipid peroxidation products. Two C. subvermispora genes were predicted to encode peroxidases structurally similar to P. chrysosporium lignin peroxidase and, following heterologous expression in Escherichia coli, the enzymes were shown to oxidize high redox potential substrates, but not Mn2. Apart from oxidative lignin degradation, we also examined cellulolytic and hemicellulolytic systems in both fungi. In summary, the C. subvermispora genetic inventory and expression patterns exhibit increased oxidoreductase potential and diminished cellulolytic capability relative to P. chrysosporium.

  15. Transcriptomics provides insight into Mytilus galloprovincialis (Mollusca: Bivalvia) mantle function and its role in biomineralisation.

    PubMed

    Bjärnmark, Nadège A; Yarra, T; Churcher, A M; Felix, R C; Clark, M S; Power, D M

    2016-06-01

    The mantle is an organ common to all molluscs and is at the forefront of the biomineralisation process. The present study used the Mediterranean mussel (Mytilus galloprovincialis) as a model species to investigate the structural and functional role of the mantle in shell formation. The transcriptomes of three regions of the mantle edge (umbo to posterior edge) were sequenced using Illumina technology which yielded a total of 61,674,325 reads after adapter trimming and filtering. The raw reads assembled into 179,879 transcripts with an N50 value of 1086bp. A total of 1363 transcripts (321, 223 and 816 in regions 1, 2 and 3, respectively) that differed in abundance in the three mantle regions were identified and putative function was assigned to 54% using BLAST sequence similarity searches (cut-off less than 1e(-10)). Morphological differences detected by histology of the three mantle regions was linked to functional heterogeneity by selecting the top five most abundant Pfam domains in the annotated 1363 differentially abundant transcripts across the three mantle regions. Calcium binding domains dominated region two (middle segment of the mantle edge). Candidate biomineralisation genes were mined and tested by qPCR. This revealed that Flp-like, a penicillin binding protein potentially involved in shell matrix maintenance of the Pacific oyster (Crassostrea gigas), had significantly higher expression in the posterior end of the mantle edge (region one). Our findings are intriguing as they indicate that the mantle edge appears to be a heterogeneous tissue, displaying structural and functional bias.

  16. Stable isotopes provide new insights into vestimentiferan physiological ecology at Gulf of Mexico cold seeps.

    PubMed

    Becker, Erin Leigh; Macko, Stephen A; Lee, Raymond W; Fisher, Charles R

    2011-02-01

    On the otherwise low-biomass seafloor of the Gulf of Mexico (GoM) continental slope, natural oil and gas seeps are oases of local primary production that support lush animal communities. Hundreds of seep communities have been documented on the continental slope, and nutrition derived from seeps could be an important link in the overall GoM food web. Here, we present a uniquely large and cohesive data set of δ(13)C, δ(15)N, and δ(34)S compositions of the vestimentiferan tubeworms Escarpia laminata and Lamellibrachia sp. 1, which dominate biomass at GoM seeps and provide habitat for hundreds of other species. Our sampling design encompassed an entire region of the GoM lower slope, allowing us for the first time to assess spatial variability in isotope compositions and to robustly address long-standing hypotheses about how vestimentiferans acquire and cycle nutrients over their long lifespan (200+ years). Tissue δ(13)C values provided strong evidence that larger adult vestimentiferans use their buried roots to take up dissolved inorganic carbon from sediment pore water, while very small individuals use their plume to take up carbon dioxide from the seawater. δ(34)S values were extremely variable among individuals of the same species within one location (<1 m(2) area), indicating high variability in the inorganic sulfur pools on a very small spatial scale. This finding supports the hypothesis that vestimentiferans use their roots to cycle sulfate and sulfide between their symbionts and free-living consortia of sulfate-reducing archaea in the sediment. Finally, consistent differences in δ(15)N between two cooccurring vestimentiferan species provided the first strong evidence for partitioning of inorganic resources, which has significant implications for the ecology and evolution of this taxonomic group.

  17. Provider Decisions to Treat Respiratory Illnesses with Antibiotics: Insights from a Randomized Controlled Trial

    PubMed Central

    Branche, Angela R.; Walsh, Edward E.; Jadhav, Nagesh; Karmally, Rachel; Baran, Andrea; Peterson, Derick R.; Falsey, Ann R.

    2016-01-01

    Rationale Lower respiratory tract illness (LRTI) frequently causes adult hospitalization and antibiotic overuse. Procalcitonin (PCT) treatment algorithms have been used successfully in Europe to safely reduce antibiotic use for LRTI but have not been adopted in the United States. We recently performed a feasibility study for a randomized clinical trial (RCT) of PCT and viral testing to guide therapy for non-pneumonic LRTI. Objective The primary objective of the current study was to understand factors influencing PCT algorithm adherence during the RCT and evaluate factors influencing provider antibiotic prescribing practices for LRTI. Study Design From October 2013-April 2014, 300 patients hospitalized at a community teaching hospital with non-pneumonic LRTI were randomized to standard or PCT-guided care with viral PCR testing. Algorithm adherence data was collected and multivariate stepwise logistic regression of clinical variables used to model algorithm compliance. 134 providers were surveyed anonymously before and after the trial to assess knowledge of biomarkers and viral testing and antibiotic prescribing practices. Results Diagnosis of pneumonia on admission was the only variable significantly associated with non-adherence [7% (adherence) vs. 26% (nonadherence), p = 0.01]. Surveys confirmed possible infiltrate on chest radiograph as important for provider decisions, as were severity of illness, positive sputum culture, abnormal CBC and fever. However, age, patient expectations and medical-legal concerns were also at least somewhat important to prescribing practices. Physician agreement with the importance of viral and PCT testing increased from 42% to 64% (p = 0.007) and 49% to 74% (p = 0.001), respectively, after the study. Conclusions Optimal algorithm adherence will be important for definitive PCT intervention trials in the US to determine if PCT guided algorithms result in better outcomes than reliance on traditional clinical variables. Factors

  18. Using Health Provider Insights to Inform Pediatric HIV Disclosure: A Qualitative Study and Practice Framework from Kenya

    PubMed Central

    John-Stewart, Grace; Shah, Brandi; Wamalwa, Dalton; Maleche-Obimbo, Elizabeth; Kelley, Maureen

    2014-01-01

    Abstract Optimal pediatric HIV disclosure impacts illness and developmental experiences while improving access to timely treatment. However, disclosure rates in high HIV prevalence countries remain low and there are limited data on best practices. We conducted a qualitative study of disclosure practices and interviewed healthcare providers from five pediatric HIV clinics in Kenya. We identified themes central to disclosure practices, rationale for approaches, barriers to implementing disclosure, and creative strategies to overcome challenges. We used these insights to develop a practice-based framework for disclosure that is sensitive to practical challenges. Overall, providers had limited training but extensive experience in disclosure, endorsed individualized disclosure practices, invested substantial time on disclosure despite clinical burden, and noted adverse outcomes associated with unplanned or abrupt disclosure. Providers advocated for an approach to disclosure that is child-centered but respects caregiver fears and values. Caregiver support was provided to enable caregivers to be the person who ultimately disclosed HIV status to children. Unplanned or abrupt disclosure to children was reported to have severe and persistent adverse impact and was a stimulus to accelerate disclosure in scenarios when providers believed children may be suspecting their diagnosis. Based on these expert insights, the framework we developed incorporates concurrent evaluation of child and caregiver readiness, identifies cues to prompt disclosure discussions, includes caregiver education and support, and utilizes a gradual approach of unveiling HIV diagnosis to the child. PMID:25216105

  19. Whole-genome sequencing of cultivated and wild peppers provides insights into Capsicum domestication and specialization

    PubMed Central

    Qin, Cheng; Yu, Changshui; Shen, Yaou; Fang, Xiaodong; Chen, Lang; Min, Jiumeng; Cheng, Jiaowen; Zhao, Shancen; Xu, Meng; Luo, Yong; Yang, Yulan; Wu, Zhiming; Mao, Likai; Wu, Haiyang; Ling-Hu, Changying; Zhou, Huangkai; Lin, Haijian; González-Morales, Sandra; Trejo-Saavedra, Diana L.; Tian, Hao; Tang, Xin; Zhao, Maojun; Huang, Zhiyong; Zhou, Anwei; Yao, Xiaoming; Cui, Junjie; Li, Wenqi; Chen, Zhe; Feng, Yongqiang; Niu, Yongchao; Bi, Shimin; Yang, Xiuwei; Li, Weipeng; Cai, Huimin; Luo, Xirong; Montes-Hernández, Salvador; Leyva-González, Marco A.; Xiong, Zhiqiang; He, Xiujing; Bai, Lijun; Tan, Shu; Tang, Xiangqun; Liu, Dan; Liu, Jinwen; Zhang, Shangxing; Chen, Maoshan; Zhang, Lu; Zhang, Li; Zhang, Yinchao; Liao, Weiqin; Zhang, Yan; Wang, Min; Lv, Xiaodan; Wen, Bo; Liu, Hongjun; Luan, Hemi; Zhang, Yonggang; Yang, Shuang; Wang, Xiaodian; Xu, Jiaohui; Li, Xueqin; Li, Shuaicheng; Wang, Junyi; Palloix, Alain; Bosland, Paul W.; Li, Yingrui; Krogh, Anders; Rivera-Bustamante, Rafael F.; Herrera-Estrella, Luis; Yin, Ye; Yu, Jiping; Hu, Kailin; Zhang, Zhiming

    2014-01-01

    As an economic crop, pepper satisfies people’s spicy taste and has medicinal uses worldwide. To gain a better understanding of Capsicum evolution, domestication, and specialization, we present here the genome sequence of the cultivated pepper Zunla-1 (C. annuum L.) and its wild progenitor Chiltepin (C. annuum var. glabriusculum). We estimate that the pepper genome expanded ∼0.3 Mya (with respect to the genome of other Solanaceae) by a rapid amplification of retrotransposons elements, resulting in a genome comprised of ∼81% repetitive sequences. Approximately 79% of 3.48-Gb scaffolds containing 34,476 protein-coding genes were anchored to chromosomes by a high-density genetic map. Comparison of cultivated and wild pepper genomes with 20 resequencing accessions revealed molecular footprints of artificial selection, providing us with a list of candidate domestication genes. We also found that dosage compensation effect of tandem duplication genes probably contributed to the pungent diversification in pepper. The Capsicum reference genome provides crucial information for the study of not only the evolution of the pepper genome but also, the Solanaceae family, and it will facilitate the establishment of more effective pepper breeding programs. PMID:24591624

  20. The Genome of the "Great Speciator" Provides Insights into Bird Diversification.

    PubMed

    Cornetti, Luca; Valente, Luis M; Dunning, Luke T; Quan, Xueping; Black, Richard A; Hébert, Olivier; Savolainen, Vincent

    2015-09-02

    Among birds, white-eyes (genus Zosterops) have diversified so extensively that Jared Diamond and Ernst Mayr referred to them as the "great speciator." The Zosterops lineage exhibits some of the fastest rates of species diversification among vertebrates, and its members are the most prolific passerine island colonizers. We present a high-quality genome assembly for the silvereye (Zosterops lateralis), a white-eye species consisting of several subspecies distributed across multiple islands. We investigate the genetic basis of rapid diversification in white-eyes by conducting genomic analyses at varying taxonomic levels. First, we compare the silvereye genome with those of birds from different families and searched for genomic features that may be unique to Zosterops. Second, we compare the genomes of different species of white-eyes from Lifou island (South Pacific), using whole genome resequencing and restriction site associated DNA. Third, we contrast the genomes of two subspecies of silvereye that differ in plumage color. In accordance with theory, we show that white-eyes have high rates of substitutions, gene duplication, and positive selection relative to other birds. Below genus level, we find that genomic differentiation accumulates rapidly and reveals contrasting demographic histories between sympatric species on Lifou, indicative of past interspecific interactions. Finally, we highlight genes possibly involved in color polymorphism between the subspecies of silvereye. By providing the first whole-genome sequence resources for white-eyes and by conducting analyses at different taxonomic levels, we provide genomic evidence underpinning this extraordinary bird radiation.

  1. Comparative Transcriptomics of Strawberries (Fragaria spp.) Provides Insights into Evolutionary Patterns

    PubMed Central

    Qiao, Qin; Xue, Li; Wang, Qia; Sun, Hang; Zhong, Yang; Huang, Jinling; Lei, Jiajun; Zhang, Ticao

    2016-01-01

    Multiple closely related species with genomic sequences provide an ideal system for studies on comparative and evolutionary genomics, as well as the mechanism of speciation. The whole genome sequences of six strawberry species (Fragaria spp.) have been released, which provide one of the richest genomic resources of any plant genus. In this study, we first generated seven transcriptome sequences of Fragaria species de novo, with a total of 48,557–82,537 unigenes per species. Combined with 13 other species genomes in Rosales, we reconstructed a phylogenetic tree at the genomic level. The phylogenic tree shows that Fragaria closed grouped with Rubus and the Fragaria clade is divided into three subclades. East Asian species appeared in every subclade, suggesting that the genus originated in this area at ∼7.99 Mya. Four species found in mountains of Southwest China originated at ∼3.98 Mya, suggesting that rapid speciation occurred to adapt to changing environments following the uplift of the Qinghai–Tibet Plateau. Moreover, we identified 510 very significantly positively selected genes in the cultivated species F. × ananassa genome. This set of genes was enriched in functions related to specific agronomic traits, such as carbon metabolism and plant hormone signal transduction processes, which are directly related to fruit quality and flavor. These findings illustrate comprehensive evolutionary patterns in Fragaria and the genetic basis of fruit domestication of cultivated strawberry at the genomic/transcriptomic level. PMID:28018379

  2. Transcriptome analysis of near-isogenic lines provides molecular insights into starch biosynthesis in maize kernel.

    PubMed

    Xiao, Yingni; Thatcher, Shawn; Wang, Min; Wang, Tingting; Beatty, Mary; Zastrow-Hayes, Gina; Li, Lin; Li, Jiansheng; Li, Bailin; Yang, Xiaohong

    2016-08-01

    Starch is the major component in maize kernels, providing a stable carbohydrate source for humans and livestock as well as raw material for the biofuel industry. Increasing maize kernel starch content will help meet industry demands and has the potential to increase overall yields. We developed a pair of maize near-isogenic lines (NILs) with different alleles for a starch quantitative trait locus on chromosome 3 (qHS3), resulting in different kernel starch content. To investigate the candidate genes for qHS3 and elucidate their effects on starch metabolism, RNA-Seq was performed for the developing kernels of the NILs at 14 and 21 d after pollination (DAP). Analysis of genomic and transcriptomic data identified 76 genes with nonsynonymous single nucleotide polymorphisms and 384 differentially expressed genes (DEGs) in the introgressed fragment, including a hexokinase gene, ZmHXK3a, which catalyzes the conversion of glucose to glucose-6-phosphate and may play a key role in starch metabolism. The expression pattern of all DEGs in starch metabolism shows that altered expression of the candidate genes for qHS3 promoted starch synthesis, with positive consequences for kernel starch content. These results expand the current understanding of starch biosynthesis and accumulation in maize kernels and provide potential candidate genes to increase starch content.

  3. Characterization of duplicated Dunaliella viridis SPT1 genes provides insights into early gene divergence after duplication.

    PubMed

    Guan, Zhenwei; Meng, Xiangzong; Sun, Zhenhua; Xu, Zhengkai; Song, Rentao

    2008-10-15

    The sodium-dependent phosphate transporter gene from unicellular green algae Dunaliella viridis, DvSPT1, shares similarity with members of Pi transporter family. Sequencing analysis of D. viridis BAC clone containing the DvSPT1 gene revealed two inverted duplicated copies of this gene (DvSPT1 and DvSPT1-2 respectively). The duplication covered most of both genes except for their 3' downstream region. The duplicated genomic sequences exhibited 97.9% identity with a synonymous divergence of Ks=0.0126 in the coding region. This data indicated very recent gene duplication in D. viridis genome, providing an excellent opportunity to investigate sequence and expression divergence of duplicated genes at an early stage. Scattered point mutations and length polymorphism of simple sequence repeats (SSRs) were predominant among the sequence divergence soon after gene duplication. Due to sequence divergence in the 5' regulatory regions and a swap of the entire 3' downstream regions (3'-UTR), DvSPT1 and DvSPT1-2 showed expression divergence in response to extra-cellular NaCl concentration changes. According to their expression patterns, the two diverged gene copies would provide better adaptation to a broader range of extra-cellular NaCl concentration. Furthermore, Southern blot analysis indicated that there might be a large phosphate transporter gene family in D. viridis.

  4. Insights from the sea: structural biology of marine polyketide synthases.

    PubMed

    Akey, David L; Gehret, Jennifer J; Khare, Dheeraj; Smith, Janet L

    2012-10-01

    The world's oceans are a rich source of natural products with extremely interesting chemistry. Biosynthetic pathways have been worked out for a few, and the story is being enriched with crystal structures of interesting pathway enzymes. By far, the greatest number of structural insights from marine biosynthetic pathways has originated with studies of curacin A, a poster child for interesting marine chemistry with its cyclopropane and thiazoline rings, internal cis double bond, and terminal alkene. Using the curacin A pathway as a model, structural details are now available for a novel loading enzyme with remarkable dual decarboxylase and acetyltransferase activities, an Fe(2+)/α-ketoglutarate-dependent halogenase that dictates substrate binding order through conformational changes, a decarboxylase that establishes regiochemistry for cyclopropane formation, and a thioesterase with specificity for β-sulfated substrates that lead to terminal alkene offloading. The four curacin A pathway dehydratases reveal an intrinsic flexibility that may accommodate bulky or stiff polyketide intermediates. In the salinosporamide A pathway, active site volume determines the halide specificity of a halogenase that catalyzes for the synthesis of a halogenated building block. Structures of a number of putative polyketide cyclases may help in understanding reaction mechanisms and substrate specificities although their substrates are presently unknown.

  5. Insights from the Sea: Structural Biology of Marine Polyketide Synthases

    PubMed Central

    Akey, David L.; Gehret, Jennifer J.; Khare, Dheeraj; Smith, Janet L.

    2013-01-01

    The world’s oceans are a rich source of natural products with extremely interesting chemistry. Biosynthetic pathways have been worked out for a few, and the story is being enriched with crystal structures of interesting pathway enzymes. By far, the greatest number of structural insights from marine biosynthetic pathways has originated with studies of curacin A, a poster child for interesting marine chemistry with its cyclopropane and thiazoline rings, internal cis double bond, and terminal alkene. Using the curacin A pathway as a model, structural details are now available for a novel loading enzyme with remarkable dual decarboxylase and acetyltransferase activities, an Fe2+/α-ketoglutarate-dependent halogenase that dictates substrate binding order through conformational changes, a decarboxylase that establishes regiochemistry for cyclopropane formation, and a thioesterase with specificity for β-sulfated substrates that lead to terminal alkene offloading. The four curacin A pathway dehydratases reveal an intrinsic flexibility that may accommodate bulky or stiff polyketide intermediates. In the salinosporamide A pathway, active site volume determines the halide specificity of a halogenase that catalyzes for the synthesis of a halogenated building block. Structures of a number of putative polyketide cyclases may help in understanding reaction mechanisms and substrate specificities although their substrates are presently unknown. PMID:22498975

  6. Environmental Metabolomics of the Tomato Plant Surface Provides Insights on Salmonella enterica Colonization

    PubMed Central

    Han, Sanghyun

    2016-01-01

    ABSTRACT Foodborne illness-causing enteric bacteria are able to colonize plant surfaces without causing infection. We lack an understanding of how epiphytic persistence of enteric bacteria occurs on plants, possibly as an adaptive transit strategy to maximize chances of reentering herbivorous hosts. We used tomato (Solanum lycopersicum) cultivars that have exhibited differential susceptibilities to Salmonella enterica colonization to investigate the influence of plant surface compounds and exudates on enteric bacterial populations. Tomato fruit, shoot, and root exudates collected at different developmental stages supported growth of S. enterica to various degrees in a cultivar- and plant organ-dependent manner. S. enterica growth in fruit exudates of various cultivars correlated with epiphytic growth data (R2 = 0.504; P = 0.006), providing evidence that plant surface compounds drive bacterial colonization success. Chemical profiling of tomato surface compounds with gas chromatography-time of flight mass spectrometry (GC-TOF-MS) provided valuable information about the metabolic environment on fruit, shoot, and root surfaces. Hierarchical cluster analysis of the data revealed quantitative differences in phytocompounds among cultivars and changes over a developmental course and by plant organ (P < 0.002). Sugars, sugar alcohols, and organic acids were associated with increased S. enterica growth, while fatty acids, including palmitic and oleic acids, were negatively correlated. We demonstrate that the plant surface metabolite landscape has a significant impact on S. enterica growth and colonization efficiency. This environmental metabolomics approach provides an avenue to understand interactions between human pathogens and plants that could lead to strategies to identify or breed crop cultivars for microbiologically safer produce. IMPORTANCE In recent years, fresh produce has emerged as a leading food vehicle for enteric pathogens. Salmonella-contaminated tomatoes

  7. Molecular Taxonomy Provides New Insights into Anopheles Species of the Neotropical Arribalzagia Series

    PubMed Central

    Gómez, Giovan F.; Bickersmith, Sara A.; González, Ranulfo; Conn, Jan E.; Correa, Margarita M.

    2015-01-01

    Phylogenetic analysis of partial mitochondrial cytochrome oxidase c subunit I (COI) and nuclear internal transcribed spacer 2 (ITS2) sequences were used to evaluate initial identification and to investigate phylogenetic relationships of seven Anopheles morphospecies of the Arribalzagia Series from Colombia. Phylogenetic trees recovered highly supported clades for An. punctimaculas.s., An. calderoni, An. malefactor s.l., An. neomaculipalpus, An. apicimacula s.l., An. mattogrossensis and An. peryassui. This study provides the first molecular confirmation of An. malefactorfrom Colombia and discovered conflicting patterns of divergence for the molecular markers among specimens from northeast and northern Colombia suggesting the presence of two previously unrecognized Molecular Operational Taxonomic Units (MOTUs). Furthermore, two highly differentiated An. apicimacula MOTUs previously found in Panama were detected. Overall, the combined molecular dataset facilitated the detection of known and new Colombian evolutionary lineages, and constitutes the baseline for future research on their bionomics, ecology and potential role as malaria vectors. PMID:25774795

  8. Single-filament kinetic studies provide novel insights into regulation of actin-based motility

    PubMed Central

    Shekhar, Shashank; Carlier, Marie-France

    2016-01-01

    Polarized assembly of actin filaments forms the basis of actin-based motility and is regulated both spatially and temporally. Cells use a variety of mechanisms by which intrinsically slower processes are accelerated, and faster ones decelerated, to match rates observed in vivo. Here we discuss how kinetic studies of individual reactions and cycles that drive actin remodeling have provided a mechanistic and quantitative understanding of such processes. We specifically consider key barbed-end regulators such as capping protein and formins as illustrative examples. We compare and contrast different kinetic approaches, such as the traditional pyrene-polymerization bulk assays, as well as more recently developed single-filament and single-molecule imaging approaches. Recent development of novel biophysical methods for sensing and applying forces will in future allow us to address the very important relationship between mechanical stimulus and kinetics of actin-based motility. PMID:26715420

  9. Characterisation of echidna IgM provides insights into the time of divergence of extant mammals.

    PubMed

    Belov, Katherine; Hellman, Lars; Cooper, Desmond W

    2002-11-01

    The immunobiology of monotremes is poorly understood. In this paper, we describe the characterisation of the heavy chain of IgM from Tachyglossus aculeatus, the short-beaked echidna. The echidna heavy chain constant region of IgM (Cmu)was isolated from a spleen cDNA library using a Trichosurus vulpecula probe. It has approximately 46.5% amino acid identity to marsupial and eutherian Cmus, and approximately 30% amino acid identity with Cmu from birds and reptiles. Phylogenetic analysis of mammalian Cmu provides strong support for the Theria hypothesis, with a sister grouping of the eutherians and marsupials to the exclusion of the monotremes. Cmu sequences suggest that monotremes and therians separated approximately 170 million years ago (mya), marsupials and eutherians separated approximately 130mya, and Australian and American marsupials separated approximately 65mya.

  10. Molecular developmental mechanism in polypterid fish provides insight into the origin of vertebrate lungs

    PubMed Central

    Tatsumi, Norifumi; Kobayashi, Ritsuko; Yano, Tohru; Noda, Masatsugu; Fujimura, Koji; Okada, Norihiro; Okabe, Masataka

    2016-01-01

    The lung is an important organ for air breathing in tetrapods and originated well before the terrestrialization of vertebrates. Therefore, to better understand lung evolution, we investigated lung development in the extant basal actinopterygian fish Senegal bichir (Polypterus senegalus). First, we histologically confirmed that lung development in this species is very similar to that of tetrapods. We also found that the mesenchymal expression patterns of three genes that are known to play important roles in early lung development in tetrapods (Fgf10, Tbx4, and Tbx5) were quite similar to those of tetrapods. Moreover, we found a Tbx4 core lung mesenchyme-specific enhancer (C-LME) in the genomes of bichir and coelacanth (Latimeria chalumnae) and experimentally confirmed that these were functional in tetrapods. These findings provide the first molecular evidence that the developmental program for lung was already established in the common ancestor of actinopterygians and sarcopterygians. PMID:27466206

  11. The Opisthorchis viverrini genome provides insights into life in the bile duct.

    PubMed

    Young, Neil D; Nagarajan, Niranjan; Lin, Suling Joyce; Korhonen, Pasi K; Jex, Aaron R; Hall, Ross S; Safavi-Hemami, Helena; Kaewkong, Worasak; Bertrand, Denis; Gao, Song; Seet, Qihui; Wongkham, Sopit; Teh, Bin Tean; Wongkham, Chaisiri; Intapan, Pewpan Maleewong; Maleewong, Wanchai; Yang, Xinhua; Hu, Min; Wang, Zuo; Hofmann, Andreas; Sternberg, Paul W; Tan, Patrick; Wang, Jun; Gasser, Robin B

    2014-07-09

    Opisthorchiasis is a neglected, tropical disease caused by the carcinogenic Asian liver fluke, Opisthorchis viverrini. This hepatobiliary disease is linked to malignant cancer (cholangiocarcinoma, CCA) and affects millions of people in Asia. No vaccine is available, and only one drug (praziquantel) is used against the parasite. Little is known about O. viverrini biology and the diseases that it causes. Here we characterize the draft genome (634.5 Mb) and transcriptomes of O. viverrini, elucidate how this fluke survives in the hostile environment within the bile duct and show that metabolic pathways in the parasite are highly adapted to a lipid-rich diet from bile and/or cholangiocytes. We also provide additional evidence that O. viverrini and other flukes secrete proteins that directly modulate host cell proliferation. Our molecular resources now underpin profound explorations of opisthorchiasis/CCA and the design of new interventions.

  12. Mouse tetrad analysis provides insights into recombination mechanisms and hotspot evolutionary dynamics.

    PubMed

    Cole, Francesca; Baudat, Frédéric; Grey, Corinne; Keeney, Scott; de Massy, Bernard; Jasin, Maria

    2014-10-01

    The ability to examine all chromatids from a single meiosis in yeast tetrads has been indispensable for defining the mechanisms of homologous recombination initiated by DNA double-strand breaks (DSBs). Using a broadly applicable strategy for the analysis of chromatids from a single meiosis at two recombination hotspots in mouse oocytes and spermatocytes, we demonstrate here the unidirectional transfer of information-gene conversion-in both crossovers and noncrossovers. Whereas gene conversion in crossovers is associated with reciprocal exchange, the unbroken chromatid is not altered in noncrossover gene conversion events, providing strong evidence that noncrossovers arise from a distinct pathway. Gene conversion frequently spares the binding site of the hotspot-specifying protein PRDM9, with the result that erosion of the hotspot is slowed. Thus, mouse tetrad analysis demonstrates how unique aspects of mammalian recombination mechanisms shape hotspot evolutionary dynamics.

  13. Plasmodium cynomolgi genome sequences provide insight into Plasmodium vivax and the monkey malaria clade

    PubMed Central

    Tachibana, Shin-Ichiro; Sullivan, Steven A.; Kawai, Satoru; Nakamura, Shota; Kim, Hyunjae R.; Goto, Naohisa; Arisue, Nobuko; Palacpac, Nirianne M. Q.; Honma, Hajime; Yagi, Masanori; Tougan, Takahiro; Katakai, Yuko; Kaneko, Osamu; Mita, Toshihiro; Kita, Kiyoshi; Yasutomi, Yasuhiro; Sutton, Patrick L.; Shakhbatyan, Rimma; Horii, Toshihiro; Yasunaga, Teruo; Barnwell, John W.; Escalante, Ananias A.; Carlton, Jane M.; Tanabe, Kazuyuki

    2013-01-01

    Plasmodium cynomolgi, a malaria parasite of Asian Old World monkeys, is the sister taxon of Plasmodium vivax, the most prevalent human malaria species outside Africa. Since P. cynomolgi shares many phenotypic, biologic and genetic characteristics of P. vivax, we generated draft genome sequences of three P. cynomolgi strains and performed comparative genomic analysis between them and P. vivax, as well as a third previously sequenced simian parasite, Plasmodium knowlesi. Here we show that genomes of the monkey malaria clade can be characterized by CNVs in multigene families involved in evasion of the human immune system and invasion of host erythrocytes. We identify genome-wide SNPs, microsatellites, and CNVs in the P. cynomolgi genome, providing a map of genetic variation for mapping parasite traits and studying parasite populations. The P. cynomolgi genome is a critical step in developing a model system for P. vivax research, and to counteract the neglect of P. vivax. PMID:22863735

  14. Analyzing Arabidopsis thaliana root proteome provides insights into the molecular bases of enantioselective imazethapyr toxicity

    NASA Astrophysics Data System (ADS)

    Qian, Haifeng; Lu, Haiping; Ding, Haiyan; Lavoie, Michel; Li, Yali; Liu, Weiping; Fu, Zhengwei

    2015-07-01

    Imazethapyr (IM) is a widely used chiral herbicide that inhibits the synthesis of branched-chain amino acids (BCAAs). IM is thought to exert its toxic effects on amino acid synthesis mainly through inhibition of acetolactate synthase activity, but little is known about the potential effects of IM on other key biochemical pathways. Here, we exposed the model plant Arabidospsis thaliana to trace S- and R-IM enantiomer concentrations and examined IM toxicity effects on the root proteome using iTRAQ. Conventional analyses of root carbohydrates, organic acids, and enzyme activities were also performed. We discovered several previously unknown key biochemical pathways targeted by IM in Arabidospsis. 1,322 and 987 proteins were differentially expressed in response to R- and S-IM treatments, respectively. Bioinformatics and physiological analyses suggested that IM reduced the BCAA tissue content not only by strongly suppressing BCAA synthesis but also by increasing BCAA catabolism. IM also affected sugar and starch metabolism, changed the composition of root cell walls, increased citrate production and exudation, and affected the microbial community structure of the rhizosphere. The present study shed new light on the multiple toxicity mechanisms of a selective herbicide on a model plant.

  15. Analyzing Arabidopsis thaliana root proteome provides insights into the molecular bases of enantioselective imazethapyr toxicity.

    PubMed

    Qian, Haifeng; Lu, Haiping; Ding, Haiyan; Lavoie, Michel; Li, Yali; Liu, Weiping; Fu, Zhengwei

    2015-07-08

    Imazethapyr (IM) is a widely used chiral herbicide that inhibits the synthesis of branched-chain amino acids (BCAAs). IM is thought to exert its toxic effects on amino acid synthesis mainly through inhibition of acetolactate synthase activity, but little is known about the potential effects of IM on other key biochemical pathways. Here, we exposed the model plant Arabidospsis thaliana to trace S- and R-IM enantiomer concentrations and examined IM toxicity effects on the root proteome using iTRAQ. Conventional analyses of root carbohydrates, organic acids, and enzyme activities were also performed. We discovered several previously unknown key biochemical pathways targeted by IM in Arabidospsis. 1,322 and 987 proteins were differentially expressed in response to R- and S-IM treatments, respectively. Bioinformatics and physiological analyses suggested that IM reduced the BCAA tissue content not only by strongly suppressing BCAA synthesis but also by increasing BCAA catabolism. IM also affected sugar and starch metabolism, changed the composition of root cell walls, increased citrate production and exudation, and affected the microbial community structure of the rhizosphere. The present study shed new light on the multiple toxicity mechanisms of a selective herbicide on a model plant.

  16. Comparative analysis of a teleost skeleton transcriptome provides insight into its regulation.

    PubMed

    Vieira, Florbela A; Thorne, M A S; Stueber, K; Darias, M; Reinhardt, R; Clark, M S; Gisbert, E; Power, D M

    2013-09-15

    An articulated endoskeleton that is calcified is a unifying innovation of the vertebrates, however the molecular basis of the structural divergence between terrestrial and aquatic vertebrates, such as teleost fish, has not been determined. In the present study long-read next generation sequencing (NGS, Roche 454 platform) was used to characterize acellular perichondral bone (vertebrae) and chondroid bone (gill arch) in the gilthead sea bream (Sparus auratus). A total of 15.97 and 14.53Mb were produced, respectively from vertebrae and gill arch cDNA libraries and yielded 32,374 and 28,371 contigs (consensus sequences) respectively. 10,455 contigs from vertebrae and 10,625 contigs from gill arches were annotated with gene ontology terms. Comparative analysis of the global transcriptome revealed 4249 unique transcripts in vertebrae, 4201 unique transcripts in the gill arches and 3700 common transcripts. Several core gene networks were conserved between the gilthead sea bream and mammalian skeleton. Transcripts for putative endocrine factors were identified in acellular gilthead sea bream bone suggesting that in common with mammalian bone it can act as an endocrine tissue. The acellular bone of the vertebra, in contrast to current opinion based on histological analysis, was responsive to a short fast and significant (p<0.05) down-regulation of several transcripts identified by NGS, osteonectin, osteocalcin, cathepsin K and IGFI occurred. In gill arches fasting caused a significant (p<0.05) down-regulation of osteocalcin and up-regulation of MMP9.

  17. New specimen of Archaeopteryx provides insights into the evolution of pennaceous feathers.

    PubMed

    Foth, Christian; Tischlinger, Helmut; Rauhut, Oliver W M

    2014-07-03

    Discoveries of bird-like theropod dinosaurs and basal avialans in recent decades have helped to put the iconic 'Urvogel' Archaeopteryx into context and have yielded important new data on the origin and early evolution of feathers. However, the biological context under which pennaceous feathers evolved is still debated. Here we describe a new specimen of Archaeopteryx with extensive feather preservation, not only on the wings and tail, but also on the body and legs. The new specimen shows that the entire body was covered in pennaceous feathers, and that the hindlimbs had long, symmetrical feathers along the tibiotarsus but short feathers on the tarsometatarsus. Furthermore, the wing plumage demonstrates that several recent interpretations are problematic. An analysis of the phylogenetic distribution of pennaceous feathers on the tail, hindlimb and arms of advanced maniraptorans and basal avialans strongly indicates that these structures evolved in a functional context other than flight, most probably in relation to display, as suggested by some previous studies. Pennaceous feathers thus represented an exaptation and were later, in several lineages and following different patterns, recruited for aerodynamic functions. This indicates that the origin of flight in avialans was more complex than previously thought and might have involved several convergent achievements of aerial abilities.

  18. Analyzing Arabidopsis thaliana root proteome provides insights into the molecular bases of enantioselective imazethapyr toxicity

    PubMed Central

    Qian, Haifeng; Lu, Haiping; Ding, Haiyan; Lavoie, Michel; Li, Yali; Liu, Weiping; Fu, Zhengwei

    2015-01-01

    Imazethapyr (IM) is a widely used chiral herbicide that inhibits the synthesis of branched-chain amino acids (BCAAs). IM is thought to exert its toxic effects on amino acid synthesis mainly through inhibition of acetolactate synthase activity, but little is known about the potential effects of IM on other key biochemical pathways. Here, we exposed the model plant Arabidospsis thaliana to trace S- and R-IM enantiomer concentrations and examined IM toxicity effects on the root proteome using iTRAQ. Conventional analyses of root carbohydrates, organic acids, and enzyme activities were also performed. We discovered several previously unknown key biochemical pathways targeted by IM in Arabidospsis. 1,322 and 987 proteins were differentially expressed in response to R- and S-IM treatments, respectively. Bioinformatics and physiological analyses suggested that IM reduced the BCAA tissue content not only by strongly suppressing BCAA synthesis but also by increasing BCAA catabolism. IM also affected sugar and starch metabolism, changed the composition of root cell walls, increased citrate production and exudation, and affected the microbial community structure of the rhizosphere. The present study shed new light on the multiple toxicity mechanisms of a selective herbicide on a model plant. PMID:26153126

  19. Large-scale genetic survey provides insights into the captive management and reintroduction of giant pandas.

    PubMed

    Shan, Lei; Hu, Yibo; Zhu, Lifeng; Yan, Li; Wang, Chengdong; Li, Desheng; Jin, Xuelin; Zhang, Chenglin; Wei, Fuwen

    2014-10-01

    The captive genetic management of threatened species strives to preserve genetic diversity and avoid inbreeding to ensure populations remain available, healthy, and viable for future reintroduction. Determining and responding to the genetic status of captive populations is therefore paramount to these programs. Here, we genotyped 19 microsatellite loci for 240 captive giant pandas (Ailuropoda melanoleuca) (∼64% of the captive population) from four breeding centers, Wolong (WL), Chengdu (CD), Louguantai (LGT), and Beijing (BJ), and analyzed 655 bp of mitochondrial DNA control region sequence for 220 of these animals. High levels of genetic diversity and low levels of inbreeding were estimated in the breeding centers, indicating that the captive population is genetically healthy and deliberate further genetic input from wild animals is unnecessary. However, the LGT population faces a higher risk of inbreeding, and significant genetic structure was detected among breeding centers, with LGT-CD and WL-BJ clustering separately. Based on these findings, we highlight that: 1) the LGT population should be managed as an independent captive population to resemble the genetic distinctness of their Qinling Mountain origins; 2) exchange between CD and WL should be encouraged because of similar wild founder sources; 3) the selection of captive individuals for reintroduction should consider their geographic origin, genetic background, and genetic contribution to wild populations; and 4) combining our molecular genetic data with existing pedigree data will better guide giant panda breeding and further reduce inbreeding into the future.

  20. Constrictor: Flux Balance Analysis Constraint Modification Provides Insight for Design of Biochemical Networks

    NASA Astrophysics Data System (ADS)

    Erickson, Keesha; Chatterjee, Anushree

    2014-03-01

    The use of in silico methods has become standard practice to correlate the structure of a biochemical network to the expression of a desired phenotype. Flux balance analysis (FBA) is one of the most prevalent techniques for modeling metabolism. FBA models have been successfully applied to obtain growth predictions, theoretical product yields from heterologous pathways, and genome engineering targets. We take inspiration from high-throughput recombineering techniques, which show that combinatorial exploration can reveal optimal mutants, and apply the advantages of computational techniques to analyze these combinations. We introduce Constrictor, an in silico tool for FBA that allows gene mutations to be analyzed in a combinatorial fashion, by applying simulated constraints accounting for regulation of gene expression. We apply this algorithm to study ethylene production in E. coli through the addition of the heterologous ethylene-forming enzyme from P. syringae. Targeting individual reactions as well as sets of reactions results in theoretical ethylene yields that are as much 65% greater than yields calculated using typical FBA. Constrictor is an adaptable technique that can be used to generate and analyze disparate populations of in silico mutants & select gene expression levels.

  1. Hunter-Gatherer Color Naming Provides New Insight into the Evolution of Color Terms.

    PubMed

    Lindsey, Delwin T; Brown, Angela M; Brainard, David H; Apicella, Coren L

    2015-09-21

    Most people name the myriad colors in the environment using between two and about a dozen color terms, with great variation within and between languages. Investigators generally agree that color lexicons evolve from fewer terms to more terms, as technology advances and color communication becomes increasingly important. However, little is understood about the color naming systems at the least technologically advanced end of the continuum. The Hadza people of Tanzania are nomadic hunter-gatherers who live a subsistence lifestyle that was common before the advent of agriculture (see Supplemental Experimental Procedures, section I;), suggesting that the Hadzane language should be at an early stage of color lexicon evolution. When Hadza, Somali, and US informants named 23 color samples, Hadza informants named only the black, white, and red samples with perfect consensus. Otherwise, they used low-consensus terms or responded "don't know." However, even low-consensus color terms grouped test colors into lexical categories that aligned with those found in other world languages. Furthermore, information-theoretic analysis showed that color communication efficiency within the Hadza, Somali, and US language communities falls on the same continuum as other world languages. Thus, the structure of color categories is in place in Hadzane, even though words for many of the categories are not in general use. These results suggest that even very simple color lexicons include precursors of many color categories but that these categories are initially represented in a diverse and distributed fashion.

  2. Genomic and Proteomic Analyses of the Fungus Arthrobotrys oligospora Provide Insights into Nematode-Trap Formation

    PubMed Central

    Feng, Yun; Li, Xiaomin; Zou, Chenggang; Xu, Jianping; Ren, Yan; Mi, Qili; Wu, Junli; Liu, Shuqun; Liu, Yu; Huang, Xiaowei; Wang, Haiyan; Niu, Xuemei; Li, Juan; Liang, Lianming; Luo, Yanlu; Ji, Kaifang; Zhou, Wei; Yu, Zefen; Li, Guohong; Liu, Yajun; Li, Lei; Qiao, Min; Feng, Lu; Zhang, Ke-Qin

    2011-01-01

    Nematode-trapping fungi are “carnivorous” and attack their hosts using specialized trapping devices. The morphological development of these traps is the key indicator of their switch from saprophytic to predacious lifestyles. Here, the genome of the nematode-trapping fungus Arthrobotrys oligospora Fres. (ATCC24927) was reported. The genome contains 40.07 Mb assembled sequence with 11,479 predicted genes. Comparative analysis showed that A. oligospora shared many more genes with pathogenic fungi than with non-pathogenic fungi. Specifically, compared to several sequenced ascomycete fungi, the A. oligospora genome has a larger number of pathogenicity-related genes in the subtilisin, cellulase, cellobiohydrolase, and pectinesterase gene families. Searching against the pathogen-host interaction gene database identified 398 homologous genes involved in pathogenicity in other fungi. The analysis of repetitive sequences provided evidence for repeat-induced point mutations in A. oligospora. Proteomic and quantitative PCR (qPCR) analyses revealed that 90 genes were significantly up-regulated at the early stage of trap-formation by nematode extracts and most of these genes were involved in translation, amino acid metabolism, carbohydrate metabolism, cell wall and membrane biogenesis. Based on the combined genomic, proteomic and qPCR data, a model for the formation of nematode trapping device in this fungus was proposed. In this model, multiple fungal signal transduction pathways are activated by its nematode prey to further regulate downstream genes associated with diverse cellular processes such as energy metabolism, biosynthesis of the cell wall and adhesive proteins, cell division, glycerol accumulation and peroxisome biogenesis. This study will facilitate the identification of pathogenicity-related genes and provide a broad foundation for understanding the molecular and evolutionary mechanisms underlying fungi-nematodes interactions. PMID:21909256

  3. Spatiotemporal Transcriptome Analysis Provides Insights into Bicolor Tepal Development in Lilium “Tiny Padhye”

    PubMed Central

    Xu, Leifeng; Yang, Panpan; Feng, Yayan; Xu, Hua; Cao, Yuwei; Tang, Yuchao; Yuan, Suxia; Liu, Xinyan; Ming, Jun

    2017-01-01

    The bicolor Asiatic hybrid lily cultivar “Tiny Padhye” is an attractive variety because of its unique color pattern. During its bicolor tepal development, the upper tepals undergo a rapid color change from green to white, while the tepal bases change from green to purple. However, the molecular mechanisms underlying these changes remain largely uncharacterized. To systematically investigate the dynamics of the lily bicolor tepal transcriptome during development, we generated 15 RNA-seq libraries from the upper tepals (S2-U) and basal tepals (S1-D, S2-D, S3-D, and S4-D) of Lilium “Tiny Padhye.” Utilizing the Illumina platform, a total of 295,787 unigenes were obtained from 713.12 million high-quality paired-end reads. A total of 16,182 unigenes were identified as differentially expressed genes during tepal development. Using Kyoto Encyclopedia of Genes and Genomes pathway analysis, candidate genes involved in the anthocyanin biosynthetic pathway (61 unigenes), and chlorophyll metabolic pathway (106 unigenes) were identified. Further analyses showed that most anthocyanin biosynthesis genes were transcribed coordinately in the tepal bases, but not in the upper tepals, suggesting that the bicolor trait of “Tiny Padhye” tepals is caused by the transcriptional regulation of anthocyanin biosynthetic genes. Meanwhile, the high expression level of chlorophyll degradation genes and low expression level of chlorophyll biosynthetic genes resulted in the absence of chlorophylls from “Tiny Padhye” tepals after flowering. Transcription factors putatively involved in the anthocyanin biosynthetic pathway and chlorophyll metabolism in lilies were identified using a weighted gene co-expression network analysis and their possible roles in lily bicolor tepal development were discussed. In conclusion, these extensive transcriptome data provide a platform for elucidating the molecular mechanisms of bicolor tepals in lilies and provide a basis for similar research in other

  4. The Genome of the “Great Speciator” Provides Insights into Bird Diversification

    PubMed Central

    Cornetti, Luca; Valente, Luis M.; Dunning, Luke T.; Quan, Xueping; Black, Richard A.; Hébert, Olivier; Savolainen, Vincent

    2015-01-01

    Among birds, white-eyes (genus Zosterops) have diversified so extensively that Jared Diamond and Ernst Mayr referred to them as the “great speciator.” The Zosterops lineage exhibits some of the fastest rates of species diversification among vertebrates, and its members are the most prolific passerine island colonizers. We present a high-quality genome assembly for the silvereye (Zosterops lateralis), a white-eye species consisting of several subspecies distributed across multiple islands. We investigate the genetic basis of rapid diversification in white-eyes by conducting genomic analyses at varying taxonomic levels. First, we compare the silvereye genome with those of birds from different families and searched for genomic features that may be unique to Zosterops. Second, we compare the genomes of different species of white-eyes from Lifou island (South Pacific), using whole genome resequencing and restriction site associated DNA. Third, we contrast the genomes of two subspecies of silvereye that differ in plumage color. In accordance with theory, we show that white-eyes have high rates of substitutions, gene duplication, and positive selection relative to other birds. Below genus level, we find that genomic differentiation accumulates rapidly and reveals contrasting demographic histories between sympatric species on Lifou, indicative of past interspecific interactions. Finally, we highlight genes possibly involved in color polymorphism between the subspecies of silvereye. By providing the first whole-genome sequence resources for white-eyes and by conducting analyses at different taxonomic levels, we provide genomic evidence underpinning this extraordinary bird radiation. PMID:26338191

  5. Biophysical Coarse-Grained Modeling Provides Insights into Transport through the Nuclear Pore Complex

    PubMed Central

    Moussavi-Baygi, R.; Jamali, Y.; Karimi, R.; Mofrad, M.R.K.

    2011-01-01

    The nuclear pore complex (NPC) is the gatekeeper of the nucleus, capable of actively discriminating between the active and inert cargo while accommodating a high rate of translocations. The biophysical mechanisms underlying transport, however, remain unclear due to the lack of information about biophysical factors playing role in transport. Based on published experimental data, we have established a coarse-grained model of an intact NPC structure to examine nucleocytoplasmic transport with refined spatial and temporal resolutions. Using our model, we estimate the transport time versus cargo sizes. Our findings suggest that the mean transport time of cargos smaller than 15 nm is independent of size, while beyond this size, there is a sharp increase in the mean transport time. The model confirms that kap-FG hydrophobicity is sufficient for active cargo transport. Moreover, our model predicts that during translocation, small and large cargo-complexes are hydrophobically attached to FG-repeat domains for 86 and 96% of their transport time, respectively. Inside the central channel FG-repeats form a thick layer on the wall leaving an open tube. The cargo-complex is almost always attached to this layer and diffuses back and forth, regardless of the cargo size. Finally, we propose a plausible model for transport in which the NPC can be viewed as a lubricated gate. This model incorporates basic assumptions underlying virtual-gate and reduction-of-dimensionality models with the addition of the FG-layer inside the central channel acting as a lubricant. PMID:21402022

  6. Hunter-gatherer color naming provides new insight into the evolution of color terms

    PubMed Central

    Lindsey, Delwin T.; Brown, Angela M.; Brainard, David H.; Apicella, Coren L.

    2015-01-01

    SUMMARY Most people name the myriad colors in the environment using between two and about a dozen color terms [1], with great variation within and between languages [2]. Investigators generally agree that color lexicons evolve from fewer terms to more terms, as technology advances and color communication becomes increasingly important [3]. However, little is understood about the color naming systems at the least technologically-advanced end of the continuum. The Hadza people of Tanzania are nomadic hunter-gatherers who live a subsistence lifestyle that was common before the advent of agriculture (see SM-I, [4]), suggesting that the Hadzane language should be at an early stage of color lexicon evolution. When Hadza, Somali, and U.S. informants named 23 color samples, Hadza informants named only the black, white and red samples with perfect consensus. Otherwise, they used low-consensus terms, or responded “don’t know”. However, even low-consensus color terms grouped test colors into lexical categories that aligned with those found in other world languages [5]. Furthermore, information-theoretic analysis showed that color communication efficiency within the Hadza, Somali, and U.S. language communities falls on the same continuum as other world languages. Thus, the structure of color categories is in place in Hadzane, even though words for many of the categories are not in general use. These results suggest that even very simple color lexicons include precursors of many color categories, but that these categories are initially represented in a diverse and distributed fashion. PMID:26365254

  7. Glycation of Lysozyme by Glycolaldehyde Provides New Mechanistic Insights in Diabetes-Related Protein Aggregation.

    PubMed

    Mariño, Laura; Maya-Aguirre, Carlos Andrés; Pauwels, Kris; Vilanova, Bartolomé; Ortega-Castro, Joaquin; Frau, Juan; Donoso, Josefa; Adrover, Miquel

    2017-03-14

    Glycation occurs in vivo as a result of the nonenzymatic reaction of carbohydrates (and/or their autoxidation products) with proteins, DNA, or lipids. Protein glycation causes loss-of-function and, consequently, the development of diabetic-related diseases. Glycation also boosts protein aggregation, which can be directly related with the higher prevalence of aggregating diseases in diabetic people. However, the molecular mechanism connecting glycation with aggregation still remains unclear. Previously we described mechanistically how glycation of hen egg-white lysozyme (HEWL) with ribose induced its aggregation. Here we address the question of whether the ribose-induced aggregation is a general process or it depends on the chemical nature of the glycating agent. Glycation of HEWL with glycolaldehyde occurs through two different scenarios depending on the HEWL concentration regime (both within the micromolar range). At low HEWL concentration, non-cross-linking fluorescent advanced glycation end-products (AGEs) are formed on Lys side chains, which do not change the protein structure but inhibit its enzymatic activity. These AGEs have little impact on HEWL surface hydrophobicity and, therefore, a negligible effect on its aggregation propensity. Upon increasing HEWL concentration, the glycation mechanism shifts toward the formation of intermolecular cross-links, which triggers a polymerization cascade involving the formation of insoluble spherical-like aggregates. These results notably differ with the aggregation-modulation mechanism of ribosylated HEWL directed by hydrophobic interactions. Additionally, their comparison constitutes the first experimental evidence showing that the mechanism underlying the aggregation of a glycated protein depends on the chemical nature of the glycating agent.

  8. Computational predictions provide insights into the biology of TAL effector target sites.

    PubMed

    Grau, Jan; Wolf, Annett; Reschke, Maik; Bonas, Ulla; Posch, Stefan; Boch, Jens

    2013-01-01

    Transcription activator-like (TAL) effectors are injected into host plant cells by Xanthomonas bacteria to function as transcriptional activators for the benefit of the pathogen. The DNA binding domain of TAL effectors is composed of conserved amino acid repeat structures containing repeat-variable diresidues (RVDs) that determine DNA binding specificity. In this paper, we present TALgetter, a new approach for predicting TAL effector target sites based on a statistical model. In contrast to previous approaches, the parameters of TALgetter are estimated from training data computationally. We demonstrate that TALgetter successfully predicts known TAL effector target sites and often yields a greater number of predictions that are consistent with up-regulation in gene expression microarrays than an existing approach, Target Finder of the TALE-NT suite. We study the binding specificities estimated by TALgetter and approve that different RVDs are differently important for transcriptional activation. In subsequent studies, the predictions of TALgetter indicate a previously unreported positional preference of TAL effector target sites relative to the transcription start site. In addition, several TAL effectors are predicted to bind to the TATA-box, which might constitute one general mode of transcriptional activation by TAL effectors. Scrutinizing the predicted target sites of TALgetter, we propose several novel TAL effector virulence targets in rice and sweet orange. TAL-mediated induction of the candidates is supported by gene expression microarrays. Validity of these targets is also supported by functional analogy to known TAL effector targets, by an over-representation of TAL effector targets with similar function, or by a biological function related to pathogen infection. Hence, these predicted TAL effector virulence targets are promising candidates for studying the virulence function of TAL effectors. TALgetter is implemented as part of the open-source Java library

  9. Whole genome sequencing of 35 individuals provides insights into the genetic architecture of Korean population

    PubMed Central

    2014-01-01

    Background Due to a significant decline in the costs associated with next-generation sequencing, it has become possible to decipher the genetic architecture of a population by sequencing a large number of individuals to a deep coverage. The Korean Personal Genomes Project (KPGP) recently sequenced 35 Korean genomes at high coverage using the Illumina Hiseq platform and made the deep sequencing data publicly available, providing the scientific community opportunities to decipher the genetic architecture of the Korean population. Methods In this study, we used two single nucleotide variant (SNV) calling pipelines: mapping the raw reads obtained from whole genome sequencing of 35 Korean individuals in KPGP using BWA and SOAP2 followed by SNV calling using SAMtools and SOAPsnp, respectively. The consensus SNVs obtained from the two SNV pipelines were used to represent the SNVs of the Korean population. We compared these SNVs to those from 17 other populations provided by the HapMap consortium and the 1000 Genomes Project (1KGP) and identified SNVs that were only present in the Korean population. We studied the mutation spectrum and analyzed the genes of non-synonymous SNVs only detected in the Korean population. Results We detected a total of 8,555,726 SNVs in the 35 Korean individuals and identified 1,213,613 SNVs detected in at least one Korean individual (SNV-1) and 12,640 in all of 35 Korean individuals (SNV-35) but not in 17 other populations. In contrast with the SNVs common to other populations in HapMap and 1KGP, the Korean only SNVs had high percentages of non-silent variants, emphasizing the unique roles of these Korean only SNVs in the Korean population. Specifically, we identified 8,361 non-synonymous Korean only SNVs, of which 58 SNVs existed in all 35 Korean individuals. The 5,754 genes of non-synonymous Korean only SNVs were highly enriched in some metabolic pathways. We found adhesion is the top disease term associated with SNV-1 and Nelson syndrome is

  10. Magnetic apatite for structural insights on the plasma membrane.

    PubMed

    Stanca, Sarmiza E; Müller, Robert; Dellith, Jan; Nietzsche, Sandor; Stöckel, Stephan; Biskup, Christoph; Deckert, Volker; Krafft, Christoph; Popp, Jürgen; Fritzsche, Wolfgang

    2015-01-21

    The iron oxide-hydroxyapatite (FeOxHA) nanoparticles reported here differ from those reported before by their advantage of homogeneity and simple preparation; moreover, the presence of carboxymethyldextran (CMD), together with hydroxyapatite (HA), allows access to the cellular membrane, which makes our magnetic apatite unique. These nanoparticles combine magnetic behavior, Raman label ability and the property of interaction with the cellular membrane; they therefore represent an interesting material for structural differentiation of the cell membrane. It was observed by Raman spectroscopy, scanning electron microscopy (SEM) and fluorescence microscopy that FeOxHA adheres to the plasma membrane and does not penetrate the membrane. These insights make the nanoparticles a promising material for magnetic cell sorting, e.g. in microfluidic device applications.

  11. Magnetic apatite for structural insights on the plasma membrane

    NASA Astrophysics Data System (ADS)

    Stanca, Sarmiza E.; Müller, Robert; Dellith, Jan; Nietzsche, Sandor; Stöckel, Stephan; Biskup, Christoph; Deckert, Volker; Krafft, Christoph; Popp, Jürgen; Fritzsche, Wolfgang

    2015-01-01

    The iron oxide-hydroxyapatite (FeOxHA) nanoparticles reported here differ from those reported before by their advantage of homogeneity and simple preparation; moreover, the presence of carboxymethyldextran (CMD), together with hydroxyapatite (HA), allows access to the cellular membrane, which makes our magnetic apatite unique. These nanoparticles combine magnetic behavior, Raman label ability and the property of interaction with the cellular membrane; they therefore represent an interesting material for structural differentiation of the cell membrane. It was observed by Raman spectroscopy, scanning electron microscopy (SEM) and fluorescence microscopy that FeOxHA adheres to the plasma membrane and does not penetrate the membrane. These insights make the nanoparticles a promising material for magnetic cell sorting, e.g. in microfluidic device applications.

  12. Aging and fertility patterns in wild chimpanzees provide insights into the evolution of menopause

    PubMed Central

    Thompson, Melissa Emery; Jones, James H.; Pusey, Anne E.; Brewer-Marsden, Stella; Goodall, Jane; Marsden, David; Matsuzawa, Tetsuro; Nishida, Toshisada; Reynolds, Vernon; Sugiyama, Yukimaru; Wrangham, Richard W.

    2008-01-01

    Summary Human menopause is remarkable in that reproductive senescence is markedly accelerated relative to somatic aging, leaving an extended post-reproductive period for a large proportion of women [1, 2]. Functional explanations for this are debated [4-11], in part because comparative data from closely-related species are inadequate. Existing studies of chimpanzees are based on very small samples and have not provided clear conclusions about the reproductive function of aging females [12-19]. These studies have not examined whether reproductive senescence in chimpanzees exceeds the pace of general aging, as in humans, or occurs in parallel with declines in overall health, as in many other animals [20, 21]. In order to remedy these problems, we examined fertility and mortality patterns in 6 free-living chimpanzee populations. Chimpanzee and human birth rates show similar patterns of decline beginning in the 4th decade, suggesting that the physiology of reproductive senescence was relatively conserved in human evolution. However, in contrast to humans, chimpanzee fertility declines are consistent with declines in survivorship, and healthy females maintain high birth rates late into life. Thus, in contrast to recent claims [16], we find no evidence that menopause is a typical characteristic of chimpanzee life histories. PMID:18083515

  13. Genomes of Ellobius species provide insight into the evolutionary dynamics of mammalian sex chromosomes

    PubMed Central

    Mulugeta, Eskeatnaf; Wassenaar, Evelyne; Sleddens-Linkels, Esther; van IJcken, Wilfred F.J.; Heard, Edith; Grootegoed, J. Anton; Just, Walter; Gribnau, Joost; Baarends, Willy M.

    2016-01-01

    The X and Y sex chromosomes of placental mammals show hallmarks of a tumultuous evolutionary past. The X Chromosome has a rich and conserved gene content, while the Y Chromosome has lost most of its genes. In the Transcaucasian mole vole Ellobius lutescens, the Y Chromosome including Sry has been lost, and both females and males have a 17,X diploid karyotype. Similarly, the closely related Ellobius talpinus, has a 54,XX karyotype in both females and males. Here, we report the sequencing and assembly of the E. lutescens and E. talpinus genomes. The results indicate that the loss of the Y Chromosome in E. lutescens and E. talpinus occurred in two independent events. Four functional homologs of mouse Y-Chromosomal genes were detected in both female and male E. lutescens, of which three were also detected in the E. talpinus genome. One of these is Eif2s3y, known as the only Y-derived gene that is crucial for successful male meiosis. Female and male E. lutescens can carry one and the same X Chromosome with a largely conserved gene content, including all genes known to function in X Chromosome inactivation. The availability of the genomes of these mole vole species provides unique models to study the dynamics of sex chromosome evolution. PMID:27510564

  14. A recovery principle provides insight into auxin pattern control in the Arabidopsis root.

    PubMed

    Moore, Simon; Liu, Junli; Zhang, Xiaoxian; Lindsey, Keith

    2017-02-21

    Regulated auxin patterning provides a key mechanism for controlling root growth and development. We have developed a data-driven mechanistic model using realistic root geometry and formulated a principle to theoretically investigate quantitative auxin pattern recovery following auxin transport perturbation. This principle reveals that auxin patterning is potentially controlled by multiple combinations of interlinked levels and localisation of influx and efflux carriers. We demonstrate that (1) when efflux carriers maintain polarity but change levels, maintaining the same auxin pattern requires non-uniform and polar distribution of influx carriers; (2) the emergence of the same auxin pattern, from different levels of influx carriers with the same nonpolar localisation, requires simultaneous modulation of efflux carrier level and polarity; and (3) multiple patterns of influx and efflux carriers for maintaining an auxin pattern do not have spatially proportional correlation. This reveals that auxin pattern formation requires coordination between influx and efflux carriers. We further show that the model makes various predictions that can be experimentally validated.

  15. Spatiotemporal transcriptome provides insights into early fruit development of tomato (Solanum lycopersicum)

    PubMed Central

    Zhang, Shuaibin; Xu, Meng; Qiu, Zhengkun; Wang, Ketao; Du, Yongchen; Gu, Lianfeng; Cui, Xia

    2016-01-01

    Early fruit development is crucial for crop production in tomato. After fertilization, the ovary undergoes cell division and cell expansion before maturation. Although the roles of regulatory signals such as hormone and carbohydrate during early fruit development have been studied, the spatial distribution and the sequential initiation of these regulatory signals still need to be explored. Using the tomato cultivar ‘Moneymaker’, we analyzed the transcriptome of the ovule and the ovary wall/pericarp dissected from four different stages of the early developing fruits by stereoscope. These datasets give us the whole picture about the spatial and temporal signal distribution in early development of ovule and pericarp. Our results indicate that the hormone signal was initiated in both ovule and pericarp after fertilization. After that, different signals were activated in ovule and pericarp due to their distinct developmental processes. Our study provides spatiotemporal regulatory landscape of gene expression with sequential information which was not studied by previous work and further strengthens the comprehension of the regulatory and metabolic events controlling early fruit development. PMID:26988970

  16. A recovery principle provides insight into auxin pattern control in the Arabidopsis root

    PubMed Central

    Moore, Simon; Liu, Junli; Zhang, Xiaoxian; Lindsey, Keith

    2017-01-01

    Regulated auxin patterning provides a key mechanism for controlling root growth and development. We have developed a data-driven mechanistic model using realistic root geometry and formulated a principle to theoretically investigate quantitative auxin pattern recovery following auxin transport perturbation. This principle reveals that auxin patterning is potentially controlled by multiple combinations of interlinked levels and localisation of influx and efflux carriers. We demonstrate that (1) when efflux carriers maintain polarity but change levels, maintaining the same auxin pattern requires non-uniform and polar distribution of influx carriers; (2) the emergence of the same auxin pattern, from different levels of influx carriers with the same nonpolar localisation, requires simultaneous modulation of efflux carrier level and polarity; and (3) multiple patterns of influx and efflux carriers for maintaining an auxin pattern do not have spatially proportional correlation. This reveals that auxin pattern formation requires coordination between influx and efflux carriers. We further show that the model makes various predictions that can be experimentally validated. PMID:28220889

  17. Moments of action provide insight into critical times for advection-diffusion-reaction processes.

    PubMed

    Ellery, Adam J; Simpson, Matthew J; McCue, Scott W; Baker, Ruth E

    2012-09-01

    Berezhkovskii and co-workers introduced the concept of local accumulation time as a finite measure of the time required for the transient solution of a reaction-diffusion equation to effectively reach steady state [Biophys J. 99, L59 (2010); Phys. Rev. E 83, 051906 (2011)]. Berezhkovskii's approach is a particular application of the concept of mean action time (MAT) that was introduced previously by McNabb [IMA J. Appl. Math. 47, 193 (1991)]. Here, we generalize these previous results by presenting a framework to calculate the MAT, as well as the higher moments, which we call the moments of action. The second moment is the variance of action time, the third moment is related to the skew of action time, and so on. We consider a general transition from some initial condition to an associated steady state for a one-dimensional linear advection-diffusion-reaction partial differential equation (PDE). Our results indicate that it is possible to solve for the moments of action exactly without requiring the transient solution of the PDE. We present specific examples that highlight potential weaknesses of previous studies that have considered the MAT alone without considering higher moments. Finally, we also provide a meaningful interpretation of the moments of action by presenting simulation results from a discrete random-walk model together with some analysis of the particle lifetime distribution. This work shows that the moments of action are identical to the moments of the particle lifetime distribution for certain transitions.

  18. Location, location, location! Monotremes provide unique insights into the evolution of sex chromosome silencing in mammals.

    PubMed

    Daish, Tasman; Grützner, Frank

    2009-02-01

    Platypus and echidnas are the only living representative of the egg-laying mammals that diverged 166 million years ago from the mammalian lineage. Despite occupying a key spot in mammalian phylogeny, research on monotremes has been limited by access to material and lack of molecular genetic resources. This has changed recently, and the sequencing of the platypus genome has promoted monotremes into a generally accessible tool in comparative genomics. The most extraordinary aspect of the monotreme genome is an amazingly complex sex chromosomes system that shares extensive homology with bird sex chromosomes and no homology with sex chromosomes of other mammals. This raises important questions about dosage compensation of the five pairs of sex chromosomes in females and meiotic silencing in males, and we are only beginning to unravel possible mechanisms and pathways that may be involved. The homology between monotreme and bird sex chromosomes makes comparison between those species worthwhile, also as they provide a well-defined example where the same sex chromosomes changed from female heterogamety (chicken) to male heterogamety (monotremes). We summarize recent research on monotreme and chicken sex chromosomes and discuss possible mechanisms that may contribute to sex chromosome silencing in monotremes.

  19. Molecular underpinnings of prefrontal cortex development in rodents provide insights into the etiology of neurodevelopmental disorders.

    PubMed

    Schubert, D; Martens, G J M; Kolk, S M

    2015-07-01

    The prefrontal cortex (PFC), seat of the highest-order cognitive functions, constitutes a conglomerate of highly specialized brain areas and has been implicated to have a role in the onset and installation of various neurodevelopmental disorders. The development of a properly functioning PFC is directed by transcription factors, guidance cues and other regulatory molecules and requires the intricate and temporal orchestration of a number of developmental processes. Disturbance or failure of any of these processes causing neurodevelopmental abnormalities within the PFC may contribute to several of the cognitive deficits seen in patients with neurodevelopmental disorders. In this review, we elaborate on the specific processes underlying prefrontal development, such as induction and patterning of the prefrontal area, proliferation, migration and axonal guidance of medial prefrontal progenitors, and their eventual efferent and afferent connections. We furthermore integrate for the first time the available knowledge from genome-wide studies that have revealed genes linked to neurodevelopmental disorders with experimental molecular evidence in rodents. The integrated data suggest that the pathogenic variants in the neurodevelopmental disorder-associated genes induce prefrontal cytoarchitectonical impairments. This enhances our understanding of the molecular mechanisms of prefrontal (mis)development underlying the four major neurodevelopmental disorders in humans, that is, intellectual disability, autism spectrum disorders, attention deficit hyperactivity disorder and schizophrenia, and may thus provide clues for the development of novel therapies.

  20. In situ biodeposition measurements on a Modiolus modiolus (horse mussel) reef provide insights into ecosystem services

    NASA Astrophysics Data System (ADS)

    Kent, Flora E. A.; Last, Kim S.; Harries, Daniel B.; Sanderson, William G.

    2017-01-01

    Horse mussel (Modiolus modiolus) shellfish reefs are a threatened and declining habitat in the North East Atlantic and support high levels of biodiversity. Shellfish can influence the surrounding water column and modify the quality of material that reaches the seabed by filtering water, actively depositing particles and changing the benthic boundary layer due to surface roughness. In the present study M. modiolus biodeposition was measured in a field location for the first time. The results show that M. modiolus enhance sedimentation and contribute to the downward flux of material to the seabed. Approximately 30% of the total sediment deposition was attributed to active filter feeding and overall, the presence of horse mussels enhanced deposition two fold. The results are discussed in terms of the potential for horse mussel reefs to provide ecosystem services to society, through functions such as benthopelagic coupling and sediment stabilisation. Highlighting the societal benefits supplied by marine habitats can help prioritise conservation efforts and feed into the sustainable management of coastal water bodies.

  1. Transcriptome Analysis of Thapsia laciniata Rouy Provides Insights into Terpenoid Biosynthesis and Diversity in Apiaceae

    PubMed Central

    Drew, Damian Paul; Dueholm, Bjørn; Weitzel, Corinna; Zhang, Ye; Sensen, Christoph W.; Simonsen, Henrik Toft

    2013-01-01

    Thapsia laciniata Rouy (Apiaceae) produces irregular and regular sesquiterpenoids with thapsane and guaiene carbon skeletons, as found in other Apiaceae species. A transcriptomic analysis utilizing Illumina next-generation sequencing enabled the identification of novel genes involved in the biosynthesis of terpenoids in Thapsia. From 66.78 million HQ paired-end reads obtained from T. laciniata roots, 64.58 million were assembled into 76,565 contigs (N50: 1261 bp). Seventeen contigs were annotated as terpene synthases and five of these were predicted to be sesquiterpene synthases. Of the 67 contigs annotated as cytochromes P450, 18 of these are part of the CYP71 clade that primarily performs hydroxylations of specialized metabolites. Three contigs annotated as aldehyde dehydrogenases grouped phylogenetically with the characterized ALDH1 from Artemisia annua and three contigs annotated as alcohol dehydrogenases grouped with the recently described ADH1 from A. annua. ALDH1 and ADH1 were characterized as part of the artemisinin biosynthesis. We have produced a comprehensive EST dataset for T. laciniata roots, which contains a large sample of the T. laciniata transcriptome. These transcriptome data provide the foundation for future research into the molecular basis for terpenoid biosynthesis in Thapsia and on the evolution of terpenoids in Apiaceae. PMID:23698765

  2. Global epigenomic analysis of primary human pancreatic islets provides insights into type 2 diabetes susceptibility loci

    PubMed Central

    Stitzel, Michael L.; Sethupathy, Praveen; Pearson, Daniel S.; Chines, Peter S.; Song, Lingyun; Erdos, Michael R.; Welch, Ryan; Parker, Stephen C. J.; Boyle, Alan P.; Scott, Laura J.; Margulies, Elliott H.; Boehnke, Michael; Furey, Terrence S.; Crawford, Gregory E.; Collins, Francis S.

    2010-01-01

    Summary Identifying cis-regulatory elements is important to understand how human pancreatic islets modulate gene expression in physiologic or pathophysiologic (e.g., diabetic) conditions. We conducted genome-wide analysis of DNase I hypersensitive sites, histone H3 lysine methylation modifications (K4me1, K4me3, K79me2), and CCCTC factor (CTCF) binding in human islets. This identified ~18,000 putative promoters (several hundred unannotated and islet-active). Surprisingly, active promoter modifications were absent at genes encoding islet-specific hormones, suggesting a distinct regulatory mechanism. Of 34,039 distal (non-promoter) regulatory elements, 47% are islet-unique and 22% are CTCF-bound. In the 18 type 2 diabetes (T2D)-associated loci, we identified 118 putative regulatory elements and confirmed enhancer activity for 12/33 tested. Among 6 regulatory elements harboring T2D-associated variants, 2 exhibit significant allele-specific differences in activity. These findings present a global snapshot of the human islet epigenome and should provide functional context for non-coding variants emerging from genetic studies of T2D and other islet disorders. PMID:21035756

  3. Transcriptome-wide sequencing provides insights into geocarpy in