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Sample records for provide structural insight

  1. Structure of CD84 Provides Insight into SLAM Family Function

    SciTech Connect

    Yan,Q.; Malashkevich, V.; Fedorov, A.; Fedorov, E.; Cao, E.; Lary, J.; Cole, J.; Nathenson, S.; Almo, S.

    2007-01-01

    The signaling lymphocyte activation molecule (SLAM) family includes homophilic and heterophilic receptors that modulate both adaptive and innate immune responses. These receptors share a common ectodomain organization: a membrane-proximal immunoglobulin constant domain and a membrane-distal immunoglobulin variable domain that is responsible for ligand recognition. CD84 is a homophilic family member that enhances IFN-{gamma} secretion in activated T cells. Our solution studies revealed that CD84 strongly self-associates with a K{sub d} in the submicromolar range. These data, in combination with previous reports, demonstrate that the SLAM family homophilic affinities span at least three orders of magnitude and suggest that differences in the affinities may contribute to the distinct signaling behavior exhibited by the individual family members. The 2.0 {angstrom} crystal structure of the human CD84 immunoglobulin variable domain revealed an orthogonal homophilic dimer with high similarity to the recently reported homophilic dimer of the SLAM family member NTB-A. Structural and chemical differences in the homophilic interfaces provide a mechanism to prevent the formation of undesired heterodimers among the SLAM family homophilic receptors. These structural data also suggest that, like NTB-A, all SLAM family homophilic dimers adopt a highly kinked organization spanning an end-to-end distance of {approx}140 {angstrom}. This common molecular dimension provides an opportunity for all two-domain SLAM family receptors to colocalize within the immunological synapse and bridge the T cell and antigen-presenting cell.

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

    PubMed

    Olal, Daniel; Daumke, Oliver

    2016-03-01

    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. PMID:26923588

  3. 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

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

    PubMed

    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

  5. 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.

  6. 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. PMID:27498030

  7. 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.

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

    PubMed Central

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

    2014-01-01

    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. PMID:24816101

  9. 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

    2015-02-01

    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. PMID:25492868

  10. Characterization of Gain-of-Function Mutant Provides New Insights into ClpP Structure.

    PubMed

    Ni, Tengfeng; Ye, Fei; Liu, Xing; Zhang, Jie; Liu, Hongchuan; Li, Jiahui; Zhang, Yingyi; Sun, Yinqiang; Wang, Meining; Luo, Cheng; Jiang, Hualiang; Lan, Lefu; Gan, Jianhua; Zhang, Ao; Zhou, Hu; Yang, Cai-Guang

    2016-07-15

    ATP-dependent Clp protease (ClpP), a highly conserved serine protease in vast bacteria, could be converted into a noncontrollable enzyme capable of degrading mature proteins in the presence of acyldepsipeptides (ADEPs). Here, we design such a gain-of-function mutant of Staphylococcus aureus ClpP (SaClpP) capable of triggering the same level of dysfunctional activity that occurs upon ADEPs treatment. The SaClpPY63A mutant degrades FtsZ in vivo and inhibits staphylococcal growth. The crystal structure of SaClpPY63A indicates that Asn42 would be an important domino to fall for further activation of ClpP. Indeed, the SaClpPN42AY63A mutant demonstrates promoted self-activated proteolysis, which is a result of an enlarged entrance pore as observed in cryo-electron microscopy images. In addition, the expression of the engineered clpP allele phenocopies treatment with ADEPs; inhibition of cell division occurs as does showing sterilizing with rifampicin antibiotics. Collectively, we show that the gain-of-function SaClpPN42AY63A mutant becomes a fairly nonspecific protease and kills persisters by degrading over 500 proteins, thus providing new insights into the structure of the ClpP protease. PMID:27171654

  11. Identification of O-glycan Structures from Chicken Intestinal Mucins Provides Insight into Campylobactor jejuni Pathogenicity*

    PubMed Central

    Struwe, Weston B.; Gough, Ronan; Gallagher, Mary E.; Kenny, Diarmuid T.; Carrington, Stephen D.; Karlsson, Niclas G.; Rudd, Pauline M.

    2015-01-01

    The Gram-negative bacteria Campylobactor jejuni is the primary bacteria responsible for food poisoning in industrialized countries, and acute diarrheal illness is a leading cause of mortality among children in developing countries. C. jejuni are commensal in chickens. They are particularly abundant in the caecal crypts, and poultry products are commonly infected as a result of cross-contamination during processing. The interactions between C. jejuni and chicken intestinal tissues as well as the pathogenic molecular mechanisms of colonization in humans are unknown, but identifying these factors could provide potential targets to reduce the incidence of campylobacteriosis. Recently, purified chicken intestinal mucin was shown to attenuate adherence and invasion of C. jejuni in the human colorectal adenocarcinoma cell line HCT-8 in vitro, and this effect was attributed to mucin O-glycosylation. Mucins from different regions of the chicken intestine inhibited C. jejuni binding and internalization differentially, with large intestine>small intestine>caecum. Here, we use LC-MS to perform a detailed structural analysis of O-glycans released from mucins purified from chicken large intestine, small intestine, and caecum. The O-glycans identified were abundantly sulfated compared with the human intestines, and sulfate moieties were present throughout the chicken intestinal tract. Interestingly, alpha 1–2 linked fucose residues, which have a high binding affinity to C. jejuni, were identified in the small and large intestines. Additionally, N-glycolylneuraminic/N-acetylneuraminic acid containing structures present as Sda-like epitopes were identified in large intestine samples but not small intestine or caecum. O-glycan structural characterization of chicken intestinal mucins provides insights into adherence and invasion properties of C. jejuni, and may offer prospective candidate molecules aimed at reducing the incidence of infection. PMID:25776888

  12. 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. PMID:17597061

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

    PubMed Central

    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. PMID:27018598

  14. 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.

  15. 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

  16. In vitro oxidative footprinting provides insight into apolipoprotein B-100 structure in low density lipoprotein

    PubMed Central

    Chakraborty, Sourav; Cai, Yang; Tarr, Matthew A.

    2015-01-01

    Low density lipoprotein (LDL) is a major cholesterol carrier in human blood. Oxidations of apolipoprotein B-100 (apo B-100, LDL protein) could be pro-atherogenic and play critical roles in early stages of plaque formation in the arterial wall. The structure of apo B-100 is still poorly understood, partially due to its size (550 KDa, 4563 amino acids). To gain an insight into LDL structure, we mapped the regions of apo B-100 in human LDL which were prone to oxidation using peroxynitrite and hypochlorite as probes. In this study, LDL was incubated with various concentrations of peroxynitrite and sodium hypochlorite in bicarbonate buffer. The LDL protein apo B-100 was delipidated, denatured, alkylated and subjected to tryptic digestion. Tryptic peptides were analyzed employing liquid chromatography – tandem mass spectrometry (LC-MS/MS). Database search was performed against the apo B-100 database (P04114) using “SEQUEST” algorithm to identify peroxynitrite and hypochlorite mediated oxidations markers nitrotyrosine, nitrotryptophan, hydroxy-tryptophan and 3-chlorotyrosine. Several site specific oxidations were identified in apo B-100 after treatment of intact LDL particles with the oxidants. We hypothesize that these regions could be accessible to oxidant and critical for early events in atherosclerotic plaque deposition. PMID:25176030

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. Stable Isotopes Provide Insight into Population Structure and Segregation in Eastern North Atlantic Sperm Whales

    PubMed Central

    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 (δ18O) in the inorganic component (hydroxyapatite), and nitrogen and carbon isotope ratios (δ15N: δ13C) in the organic component (primarily collagenous). We found significant differences between Denmark and NW Spain in δ15N and δ18O values in the layer deposited at age 3, considered to be the one best representing the baseline of the breeding ground, in δ15N, δ13C and δ18O values in the period up to age 20, and in the ontogenetic variation of δ15N and δ18O 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. PMID:24324782

  2. 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. PMID:24324782

  3. Neristatin 1 provides critical insight into bryostatin 1 structure-function relationships.

    PubMed

    Kedei, Noemi; Kraft, Matthew B; Keck, Gary E; Herald, Cherry L; Melody, Noeleen; Pettit, George R; Blumberg, Peter M

    2015-04-24

    Bryostatin 1, a complex macrocyclic lactone isolated from Bugula neritina, has been the subject of multiple clinical trials for cancer. Although it functions as an activator of protein kinase C (PKC) in vitro, bryostatin 1 paradoxically antagonizes most responses to the prototypical PKC activator, the phorbol esters. The bottom half of the bryostatin 1 structure has been shown to be sufficient to confer binding to PKC. In contrast, we have previously shown that the top half of the bryostatin 1 structure is necessary for its unique biological behavior to antagonize phorbol ester responses. Neristatin 1 comprises a top half similar to that of bryostatin 1 together with a distinct bottom half that confers PKC binding. We report here that neristatin 1 is bryostatin 1-like, not phorbol ester-like, in its biological activity on U937 promyelocytic leukemia cells. We conclude that the top half of the bryostatin 1 structure is largely sufficient for bryostatin 1-like activity, provided the molecule also possesses an appropriate PKC binding domain. PMID:25808573

  4. 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

  5. Structure of LIMP-2 provides functional insights with implications for SR-BI and CD36.

    PubMed

    Neculai, Dante; Schwake, Michael; Ravichandran, Mani; Zunke, Friederike; Collins, Richard F; Peters, Judith; Neculai, Mirela; Plumb, Jonathan; Loppnau, Peter; Pizarro, Juan Carlos; Seitova, Alma; Trimble, William S; Saftig, Paul; Grinstein, Sergio; Dhe-Paganon, Sirano

    2013-12-01

    Members of the CD36 superfamily of scavenger receptor proteins are important regulators of lipid metabolism and innate immunity. They recognize normal and modified lipoproteins, as well as pathogen-associated molecular patterns. The family consists of three members: SR-BI (which delivers cholesterol to the liver and steroidogenic organs and is a co-receptor for hepatitis C virus), LIMP-2/LGP85 (which mediates lysosomal delivery of β-glucocerebrosidase and serves as a receptor for enterovirus 71 and coxsackieviruses) and CD36 (a fatty-acid transporter and receptor for phagocytosis of effete cells and Plasmodium-infected erythrocytes). Notably, CD36 is also a receptor for modified lipoproteins and β-amyloid, and has been implicated in the pathogenesis of atherosclerosis and of Alzheimer's disease. Despite their prominent roles in health and disease, understanding the function and abnormalities of the CD36 family members has been hampered by the paucity of information about their structure. Here we determine the crystal structure of LIMP-2 and infer, by homology modelling, the structure of SR-BI and CD36. LIMP-2 shows a helical bundle where β-glucocerebrosidase binds, and where ligands are most likely to bind to SR-BI and CD36. Remarkably, the crystal structure also shows the existence of a large cavity that traverses the entire length of the molecule. Mutagenesis of SR-BI indicates that the cavity serves as a tunnel through which cholesterol(esters) are delivered from the bound lipoprotein to the outer leaflet of the plasma membrane. We provide evidence supporting a model whereby lipidic constituents of the ligands attached to the receptor surface are handed off to the membrane through the tunnel, accounting for the selective lipid transfer characteristic of SR-BI and CD36. PMID:24162852

  6. Single nucleotide polymorphism discovery in albacore and Atlantic bluefin tuna provides insights into worldwide population structure.

    PubMed

    Albaina, A; Iriondo, M; Velado, I; Laconcha, U; Zarraonaindia, I; Arrizabalaga, H; Pardo, M A; Lutcavage, M; Grant, W S; Estonba, A

    2013-12-01

    The optimal management of the commercially important, but mostly over-exploited, pelagic tunas, albacore (Thunnus alalunga Bonn., 1788) and Atlantic bluefin tuna (BFT; Thunnus thynnus L., 1758), requires a better understanding of population structure than has been provided by previous molecular methods. Despite numerous studies of both species, their population structures remain controversial. This study reports the development of single nucleotide polymorphisms (SNPs) in albacore and BFT and the application of these SNPs to survey genetic variability across the geographic ranges of these tunas. A total of 616 SNPs were discovered in 35 albacore tuna by comparing sequences of 54 nuclear DNA fragments. A panel of 53 SNPs yielded FST values ranging from 0.0 to 0.050 between samples after genotyping 460 albacore collected throughout the distribution of this species. No significant heterogeneity was detected within oceans, but between-ocean comparisons (Atlantic, Pacific and Indian oceans along with Mediterranean Sea) were significant. Additionally, a 17-SNP panel was developed in Atlantic BFT by cross-species amplification in 107 fish. This limited number of SNPs discriminated between samples from the two major spawning areas of Atlantic BFT (FST  = 0.116). The SNP markers developed in this study can be used to genotype large numbers of fish without the need for standardizing alleles among laboratories. PMID:23668670

  7. 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

    2016-01-01

    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 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. PMID:26975884

  8. 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

  9. 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. PMID:27211345

  10. Variations in the colchicine-binding domain provide insight into the structural switch of tubulin

    PubMed Central

    Dorléans, Audrey; Gigant, Benoît; Ravelli, Raimond B. G.; Mailliet, Patrick; Mikol, Vincent; Knossow, Marcel

    2009-01-01

    Structural changes occur in the αβ-tubulin heterodimer during the microtubule assembly/disassembly cycle. Their most prominent feature is a transition from a straight, microtubular structure to a curved structure. There is a broad range of small molecule compounds that disturbs the microtubule cycle, a class of which targets the colchicine-binding site and prevents microtubule assembly. This class includes compounds with very different chemical structures, and it is presently unknown whether they prevent tubulin polymerization by the same mechanism. To address this issue, we have determined the structures of tubulin complexed with a set of such ligands and show that they interfere with several of the movements of tubulin subunits structural elements upon its transition from curved to straight. We also determined the structure of tubulin unliganded at the colchicine site; this reveals that a β-tubulin loop (termed T7) flips into this site. As with colchicine site ligands, this prevents a helix which is at the interface with α-tubulin from stacking onto a β-tubulin β sheet as in straight protofilaments. Whereas in the presence of these ligands the interference with microtubule assembly gets frozen, by flipping in and out the β-subunit T7 loop participates in a reversible way in the resistance to straightening that opposes microtubule assembly. Our results suggest that it thereby contributes to microtubule dynamic instability. PMID:19666559

  11. Crystal structure of an acetyl esterase complexed with acetate ion provides insights into the catalytic mechanism.

    PubMed

    Uechi, Keiko; Kamachi, Saori; Akita, Hironaga; Mine, Shouhei; Watanabe, Masahiro

    2016-08-26

    We previously reported the crystal structure of an acetyl esterase (TcAE206) belonging to carbohydrate esterase family 3 from Talaromyces cellulolyticus. In this study, we solved the crystal structure of an S10A mutant of TcAE206 complexed with an acetate ion. The acetate ion was stabilized by three hydrogen bonds in the oxyanion hole instead of a water molecule as in the structure of wild-type TcAE206. Furthermore, the catalytic triad residue His182 moved 0.8 Å toward the acetate ion upon substrate entering the active site, suggesting that this movement is necessary for completion of the catalytic reaction. PMID:27329813

  12. Structural analysis of mevalonate-3-kinase provides insight into the mechanisms of isoprenoid pathway decarboxylases

    PubMed Central

    Vinokur, Jeffrey M; Korman, Tyler P; Sawaya, Michael R; Collazo, Michael; Cascio, Duillio; Bowie, James U

    2015-01-01

    In animals, cholesterol is made from 5-carbon building blocks produced by the mevalonate pathway. Drugs that inhibit the mevalonate pathway such as atorvastatin (lipitor) have led to successful treatments for high cholesterol in humans. Another potential target for the inhibition of cholesterol synthesis is mevalonate diphosphate decarboxylase (MDD), which catalyzes the phosphorylation of (R)-mevalonate diphosphate, followed by decarboxylation to yield isopentenyl pyrophosphate. We recently discovered an MDD homolog, mevalonate-3-kinase (M3K) from Thermoplasma acidophilum, which catalyzes the identical phosphorylation of (R)-mevalonate, but without concomitant decarboxylation. Thus, M3K catalyzes half the reaction of the decarboxylase, allowing us to separate features of the active site that are required for decarboxylation from features required for phosphorylation. Here we determine the crystal structure of M3K in the apo form, and with bound substrates, and compare it to MDD structures. Structural and mutagenic analysis reveals modifications that allow M3K to bind mevalonate rather than mevalonate diphosphate. Comparison to homologous MDD structures show that both enzymes employ analogous Arg or Lys residues to catalyze phosphate transfer. However, an invariant active site Asp/Lys pair of MDD previously thought to play a role in phosphorylation is missing in M3K with no functional replacement. Thus, we suggest that the invariant Asp/Lys pair in MDD may be critical for decarboxylation rather than phosphorylation. PMID:25422158

  13. 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...

  14. Structural Studies of Streptococcus pyogenes Streptolysin O Provide Insights into the Early Steps of Membrane Penetration

    PubMed Central

    Feil, Susanne C.; Ascher, David B.; Kuiper, Michael J.; Tweten, Rodney K.; Parker, Michael W.

    2015-01-01

    Cholesterol-dependent cytolysins (CDCs) are a large family of bacterial toxins that exhibit a dependence on the presence of membrane cholesterol in forming large pores in cell membranes. Significant changes in the three-dimensional structure of these toxins are necessary to convert the soluble monomeric protein into a membrane pore. We have determined the crystal structure of the archetypical member of the CDC family, streptolysin O (SLO), a virulence factor from Streptococcus pyogenes. The overall fold is similar to previously reported CDC structures, although the C-terminal domain is in a different orientation with respect to the rest of the molecule. Surprisingly, a signature stretch of CDC sequence called the undecapeptide motif, a key region involved in membrane recognition, adopts a very different structure in SLO to that of the well-characterized CDC perfringolysin O (PFO), although the sequences in this region are identical. An analysis reveals that, in PFO, there are complementary interactions between the motif and the rest of domain 4 that are lost in SLO. Molecular dynamics simulations suggest that the loss of a salt bridge in SLO and a cation–pi interaction are determining factors in the extended conformation of the motif, which in turn appears to result in a greater flexibility of the neighboring L1 loop that houses a cholesterol-sensing motif. These differences may explain the differing abilities of SLO and PFO to efficiently penetrate target cell membranes in the first step of toxin insertion into the membrane. PMID:24316049

  15. 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...

  16. High-resolution structures of cholesterol oxidase in the reduced state provide insights into redox stabilization.

    PubMed

    Golden, Emily; Karton, Amir; Vrielink, Alice

    2014-12-01

    Cholesterol oxidase (CO) is a flavoenzyme that catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one. The reductive half reaction occurs via a hydride transfer from the substrate to the FAD cofactor. The structures of CO reduced with dithionite under aerobic conditions and in the presence of the substrate 2-propanol under both aerobic and anaerobic conditions are presented. The 1.32 Å resolution structure of the dithionite-reduced enzyme reveals a sulfite molecule covalently bound to the FAD cofactor. The isoalloxazine ring system displays a bent structure relative to that of the oxidized enzyme, and alternate conformations of a triad of aromatic residues near to the cofactor are evident. A 1.12 Å resolution anaerobically trapped reduced enzyme structure in the presence of 2-propanol does not show a similar bending of the flavin ring system, but does show alternate conformations of the aromatic triad. Additionally, a significant difference electron-density peak is observed within a covalent-bond distance of N5 of the flavin moiety, suggesting that a hydride-transfer event has occurred as a result of substrate oxidation trapping the flavin in the electron-rich reduced state. The hydride transfer generates a tetrahedral geometry about the flavin N5 atom. High-level density-functional theory calculations were performed to correlate the crystallographic findings with the energetics of this unusual arrangement of the flavin moiety. These calculations suggest that strong hydrogen-bond interactions between Gly120 and the flavin N5 centre may play an important role in these structural features. PMID:25478834

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

    PubMed

    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. PMID:27529188

  18. 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.

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

    PubMed Central

    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.

    2015-01-01

    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. PMID:26446437

  20. Structural Studies of Neuropilin/Antibody Complexes Provide Insights Into Semaphorin And VEGF Binding

    SciTech Connect

    Appleton, B.A.; Wu, P.; Maloney, J.; Yin, J.; Liang, W.-C.; Stawicki, S.; Mortara, K.; Bowman, K.A.; Elliott, J.Michael.; Desmarais, W.; Koch, A.W.; Wu, Y.; Watts, R.J.; Wiesmann, C.

    2009-06-01

    Neuropilins (Nrps) are co-receptors for class 3 semaphorins and vascular endothelial growth factors and important for the development of the nervous system and the vasculature. The extracellular portion of Nrp is composed of two domains that are essential for semaphorin binding (a1a2), two domains necessary for VEGF binding (b1b2), and one domain critical for receptor dimerization (c). We report several crystal structures of Nrp1 and Nrp2 fragments alone and in complex with antibodies that selectively block either semaphorin or vascular endothelial growth factor (VEGF) binding. In these structures, Nrps adopt an unexpected domain arrangement in which the a2, b1, and b2 domains form a tightly packed core that is only loosely connected to the a1 domain. The locations of the antibody epitopes together with in vitro experiments indicate that VEGF and semaphorin do not directly compete for Nrp binding. Based upon our structural and functional data, we propose possible models for ligand binding to neuropilins.

  1. Structures of RabGGTase-substrate/product complexes provide insights into the evolution of protein prenylation.

    PubMed

    Guo, Zhong; Wu, Yao-Wen; Das, Debapratim; Delon, Christine; Cramer, Janinna; Yu, Shen; Thuns, Sandra; Lupilova, Nataliya; Waldmann, Herbert; Brunsveld, Luc; Goody, Roger S; Alexandrov, Kirill; Blankenfeldt, Wulf

    2008-09-17

    Post-translational isoprenylation of proteins is carried out by three related enzymes: farnesyltransferase, geranylgeranyl transferase-I, and Rab geranylgeranyl transferase (RabGGTase). Despite the fact that the last one is responsible for the largest number of individual protein prenylation events in the cell, no structural information is available on its interaction with substrates and products. Here, we present structural and biophysical analyses of RabGGTase in complex with phosphoisoprenoids as well as with the prenylated peptides that mimic the C terminus of Rab7 GTPase. The data demonstrate that, unlike other protein prenyl transferases, both RabGGTase and its substrate RabGTPases completely 'outsource' their specificity for each other to an accessory subunit, the Rab escort protein (REP). REP mediates the placement of the C terminus of RabGTPase into the active site of RabGGTase through a series protein-protein interactions of decreasing strength and selectivity. This arrangement enables RabGGTase to prenylate any cysteine-containing sequence. On the basis of our structural and thermodynamic data, we propose that RabGGTase has evolved from a GGTase-I-like molecule that 'learned' to interact with a recycling factor (GDI) that, in turn, eventually gave rise to REP. PMID:18756270

  2. 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

  3. 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.

  4. Structures of E. coli σS-transcription initiation complexes provide new insights into polymerase mechanism.

    PubMed

    Liu, Bin; Zuo, Yuhong; Steitz, Thomas A

    2016-04-12

    In bacteria, multiple σ factors compete to associate with the RNA polymerase (RNAP) core enzyme to form a holoenzyme that is required for promoter recognition. During transcription initiation RNAP remains associated with the upstream promoter DNA via sequence-specific interactions between the σ factor and the promoter DNA while moving downstream for RNA synthesis. As RNA polymerase repetitively adds nucleotides to the 3'-end of the RNA, a pyrophosphate ion is generated after each nucleotide incorporation. It is currently unknown how the release of pyrophosphate affects transcription. Here we report the crystal structures of E coli transcription initiation complexes (TICs) containing the stress-responsive σ(S) factor, a de novo synthesized RNA oligonucleotide, and a complete transcription bubble (σ(S)-TIC) at about 3.9-Å resolution. The structures show the 3D topology of the σ(S) factor and how it recognizes the promoter DNA, including likely specific interactions with the template-strand residues of the -10 element. In addition, σ(S)-TIC structures display a highly stressed pretranslocated initiation complex that traps a pyrophosphate at the active site that remains closed. The position of the pyrophosphate and the unusual phosphodiester linkage between the two terminal RNA residues suggest an unfinished nucleotide-addition reaction that is likely at equilibrium between nucleotide addition and pyrophosphorolysis. Although these σ(S)-TIC crystals are enzymatically active, they are slow in nucleotide addition, as suggested by an NTP soaking experiment. Pyrophosphate release completes the nucleotide addition reaction and is associated with extensive conformational changes around the secondary channel but causes neither active site opening nor transcript translocation. PMID:27035955

  5. Structures of E. coli σS-transcription initiation complexes provide new insights into polymerase mechanism

    PubMed Central

    Liu, Bin; Zuo, Yuhong; Steitz, Thomas A.

    2016-01-01

    In bacteria, multiple σ factors compete to associate with the RNA polymerase (RNAP) core enzyme to form a holoenzyme that is required for promoter recognition. During transcription initiation RNAP remains associated with the upstream promoter DNA via sequence-specific interactions between the σ factor and the promoter DNA while moving downstream for RNA synthesis. As RNA polymerase repetitively adds nucleotides to the 3′-end of the RNA, a pyrophosphate ion is generated after each nucleotide incorporation. It is currently unknown how the release of pyrophosphate affects transcription. Here we report the crystal structures of E. coli transcription initiation complexes (TICs) containing the stress-responsive σS factor, a de novo synthesized RNA oligonucleotide, and a complete transcription bubble (σS-TIC) at about 3.9-Å resolution. The structures show the 3D topology of the σS factor and how it recognizes the promoter DNA, including likely specific interactions with the template-strand residues of the −10 element. In addition, σS-TIC structures display a highly stressed pretranslocated initiation complex that traps a pyrophosphate at the active site that remains closed. The position of the pyrophosphate and the unusual phosphodiester linkage between the two terminal RNA residues suggest an unfinished nucleotide-addition reaction that is likely at equilibrium between nucleotide addition and pyrophosphorolysis. Although these σS-TIC crystals are enzymatically active, they are slow in nucleotide addition, as suggested by an NTP soaking experiment. Pyrophosphate release completes the nucleotide addition reaction and is associated with extensive conformational changes around the secondary channel but causes neither active site opening nor transcript translocation. PMID:27035955

  6. Crystal structure of measles virus hemagglutinin provides insight into effective vaccines

    PubMed Central

    Hashiguchi, Takao; Kajikawa, Mizuho; Maita, Nobuo; Takeda, Makoto; Kuroki, Kimiko; Sasaki, Kaori; Kohda, Daisuke; Yanagi, Yusuke; Maenaka, Katsumi

    2007-01-01

    Measles still remains a major cause of childhood morbidity and mortality worldwide. Measles virus (MV) vaccines are highly successful, but the mechanism underlying their efficacy has been unclear. Here we report the crystal structure of the MV attachment protein, hemagglutinin, responsible for MV entry. The receptor-binding head domain exhibits a cubic-shaped β-propeller structure and forms a homodimer. N-linked sugars appear to mask the broad regions and cause the two molecules forming the dimer to tilt oppositely toward the horizontal plane. Accordingly, residues of the putative receptor-binding site, highly conserved among MV strains, are strategically positioned in the unshielded area of the protein. These conserved residues also serve as epitopes for neutralizing antibodies, ensuring the serological monotype, a basis for effective MV vaccines. Our findings suggest that sugar moieties in the MV hemagglutinin critically modulate virus–receptor interaction as well as antiviral antibody responses, differently from sugars of the HIV gp120, which allow for immune evasion. PMID:18003910

  7. 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.

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

    PubMed

    Strecker, Angela L; Olden, Julian D

    2014-03-01

    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

  9. Solution structure of DinI provides insight into its mode of RecA inactivation.

    PubMed Central

    Ramirez, B. E.; Voloshin, O. N.; Camerini-Otero, R. D.; Bax, A.

    2000-01-01

    The Escherichia coli RecA protein triggers both DNA repair and mutagenesis in a process known as the SOS response. The 81-residue E. coli protein DinI inhibits activity of RecA in vivo. The solution structure of DinI has been determined by multidimensional triple resonance NMR spectroscopy, using restraints derived from two sets of residual dipolar couplings, obtained in bicelle and phage media, supplemented with J couplings and a moderate number of NOE restraints. DinI has an alpha/beta fold comprised of a three-stranded beta-sheet and two alpha-helices. The beta-sheet topology is unusual: the central strand is flanked by a parallel and an antiparallel strand and the sheet is remarkably flat. The structure of DinI shows that six negatively charged Glu and Asp residues on DinI's kinked C-terminal alpha-helix form an extended, negatively charged ridge. We propose that this ridge mimics the electrostatic character of the DNA phospodiester backbone, thereby enabling DinI to compete with single-stranded DNA for RecA binding. Biochemical data confirm that DinI is able to displace ssDNA from RecA. PMID:11152126

  10. 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

  11. 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

  12. 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.

  13. 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.

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

    PubMed

    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; Ashish; Nihalani, Deepak

    2016-06-01

    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

  15. Structural analysis of the α-glucosidase HaG provides new insights into substrate specificity and catalytic mechanism.

    PubMed

    Shen, Xing; Saburi, Wataru; Gai, Zuoqi; Kato, Koji; Ojima-Kato, Teruyo; Yu, Jian; Komoda, Keisuke; Kido, Yusuke; Matsui, Hirokazu; Mori, Haruhide; Yao, Min

    2015-06-01

    α-Glucosidases, which catalyze the hydrolysis of the α-glucosidic linkage at the nonreducing end of the substrate, are important for the metabolism of α-glucosides. Halomonas sp. H11 α-glucosidase (HaG), belonging to glycoside hydrolase family 13 (GH13), only has high hydrolytic activity towards the α-(1 → 4)-linked disaccharide maltose among naturally occurring substrates. Although several three-dimensional structures of GH13 members have been solved, the disaccharide specificity and α-(1 → 4) recognition mechanism of α-glucosidase are unclear owing to a lack of corresponding substrate-bound structures. In this study, four crystal structures of HaG were solved: the apo form, the glucosyl-enzyme intermediate complex, the E271Q mutant in complex with its natural substrate maltose and a complex of the D202N mutant with D-glucose and glycerol. These structures explicitly provide insights into the substrate specificity and catalytic mechanism of HaG. A peculiar long β → α loop 4 which exists in α-glucosidase is responsible for the strict recognition of disaccharides owing to steric hindrance. Two residues, Thr203 and Phe297, assisted with Gly228, were found to determine the glycosidic linkage specificity of the substrate at subsite +1. Furthermore, an explanation of the α-glucosidase reaction mechanism is proposed based on the glucosyl-enzyme intermediate structure. PMID:26057678

  16. Structural Characterization of the Crimean-Congo Hemorrhagic Fever Virus Gn Tail Provides Insight into Virus Assembly*

    PubMed Central

    Estrada, D. Fernando; De Guzman, Roberto N.

    2011-01-01

    The RNA virus that causes the Crimean Congo Hemorrhagic Fever (CCHF) is a tick-borne pathogen of the Nairovirus genus, family Bunyaviridae. Unlike many zoonotic viruses that are only passed between animals and humans, the CCHF virus can also be transmitted from human to human with an overall mortality rate approaching 30%. Currently, there are no atomic structures for any CCHF virus proteins or for any Nairovirus proteins. A critical component of the virus is the envelope Gn glycoprotein, which contains a C-terminal cytoplasmic tail. In other Bunyaviridae viruses, the Gn tail has been implicated in host-pathogen interaction and viral assembly. Here we report the NMR structure of the CCHF virus Gn cytoplasmic tail, residues 729–805. The structure contains a pair of tightly arranged dual ββα zinc fingers similar to those found in the Hantavirus genus, with which it shares about 12% sequence identity. Unlike Hantavirus zinc fingers, however, the CCHF virus zinc fingers bind viral RNA and contain contiguous clusters of conserved surface electrostatics. Our results provide insight into a likely role of the CCHF virus Gn zinc fingers in Nairovirus assembly. PMID:21507948

  17. Mitochondrial ribonuclease P structure provides insight into the evolution of catalytic strategies for precursor-tRNA 5′ processing

    PubMed Central

    Howard, Michael J.; Lim, Wan Hsin; Fierke, Carol A.; Koutmos, Markos

    2012-01-01

    Ribonuclease P (RNase P) catalyzes the maturation of the 5′ end of tRNA precursors. Typically these enzymes are ribonucleoproteins with a conserved RNA component responsible for catalysis. However, protein-only RNase P (PRORP) enzymes process precursor tRNAs in human mitochondria and in all tRNA-using compartments of Arabidopsis thaliana. PRORP enzymes are nuclear encoded and conserved among many eukaryotes, having evolved recently as yeast mitochondrial genomes encode an RNase P RNA. Here we report the crystal structure of PRORP1 from A. thaliana at 1.75 Å resolution, revealing a prototypical metallonuclease domain tethered to a pentatricopeptide repeat (PPR) domain by a structural zinc-binding domain. The metallonuclease domain is a unique high-resolution structure of a Nedd4-BP1, YacP Nucleases (NYN) domain that is a member of the PIN domain-like fold superfamily, including the FLAP nuclease family. The structural similarity between PRORP1 and the FLAP nuclease family suggests that they evolved from a common ancestor. Biochemical data reveal that conserved aspartate residues in PRORP1 are important for catalytic activity and metal binding and that the PPR domain also enhances activity, likely through an interaction with pre-tRNA. These results provide a foundation for understanding tRNA maturation in organelles. Furthermore, these studies allow for a molecular-level comparison of the catalytic strategies used by the only known naturally evolved protein and RNA-based catalysts that perform the same biological function, pre-tRNA maturation, thereby providing insight into the differences between the prebiotic RNA world and the present protein-dominated world. PMID:22991464

  18. Genomic-scale comparison of sequence- and structure-based methods of function prediction: Does structure provide additional insight?

    PubMed Central

    Fetrow, Jacquelyn S.; Siew, Naomi; Di Gennaro, Jeannine A.; Martinez-Yamout, Maria; Dyson, H. Jane; Skolnick, Jeffrey

    2001-01-01

    A function annotation method using the sequence-to-structure-to-function paradigm is applied to the identification of all disulfide oxidoreductases in the Saccharomyces cerevisiae genome. The method identifies 27 sequences as potential disulfide oxidoreductases. All previously known thioredoxins, glutaredoxins, and disulfide isomerases are correctly identified. Three of the 27 predictions are probable false-positives. Three novel predictions, which subsequently have been experimentally validated, are presented. Two additional novel predictions suggest a disulfide oxidoreductase regulatory mechanism for two subunits (OST3 and OST6) of the yeast oligosaccharyltransferase complex. Based on homology, this prediction can be extended to a potential tumor suppressor gene, N33, in humans, whose biochemical function was not previously known. Attempts to obtain a folded, active N33 construct to test the prediction were unsuccessful. The results show that structure prediction coupled with biochemically relevant structural motifs is a powerful method for the function annotation of genome sequences and can provide more detailed, robust predictions than function prediction methods that rely on sequence comparison alone. PMID:11316881

  19. Crystal structure of the Retinoblastoma protein N-domain provides insight into tumor suppression, ligand interaction and holoprotein architecture

    PubMed Central

    Hassler, Markus; Singh, Shradha; Yu, Wyatt W.; Luczynski, Maciej; Lakbir, Rachid; Sanchez-Sanchez, Francisco; Bader, Thomas; Pearl, Laurence H.; Mittnacht, Sibylle

    2016-01-01

    Summary The retinoblastoma susceptibility protein, Rb, has a key role in regulating cell cycle progression via interactions involving the central 'pocket' and C-terminal regions. While the N-terminal domain of Rb is dispensable for this function, it is nonetheless strongly conserved, and harbours many missense mutations found in hereditary retinoblastoma, indicating that disruption of its function is oncogenic. The crystal structure of the Rb N-terminal domain (RbN), reveals a single globular entity formed by two cyclin-like folds. The intrinsic similarity of RbN to the A and B boxes of the Rb pocket domain suggests that Rb evolved through domain duplication. Structural and functional analysis provides insight into oncogenicity of mutations in RbN and identifies a unique phosphorylation-regulated site of protein interaction. Additionally, this analysis suggests a coherent conformation for the Rb holoprotein in which RbN and pocket domains directly interact, and which can be modulated through ligand binding and possibly Rb phosphorylation. PMID:17996702

  20. 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. PMID:26527682

  1. Structure of the CED-4-CED-9 Complex Provides Insights into Programmed Cell Death in Caenorhabditis elegans

    SciTech Connect

    Yan,N.; Chai, J.; Lee, E.; Gu, L.; Liu, Q.; He, J.; Wu, J.; Kokel, D.; Li, H.; et al.

    2005-01-01

    Interplay among four genes-egl-1, ced-9, ced-4 and ced-3-controls the onset of programmed cell death in the nematode Caenorhabditis elegans. Activation of the cell-killing protease CED-3 requires CED-4. However, CED-4 is constitutively inhibited by CED-9 until its release by EGL-1. Here we report the crystal structure of the CED-4-CED-9 complex at 2.6 Angstrom resolution, and a complete reconstitution of the CED-3 activation pathway using homogeneous proteins of CED-4, CED-9 and EGL-1. One molecule of CED-9 binds to an asymmetric dimer of CED-4, but specifically recognizes only one of the two CED-4 molecules. This specific interaction prevents CED-4 from activating CED-3. EGL-1 binding induces pronounced conformational changes in CED-9 that result in the dissociation of the CED-4 dimer from CED-9. The released CED-4 dimer further dimerizes to form a tetramer, which facilitates the autoactivation of CED-3. Together, our studies provide important insights into the regulation of cell death activation in C. elegans.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26527682

  3. Kinetic analysis of Pseudomonas aeruginosa arginine deiminase mutants and alternate substrates provides insight into structural determinants of function.

    PubMed

    Lu, Xuefeng; Li, Ling; Wu, Rui; Feng, Xiaohua; Li, Zhimin; Yang, Heyi; Wang, Canhui; Guo, Hua; Galkin, Andrey; Herzberg, Osnat; Mariano, Patrick S; Martin, Brian M; Dunaway-Mariano, Debra

    2006-01-31

    L-Arginine deiminase from Pseudomonas aeruginosa (PaADI) catalyzes the hydrolysis of arginine to citrulline and ammonia. PaADI belongs to the guanidino group-modifying enzyme superfamily (GMSF), which conserves backbone fold and a Cys-, His-, and Asp-based catalytic core. In this paper the contributions made by the PaADI core residues Cys406, His278, and Asp166 and the contribution from the neighboring Asp280 (conserved in most but not all GMSF members) to catalysis of the formation and hydrolysis of the Cys406-alkyluronium intermediate were accessed by kinetic analysis of site-directed mutants. In addition, solution hydrolysis in a chemical model of the S-alkylthiouronium intermediate was examined to reveal the importance of general base catalysis in the enzymatic reaction. Substitutions of the active site gating residue Arg401, the l-arginine C(alpha)NH(3)(+)(COO(-)) binding residues, Arg185, Arg243, and Asn160, or the His278 hydrogen bond partner, Glu224, were found to cause dramatic reductions in the enzyme turnover rate. These results are interpreted to suggest that electrostatic interactions play a dominant role in PaADI catalysis. Structural variations observed in P. aeruginosa GMSF enzymes PaADI, agmatine deiminase (PaAgDI), and N(omega),N(omega)-dimethylarginine dimethylaminohydrolase (PaDDAH) indicate an early divergence of the encoding genes. Arginine analogues that are known substrates for PaAgDI and PaDDAH were tested with PaADI to define clear boundaries of biochemical function in the three hydrolases. The conservation of a catalytic core associated with the common chemical function and the divergence of substrate-binding residues (as well as one key catalytic residue) to expand the substrate range provide insight into the evolution of the catalysts that form the GMSF. PMID:16430212

  4. Crystal structure analysis of ornithine transcarbamylase from Thermus thermophilus --HB8 provides insights on the plasticity of the active site.

    PubMed

    Sundaresan, Ramya; Ebihara, Akio; Kuramitsu, Seiki; Yokoyama, Shigeyuki; Kumarevel, Thirumananseri; Ponnuraj, Karthe

    2015-09-18

    The enzymatic biosynthesis of L-arginine involves complex, sequential action of many enzymes and ornithine transcarbamylase (OTCase) is one of the essential enzymes in the pathway. In mammals OTCase is part of the urea cycle. Arginine is used in a variety of pharmaceutical and industrial applications and therefore engineering arginine biosynthesis pathway for overproduction of arginine has gained importance. On the other hand, it was found that detrimental mutations in the human OTCase gene resulted clinical hyperammonemia, with subsequent neurological damage. Therefore a better understanding of the structure-function relationship of this enzyme from various sources could be useful for modifying its enzymatic action. Here we report the structure of ornithine transcarbamylase of Thermus thermophilus HB8 (aTtOTCase) at 2.0 Å resolution. On comparison with its homologs, aTtOTCase showed maximum variation at the substrate binding loops namely 80s and SMG/240s loops. The active site geometry of aTtOTCase is unique among its homologs where the side chain of certain residues (Leu57, Arg58 and Arg288) is oriented differently. To study the structural insights of substrate binding in aTtOTCase, docking of carbamoyl phosphate (CP) and ornithine (Orn) was carried out sequentially. Both substrates were unable to bind in a proper orientation in the active site pocket and this could be due to the differently oriented side chains. This suggests that the active site geometry should also undergo fine tuning besides the large structural changes as the enzyme switches from completely open to a substrate bound closed state. PMID:26210451

  5. A Novel Family of Soluble Minimal Scaffolds Provides Structural Insight into the Catalytic Domains of Integral Membrane Metallopeptidases*

    PubMed Central

    López-Pelegrín, Mar; Cerdà-Costa, Núria; Martínez-Jiménez, Francisco; Cintas-Pedrola, Anna; Canals, Albert; Peinado, Juan R.; Marti-Renom, Marc A.; López-Otín, Carlos; Arolas, Joan L.; Gomis-Rüth, F. Xavier

    2013-01-01

    In the search for structural models of integral-membrane metallopeptidases (MPs), we discovered three related proteins from thermophilic prokaryotes, which we grouped into a novel family called “minigluzincins.” We determined the crystal structures of the zymogens of two of these (Pyrococcus abyssi proabylysin and Methanocaldococcus jannaschii projannalysin), which are soluble and, with ∼100 residues, constitute the shortest structurally characterized MPs to date. Despite relevant sequence and structural similarity, the structures revealed two unique mechanisms of latency maintenance through the C-terminal segments previously unseen in MPs as follows: intramolecular, through an extended tail, in proabylysin, and crosswise intermolecular, through a helix swap, in projannalysin. In addition, structural and sequence comparisons revealed large similarity with MPs of the gluzincin tribe such as thermolysin, leukotriene A4 hydrolase relatives, and cowrins. Noteworthy, gluzincins mostly contain a glutamate as third characteristic zinc ligand, whereas minigluzincins have a histidine. Sequence and structural similarity further allowed us to ascertain that minigluzincins are very similar to the catalytic domains of integral membrane MPs of the MEROPS database families M48 and M56, such as FACE1, HtpX, Oma1, and BlaR1/MecR1, which are provided with trans-membrane helices flanking or inserted into a minigluzincin-like catalytic domain. In a time where structural biochemistry of integral-membrane proteins in general still faces formidable challenges, the minigluzincin soluble minimal scaffold may contribute to our understanding of the working mechanisms of these membrane MPs and to the design of novel inhibitors through structure-aided rational drug design approaches. PMID:23733187

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

    PubMed Central

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

    2013-01-01

    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. PMID:24127570

  7. 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. PMID:24127570

  8. 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

  9. Structural and Mutational Analysis of Escherichia coli AlkB Provides Insight into Substrate Specificity and DNA Damage Searching

    SciTech Connect

    Holland, P.; Hollis, T

    2010-01-01

    In Escherichia coli, cytotoxic DNA methyl lesions on the N1 position of purines and N3 position of pyrimidines are primarily repaired by the 2-oxoglutarate (2-OG) iron(II) dependent dioxygenase, AlkB. AlkB repairs 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) lesions, but it also repairs 1-methylguanine (1-meG) and 3-methylthymine (3-meT) at a much less efficient rate. How the AlkB enzyme is able to locate and identify methylated bases in ssDNA has remained an open question. We determined the crystal structures of the E. coli AlkB protein holoenzyme and the AlkB-ssDNA complex containing a 1-meG lesion. We coupled this to site-directed mutagenesis of amino acids in and around the active site, and tested the effects of these mutations on the ability of the protein to bind both damaged and undamaged DNA, as well as catalyze repair of a methylated substrate. A comparison of our substrate-bound AlkB-ssDNA complex with our unliganded holoenzyme reveals conformational changes of residues within the active site that are important for binding damaged bases. Site-directed mutagenesis of these residues reveals novel insight into their roles in DNA damage recognition and repair. Our data support a model that the AlkB protein utilizes at least two distinct conformations in searching and binding methylated bases within DNA: a 'searching' mode and 'repair' mode. Moreover, we are able to functionally separate these modes through mutagenesis of residues that affect one or the other binding state. Finally, our mutagenesis experiments show that amino acid D135 of AlkB participates in both substrate specificity and catalysis.

  10. Crystal structures of malonyl-coenzyme A decarboxylase provide insights into its catalytic mechanism and disease-causing mutations.

    PubMed

    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

    2013-07-01

    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

  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. 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

    2016-06-01

    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. PMID:27161976

  13. Crystal structures of Salmonella typhimurium biodegradative threonine deaminase and its complex with CMP provide structural insights into ligand-induced oligomerization and enzyme activation.

    PubMed

    Simanshu, Dhirendra K; Savithri, Handanahal S; Murthy, Mathur R N

    2006-12-22

    Two different pyridoxal 5'-phosphate-containing l-threonine deaminases (EC 4.3.1.19), biosynthetic and biodegradative, which catalyze the deamination of l-threonine to alpha-ketobutyrate, are present in Escherichia coli and Salmonella typhimurium. Biodegradative threonine deaminase (TdcB) catalyzes the first reaction in the anaerobic breakdown of l-threonine to propionate. TdcB, unlike the biosynthetic threonine deaminase, is insensitive to l-isoleucine and is activated by AMP. In the present study, TdcB from S. typhimurium was cloned and overexpressed in E. coli. In the presence of AMP or CMP, the recombinant enzyme was converted to the tetrameric form accompanied by significant enzyme activation. To provide insights into ligand-mediated oligomerization and enzyme activation, crystal structures of S. typhimurium TdcB and its complex with CMP were determined. In the native structure, TdcB is in a dimeric form, whereas in the TdcB.CMP complex, it exists in a tetrameric form with 222 symmetry and appears as a dimer of dimers. Tetrameric TdcB binds to four molecules of CMP, two at each of the dimer interfaces. Comparison of the dimer structure in the ligand (CMP)-free and -bound forms suggests that the changes induced by ligand binding at the dimer interface are essential for tetramerization. The differences observed in the tertiary and quaternary structures of TdcB in the absence and presence of CMP appear to account for enzyme activation and increased binding affinity for l-threonine. Comparison of TdcB with related pyridoxal 5'-phosphate-dependent enzymes points to structural and mechanistic similarities. PMID:17046821

  14. Whole genome sequencing of multiple Leishmania donovani clinical isolates provides insights into population structure and mechanisms of drug resistance.

    PubMed

    Downing, Tim; Imamura, Hideo; Decuypere, Saskia; Clark, Taane G; Coombs, Graham H; Cotton, James A; Hilley, James D; de Doncker, Simonne; Maes, Ilse; Mottram, Jeremy C; Quail, Mike A; Rijal, Suman; Sanders, Mandy; Schönian, Gabriele; Stark, Olivia; Sundar, Shyam; Vanaerschot, Manu; Hertz-Fowler, Christiane; Dujardin, Jean-Claude; Berriman, Matthew

    2011-12-01

    Visceral leishmaniasis is a potentially fatal disease endemic to large parts of Asia and Africa, primarily caused by the protozoan parasite Leishmania donovani. Here, we report a high-quality reference genome sequence for a strain of L. donovani from Nepal, and use this sequence to study variation in a set of 16 related clinical lines, isolated from visceral leishmaniasis patients from the same region, which also differ in their response to in vitro drug susceptibility. We show that whole-genome sequence data reveals genetic structure within these lines not shown by multilocus typing, and suggests that drug resistance has emerged multiple times in this closely related set of lines. Sequence comparisons with other Leishmania species and analysis of single-nucleotide diversity within our sample showed evidence of selection acting in a range of surface- and transport-related genes, including genes associated with drug resistance. Against a background of relative genetic homogeneity, we found extensive variation in chromosome copy number between our lines. Other forms of structural variation were significantly associated with drug resistance, notably including gene dosage and the copy number of an experimentally verified circular episome present in all lines and described here for the first time. This study provides a basis for more powerful molecular profiling of visceral leishmaniasis, providing additional power to track the drug resistance and epidemiology of an important human pathogen. PMID:22038251

  15. Crystal structure of hGEF-H1 PH domain provides insight into incapability in phosphoinositide binding.

    PubMed

    Jiang, Yan; Jiang, Heli; Zhou, Shaoyang; Meng, Bing; Liu, Zhi-Jie; Ouyang, Songying

    2016-03-18

    The guanine nucleotide exchange factor GEF-H1 (also known as ARHGEF2) is identified as a member of the Dbl family of GEFs. It regulates RhoA-dependent cell signaling pathways and plays important roles in biological processes. GEF-H1 contains an N-terminal zinc finger domain, a Dbl-homologous (DH) domain followed by a Pleckstrin homology (PH) domain, and a C-terminal domain. The specific roles of its PH domain are poorly understood. Here we report the crystal structure of human GEF-H1 PH domain to 2.45 Å resolution. A conserved surface is formed by β8, β9, β10, and it may mediate protein-protein interactions. Although the folding resembles other PH domains that have defined structures, superposition of different PH domains clearly shows that the loop between β6/β7 and the loop between β3/β4 are so close that they will prevent its binding with phosphoinositide due to steric hindrance, and this has been proved by isothermal titration calorimetry (ITC) and thermal shift assay (TSA). Our studies provide a structural framework for further work on the function of GEF-H1. PMID:26820534

  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

    2010-01-01

    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. PMID:20941397

  17. The Crystal Structure of Burkholderia cenocepacia DfsA Provides Insights into Substrate Recognition and Quorum Sensing Fatty Acid Biosynthesis.

    PubMed

    Spadaro, Francesca; Scoffone, Viola C; Chiarelli, Laurent R; Fumagalli, Marco; Buroni, Silvia; Riccardi, Giovanna; Forneris, Federico

    2016-06-14

    Burkholderia cenocepacia is a major concern among respiratory tract infections in cystic fibrosis patients. This pathogen is particularly difficult to treat because of its high level of resistance to the clinically relevant antimicrobial agents. In B. cenocepacia, the quorum sensing cell-cell communication system is involved in different processes that are important for bacterial virulence, such as biofilm formation and protease and siderophore production. Targeting the enzymes involved in this process represents a promising therapeutic approach. With the aim of finding effective quorum sensing inhibitors, we have determined the three-dimensional structure of B. cenocepacia diffusible factor synthase A, DfsA. This bifunctional crotonase (dehydratase/thioesterase) produces the characteristic quorum sensing molecule of B. cenocepacia, cis-2-dodecenoic acid or BDSF, starting from 3-hydroxydodecanoyl-acyl carrier protein. Unexpectedly, the crystal structure revealed the presence of a lipid molecule in the catalytic site of the enzyme, which was identified as dodecanoic acid. Our biochemical characterization shows that DfsA is able to use dodecanoyl-acyl carrier protein as a substrate, demonstrating that dodecanoic acid, the product of this reaction, is released very slowly from the DfsA active site, therefore acting as a DfsA inhibitor. This molecule shows an unprecedented conformational arrangement inside the DfsA active site. In contrast with previous hypotheses, our data illustrate how DfsA and closely related homologous enzymes can recognize long hydrophobic substrates without large conformational changes or assistance by additional regulator molecules. The elucidation of the substrate binding mode in DfsA provides the starting point for structure-based drug discovery studies targeting B. cenocepacia quorum sensing-assisted virulence. PMID:27198181

  18. NMR structure of a lytic polysaccharide monooxygenase provides insight into copper binding, protein dynamics, and substrate interactions.

    PubMed

    Aachmann, Finn L; Sørlie, Morten; Skjåk-Bræk, Gudmund; Eijsink, Vincent G H; Vaaje-Kolstad, Gustav

    2012-11-13

    Lytic polysaccharide monooxygenases currently classified as carbohydrate binding module family 33 (CBM33) and glycoside hydrolase family 61 (GH61) are likely to play important roles in future biorefining. However, the molecular basis of their unprecedented catalytic activity remains largely unknown. We have used NMR techniques and isothermal titration calorimetry to address structural and functional aspects of CBP21, a chitin-active CBM33. NMR structural and relaxation studies showed that CBP21 is a compact and rigid molecule, and the only exception is the catalytic metal binding site. NMR data further showed that His28 and His114 in the catalytic center bind a variety of divalent metal ions with a clear preference for Cu(2+) (K(d) = 55 nM; from isothermal titration calorimetry) and higher preference for Cu(1+) (K(d) ∼ 1 nM; from the experimentally determined redox potential for CBP21-Cu(2+) of 275 mV using a thermodynamic cycle). Strong binding of Cu(1+) was also reflected in a reduction in the pK(a) values of the histidines by 3.6 and 2.2 pH units, respectively. Cyanide, a mimic of molecular oxygen, was found to bind to the metal ion only. These data support a model where copper is reduced on the enzyme by an externally provided electron and followed by oxygen binding and activation by internal electron transfer. Interactions of CBP21 with a crystalline substrate were mapped in a (2)H/(1)H exchange experiment, which showed that substrate binding involves an extended planar binding surface, including the metal binding site. Such a planar catalytic surface seems well-suited to interact with crystalline substrates. PMID:23112164

  19. NMR structure of a lytic polysaccharide monooxygenase provides insight into copper binding, protein dynamics, and substrate interactions

    PubMed Central

    Aachmann, Finn L.; Sørlie, Morten; Skjåk-Bræk, Gudmund; Eijsink, Vincent G. H.; Vaaje-Kolstad, Gustav

    2012-01-01

    Lytic polysaccharide monooxygenases currently classified as carbohydrate binding module family 33 (CBM33) and glycoside hydrolase family 61 (GH61) are likely to play important roles in future biorefining. However, the molecular basis of their unprecedented catalytic activity remains largely unknown. We have used NMR techniques and isothermal titration calorimetry to address structural and functional aspects of CBP21, a chitin-active CBM33. NMR structural and relaxation studies showed that CBP21 is a compact and rigid molecule, and the only exception is the catalytic metal binding site. NMR data further showed that His28 and His114 in the catalytic center bind a variety of divalent metal ions with a clear preference for Cu2+ (Kd = 55 nM; from isothermal titration calorimetry) and higher preference for Cu1+ (Kd ∼ 1 nM; from the experimentally determined redox potential for CBP21-Cu2+ of 275 mV using a thermodynamic cycle). Strong binding of Cu1+ was also reflected in a reduction in the pKa values of the histidines by 3.6 and 2.2 pH units, respectively. Cyanide, a mimic of molecular oxygen, was found to bind to the metal ion only. These data support a model where copper is reduced on the enzyme by an externally provided electron and followed by oxygen binding and activation by internal electron transfer. Interactions of CBP21 with a crystalline substrate were mapped in a 2H/1H exchange experiment, which showed that substrate binding involves an extended planar binding surface, including the metal binding site. Such a planar catalytic surface seems well-suited to interact with crystalline substrates. PMID:23112164

  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. 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

    2015-01-01

    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

  3. 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

  4. Structural and mechanistic insights into bisphenols action provide guidelines for risk assessment and discovery of bisphenol A substitutes.

    PubMed

    Delfosse, Vanessa; Grimaldi, Marina; Pons, Jean-Luc; Boulahtouf, Abdelhay; le Maire, Albane; Cavailles, Vincent; Labesse, Gilles; Bourguet, William; Balaguer, Patrick

    2012-09-11

    Bisphenol A (BPA) is an industrial compound and a well known endocrine-disrupting chemical with estrogenic activity. The widespread exposure of individuals to BPA is suspected to affect a variety of physiological functions, including reproduction, development, and metabolism. Here we report that the mechanisms by which BPA and two congeners, bisphenol AF and bisphenol C (BPC), bind to and activate estrogen receptors (ER) α and β differ from that used by 17β-estradiol. We show that bisphenols act as partial agonists of ERs by activating the N-terminal activation function 1 regardless of their effect on the C-terminal activation function 2, which ranges from weak agonism (with BPA) to antagonism (with BPC). Crystallographic analysis of the interaction between bisphenols and ERs reveals two discrete binding modes, reflecting the different activities of compounds on ERs. BPA and 17β-estradiol bind to ERs in a similar fashion, whereas, with a phenol ring pointing toward the activation helix H12, the orientation of BPC accounts for the marked antagonist character of this compound. Based on structural data, we developed a protocol for in silico evaluation of the interaction between bisphenols and ERs or other members of the nuclear hormone receptor family, such as estrogen-related receptor γ and androgen receptor, which are two known main targets of bisphenols. Overall, this study provides a wealth of tools and information that could be used for the development of BPA substitutes devoid of nuclear hormone receptor-mediated activity and more generally for environmental risk assessment. PMID:22927406

  5. Structural and mechanistic insights into bisphenols action provide guidelines for risk assessment and discovery of bisphenol A substitutes

    PubMed Central

    Delfosse, Vanessa; Grimaldi, Marina; Pons, Jean-Luc; Boulahtouf, Abdelhay; le Maire, Albane; Cavailles, Vincent; Labesse, Gilles; Bourguet, William; Balaguer, Patrick

    2012-01-01

    Bisphenol A (BPA) is an industrial compound and a well known endocrine-disrupting chemical with estrogenic activity. The widespread exposure of individuals to BPA is suspected to affect a variety of physiological functions, including reproduction, development, and metabolism. Here we report that the mechanisms by which BPA and two congeners, bisphenol AF and bisphenol C (BPC), bind to and activate estrogen receptors (ER) α and β differ from that used by 17β-estradiol. We show that bisphenols act as partial agonists of ERs by activating the N-terminal activation function 1 regardless of their effect on the C-terminal activation function 2, which ranges from weak agonism (with BPA) to antagonism (with BPC). Crystallographic analysis of the interaction between bisphenols and ERs reveals two discrete binding modes, reflecting the different activities of compounds on ERs. BPA and 17β-estradiol bind to ERs in a similar fashion, whereas, with a phenol ring pointing toward the activation helix H12, the orientation of BPC accounts for the marked antagonist character of this compound. Based on structural data, we developed a protocol for in silico evaluation of the interaction between bisphenols and ERs or other members of the nuclear hormone receptor family, such as estrogen-related receptor γ and androgen receptor, which are two known main targets of bisphenols. Overall, this study provides a wealth of tools and information that could be used for the development of BPA substitutes devoid of nuclear hormone receptor-mediated activity and more generally for environmental risk assessment. PMID:22927406

  6. High-resolution structure of the nitrile reductase QueF combined with molecular simulations provide insight into enzyme mechanism.

    SciTech Connect

    Kim, Y.; Zhou, M.; Moy, S.; Morales, J.; Cunningham, M.; Joachimiak, A.; Biosciences Division; Univ. of Texas-Pan American

    2010-01-01

    Here, we report the 1.53-{angstrom} crystal structure of the enzyme 7-cyano-7-deazaguanine reductase (QueF) from Vibrio cholerae, which is responsible for the complete reduction of a nitrile (C {triple_bond} N) bond to a primary amine (H{sub 2}C-NH{sub 2}). At present, this is the only example of a biological pathway that includes reduction of a nitrile bond, establishing QueF as particularly noteworthy. The structure of the QueF monomer resembles two connected ferrodoxin-like domains that assemble into dimers. Ligands identified in the crystal structure suggest the likely binding conformation of the native substrates NADPH and 7-cyano-7-deazaguanine. We also report on a series of numerical simulations that have shed light on the mechanism by which this enzyme affects the transfer of four protons (and electrons) to the 7-cyano-7-deazaguanine substrate. In particular, the simulations suggest that the initial step of the catalytic process is the formation of a covalent adduct with the residue Cys194, in agreement with previous studies. The crystal structure also suggests that two conserved residues (His233 and Asp102) play an important role in the delivery of a fourth proton to the substrate.

  7. Structure-guided studies of the SHP-1/JAK1 interaction provide new insights into phosphatase catalytic domain substrate recognition

    PubMed Central

    Alicea-Velázquez, Nilda L.; Jakoncic, Jean; Boggon, Titus J.

    2013-01-01

    SHP-1 (PTPN6) is a member of the SHP sub-family of protein tyrosine phosphatases and plays a critical role in the regulation of the JAK/STAT signaling pathway. Previous studies suggested that SHP-1 contains a PTP1B-like second phosphotyrosine pocket that allows for binding of tandem phosphotyrosine residues, such as those found in the activation loop of JAK kinases. To discover the structural nature of the interaction between SHP-1 and the JAK family member, JAK1, we determined the 1.8 Å co-crystal structure of the SHP-1 catalytic domain and a JAK1-derived substrate peptide. This structure reveals electron density for only one bound phosphotyrosine residue. To investigate the role of the predicted second site pocket we determined the structures of SHP-1 in complex with phosphate and sulfate to 1.37 Å and 1.7 Å, respectively, and performed anomalous scattering experiments for a selenate-soaked crystal. These crystallographic data suggest that SHP-1 does not contain a PTP1B-like second site pocket. This conclusion is further supported by analysis of the relative dephosphorylation and binding affinities of mono-and tandem-phosphorylated peptide substrates. The crystal structures instead indicate that SHP-1 contains an extended C-terminal helix α2′ incompatible with the predicted second phosphotyrosine binding site. This study suggests that SHP-1 defines a new category of PTP1B-like protein tyrosine phosphatases with a hindered second phosphotyrosine pocket. PMID:23296072

  8. 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. PMID:24338896

  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

    2016-01-01

    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

  10. The structure of TAX1BP1 UBZ1+2 provides insight into target specificity and adaptability.

    PubMed

    Ceregido, M Angeles; Spínola Amilibia, Mercedes; Buts, Lieven; Rivera-Torres, José; Garcia-Pino, Abel; Bravo, Jerónimo; van Nuland, Nico A J

    2014-02-01

    TAX1BP1 is a novel ubiquitin-binding adaptor protein involved in the negative regulation of the NF-kappaB transcription factor, which is a key player in inflammatory responses, immunity and tumorigenesis. TAX1BP1 recruits A20 to the ubiquitinated signaling proteins TRAF6 and RIP1, leading to their A20-mediated deubiquitination and the disruption of IL-1-induced and TNF-induced NF-kappaB signaling, respectively. The two zinc fingers localized at its C-terminus function as novel ubiquitin-binding domains (UBZ, ubiquitin-binding zinc finger). Here we present for the first time both the solution and crystal structures of two classical UBZ domains in tandem within the human TAX1BP1. The relative orientation of the two domains is slightly different in the X-ray structure with respect to the NMR structure, indicating some degree of conformational flexibility, which is rationalized by NMR relaxation data. The observed degree of flexibility and stability between the two UBZ domains might have consequences on the recognition mechanism of interacting partners. PMID:24239949

  11. 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

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

    PubMed

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

    2015-09-14

    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

  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. Crystal Structure of Insulin-Regulated Aminopeptidase with Bound Substrate Analogue Provides Insight on Antigenic Epitope Precursor Recognition and Processing.

    PubMed

    Mpakali, Anastasia; Saridakis, Emmanuel; Harlos, Karl; Zhao, Yuguang; Papakyriakou, Athanasios; Kokkala, Paraskevi; Georgiadis, Dimitris; Stratikos, Efstratios

    2015-09-15

    Aminopeptidases that generate antigenic peptides influence immunodominance and adaptive cytotoxic immune responses. The mechanisms that allow these enzymes to efficiently process a vast number of different long peptide substrates are poorly understood. In this work, we report the structure of insulin-regulated aminopeptidase, an enzyme that prepares antigenic epitopes for cross-presentation in dendritic cells, in complex with an antigenic peptide precursor analog. Insulin-regulated aminopeptidase is found in a semiclosed conformation with an extended internal cavity with limited access to the solvent. The N-terminal moiety of the peptide is located at the active site, positioned optimally for catalysis, whereas the C-terminal moiety of the peptide is stabilized along the extended internal cavity lodged between domains II and IV. Hydrophobic interactions and shape complementarity enhance peptide affinity beyond the catalytic site and support a limited selectivity model for antigenic peptide selection that may underlie the generation of complex immunopeptidomes. PMID:26259583

  15. Structure of Gαi1 bound to a GDP-selective peptide provides insight into guanine nucleotide exchange

    PubMed Central

    Johnston, Christopher A.; Willard, Francis S.; Jezyk, Mark R.; Fredericks, Zoey; Bodor, Erik T.; Jones, Miller B.; Blaesius, Rainer; Watts, Val J.; Harden, T. Kendall; Sondek, John; Ramer, J. Kevin; Siderovski, David P.

    2005-01-01

    Heterotrimeric G-proteins are molecular switches that regulate numerous signaling pathways involved in cellular physiology. This characteristic is achieved by the adoption of two principal states: an inactive, GDP-bound and an active, GTP-bound state. Under basal conditions G-proteins exist in the inactive GDP-bound state, thus nucleotide exchange is crucial to the onset of signaling. Despite our understanding of G-protein signaling pathways, the mechanism of nucleotide exchange remains elusive. We employed phage display technology to identify nucleotide-state-dependent Gα binding peptides. Herein, we report a GDP-selective Gα-binding peptide, KB-752, that enhances spontaneous nucleotide exchange of Gαi subunits. Structural determination of the Gαi1/peptide complex reveals unique changes in the Gα switch regions predicted to enhance nucleotide exchange by creating a GDP dissociation route. Our results cast light onto a potential mechanism by which Gα subunits adopt a conformation suitable for nucleotide exchange. PMID:16004878

  16. 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

  17. Biophysical and Biochemical Characterization of Avian Secretory Component Provides Structural Insights into the Evolution of the Polymeric Ig Receptor.

    PubMed

    Stadtmueller, Beth M; Yang, Zhongyu; Huey-Tubman, Kathryn E; Roberts-Mataric, Helena; Hubbell, Wayne L; Bjorkman, Pamela J

    2016-08-15

    The polymeric Ig receptor (pIgR) transports polymeric Abs across epithelia to the mucosa, where proteolytic cleavage releases the ectodomain (secretory component [SC]) as an integral component of secretory Abs, or as an unliganded protein that can mediate interactions with bacteria. SC is conserved among vertebrates, but domain organization is variable: mammalian SC has five domains (D1-D5), whereas avian, amphibian, and reptilian SC lack the D2 domain, and fish SC lacks domains D2-D4. In this study, we used double electron-electron resonance spectroscopy and surface plasmon resonance binding studies to characterize the structure, dynamics, and ligand binding properties of avian SC, avian SC domain variants, and a human SC (hSC) variant lacking the D2 domain. These experiments demonstrated that, unlike hSC, which adopts a compact or "closed" domain arrangement, unliganded avian SC is flexible and exists in both closed and open states, suggesting that the mammalian SC D2 domain stabilizes the closed conformation observed for hSC D1-D5. Experiments also demonstrated that avian and mammalian pIgR share related, but distinct, mechanisms of ligand binding. Together, our data reveal differences in the molecular recognition mechanisms associated with evolutionary changes in the pIgR protein. PMID:27412418

  18. 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

    2016-01-01

    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. Structure-Function Analysis of the Drosophila melanogaster Caudal Transcription Factor Provides Insights into Core Promoter-preferential Activation.

    PubMed

    Shir-Shapira, Hila; Sharabany, Julia; Filderman, Matan; Ideses, Diana; Ovadia-Shochat, Avital; Mannervik, Mattias; Juven-Gershon, Tamar

    2015-07-10

    Regulation of RNA polymerase II transcription is critical for the proper development, differentiation, and growth of an organism. The RNA polymerase II core promoter is the ultimate target of a multitude of transcription factors that control transcription initiation. Core promoters encompass the RNA start site and consist of functional elements such as the TATA box, initiator, and downstream core promoter element (DPE), which confer specific properties to the core promoter. We have previously discovered that Drosophila Caudal, which is a master regulator of genes involved in development and differentiation, is a DPE-specific transcriptional activator. Here, we show that the mouse Caudal-related homeobox (Cdx) proteins (mCdx1, mCdx2, and mCdx4) are also preferential core promoter transcriptional activators. To elucidate the mechanism that enables Caudal to preferentially activate DPE transcription, we performed structure-function analysis. Using a systematic series of deletion mutants (all containing the intact DNA-binding homeodomain) we discovered that the C-terminal region of Caudal contributes to the preferential activation of the fushi tarazu (ftz) Caudal target gene. Furthermore, the region containing both the homeodomain and the C terminus of Caudal was sufficient to confer core promoter-preferential activation to the heterologous GAL4 DNA-binding domain. Importantly, we discovered that Drosophila CREB-binding protein (dCBP) is a co-activator for Caudal-regulated activation of ftz. Strikingly, dCBP conferred the ability to preferentially activate the DPE-dependent ftz reporter to mini-Caudal proteins that were unable to preferentially activate ftz transcription themselves. Taken together, it is the unique combination of dCBP and Caudal that enables the co-activation of ftz in a core promoter-preferential manner. PMID:26018075

  20. 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

  1. 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

  2. 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

    2015-10-01

    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. PMID:26320580

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

    PubMed Central

    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

    2015-01-01

    Summary 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. PMID:26320580

  4. 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. PMID:23710935

  5. 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

  6. The Crystal Structure of N-Acetyl-l-glutamate Synthase from Neisseria gonorrhoeae Provides Insights into Mechanisms of Catalysis and Regulation*†

    PubMed Central

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

    2014-01-01

    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-acetylgluta-mate have been determined at 2.5- and 2.6-Å 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-Å 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 100Å, respectively, and a height of 110Å. 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 argi-nine. This is the first reported crystal structure of any NAGS, and it provides insights into the catalytic function and arginine regulation of NAGS enzymes. PMID:18184660

  7. The crystal structure of N-acetyl-L-glutamate synthase from Neisseria gonorrhoeae provides insights into mechanisms of catalysis and regulation.

    PubMed

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

    2008-03-14

    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. PMID:18184660

  8. Directed evolution and structural analysis of N-carbamoyl-D-amino acid amidohydrolase provide insights into recombinant protein solubility in Escherichia coli

    SciTech Connect

    Jiang, Shimin; Li, Chunhong; Zhang, Weiwen; Cai, Yuanheng; Yang, Yunlin; Yang, Sheng; Jiang, Weihong

    2007-03-15

    One of the greatest bottlenecks in producing recombinant proteins in Escherichia coli is that over-expressed target proteins are mostly present in an insoluble form without any biological activity. N-carbamoyl-D-amino-acid amidohydrolase (DCase) is an important enzyme involved in semi-synthesis of β-lactam antibiotics in industry. In this study, in order to determine the amino acid sites responsible for solubility in DCase, error-prone PCR and DNA shuffling techniques are applied to randomly mutate its encoding sequence, followed by an efficient screening based on structural complementation. Several mutants of DCase with reduced aggregation are isolated. Solubility tests of these mutants and several other mutants generated by site-directed mutagenesis indicate that three amino acid residues of DCase (A18, Y30 and K34) are related to the protein solubility in DCase. In silico structural modeling analyses further suggest that hydrophilicity and/or negative charge at these three residues may be responsible for the increased solubility of DCase proteins in E. coli. Based on the information, multiple engineering designated mutants were constructed by site-directed mutagenesis; among them, a triple mutant A18T/Y30N/K34E (named as DCase-M3) can be over-expressed in E. coli with up to 80% of DCase-M3 protein as soluble. DCase-M3 is purified to homogeneity and a comparative analysis with WT DCase demonstrates that DCase-M3 enzyme is similar to the native DCase in terms of its kinetic and thermodynamic properties. The study provides new insights on recombinant protein solubility in E. coli.

  9. 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.

  10. A structural snapshot of CYP2B4 in complex with paroxetine provides insights into ligand binding and clusters of conformational states.

    PubMed

    Shah, Manish B; Kufareva, Irina; Pascual, Jaime; Zhang, Qinghai; Stout, C David; Halpert, James R

    2013-07-01

    An X-ray crystal structure of CYP2B4 in complex with the drug paroxetine [(3S,4R)-3-[(2H-1,3-benzodioxol-5-yloxy)methyl]-4-(4-fluorophenyl)piperidine] was solved at 2.14 Å resolution. The structure revealed a conformation intermediate to that of the recently solved complex with amlodipine and that of the more compact complex with 4-(4-chlorophenyl)imidazole in terms of the placement of the F-G cassette. Moreover, comparison of the new structure with 15 previously solved structures of CYP2B4 revealed some new insights into the determinants of active-site size and shape. The 2B4-paroxetine structure is nearly superimposable on a previously solved closed structure in a ligand-free state. Despite the overall conformational similarity among multiple closed structures, the active-site cavity volume of the paroxetine complex is enlarged. Further analysis of the accessible space and binding pocket near the heme reveals a new subchamber that resulted from the movement of secondary structural elements and rearrangements of active-site side chains. Overall, the results from the comparison of all 16 structures of CYP2B4 demonstrate a cluster of protein conformations that were observed in the presence or absence of various ligands. PMID:23633618

  11. 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...

  12. 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.

  13. Crystal structure of a 117 kDa glucansucrase fragment provides insight into evolution and product specificity of GH70 enzymes.

    PubMed

    Vujicic-Zagar, Andreja; Pijning, Tjaard; Kralj, Slavko; López, Cesar A; Eeuwema, Wieger; Dijkhuizen, Lubbert; Dijkstra, Bauke W

    2010-12-14

    Glucansucrases are large enzymes belonging to glycoside hydrolase family 70, which catalyze the cleavage of sucrose into fructose and glucose, with the concomitant transfer of the glucose residue to a growing α-glucan polymer. Among others, plaque-forming oral bacteria secrete these enzymes to produce α-glucans, which facilitate the adhesion of the bacteria to the tooth enamel. We determined the crystal structure of a fully active, 1,031-residue fragment encompassing the catalytic and C-terminal domains of GTF180 from Lactobacillus reuteri 180, both in the native state, and in complexes with sucrose and maltose. These structures show that the enzyme has an α-amylase-like (β/α)(8)-barrel catalytic domain that is circularly permuted compared to the catalytic domains of members of glycoside hydrolase families 13 and 77, which belong to the same GH-H superfamily. In contrast to previous suggestions, the enzyme has only one active site and one nucleophilic residue. Surprisingly, in GTF180 the peptide chain follows a "U"-path, such that four of the five domains are made up from discontiguous N- and C-terminal stretches of the peptide chain. Finally, the structures give insight into the factors that determine the different linkage types in the polymeric product. PMID:21118988

  14. Crystal structure of a 117 kDa glucansucrase fragment provides insight into evolution and product specificity of GH70 enzymes

    PubMed Central

    Vujičić-Žagar, Andreja; Pijning, Tjaard; Kralj, Slavko; López, Cesar A.; Eeuwema, Wieger; Dijkhuizen, Lubbert; Dijkstra, Bauke W.

    2010-01-01

    Glucansucrases are large enzymes belonging to glycoside hydrolase family 70, which catalyze the cleavage of sucrose into fructose and glucose, with the concomitant transfer of the glucose residue to a growing α-glucan polymer. Among others, plaque-forming oral bacteria secrete these enzymes to produce α-glucans, which facilitate the adhesion of the bacteria to the tooth enamel. We determined the crystal structure of a fully active, 1,031-residue fragment encompassing the catalytic and C-terminal domains of GTF180 from Lactobacillus reuteri 180, both in the native state, and in complexes with sucrose and maltose. These structures show that the enzyme has an α-amylase-like (β/α)8-barrel catalytic domain that is circularly permuted compared to the catalytic domains of members of glycoside hydrolase families 13 and 77, which belong to the same GH-H superfamily. In contrast to previous suggestions, the enzyme has only one active site and one nucleophilic residue. Surprisingly, in GTF180 the peptide chain follows a “U”-path, such that four of the five domains are made up from discontiguous N- and C-terminal stretches of the peptide chain. Finally, the structures give insight into the factors that determine the different linkage types in the polymeric product. PMID:21118988

  15. Crystal structure of the AmpR effector binding domain provides insight into the molecular regulation of inducible ampc beta-lactamase.

    PubMed

    Balcewich, Misty D; Reeve, Thomas M; Orlikow, Evan A; Donald, Lynda J; Vocadlo, David J; Mark, Brian L

    2010-07-30

    Hyperproduction of AmpC beta-lactamase (AmpC) is a formidable mechanism of resistance to penicillins and cephalosporins in Gram-negative bacteria such as Pseudomonas aeruginosa and Enterobacteriaceae. AmpC expression is regulated by the LysR-type transcriptional regulator AmpR. ampR and ampC genes form a divergent operon with overlapping promoters to which AmpR binds and regulates the transcription of both genes. AmpR induces ampC by binding to one member of the family of 1,6-anhydro-N-acetylmuramyl peptides, which are cytosolic catabolites of peptidoglycan that accumulate during beta-lactam challenge. To gain structural insights into AmpR regulation, we determined the crystal structure of the effector binding domain (EBD) of AmpR from Citrobacter freundii up to 1.83 A resolution. The AmpR EBD is dimeric and each monomer comprises two subdomains that adopt alpha/beta Rossmann-like folds. Located between the monomer subdomains is a pocket that was found to bind the crystallization buffer molecule 2-(N-morpholino)ethanesulfonic acid. The pocket, together with a groove along the surface of subdomain I, forms a putative effector binding site into which a molecule of 1,6-anhydro-N-acetylmuramyl pentapeptide could be modeled. Amino acid substitutions at the base of the interdomain pocket either were found to render AmpR incapable of inducing ampC (Thr103Val, Ser221Ala and Tyr264Phe) or resulted in constitutive ampC expression (Gly102Glu). While the substitutions that prevented ampC induction did not alter the overall AmpR EBD structure, circular dichroism spectroscopy revealed that the nonconservative Gly102Glu mutation affected EBD secondary structure, confirming previous work suggesting that Gly102Glu induces a conformational change to result in constitutive AmpC production. PMID:20594961

  16. Functional Insights from Structural Genomics

    SciTech Connect

    Forouhar,F.; Kuzin, A.; Seetharaman, J.; Lee, I.; Zhou, W.; Abashidze, M.; Chen, Y.; Montelione, G.; Tong, L.; et al

    2007-01-01

    Structural genomics efforts have produced structural information, either directly or by modeling, for thousands of proteins over the past few years. While many of these proteins have known functions, a large percentage of them have not been characterized at the functional level. The structural information has provided valuable functional insights on some of these proteins, through careful structural analyses, serendipity, and structure-guided functional screening. Some of the success stories based on structures solved at the Northeast Structural Genomics Consortium (NESG) are reported here. These include a novel methyl salicylate esterase with important role in plant innate immunity, a novel RNA methyltransferase (H. influenzae yggJ (HI0303)), a novel spermidine/spermine N-acetyltransferase (B. subtilis PaiA), a novel methyltransferase or AdoMet binding protein (A. fulgidus AF{_}0241), an ATP:cob(I)alamin adenosyltransferase (B. subtilis YvqK), a novel carboxysome pore (E. coli EutN), a proline racemase homolog with a disrupted active site (B. melitensis BME11586), an FMN-dependent enzyme (S. pneumoniae SP{_}1951), and a 12-stranded {beta}-barrel with a novel fold (V. parahaemolyticus VPA1032).

  17. Structure of the 26S proteasome with ATP-γS bound provides insights into the mechanism of nucleotide-dependent substrate translocation

    PubMed Central

    Śledź, Paweł; Unverdorben, Pia; Beck, Florian; Pfeifer, Günter; Schweitzer, Andreas; Förster, Friedrich; Baumeister, Wolfgang

    2013-01-01

    The 26S proteasome is a 2.5-MDa, ATP-dependent multisubunit proteolytic complex that processively destroys proteins carrying a degradation signal. The proteasomal ATPase heterohexamer is a key module of the 19S regulatory particle; it unfolds substrates and translocates them into the 20S core particle where degradation takes place. We used cryoelectron microscopy single-particle analysis to obtain insights into the structural changes of 26S proteasome upon the binding and hydrolysis of ATP. The ATPase ring adopts at least two distinct helical staircase conformations dependent on the nucleotide state. The transition from the conformation observed in the presence of ATP to the predominant conformation in the presence of ATP-γS induces a sliding motion of the ATPase ring over the 20S core particle ring leading to an alignment of the translocation channels of the ATPase and the core particle gate, a conformational state likely to facilitate substrate translocation. Two types of intersubunit modules formed by the large ATPase domain of one ATPase subunit and the small ATPase domain of its neighbor exist. They resemble the contacts observed in the crystal structures of ClpX and proteasome-activating nucleotidase, respectively. The ClpX-like contacts are positioned consecutively and give rise to helical shape in the hexamer, whereas the proteasome-activating nucleotidase-like contact is required to close the ring. Conformational switching between these forms allows adopting different helical conformations in different nucleotide states. We postulate that ATP hydrolysis by the regulatory particle ATPase (Rpt) 5 subunit initiates a cascade of conformational changes, leading to pulling of the substrate, which is primarily executed by Rpt1, Rpt2, and Rpt6. PMID:23589842

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

    PubMed Central

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

    2010-01-01

    Nicotinamidases are salvage enzymes that convert nicotinamide to nicotinic acid. These enzymes are essential for the recycling of nicotinamide into NAD+ in most prokaryotes, most single cell and multicellular eukaryotes, but not in mammals. The significance of these enzymes for nicotinamide salvage and for NAD+ homeostasis has increased interest in nicotinamidases as possible antibiotic targets. Nicotinamidases are also regulators of intracellular nicotinamide concentrations, thereby regulating signaling of downstream NAD+ consuming enzymes, such as the NAD+-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 complexed 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 which 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 for several proposed reaction intermediates and allow for a more complete understanding of the catalytic mechanism of this enzyme. PMID:20853856

  19. 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.

  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

    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. PMID:26124200

  1. 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

  2. The glove-like structure of the conserved membrane protein TatC provides insight into signal sequence recognition in twin-arginine translocation.

    PubMed

    Ramasamy, Sureshkumar; Abrol, Ravinder; Suloway, Christian J M; Clemons, William M

    2013-05-01

    In bacteria, two signal-sequence-dependent secretion pathways translocate proteins across the cytoplasmic membrane. Although the mechanism of the ubiquitous general secretory pathway is becoming well understood, that of the twin-arginine translocation pathway, responsible for translocation of folded proteins across the bilayer, is more mysterious. TatC, the largest and most conserved of three integral membrane components, provides the initial binding site of the signal sequence prior to pore assembly. Here, we present two crystal structures of TatC from the thermophilic bacteria Aquifex aeolicus at 4.0 Å and 6.8 Å resolution. The membrane architecture of TatC includes a glove-shaped structure with a lipid-exposed pocket predicted by molecular dynamics to distort the membrane. Correlating the biochemical literature to these results suggests that the signal sequence binds in this pocket, leading to structural changes that facilitate higher order assemblies. PMID:23583035

  3. The glove-like structure of the conserved membrane protein TatC provides insight into signal sequence recognition in twin-arginine translocation

    PubMed Central

    Ramasamy, Sureshkumar; Abrol, Ravinder; Suloway, Christian J.M.; Clemons, William M.

    2013-01-01

    SUMMARY In bacteria, two signal sequence dependent secretion pathways translocate proteins across the cytoplasmic membrane. While the mechanism of the ubiquitous general secretory pathway (SEC) is becoming well understood, that of the twin-arginine translocation pathway (TAT), responsible for translocation of folded proteins across the bilayer, is more mysterious. TatC, the largest and most conserved of three integral membrane components, provides the initial binding site of the signal sequence prior to pore assembly. Here, we present two crystal structures of TatC from the thermophilic bacteria Aquifex aeolicus at 4.0Å and 6.8Å resolution. The novel membrane architecture of TatC includes a glove-shaped structure with a lipid-exposed pocket predicted by molecular dynamics to distort the membrane. Correlating the biochemical literature to these results suggests that the signal sequence binds in this pocket leading to structural changes that facilitate higher order assemblies. PMID:23583035

  4. 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.

  5. 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

  6. 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. PMID:27493782

  7. Structure of the Sgt2/Get5 complex provides insights into GET-mediated targeting of tail-anchored membrane proteins

    PubMed Central

    Simon, Aline C.; Simpson, Peter J.; Goldstone, Rachael M.; Krysztofinska, Ewelina M.; Murray, James W.; High, Stephen; Isaacson, Rivka L.

    2013-01-01

    Small, glutamine-rich, tetratricopeptide repeat protein 2 (Sgt2) is the first known port of call for many newly synthesized tail-anchored (TA) proteins released from the ribosome and destined for the GET (Guided Entry of TA proteins) pathway. This leads them to the residential membrane of the endoplasmic reticulum via an alternative to the cotranslational, signal recognition particle-dependent mechanism that their topology denies them. In yeast, the first stage of the GET pathway involves Sgt2 passing TA proteins on to the Get4/Get5 complex through a direct interaction between the N-terminal (NT) domain of Sgt2 and the ubiquitin-like (UBL) domain of Get5. Here we characterize this interaction at a molecular level by solving both a solution structure of Sgt2_NT, which adopts a unique helical fold, and a crystal structure of the Get5_UBL. Furthermore, using reciprocal chemical shift perturbation data and experimental restraints, we solve a structure of the Sgt2_NT/Get5_UBL complex, validate it via site-directed mutagenesis, and empirically determine its stoichiometry using relaxation experiments and isothermal titration calorimetry. Taken together, these data provide detailed structural information about the interaction between two key players in the coordinated delivery of TA protein substrates into the GET pathway. PMID:23297211

  8. 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

  9. 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. PMID:26655022

  10. 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.

  11. The Crystal Structure of D-Threonine Aldolase from Alcaligenes xylosoxidans Provides Insight into a Metal Ion Assisted PLP-Dependent Mechanism

    PubMed Central

    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. PMID:25884707

  12. 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

    2016-01-01

    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

  13. 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

  14. Structure of a Ca2+/CaM:Kv7.4 (KCNQ4) B helix complex provides insight into M-current modulation

    PubMed Central

    Xu, Qiang; Chang, Aram; Tolia, Alexandra; Minor, Daniel L.

    2012-01-01

    Calmodulin (CaM) is an important regulator of Kv7.x (KCNQx) voltage-gated potassium channels. Channels from this family produce neuronal M-currents and cardiac and auditory IKS currents, and harbor mutations that cause arrhythmias, epilepsy, and deafness. Despite extensive functional characterization, biochemical and structural details of the interaction between CaM and the channel have remained elusive. Here, we show that both apo-CaM and Ca2+/CaM bind to the C-terminal tail of the neuronal channel Kv7.4 (KCNQ4), which is involved both hearing and mechanosensation. Interactions between apo-CaM and the Kv7.4 tail involve two C-terminal tail segments, known as the A and B segments, whereas the interaction between Ca2+/CaM and the Kv7.4 C-terminal tail requires only the B segment. Biochemical studies show that the calcium dependence of the CaM:B segment interaction is conserved in all Kv7 subtypes. X-ray crystallographic determination of the structure of the Ca2+/CaM:Kv7.4 B segment complex shows that Ca2+/CaM wraps around the Kv7.4 B segment, which forms an α-helix, in an antiparallel orientation that embodies a variation of the classic 1-14 Ca2+/CaM interaction motif. Taken together with the context of prior studies, our data suggest a model for modulation of neuronal Kv7 channels involving a calcium-dependent conformational switch from an apo-CaM form that bridges the A and B segments to a Ca2+/CaM form bound to the B-helix. The structure presented here also provides a context for a number of disease causing mutations and for further dissection of the mechanisms by which CaM controls Kv7 function. PMID:23178170

  15. Structure of the Proline Utilization A Proline Dehydrogenase Domain Inactivated by N-propargylglycine Provides Insight into Conformational Changes Induced by Substrate Binding and Flavin Reduction†,‡

    PubMed Central

    Srivastava, Dhiraj; Zhu, Weidong; Johnson, William H.; Whitman, Christian P.; Becker, Donald F.; Tanner, John J.

    2013-01-01

    Proline utilization A (PutA) from Escherichia coli is a flavoprotein that has mutually exclusive roles as a transcriptional repressor of the put regulon and a membrane-associated enzyme that catalyzes the oxidation of proline to glutamate. Previous studies have shown that the binding of proline in the proline dehydrogenase (PRODH) active site and subsequent reduction of the FAD trigger global conformational changes that enhance PutA-membrane affinity. These events cause PutA to switch from its repressor to enzymatic role, but the mechanism by which this signal is propagated from the active site to the distal membrane-binding domain is largely unknown. Here, it is shown that N-propargylglycine irreversibly inactivates PutA by covalently linking the flavin N(5) atom to the ε-amino of Lys329. Furthermore, inactivation locks PutA into a conformation that may mimic the proline reduced, membrane-associated form. The 2.15 Å resolution structure of the inactivated PRODH domain suggests that the initial events involved in broadcasting the reduced flavin state to the distal membrane binding domain include major reorganization of the flavin ribityl chain, severe (35 degree) butterfly bending of the isoalloxazine ring, and disruption of an electrostatic network involving the flavin N(5), Arg431, and Asp370. The structure also provides information about conformational changes associated with substrate binding. This analysis suggests that the active site is incompletely assembled in the absence of the substrate, and the binding of proline draws together conserved residues in helix 8 and the β1-αl loop to complete the active site. PMID:19994913

  16. 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.

  17. 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. PMID:27040018

  18. Modern Statistical Graphs that Provide Insight in Research Results

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Modern statistical graphics offer insight in assessing the results of many common statistical analyses. These ideas, however, are rarely employed in agronomic research articles. This work presents several commonly used graphs, offers one or more alternatives for each, and provides the reasons for pr...

  19. Comparative genomics provides insight into maize adaptation in temperate regions.

    PubMed

    Hufford, Matthew B

    2016-01-01

    A new study provides insights into the evolution of maize during its global spread into temperate regions from its origin in coastal Mexico.Please see related Research article: http://genomebiology.biomedcentral.com/articles/10.1186/s13059-016-1009-x. PMID:27411931

  20. The three-dimensional structure of "Lonely Guy" from Claviceps purpurea provides insights into the phosphoribohydrolase function of Rossmann fold-containing lysine decarboxylase-like proteins.

    PubMed

    Dzurová, Lenka; Forneris, Federico; Savino, Simone; Galuszka, Petr; Vrabka, Josef; Frébort, Ivo

    2015-08-01

    The recently discovered cytokinin (CK)-specific phosphoribohydrolase "Lonely Guy" (LOG) is a key enzyme of CK biosynthesis, converting inactive CK nucleotides into biologically active free bases. We have determined the crystal structures of LOG from Claviceps purpurea (cpLOG) and its complex with the enzymatic product phosphoribose. The structures reveal a dimeric arrangement of Rossmann folds, with the ligands bound to large pockets at the interface between cpLOG monomers. Structural comparisons highlight the homology of cpLOG to putative lysine decarboxylases. Extended sequence analysis enabled identification of a distinguishing LOG sequence signature. Taken together, our data suggest phosphoribohydrolase activity for several proteins of unknown function. PMID:26010010

  1. 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

  2. Crystal and EM Structures of Human Phosphoribosyl Pyrophosphate Synthase I (PRS1) Provide Novel Insights into the Disease-Associated Mutations

    PubMed Central

    Chen, Peng; Liu, Zheng; Wang, Xuejuan; Peng, Junhui; Sun, Qianqian; Li, Jianzhong; Wang, Mingxing; Niu, Liwen; Zhang, Zhiyong; Cai, Gang; Teng, Maikun; Li, Xu

    2015-01-01

    Human PRS1, which is indispensable for the biosynthesis of nucleotides, deoxynucleotides and their derivatives, is associated directly with multiple human diseases because of single base mutation. However, a molecular understanding of the effect of these mutations is hampered by the lack of understanding of its catalytic mechanism. Here, we reconstruct the 3D EM structure of the PRS1 apo state. Together with the native stain EM structures of AMPNPP, AMPNPP and R5P, ADP and the apo states with distinct conformations, we suggest the hexamer is the enzymatically active form. Based on crystal structures, sequence analysis, mutagenesis, enzyme kinetics assays, and MD simulations, we reveal the conserved substrates binding motifs and make further analysis of all pathogenic mutants. PMID:25781187

  3. Crystal and EM structures of human phosphoribosyl pyrophosphate synthase I (PRS1) provide novel insights into the disease-associated mutations.

    PubMed

    Chen, Peng; Liu, Zheng; Wang, Xuejuan; Peng, Junhui; Sun, Qianqian; Li, Jianzhong; Wang, Mingxing; Niu, Liwen; Zhang, Zhiyong; Cai, Gang; Teng, Maikun; Li, Xu

    2015-01-01

    Human PRS1, which is indispensable for the biosynthesis of nucleotides, deoxynucleotides and their derivatives, is associated directly with multiple human diseases because of single base mutation. However, a molecular understanding of the effect of these mutations is hampered by the lack of understanding of its catalytic mechanism. Here, we reconstruct the 3D EM structure of the PRS1 apo state. Together with the native stain EM structures of AMPNPP, AMPNPP and R5P, ADP and the apo states with distinct conformations, we suggest the hexamer is the enzymatically active form. Based on crystal structures, sequence analysis, mutagenesis, enzyme kinetics assays, and MD simulations, we reveal the conserved substrates binding motifs and make further analysis of all pathogenic mutants. PMID:25781187

  4. Further examples of the failure of surrogates to properly model the structural and hydrothermal chemistry of transuranium elements: Insights provided by uranium and neptunium diphosphonates

    SciTech Connect

    Nelson, Anna-Gay D; Bray, Travis H; Zhang, Wei; Haire, Richard G.; Sayler, Todd S.; Albrecht-Schmitt, Thomas E

    2008-01-01

    In situ hydrothermal reduction of Np(VI) to Np(IV) in the presence of methylenediphosphonic acid (C1P2) results in the crystallization of Np[CH2(PO3)2](H2O)2 (NpC1P2-1). Similar reactions have been explored with U(VI) resulting in the isolation of the U(IV) diphosphonate U[CH2(PO3)2](H2O) (UC1P2-1), and the two U(VI) diphosphonates (UO2)2[CH2(PO3)2](H2O)3 H2O (UC1P2-2) and UO2[CH2(PO3H)2](H2O) (UC1P2-3). Single crystal diffraction studies of NpC1P2-1 reveal that it consists of eight-coordinate Np(IV) bound by diphosphonate anions and two coordinating water molecules to create a polar three-dimensional framework structure wherein the water molecules reside in channels. The structure of UC1P2-1 is similar to that of NpC1P2-1 in that it also adopts a three-dimensional structure. However, the U(IV) centers are seven-coordinate with only a single bound water molecule. UC1P2-2 and UC1P2-3 both contain U(VI). Nevertheless, their structures are quite distinct with UC1P2-2 being composed of corrugated layers containing UO6 and UO7 units bridged by C1P2; whereas, UC1P2-3 is found as a polar three-dimensional network structure containing only pentagonal bipyramidal U(VI). Fluorescence measurements on UC1P2-2 and UC1P2-3 exhibit emission from the uranyl moieties with classical vibronic fine-structure.

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

    DOE PAGESBeta

    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 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

  6. 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

  7. 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. PMID:22751668

  8. 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.

  9. 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

  10. Two Structures of a Thiazolinyl Imine Reductase from Yersinia enterocolitica (Irp3) Provide Insight for Catalysis and Binding to the Nonribosomal Peptide Synthetase Module of HMWP1

    PubMed Central

    Meneely, Kathleen M.; Lamb, Audrey L.

    2012-01-01

    The thiazolinyl imine reductase from Yersinia enterocolitica (Irp3) catalyzes the NADPH-dependent reduction of a thiazoline ring in an intermediate for the formation of the siderophore yersiniabactin. Two structures of Irp3 were determined in the apo- (1.85 Å) and NADP+-bound (2.31 Å) forms. Irp3 shows structural homology to sugar oxidoreductases such as glucose-fructose oxidoreductase and 1,5-anhydro-D-fructose reductase, as well as to biliverdin reductase. A homology model of the thiazolinyl imine reductase from Pseudomonas aeruginosa (PchG) was generated. Extensive loop insertions are observed in the C-terminal domain that are unique to Irp3 and PchG and not found in the structural homologs that recognize small molecular substrates. These loops are hypothesized to be important for binding of the nonribosomal peptide synthetase modules (found in HMWP1 and PchF, respectively) to which the substrate of the reductase is covalently attached. A catalytic mechanism of proton donation from a general acid (either histidine-101 or tyrosine-128) and hydride donation from C4 of nicotinamide of the NADPH cofactor is proposed for reduction of the carbon-nitrogen double bond of the thiazoline. PMID:23066849

  11. Crystal Structure of SmcR, a Quorum-sensing Master Regulator of Vibrio vulnificus, Provides Insight into Its Regulation of Transcription*

    PubMed Central

    Kim, Yoonjeong; Kim, Byoung Sik; Park, Yu Jin; Choi, Won-Chan; Hwang, Jungwon; Kang, Beom Sik; Oh, Tae-Kwang; Choi, Sang Ho; Kim, Myung Hee

    2010-01-01

    Quorum sensing has been implicated as an important global regulatory system controlling the expression of numerous virulence factors in bacterial pathogens. SmcR, a homologue of Vibrio harveyi LuxR, has been proposed as a quorum-sensing master regulator of Vibrio vulnificus, an opportunistic human pathogen. Previous studies demonstrated that SmcR is essential for the survival and pathogenesis of V. vulnificus, indicating that inhibiting SmcR is an attractive approach to combat infections by the bacteria. Here, we determined the crystal structure of SmcR at 2.1 Å resolution. The protein structure reveals a typical TetR superfamily fold consisting of an N-terminal DNA binding domain and a C-terminal dimerization domain. In vivo and in vitro functional analysis of the dimerization domain suggested that dimerization of SmcR is vital for its biological regulatory function. The N-terminal DNA recognition and binding residues were assigned based on the protein structure and the results of in vivo and in vitro mutagenesis experiments. Furthermore, protein-DNA interaction experiments suggested that SmcR may have a sophisticated mechanism that enables the protein to recognize each of its many target operators with different affinities. PMID:20178981

  12. Structure of the N-terminal oligomerization domain of DnaD reveals a unique tetramerization motif and provides insights into scaffold formation.

    PubMed

    Schneider, S; Zhang, W; Soultanas, P; Paoli, M

    2008-03-01

    DnaD is a primosomal protein that remodels supercoiled plasmids. It binds to supercoiled forms and converts them to open forms without nicking. During this remodeling process, all the writhe is converted to twist and the plasmids are held around the periphery of large scaffolds made up of DnaD molecules. This DNA-remodeling function is the sum of a scaffold-forming activity on the N-terminal domain and a DNA-dependent oligomerization activity on the C-terminal domain. We have determined the crystal structure of the scaffold-forming N-terminal domain, which reveals a winged-helix architecture, with additional structural elements extending from both N- and C-termini. Four monomers form dimers that join into a tetramer. The N-terminal extension mediates dimerization and tetramerization, with extensive interactions and distinct interfaces. The wings and helices of the winged-helix domains remain exposed on the surface of the tetramer. Structure-guided mutagenesis and atomic force microscopy imaging indicate that these elements, together with the C-terminal extension, are involved in scaffold formation. Based upon our data, we propose a model for the DnaD-mediated scaffold formation. PMID:18206906

  13. Crystal structures and kinetics of monofunctional proline dehydrogenase provide insight into substrate recognition and conformational changes associated with flavin reduction and product release.

    PubMed

    Luo, Min; Arentson, Benjamin W; Srivastava, Dhiraj; Becker, Donald F; Tanner, John J

    2012-12-18

    Proline dehydrogenase (PRODH) catalyzes the FAD-dependent oxidation of proline to Δ(1)-pyrroline-5-carboxylate, which is the first step of proline catabolism. Here, we report the structures of proline dehydrogenase from Deinococcus radiodurans in the oxidized state complexed with the proline analogue L-tetrahydrofuroic acid and in the reduced state with the proline site vacant. The analogue binds against the si face of the FAD isoalloxazine and is protected from bulk solvent by helix α8 and the β1-α1 loop. The FAD ribityl chain adopts two conformations in the E-S complex, which is unprecedented for flavoenzymes. One of the conformations is novel for the PRODH superfamily and may contribute to the low substrate affinity of Deinococcus PRODH. Reduction of the crystalline enzyme-inhibitor complex causes profound structural changes, including 20° butterfly bending of the isoalloxazine, crankshaft rotation of the ribityl, shifting of α8 by 1.7 Å, reconfiguration of the β1-α1 loop, and rupture of the Arg291-Glu64 ion pair. These changes dramatically open the active site to facilitate product release and allow electron acceptors access to the reduced flavin. The structures suggest that the ion pair, which is conserved in the PRODH superfamily, functions as the active site gate. Mutagenesis of Glu64 to Ala decreases the catalytic efficiency 27-fold, which demonstrates the importance of the gate. Mutation of Gly63 decreases the efficiency 140-fold, which suggests that flexibility of the β1-α1 loop is essential for optimal catalysis. The large conformational changes that are required to form the E-S complex suggest that conformational selection plays a role in substrate recognition. PMID:23151026

  14. Crystal Structures and Kinetics of Monofunctional Proline Dehydrogenase Provide Insight into Substrate Recognition and Conformational Changes Associated With Flavin Reduction and Product Release

    PubMed Central

    Luo, Min; Arentson, Benjamin W.; Srivastava, Dhiraj; Becker, Donald F.; Tanner, John J.

    2012-01-01

    Proline dehydrogenase catalyzes the FAD-dependent oxidation of proline to Δ1- pyrroline-5-carboxylate, which is the first step of proline catabolism. Here, we report the structures of proline dehydrogenase from Deinococcus radiodurans in the oxidized state complexed with the proline analog L-tetrahydrofuroic acid and in the reduced state with the proline site vacant. The analog binds against the si face of the FAD isoalloxazine and is protected from bulk solvent by the α8 helix and the β1-α1 loop. The FAD ribityl chain adopts two conformations in the E-S complex, which is unprecedented for flavoenzymes. One of the conformations is novel for the PRODH superfamily and may contribute to the low substrate affinity of Deinococcus PRODH. Reduction of the crystalline enzyme-inhibitor complex causes profound structural changes, including 20° butterfly bending of the isoalloxazine, crankshaft rotation of the ribityl, shifting of α8 by 1.7 Å, reconfiguration of the β1-α1 loop, and rupture of the Arg291-Glu64 ion pair. These changes dramatically open the active site to facilitate product release and allow electron acceptors access to the reduced flavin. The structures suggest that the ion pair, which is conserved in the PRODH superfamily, functions as the active site gate. Mutagenesis of Glu64 to Ala decreases catalytic efficiency 27-fold, which demonstrates the importance of the gate. Mutation of Gly63 decreases efficiency 140-fold, which suggests that flexibility of the β1-α1 loop is essential for optimal catalysis. The large conformational changes that are required to form the E-S complex suggest that conformational selection plays a role in substrate recognition. PMID:23151026

  15. Structure of the Small Dictyostelium discoideum Myosin Light Chain MlcB Provides Insights into MyoB IQ Motif Recognition*

    PubMed Central

    Liburd, Janine; Chitayat, Seth; Crawley, Scott W.; Munro, Kim; Miller, Emily; Denis, Chris M.; Spencer, Holly L.; Côté, Graham P.; Smith, Steven P.

    2014-01-01

    Dictyostelium discoideum MyoB is a class I myosin involved in the formation and retraction of membrane projections, cortical tension generation, membrane recycling, and phagosome maturation. The MyoB-specific, single-lobe EF-hand light chain MlcB binds the sole IQ motif of MyoB with submicromolar affinity in the absence and presence of Ca2+. However, the structural features of this novel myosin light chain and its interaction with its cognate IQ motif remain uncharacterized. Here, we describe the NMR-derived solution structure of apoMlcB, which displays a globular four-helix bundle. Helix 1 adopts a unique orientation when compared with the apo states of the EF-hand calcium-binding proteins calmodulin, S100B, and calbindin D9k. NMR-based chemical shift perturbation mapping identified a hydrophobic MyoB IQ binding surface that involves amino acid residues in helices I and IV and the functional N-terminal Ca2+ binding loop, a site that appears to be maintained when MlcB adopts the holo state. Complementary mutagenesis and binding studies indicated that residues Ile-701, Phe-705, and Trp-708 of the MyoB IQ motif are critical for recognition of MlcB, which together allowed the generation of a structural model of the apoMlcB-MyoB IQ complex. We conclude that the mode of IQ motif recognition by the novel single-lobe MlcB differs considerably from that of stereotypical bilobal light chains such as calmodulin. PMID:24790102

  16. Structural Characterization of Carbohydrate Binding by LMAN1 Protein Provides New Insight into the Endoplasmic Reticulum Export of Factors V (FV) and VIII (FVIII)*

    PubMed Central

    Zheng, Chunlei; Page, Richard C.; Das, Vaijayanti; Nix, Jay C.; Wigren, Edvard; Misra, Saurav; Zhang, Bin

    2013-01-01

    LMAN1 (ERGIC-53) is a key mammalian cargo receptor responsible for the export of a subset of glycoproteins from the endoplasmic reticulum. Together with its soluble coreceptor MCFD2, LMAN1 transports coagulation factors V (FV) and VIII (FVIII). Mutations in LMAN1 or MCFD2 cause the genetic bleeding disorder combined deficiency of FV and FVIII (F5F8D). The LMAN1 carbohydrate recognition domain (CRD) binds to both glycoprotein cargo and MCFD2 in a Ca2+-dependent manner. To understand the biochemical basis and regulation of LMAN1 binding to glycoprotein cargo, we solved crystal structures of the LMAN1-CRD bound to Man-α-1,2-Man, the terminal carbohydrate moiety of high mannose glycans. Our structural data, combined with mutagenesis and in vitro binding assays, define the central mannose-binding site on LMAN1 and pinpoint histidine 178 and glycines 251/252 as critical residues for FV/FVIII binding. We also show that mannobiose binding is relatively independent of pH in the range relevant for endoplasmic reticulum-to-Golgi traffic, but is sensitive to lowered Ca2+ concentrations. The distinct LMAN1/MCFD2 interaction is maintained at these lowered Ca2+ concentrations. Our results suggest that compartmental changes in Ca2+ concentration regulate glycoprotein cargo binding and release from the LMAN1·MCFD2 complex in the early secretory pathway. PMID:23709226

  17. 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.

  18. Crystal structure of [alpha]-COP in complex with [epsilon]-COP provides insight into the architecture of the COPI vesicular coat

    SciTech Connect

    Hsia, Kuo-Chiang; Hoelz, André

    2010-07-23

    The heptameric coatomer complex forms the protein shell of membrane-bound vesicles that are involved in transport from the Golgi to the endoplasmatic reticulum and in intraGolgi trafficking. The heptamer can be dissected into a heterotetrameric F-subcomplex, which displays similarities to the adapter complex of the 'inner' coat in clathrin-coated vesicles, and a heterotrimeric B-subcomplex, which is believed to form an 'outer' coat with a morphology distinct from that of clathrin-coated vesicles. We have determined the crystal structure of the complex between the C-terminal domain (CTD) of {alpha}-COP and full-length {epsilon}-COP, two components of the B-subcomplex, at a 2.9 {angstrom} resolution. The {alpha}-COP{sup CTD} {center_dot} {epsilon}-COP heterodimer forms a rod-shaped structure, in which {epsilon}-COP adopts a tetratricopeptide repeat (TPR) fold that deviates substantially from the canonical superhelical conformation. The {alpha}-COP CTD adopts a U-shaped architecture that complements the TPR fold of {epsilon}-COP. The {epsilon}-COP TPRs form a circular bracelet that wraps around a protruding {beta}-hairpin of the {alpha}-COP CTD, thus interlocking the two proteins. The {alpha}-COPCTD {center_dot} {epsilon}-COP complex forms heterodimers in solution, and we demonstrate biochemically that the heterodimer directly interacts with the Dsl1 tethering complex. These data suggest that the heterodimer is exposed on COPI vesicles, while the remaining part of the B-subcomplex oligomerizes underneath into a cage.

  19. 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

  20. Structure and Dynamics of the Second CARD of Human RIG-I Provide Mechanistic Insights into Regulation of RIG-I Activation

    PubMed Central

    Ferrage, Fabien; Dutta, Kaushik; Nistal-Villán, Estanislao; Patel, Jenish R.; Sánchez-Aparicio, María T.; De Ioannes, Pablo; Buku, Angeliki; Aseguinolaza, Gloria González; García-Sastre, Adolfo; Aggarwal, Aneel K.

    2012-01-01

    SUMMARY RIG-I is a cytosolic sensor of viral RNA, comprised of two N-terminal CARDs followed by helicase and C-terminal regulatory domains (helicase-CTD). Viral RNA binds to the helicase-CTD and “exposes” the CARDs for downstream signaling. The role of the second CARD (CARD2) is essential as RIG-I activation requires dephosphorylation of Thr170 followed by ubiquitination at Lys172. Here, we present the solution structure and dynamics of human RIG-I CARD2. Surprisingly, we find that Thr170 is mostly buried. Parallel studies on the phosphomimetic T170E mutant suggest that the loss of function upon Thr170 phosphorylation is likely associated with changes in the CARD1–CARD2 interface that may prevent Lys172 ubiquitination and/or binding to free K63-linked polyubiquitin. We also demonstrate a strong interaction between CARD2 and the helicase-CTD, and show that mutations at the interface result in constitutive activation of RIG-I. Collectively, our data suggests a close interplay between phosphorylation, ubiquitination, and activation of human RIG-I, all mediated by CARD2. PMID:23063562

  1. 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

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

    2016-01-15

    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

  2. Cryo-EM structure of fatty acid synthase (FAS) from Rhodosporidium toruloides provides insights into the evolutionary development of fungal FAS

    PubMed Central

    Fischer, Manuel; Rhinow, Daniel; Zhu, Zhiwei; Mills, Deryck J; Zhao, Zongbao K; Vonck, Janet; Grininger, Martin

    2015-01-01

    Fungal fatty acid synthases Type I (FAS I) are up to 2.7 MDa large molecular machines composed of large multifunctional polypeptides. Half of the amino acids in fungal FAS I are involved in structural elements that are responsible for scaffolding the elaborate barrel-shaped architecture and turning fungal FAS I into highly efficient de novo producers of fatty acids. Rhodosporidium toruloides is an oleaginous fungal species and renowned for its robust conversion of carbohydrates into lipids to over 70% of its dry cell weight. Here, we use cryo-EM to determine a 7.8-Å reconstruction of its FAS I that reveals unexpected features; its novel form of splitting the multifunctional polypeptide chain into the two subunits α and β, and its duplicated ACP domains. We show that the specific distribution into α and β occurs by splitting at one of many possible sites that can be accepted by fungal FAS I. While, therefore, the specific distribution in α and β chains in R. toruloides FAS I is not correlated to increased protein activities, we also show that the duplication of ACP is an evolutionary late event and argue that duplication is beneficial for the lipid overproduction phenotype. PMID:25761671

  3. Oligomeric states of the Shigella translocator protein IpaB provide structural insights into formation of the type III secretion translocon

    PubMed Central

    Dickenson, Nicholas E; Choudhari, Shyamal P; Adam, Philip R; Kramer, Ryan M; Joshi, Sangeeta B; Middaugh, C Russell; Picking, Wendy L; Picking, William D

    2013-01-01

    The Shigella flexneri Type III secretion system (T3SS) senses contact with human intestinal cells and injects effector proteins that promote pathogen entry as the first step in causing life threatening bacillary dysentery (shigellosis). The Shigella Type III secretion apparatus (T3SA) consists of an anchoring basal body, an exposed needle, and a temporally assembled tip complex. Exposure to environmental small molecules recruits IpaB, the first hydrophobic translocator protein, to the maturing tip complex. IpaB then senses contact with a host cell membrane, forming the translocon pore through which effectors are delivered to the host cytoplasm. Within the bacterium, IpaB exists as a heterodimer with its chaperone IpgC; however, IpaB's structural state following secretion is unknown due to difficulties isolating stable protein. We have overcome this by coexpressing the IpaB/IpgC heterodimer and isolating IpaB by incubating the complex in mild detergents. Interestingly, preparation of IpaB with n-octyl-oligo-oxyethylene (OPOE) results in the assembly of discrete oligomers while purification in N,N-dimethyldodecylamine N-oxide (LDAO) maintains IpaB as a monomer. In this study, we demonstrate that IpaB tetramers penetrate phospholipid membranes to allow a size-dependent release of small molecules, suggesting the formation of discrete pores. Monomeric IpaB also interacts with liposomes but fails to disrupt them. From these and additional findings, we propose that IpaB can exist as a tetramer having inherent flexibility, which allows it to cooperatively interact with and insert into host cell membranes. This event may then lay the foundation for formation of the Shigella T3SS translocon pore. PMID:23456854

  4. 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...

  5. 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

  6. 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

  7. 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

  8. Genomic analyses provide insights into the history of tomato breeding.

    PubMed

    Lin, Tao; Zhu, Guangtao; Zhang, Junhong; Xu, Xiangyang; Yu, Qinghui; Zheng, Zheng; Zhang, Zhonghua; Lun, Yaoyao; Li, Shuai; Wang, Xiaoxuan; Huang, Zejun; Li, Junming; Zhang, Chunzhi; Wang, Taotao; Zhang, Yuyang; Wang, Aoxue; Zhang, Yancong; Lin, Kui; Li, Chuanyou; Xiong, Guosheng; Xue, Yongbiao; Mazzucato, Andrea; Causse, Mathilde; Fei, Zhangjun; Giovannoni, James J; Chetelat, Roger T; Zamir, Dani; Städler, Thomas; Li, Jingfu; Ye, Zhibiao; Du, Yongchen; Huang, Sanwen

    2014-11-01

    The histories of crop domestication and breeding are recorded in genomes. Although tomato is a model species for plant biology and breeding, the nature of human selection that altered its genome remains largely unknown. Here we report a comprehensive analysis of tomato evolution based on the genome sequences of 360 accessions. We provide evidence that domestication and improvement focused on two independent sets of quantitative trait loci (QTLs), resulting in modern tomato fruit ∼100 times larger than its ancestor. Furthermore, we discovered a major genomic signature for modern processing tomatoes, identified the causative variants that confer pink fruit color and precisely visualized the linkage drag associated with wild introgressions. This study outlines the accomplishments as well as the costs of historical selection and provides molecular insights toward further improvement. PMID:25305757

  9. 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

  10. Providing Structure: The Critical Element.

    ERIC Educational Resources Information Center

    Miller, Judith E.; And Others

    1996-01-01

    Discussion of course structure in active learning at the college level looks at ways level and type of structure can be varied and manipulated to meet challenges presented by a diverse student body. Issues discussed include the relationship of structure to cognitive style and development, fitting structure to content and objectives, and what can…

  11. 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

  12. Structural Insight into Proteorhodopsin Oligomers

    PubMed Central

    Stone, Katherine M.; Voska, Jeda; Kinnebrew, Maia; Pavlova, Anna; Junk, Matthias J.N.; Han, Songi

    2013-01-01

    Oligomerization has important functional implications for many membrane proteins. However, obtaining structural insight into oligomeric assemblies is challenging, as they are large and resist crystallization. We focus on proteorhodopsin (PR), a protein with seven transmembrane α-helices that was found to assemble to hexamers in densely packed lipid membrane, or detergent-solubilized environments. Yet, the structural organization and the subunit interface of these PR oligomers were unknown. We used site-directed spin-labeling together with electron spin-resonance lineshape and Overhauser dynamic nuclear polarization analysis to construct a model for the specific orientation of PR subunits within the hexameric complex. We found intersubunit distances to average 16 Å between neighboring 55 residues and that residues 177 are >20 Å apart from each other. These distance constraints show that PR has a defined and radial orientation within a hexamer, with the 55-site of the A-B loop facing the hexamer core and the 177-site of the E-F loop facing the hexamer exterior. Dynamic nuclear polarization measurements of the local solvent dynamics complement the electron spin-resonance-based distance analysis, by resolving whether protein surfaces at positions 55, 58, and 177 are exposed to solvent, or covered by protein-protein or protein-detergent contacts. PMID:23442869

  13. 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.

  14. Crystal Structure of the N-Acetyltransferase Domain of Human N-Acetyl-L-Glutamate Synthase in Complex with N-Acetyl-L-Glutamate Provides Insights into Its Catalytic and Regulatory Mechanisms

    PubMed Central

    Zhao, Gengxiang; Jin, Zhongmin; Allewell, Norma M.; Tuchman, Mendel; Shi, Dashuang

    2013-01-01

    N-acetylglutamate synthase (NAGS) catalyzes the conversion of AcCoA and L-glutamate to CoA and N-acetyl-L-glutamate (NAG), an obligate cofactor for carbamyl phosphate synthetase I (CPSI) in the urea cycle. NAGS deficiency results in elevated levels of plasma ammonia which is neurotoxic. We report herein the first crystal structure of human NAGS, that of the catalytic N-acetyltransferase (hNAT) domain with N-acetyl-L-glutamate bound at 2.1 Å resolution. Functional studies indicate that the hNAT domain retains catalytic activity in the absence of the amino acid kinase (AAK) domain. Instead, the major functions of the AAK domain appear to be providing a binding site for the allosteric activator, L-arginine, and an N-terminal proline-rich motif that is likely to function in signal transduction to CPS1. Crystalline hNAT forms a dimer similar to the NAT-NAT dimers that form in crystals of bifunctional N-acetylglutamate synthase/kinase (NAGS/K) from Maricaulis maris and also exists as a dimer in solution. The structure of the NAG binding site, in combination with mutagenesis studies, provide insights into the catalytic mechanism. We also show that native NAGS from human and mouse exists in tetrameric form, similar to those of bifunctional NAGS/K. PMID:23894642

  15. Crystal structure of the N-acetyltransferase domain of human N-acetyl-L-glutamate synthase in complex with N-acetyl-L-glutamate provides insights into its catalytic and regulatory mechanisms.

    PubMed

    Zhao, Gengxiang; Jin, Zhongmin; Allewell, Norma M; Tuchman, Mendel; Shi, Dashuang

    2013-01-01

    N-acetylglutamate synthase (NAGS) catalyzes the conversion of AcCoA and L-glutamate to CoA and N-acetyl-L-glutamate (NAG), an obligate cofactor for carbamyl phosphate synthetase I (CPSI) in the urea cycle. NAGS deficiency results in elevated levels of plasma ammonia which is neurotoxic. We report herein the first crystal structure of human NAGS, that of the catalytic N-acetyltransferase (hNAT) domain with N-acetyl-L-glutamate bound at 2.1 Å resolution. Functional studies indicate that the hNAT domain retains catalytic activity in the absence of the amino acid kinase (AAK) domain. Instead, the major functions of the AAK domain appear to be providing a binding site for the allosteric activator, L-arginine, and an N-terminal proline-rich motif that is likely to function in signal transduction to CPS1. Crystalline hNAT forms a dimer similar to the NAT-NAT dimers that form in crystals of bifunctional N-acetylglutamate synthase/kinase (NAGS/K) from Maricaulis maris and also exists as a dimer in solution. The structure of the NAG binding site, in combination with mutagenesis studies, provide insights into the catalytic mechanism. We also show that native NAGS from human and mouse exists in tetrameric form, similar to those of bifunctional NAGS/K. PMID:23894642

  16. 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

  17. 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

  18. Marsupial genome sequences: providing insight into evolution and disease.

    PubMed

    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

  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. Genetic Determinants of Epigenetic Patterns: Providing Insight into Disease

    PubMed Central

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

    2015-01-01

    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. PMID:25822796

  1. 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

  2. Proteomic Analysis Provides Insights on Venom Processing in Conus textile

    PubMed Central

    Tayo, Lemmuel L.; Lu, Bingwen; Cruz, Lourdes J.; Yates, John R.

    2010-01-01

    Conus species of marine snails deliver a potent collection of toxins from the venom duct via a long proboscis attached to a harpoon tooth. Conotoxins are known to possess powerful neurological effects and some have been developed for therapeutic uses. Using mass-spectrometry based proteomics, qualitative and quantitative differences in conotoxin components were found in the proximal, central and distal sections of the C. textile venom duct suggesting specialization of duct sections for biosynthesis of particular conotoxins. Reversed phase HPLC followed by Orbitrap mass spectrometry and data analysis using SEQUEST and ProLuCID identified 31 conotoxin sequences and 25 post-translational modification (PTM) variants with King-Kong 2 peptide being the most abundant. Several previously unreported variants of known conopeptides and were found and this is the first time that HyVal is reported for a disulfide rich Conus peptide. Differential expression along the venom duct, production of PTM variants, alternative proteolytic cleavage sites, and venom processing enroute to the proboscis all appear to contribute to enriching the combinatorial pool of conopeptides and producing the appropriate formulation for a particular hunting situation. The complimentary tools of mass spectrometry-based proteomics and molecular biology can greatly accelerate the discovery of Conus peptides and provide insights on envenomation and other biological strategies of cone snails. PMID:20334424

  3. Insight into nucleon structure from generalized parton distributions

    SciTech Connect

    J.W. Negele; R.C. Brower; P. Dreher; R. Edwards; G. Fleming; Ph. Hagler; Th. Lippert; A.V.Pochinsky; D.B. Renner; D. Richards; K. Schilling; W. Schroers

    2004-03-01

    The lowest three moments of generalized parton distributions are calculated in full QCD and provide new insight into the behavior of nucleon electromagnetic form factors, the origin of the nucleon spin, and the transverse structure of the nucleon.

  4. Combined EXAFS and DFT Structure Calculations Provide Structural Insights into the 1:1 Multi-Histidine Complexes of CuII, CuI and ZnII with the Tandem Octarepeats of the Mammalian Prion Protein

    PubMed Central

    Pushie, M. Jake; Nienaber, Kurt H.; McDonald, Alex; Millhauser, Glenn L.; George, Graham N.

    2014-01-01

    The metal coordinating properties of the prion protein (PrP) have been the subject of intense focus and debate since the first reports of copper interaction with PrP just before the turn of the century. The picture of metal coordination to PrP has been improved and refined over the past decade, and yet the structural details of the various metal coordination modes have not been fully elucidated in some cases. Herein we employ X-ray absorption near edge spectroscopy as well as extended X-ray absorption fine structure (EXAFS) spectroscopy to structurally characterize the dominant 1:1 coordination modes for CuII, CuI and ZnII with an N-terminal fragment of PrP. The PrP fragment constitutes four tandem repeats representative of the mammalian octarepeat domain, designated OR4, which is also the most studied PrP fragment for metal interactions, making our findings applicable to a large body of previous work. Density functional theory (DFT) calculations provide additional structural and thermodynamic data, and candidate structures are used to inform EXAFS data analysis. The optimized geometries from DFT calculations are used to identify potential coordination complexes for multi-histidine coordination of CuII, CuI and ZnII in an aqueous medium, modeled using 4-methylimidazole to represent the histidine side chain. Through a combination of in silico coordination chemistry as well as rigorous EXAFS curve fitting, using full multiple scattering on candidate structures from DFT calculations, we have characterized the predominant coordination modes for the 1:1 complexes of CuII, CuI and ZnII with the OR4 peptide at pH 7.4 at atomic resolution, which are best represented as a square planar [CuII(His)4]2+, digonal [CuI(His)2]+ and tetrahedral [ZnII(His)3(OH2)]2+, respectively. PMID:25042361

  5. A logical model provides insights into T cell receptor signaling.

    PubMed

    Saez-Rodriguez, Julio; Simeoni, Luca; Lindquist, Jonathan A; Hemenway, Rebecca; Bommhardt, Ursula; Arndt, Boerge; Haus, Utz-Uwe; Weismantel, Robert; Gilles, Ernst D; Klamt, Steffen; Schraven, Burkhart

    2007-08-01

    Cellular decisions are determined by complex molecular interaction networks. Large-scale signaling networks are currently being reconstructed, but the kinetic parameters and quantitative data that would allow for dynamic modeling are still scarce. Therefore, computational studies based upon the structure of these networks are of great interest. Here, a methodology relying on a logical formalism is applied to the functional analysis of the complex signaling network governing the activation of T cells via the T cell receptor, the CD4/CD8 co-receptors, and the accessory signaling receptor CD28. Our large-scale Boolean model, which comprises 94 nodes and 123 interactions and is based upon well-established qualitative knowledge from primary T cells, reveals important structural features (e.g., feedback loops and network-wide dependencies) and recapitulates the global behavior of this network for an array of published data on T cell activation in wild-type and knock-out conditions. More importantly, the model predicted unexpected signaling events after antibody-mediated perturbation of CD28 and after genetic knockout of the kinase Fyn that were subsequently experimentally validated. Finally, we show that the logical model reveals key elements and potential failure modes in network functioning and provides candidates for missing links. In summary, our large-scale logical model for T cell activation proved to be a promising in silico tool, and it inspires immunologists to ask new questions. We think that it holds valuable potential in foreseeing the effects of drugs and network modifications. PMID:17722974

  6. Structural and mechanistic insights on nitrate reductases.

    PubMed

    Coelho, Catarina; Romão, Maria João

    2015-12-01

    Nitrate reductases (NR) belong to the DMSO reductase family of Mo-containing enzymes and perform key roles in the metabolism of the nitrogen cycle, reducing nitrate to nitrite. Due to variable cell location, structure and function, they have been divided into periplasmic (Nap), cytoplasmic, and membrane-bound (Nar) nitrate reductases. The first crystal structure obtained for a NR was that of the monomeric NapA from Desulfovibrio desulfuricans in 1999. Since then several new crystal structures were solved providing novel insights that led to the revision of the commonly accepted reaction mechanism for periplasmic nitrate reductases. The two crystal structures available for the NarGHI protein are from the same organism (Escherichia coli) and the combination with electrochemical and spectroscopic studies also lead to the proposal of a reaction mechanism for this group of enzymes. Here we present an overview on the current advances in structural and functional aspects of bacterial nitrate reductases, focusing on the mechanistic implications drawn from the crystallographic data. PMID:26362109

  7. 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. PMID:24699014

  8. Structural insight into the specificity of the B3 DNA-binding domains provided by the co-crystal structure of the C-terminal fragment of BfiI restriction enzyme

    PubMed Central

    Golovenko, Dmitrij; Manakova, Elena; Zakrys, Linas; Zaremba, Mindaugas; Sasnauskas, Giedrius; Gražulis, Saulius; Siksnys, Virginijus

    2014-01-01

    The B3 DNA-binding domains (DBDs) of plant transcription factors (TF) and DBDs of EcoRII and BfiI restriction endonucleases (EcoRII-N and BfiI-C) share a common structural fold, classified as the DNA-binding pseudobarrel. The B3 DBDs in the plant TFs recognize a diverse set of target sequences. The only available co-crystal structure of the B3-like DBD is that of EcoRII-N (recognition sequence 5′-CCTGG-3′). In order to understand the structural and molecular mechanisms of specificity of B3 DBDs, we have solved the crystal structure of BfiI-C (recognition sequence 5′-ACTGGG-3′) complexed with 12-bp cognate oligoduplex. Structural comparison of BfiI-C–DNA and EcoRII-N–DNA complexes reveals a conserved DNA-binding mode and a conserved pattern of interactions with the phosphodiester backbone. The determinants of the target specificity are located in the loops that emanate from the conserved structural core. The BfiI-C–DNA structure presented here expands a range of templates for modeling of the DNA-bound complexes of the B3 family of plant TFs. PMID:24423868

  9. Studies provide new insights into Japan's March 2011 tsunami

    NASA Astrophysics Data System (ADS)

    Balcerak, Ernie

    2011-12-01

    New data, field studies, modeling efforts, and surveys are revealing a wealth of information about the devastating tsunami that followed the 11 March 2011 Tohoku earthquake. The earthquake and resulting tsunami caused massive destruction and the loss of about 20,000 lives. At the AGU Fall Meeting last week in San Francisco, Calif., scientists reported a variety of new insights about the tsunami and its effects and about tsunami defense mechanisms, ways to forecast tsunami inundation, and public perception of risk. For example, part of the reason the tsunami generated by the Tohoku quake was so large and destructive was that it was a "merging tsunami." Merging tsunamis, which have been hypothesized but not previously observed with certainty, grow in size dramatically when seafloor topography alters the direction in which the waves are traveling and causes two waves to merge into a larger wave. The alteration of wave paths by complex ocean-floor topography makes it difficult to predict where the largest waves will strike land. "Tsunamis often destroy some coastal areas while leaving others with little damage," explained Tony Song of the NASA Jet Propulsion Laboratory, California Institute of Technology, at a 5 December press conference.

  10. NIH scientists provide new insight into rare kidney cancer

    Cancer.gov

    NIH scientists have discovered a unique feature of a rare, hereditary form of kidney cancer that may provide a better understanding of its progression and metastasis, possibly laying the foundation for the development of new targeted therapies.

  11. 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.

  12. Structural insights into ribosome translocation.

    PubMed

    Ling, Clarence; Ermolenko, Dmitri N

    2016-09-01

    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. Recent history provides sustainable African water quality project insight.

    PubMed

    Holm, Rochelle H

    2012-01-01

    Small-scale projects to provide clean drinking water undertaken in the developing world can contribute to significantly improving the livelihood of rural communities. There has been a historical tendency to poorly plan such projects leading to an unsustainable future. Recent history indicates three simple steps to ensuring successful and enduring clean water projects. First, identification of need by the indigenous community provides ownership in the project. Second, a partnership between key individuals in the indigenous community with the donor provides for ambassadors on both sides of the project. Finally, an exit strategy by the donors for the indigenous communities ensures local sustainability for the future. The study site is the village of Geisha in northern Malawi, Africa. Sustainable implementation approaches are discussed in this case study as well as the various lessons learned. Improved project processes ensure sustainable small-scale water quality projects by donor organizations in developing countries. PMID:22070428

  14. 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 ...

  15. Solid phase synthesis, NMR structure determination of α-KTx3.8, its in silico docking to Kv1.x potassium channels, and electrophysiological analysis provide insights into toxin-channel selectivity.

    PubMed

    Kohl, Bastian; Rothenberg, Ina; Ali, Syed Abid; Alam, Mehtab; Seebohm, Guiscard; Kalbacher, Hubert; Voelter, Wolfgang; Stoll, Raphael

    2015-07-01

    Animal venoms, such as those from scorpions, are a potent source for new pharmacological substances. In this study we have determined the structure of the α-KTx3.8 (named as Bs6) scorpion toxin by multidimensional (1)H homonuclear NMR spectroscopy and investigated its function by molecular dynamics (MD) simulations and electrophysiological measurements. Bs6 is a potent inhibitor of the Kv1.3 channel which plays an important role during the activation and proliferation of memory T-cells (TEM), which play an important role in autoimmune diseases. Therefore, it could be an interesting target for treatment of autoimmune diseases. In this study, Bs6 was synthesised by solid phase synthesis and its three-dimensional (3D) structure has been determined. To gain a deeper insight into the interaction of Bs6 with different potassium channels like hKv1.1 and hKv1.3, the protein-protein complex was modelled based on known toxin-channel structures and tested for stability in MD simulations using GROMACS. The toxin-channel interaction was further analysed by electrophysiological measurements of different potassium channels like hKv1.3 and hKv7.1. As potassium channel inhibitors could play an important role to overcome autoimmune diseases like multiple sclerosis and type-1 diabetes mellitus, our data contributes to the understanding of the molecular mechanism of action and will ultimately help to develop new potent inhibitors in future. PMID:25953725

  16. New Biological Insights from Better Structure Models.

    PubMed

    Touw, Wouter G; Joosten, Robbie P; Vriend, Gert

    2016-03-27

    Structure validation is a key component of all steps in the structure determination process, from structure building, refinement, deposition, and evaluation all the way to post-deposition optimisation of structures in the Protein Data Bank (PDB) by re-refinement and re-building. Today, many aspects of protein structures are understood better than 10years ago, and combined with improved software and more computing power, the automated PDB_REDO procedure can significantly improve about 85% of all X-ray structures ever deposited in the PDB. We review structure validation, structure improvement, and a series of validation resources and facilities that give access to improved PDB files and to reports on the quality of the original and the improved structures. Post-deposition optimisation generally leads to improved protein structures and a series of examples will illustrate how that, in turn, leads to improved or even novel biological insights. PMID:26869101

  17. Daphnia magna ecotoxicogenomics provides mechanistic insights into metal toxicity.

    PubMed

    Poynton, Helen C; Varshavsky, Julia R; Chang, Bonnie; Cavigiolio, Giorgio; Chan, Sarah; Holman, Patricia S; Loguinov, Alexandre V; Bauer, Darren J; Komachi, Kelly; Theil, Elizabeth C; Perkins, Edward J; Hughes, Owen; Vulpe, Chris D

    2007-02-01

    Toxicogenomics has provided innovative approaches to chemical screening, risk assessment, and predictive toxicology. If applied to ecotoxicology, genomics tools could greatly enhance the ability to understand the modes of toxicity in environmentally relevant organisms. Daphnia magna, a small aquatic crustacean, is considered a "keystone" species in ecological food webs and is an indicator species for toxicant exposure. Our objective was to demonstrate the potential utility of gene expression profiling in ecotoxicology by identifying novel biomarkers and uncovering potential modes of action in D. magna. Using a custom D. magna cDNA microarray, we identified distinct expression profiles in response to sublethal copper, cadmium, and zinc exposures and discovered specific biomarkers of exposure including two probable metallothioneins, and a ferritin mRNA with a functional IRE. The gene expression patterns support known mechanisms of metal toxicity and reveal novel modes of action including zinc inhibition of chitinase activity. By integrating gene expression profiling into an environmentally important organism, this study provides experimental support for the utility of ecotoxicogenomics. PMID:17328222

  18. Noise provides new insights on contrast sensitivity function.

    PubMed

    Chen, Ge; Hou, Fang; Yan, Fang-Fang; Zhang, Pan; Xi, Jie; Zhou, Yifeng; Lu, Zhong-Lin; Huang, Chang-Bing

    2014-01-01

    Sensitivity to luminance difference, or contrast sensitivity, is critical for animals to survive in and interact with the external world. The contrast sensitivity function (CSF), which measures visual sensitivity to spatial patterns over a wide range of spatial frequencies, provides a comprehensive characterization of the visual system. Despite its popularity and significance in both basic research and clinical practice, it hasn't been clear what determines the CSF and how the factors underlying the CSF change in different conditions. In the current study, we applied the external noise method and perceptual template model to a wide range of external noise and spatial frequency (SF) conditions, and evaluated how the various sources of observer inefficiency changed with SF and determined the limiting factors underlying the CSF. We found that only internal additive noise and template gain changed significantly with SF, while the transducer non-linearity and coefficient for multiplicative noise were constant. The 12-parameter model provided a very good account of all the data in the 200 tested conditions (86.5%, 86.2%, 89.5%, and 96.4% for the four subjects, respectively). Our results suggest a re-consideration of the popular spatial vision model that employs the CSF as the front-end filter and constant internal additive noise across spatial frequencies. The study will also be of interest to scientists and clinicians engaged in characterizing spatial vision deficits and/or developing rehabilitation methods to restore spatial vision in clinical populations. PMID:24626135

  19. Monoclonal regulatory T cells provide insights into T cell suppression

    PubMed Central

    Gubser, Céline; Schmaler, Mathias; Rossi, Simona W.; Palmer, Ed

    2016-01-01

    Regulatory T cells (Tregs) have a crucial role in maintaining lymphocyte homeostasis. However an understanding of how Tregs function at a cellular and molecular level has not yet been fully elucidated. Here, we make use of a T cell receptor (TCR) transgenic, Rag−/− mouse expressing a Forkhead-Box-Protein P3 (Foxp3) transgene. This mouse provides a source of monoclonal CD4+ Foxp3+ T cells with a defined specificity. Here we show that monoclonal B3K506 Tregs are functional in vitro and in vivo and clearly require cognate antigen to be suppressive. We further show that the strength of Treg stimulation determines the strength of Treg mediated suppression. Finally we analysed various suppressive mechanisms used by monoclonal Tregs and found that Treg-Tconv proximity is a parameter, which correlates with enhanced suppression. PMID:27210828

  20. Monoclonal regulatory T cells provide insights into T cell suppression.

    PubMed

    Gubser, Céline; Schmaler, Mathias; Rossi, Simona W; Palmer, Ed

    2016-01-01

    Regulatory T cells (Tregs) have a crucial role in maintaining lymphocyte homeostasis. However an understanding of how Tregs function at a cellular and molecular level has not yet been fully elucidated. Here, we make use of a T cell receptor (TCR) transgenic, Rag(-/-) mouse expressing a Forkhead-Box-Protein P3 (Foxp3) transgene. This mouse provides a source of monoclonal CD4(+) Foxp3(+) T cells with a defined specificity. Here we show that monoclonal B3K506 Tregs are functional in vitro and in vivo and clearly require cognate antigen to be suppressive. We further show that the strength of Treg stimulation determines the strength of Treg mediated suppression. Finally we analysed various suppressive mechanisms used by monoclonal Tregs and found that Treg-Tconv proximity is a parameter, which correlates with enhanced suppression. PMID:27210828

  1. Arabidopsis thaliana nucleosidase mutants provide new insights into nucleoside degradation

    PubMed Central

    Riegler, Heike; Geserick, Claudia; Zrenner, Rita

    2011-01-01

    A central step in nucleoside and nucleobase salvage pathways is the hydrolysis of nucleosides to their respective nucleobases. In plants this is solely accomplished by nucleosidases (EC 3.2.2.x). To elucidate the importance of nucleosidases for nucleoside degradation, general metabolism, and plant growth, thorough phenotypic and biochemical analyses were performed using Arabidopsis thaliana T-DNA insertion mutants lacking expression of the previously identified genes annotated as uridine ribohydrolases (URH1 and URH2). Comprehensive functional analyses of single and double mutants demonstrated that both isoforms are unimportant for seedling establishment and plant growth, while one participates in uridine degradation. Rather unexpectedly, nucleoside and nucleotide profiling and nucleosidase activity screening of soluble crude extracts revealed a deficiency of xanthosine and inosine hydrolysis in the single mutants, with substantial accumulation of xanthosine in one of them. Mixing of the two mutant extracts, and by in vitro activity reconstitution using a mixture of recombinant URH1 and URH2 proteins, both restored activity, thus providing biochemical evidence that at least these two isoforms are needed for inosine and xanthosine hydrolysis. This mutant study demonstrates the utility of in vivo systems for the examination of metabolic activities, with the discovery of the new substrate xanthosine and elucidation of a mechanism for expanding the nucleosidase substrate spectrum. PMID:21599668

  2. 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. PMID:27088604

  3. Chromosomal passports provide new insights into diffusion of emmer wheat.

    PubMed

    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

  4. 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. PMID:23598338

  5. 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

  6. 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

  7. Elephant Transcriptome Provides Insights into the Evolution of Eutherian Placentation

    PubMed Central

    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. PMID:22546564

  8. 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

  9. 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.

  10. 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. PMID:26969680

  11. 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. PMID:26432641

  12. Molecular fossils in modern genomes provide physiological and geochemical insights to the ancient earth (Invited)

    NASA Astrophysics Data System (ADS)

    Dupont, C.; Caetano-Anolles, G.

    2010-12-01

    The genomes of extant organisms are ultimately derived from ancient life, thus theoretically contain insight to ancient physiology, ecology, and environments. In particular, metalloenzymes may be particularly insightful. The fundamental chemistry of trace elements dictates the molecular speciation and reactivity both within cells and the environment at large. Using protein structure and comparative genomics, we elucidate several major influences this chemistry has had upon biology. All of life exhibits the same proteome size-dependent scaling for the number of metal-binding proteins within a proteome. This fundamental evolutionary constant shows that the selection of one element occurs at the exclusion of another, with the eschewal of Fe for Zn and Ca being a defining feature of eukaryotic pro- teomes. Early life lacked both the structures required to control intracellular metal concentrations and the metal-binding proteins that catalyze electron transport and redox transformations. The development of protein structures for metal homeostasis coincided with the emergence of metal-specific structures, which predomi- nantly bound metals abundant in the Archean ocean. Potentially, this promoted the diversification of emerging lineages of Archaea and Bacteria through the establishment of biogeochemical cycles. In contrast, structures binding Cu and Zn evolved much later, pro- viding further evidence that environmental availability influenced the selection of the elements. The late evolving Zn-binding proteins are fundamental to eukaryotic cellular biology, and Zn bioavailabil- ity may have been a limiting factor in eukaryotic evolution. The results presented here provide an evolutionary timeline based on genomic characteristics, and key hypotheses can be tested by alternative geochemical methods.

  13. 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. PMID:21524250

  14. Study Provides Insights into Diagnosis, Treatment of Rare Immune Disease: Autoimmmune Lymphoproliferative Syndrome ...

    MedlinePlus

    ... Related Links​ ALPS Unit, Laboratory of Immunology Autoimmune Diseases Immune System Primary Immune Deficiency Diseases National Library of ... Study Provides Insights Into Diagnosis, Treatment of Rare Immune Disease NIH Scientists Report Findings From 20 Years of ...

  15. Structural insights into eukaryotic aquaporin regulation.

    PubMed

    Törnroth-Horsefield, Susanna; Hedfalk, Kristina; Fischer, Gerhard; Lindkvist-Petersson, Karin; Neutze, Richard

    2010-06-18

    Aquaporin-mediated water transport across cellular membranes is an ancient, ubiquitous mechanism within cell biology. This family of integral membrane proteins includes both water selective pores (aquaporins) and transport facilitators of other small molecules such as glycerol and urea (aquaglyceroporins). Eukaryotic aquaporins are frequently regulated post-translationally by gating, whereby the rate of flux through the channel is controlled, or by trafficking, whereby aquaporins are shuttled from intracellular storage sites to the plasma membrane. A number of high-resolution X-ray structures of eukaryotic aquaporins have recently been reported and the new structural insights into gating and trafficking that emerged from these studies are described. Basic structural themes reoccur, illustrating how the problem of regulation in diverse biological contexts builds upon a limited set of possible solutions. PMID:20416297

  16. 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

    2014-01-01

    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. PMID:25025742

  17. 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

  18. A Parameterized Model of Amylopectin Synthesis Provides Key Insights into the Synthesis of Granular Starch

    PubMed Central

    Wu, Alex Chi; Morell, Matthew K.; Gilbert, Robert G.

    2013-01-01

    A core set of genes involved in starch synthesis has been defined by genetic studies, but the complexity of starch biosynthesis has frustrated attempts to elucidate the precise functional roles of the enzymes encoded. The chain-length distribution (CLD) of amylopectin in cereal endosperm is modeled here on the basis that the CLD is produced by concerted actions of three enzyme types: starch synthases, branching and debranching enzymes, including their respective isoforms. The model, together with fitting to experiment, provides four key insights. (1) To generate crystalline starch, defined restrictions on particular ratios of enzymatic activities apply. (2) An independent confirmation of the conclusion, previously reached solely from genetic studies, of the absolute requirement for debranching enzyme in crystalline amylopectin synthesis. (3) The model provides a mechanistic basis for understanding how successive arrays of crystalline lamellae are formed, based on the identification of two independent types of long amylopectin chains, one type remaining in the amorphous lamella, while the other propagates into, and is integral to the formation of, an adjacent crystalline lamella. (4) The model provides a means by which a small number of key parameters defining the core enzymatic activities can be derived from the amylopectin CLD, providing the basis for focusing studies on the enzymatic requirements for generating starches of a particular structure. The modeling approach provides both a new tool to accelerate efforts to understand granular starch biosynthesis and a basis for focusing efforts to manipulate starch structure and functionality using a series of testable predictions based on a robust mechanistic framework. PMID:23762422

  19. Insights into the Mechanisms of Absence Seizure Generation Provided by EEG with Functional MRI

    PubMed Central

    Carney, Patrick W.; Jackson, Graeme D.

    2014-01-01

    Absence seizures (AS) are brief epileptic events characterized by loss of awareness with subtle motor features. They may be very frequent, and impact on attention, learning, and memory. A number of pathophysiological models have been developed to explain the mechanism of absence seizure generation, which relies heavily on observations from animal studies. Studying the structural and functional relationships between large-scale brain networks in humans is only practical with non-invasive whole brain techniques. EEG with functional MRI (EEG-fMRI) is one such technique that provides an opportunity to explore the interactions between brain structures involved in AS generation. A number of fMRI techniques including event-related analysis, time-course analysis, and functional connectivity (FC) have identified a common network of structures involved in AS. This network comprises the thalamus, midline, and lateral parietal cortex [the default mode network (DMN)], caudate nuclei, and the reticular structures of the pons. The main component displaying an increase in blood oxygen level dependent (BOLD) signal relative to the resting state, in group studies, is the thalamus while the most consistent cortical change is reduced BOLD signal in the DMN. Time-course analysis shows that, rather than some structures being activated or inactivated during AS, there appears to be increase in activity across components of the network preceding or following the electro-clinical onset of the seizure. The earliest change in BOLD signal occurs in the DMN, prior to the onset of epileptiform events. This region also shows altered FC in patients with AS. Hence, it appears that engagement of this network is central to AS. In this review, we will explore the insights of EEG-fMRI studies into the mechanisms of AS and consider how the DMN is likely to be the major large-scale brain network central to both seizure generation and seizure manifestations. PMID:25225491

  20. Inter-chromosomal contact networks provide insights into Mammalian chromatin organization.

    PubMed

    Kaufmann, Stefanie; Fuchs, Christiane; Gonik, Mariya; Khrameeva, Ekaterina E; Mironov, Andrey A; Frishman, Dmitrij

    2015-01-01

    The recent advent of conformation capture techniques has provided unprecedented insights into the spatial organization of chromatin. We present a large-scale investigation of the inter-chromosomal segment and gene contact networks in embryonic stem cells of two mammalian organisms: humans and mice. Both interaction networks are characterized by a high degree of clustering of genome regions and the existence of hubs. Both genomes exhibit similar structural characteristics such as increased flexibility of certain Y chromosome regions and co-localization of centromere-proximal regions. Spatial proximity is correlated with the functional similarity of genes in both species. We also found a significant association between spatial proximity and the co-expression of genes in the human genome. The structural properties of chromatin are also species specific, including the presence of two highly interactive regions in mouse chromatin and an increased contact density on short, gene-rich human chromosomes, thereby indicating their central nuclear position. Trans-interacting segments are enriched in active marks in human and had no distinct feature profile in mouse. Thus, in contrast to interactions within individual chromosomes, the inter-chromosomal interactions in human and mouse embryonic stem cells do not appear to be conserved. PMID:25961318

  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. PMID:25106134

  2. 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. PMID:21707800

  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. Analysis of Imatinib and Sorafenib Binding to p38 Compared with c-Abl and b-Raf Provides Structural Insights for Understanding the Selectivity of Inhibitors Targeting the DFG-Out Form of Protein Kinases

    SciTech Connect

    Namboodiri, H.; Bukhtiyarova, M; Ramcharan, J; Karpusas, M; Lee, Y; Springman, E

    2010-01-01

    Protein kinases c-Abl, b-Raf, and p38{alpha} are recognized as important targets for therapeutic intervention. c-Abl and b-Raf are major targets of marketed oncology drugs Imatinib (Gleevec) and Sorafenib (Nexavar), respectively, and BIRB-796 is a p38{alpha} inhibitor that reached Phase II clinical trials. A shared feature of these drugs is the fact that they bind to the DFG-out forms of their kinase targets. Although the discovery of this class of kinase inhibitors has increased the level of emphasis on the design of DFG-out inhibitors, the structural determinants for their binding and stabilization of the DFG-out conformation remain unclear. To improve our understanding of these determinants, we determined cocrystal structures of Imatinib and Sorafenib with p38{alpha}. We also conducted a detailed analysis of Imatinib and Sorafenib binding to p38{alpha} in comparison with BIRB-796, including binding kinetics, binding interactions, the solvent accessible surface area (SASA) of the ligands, and stabilization of key structural elements of the protein upon ligand binding. Our results yield an improved understanding of the structural requirements for stabilizing the DFG-out form and a rationale for understanding the genesis of ligand selectivity among DFG-out inhibitors of protein kinases.

  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. 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 Central

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

    2012-01-01

    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 Ni2+ and Fe3+ 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 Co2+ and Ni2+ under aerobic conditions, and Co2+, Ni2+ and Fe2+ under anaerobic conditions, but decreased activity with Zn2+. 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. PMID:22810235

  7. 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

  8. Structure-informed insights for NLR functioning in plant immunity.

    PubMed

    Sukarta, Octavina C A; Slootweg, Erik J; Goverse, Aska

    2016-08-01

    To respond to foreign invaders, plants have evolved a cell autonomous multilayered immune system consisting of extra- and intracellular immune receptors. Nucleotide binding and oligomerization domain (NOD)-like receptors (NLRs) mediate recognition of pathogen effectors inside the cell and trigger a host specific defense response, often involving controlled cell death. NLRs consist of a central nucleotide-binding domain, which is flanked by an N-terminal CC or TIR domain and a C-terminal leucine-rich repeat domain (LRR). These multidomain proteins function as a molecular switch and their activity is tightly controlled by intra and inter-molecular interactions. In contrast to metazoan NLRs, the structural basis underlying NLR functioning as a pathogen sensor and activator of immune responses in plants is largely unknown. However, the first crystal structures of a number of plant NLR domains were recently obtained. In addition, biochemical and structure-informed analyses revealed novel insights in the cooperation between NLR domains and the formation of pre- and post activation complexes, including the coordinated activity of NLR pairs as pathogen sensor and executor of immune responses. Moreover, the discovery of novel integrated domains underscores the structural diversity of NLRs and provides alternative models for how these immune receptors function in plants. In this review, we will highlight these recent advances to provide novel insights in the structural, biochemical and molecular aspects involved in plant NLR functioning. PMID:27208725

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

    PubMed

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

    2016-02-23

    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

  10. 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

  11. 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

  12. 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. PMID:26474847

  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

    Bijelic, Aleksandar; Theiner, Sarah; Keppler, Bernhard K; Rompel, Annette

    2016-06-23

    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. 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

  15. 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.

  16. Forward Dynamics Simulations Provide insight into Muscle Mechanical Work during Human Locomotion

    PubMed Central

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

    2009-01-01

    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. PMID:19955870

  17. 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

  18. New insights into nucleolar structure and function

    PubMed Central

    Lam, Yun Wah

    2015-01-01

    The nucleolus is a non-membrane-bound nuclear organelle found in all eukaryotes. It is the quintessential ‘RNA-seeded’ nuclear body, forming around specific chromosomal features called nucleolar organizing regions that contain arrays of ribosomal DNA. Assembly is triggered by activation of RNA polymerase I-mediated transcription and regulated in mammalian cells in a cell cycle-dependent manner. Although the nucleolus is best known for its role in coordinating ribosome biogenesis, biochemical and proteomic analyses have revealed a much wider functional complexity than previously appreciated, including roles in cell cycle regulation, DNA damage sensing and repair, pre-mRNA processing, telomere metabolism, processing of non-coding RNAs, and coordination of the cellular response to various stresses. Despite these advances, much remains to be learned about the full range of biological processes that occur within, or involve, this organelle and how its assembly/disassembly and functional reorganization in response to various stimuli are regulated. Here, we review the impact of recent studies that provide major insights into these fundamental questions, and we highlight the therapeutic potential of targeting nucleolar pathways. PMID:26097721

  19. 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. PMID:26407004

  20. 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

  1. Structural insights into protein-metal ion partnerships.

    PubMed

    Barondeau, David P; Getzoff, Elizabeth D

    2004-12-01

    New metalloprotein structures continue to provide discoveries regarding protein-metal ion partnerships. Many recent structures reveal metal ion sites that control or are controlled by protein conformational change, including modulation by alternative splice variants and striking conformational changes. Only a few novel catalytic metal centers have been revealed recently, such as the surprising Ni-hook superoxide dismutase catalytic site and the cubane-like Mn(3)CaO(4) photosynthetic oxygen-evolving center. However, important new variations on old heme themes, breakthroughs in the fields of metal ion regulation and metallochaperones, and captivating insights into partnerships between proteins and minerals have also been described. Very high resolution metal site structures and metalloprotein design will be increasingly important in order to leverage the wealth of native metalloprotein structures into a deep understanding of metal ion site specificity and activity. PMID:15582401

  2. 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

  3. 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

  4. 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. PMID:27460707

  5. 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

  6. The structural biology of HIV-1: mechanistic and therapeutic insights

    PubMed Central

    Engelman, Alan; Cherepanov, Peter

    2013-01-01

    Three-dimensional molecular structures can provide detailed information on biological mechanisms and, in cases where molecular function impacts on human health, significantly aid in the development of therapeutic interventions. Over the past 23 years, key components of the lentivirus HIV-1, including its envelope glycoproteins and capsid, and the replication enzymes reverse transcriptase, integrase and protease, have accordingly been scrutinized to near atomic scale resolution. Structural analyses of the interactions between viral and host cell components have moreover yielded key insights into the mechanisms of virus entry, chromosomal integration, transcription and egress from cells. Here, we review recent advances in HIV-1 structural biology, focusing on the impact these results have had on our understanding of virus replication and the development of new therapeutics. PMID:22421880

  7. Functional Insights from Glutamate Receptor Ion Channel Structures

    PubMed Central

    Kumar, Janesh; Mayer, Mark L.

    2014-01-01

    X-ray crystal structures for the soluble amino terminal and ligand binding domains of glutamate receptor ion channels, combined with a 3.6 Å resolution structure of the full length AMPA receptor GluA2 homotetramer, provide unique insights into the mechanisms of iGluR assembly and function. Increasingly sophisticated biochemical, computational and electrophysiological experiments are beginning to reveal the mechanism of action of partial agonists, and yield new models for the mechanism of action of allosteric modulators. Newly identified NMDA receptor ligands acting at novel sites offer hope for development of subtype selective modulators. Many issues remain unsolved, including the role of the ATD in AMPA receptor signaling, and the mechanisms by which auxiliary proteins regulate receptor activity. The structural basis for ion permeation and ion channel block also remain areas of uncertainty, and despite substantial progress, molecular dynamics simulations have yet to reveal how binding of glutamate opens the ion channel pore. PMID:22974439

  8. Biological Insights from Structures of Two-Component Proteins

    PubMed Central

    Gao, Rong; Stock, Ann M.

    2013-01-01

    Two-component signal transduction based on phosphotransfer from a histidine protein kinase to a response regulator protein is a prevalent strategy for coupling environmental stimuli to adaptive responses in bacteria. In both histidine kinases and response regulators, modular domains with conserved structures and biochemical activities adopt different conformational states in the presence of stimuli or upon phosphorylation, enabling a diverse array of regulatory mechanisms based on inhibitory and/or activating protein-protein interactions imparted by different domain arrangements. This review summarizes some of the recent structural work that has provided insight to the functioning of bacterial histidine kinases and response regulators. Particular emphasis is placed on identifying features that are expected to be conserved among different two-component proteins from those that are expected to differ, with the goal of defining the extent to which knowledge of previously characterized two-component proteins can be applied to newly discovered systems. PMID:19575571

  9. 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

  10. 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

  11. 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

  12. Sequencing of the sea lamprey (Petromyzon marinus) genome provides insights into vertebrate evolution.

    PubMed

    Smith, Jeramiah J; Kuraku, Shigehiro; Holt, Carson; Sauka-Spengler, Tatjana; Jiang, Ning; Campbell, Michael S; Yandell, Mark D; Manousaki, Tereza; Meyer, Axel; Bloom, Ona E; Morgan, Jennifer R; Buxbaum, Joseph D; Sachidanandam, Ravi; Sims, Carrie; Garruss, Alexander S; Cook, Malcolm; Krumlauf, Robb; Wiedemann, Leanne M; Sower, Stacia A; Decatur, Wayne A; Hall, Jeffrey A; Amemiya, Chris T; Saha, Nil R; Buckley, Katherine M; Rast, Jonathan P; Das, Sabyasachi; Hirano, Masayuki; McCurley, Nathanael; Guo, Peng; Rohner, Nicolas; Tabin, Clifford J; Piccinelli, Paul; Elgar, Greg; Ruffier, Magali; Aken, Bronwen L; Searle, Stephen M J; Muffato, Matthieu; Pignatelli, Miguel; Herrero, Javier; Jones, Matthew; Brown, C Titus; Chung-Davidson, Yu-Wen; Nanlohy, Kaben G; Libants, Scot V; Yeh, Chu-Yin; McCauley, David W; Langeland, James A; Pancer, Zeev; Fritzsch, Bernd; de Jong, Pieter J; Zhu, Baoli; Fulton, Lucinda L; Theising, Brenda; Flicek, Paul; Bronner, Marianne E; Warren, Wesley C; Clifton, Sandra W; Wilson, Richard K; Li, Weiming

    2013-04-01

    Lampreys are representatives of an ancient vertebrate lineage that diverged from our own ∼500 million years ago. By virtue of this deeply shared ancestry, the sea lamprey (P. marinus) genome is uniquely poised to provide insight into the ancestry of vertebrate genomes and the underlying principles of vertebrate biology. Here, we present the first lamprey whole-genome sequence and assembly. We note challenges faced owing to its high content of repetitive elements and GC bases, as well as the absence of broad-scale sequence information from closely related species. Analyses of the assembly indicate that two whole-genome duplications likely occurred before the divergence of ancestral lamprey and gnathostome lineages. Moreover, the results help define key evolutionary events within vertebrate lineages, including the origin of myelin-associated proteins and the development of appendages. The lamprey genome provides an important resource for reconstructing vertebrate origins and the evolutionary events that have shaped the genomes of extant organisms. PMID:23435085

  13. 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

  14. Sequencing of the sea lamprey (Petromyzon marinus) genome provides insights into vertebrate evolution

    PubMed Central

    Smith, Jeramiah J; Kuraku, Shigehiro; Holt, Carson; Sauka-Spengler, Tatjana; Jiang, Ning; Campbell, Michael S; Yandell, Mark D; Manousaki, Tereza; Meyer, Axel; Bloom, Ona E; Morgan, Jennifer R; Buxbaum, Joseph D; Sachidanandam, Ravi; Sims, Carrie; Garruss, Alexander S; Cook, Malcolm; Krumlauf, Robb; Wiedemann, Leanne M; Sower, Stacia A; Decatur, Wayne A; Hall, Jeffrey A; Amemiya, Chris T; Saha, Nil R; Buckley, Katherine M; Rast, Jonathan P; Das, Sabyasachi; Hirano, Masayuki; McCurley, Nathanael; Guo, Peng; Rohner, Nicolas; Tabin, Clifford J; Piccinelli, Paul; Elgar, Greg; Ruffier, Magali; Aken, Bronwen L; Searle, Stephen MJ; Muffato, Matthieu; Pignatelli, Miguel; Herrero, Javier; Jones, Matthew; Brown, C Titus; Chung-Davidson, Yu-Wen; Nanlohy, Kaben G; Libants, Scot V; Yeh, Chu-Yin; McCauley, David W; Langeland, James A; Pancer, Zeev; Fritzsch, Bernd; de Jong, Pieter J; Zhu, Baoli; Fulton, Lucinda L; Theising, Brenda; Flicek, Paul; Bronner, Marianne E; Warren, Wesley C; Clifton, Sandra W; Wilson, Richard K; Li, Weiming

    2013-01-01

    Lampreys are representatives of an ancient vertebrate lineage that diverged from our own ~500 million years ago. By virtue of this deeply shared ancestry, the sea lamprey (P. marinus) genome is uniquely poised to provide insight into the ancestry of vertebrate genomes and the underlying principles of vertebrate biology. Here, we present the first lamprey whole-genome sequence and assembly. We note challenges faced owing to its high content of repetitive elements and GC bases, as well as the absence of broad-scale sequence information from closely related species. Analyses of the assembly indicate that two whole-genome duplications likely occurred before the divergence of ancestral lamprey and gnathostome lineages. Moreover, the results help define key evolutionary events within vertebrate lineages, including the origin of myelin-associated proteins and the development of appendages. The lamprey genome provides an important resource for reconstructing vertebrate origins and the evolutionary events that have shaped the genomes of extant organisms. PMID:23435085

  15. 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

    2015-12-01

    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

  16. New insight into structural heterogeneity beneath Taiwan

    NASA Astrophysics Data System (ADS)

    Wang, Z.

    2007-12-01

    To know whether the Eurasian lithosphere subducts beneath Taiwan is an important issue for a better understanding of mountain building, arc magmatism and plate collision in the western Pacific region. High- resolution 3-D velocity images are estimated at depths of 0-400 km beneath Taiwan by inverting a large number of arrival times from local and teleseismic events simultaneously. We used 215,676 P-wave arrival time data from 6782 shallow and intermediate-depth earthquakes that are located in and around the Taiwan Island. We also used 12,078 P-wave arrival times that are collected from 3-componenet seismograms of 1108 teleseismic events recorded by the networks installed by Taiwan, Japan and China. Our tomographic images provide further direct geophysical evidence for the tectonic models proposed by previous studies and revealed some new features of structural heterogeneity related to the subducted Eurasian lithosphere and the subducting Philippine Sea slab. Low-velocity anomalies beneath the active volcanoes are visible in the subduction zone of Taiwan, which might caused by the collision between the subducted Eurasian plate and the subducting Philippine Sea slab. In the southern portion of Taiwan, the Eurasian lithosphere is clearly imaged as a high velocity zone with a thickness of 65-80 km and subducted down to a depth of 300 km, whilst it has not been observed beneath North Taiwan. Despite that the existence of subducted Eurasia slab beneath Taiwan has been documented by Lellamant et al. (2001), the present study is the first one to provide high-resolution image and indicate that the Eurasian lithosphere stops at the depth of 300 km beneath South Taiwan but not under North Taiwan. Meanwhile, the present tomographic results are also coherent well with the geology and with plate reconstructions in the region. The previous study proposed that the plate convergence rate is constant at about 7 cm/yr (Seno et al., 1993), it takes about 4-5 Ma for the subducted slab

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

    DOE PAGESBeta

    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; et al

    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

  18. 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.

  19. 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

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

    PubMed

    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)-N(9)-(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

  1. 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.

  2. Caught in the act: RNA-Seq provides novel insights into mRNA degradation.

    PubMed

    Gerwig, Jan; Stülke, Jörg

    2014-10-01

    RNA degradation is a major mechanism of post-transcriptional control of gene expression. Moreover, the rapid turnover of bacterial mRNAs is central to the fast adaptation of these organisms to changing environmental conditions by the regulation of transcription initiation. In most bacteria, RNA processing and degradation require the concerted action of endo- and exoribonucleases. In Molecular Microbiology, Liu et al. (2014) have analysed RNA processing by polynucleotide phosphorylase, the major 3'-5' exonuclease in Bacillus subtilis. For the first time, they were able to study RNA degradation by this enzyme at single nucleotide resolution. The work provides novel insights into the mechanism by which RNA degradation acts in B. subtilis. Moreover, Liu et al. demonstrate that the post-transcriptional control of central regulators affects the expression of whole regulons. PMID:25155548

  3. 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

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

    PubMed

    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

  5. 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

  6. Comparative analysis of bat genomes provides insight into the evolution of flight and immunity.

    PubMed

    Zhang, Guojie; Cowled, Christopher; Shi, Zhengli; Huang, Zhiyong; Bishop-Lilly, Kimberly A; Fang, Xiaodong; Wynne, James W; Xiong, Zhiqiang; Baker, Michelle L; Zhao, Wei; Tachedjian, Mary; Zhu, Yabing; Zhou, Peng; Jiang, Xuanting; Ng, Justin; Yang, Lan; Wu, Lijun; Xiao, Jin; Feng, Yue; Chen, Yuanxin; Sun, Xiaoqing; Zhang, Yong; Marsh, Glenn A; Crameri, Gary; Broder, Christopher C; Frey, Kenneth G; Wang, Lin-Fa; Wang, Jun

    2013-01-25

    Bats are the only mammals capable of sustained flight and are notorious reservoir hosts for some of the world's most highly pathogenic viruses, including Nipah, Hendra, Ebola, and severe acute respiratory syndrome (SARS). To identify genetic changes associated with the development of bat-specific traits, we performed whole-genome sequencing and comparative analyses of two distantly related species, fruit bat Pteropus alecto and insectivorous bat Myotis davidii. We discovered an unexpected concentration of positively selected genes in the DNA damage checkpoint and nuclear factor κB pathways that may be related to the origin of flight, as well as expansion and contraction of important gene families. Comparison of bat genomes with other mammalian species has provided new insights into bat biology and evolution. PMID:23258410

  7. New developments providing mechanistic insight into the impact of the microbiota on allergic disease.

    PubMed

    McCoy, Kathy D; Köller, Yasmin

    2015-08-01

    The increase in allergic diseases over the past several decades is correlated with changes in the composition and diversity of the intestinal microbiota. Microbial-derived signals are critical for instructing the developing immune system and conversely, immune regulation can impact the microbiota. Perturbations in the microbiota composition may be especially important during early-life when the immune system is still developing, resulting in a critical window of opportunity for instructing the immune system. This review highlights recent studies investigating the role of the microbiome in susceptibility or development of allergic diseases with a focus on animal models that provide insight into the mechanisms and pathways involved. Identification of a causal link between reduced microbial diversity or altered microbial composition and increased susceptibility to immune-mediated diseases will hopefully pave the way for better preventive therapies. PMID:25988860

  8. Structural Insights into Reelin Function: Present and Future

    PubMed Central

    Ranaivoson, Fanomezana M.; von Daake, Sventja; Comoletti, Davide

    2016-01-01

    Reelin is a neuronal glycoprotein secreted by the Cajal-Retzius cells in marginal regions of the cerebral cortex and the hippocampus where it plays important roles in the control of neuronal migration and the formation of cellular layers during brain development. This 3461 residue-long protein is composed of a signal peptide, an F-spondin-like domain, eight Reelin repeats (RR1–8), and a positively charged sequence at the C-terminus. Biochemical data indicate that the central region of Reelin binds to the low-density lipoprotein receptors apolipoprotein E receptor 2 (ApoER2) and the very-low-density lipoprotein receptor (VLDLR), leading to the phosphorylation of the intracellular adaptor protein Dab1. After secretion, Reelin is rapidly degraded in three major fragments, but the functional significance of this degradation is poorly understood. Probably due to its large mass and the complexity of its architecture, the high-resolution, three-dimensional structure of Reelin has never been determined. However, the crystal structures of some of the RRs have been solved, providing important insights into their fold and the interaction with the ApoER2 receptor. This review discusses the current findings on the structure of Reelin and its binding to the ApoER2 and VLDLR receptors, and we discuss some areas where proteomics and structural biology can help understanding Reelin function in brain development and human health. PMID:27303268

  9. Vertebrate Membrane Proteins: Structure, Function, and Insights from Biophysical Approaches

    PubMed Central

    MÜLLER, DANIEL J.; WU, NAN; PALCZEWSKI, KRZYSZTOF

    2008-01-01

    Membrane proteins are key targets for pharmacological intervention because they are vital for cellular function. Here, we analyze recent progress made in the understanding of the structure and function of membrane proteins with a focus on rhodopsin and development of atomic force microscopy techniques to study biological membranes. Membrane proteins are compartmentalized to carry out extra- and intracellular processes. Biological membranes are densely populated with membrane proteins that occupy approximately 50% of their volume. In most cases membranes contain lipid rafts, protein patches, or paracrystalline formations that lack the higher-order symmetry that would allow them to be characterized by diffraction methods. Despite many technical difficulties, several crystal structures of membrane proteins that illustrate their internal structural organization have been determined. Moreover, high-resolution atomic force microscopy, near-field scanning optical microscopy, and other lower resolution techniques have been used to investigate these structures. Single-molecule force spectroscopy tracks interactions that stabilize membrane proteins and those that switch their functional state; this spectroscopy can be applied to locate a ligand-binding site. Recent development of this technique also reveals the energy landscape of a membrane protein, defining its folding, reaction pathways, and kinetics. Future development and application of novel approaches during the coming years should provide even greater insights to the understanding of biological membrane organization and function. PMID:18321962

  10. Structural insights into ABC transporter mechanism

    SciTech Connect

    Oldham, Michael L.; Davidson, Amy L.; Chen, Jue

    2010-07-27

    ATP-binding cassette (ABC) transporters utilize the energy from ATP hydrolysis to transport substances across the membrane. In recent years, crystal structures of several ABC transporters have become available. These structures show that both importers and exporters oscillate between two conformations: an inward-facing conformation with the substrate translocation pathway open to the cytoplasm and an outward-facing conformation with the translocation pathway facing the opposite side of the membrane. In this review, conformational differences found in the structures of homologous ABC transporters are analyzed to understand how alternating-access is achieved. It appears that rigid-body rotations of the transmembrane subunits, coinciding with the opening and closing of the nucleotide-binding subunits, couples ATP hydrolysis to substrate translocation.

  11. The Pion cloud: Insights into hadron structure

    SciTech Connect

    A.W. Thomas

    2007-11-01

    Modern nuclear theory presents a fascinating study in contrasting approaches to the structure of hadrons and nuclei. Nowhere is this more apparent than in the treatment of the pion cloud. As this discussion really begins with Yukawa, it is entirely appropriate that this invited lecture at the Yukawa Institute in Kyoto should deal with the issue.

  12. Structural insights into the translational infidelity mechanism

    PubMed Central

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

    2015-01-01

    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. PMID:26037619

  13. Comparative Analysis of Proteomes and Functionomes Provides Insights into Origins of Cellular Diversification

    PubMed Central

    Caetano-Anollés, Gustavo

    2013-01-01

    Reconstructing the evolutionary history of modern species is a difficult problem complicated by the conceptual and technical limitations of phylogenetic tree building methods. Here, we propose a comparative proteomic and functionomic inferential framework for genome evolution that allows resolving the tripartite division of cells and sketching their history. Evolutionary inferences were derived from the spread of conserved molecular features, such as molecular structures and functions, in the proteomes and functionomes of contemporary organisms. Patterns of use and reuse of these traits yielded significant insights into the origins of cellular diversification. Results uncovered an unprecedented strong evolutionary association between Bacteria and Eukarya while revealing marked evolutionary reductive tendencies in the archaeal genomic repertoires. The effects of nonvertical evolutionary processes (e.g., HGT, convergent evolution) were found to be limited while reductive evolution and molecular innovation appeared to be prevalent during the evolution of cells. Our study revealed a strong vertical trace in the history of proteins and associated molecular functions, which was reliably recovered using the comparative genomics approach. The trace supported the existence of a stem line of descent and the very early appearance of Archaea as a diversified superkingdom, but failed to uncover a hidden canonical pattern in which Bacteria was the first superkingdom to deploy superkingdom-specific structures and functions. PMID:24492748

  14. 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.

  15. 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

  16. Structural insights of a hormone sensitive lipase homologue Est22

    PubMed Central

    Huang, Jing; Huo, Ying-Yi; Ji, Rui; Kuang, Siyun; Ji, Chaoneng; Xu, Xue-Wei; Li, Jixi

    2016-01-01

    Hormone sensitive lipase (HSL) catalyzes the hydrolysis of triacylglycerols into fatty acids and glycerol, thus playing key roles in energy homeostasis. However, the application of HSL serving as a pharmaceutical target and an industrial biocatalyst is largely hampered due to the lack of high-resolution structural information. Here we report biochemical properties and crystal structures of a novel HSL homologue esterase Est22 from a deep-sea metagenomic library. Est22 prefers short acyl chain esters and has a very high activity with substrate p-nitrophenyl butyrate. The crystal structures of wild type and mutated Est22 with its product p-nitrophenol are solved with resolutions ranging from 1.4 Å to 2.43 Å. The Est22 exhibits a α/β-hydrolase fold consisting with a catalytic domain and a substrate-recognizing cap domain. Residues Ser188, Asp287, and His317 comprise the catalytic triad in the catalytic domain. The p-nitrophenol molecule occupies the substrate binding pocket and forms hydrogen bonds with adjacent residues Gly108, Gly109, and Gly189. Est22 exhibits a dimeric form in solution, whereas mutants D287A and H317A change to polymeric form, which totally abolished its enzymatic activities. Our study provides insights into the catalytic mechanism of HSL family esterase and facilitates the understanding for further industrial and biotechnological applications of esterases. PMID:27328716

  17. Structural Insights into the Functional Versatility of WWOX Tumor Suppressor

    PubMed Central

    Farooq, Amjad

    2014-01-01

    Recent work on WWOX tumor suppressor is beginning to shed new light on both the molecular mechanism of action of its WW domains as well as the contiguous catalytic domain. Herein, the structural basis underlying the ability of WW1 domain to bind to various physiological ligands and how the orphan WW2 tandem partner synergizes its ligand binding in the context of WW1-WW2 tandem module of WWOX is discussed. Notably, the WW domains within the WW1-WW2 tandem module physically associate so as to adopt a fixed spatial orientation relative to each other. In this manner, the association of WW2 domain with WW1 hinders ligand binding to the latter. Consequently, ligand binding to WW1 domain not only results in the displacement of WW2 lid but also disrupts the fixed orientation of WW domains in the liganded conformation. Equally importantly, structure-guided functional approach suggests that the catalytic domain of WWOX likely serves as a retinal oxidoreductase that catalyzes the reversible oxidation and reduction of all-trans-retinal. Collectively, this review provides structural insights into the functional versatility of a key signaling protein with important implications on its biology. PMID:25662954

  18. Structural insights of a hormone sensitive lipase homologue Est22.

    PubMed

    Huang, Jing; Huo, Ying-Yi; Ji, Rui; Kuang, Siyun; Ji, Chaoneng; Xu, Xue-Wei; Li, Jixi

    2016-01-01

    Hormone sensitive lipase (HSL) catalyzes the hydrolysis of triacylglycerols into fatty acids and glycerol, thus playing key roles in energy homeostasis. However, the application of HSL serving as a pharmaceutical target and an industrial biocatalyst is largely hampered due to the lack of high-resolution structural information. Here we report biochemical properties and crystal structures of a novel HSL homologue esterase Est22 from a deep-sea metagenomic library. Est22 prefers short acyl chain esters and has a very high activity with substrate p-nitrophenyl butyrate. The crystal structures of wild type and mutated Est22 with its product p-nitrophenol are solved with resolutions ranging from 1.4 Å to 2.43 Å. The Est22 exhibits a α/β-hydrolase fold consisting with a catalytic domain and a substrate-recognizing cap domain. Residues Ser188, Asp287, and His317 comprise the catalytic triad in the catalytic domain. The p-nitrophenol molecule occupies the substrate binding pocket and forms hydrogen bonds with adjacent residues Gly108, Gly109, and Gly189. Est22 exhibits a dimeric form in solution, whereas mutants D287A and H317A change to polymeric form, which totally abolished its enzymatic activities. Our study provides insights into the catalytic mechanism of HSL family esterase and facilitates the understanding for further industrial and biotechnological applications of esterases. PMID:27328716

  19. Quantitative Structural Insight into Human Variegate Porphyria Disease*

    PubMed Central

    Wang, Baifan; Wen, Xin; Qin, Xiaohong; Wang, Zhifang; Tan, Ying; Shen, Yuequan; Xi, Zhen

    2013-01-01

    Defects in the human protoporphyrinogen oxidase (hPPO) gene, resulting in ∼50% decreased activity of hPPO, is responsible for the dominantly inherited disorder variegate porphyria (VP). To understand the molecular mechanism of VP, we employed the site-directed mutagenesis, biochemical assays, structural biology, and molecular dynamics simulation studies to investigate VP-causing hPPO mutants. We report here the crystal structures of R59Q and R59G mutants in complex with acifluorfen at a resolution of 2.6 and 2.8 Å. The r.m.s.d. of the Cα atoms of the active site structure of R59G and R59Q with respect to the wild-type was 0.20 and 0.15 Å, respectively. However, these highly similar static crystal structures of mutants with the wild-type could not quantitatively explain the observed large differences in their enzymatic activity. To understand how the hPPO mutations affect their catalytic activities, we combined molecular dynamics simulation and statistical analysis to quantitatively understand the molecular mechanism of VP-causing mutants. We have found that the probability of the privileged conformations of hPPO can be correlated very well with the kcat/Km of PPO (correlation coefficient, R2 > 0.9), and the catalytic activity of 44 clinically reported VP-causing mutants can be accurately predicted. These results indicated that the VP-causing mutation affect the catalytic activity of hPPO by affecting the ability of hPPO to sample the privileged conformations. The current work, together with our previous crystal structure study on the wild-type hPPO, provided the quantitative structural insight into human variegate porphyria disease. PMID:23467411

  20. Structural Insights into Sulfite Oxidase Deficiency

    SciTech Connect

    Karakas,E.; Wilson, H.; Graf, T.; Xiang, S.; Jaramillo-Busquets, S.; Rajagopalan, K.; Kisker, C.

    2005-01-01

    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.

  1. Proteomic profiling of serum samples from chikungunya-infected patients provides insights into host response

    PubMed Central

    2013-01-01

    Background Chikungunya is a highly debilitating febrile illness caused by Chikungunya virus, a single-stranded RNA virus, which is transmitted by Aedes aegypti or Aedes albopictus mosquito species. The pathogenesis and host responses in individuals infected with the chikungunya virus are not well understood at the molecular level. We carried out proteomic profiling of serum samples from chikungunya patients in order to identify molecules associated with the host response to infection by this virus. Results Proteomic profiling of serum obtained from the infected individuals resulted in identification of 569 proteins. Of these, 63 proteins were found to be differentially expressed (≥ 2-fold) in patient as compared to control sera. These differentially expressed proteins were involved in various processes such as lipid metabolism, immune response, transport, signal transduction and apoptosis. Conclusions This is the first report providing a global proteomic profile of serum samples from individuals infected with the chikungunya virus. Our data provide an insight into the proteins that are involved as host response factors during an infection. These proteins include clusterin, apolipoproteins and S100A family of proteins. PMID:24124767

  2. 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

  3. 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.

  4. 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

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

    PubMed

    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

  6. 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. PMID:19025964

  7. Comparative MD analysis of the stability of transthyretin providing insight into the fibrillation mechanism.

    PubMed

    Sørensen, Jesper; Hamelberg, Donald; Schiøtt, Birgit; McCammon, J Andrew

    2007-05-01

    Proteins can misfold and aggregate, which is believed to be the cause of a variety of diseases, affecting very diverse organs in the body. Many questions about the nature of aggregation and the proteins that are involved in these events are still left unanswered. One of the proteins that is known to form amyloids is transthyretin (TTR), the secondary transporter of thyroxine, and transporter of retinol-binding protein. Several experimental results have helped to explain this aberrant behavior of TTR; however, structural insights of the amyloidgenic process are still lacking. Therefore, we have used all-atom MD simulation and free energy calculations to study the initial phase of this process. We have calculated the free energy changes of the initial tetramer dissociation under different conditions and in the presence of thyroxine. We show that tetramer formation is indeed only thermodynamically favorable in neutral pH conditions. We find that binding of two thyroxine molecules stabilizes the complex, and that this occurs with negative cooperativity. In addition to the energetic calculations, we have also investigated the dominant motions of the TTR and found that only the dimeric form of the protein could undergo the initial fibril formation. PMID:17315201

  8. 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. PMID:26487042

  9. 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

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

    2016-07-27

    N(2)-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 N(2)-methylguanosine and N(2),N(2)-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

  10. Global and local molecular dynamics of a bacterial carboxylesterase provide insight into its catalytic mechanism

    PubMed Central

    Yu, Xiaozhen; Sigler, Sara C.; Hossain, Delwar; Wierdl, Monika; Gwaltney, Steven R.; Potter, Philip M.; Wadkins, Randy M.

    2013-01-01

    Carboxylesterases (CEs) are ubiquitous enzymes responsible for the detoxification of xenobiotics. In humans, substrates for these enzymes are far-ranging, and include the street drug heroin and the anticancer agent irinotecan. Hence, their ability to bind and metabolize substrates is of broad interest to biomedical science. In this study, we focused our attention on dynamic motions of a CE from B. subtilis (pnbCE), with emphasis on the question of what individual domains of the enzyme might contribute to its catalytic activity. We used a 10 ns all-atom molecular dynamics simulation, normal mode calculations, and enzyme kinetics to understand catalytic consequences of structural changes within this enzyme. Our results shed light on how molecular motions are coupled with catalysis. During molecular dynamics, we observed a distinct C-C bond rotation between two conformations of Glu310. Such a bond rotation would alternately facilitate and impede protonation of the active site His399 and act as a mechanism by which the enzyme alternates between its active and inactive conformation. Our normal mode results demonstrate that the distinct low-frequency motions of two loops in pnbCE, coil_5 and coil_21, are important in substrate conversion and seal the active site. Mutant CEs lacking these external loops show significantly reduced rates of substrate conversion, suggesting this sealing motion prevents escape of substrate. Overall, the results of our studies give new insight into the structure-function relationship of CEs and have implications for the entire family of α/β fold family of hydrolases, of which this CE is a member. PMID:22127613

  11. 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

  12. 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

  13. Comparative Genomic and Sequence Analysis Provides Insight into the Molecular Functionality of NOD1 and NOD2

    PubMed Central

    Boyle, Joseph P.; Mayle, Sophie; Parkhouse, Rhiannon; Monie, Tom P.

    2013-01-01

    Amino acids with functional or key structural roles display higher degrees of conservation through evolution. The comparative analysis of protein sequences from multiple species and/or between homologous proteins can be highly informative in the identification of key structural and functional residues. Residues which in turn provide insight into the molecular mechanisms of protein function. We have explored the genomic and amino acid conservation of the prototypic innate immune genes NOD1 and NOD2. NOD1 orthologs were found in all vertebrate species analyzed, whilst NOD2 was absent from the genomes of avian, reptilian and amphibian species. Evolutionary trace analysis was used to identify highly conserved regions of NOD1 and NOD2 across multiple species. Consistent with the known functions of NOD1 and NOD2 highly conserved patches were identified that matched the Walker A and B motifs and provided interaction surfaces for the adaptor protein RIP2. Other patches of high conservation reflect key structural functions as predicted by homology models. In addition, the pattern of residue conservation within the leucine-rich repeat (LRR) region of NOD1 and NOD2 is indicative of a conserved mechanism of ligand recognition involving the concave surface of the LRRs. PMID:24109482

  14. Can Transcriptomics Provide Insight into the Chemopreventive Mechanisms of Complex Mixtures of Phytochemicals in Humans?

    PubMed Central

    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.

    2014-01-01

    Abstract 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. Antioxid. Redox Signal. 20, 2107–2113. PMID:24328558

  15. Transcriptome analysis in different rice cultivars provides novel insights into desiccation and salinity stress responses.

    PubMed

    Shankar, Rama; Bhattacharjee, Annapurna; Jain, Mukesh

    2016-01-01

    Drought and salinity are the major environmental factors that affect rice productivity. Comparative transcriptome analysis between tolerant and sensitive rice cultivars can provide insights into the regulatory mechanisms involved in these stress responses. In this study, the comparison of transcriptomes of a drought-tolerant [Nagina 22 (N22)] and a salinity-tolerant (Pokkali) rice cultivar with IR64 (susceptible cultivar) revealed variable transcriptional responses under control and stress conditions. A total of 801 and 507 transcripts were exclusively differentially expressed in N22 and Pokkali rice cultivars, respectively, under stress conditions. Gene ontology analysis suggested the enrichment of transcripts involved in response to abiotic stress and regulation of gene expression in stress-tolerant rice cultivars. A larger number of transcripts encoding for members of NAC and DBP transcription factor (TF) families in N22 and members of bHLH and C2H2 TF families in Pokkali exhibited differential regulation under desiccation and salinity stresses, respectively. Transcripts encoding for thioredoxin and involved in phenylpropanoid metabolism were up-regulated in N22, whereas transcripts involved in wax and terpenoid metabolism were up-regulated in Pokkali. Overall, common and cultivar-specific stress-responsive transcripts identified in this study can serve as a helpful resource to explore novel candidate genes for abiotic stress tolerance in rice. PMID:27029818

  16. 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

  17. Neanderthal brain size at birth provides insights into the evolution of human life history

    PubMed Central

    Ponce de León, Marcia S.; Golovanova, Lubov; Doronichev, Vladimir; Romanova, Galina; Akazawa, Takeru; Kondo, Osamu; Ishida, Hajime; Zollikofer, Christoph P. E.

    2008-01-01

    From birth to adulthood, the human brain expands by a factor of 3.3, compared with 2.5 in chimpanzees [DeSilva J and Lesnik J (2006) Chimpanzee neonatal brain size: Implications for brain growth in Homo erectus. J Hum Evol 51: 207–212]. How the required extra amount of human brain growth is achieved and what its implications are for human life history and cognitive development are still a matter of debate. Likewise, because comparative fossil evidence is scarce, when and how the modern human pattern of brain growth arose during evolution is largely unknown. Virtual reconstructions of a Neanderthal neonate from Mezmaiskaya Cave (Russia) and of two Neanderthal infant skeletons from Dederiyeh Cave (Syria) now provide new comparative insights: Neanderthal brain size at birth was similar to that in recent Homo sapiens and most likely subject to similar obstetric constraints. Neanderthal brain growth rates during early infancy were higher, however. This pattern of growth resulted in larger adult brain sizes but not in earlier completion of brain growth. Because large brains growing at high rates require large, late-maturing, mothers [Leigh SR and Blomquist GE (2007) in Campbell CJ et al. Primates in perspective; pp 396–407], it is likely that Neanderthal life history was similarly slow, or even slower-paced, than in recent H. sapiens. PMID:18779579

  18. Morphological evolution in sea urchin development: hybrids provide insights into the pace of evolution.

    PubMed

    Byrne, Maria; Voltzow, Janice

    2004-04-01

    Hybridisations between related species with divergent ontogenies can provide insights into the bases for evolutionary change in development. One example of such hybridisations involves sea urchin species that exhibit either standard larval (pluteal) stages or those that develop directly from embryo to adult without an intervening feeding larval stage. In such crosses, pluteal features were found to be restored in fertilisations of the eggs of some direct developing sea urchins (Heliocidaris erythrogramma) with the sperm of closely (Heliocidaris tuberculata) and distantly (Pseudoboletia maculata) related species with feeding larvae. Such results can be argued to support the punctuated equilibrium model-conservation in pluteal regulatory systems and a comparatively rapid switch to direct development in evolution.1,2 Generation of hybrids between distantly related direct developers may, however, indicate evolutionary convergence. The 'rescue' of pluteal features by paternal genomes may require maternal factors from H. erythrogramma because the larva of this species has pluteal features. In contrast, pluteal features were not restored in hybridisations with the eggs of Holopneustes purpurescens, which lacks pluteal features. How much of pluteal development can be lost before it cannot be rescued in such crosses? The answer awaits hybridisations among indirect and direct developing sea urchins differing in developmental phenotype, in parallel with investigations of the genetic programs involved. PMID:15057932

  19. 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. PMID:19834890

  20. The tiny eukaryote Ostreococcus provides genomic insights into the paradox of plankton speciation

    PubMed Central

    Palenik, Brian; Grimwood, Jane; Aerts, Andrea; Rouzé, Pierre; Salamov, Asaf; Putnam, Nicholas; Dupont, Chris; Jorgensen, Richard; Derelle, Evelyne; Rombauts, Stephane; Zhou, Kemin; Otillar, Robert; Merchant, Sabeeha S.; Podell, Sheila; Gaasterland, Terry; Napoli, Carolyn; Gendler, Karla; Manuell, Andrea; Tai, Vera; Vallon, Olivier; Piganeau, Gwenael; Jancek, Séverine; Heijde, Marc; Jabbari, Kamel; Bowler, Chris; Lohr, Martin; Robbens, Steven; Werner, Gregory; Dubchak, Inna; Pazour, Gregory J.; Ren, Qinghu; Paulsen, Ian; Delwiche, Chuck; Schmutz, Jeremy; Rokhsar, Daniel; Van de Peer, Yves; Moreau, Hervé; Grigoriev, Igor V.

    2007-01-01

    The smallest known eukaryotes, at ≈1-μm diameter, are Ostreococcus tauri and related species of marine phytoplankton. The genome of Ostreococcus lucimarinus has been completed and compared with that of O. tauri. This comparison reveals surprising differences across orthologous chromosomes in the two species from highly syntenic chromosomes in most cases to chromosomes with almost no similarity. Species divergence in these phytoplankton is occurring through multiple mechanisms acting differently on different chromosomes and likely including acquisition of new genes through horizontal gene transfer. We speculate that this latter process may be involved in altering the cell-surface characteristics of each species. In addition, the genome of O. lucimarinus provides insights into the unique metal metabolism of these organisms, which are predicted to have a large number of selenocysteine-containing proteins. Selenoenzymes are more catalytically active than similar enzymes lacking selenium, and thus the cell may require less of that protein. As reported here, selenoenzymes, novel fusion proteins, and loss of some major protein families including ones associated with chromatin are likely important adaptations for achieving a small cell size. PMID:17460045

  1. Transcriptome analysis in different rice cultivars provides novel insights into desiccation and salinity stress responses

    PubMed Central

    Shankar, Rama; Bhattacharjee, Annapurna; Jain, Mukesh

    2016-01-01

    Drought and salinity are the major environmental factors that affect rice productivity. Comparative transcriptome analysis between tolerant and sensitive rice cultivars can provide insights into the regulatory mechanisms involved in these stress responses. In this study, the comparison of transcriptomes of a drought-tolerant [Nagina 22 (N22)] and a salinity-tolerant (Pokkali) rice cultivar with IR64 (susceptible cultivar) revealed variable transcriptional responses under control and stress conditions. A total of 801 and 507 transcripts were exclusively differentially expressed in N22 and Pokkali rice cultivars, respectively, under stress conditions. Gene ontology analysis suggested the enrichment of transcripts involved in response to abiotic stress and regulation of gene expression in stress-tolerant rice cultivars. A larger number of transcripts encoding for members of NAC and DBP transcription factor (TF) families in N22 and members of bHLH and C2H2 TF families in Pokkali exhibited differential regulation under desiccation and salinity stresses, respectively. Transcripts encoding for thioredoxin and involved in phenylpropanoid metabolism were up-regulated in N22, whereas transcripts involved in wax and terpenoid metabolism were up-regulated in Pokkali. Overall, common and cultivar-specific stress-responsive transcripts identified in this study can serve as a helpful resource to explore novel candidate genes for abiotic stress tolerance in rice. PMID:27029818

  2. Multi-omics analysis of niche specificity provides new insights into ecological adaptation in bacteria.

    PubMed

    Zhu, Bo; Ibrahim, Muhammad; Cui, Zhouqi; Xie, Guanlin; Jin, Gulei; Kube, Michael; Li, Bin; Zhou, Xueping

    2016-08-01

    Different lifestyles, ranging from a saprophyte to a pathogen, have been reported in bacteria of one species. Here, we performed genome-wide survey of the ecological adaptation in four Burkholderia seminalis strains, distinguished by their origin as part of the saprophytic microbial community of soil or water but also including human and plant pathogens. The results indicated that each strain is separated from the others by increased fitness in medium simulating its original niche corresponding to the difference between strains in metabolic capacities. Furthermore, strain-specific metabolism and niche survival was generally linked with genomic variants and niche-dependent differential expression of the corresponding genes. In particular, the importance of iron, trehalose and d-arabitol utilization was highlighted by the involvement of DNA-methylation and horizontal gene transfer in niche-adapted regulation of the corresponding operons based on the integrated analysis of our multi-omics data. Overall, our results provided insights of niche-specific adaptation in bacteria. PMID:26859773

  3. A Molecular Analysis Provides Novel Insights into Androgen Receptor Signalling in Breast Cancer

    PubMed Central

    Mehta, Jatin; Asthana, Shailendra; Mandal, Chandi Charan; Saxena, Sunita

    2015-01-01

    Background Androgen Receptor (AR) is an essential transcription factor for the development of secondary sex characteristics, spermatogenesis and carcinogenesis. Recently AR has been implicated in the development and progression of breast and prostate cancers. Although some of the functions of the AR are known but the mechanistic details of these divergent processes are still not clear. Therefore understanding the regulatory mechanisms of the functioning of the AR in ER-/AR+ breast cancer will provide many novel targets for the purpose of therapeutic intervention. Methods/Results Using bioinformatics tools, we have identified 75 AR targets having prominent roles in cell cycle, apoptosis and metabolism. Herein, we validated 10 genes as AR targets by studying the regulation of these genes in MDA-MB-453 cell line on stimulation by androgens like 5α-dihydrotestosterone (DHT), using RT-qPCR and ChIP assay. It was observed that all the identified genes involved in cell cycle except MAD1L1 were found to be up regulated whereas expression of apoptosis related genes was decreased in response to DHT treatment. We performed an exhaustive, rigid-body docking between individual ARE and DNA binding domain (DBD) of the AR protein and it was found that novel residues K567, K588, K591 and R592 are involved in the process of DNA binding. To verify these specific DNA-protein interactions electrostatic energy term calculations for each residue was determined using the linearized Poisson–Boltzmann equation. Our experimental data showed that treatment of breast cancer cells with DHT promotes cell proliferation and decreases apoptosis. It was observed that bicalutamide treatment was able to reverse the effect of DHT. Conclusion Taken together, our results provide new insights into the mechanism by which AR promotes breast cancer progression. Moreover our work proposes to use bicalutamide along with taxanes as novel therapy for the treatment of TNBCs, which are positive for downstream

  4. Sex-linked and autosomal microsatellites provide new insights into island populations of the tammar wallaby

    PubMed Central

    MacDonald, A J; FitzSimmons, N N; Chambers, B; Renfree, M B; Sarre, S D

    2014-01-01

    The emerging availability of microsatellite markers from mammalian sex chromosomes provides opportunities to investigate both male- and female-mediated gene flow in wild populations, identifying patterns not apparent from the analysis of autosomal markers alone. Tammar wallabies (Macropus eugenii), once spread over the southern mainland, have been isolated on several islands off the Western Australian and South Australian coastlines for between 10 000 and 13 000 years. Here, we combine analyses of autosomal, Y-linked and X-linked microsatellite loci to investigate genetic variation in populations of this species on two islands (Kangaroo Island, South Australia and Garden Island, Western Australia). All measures of diversity were higher for the larger Kangaroo Island population, in which genetic variation was lowest at Y-linked markers and highest at autosomal markers (θ=3.291, 1.208 and 0.627 for autosomal, X-linked and Y-linked data, respectively). Greater relatedness among females than males provides evidence for male-biased dispersal in this population, while sex-linked markers identified genetic lineages not apparent from autosomal data alone. Overall genetic diversity in the Garden Island population was low, especially on the Y chromosome where most males shared a common haplotype, and we observed high levels of inbreeding and relatedness among individuals. Our findings highlight the utility of this approach for management actions, such as the selection of animals for translocation or captive breeding, and the ecological insights that may be gained by combining analyses of microsatellite markers on sex chromosomes with those derived from autosomes. PMID:24169646

  5. 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. PMID:26941746

  6. 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

  7. Mitochondrial Genome and Nuclear Markers Provide New Insight into the Evolutionary History of Macaques.

    PubMed

    Jiang, Juan; Yu, Jianqiu; Li, Jing; Li, Peng; Fan, Zhenxin; Niu, Lili; Deng, Jiabo; Yue, Bisong; Li, Jing

    2016-01-01

    The evolutionary history of macaques, genus Macaca, has been under debate due to the short times of divergence. In this study, maternal, paternal, and biparental genetic systems were applied to infer phylogenetic relationships among macaques and to trace ancient hybridization events in their evolutionary history. Using a PCR display method, 17 newly phylogenetically informative Alu insertions were identified from M. assamensis. We combined presence/absence analysis of 84 Alu elements with mitochondrial genomes as well as nuclear sequences (five autosomal genes, two Y chromosomal genes, and one X chromosomal fragment) to reconstruct a robust macaque phylogeny. Topologies generated from different inherited markers were similar supporting six well defined species groups and a close relationship of M. assamensis and M. thibetana, but differed in the placing of M. arctoides. Both Alu elements and nuclear genes supported that M. arctoides was close to the sinica group, whereas the mitochondrial data clustered it into the fascicularis/mulatta lineage. Our results reveal that a sex-biased hybridization most likely occurred in the evolutionary history of M. arctoides, and suggest an introgressive pattern of male-mediated gene flow from the ancestors of M. arctoides to the M. mulatta population followed by nuclear swamping. According to the estimation of divergence dates, the hybridization occurred around 0.88~1.77 mya (nuclear data) or 1.38~2.56 mya (mitochondrial data). In general, our study indicates that a combination of various molecular markers could help explain complicated evolutionary relationships. Our results have provided new insights into the evolutionary history of macaques and emphasize that hybridization might play an important role in macaque evolution. PMID:27135608

  8. Yeast nutrient transceptors provide novel insight in the functionality of membrane transporters.

    PubMed

    Schothorst, Joep; Kankipati, Harish Nag; Conrad, Michaela; Samyn, Dieter R; Van Zeebroeck, Griet; Popova, Yulia; Rubio-Texeira, Marta; Persson, Bengt L; Thevelein, Johan M

    2013-11-01

    In the yeast Saccharomyces cerevisiae several nutrient transporters have been identified that possess an additional function as nutrient receptor. These transporters are induced when yeast cells are starved for their substrate, which triggers entry into stationary phase and acquirement of a low protein kinase A (PKA) phenotype. Re-addition of the lacking nutrient triggers exit from stationary phase and sudden activation of the PKA pathway, the latter being mediated by the nutrient transceptors. At the same time, the transceptors are ubiquitinated, endocytosed and sorted to the vacuole for breakdown. Investigation of the signaling function of the transceptors has provided a new read-out and new tools for gaining insight into the functionality of transporters. Identification of amino acid residues that bind co-transported ions in symporters has been challenging because the inactivation of transport by site-directed mutagenesis is not conclusive with respect to the cause of the inactivation. The discovery of nontransported agonists of the signaling function in transceptors has shown that transport is not required for signaling. Inactivation of transport with maintenance of signaling in transceptors supports that a true proton-binding residue was mutagenised. Determining the relationship between transport and induction of endocytosis has also been challenging, since inactivation of transport by mutagenesis easily causes loss of all affinity for the substrate. The use of analogues with different combinations of transport and signaling capacities has revealed that transport, ubiquitination and endocytosis can be uncoupled in several unexpected ways. The results obtained are consistent with transporters undergoing multiple substrate-induced conformational changes, which allow interaction with different accessory proteins to trigger specific downstream events. PMID:24114446

  9. Multiple Kisspeptin Receptors in Early Osteichthyans Provide New Insights into the Evolution of This Receptor Family

    PubMed Central

    Pasquier, Jérémy; Lafont, Anne-Gaëlle; Jeng, Shan-Ru; Morini, Marina; Dirks, Ron; van den Thillart, Guido; Tomkiewicz, Jonna; Tostivint, Hervé; Chang, Ching-Fong; Rousseau, Karine; Dufour, Sylvie

    2012-01-01

    Deorphanization of GPR54 receptor a decade ago led to the characterization of the kisspeptin receptor (Kissr) in mammals and the discovery of its major role in the brain control of reproduction. While a single gene encodes for Kissr in eutherian mammals including human, other vertebrates present a variable number of Kissr genes, from none in birds, one or two in teleosts, to three in an amphibian, xenopus. In order to get more insight into the evolution of Kissr gene family, we investigated the presence of Kissr in osteichthyans of key-phylogenetical positions: the coelacanth, a representative of early sarcopterygians, the spotted gar, a non-teleost actinopterygian, and the European eel, a member of an early group of teleosts (elopomorphs). We report the occurrence of three Kissr for the first time in a teleost, the eel. As measured by quantitative RT-PCR, the three eel Kissr were differentially expressed in the brain-pituitary-gonadal axis, and differentially regulated in experimentally matured eels, as compared to prepubertal controls. Subfunctionalisation, as shown by these differences in tissue distribution and regulation, may have represented significant evolutionary constraints for the conservation of multiple Kissr paralogs in this species. Furthermore, we identified four Kissr in both coelacanth and spotted gar genomes, providing the first evidence for the presence of four Kissr in vertebrates. Phylogenetic and syntenic analyses supported the existence of four Kissr paralogs in osteichthyans and allowed to propose a clarified nomenclature of Kissr (Kissr-1 to -4) based on these paralogs. Syntenic analysis suggested that the four Kissr paralogs arose through the two rounds of whole genome duplication (1R and 2R) in early vertebrates, followed by multiple gene loss events in the actinopterygian and sarcopterygian lineages. Due to gene loss there was no impact of the teleost-specific whole genome duplication (3R) on the number of Kissr paralogs in current

  10. Comparative Genome Analysis Provides Insights into the Pathogenicity of Flavobacterium psychrophilum.

    PubMed

    Castillo, Daniel; Christiansen, Rói Hammershaimb; Dalsgaard, Inger; Madsen, Lone; Espejo, Romilio; Middelboe, Mathias

    2016-01-01

    phenotypic properties may provide new insights to the mechanisms of pathogenicity in F. psychrophilum. PMID:27071075

  11. Mitochondrial Genome and Nuclear Markers Provide New Insight into the Evolutionary History of Macaques

    PubMed Central

    Jiang, Juan; Yu, Jianqiu; Li, Jing; Li, Peng; Fan, Zhenxin; Niu, Lili; Deng, Jiabo; Yue, Bisong; Li, Jing

    2016-01-01

    The evolutionary history of macaques, genus Macaca, has been under debate due to the short times of divergence. In this study, maternal, paternal, and biparental genetic systems were applied to infer phylogenetic relationships among macaques and to trace ancient hybridization events in their evolutionary history. Using a PCR display method, 17 newly phylogenetically informative Alu insertions were identified from M. assamensis. We combined presence/absence analysis of 84 Alu elements with mitochondrial genomes as well as nuclear sequences (five autosomal genes, two Y chromosomal genes, and one X chromosomal fragment) to reconstruct a robust macaque phylogeny. Topologies generated from different inherited markers were similar supporting six well defined species groups and a close relationship of M. assamensis and M. thibetana, but differed in the placing of M. arctoides. Both Alu elements and nuclear genes supported that M. arctoides was close to the sinica group, whereas the mitochondrial data clustered it into the fascicularis/mulatta lineage. Our results reveal that a sex-biased hybridization most likely occurred in the evolutionary history of M. arctoides, and suggest an introgressive pattern of male-mediated gene flow from the ancestors of M. arctoides to the M. mulatta population followed by nuclear swamping. According to the estimation of divergence dates, the hybridization occurred around 0.88~1.77 mya (nuclear data) or 1.38~2.56 mya (mitochondrial data). In general, our study indicates that a combination of various molecular markers could help explain complicated evolutionary relationships. Our results have provided new insights into the evolutionary history of macaques and emphasize that hybridization might play an important role in macaque evolution. PMID:27135608

  12. 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

  13. 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

  14. Modelling the self-assembly of elastomeric proteins provides insights into the evolution of their domain architectures.

    PubMed

    Song, Hongyan; Parkinson, John

    2012-01-01

    Elastomeric proteins have evolved independently multiple times through evolution. Produced as monomers, they self-assemble into polymeric structures that impart properties of stretch and recoil. They are composed of an alternating domain architecture of elastomeric domains interspersed with cross-linking elements. While the former provide the elasticity as well as help drive the assembly process, the latter serve to stabilise the polymer. Changes in the number and arrangement of the elastomeric and cross-linking regions have been shown to significantly impact their assembly and mechanical properties. However, to date, such studies are relatively limited. Here we present a theoretical study that examines the impact of domain architecture on polymer assembly and integrity. At the core of this study is a novel simulation environment that uses a model of diffusion limited aggregation to simulate the self-assembly of rod-like particles with alternating domain architectures. Applying the model to different domain architectures, we generate a variety of aggregates which are subsequently analysed by graph-theoretic metrics to predict their structural integrity. Our results show that the relative length and number of elastomeric and cross-linking domains can significantly impact the morphology and structural integrity of the resultant polymeric structure. For example, the most highly connected polymers were those constructed from asymmetric rods consisting of relatively large cross-linking elements interspersed with smaller elastomeric domains. In addition to providing insights into the evolution of elastomeric proteins, simulations such as those presented here may prove valuable for the tuneable design of new molecules that may be exploited as useful biomaterials. PMID:22396636

  15. 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

  16. Quantitative Hydraulic Models Of Early Land Plants Provide Insight Into Middle Paleozoic Terrestrial Paleoenvironmental Conditions

    NASA Astrophysics Data System (ADS)

    Wilson, J. P.; Fischer, W. W.

    2010-12-01

    Fossil plants provide useful proxies of Earth’s climate because plants are closely connected, through physiology and morphology, to the environments in which they lived. Recent advances in quantitative hydraulic models of plant water transport provide new insight into the history of climate by allowing fossils to speak directly to environmental conditions based on preserved internal anatomy. We report results of a quantitative hydraulic model applied to one of the earliest terrestrial plants preserved in three dimensions, the ~396 million-year-old vascular plant Asteroxylon mackei. This model combines equations describing the rate of fluid flow through plant tissues with detailed observations of plant anatomy; this allows quantitative estimates of two critical aspects of plant function. First and foremost, results from these models quantify the supply of water to evaporative surfaces; second, results describe the ability of plant vascular systems to resist tensile damage from extreme environmental events, such as drought or frost. This approach permits quantitative comparisons of functional aspects of Asteroxylon with other extinct and extant plants, informs the quality of plant-based environmental proxies, and provides concrete data that can be input into climate models. Results indicate that despite their small size, water transport cells in Asteroxylon could supply a large volume of water to the plant's leaves--even greater than cells from some later-evolved seed plants. The smallest Asteroxylon tracheids have conductivities exceeding 0.015 m^2 / MPa * s, whereas Paleozoic conifer tracheids do not reach this threshold until they are three times wider. However, this increase in conductivity came at the cost of little to no adaptations for transport safety, placing the plant’s vegetative organs in jeopardy during drought events. Analysis of the thickness-to-span ratio of Asteroxylon’s tracheids suggests that environmental conditions of reduced relative

  17. Neristatin 1 Provides Critical Insight into Bryostatin 1 Structure–Function Relationships

    PubMed Central

    2015-01-01

    Bryostatin 1, a complex macrocyclic lactone isolated from Bugula neritina, has been the subject of multiple clinical trials for cancer. Although it functions as an activator of protein kinase C (PKC) in vitro, bryostatin 1 paradoxically antagonizes most responses to the prototypical PKC activator, the phorbol esters. The bottom half of the bryostatin 1 structure has been shown to be sufficient to confer binding to PKC. In contrast, we have previously shown that the top half of the bryostatin 1 structure is necessary for its unique biological behavior to antagonize phorbol ester responses. Neristatin 1 comprises a top half similar to that of bryostatin 1 together with a distinct bottom half that confers PKC binding. We report here that neristatin 1 is bryostatin 1-like, not phorbol ester-like, in its biological activity on U937 promyelocytic leukemia cells. We conclude that the top half of the bryostatin 1 structure is largely sufficient for bryostatin 1-like activity, provided the molecule also possesses an appropriate PKC binding domain. PMID:25808573

  18. 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.

  19. Phylogenies and traits provide distinct insights about the historical and contemporary assembly of aquatic insect communities.

    PubMed

    Saito, Victor S; Cianciaruso, Marcus Vinicius; Siqueira, Tadeu; Fonseca-Gessner, Alaide A; Pavoine, Sandrine

    2016-05-01

    The assumption that traits and phylogenies can be used as proxies of species niche has faced criticisms. Evidence suggested that phylogenic relatedness is a weak proxy of trait similarity. Moreover, different processes can select different traits, giving opposing signals in null model analyses. To circumvent these criticisms, we separated traits of stream insects based on the concept of α and β niches, which should give clues about assembling pressures expected to act independently of each other. We investigated the congruence between the phylogenetic structure and trait structure of communities using all available traits and all possible combinations of traits (4095 combinations). To account for hierarchical assembling processes, we analyzed patterns on two spatial scales with three pools of genera. Beta niche traits selected a priori - i.e., traits related to environmental variation (e.g., respiration type) - were consistently clustered on the smaller scale, suggesting environmental filtering, while α niche traits - i.e., traits related to resource use (e.g., trophic position) - did not display the expected overdispersion, suggesting a weak role of competition. Using all traits together provided random patterns and the analysis of all possible combinations of traits provided scenarios ranging from strong clustering to overdispersion. Communities were phylogenetically overdispersed, a pattern previously interpreted as phylogenetic limiting similarity. However, our results likely reflect the co-occurrence of ancient clades due to the stability of stream habitats along the evolutionary scale. We advise ecologists to avoid using combinations of all available traits but rather carefully traits based on the objective under consideration. Both trait and phylogenetic approaches should be kept in the ecologist toolbox, but phylogenetic distances should not be used as proxies of traits differences. Although the phylogenetic structure revealed processes operating at the

  20. 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

  1. Characterizing dynamic processes in the Critical Zone: Crazy new tools provide crazy new insights

    NASA Astrophysics Data System (ADS)

    Selker, J. S.; Sayde, C.; Thomas, C. K.

    2015-12-01

    The dynamics of the critical zone are in many aspects poorly understood, resulting largely from the difficulty of observing key processes. Key stores and fluxes are invisible, from the energy budget in air and soil, to the fluxes of water in soils, air and plants. Fortunately we are poised to remove the blinkers, and reveal the spatial and temporal structure of these terms. This will be achieved by novel combinations of observation technologies and high-performance computing. Fiber optic technology allows 1 s measurement of temperature at 0.25 m resolution to 0.1 C. By injecting heat in the fiber, we can quantify stores of soil water, fluxes in soils and boreholes, and movement of the atmosphere. Air-born digital imagining is shown to facilitate cm-level observation of landscapes at the km-scale, as well as the potential to monitor the temporal dynamics of canopy interception. These data can be used to connect specific structures and plant assemblages to key fluxes. Many of these methods are being developed and provided at low cost to the community by the Center for Transformative Environmental Monitoring Programs (CTEMPs.org). A parallel effort, the Open-Source Published Environmental Sensing (OPENS.org) laboratory, provides a forum for the development and publication of user-producible instruments. In all cases the developments are characterized by combining low-cost per data point at hundreds to thousands of locations to provide a comprehensive view of the spacio-temporal dynamics of the crucial zone.

  2. Insight into nucleon structure from lattice calculations of moments of parton and generalized parton distributions

    SciTech Connect

    J.W. Negele; R.C. Brower; P. Dreher; R. Edwards; G. Fleming; Ph. Hagler; U.M. Heller; Th. Lippert; A.V.Pochinsky; D.B. Renner; D. Richards; K. Schilling; W. Schroers

    2004-04-01

    This talk presents recent calculations in full QCD of the lowest three moments of generalized parton distributions and the insight they provide into the behavior of nucleon electromagnetic form factors, the origin of the nucleon spin, and the transverse structure of the nucleon. In addition, new exploratory calculations in the chiral regime of full QCD are discussed.

  3. 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…

  4. High-density interspecific genetic linkage mapping provides insights into genomic incompatibility between channel catfish and blue catfish.

    PubMed

    Liu, S; Li, Y; Qin, Z; Geng, X; Bao, L; Kaltenboeck, L; Kucuktas, H; Dunham, R; Liu, Z

    2016-02-01

    Catfish is the leading aquaculture species in the United States. The interspecific hybrid catfish produced by mating female channel catfish with male blue catfish outperform both of their parent species in a number of traits. However, mass production of the hybrids has been difficult because of reproductive isolation. Investigations of genome structure and organization of the hybrids provide insights into the genetic basis for maintenance of species divergence in the face of gene flow, thereby helping develop strategies for introgression and efficient production of the hybrids for aquaculture. In this study, we constructed a high-density genetic linkage map using the hybrid catfish system with the catfish 250K SNP array. A total of 26,238 SNPs were mapped to 29 linkage groups, with 12,776 unique marker positions. The linkage map spans approximately 3240 cM with an average intermarker distance of 0.25 cM. A fraction of markers (986 of 12,776) exhibited significant deviation from the expected Mendelian ratio of segregation, and they were clustered in major genomic blocks across 15 LGs, most notably LG9 and LG15. The distorted markers exhibited significant bias for maternal alleles among the backcross progenies, suggesting strong selection against the blue catfish alleles. The clustering of distorted markers within genomic blocks should lend insights into speciation as marked by incompatibilities between the two species. Such findings should also have profound implications for understanding the genomic evolution of closely related species as well as the introgression of hybrid production programs in aquaculture. PMID:26537786

  5. Aquatic proteins with repetitive motifs provide insights to bioengineering of novel biomaterials.

    PubMed

    Yang, Yun Jung; Jung, Dooyup; Yang, Byeongseon; Hwang, Byeong Hee; Cha, Hyung Joon

    2014-12-01

    Proteins with repetitive motifs play vital structural and adhesive functions in nature. Some repeat proteins in particular have adapted to harsh aquatic surroundings to support the survival and reproduction of organisms. Significant effort has been made to identify aquatic repeat proteins with attractive properties and functions to be used as novel biomaterials. Examples of such proteins include matrix proteins from pearl oysters, minicollagens from sea anemones, cement proteins from sandcastle worms, and byssal proteins from marine mussels. Here, several repetitive motifs from aquatic proteins are reviewed, and their characteristic properties are linked to practical uses in three aspects of aquatic life: defense, shelter, and attachment. Some repetitive motifs interact with minerals and consequently generate strong outer cover of shells, and some motifs relate with sticky nature, which contribute to organisms' habitation by adhering themselves in harsh aquatic environments. Other motifs, such as silk- or collagen-like motifs, are also involved in structural rigidity as shown in mussel's byssus and egg membrane. Thus, understanding aquatic repetitive motifs will provide clues about biomedical and biotechnological applications of engineered biomaterials in wet environments. PMID:25208823

  6. The channel catfish genome sequence provides insights into the evolution of scale formation in teleosts.

    PubMed

    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

  7. 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

  8. 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

  9. 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. PMID:21194241

  10. 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.

  11. 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 adjustable…

  12. 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

  13. Neuroanatomy of the Vestimentiferan Tubeworm Lamellibrachia satsuma Provides Insights into the Evolution of the Polychaete Nervous System

    PubMed Central

    Miyamoto, Norio; Shinozaki, Ayuta; Fujiwara, Yoshihiro

    2013-01-01

    Vestimentiferan tubeworms are marine invertebrates that inhabit chemosynthetic environments, and although recent molecular phylogenetic analyses have suggested that vestimentiferan tubeworms are derived from polychaete annelids, they show some morphological features that are different from other polychaetes. For example, vestimentiferans lack a digestive tract and have less body segments and comparative neuroanatomy can provide essential insight into the vestimentiferan body plan and its evolution. In the present study, we investigated the adult nervous system in the vestimentiferan Lamellibrachia satsuma using antibodies against synapsin, serotonin, FMRMamide and acetylated α-tubulin. We also examined the expressions of neural marker genes, elav and synaptotagmin to reveal the distribution of neuronal cell bodies. Brain anatomy shows simple organization in Lamellibrachia compared to other polychaetes. This simplification is probably due to the loss of the digestive tract, passing through the body between the brain and the subesophageal ganglion. In contrast, the ventral nerve cord shows a repeated organizational structure as in the other polychaetes, despite the absence of the multiple segmentation of the trunk. These results suggest that the brain anatomy is variable depending on the function and the condition of surrounding tissues, and that the formation of the rope ladder-like nervous system of the ventral nerve cord is independent from segmentation in polychaetes. PMID:23372830

  14. Analysis of the Phlebiopsis gigantea Genome, Transcriptome and Secretome Provides Insight into Its Pioneer Colonization Strategies of Wood

    DOE PAGESBeta

    Hori, Chiaki; Ishida, Takuya; Igarashi, Kiyohiko; Samejima, Masahiro; Suzuki, Hitoshi; Master, Emma; Ferreira, Patricia; Ruiz-Dueñas, Francisco J.; Held, Benjamin; Canessa, Paulo; et al

    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

    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

  16. 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.

  17. Neuroanatomy of the vestimentiferan tubeworm Lamellibrachia satsuma provides insights into the evolution of the polychaete nervous system.

    PubMed

    Miyamoto, Norio; Shinozaki, Ayuta; Fujiwara, Yoshihiro

    2013-01-01

    Vestimentiferan tubeworms are marine invertebrates that inhabit chemosynthetic environments, and although recent molecular phylogenetic analyses have suggested that vestimentiferan tubeworms are derived from polychaete annelids, they show some morphological features that are different from other polychaetes. For example, vestimentiferans lack a digestive tract and have less body segments and comparative neuroanatomy can provide essential insight into the vestimentiferan body plan and its evolution. In the present study, we investigated the adult nervous system in the vestimentiferan Lamellibrachia satsuma using antibodies against synapsin, serotonin, FMRMamide and acetylated α-tubulin. We also examined the expressions of neural marker genes, elav and synaptotagmin to reveal the distribution of neuronal cell bodies. Brain anatomy shows simple organization in Lamellibrachia compared to other polychaetes. This simplification is probably due to the loss of the digestive tract, passing through the body between the brain and the subesophageal ganglion. In contrast, the ventral nerve cord shows a repeated organizational structure as in the other polychaetes, despite the absence of the multiple segmentation of the trunk. These results suggest that the brain anatomy is variable depending on the function and the condition of surrounding tissues, and that the formation of the rope ladder-like nervous system of the ventral nerve cord is independent from segmentation in polychaetes. PMID:23372830

  18. Analysis of the Phlebiopsis gigantea genome, transcriptome and secretome provides insight into its pioneer colonization strategies of wood.

    PubMed

    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

    2014-12-01

    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

  19. Multi-scale Detection of Organic and Inorganic Signatures Provides Insights into Gas Shale Properties and Evolution

    SciTech Connect

    Bernard, S.; Horsfield, B; Schultz, H; Schreiber, A; Wirth, R; Thi AnhVu, T; Perssen, F; Konitzer, S; Volk, H; et. al.

    2010-01-01

    Organic geochemical analyses, including solvent extraction or pyrolysis, followed by gas chromatography and mass spectrometry, are generally conducted on bulk gas shale samples to evaluate their source and reservoir properties. While organic petrology has been directed at unravelling the matrix composition and textures of these economically important unconventional resources, their spatial variability in chemistry and structure is still poorly documented at the sub-micrometre scale. Here, a combination of techniques including transmission electron microscopy and a synchrotron-based microscopy tool, scanning transmission X-ray microscopy, have been used to characterize at a multiple length scale an overmature organic-rich calcareous mudstone from northern Germany. We document multi-scale chemical and mineralogical heterogeneities within the sample, from the millimetre down to the nanometre-scale. From the detection of different types of bitumen and authigenic minerals associated with the organic matter, we show that the multi-scale approach used in this study may provide new insights into gaseous hydrocarbon generation/retention processes occurring within gas shales and may shed new light on their thermal history.

  20. 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

  1. Spider genomes provide insight into composition and evolution of venom and silk.

    PubMed

    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

  2. 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

  3. 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

  4. Insights into the serine protease mechanism from atomic resolution structures of trypsin reaction intermediates

    PubMed Central

    Radisky, Evette S.; Lee, Justin M.; Lu, Chia-Jung Karen; Koshland, Daniel E.

    2006-01-01

    Atomic resolution structures of trypsin acyl-enzymes and a tetrahedral intermediate analog, along with previously solved structures representing the Michaelis complex, are used to reconstruct events in the catalytic cycle of this classic serine protease. Structural comparisons provide insight into active site adjustments involved in catalysis. Subtle motions of the catalytic serine and histidine residues coordinated with translation of the substrate reaction center are seen to favor the forward progress of the acylation reaction. The structures also clarify the attack trajectory of the hydrolytic water in the deacylation reaction. PMID:16636277

  5. 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). PMID:25234435

  6. The locust genome provides insight into swarm formation and long-distance flight

    PubMed Central

    Wang, Xianhui; Fang, Xiaodong; Yang, Pengcheng; Jiang, Xuanting; Jiang, Feng; Zhao, Dejian; Li, Bolei; Cui, Feng; Wei, Jianing; Ma, Chuan; Wang, Yundan; He, Jing; Luo, Yuan; Wang, Zhifeng; Guo, Xiaojiao; Guo, Wei; Wang, Xuesong; Zhang, Yi; Yang, Meiling; Hao, Shuguang; Chen, Bing; Ma, Zongyuan; Yu, Dan; Xiong, Zhiqiang; Zhu, Yabing; Fan, Dingding; Han, Lijuan; Wang, Bo; Chen, Yuanxin; Wang, Junwen; Yang, Lan; Zhao, Wei; Feng, Yue; Chen, Guanxing; Lian, Jinmin; Li, Qiye; Huang, Zhiyong; Yao, Xiaoming; Lv, Na; Zhang, Guojie; Li, Yingrui; Wang, Jian; Wang, Jun; Zhu, Baoli; Kang, Le

    2014-01-01

    Locusts are one of the world’s most destructive agricultural pests and represent a useful model system in entomology. Here we present a draft 6.5 Gb genome sequence of Locusta migratoria, which is the largest animal genome sequenced so far. Our findings indicate that the large genome size of L. migratoria is likely to be because of transposable element proliferation combined with slow rates of loss for these elements. Methylome and transcriptome analyses reveal complex regulatory mechanisms involved in microtubule dynamic-mediated synapse plasticity during phase change. We find significant expansion of gene families associated with energy consumption and detoxification, consistent with long-distance flight capacity and phytophagy. We report hundreds of potential insecticide target genes, including cys-loop ligand-gated ion channels, G-protein-coupled receptors and lethal genes. The L. migratoria genome sequence offers new insights into the biology and sustainable management of this pest species, and will promote its wide use as a model system. PMID:24423660

  7. Fungal evolutionary genomics provides insight into the mechanisms of adaptive divergence in eukaryotes.

    PubMed

    Gladieux, Pierre; Ropars, Jeanne; Badouin, Hélène; Branca, Antoine; Aguileta, Gabriela; de Vienne, Damien M; Rodríguez de la Vega, Ricardo C; Branco, Sara; Giraud, Tatiana

    2014-02-01

    Fungi are ideal model organisms for dissecting the genomic bases of adaptive divergence in eukaryotes. They have simple morphologies and small genomes, occupy contrasting, well-identified ecological niches and tend to have short generation times, and many are amenable to experimental approaches. Fungi also display diverse lifestyles, from saprotrophs to pathogens or mutualists, and they play extremely important roles in both ecosystems and human activities, as wood decayers, mycorrhizal fungi, lichens, endophytes, plant and animal pathogens, and in fermentation or drug production. We review here recent insights into the patterns and mechanisms of adaptive divergence in fungi, including sources of divergence, genomic variation and, ultimately, speciation. We outline the various ecological sources of divergent selection and genomic changes, showing that gene loss and changes in gene expression and in genomic architecture are important adaptation processes, in addition to the more widely recognized processes of amino acid substitution and gene duplication. We also review recent findings regarding the interspecific acquisition of genomic variation and suggesting an important role for introgression, hybridization and horizontal gene transfers (HGTs). We show that transposable elements can mediate several of these genomic changes, thus constituting important factors for adaptation. Finally, we review the consequences of divergent selection in terms of speciation, arguing that genetic incompatibilities may not be as widespread as generally thought and that pleiotropy between adaptation and reproductive isolation is an important route of speciation in fungal pathogens. PMID:24341913

  8. 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

  9. 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. PMID:24486486

  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. PMID:26864641

  11. Structural insights into bacterial recognition of intestinal mucins.

    PubMed

    Etzold, Sabrina; Juge, Nathalie

    2014-10-01

    The mucosal layer covering our gut epithelium represents the first line of host defenses against the luminal content, while enabling contacts between the resident microbiota and the host. Mucus is mainly composed of mucins, large glycoproteins containing a protein core and a high number of O-linked oligosaccharides. Mucin glycans act as binding sites or carbon sources for the intestinal microbes, thereby functioning as a host-specific determinant affecting the microbiota composition and human health. Reflecting the structural diversity of mucin glycans and their prime location, commensal and pathogenic microbes have evolved a range of adhesins allowing their interaction with the host. However, despite the recognised importance of mucin glycans in modulating intestinal homeostasis, information on carbohydrate-binding proteins from gut bacteria is disparate. This review is focussed on recent structural insights into host-microbe interactions mediated by mucins. PMID:25106027

  12. Calorimetric, spectroscopic, and model studies provide insight into the transport of Ti(IV) by human serum transferrin.

    PubMed

    Tinoco, Arthur D; Incarvito, Christopher D; Valentine, Ann M

    2007-03-21

    Evidence suggests that transferrin can bind Ti(IV) in an unhydrolyzed form (without bound hydroxide or oxide) or in a hydrolyzed form. Ti(IV) coordination by N,N'-di(o-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED) at different pH values models the two forms of Ti(IV)-loaded transferrin spectrally and structurally. 13C NMR and stopped-flow kinetic experiments reveal that when the metal is delivered to the protein using an unhydrolyzed source, Ti(IV) can coordinate in the typical distorted octahedral environment with a bound synergistic anion. The crystal structure of TiHBED obtained at low pH models this type of coordination. The solution structure of the complex compares favorably with the solid state from pH 3.0 to 4.0, and the complex can be reduced with E1/2 = -641 mV vs NHE. Kinetic and thermodynamic competition studies at pH 3.0 reveal that Ti(citrate)3 reacts with HBED via a dissociative mechanism and that the stability of TiHBED (log beta = 34.024) is weaker than that of the Fe(III) complex. pH stability studies show that Ti(IV) hydrolyzes ligand waters at higher pH but still remains bound to HBED until pH 9.5. Similarly, at a pH greater than 8.0 the synergistic anion that binds Ti(IV) in transferrin is readily displaced by irreversible metal hydrolysis although the metal remains bound to the protein until pH 9.5. Thermal denaturation studies conducted optically and by differential scanning calorimetry reveal that Ti(IV)-bound transferrin experiences only minimal enhanced thermal stability unlike when Fe(III) is bound. The C- and N-lobe transition Tm values shift to a few degrees higher. The stability, competition, and redox studies performed provide insight into the possible mechanism of Ti2-Tf transport in cells. PMID:17315875

  13. Community proteomics provides functional insight into polyhydroxyalkanoate production by a mixed microbial culture cultivated on fermented dairy manure.

    PubMed

    Hanson, Andrea J; Guho, Nicholas M; Paszczynski, Andrzej J; Coats, Erik R

    2016-09-01

    Polyhydroxyalkanoates (PHAs) are bio-based, biodegradable polyesters that can be produced from organic-rich waste streams using mixed microbial cultures (MMCs). To maximize PHA production, MMCs are enriched for bacteria with a high polymer storage capacity through the application of aerobic dynamic feeding (ADF) in a sequencing batch reactor (SBR), which consequently induces a feast-famine metabolic response. Though the feast-famine response is generally understood empirically at a macro-level, the molecular level is less refined. The objective of this study was to investigate the microbial community composition and proteome profile of an enriched MMC cultivated on fermented dairy manure. The enriched MMC exhibited a feast-famine response and was capable of producing up to 40 % (wt. basis) PHA in a fed-batch reactor. High-throughput 16S rRNA gene sequencing revealed a microbial community dominated by Meganema, a known PHA-producing genus not often observed in high abundance in enrichment SBRs. The application of the proteomic methods two-dimensional electrophoresis and LC-MS/MS revealed PHA synthesis, energy generation, and protein synthesis prominently occurring during the feast phase, corroborating bulk solution variable observations and theoretical expectations. During the famine phase, nutrient transport, acyl-CoA metabolism, additional energy generation, and housekeeping functions were more pronounced, informing previously under-determined MMC functionality under famine conditions. During fed-batch PHA production, acetyl-CoA acetyltransferase and PHA granule-bound phasin proteins were in increased abundance relative to the SBR, supporting the higher PHA content observed. Collectively, the results provide unique microbial community structural and functional insight into feast-famine PHA production from waste feedstocks using MMCs. PMID:27147532

  14. 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. PMID:26976571

  15. 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

  16. 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

  17. Macrotene chromosomes provide insights to a new mechanism of high-order gene amplification in eukaryotes

    PubMed Central

    Thierry, Agnès; Khanna, Varun; Créno, Sophie; Lafontaine, Ingrid; Ma, Laurence; Bouchier, Christiane; Dujon, Bernard

    2015-01-01

    Copy number variation of chromosomal segments is now recognized as a major source of genetic polymorphism within natural populations of eukaryotes, as well as a possible cause of genetic diseases in humans, including cancer, but its molecular bases remain incompletely understood. In the baker’s yeast Saccharomyces cerevisiae, a variety of low-order amplifications (segmental duplications) were observed after adaptation to limiting environmental conditions or recovery from gene dosage imbalance, and interpreted in terms of replication-based mechanisms associated or not with homologous recombination. Here we show the emergence of novel high-order amplification structures, with corresponding overexpression of embedded genes, during evolution under favourable growth conditions of severely unfit yeast cells bearing genetically disabled genomes. Such events form massively extended chromosomes, which we propose to call macrotene, whose characteristics suggest the products of intrachromosomal rolling-circle type of replication structures, probably initiated by increased accidental template switches under important cellular stress conditions. PMID:25635677

  18. Reconstitution of the augmin complex provides insights into its architecture and function.

    PubMed

    Hsia, Kuo-Chiang; Wilson-Kubalek, Elizabeth M; Dottore, Alejandro; Hao, Qi; Tsai, Kuang-Lei; Forth, Scott; Shimamoto, Yuta; Milligan, Ronald A; Kapoor, Tarun M

    2014-09-01

    Proper microtubule nucleation during cell division requires augmin, a microtubule-associated hetero-octameric protein complex. In current models, augmin recruits γ-tubulin, through the carboxyl terminus of its hDgt6 subunit 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 that 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 with octameric complexes. Together, our findings shed light on augmin's structural organization and microtubule-binding properties, and define subunits required for its function in organizing microtubule-based structures. PMID:25173975

  19. Human evolutionary history and contemporary evolutionary theory provide insight when assessing cultural group selection.

    PubMed

    Fuentes, Agustin; Kissel, Marc

    2016-01-01

    Richerson et al. provide a much needed roadmap for assessing cultural group selection (CGS) theory and for applying it to understanding variation between contemporary human groups. However, the current proposal lacks connection to relevant evidence from the human evolutionary record and requires a better integration with contemporary evolutionary theory. The article also misapplies the F st statistic. PMID:27562510

  20. Neotenic Phenotype and Sex Ratios Provide Insight into Developmental Pathways in Reticulitermes flavipes (Isoptera: Rhinotermitidae)

    PubMed Central

    Hu, Jian; Forschler, Brian T.

    2012-01-01

    Several thousand Reticulitermes flavipes (Kollar) including worker, nymph, soldier, neotenic and alate castes were collected from three pine logs brought into the laboratory on dates five years apart. The neotenics, all nymphoid, were divided into three groups based on the extent of cuticle pigmentation and termed regular neotenics (RN), black-headed neotenics (BHN) or black neotenics (BN). All castes, from Log A, in 2008, provided a neutral sex ratio except BHN (N = 378) and BN (N = 51) which were exclusively male while the soldiers (N = 466) were female-biased. This information suggests that there is a sex-linked bifurcation along the path for termite development with a male-biased neotenic or female-biased soldier as the choice. In contrast, termites collected in 2004 from Log B provided sex ratios that included a female biased RN (N = 1017), a neutral soldier (N = 258) and male biased BHN (N = 99) and workers (N = 54). Log C, collected in 2009, provided female biased soldiers (N = 32), RNs (N = 18) and BHNs (N = 4) and only male BN (N = 5). Eight laboratory cultures, ranging in age from five to 14 years old, also were sampled and all castes sexed. The census included a 14-year old queen-right colony, an 8-year old polyandrous colony and six colonies provided nymphs and male-biased worker populations. Together these data indicate a flexible caste determination system providing a unique opportunity for a better understanding of the flexible developmental options available in R. flavipes that we discuss relative to the literature on Reticulitermes ontogeny. PMID:26466543

  1. 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

  2. Structural insights into the architecture of the hyperthermophilic Fusellovirus SSV1.

    PubMed

    Stedman, Kenneth M; DeYoung, Melissa; Saha, Mitul; Sherman, Michael B; Morais, Marc C

    2015-01-01

    The structure and assembly of many icosahedral and helical viruses are well-characterized. However, the molecular basis for the unique spindle-shaped morphology of many viruses that infect Archaea remains unknown. To understand the architecture and assembly of these viruses, the spindle-shaped virus SSV1 was examined using cryo-EM, providing the first 3D-structure of a spindle-shaped virus as well as insight into SSV1 biology, assembly and evolution. Furthermore, a geometric framework underlying the distinct spindle-shaped structure is proposed. PMID:25463608

  3. Mudskipper genomes provide insights into the terrestrial adaptation of amphibious fishes

    PubMed Central

    You, Xinxin; Bian, Chao; Zan, Qijie; Xu, Xun; Liu, Xin; Chen, Jieming; Wang, Jintu; Qiu, Ying; Li, Wujiao; Zhang, Xinhui; Sun, Ying; Chen, Shixi; Hong, Wanshu; Li, Yuxiang; Cheng, Shifeng; Fan, Guangyi; Shi, Chengcheng; Liang, Jie; Tom Tang, Y.; Yang, Chengye; Ruan, Zhiqiang; Bai, Jie; Peng, Chao; Mu, Qian; Lu, Jun; Fan, Mingjun; Yang, Shuang; Huang, Zhiyong; Jiang, Xuanting; Fang, Xiaodong; Zhang, Guojie; Zhang, Yong; Polgar, Gianluca; Yu, Hui; Li, Jia; Liu, Zhongjian; Zhang, Guoqiang; Ravi, Vydianathan; Coon, Steven L.; Wang, Jian; Yang, Huanming; Venkatesh, Byrappa; Wang, Jun; Shi, Qiong

    2014-01-01

    Mudskippers are amphibious fishes that have developed morphological and physiological adaptations to match their unique lifestyles. Here we perform whole-genome sequencing of four representative mudskippers to elucidate the molecular mechanisms underlying these adaptations. We discover an expansion of innate immune system genes in the mudskippers that may provide defence against terrestrial pathogens. Several genes of the ammonia excretion pathway in the gills have experienced positive selection, suggesting their important roles in mudskippers’ tolerance to environmental ammonia. Some vision-related genes are differentially lost or mutated, illustrating genomic changes associated with aerial vision. Transcriptomic analyses of mudskippers exposed to air highlight regulatory pathways that are up- or down-regulated in response to hypoxia. The present study provides a valuable resource for understanding the molecular mechanisms underlying water-to-land transition of vertebrates. PMID:25463417

  4. Mudskipper genomes provide insights into the terrestrial adaptation of amphibious fishes.

    PubMed

    You, Xinxin; Bian, Chao; Zan, Qijie; Xu, Xun; Liu, Xin; Chen, Jieming; Wang, Jintu; Qiu, Ying; Li, Wujiao; Zhang, Xinhui; Sun, Ying; Chen, Shixi; Hong, Wanshu; Li, Yuxiang; Cheng, Shifeng; Fan, Guangyi; Shi, Chengcheng; Liang, Jie; Tom Tang, Y; Yang, Chengye; Ruan, Zhiqiang; Bai, Jie; Peng, Chao; Mu, Qian; Lu, Jun; Fan, Mingjun; Yang, Shuang; Huang, Zhiyong; Jiang, Xuanting; Fang, Xiaodong; Zhang, Guojie; Zhang, Yong; Polgar, Gianluca; Yu, Hui; Li, Jia; Liu, Zhongjian; Zhang, Guoqiang; Ravi, Vydianathan; Coon, Steven L; Wang, Jian; Yang, Huanming; Venkatesh, Byrappa; Wang, Jun; Shi, Qiong

    2014-01-01

    Mudskippers are amphibious fishes that have developed morphological and physiological adaptations to match their unique lifestyles. Here we perform whole-genome sequencing of four representative mudskippers to elucidate the molecular mechanisms underlying these adaptations. We discover an expansion of innate immune system genes in the mudskippers that may provide defence against terrestrial pathogens. Several genes of the ammonia excretion pathway in the gills have experienced positive selection, suggesting their important roles in mudskippers' tolerance to environmental ammonia. Some vision-related genes are differentially lost or mutated, illustrating genomic changes associated with aerial vision. Transcriptomic analyses of mudskippers exposed to air highlight regulatory pathways that are up- or down-regulated in response to hypoxia. The present study provides a valuable resource for understanding the molecular mechanisms underlying water-to-land transition of vertebrates. PMID:25463417

  5. Molecular dynamics simulations provide insights into the substrate specificity of FAOX family members.

    PubMed

    Rigoldi, Federica; Spero, Ludovica; Dalle Vedove, Andrea; Redaelli, Alberto; Parisini, Emilio; Gautieri, Alfonso

    2016-07-19

    Enzymatic assays based on Fructosyl Amino Acid Oxidases (FAOX) represent a potential, rapid and economical strategy to measure glycated hemoglobin (HbA1c), which is in turn a reliable method to monitor the insurgence and the development of diabetes mellitus. However, the engineering of naturally occurring FAOX to specifically recognize fructosyl-valine (the glycated N-terminal residue of HbA1c) has been hindered by the paucity of information on the tridimensional structures and catalytic residues of the different FAOX that exist in nature, and in general on the molecular mechanisms that regulate specificity in this class of enzymes. In this study, we use molecular dynamics simulations and advanced modeling techniques to investigate five different relevant wild-type FAOX (Amadoriase I, Amadoriase II, PnFPOX, FPOX-E and N1-1-FAOD) in order to elucidate the molecular mechanisms that drive their specificity towards polar and nonpolar substrates. Specifically, we compare these five different FAOX in terms of overall folding, ligand entry tunnels, ligand binding residues and ligand binding energies. Our work will contribute to future enzyme structure modifications aimed at the rational design of novel biosensors for the monitoring of blood glucose levels. PMID:27327839

  6. Electrochemical studies of capping agent adsorption provide insight into the formation of anisotropic gold nanocrystals.

    PubMed

    Danger, Brook R; Fan, Donna; Vivek, J P; Burgess, Ian J

    2012-12-21

    The ability of the 4-dimethylaminopyridine (DMAP) to stabilize and control the formation of anisotropic gold nanocrystals produced via the borohydride reduction of gold(III) salts is reported here. Electrochemical measurements of DMAP electrosorption on different low-index single crystal and polycrystalline electrodes is provided and shows a propensity for DMAP to preferentially adsorb on {100} facets. Measuring the electrochemical potential during nanocrystal formation shows that experimental conditions can easily be manipulated so that the growth of nanoseeds occurs at potentials that support preferential DMAP adsorption on {100} surfaces giving rise to highly anisotropic nanocrystals (nanorods, bipyramids, and nanopods). Nanopods with nearly 50 nm arm lengths are shown to form and produce a surface plasmon mode that extends well into the near IR (λ(max) ≈ 1350 nm). Evidence is provided of the slow, partial reduction of tetrachloroaurate to a DMAP stabilized Au(I) species. Shape control is achieved simply by varying the length of time, τ, that DMAP is allowed to partially reduce the Au(III) ions prior to the addition of the strong reducing agent, NaBH(4). Thus the role of DMAP in producing anisotropic particle shapes is shown to be multifunctional. A mechanism accounting for the dependence of particle shape on τ is provided. PMID:23186041

  7. Structural plasticity of Cid1 provides a basis for its distributive RNA terminal uridylyl transferase activity.

    PubMed

    Yates, Luke A; Durrant, Benjamin P; Fleurdépine, Sophie; Harlos, Karl; Norbury, Chris J; Gilbert, Robert J C

    2015-03-11

    Terminal uridylyl transferases (TUTs) are responsible for the post-transcriptional addition of uridyl residues to RNA 3' ends, leading in some cases to altered stability. The Schizosaccharomyces pombe TUT Cid1 is a model enzyme that has been characterized structurally at moderate resolution and provides insights into the larger and more complex mammalian TUTs, ZCCHC6 and ZCCHC11. Here, we report a higher resolution (1.74 Å) crystal structure of Cid1 that provides detailed evidence for uracil selection via the dynamic flipping of a single histidine residue. We also describe a novel closed conformation of the enzyme that may represent an intermediate stage in a proposed product ejection mechanism. The structural insights gained, combined with normal mode analysis and biochemical studies, demonstrate that the plasticity of Cid1, particularly about a hinge region (N164-N165), is essential for catalytic activity, and provide an explanation for its distributive uridylyl transferase activity. We propose a model clarifying observed differences between the in vitro apparently processive activity and in vivo distributive monouridylylation activity of Cid1. We suggest that modulating the flexibility of such enzymes-for example by the binding of protein co-factors-may allow them alternatively to add single or multiple uridyl residues to the 3' termini of RNA molecules. PMID:25712096

  8. Structural plasticity of Cid1 provides a basis for its distributive RNA terminal uridylyl transferase activity

    PubMed Central

    Yates, Luke A.; Durrant, Benjamin P.; Fleurdépine, Sophie; Harlos, Karl; Norbury, Chris J.; Gilbert, Robert J.C.

    2015-01-01

    Terminal uridylyl transferases (TUTs) are responsible for the post-transcriptional addition of uridyl residues to RNA 3′ ends, leading in some cases to altered stability. The Schizosaccharomyces pombe TUT Cid1 is a model enzyme that has been characterized structurally at moderate resolution and provides insights into the larger and more complex mammalian TUTs, ZCCHC6 and ZCCHC11. Here, we report a higher resolution (1.74 Å) crystal structure of Cid1 that provides detailed evidence for uracil selection via the dynamic flipping of a single histidine residue. We also describe a novel closed conformation of the enzyme that may represent an intermediate stage in a proposed product ejection mechanism. The structural insights gained, combined with normal mode analysis and biochemical studies, demonstrate that the plasticity of Cid1, particularly about a hinge region (N164–N165), is essential for catalytic activity, and provide an explanation for its distributive uridylyl transferase activity. We propose a model clarifying observed differences between the in vitro apparently processive activity and in vivo distributive monouridylylation activity of Cid1. We suggest that modulating the flexibility of such enzymes—for example by the binding of protein co-factors—may allow them alternatively to add single or multiple uridyl residues to the 3′ termini of RNA molecules. PMID:25712096

  9. 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

  10. Structural insights into SAM domain-mediated tankyrase oligomerization.

    PubMed

    DaRosa, Paul A; Ovchinnikov, Sergey; Xu, Wenqing; Klevit, Rachel E

    2016-09-01

    Tankyrase 1 (TNKS1; a.k.a. ARTD5) and tankyrase 2 (TNKS2; a.k.a ARTD6) are highly homologous poly(ADP-ribose) polymerases (PARPs) that function in a wide variety of cellular processes including Wnt signaling, Src signaling, Akt signaling, Glut4 vesicle translocation, telomere length regulation, and centriole and spindle pole maturation. Tankyrase proteins include a sterile alpha motif (SAM) domain that undergoes oligomerization in vitro and in vivo. However, the SAM domains of TNKS1 and TNKS2 have not been structurally characterized and the mode of oligomerization is not yet defined. Here we model the SAM domain-mediated oligomerization of tankyrase. The structural model, supported by mutagenesis and NMR analysis, demonstrates a helical, homotypic head-to-tail polymer that facilitates TNKS self-association. Furthermore, we show that TNKS1 and TNKS2 can form (TNKS1 SAM-TNKS2 SAM) hetero-oligomeric structures mediated by their SAM domains. Though wild-type tankyrase proteins have very low solubility, model-based mutations of the SAM oligomerization interface residues allowed us to obtain soluble TNKS proteins. These structural insights will be invaluable for the functional and biophysical characterization of TNKS1/2, including the role of TNKS oligomerization in protein poly(ADP-ribosyl)ation (PARylation) and PARylation-dependent ubiquitylation. PMID:27328430

  11. 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. PMID:22498975

  12. 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

  13. 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

  14. 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

  15. Cambrian lobopodians and extant onychophorans provide new insights into early cephalization in Panarthropoda

    PubMed Central

    Ou, Qiang; Shu, Degan; Mayer, Georg

    2012-01-01

    Cambrian lobopodians are important for understanding the evolution of arthropods, but despite their soft-bodied preservation, the organization of the cephalic region remains obscure. Here we describe new material of the early Cambrian lobopodian Onychodictyon ferox from southern China, which reveals hitherto unknown head structures. These include a proboscis with a terminal mouth, an anterior arcuate sclerite, a pair of ocellus-like eyes and branched, antenniform appendages associated with this ocular segment. These findings, combined with a comparison with other lobopodians, suggest that the head of the last common ancestor of fossil lobopodians and extant panarthropods comprized a single ocular segment with a proboscis and terminal mouth. The lack of specialized mouthparts in O. ferox and the involvement of non-homologous mouthparts in onychophorans, tardigrades and arthropods argue against a common origin of definitive mouth openings among panarthropods, whereas the embryonic stomodaeum might well be homologous at least in Onychophora and Arthropoda. PMID:23232391

  16. 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.

  17. Cambrian lobopodians and extant onychophorans provide new insights into early cephalization in Panarthropoda.

    PubMed

    Ou, Qiang; Shu, Degan; Mayer, Georg

    2012-01-01

    Cambrian lobopodians are important for understanding the evolution of arthropods, but despite their soft-bodied preservation, the organization of the cephalic region remains obscure. Here we describe new material of the early Cambrian lobopodian Onychodictyon ferox from southern China, which reveals hitherto unknown head structures. These include a proboscis with a terminal mouth, an anterior arcuate sclerite, a pair of ocellus-like eyes and branched, antenniform appendages associated with this ocular segment. These findings, combined with a comparison with other lobopodians, suggest that the head of the last common ancestor of fossil lobopodians and extant panarthropods comprized a single ocular segment with a proboscis and terminal mouth. The lack of specialized mouthparts in O. ferox and the involvement of non-homologous mouthparts in onychophorans, tardigrades and arthropods argue against a common origin of definitive mouth openings among panarthropods, whereas the embryonic stomodaeum might well be homologous at least in Onychophora and Arthropoda. PMID:23232391

  18. A Devonian predatory fish provides insights into the early evolution of modern sarcopterygians.

    PubMed

    Lu, Jing; Zhu, Min; Ahlberg, Per Erik; Qiao, Tuo; Zhu, You'an; Zhao, Wenjin; Jia, Liantao

    2016-06-01

    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

  19. 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.

  20. Insights into HLA-G Genetics Provided by Worldwide Haplotype Diversity

    PubMed Central

    Castelli, Erick C.; Ramalho, Jaqueline; Porto, Iane O. P.; Lima, Thálitta H. A.; Felício, Leandro P.; Sabbagh, Audrey; Donadi, Eduardo A.; Mendes-Junior, Celso T.

    2014-01-01

    Human leukocyte antigen G (HLA-G) belongs to the family of non-classical HLA class I genes, located within the major histocompatibility complex (MHC). HLA-G has been the target of most recent research regarding the function of class I non-classical genes. The main features that distinguish HLA-G from classical class I genes are (a) limited protein variability, (b) alternative splicing generating several membrane bound and soluble isoforms, (c) short cytoplasmic tail, (d) modulation of immune response (immune tolerance), and (e) restricted expression to certain tissues. In the present work, we describe the HLA-G gene structure and address the HLA-G variability and haplotype diversity among several populations around the world, considering each of its major segments [promoter, coding, and 3′ untranslated region (UTR)]. For this purpose, we developed a pipeline to reevaluate the 1000Genomes data and recover miscalled or missing genotypes and haplotypes. It became clear that the overall structure of the HLA-G molecule has been maintained during the evolutionary process and that most of the variation sites found in the HLA-G coding region are either coding synonymous or intronic mutations. In addition, only a few frequent and divergent extended haplotypes are found when the promoter, coding, and 3′UTRs are evaluated together. The divergence is particularly evident for the regulatory regions. The population comparisons confirmed that most of the HLA-G variability has originated before human dispersion from Africa and that the allele and haplotype frequencies have probably been shaped by strong selective pressures. PMID:25339953

  1. Analysis of Oligomerization Properties of Heme a Synthase Provides Insights into Its Function in Eukaryotes.

    PubMed

    Swenson, Samantha; Cannon, Andrew; Harris, Nicholas J; Taylor, Nicholas G; Fox, Jennifer L; Khalimonchuk, Oleh

    2016-05-01

    Heme a is an essential cofactor for function of cytochrome c oxidase in the mitochondrial electron transport chain. Several evolutionarily conserved enzymes have been implicated in the biosynthesis of heme a, including the heme a synthase Cox15. However, the structure of Cox15 is unknown, its enzymatic mechanism and the role of active site residues remain debated, and recent discoveries suggest additional chaperone-like roles for this enzyme. Here, we investigated Cox15 in the model eukaryote Saccharomyces cerevisiae via several approaches to examine its oligomeric states and determine the effects of active site and human pathogenic mutations. Our results indicate that Cox15 exhibits homotypic interactions, forming highly stable complexes dependent upon hydrophobic interactions. This multimerization is evolutionarily conserved and independent of heme levels and heme a synthase catalytic activity. Four conserved histidine residues are demonstrated to be critical for eukaryotic heme a synthase activity and cannot be substituted with other heme-ligating amino acids. The 20-residue linker region connecting the two conserved domains of Cox15 is also important; removal of this linker impairs both Cox15 multimerization and enzymatic activity. Mutations of COX15 causing single amino acid conversions associated with fatal infantile hypertrophic cardiomyopathy and the neurological disorder Leigh syndrome result in impaired stability (S344P) or catalytic function (R217W), and the latter mutation affects oligomeric properties of the enzyme. Structural modeling of Cox15 suggests these two mutations affect protein folding and heme binding, respectively. We conclude that Cox15 multimerization is important for heme a biosynthesis and/or transfer to maturing cytochrome c oxidase. PMID:26940873

  2. Fluorescently labeled bacteria provide insight on post-mortem microbial transmigration.

    PubMed

    Burcham, Z M; Hood, J A; Pechal, J L; Krausz, K L; Bose, J L; Schmidt, C J; Benbow, M E; Jordan, H R

    2016-07-01

    Microbially mediated mechanisms of human decomposition begin immediately after death, and are a driving force for the conversion of a once living organism to a resource of energy and nutrients. Little is known about post-mortem microbiology in cadavers, particularly the community structure of microflora residing within the cadaver and the dynamics of these communities during decomposition. Recent work suggests these bacterial communities undergo taxa turnover and shifts in community composition throughout the post-mortem interval. In this paper we describe how the microbiome of a living host changes and transmigrates within the body after death thus linking the microbiome of a living individual to post-mortem microbiome changes. These differences in the human post-mortem from the ante-mortem microbiome have demonstrated promise as evidence in death investigations. We investigated the post-mortem structure and function dynamics of Staphylococcus aureus and Clostridium perfringens after intranasal inoculation in the animal model Mus musculus L. (mouse) to identify how transmigration of bacterial species can potentially aid in post-mortem interval estimations. S. aureus was tracked using in vivo and in vitro imaging to determine colonization routes associated with different physiological events of host decomposition, while C. perfringens was tracked using culture-based techniques. Samples were collected at discrete time intervals associated with various physiological events and host decomposition beginning at 1h and ending at 60 days post-mortem. Results suggest that S. aureus reaches its highest concentration at 5-7 days post-mortem then begins to rapidly decrease and is undetectable by culture on day 30. The ability to track these organisms as they move in to once considered sterile space may be useful for sampling during autopsy to aid in determining post-mortem interval range estimations, cause of death, and origins associated with the geographic location of human

  3. 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. PMID:27037218

  4. 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

  5. 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

  6. Stable isotopes provide new insights into vestimentiferan physiological ecology at Gulf of Mexico cold seeps

    NASA Astrophysics Data System (ADS)

    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 δ13C, δ15N, and δ34S 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 δ13C 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. δ34S values were extremely variable among individuals of the same species within one location (<1 m2 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 δ15N 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.

  7. 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

  8. 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

    2015-09-01

    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

  9. 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. PMID:18662752

  10. Studies of alternative isoforms provide insight into TDP-43 autoregulation and pathogenesis

    PubMed Central

    D'Alton, Simon; Altshuler, Marcelle; Lewis, Jada

    2015-01-01

    TDP-43 is a soluble, nuclear protein that undergoes cytoplasmic redistribution and aggregation in the majority of cases of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. TDP-43 autoregulates the abundance of its own transcript TARDBP by binding to an intron in the 3′ untranslated region, although the mechanisms underlying this activity have been debated. Herein, we provide the most extensive analysis of TARDBP transcript yet undertaken. We detail the existence of a plethora of complex splicing events and alternative poly(A) use and provide data that explain the discrepancies reported to date regarding the autoregulatory capacity of TDP-43. Additionally, although many splice isoforms emanating from the TARDBP locus contain the regulated intron in the 3′ UTR, we find only evidence for autoregulation of the transcript encoding full-length TDP-43. Finally, we use a novel cytoplasmic isoform of TDP to induce disease-like loss of soluble, nuclear TDP-43, which results in aberrant splicing and up-regulation of endogenous TARDBP. These results reveal a previously underappreciated complexity to TDP-43 regulated splicing and suggest that loss of TDP-43 autoregulatory capacity may contribute to the pathogenesis of ALS. PMID:26089325

  11. 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. PMID:26676690

  12. Structural insights into transcription initiation by RNA polymerase II

    PubMed Central

    Grünberg, Sebastian; Hahn, Steven

    2013-01-01

    Transcriptional regulation is one of the most important steps in control of cell identity, growth, differentiation and development. Many signaling pathways controlling these processes ultimately target the core transcription machinery that, for protein coding genes, consists of RNA polymerase II (Pol II) and the general transcription factors (GTFs). New studies on the structure and mechanism of the core assembly and how it interfaces with promoter DNA and coactivator complexes have given tremendous insight into early steps in the initiation process, genome-wide binding, and mechanisms conserved for all nuclear and archaeal Pols. Here we review recent developments in dissecting the architecture of the Pol II core machinery with a focus on early and regulated steps in transcription initiation. PMID:24120742

  13. 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.

  14. Advanced electron microscopic techniques provide a deeper insight into the peculiar features of podocytes.

    PubMed

    Burghardt, Tillmann; Hochapfel, Florian; Salecker, Benjamin; Meese, Christine; Gröne, Hermann-Josef; Rachel, Reinhard; Wanner, Gerhard; Krahn, Michael P; Witzgall, Ralph

    2015-12-15

    Podocytes constitute the outer layer of the glomerular filtration barrier, where they form an intricate network of interdigitating foot processes which are connected by slit diaphragms. A hitherto unanswered puzzle concerns the question of whether slit diaphragms are established between foot processes of the same podocyte or between foot processes of different podocytes. By employing focused ion beam-scanning electron microscopy (FIB-SEM), we provide unequivocal evidence that slit diaphragms are formed between foot processes of different podocytes. We extended our investigations of the filtration slit by using dual-axis electron tomography of human and mouse podocytes as well as of Drosophila melanogaster nephrocytes. Using this technique, we not only find a single slit diaphragm which spans the filtration slit around the whole periphery of the foot processes but additional punctate filamentous contacts between adjacent foot processes. Future work will be necessary to determine the proteins constituting the two types of cell-cell contacts. PMID:26400546

  15. Molecular taxonomy provides new insights into anopheles species of the neotropical arribalzagia series.

    PubMed

    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

  16. 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

  17. Molecular developmental mechanism in polypterid fish provides insight into the origin of vertebrate lungs.

    PubMed

    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

  18. Mouse tetrad analysis provides insights into recombination mechanisms and hotspot evolutionary dynamics

    PubMed Central

    Cole, Francesca; Baudat, Frédéric; Grey, Corinne; Keeney, Scott; de Massy, Bernard; Jasin, Maria

    2014-01-01

    The ability to examine all chromatids from a single meiosis in yeast tetrads has been indispensable for defining 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 conversions, 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. PMID:25151354

  19. 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

  20. Integration-dependent bacteriophage immunity provides insights into the evolution of genetic switches

    PubMed Central

    Broussard, Gregory W.; Oldfield, Lauren M.; Villanueva, Valerie M.; Lunt, Bryce L.; Shine, Emilee E.; Hatfull, Graham F.

    2012-01-01

    SUMMARY Genetic switches are critical components of developmental circuits. Because temperate bacteriophages are vastly abundant and greatly diverse, they are rich resources for understanding the mechanisms and evolution of switches and the molecular control of genetic circuitry. Here, we describe a new class of small, compact, and simple switches that use site-specific recombination as the key decision point. The phage attachment site attP is located within the phage repressor gene such that chromosomal integration results in removal of a C-terminal tag that destabilizes the virally-encoded form of the repressor. Integration thus not only confers prophage stability, but also is a requirement for lysogenic establishment. The variety of these self-contained integration-dependent immunity systems in different genomic contexts suggests that these represent ancestral states in switch evolution from which more complex switches have evolved. They also provide a powerful toolkit for building synthetic biological circuits. PMID:23246436

  1. The Opisthorchis viverrini genome provides insights into life in the bile duct

    PubMed Central

    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.

    2014-01-01

    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. PMID:25007141

  2. Establishing a leaf proteome reference map for Ginkgo biloba provides insight into potential ethnobotanical uses.

    PubMed

    Uvackova, Lubica; Ondruskova, Emilia; Danchenko, Maksym; Skultety, Ludovit; Miernyk, Ján A; Hrubík, Pavel; Hajduch, Martin

    2014-11-26

    Although ginkgo (Maidenhair tree, Ginkgo biloba L.) is an ancient medicinal and ornamental tree, there has not previously been any systematic proteomic study of the leaves. Herein we describe results from the initial study identifying abundant ginkgo leaf proteins and present a gel reference map. Proteins were isolated from fully developed mature leaves in biological triplicate and analyzed by two-dimensional electrophoresis plus tandem mass spectrometry. Using this approach, we were able to reproducibly quantify 190 abundant protein spots, from which 157 proteins were identified. Most of identified proteins are associated with the energy and protein destination/storage categories. The reference map provides a basis for understanding the accumulation of flavonoids and other phenolic compounds in mature leaves (e.g., identification of chalcone synthase, the first committed enzyme in flavonoid biosynthesis). We additionally detected several proteins of as yet unknown function. These proteins comprise a pool of potential targets that might be useful in nontraditional medical applications. PMID:25365400

  3. The Mechanism of Variegation in immutans Provides Insight into Chloroplast Biogenesis

    PubMed Central

    Foudree, Andrew; Putarjunan, Aarthi; Kambakam, Sekhar; Nolan, Trevor; Fussell, Jenna; Pogorelko, Gennady; Rodermel, Steve

    2012-01-01

    The immutans (im) variegation mutant of Arabidopsis has green and white-sectored leaves due to the absence of fully functional plastid terminal oxidase (PTOX), a plastoquinol oxidase in thylakoid membranes. PTOX appears to be at the nexus of a growing number of biochemical pathways in the plastid, including carotenoid biosynthesis, PSI cyclic electron flow, and chlororespiration. During the early steps of chloroplast biogenesis, PTOX serves as an alternate electron sink and is a prime determinant of the redox poise of the developing photosynthetic apparatus. Whereas a lack of PTOX causes the formation of photooxidized plastids in the white sectors of im, compensating mechanisms allow the green sectors to escape the effects of the mutation. This manuscript provides an update on PTOX, the mechanism of im variegation, and findings about im compensatory mechanisms. PMID:23205022

  4. Integration-dependent bacteriophage immunity provides insights into the evolution of genetic switches.

    PubMed

    Broussard, Gregory W; Oldfield, Lauren M; Villanueva, Valerie M; Lunt, Bryce L; Shine, Emilee E; Hatfull, Graham F

    2013-01-24

    Genetic switches are critical components of developmental circuits. Because temperate bacteriophages are vastly abundant and greatly diverse, they are rich resources for understanding the mechanisms and evolution of switches and the molecular control of genetic circuitry. Here, we describe a new class of small, compact, and simple switches that use site-specific recombination as the key decision point. The phage attachment site attP is located within the phage repressor gene such that chromosomal integration results in removal of a C-terminal tag that destabilizes the virally encoded form of the repressor. Integration thus not only confers prophage stability but also is a requirement for lysogenic establishment. The variety of these self-contained integration-dependent immunity systems in different genomic contexts suggests that these represent ancestral states in switch evolution from which more-complex switches have evolved. They also provide a powerful toolkit for building synthetic biological circuits. PMID:23246436

  5. 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

  6. 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. PMID:25015646

  7. 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.

  8. High Resolution Muscle Measurements Provide Insights into Equinus Contractures in Patients with Cerebral Palsy

    PubMed Central

    Mathewson, Margie A.; Ward, Samuel R.; Chambers, Henry G.; Lieber, Richard L.

    2015-01-01

    Muscle contractures that occur after upper motor neuron lesion are often surgically released or lengthened. However, surgical manipulation of muscle length changes a muscle’s sarcomere length (Ls), which can affect force production. To predict effects of surgery, both macro- (fascicle length (Lf)) and micro- (Ls) level structural measurements are needed. Therefore, the purpose of this study was to quantify both Ls and Lf in patients with cerebral palsy (CP) as well as typically developing (TD) children. Soleus ultrasound images were obtained from children with CP and TD children. Lf was determined and, with the joint in the same position, CP biopsies were obtained, formalin fixed and Ls measured by laser diffraction. Since soleus Ls values were not measurable in TD children, TD Ls values were obtained using three independent methods. While average Lf did not differ between groups (CP=3.6±1.2 cm, TD=3.5±0.9 cm; p>0.6), Ls was dramatically longer in children with CP (4.07±0.45 μm vs. TD=2.17±0.24 μm; p<0.0001). While Lf values were similar between children with CP and TD children, this was due to highly stretched sarcomeres within the soleus muscle. Surgical manipulation of muscle-tendon unit length will thus alter muscle sarcomere length and change force generating capacity of the muscle. PMID:25242618

  9. New specimen of Archaeopteryx provides insights into the evolution of pennaceous feathers.

    PubMed

    Foth, Christian; Tischlinger, Helmut; Rauhut, Oliver W M

    2014-07-01

    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. PMID:24990749

  10. Comparative Genomic Analyses of Streptococcus pseudopneumoniae Provide Insight into Virulence and Commensalism Dynamics

    PubMed Central

    Shahinas, Dea; Thornton, Christina S.; Tamber, Gurdip Singh; Arya, Gitanjali; Wong, Andrew; Jamieson, Frances B.; Ma, Jennifer H.; Alexander, David C.; Low, Donald E.; Pillai, Dylan R.

    2013-01-01

    Streptococcus pseudopneumoniae (SPPN) is a recently described species of the viridans group streptococci (VGS). Although the pathogenic potential of S. pseudopneumoniae remains uncertain, it is most commonly isolated from patients with underlying medical conditions, such as chronic obstructive pulmonary disease. S. pseudopneumoniae can be distinguished from the closely related species, S. pneumoniae and S. mitis, by phenotypic characteristics, including optochin resistance in the presence of 5% CO2, bile insolubility, and the lack of the pneumococcal capsule. Previously, we reported the draft genome sequence of S. pseudopneumoniae IS7493, a clinical isolate obtained from an immunocompromised patient with documented pneumonia. Here, we use comparative genomics approaches to identify similarities and key differences between S. pseudopneumoniae IS7493, S. pneumoniae and S. mitis. The genome structure of S. pseudopneumoniae IS7493 is most closely related to that of S. pneumoniae R6, but several recombination events are evident. Analysis of gene content reveals numerous unique features that distinguish S. pseudopneumoniae from other streptococci. The presence of loci for competence, iron transport, pneumolysin production and antimicrobial resistance reinforce the phylogenetic position of S. pseudopneumoniae as an intermediate species between S. pneumoniae and S. mitis. Additionally, the presence of several virulence factors and antibiotic resistance mechanisms suggest the potential of this commensal species to become pathogenic or to contribute to increasing antibiotic resistance levels seen among the VGS. PMID:23840352

  11. 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

    2015-01-01

    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

  12. Reconstitution of CF IA from overexpressed subunits reveals stoichiometry and provides insights into molecular topology

    PubMed Central

    Gordon, James; Shikov, Sergei; Kuehner, Jason N.; Liriano, Melissa; Lee, Eunhee; Stafford, Walter; Poulsen, Mathias Bach; Harrison, Celia; Moore, Claire; Bohm, Andrew

    2011-01-01

    Yeast Cleavage Factor I (CF I) is an essential complex of five proteins that binds signal sequences at the 3′ end of yeast mRNA. CF I is required for correct positioning of a larger protein complex, CPF, which contains the catalytic subunits executing mRNA cleavage and polyadenylation. CF I is composed of two parts, CF IA and Hrp1. The CF IA has only four subunits, Rna14, Rna15, Pcf11, and Clp1, but the structural organization has not been fully established. Using biochemical and biophysical methods, we demonstrate that CF IA can be reconstituted from bacterially-expressed proteins and that it has 2:2:1:1 stoichiometry of its four proteins, respectively. We also describe mutations that disrupt the dimer interface of Rna14 while preserving the other subunit interactions. Based on our results and existing interaction data, we present a topological model for heterohexameric CF IA and its association with RNA and Hrp1. PMID:22026644

  13. 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.

  14. 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

  15. 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

  16. The ASCO Oncology Composite Provider Utilization File: New Data, New Insights.

    PubMed

    Barr, Thomas R; Towle, Elaine L; Barr, Thomas R; Towle, Elaine L

    2016-01-01

    As we seek to understand the changing practice environment in oncology, the need for accurate information about demand for services, distribution of the delivery system in this sector of the health economy, and other practice trends is apparent. In this article, we present analysis of the sector using one of the public use files from the Centers for Medicare & Medicaid Services in combination with other publicly available data. Medicare data are particularly useful for this analysis because cancer is associated with aging and Medicare is the primary payer in the United States for patients older than age 65. As a result, nearly all oncologists who serve adult populations are represented in these data. By combining publicly available datasets into what we call the ASCO Provider Utilization File,we can investigate a wide range of supply, demand, and practice issues. We calculate the average work performed per physician, observe regional differences in work production,and quantify the downside risk and upside potential associated with the provision of chemotherapy drugs. Comparing the supply of oncologists by state with physician work relative value units and with estimates of cancer incidence by state reveals intriguing differences in the distribution of physicians and the demand for oncology services. In addition, our analysis demonstrates significant downside practice risk associated with the provision of drug therapy to Medicare beneficiaries. The economic risk associated with the purchase and delivery of chemotherapy is of particular concern as pressure for value increases. This article provides a description of a new dataset and interesting observations from these data. PMID:26759470

  17. 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

  18. Kinetic model of the aggregation of alpha-synuclein provides insights into prion-like spreading

    PubMed Central

    Iljina, Marija; Garcia, Gonzalo A.; Horrocks, Mathew H.; Tosatto, Laura; Choi, Minee L.; Ganzinger, Kristina A.; Abramov, Andrey Y.; Gandhi, Sonia; Wood, Nicholas W.; Cremades, Nunilo; Dobson, Christopher M.; Knowles, Tuomas P. J.; Klenerman, David

    2016-01-01

    The protein alpha-synuclein (αS) self-assembles into small oligomeric species and subsequently into amyloid fibrils that accumulate and proliferate during the development of Parkinson’s disease. However, the quantitative characterization of the aggregation and spreading of αS remains challenging to achieve. Previously, we identified a conformational conversion step leading from the initially formed oligomers to more compact oligomers preceding fibril formation. Here, by a combination of single-molecule fluorescence measurements and kinetic analysis, we find that the reaction in solution involves two unimolecular structural conversion steps, from the disordered to more compact oligomers and then to fibrils, which can elongate by further monomer addition. We have obtained individual rate constants for these key microscopic steps by applying a global kinetic analysis to both the decrease in the concentration of monomeric protein molecules and the increase in oligomer concentrations over a 0.5–140-µM range of αS. The resulting explicit kinetic model of αS aggregation has been used to quantitatively explore seeding the reaction by either the compact oligomers or fibrils. Our predictions reveal that, although fibrils are more effective at seeding than oligomers, very high numbers of seeds of either type, of the order of 104, are required to achieve efficient seeding and bypass the slow generation of aggregates through primary nucleation. Complementary cellular experiments demonstrated that two orders of magnitude lower numbers of oligomers were sufficient to generate high levels of reactive oxygen species, suggesting that effective templated seeding is likely to require both the presence of template aggregates and conditions of cellular stress. PMID:26884195

  19. Proteomic analyses provide new insights into the responses of Pinus massoniana seedlings to phosphorus deficiency.

    PubMed

    Fan, Fuhua; Ding, Guijie; Wen, Xiaopeng

    2016-02-01

    Phosphorus is an essential macronutrient for plant growth and development. Plants can respond defensively to phosphorus deficiency by modifying their morphology and metabolic pathways via the differential expression of low phosphate responsive genes. To better understand the mechanisms by which the Masson pine (Pinus massoniana) adapts to phosphorus deficiency, we conducted comparative proteomic analysis using an elite line exhibiting high tolerance to phosphorus deficiency. The selected seedlings were treated with 0.5 mM KH2 PO4 (control), 0.01 mM KH2 PO4 (P1), or 0.06 mM KH2 PO4 (P2) for 48 days. Total protein samples were separated via 2DE. A total of 98 differentially expressed proteins, which displayed at least 1.7-fold change expression compared to the control levels (p ≤ 0.05), were identified by MALDI-TOF/TOF MS. These phosphate starvation responsive proteins were implicated in photosynthesis, defense, cellular organization, biosynthesis, energy metabolism, secondary metabolism, signal transduction etc. Therefore, these proteins might play important roles in facilitating internal phosphorus homeostasis. Additionally, the obtained data may be useful for the further characterization of gene function and may provide a foundation for a more comprehensive understanding of the adaptations of the Masson pine to phosphorus-deficient conditions. PMID:26603831

  20. Transcriptome Analysis of Differentially Expressed Genes Provides Insight into Stolon Formation in Tulipa edulis.

    PubMed

    Miao, Yuanyuan; Zhu, Zaibiao; Guo, Qiaosheng; Zhu, Yunhao; Yang, Xiaohua; Sun, Yuan

    2016-01-01

    Tulipa edulis (Miq.) Baker is an important medicinal plant with a variety of anti-cancer properties. The stolon is one of the main asexual reproductive organs of T. edulis and possesses a unique morphology. To explore the molecular mechanism of stolon formation, we performed an RNA-seq analysis of the transcriptomes of stolons at three developmental stages. In the present study, 15.49 Gb of raw data were generated and assembled into 74,006 unigenes, and a total of 2,811 simple sequence repeats were detected in T. edulis. Among the three libraries of stolons at different developmental stages, there were 5,119 differentially expressed genes (DEGs). A functional annotation analysis based on sequence similarity queries of the GO, COG, KEGG databases showed that these DEGs were mainly involved in many physiological and biochemical processes, such as material and energy metabolism, hormone signaling, cell growth, and transcription regulation. In addition, quantitative real-time PCR analysis revealed that the expression patterns of the DEGs were consistent with the transcriptome data, which further supported a role for the DEGs in stolon formation. This study provides novel resources for future genetic and molecular studies in T. edulis. PMID:27064558

  1. De Novo Assembly and Comparative Transcriptome Analysis Provide Insight into Lysine Biosynthesis in Toona sinensis Roem

    PubMed Central

    Zhang, Xia; Song, Zhenqiao; Liu, Tian; Guo, Linlin; Li, Xingfeng

    2016-01-01

    Toona sinensis Roem is a popular leafy vegetable in Chinese cuisine and is also used as a traditional Chinese medicine. In this study, leaf samples were collected from the same plant on two development stages and then used for high-throughput Illumina RNA-sequencing (RNA-Seq). 125,884 transcripts and 54,628 unigenes were obtained through de novo assembly. A total of 25,570 could be annotated with known biological functions, which indicated that the T. sinensis leaves and shoots were undergoing multiple developmental processes especially for active metabolic processes. Analysis of differentially expressed unigenes between the two libraries showed that the lysine biosynthesis was an enriched KEGG pathway, and candidate genes involved in the lysine biosynthesis pathway in T. sinensis leaves and shoots were identified. Our results provide a primary analysis of the gene expression files of T. sinensis leaf and shoot on different development stages and afford a valuable resource for genetic and genomic research on plant lysine biosynthesis. PMID:27376077

  2. Molecular interactions of the Saccharomyces cerevisiae Atg1 complex provide insights into assembly and regulatory mechanisms

    PubMed Central

    Chew, Leon H; Lu, Shan; Liu, Xu; Li, Franco Kk; Yu, Angela Yh; Klionsky, Daniel J; Dong, Meng-Qiu; Yip, Calvin K

    2015-01-01

    The Atg1 complex, which contains 5 major subunits: Atg1, Atg13, Atg17, Atg29, and Atg31, regulates the induction of autophagy and autophagosome formation. To gain a better understanding of the overall architecture and assembly mechanism of this essential autophagy regulatory complex, we have reconstituted a core assembly of the Saccharomyces cerevisiae Atg1 complex composed of full-length Atg17, Atg29, and Atg31, along with the C-terminal domains of Atg1 (Atg1[CTD]) and Atg13 (Atg13[CTD]). Using chemical-crosslinking coupled with mass spectrometry (CXMS) analysis we systematically mapped the intersubunit interaction interfaces within this complex. Our data revealed that the intrinsically unstructured C-terminal domain of Atg29 interacts directly with Atg17, whereas Atg17 interacts with Atg13 in 2 distinct intrinsically unstructured regions, including a previously unknown motif that encompasses several putative phosphorylation sites. The Atg1[CTD] crosslinks exclusively to the Atg13[CTD] and does not appear to make direct contact with the Atg17-Atg31-Atg29 scaffold. Finally, single-particle electron microscopy analysis revealed that both the Atg13[CTD] and Atg1[CTD] localize to the tip regions of Atg17-Atg31-Atg29 and do not alter the distinct curvature of this scaffolding subcomplex. This work provides a comprehensive understanding of the subunit interactions in the fully assembled Atg1 core complex, and uncovers the potential role of intrinsically disordered regions in regulating complex integrity. PMID:25998554

  3. Transcriptome Analysis of Differentially Expressed Genes Provides Insight into Stolon Formation in Tulipa edulis

    PubMed Central

    Miao, Yuanyuan; Zhu, Zaibiao; Guo, Qiaosheng; Zhu, Yunhao; Yang, Xiaohua; Sun, Yuan

    2016-01-01

    Tulipa edulis (Miq.) Baker is an important medicinal plant with a variety of anti-cancer properties. The stolon is one of the main asexual reproductive organs of T. edulis and possesses a unique morphology. To explore the molecular mechanism of stolon formation, we performed an RNA-seq analysis of the transcriptomes of stolons at three developmental stages. In the present study, 15.49 Gb of raw data were generated and assembled into 74,006 unigenes, and a total of 2,811 simple sequence repeats were detected in T. edulis. Among the three libraries of stolons at different developmental stages, there were 5,119 differentially expressed genes (DEGs). A functional annotation analysis based on sequence similarity queries of the GO, COG, KEGG databases showed that these DEGs were mainly involved in many physiological and biochemical processes, such as material and energy metabolism, hormone signaling, cell growth, and transcription regulation. In addition, quantitative real-time PCR analysis revealed that the expression patterns of the DEGs were consistent with the transcriptome data, which further supported a role for the DEGs in stolon formation. This study provides novel resources for future genetic and molecular studies in T. edulis. PMID:27064558

  4. Maize pan-transcriptome provides novel insights into genome complexity and quantitative trait variation

    PubMed Central

    Jin, Minliang; Liu, Haijun; He, Cheng; Fu, Junjie; Xiao, Yingjie; Wang, Yuebin; Xie, Weibo; Wang, Guoying; Yan, Jianbing

    2016-01-01

    Gene expression variation largely contributes to phenotypic diversity and constructing pan-transcriptome is considered necessary for species with complex genomes. However, the regulation mechanisms and functional consequences of pan-transcriptome is unexplored systematically. By analyzing RNA-seq data from 368 maize diverse inbred lines, we identified almost one-third nuclear genes under expression presence and absence variation, which tend to play regulatory roles and are likely regulated by distant eQTLs. The ePAV was directly used as “genotype” to perform GWAS for 15 agronomic phenotypes and 526 metabolic traits to efficiently explore the associations between transcriptomic and phenomic variations. Through a modified assembly strategy, 2,355 high-confidence novel sequences with total 1.9 Mb lengths were found absent within reference genome. Ten randomly selected novel sequences were fully validated with genomic PCR, including another two NBS_LRR candidates potentially affect flavonoids and disease-resistance. A simulation analysis suggested that the pan-transcriptome of the maize whole kernel is approaching a maximum value of 63,000 genes, and through developing two test-cross populations and surveying several most important yield traits, the dispensable genes were shown to contribute to heterosis. Novel perspectives and resources to discover maize quantitative trait variations were provided to better understand the kernel regulation networks and to enhance maize breeding. PMID:26729541

  5. Maternal separation with early weaning: A rodent model providing novel insights into neglect associated developmental deficits

    PubMed Central

    CARLYLE, BECKY C.; DUQUE, ALVARO; KITCHEN, ROBERT R.; BORDNER, KELLY A.; COMAN, DANIEL; DOOLITTLE, ELIZA; PAPADEMETRIS, XENOPHONIOS; HYDER, FAHMEED; TAYLOR, JANE R.; SIMEN, ARTHUR A.

    2013-01-01

    Child neglect is the most prevalent form of child maltreatment in the United States, and poses a serious public health concern. Children who survive such episodes go on to experience long-lasting psychological and behavioral problems, including higher rates of post-traumatic stress disorder symptoms, depression, alcohol and drug abuse, attention-deficit/hyperactivity disorder, and cognitive deficits. To date, most research into the causes of these life-long problems has focused on well-established targets such as stress responsive systems, including the hypothalamus–pituitary–adrenal axis. Using the maternal separation and early weaning model, we have attempted to provide comprehensive molecular profiling of a model of early-life neglect in an organism amenable to genomic manipulation: the mouse. In this article, we report new findings generated with this model using chromatin immunoprecipitation sequencing, diffuse tensor magnetic resonance imaging, and behavioral analyses. We also review the validity of the maternal separation and early weaning model, which reflects behavioral deficits observed in neglected humans including hyperactivity, anxiety, and attentional deficits. Finally, we summarize the molecular characterization of these animals, including RNA profiling and label-free proteomics, which highlight protein translation and myelination as novel pathways of interest. PMID:23062306

  6. Integrated network model provides new insights into castration-resistant prostate cancer

    PubMed Central

    Hu, Yanling; Gu, Yinmin; Wang, Huimin; Huang, Yuanjie; Zou, Yi Ming

    2015-01-01

    Castration-resistant prostate cancer (CRPC) is the main challenge for prostate cancer treatment. Recent studies have indicated that extending the treatments to simultaneously targeting different pathways could provide better approaches. To better understand the regulatory functions of different pathways, a system-wide study of CRPC regulation is necessary. For this purpose, we constructed a comprehensive CRPC regulatory network by integrating multiple pathways such as the MEK/ERK and the PI3K/AKT pathways. We studied the feedback loops of this network and found that AKT was involved in all detected negative feedback loops. We translated the network into a predictive Boolean model and analyzed the stable states and the control effects of genes using novel methods. We found that the stable states naturally divide into two obvious groups characterizing PC3 and DU145 cells respectively. Stable state analysis further revealed that several critical genes, such as PTEN, AKT, RAF, and CDKN2A, had distinct expression behaviors in different clusters. Our model predicted the control effects of many genes. We used several public datasets as well as FHL2 overexpression to verify our finding. The results of this study can help in identifying potential therapeutic targets, especially simultaneous targets of multiple pathways, for CRPC. PMID:26603105

  7. Transcriptome-wide sequencing provides insights into geocarpy in peanut (Arachis hypogaea L.).

    PubMed

    Chen, Xiaoping; Yang, Qingli; Li, Haifen; Li, Heying; Hong, Yanbin; Pan, Lijuan; Chen, Na; Zhu, Fanghe; Chi, Xiaoyuan; Zhu, Wei; Chen, Mingna; Liu, Haiyan; Yang, Zhen; Zhang, Erhua; Wang, Tong; Zhong, Ni; Wang, Mian; Liu, Hong; Wen, Shijie; Li, Xingyu; Zhou, Guiyuan; Li, Shaoxiong; Wu, Hong; Varshney, Rajeev; Liang, Xuanqiang; Yu, Shanlin

    2016-05-01

    A characteristic feature of peanut is the subterranean fructification, geocarpy, in which the gynophore ('peg'), a specialized organ that transitions from upward growth habit to downward outgrowth upon fertilization, drives the developing pod into the soil for subsequent development underground. As a step towards understanding this phenomenon, we explore the developmental dynamics of the peanut pod transcriptome at 11 successive stages. We identified 110 217 transcripts across developmental stages and quantified their abundance along a pod developmental gradient in pod wall. We found that the majority of transcripts were differentially expressed along the developmental gradient as well as identified temporal programs of gene expression, including hundreds of transcription factors. Thought to be an adaptation to particularly harsh subterranean environments, both up- and down-regulated gene sets in pod wall were enriched for response to a broad array of stimuli, like gravity, light and subterranean environmental factors. We also identified hundreds of transcripts associated with gravitropism and photomorphogenesis, which may be involved in the geocarpy. Collectively, this study forms a transcriptional baseline for geocarpy in peanut as well as provides a considerable body of evidence that transcriptional regulation in peanut aerial and subterranean fruits is complex. PMID:26502832

  8. Molecular underpinnings of prefrontal cortex development in rodents provide insights into the etiology of neurodevelopmental disorders

    PubMed Central

    Schubert, D; Martens, G J M; Kolk, S M

    2015-01-01

    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. PMID:25450230

  9. 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

  10. Transcriptomic analysis of the white rot fungus Polyporus brumalis provides insight into sesquiterpene biosynthesis.

    PubMed

    Lee, Su-Yeon; Kim, Myungkil; Kim, Seon-Hong; Hong, Chang-Young; Ryu, Sun-Hwa; Choi, In-Gyu

    2016-01-01

    Object of this study was to identify genes and enzymes that are involved in sesquiterpene biosynthesis in the wood rotting fungus, Polyporus brumalis. Sesquiterpenes, β-eudesmane and β-eudesmol, were produced by the mycelium of P. brumalis cultured in modified medium. However, theses final products were not observed when the fungus was grown in potato dextrose medium. We used next generation sequencing (NGS) to identify differentially expressed genes (DEGs) related to terpene metabolism. This approach generated 25,000 unigenes and 127 metabolic pathways that were assigned to Kyoto Encyclopedia Genes Groups (KEGG). Further analysis of samples from modified medium indicated significant upregulation of 8 unigenes involved in the mevalonate (MVA) and methylerythritol phosphate (MEP) biosynthetic pathways. These pathways generate isopentenyl pyrophosphate (IPP) and farnesyl pyrophosphate (FPP), which are precursors for the synthesis of sesquiterpenes. Furthermore, genes encoding germacrene A synthase, which facilitate the cyclization of FPP, were only differentially expressed in mycelium from fungi grown in modified medium. Our data provide a resource for studying the molecular mechanisms underpinning sesquiterpene biosynthesis and terpene metabolism. PMID:26686622

  11. Maternal separation with early weaning: a rodent model providing novel insights into neglect associated developmental deficits.

    PubMed

    Carlyle, Becky C; Duque, Alvaro; Kitchen, Robert R; Bordner, Kelly A; Coman, Daniel; Doolittle, Eliza; Papademetris, Xenophonios; Hyder, Fahmeed; Taylor, Jane R; Simen, Arthur A

    2012-11-01

    Child neglect is the most prevalent form of child maltreatment in the United States, and poses a serious public health concern. Children who survive such episodes go on to experience long-lasting psychological and behavioral problems, including higher rates of post-traumatic stress disorder symptoms, depression, alcohol and drug abuse, attention-deficit/hyperactivity disorder, and cognitive deficits. To date, most research into the causes of these life-long problems has focused on well-established targets such as stress responsive systems, including the hypothalamus-pituitary-adrenal axis. Using the maternal separation and early weaning model, we have attempted to provide comprehensive molecular profiling of a model of early-life neglect in an organism amenable to genomic manipulation: the mouse. In this article, we report new findings generated with this model using chromatin immunoprecipitation sequencing, diffuse tensor magnetic resonance imaging, and behavioral analyses. We also review the validity of the maternal separation and early weaning model, which reflects behavioral deficits observed in neglected humans including hyperactivity, anxiety, and attentional deficits. Finally, we summarize the molecular characterization of these animals, including RNA profiling and label-free proteomics, which highlight protein translation and myelination as novel pathways of interest. PMID:23062306

  12. Intraspecific Variation in Mitogenomes of Five Crassostrea Species Provides Insight into Oyster Diversification and Speciation.

    PubMed

    Ren, Jianfeng; Hou, Zhanhui; Wang, Haiyan; Sun, Ming-An; Liu, Xiao; Liu, Bin; Guo, Ximing

    2016-04-01

    A large number of Crassostrea oysters are found in Asia-Pacific. While analyses of interspecific variation have helped to establish historical relationships among these species, studies on intraspecific variation are necessary to understand their recent evolutionary history and current forces driving population biology. We resequenced 18 and analyzed 31 mitogenomes of five Crassostrea species from China: Crassostrea gigas, Crassostrea angulata, Crassostrea sikamea, Crassostrea ariakensis, and Crassostrea hongkongensis. Our analysis finds abundant insertions, deletions, and single-nucleotide polymorphisms in all species. Intraspecific variation varies greatly among species with polymorphic sites ranging from 54 to 293 and nucleotide diversity ranging from 0.00106 to 0.00683. In all measurements, C. hongkongensis that has the narrowest geographic distribution exhibits the least sequence diversity; C. ariakensis that has the widest distribution shows the highest diversity, and species with intermediate distribution show intermediate levels of diversity. Low sequence diversity in C. hongkongensis may reflect recent bottlenecks that are probably exacerbated by human transplantation. High diversity in C. ariakensis is likely due to divergence of northern and southern China populations that have been separated without gene flow. The significant differences in mitogenome diversity suggest that the five sister species of Crassostrea have experienced different evolutionary forces since their divergence. The recent divergence of two C. ariakensis populations and the C. gigas/angulata species complex provides evidence for continued diversification and speciation of Crassostrea species along China's coast, which are shaped by unknown mechanisms in a north-south divide. PMID:26846524

  13. Whole-genome sequencing of Berkshire (European native pig) provides insights into its origin and domestication

    PubMed Central

    Li, Mingzhou; Tian, Shilin; Yeung, Carol K. L.; Meng, Xuehong; Tang, Qianzi; Niu, Lili; Wang, Xun; Jin, Long; Ma, Jideng; Long, Keren; Zhou, Chaowei; Cao, Yinchuan; Zhu, Li; Bai, Lin; Tang, Guoqing; Gu, Yiren; Jiang, An'an; Li, Xuewei; Li, Ruiqiang

    2014-01-01

    Domesticated organisms have experienced strong selective pressures directed at genes or genomic regions controlling traits of biological, agricultural or medical importance. The genome of native and domesticated pigs provide a unique opportunity for tracing the history of domestication and identifying signatures of artificial selection. Here we used whole-genome sequencing to explore the genetic relationships among the European native pig Berkshire and breeds that are distributed worldwide, and to identify genomic footprints left by selection during the domestication of Berkshire. Numerous nonsynonymous SNPs-containing genes fall into olfactory-related categories, which are part of a rapidly evolving superfamily in the mammalian genome. Phylogenetic analyses revealed a deep phylogenetic split between European and Asian pigs rather than between domestic and wild pigs. Admixture analysis exhibited higher portion of Chinese genetic material for the Berkshire pigs, which is consistent with the historical record regarding its origin. Selective sweep analyses revealed strong signatures of selection affecting genomic regions that harbor genes underlying economic traits such as disease resistance, pork yield, fertility, tameness and body length. These discoveries confirmed the history of origin of Berkshire pig by genome-wide analysis and illustrate how domestication has shaped the patterns of genetic variation. PMID:24728479

  14. Mutational spectrum at GATA1 provides insights into mutagenesis and leukemogenesis in Down syndrome

    PubMed Central

    Cabelof, Diane C.; Patel, Hiral V.; Chen, Qing; van Remmen, Holly; Matherly, Larry H.; Ge, Yubin

    2009-01-01

    Down syndrome (DS) children have a unique genetic susceptibility to develop leukemia, in particular, acute megakaryocytic leukemia (AMkL) associated with somatic GATA1 mutations. The study of this genetic susceptibility with the use of DS as a model of leukemogenesis has broad applicability to the understanding of leukemia in children overall. On the basis of the role of GATA1 mutations in DS AMkL, we analyzed the mutational spectrum of GATA1 mutations to begin elucidating possible mechanisms by which these sequence alterations arise. Mutational analysis revealed a predominance of small insertion/deletion, duplication, and base substitution mutations, including G:C>T:A, G:C>A:T, and A:T>G:C. This mutational spectrum points to potential oxidative stress and aberrant folate metabolism secondary to genes on chromosome 21 (eg, cystathionine-β-synthase, superoxide dismutase) as potential causes of GATA1 mutations. Furthermore, DNA repair capacity evaluated in DS and non-DS patient samples provided evidence that the base excision repair pathway is compromised in DS tissues, suggesting that inability to repair DNA damage also may play a critical role in the unique susceptibility of DS children to develop leukemia. A model of leukemogenesis in DS is proposed in which mutagenesis is driven by cystathionine-β-synthase overexpression and altered folate homeostasis that becomes fixed as the ability to repair DNA damage is compromised. PMID:19633202

  15. Challenges to normal neural functioning provide insights into separability of motion processing mechanisms.

    PubMed

    Billino, Jutta; Braun, Doris I; Bremmer, Frank; Gegenfurtner, Karl R

    2011-10-01

    There is a long history of attempts to disentangle different visual processing mechanisms for physically different motion cues. However, underlying neural correlates and separability of networks are still under debate. We aimed to refine the current understanding by studying differential vulnerabilities when normal neural functioning is challenged. We investigated effects of ageing and extrastriate brain lesions on detection thresholds for motion defined by either luminance- or contrast modulations, known as first- and second-order motion. Both approaches focus on extrastriate processing changes and combine distributed as well as more focal constraints. Our ageing sample comprised 102 subjects covering an age range from 20 to 82 years. Threshold signal-to-noise ratios for detection approximately doubled across the age range for both motion types. Results suggest that ageing affects perception of both motion types to an equivalent degree and thus support overlapping processing resources. Underlying neural substrates were further qualified by testing perceptual performance of 18 patients with focal cortical brain lesions. We determined selective first-order motion deficits in three patients, selective second-order motion deficits in only one patient, and deficits for both motion types in three patients. Lesion analysis yielded support for common functional substrates in higher cortical regions. Functionally specific substrates remained ambiguous, but tended to cover earlier visual areas. We conclude that observed vulnerabilities of first- and second-order motion perception provide limited evidence for functional specialization at early extrastriate stages, but emphasize shared processing pathways at higher cortical levels. PMID:21807009

  16. Weird mammals provide insights into the evolution of mammalian sex chromosomes and dosage compensation.

    PubMed

    Graves, Jennifer A Marshall

    2015-12-01

    The deep divergence of mammalian groups 166 and 190 million years ago (MYA) provide genetic variation to explore the evolution of DNA sequence, gene arrangement and regulation of gene expression in mammals. With encouragement from the founder of the field, Mary Lyon, techniques in cytogenetics and molecular biology were progressively adapted to characterize the sex chromosomes of kangaroos and other marsupials, platypus and echidna-and weird rodent species. Comparative gene mapping reveals the process of sex chromosome evolution from their inception 190 MYA (they are autosomal in platypus) to their inevitable end (the Y has disappeared in two rodent lineages). Our X and Y are relatively young, getting their start with the evolution of the sex-determining SRY gene, which triggered progressive degradation of the Y chromosome. Even more recently, sex chromosomes of placental mammals fused with an autosomal region which now makes up most of the Y. Exploration of gene activity patterns over four decades showed that dosage compensation via X-chromosome inactivation is unique to therian mammals, and that this whole chromosome control process is different in marsupials and absent in monotremes and reptiles, and birds. These differences can be exploited to deduce how mammalian sex chromosomes and epigenetic silencing evolved. PMID:26690510

  17. Broad-scale phylogenomics provides insights into retrovirus–host evolution

    PubMed Central

    Hayward, Alexander; Grabherr, Manfred; Jern, Patric

    2013-01-01

    Genomic data provide an excellent resource to improve understanding of retrovirus evolution and the complex relationships among viruses and their hosts. In conjunction with broad-scale in silico screening of vertebrate genomes, this resource offers an opportunity to complement data on the evolution and frequency of past retroviral spread and so evaluate future risks and limitations for horizontal transmission between different host species. Here, we develop a methodology for extracting phylogenetic signal from large endogenous retrovirus (ERV) datasets by collapsing information to facilitate broad-scale phylogenomics across a wide sample of hosts. Starting with nearly 90,000 ERVs from 60 vertebrate host genomes, we construct phylogenetic hypotheses and draw inferences regarding the designation, host distribution, origin, and transmission of the Gammaretrovirus genus and associated class I ERVs. Our results uncover remarkable depths in retroviral sequence diversity, supported within a phylogenetic context. This finding suggests that current infectious exogenous retrovirus diversity may be underestimated, adding credence to the possibility that many additional exogenous retroviruses may remain to be discovered in vertebrate taxa. We demonstrate a history of frequent horizontal interorder transmissions from a rodent reservoir and suggest that rats may have acted as important overlooked facilitators of gammaretrovirus spread across diverse mammalian hosts. Together, these results demonstrate the promise of the methodology used here to analyze large ERV datasets and improve understanding of retroviral evolution and diversity for utilization in wider applications. PMID:24277832

  18. Maize pan-transcriptome provides novel insights into genome complexity and quantitative trait variation.

    PubMed

    Jin, Minliang; Liu, Haijun; He, Cheng; Fu, Junjie; Xiao, Yingjie; Wang, Yuebin; Xie, Weibo; Wang, Guoying; Yan, Jianbing

    2016-01-01

    Gene expression variation largely contributes to phenotypic diversity and constructing pan-transcriptome is considered necessary for species with complex genomes. However, the regulation mechanisms and functional consequences of pan-transcriptome is unexplored systematically. By analyzing RNA-seq data from 368 maize diverse inbred lines, we identified almost one-third nuclear genes under expression presence and absence variation, which tend to play regulatory roles and are likely regulated by distant eQTLs. The ePAV was directly used as "genotype" to perform GWAS for 15 agronomic phenotypes and 526 metabolic traits to efficiently explore the associations between transcriptomic and phenomic variations. Through a modified assembly strategy, 2,355 high-confidence novel sequences with total 1.9 Mb lengths were found absent within reference genome. Ten randomly selected novel sequences were fully validated with genomic PCR, including another two NBS_LRR candidates potentially affect flavonoids and disease-resistance. A simulation analysis suggested that the pan-transcriptome of the maize whole kernel is approaching a maximum value of 63,000 genes, and through developing two test-cross populations and surveying several most important yield traits, the dispensable genes were shown to contribute to heterosis. Novel perspectives and resources to discover maize quantitative trait variations were provided to better understand the kernel regulation networks and to enhance maize breeding. PMID:26729541

  19. European sea bass genome and its variation provide insights into adaptation to euryhalinity and speciation

    PubMed Central

    Tine, Mbaye; Kuhl, Heiner; Gagnaire, Pierre-Alexandre; Louro, Bruno; Desmarais, Erick; Martins, Rute S.T.; Hecht, Jochen; Knaust, Florian; Belkhir, Khalid; Klages, Sven; Dieterich, Roland; Stueber, Kurt; Piferrer, Francesc; Guinand, Bruno; Bierne, Nicolas; Volckaert, Filip A. M.; Bargelloni, Luca; Power, Deborah M.; Bonhomme, François; Canario, Adelino V. M.; Reinhardt, Richard

    2014-01-01

    The European sea bass (Dicentrarchus labrax) is a temperate-zone euryhaline teleost of prime importance for aquaculture and fisheries. This species is subdivided into two naturally hybridizing lineages, one inhabiting the north-eastern Atlantic Ocean and the other the Mediterranean and Black seas. Here we provide a high-quality chromosome-scale assembly of its genome that shows a high degree of synteny with the more highly derived teleosts. We find expansions of gene families specifically associated with ion and water regulation, highlighting adaptation to variation in salinity. We further generate a genome-wide variation map through RAD-sequencing of Atlantic and Mediterranean populations. We show that variation in local recombination rates strongly influences the genomic landscape of diversity within and differentiation between lineages. Comparing predictions of alternative demographic models to the joint allele-frequency spectrum indicates that genomic islands of differentiation between sea bass lineages were generated by varying rates of introgression across the genome following a period of geographical isolation. PMID:25534655

  20. SYSTEMS ANALYSIS OF TRANSCRIPTIONAL DATA PROVIDES INSIGHTS INTO MUSCLE’S BIOLOGICAL RESPONSE TO BOTULINUM TOXIN

    PubMed Central

    MUKUND, KAVITHA; MATHEWSON, MARGIE; MINAMOTO, VIVIANE; WARD, SAMUEL R.; SUBRAMANIAM, SHANKAR; LIEBER, RICHARD L.

    2014-01-01

    Introduction In this study we provide global transcriptomic profiling and analysis of botulinum toxin A (BoNT-A)–treated muscle over a 1-year period. Methods Microarray analysis was performed on rat tibialis anterior muscles from 4 groups (n =4/group) at 1, 4, 12, and 52 weeks after BoNT-A injection compared with saline-injected rats at 12 weeks. Results Dramatic transcriptional adaptation occurred at 1 week with a paradoxical increase in expression of slow and immature isoforms, activation of genes in competing pathways of repair and atrophy, impaired mitochondrial biogenesis, and increased metal ion imbalance. Adaptations of the basal lamina and fibrillar extracellular matrix (ECM) occurred by 4 weeks. The muscle transcriptome returned to its unperturbed state 12 weeks after injection. Conclusion Acute transcriptional adaptations resemble denervated muscle with some subtle differences, but resolved more quickly compared with denervation. Overall, gene expression, across time, correlates with the generally accepted BoNT-A time course and suggests that the direct action of BoNT-A in skeletal muscle is relatively rapid. PMID:24536034

  1. Genomes of Ellobius species provide insight into the evolutionary dynamics of mammalian sex chromosomes.

    PubMed

    Mulugeta, Eskeatnaf; Wassenaar, Evelyne; Sleddens-Linkels, Esther; van IJcken, Wilfred F J; Heard, Edith; Grootegoed, J Anton; Just, Walter; Gribnau, Joost; Baarends, Willy M

    2016-09-01

    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

  2. In vivo cell biology in zebrafish – providing insights into vertebrate development and disease

    PubMed Central

    Vacaru, Ana M.; Unlu, Gokhan; Spitzner, Marie; Mione, Marina; Knapik, Ela W.; Sadler, Kirsten C.

    2014-01-01

    ABSTRACT Over the past decades, studies using zebrafish have significantly advanced our understanding of the cellular basis for development and human diseases. Zebrafish have rapidly developing transparent embryos that allow comprehensive imaging of embryogenesis combined with powerful genetic approaches. However, forward genetic screens in zebrafish have generated unanticipated findings that are mirrored by human genetic studies: disruption of genes implicated in basic cellular processes, such as protein secretion or cytoskeletal dynamics, causes discrete developmental or disease phenotypes. This is surprising because many processes that were assumed to be fundamental to the function and survival of all cell types appear instead to be regulated by cell-specific mechanisms. Such discoveries are facilitated by experiments in whole animals, where zebrafish provides an ideal model for visualization and manipulation of organelles and cellular processes in a live vertebrate. Here, we review well-characterized mutants and newly developed tools that underscore this notion. We focus on the secretory pathway and microtubule-based trafficking as illustrative examples of how studying cell biology in vivo using zebrafish has broadened our understanding of the role fundamental cellular processes play in embryogenesis and disease. PMID:24481493

  3. Elephant shark (Callorhinchus milii) provides insights into the evolution of Hox gene clusters in gnathostomes.

    PubMed

    Ravi, Vydianathan; Lam, Kevin; Tay, Boon-Hui; Tay, Alice; Brenner, Sydney; Venkatesh, Byrappa

    2009-09-22

    We have sequenced and analyzed Hox gene clusters from elephant shark, a holocephalian cartilaginous fish. Elephant shark possesses 4 Hox clusters with 45 Hox genes that include orthologs for a higher number of ancient gnathostome Hox genes than the 4 clusters in tetrapods and the supernumerary clusters in teleost fishes. Phylogenetic analysis of elephant shark Hox genes from 7 paralogous groups that contain all of the 4 members indicated an ((AB)(CD)) topology for the order of Hox cluster duplication, providing support for the 2R hypothesis (i.e., 2 rounds of whole-genome duplication during the early evolution of vertebrates). Comparisons of noncoding sequences of the elephant shark and human Hox clusters have identified a large number of conserved noncoding elements (CNEs), which represent putative cis-regulatory elements that may be involved in the regulation of Hox genes. Interestingly, in fugu more than 50% of these ancient CNEs have diverged beyond recognition in the duplicated (HoxA, HoxB, and HoxD) as well as the singleton (HoxC) Hox clusters. Furthermore, the b-paralogs of the duplicated fugu Hox clusters are virtually devoid of unique ancient CNEs. In contrast to fugu Hox clusters, elephant shark and human Hox clusters have lost fewer ancient CNEs. If these ancient CNEs are indeed enhancers directing tissue-specific expression of Hox genes, divergence of their sequences in vertebrate lineages might have led to altered expression patterns and presumably the functions of their associated Hox genes. PMID:19805301

  4. Analyses of pig genomes provide insight into porcine demography and evolution

    PubMed Central

    Groenen, Martien A. M.; Archibald, Alan L.; Uenishi, Hirohide; Tuggle, Christopher K.; Takeuchi, Yasuhiro; Rothschild, Max F.; Rogel-Gaillard, Claire; Park, Chankyu; Milan, Denis; Megens, Hendrik-Jan; Li, Shengting; Larkin, Denis M.; Kim, Heebal; Frantz, Laurent A. F.; Caccamo, Mario; Ahn, Hyeonju; Aken, Bronwen L.; Anselmo, Anna; Anthon, Christian; Auvil, Loretta; Badaoui, Bouabid; Beattie, Craig W.; Bendixen, Christian; Berman, Daniel; Blecha, Frank; Blomberg, Jonas; Bolund, Lars; Bosse, Mirte; Botti, Sara; Bujie, Zhan; Bystrom, Megan; Capitanu, Boris; Silva, Denise Carvalho; Chardon, Patrick; Chen, Celine; Cheng, Ryan; Choi, Sang-Haeng; Chow, William; Clark, Richard C.; Clee, Christopher; Crooijmans, Richard P. M. A.; Dawson, Harry D.; Dehais, Patrice; De Sapio, Fioravante; Dibbits, Bert; Drou, Nizar; Du, Zhi-Qiang; Eversole, Kellye; Fadista, João; Fairley, Susan; Faraut, Thomas; Faulkner, Geoffrey J.; Fowler, Katie E.; Fredholm, Merete; Fritz, Eric; Gilbert, James G. R.; Giuffra, Elisabetta; Gorodkin, Jan; Griffin, Darren K.; Harrow, Jennifer L.; Hayward, Alexander; Howe, Kerstin; Hu, Zhi-Liang; Humphray, Sean J.; Hunt, Toby; Hornshøj, Henrik; Jeon, Jin-Tae; Jern, Patric; Jones, Matthew; Jurka, Jerzy; Kanamori, Hiroyuki; Kapetanovic, Ronan; Kim, Jaebum; Kim, Jae-Hwan; Kim, Kyu-Won; Kim, Tae-Hun; Larson, Greger; Lee, Kyooyeol; Lee, Kyung-Tai; Leggett, Richard; Lewin, Harris A.; Li, Yingrui; Liu, Wansheng; Loveland, Jane E.; Lu, Yao; Lunney, Joan K.; Ma, Jian; Madsen, Ole; Mann, Katherine; Matthews, Lucy; McLaren, Stuart; Morozumi, Takeya; Murtaugh, Michael P.; Narayan, Jitendra; Nguyen, Dinh Truong; Ni, Peixiang; Oh, Song-Jung; Onteru, Suneel; Panitz, Frank; Park, Eung-Woo; Park, Hong-Seog; Pascal, Geraldine; Paudel, Yogesh; Perez-Enciso, Miguel; Ramirez-Gonzalez, Ricardo; Reecy, James M.; Zas, Sandra Rodriguez; Rohrer, Gary A.; Rund, Lauretta; Sang, Yongming; Schachtschneider, Kyle; Schraiber, Joshua G.; Schwartz, John; Scobie, Linda; Scott, Carol; Searle, Stephen; Servin, Bertrand; Southey, Bruce R.; Sperber, Goran; Stadler, Peter; Sweedler, Jonathan V.; Tafer, Hakim; Thomsen, Bo; Wali, Rashmi; Wang, Jian; Wang, Jun; White, Simon; Xu, Xun; Yerle, Martine; Zhang, Guojie; Zhang, Jianguo; Zhang, Jie; Zhao, Shuhong; Rogers, Jane; Churcher, Carol; Schook, Lawrence B.

    2013-01-01

    For 10,000 years pigs and humans have shared a close and complex relationship. From domestication to modern breeding practices, humans have shaped the genomes of domestic pigs. Here we present the assembly and analysis of the genome sequence of a female domestic Duroc pig (Sus scrofa) and a comparison with the genomes of wild and domestic pigs from Europe and Asia. Wild pigs emerged in South East Asia and subsequently spread across Eurasia. Our results reveal a deep phylogenetic split between European and Asian wild boars ~1 million years ago, and a selective sweep analysis indicates selection on genes involved in RNA processing and regulation. Genes associated with immune response and olfaction exhibit fast evolution. Pigs have the largest repertoire of functional olfactory receptor genes, reflecting the importance of smell in this scavenging animal. The pig genome sequence provides an important resource for further improvements of this important livestock species, and our identification of many putative disease-causing variants extends the potential of the pig as a biomedical model. PMID:23151582

  5. Comparative Proteomic Analyses Provide New Insights into Low Phosphorus Stress Responses in Maize Leaves

    PubMed Central

    Zhang, Kewei; Liu, Hanhan; Tao, Peilin; Chen, Huan

    2014-01-01

    Phosphorus deficiency limits plant growth and development. To better understand the mechanisms behind how maize responds to phosphate stress, we compared the proteome analysis results of two groups of maize leaves that were treated separately with 1,000 µM (control, +P) and 5 µM of KH2PO4 (intervention group, −P) for 25 days. In total, 1,342 protein spots were detected on 2-DE maps and 15.43% had changed (P<0.05; ≥1.5-fold) significantly in quantity between the +P and −P groups. These proteins are involved in several major metabolic pathways, including photosynthesis, carbohydrate metabolism, energy metabolism, secondary metabolism, signal transduction, protein synthesis, cell rescue and cell defense and virulence. The results showed that the reduction in photosynthesis under low phosphorus treatment was due to the down-regulation of the proteins involved in CO2 enrichment, the Calvin cycle and the electron transport system. Electron transport and photosynthesis restrictions resulted in a large accumulation of peroxides. Maize has developed many different reactive oxygen species (ROS) scavenging mechanisms to cope with low phosphorus stress, including up-regulating its antioxidant content and antioxidase activity. After being subjected to phosphorus stress over a long period, maize may increase its internal phosphorus utilization efficiency by altering photorespiration, starch synthesis and lipid composition. These results provide important information about how maize responds to low phosphorus stress. PMID:24858307

  6. 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

  7. Sibling cooperation in earwig families provides insights into the early evolution of social life.

    PubMed

    Falk, Joachim; Wong, Janine W Y; Kölliker, Mathias; Meunier, Joël

    2014-04-01

    The evolutionary transition from solitary to social life is driven by direct and indirect fitness benefits of social interactions. Understanding the conditions promoting the early evolution of social life therefore requires identification of these benefits in nonderived social systems, such as animal families where offspring are mobile and able to disperse and will survive independently. Family life is well known to provide benefits to offspring through parental care, but research on sibling interactions generally focused on fitness costs to offspring due to competitive behaviors. Here we show experimentally that sibling interactions also reflect cooperative behaviors in the form of food sharing in nonderived families of the European earwig, Forficula auricularia. Food ingested by individual offspring was transferred to their siblings through mouth-to-anus contacts and active allo-coprophagy. These transfers occurred in both the presence and the absence of the tending mothers, even though the direct contact with the mothers limited sibling food sharing. Neither food deprivation or relatedness influenced the total amount of transferred food, but relatedness affected frass release and the behavioral mechanisms mediating food sharing. Related offspring obtained food predominately through allo-coprophagy, whereas unrelated offspring obtained food through mouth-to-anus contacts. Overall, this study emphasizes that sibling cooperation may be a key process promoting the early evolution of social life. PMID:24642498

  8. The amphioxus genome provides unique insight into the evolution of immunity

    PubMed Central

    Dishaw, Larry J.; Haire, Robert N.

    2012-01-01

    Immune systems evolve as essential strategies to maintain homeostasis with the environment, prevent microbial assault and recycle damaged host tissues. The immune system is composed of two components, innate and adaptive immunity. The former is common to all animals while the latter consists of a vertebrate-specific system that relies on somatically derived lymphocytes and is associated with near limitless genetic diversity as well as long-term memory. Deuterostome invertebrates provide a view of immune repertoires in phyla that immediately predate the origins of vertebrates. Genomic studies in amphioxus, a cephalochordate, have revealed homologs of genes encoding most innate immune receptors found in vertebrates; however, many of the gene families have undergone dramatic expansions, greatly increasing the innate immune repertoire. In addition, domain-swapping accounts for the innovation of new predicted pathways of receptor function. In both amphioxus and Ciona, a urochordate, the VCBPs (variable region containing chitin-binding proteins), which consist of immunoglobulin V (variable) and chitin binding domains, mediate recognition through the V domains. The V domains of VCBPs in amphioxus exhibit high levels of allelic complexity that presumably relate to functional specificity. Various features of the amphioxus immune repertoire reflect novel selective pressures, which likely have resulted in innovative strategies. Functional genomic studies underscore the value of amphioxus as a model for studying innate immunity and may help reveal how unique relationships between innate immune receptors and both pathogens and symbionts factored in the evolution of adaptive immune systems. PMID:22402506

  9. 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. PMID:25450230

  10. Fish genomes provide novel insights into the evolution of vertebrate secretin receptors and their ligand.

    PubMed

    Cardoso, João C R; Félix, Rute C; Trindade, Marlene; Power, Deborah M

    2014-12-01

    The secretin receptor (SCTR) is a member of Class 2 subfamily B1 GPCRs and part of the PAC1/VPAC receptor subfamily. This receptor has long been known in mammals but has only recently been identified in other vertebrates including teleosts, from which it was previously considered to be absent. The ligand for SCTR in mammals is secretin (SCT), an important gastrointestinal peptide, which in teleosts has not yet been isolated, or the gene identified. This study revises the evolutionary model previously proposed for the secretin-GPCRs in metazoan by analysing in detail the fishes, the most successful of the extant vertebrates. All the Actinopterygii genomes analysed and the Chondrichthyes and Sarcopterygii fish possess a SCTR gene that shares conserved sequence, structure and synteny with the tetrapod homologue. Phylogenetic clustering and gene environment comparisons revealed that fish and tetrapod SCTR shared a common origin and diverged early from the PAC1/VPAC subfamily group. In teleosts SCTR duplicated as a result of the fish specific whole genome duplication but in all the teleost genomes analysed, with the exception of tilapia (Oreochromis niloticus), one of the duplicates was lost. The function of SCTR in teleosts is unknown but quantitative PCR revealed that in both sea bass (Dicentrarchus labrax) and tilapia (Oreochromis mossambicus) transcript abundance is high in the gastrointestinal tract suggesting it may intervene in similar processes to those in mammals. In contrast, no gene encoding the ligand SCT was identified in the ray-finned fishes (Actinopterygii) although it was present in the coelacanth (lobe finned fish, Sarcopterygii) and in the elephant shark (holocephalian). The genes in linkage with SCT in tetrapods and coelacanth were also identified in ray-finned fishes supporting the idea that it was lost from their genome. At present SCTR remains an orphan receptor in ray-finned fishes and it will be of interest in the future to establish why SCT was

  11. The Genome of Laccaria Bi color Provides Insights into Mycorrhizal Symbiosis

    SciTech Connect

    Martin, F; Aerts, A.; Ahren, D; Brun, A; 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; Danchin, E; 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; 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; Tuskan, Gerald 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

  12. Echidna Venom Gland Transcriptome Provides Insights into the Evolution of Monotreme Venom

    PubMed Central

    Wong, Emily S. W.; Nicol, Stewart; Warren, Wesley C.; Belov, Katherine

    2013-01-01

    Monotremes (echidna and platypus) are egg-laying mammals. One of their most unique characteristic is that males have venom/crural glands that are seasonally active. Male platypuses produce venom during the breeding season, delivered via spurs, to aid in competition against other males. Echidnas are not able to erect their spurs, but a milky secretion is produced by the gland during the breeding season. The function and molecular composition of echidna venom is as yet unknown. Hence, we compared the deeply sequenced transcriptome of an in-season echidna crural gland to that of a platypus and searched for putative venom genes to provide clues into the function of echidna venom and the evolutionary history of monotreme venom. We found that the echidna venom gland transcriptome was markedly different from the platypus with no correlation between the top 50 most highly expressed genes. Four peptides found in the venom of the platypus were detected in the echidna transcriptome. However, these genes were not highly expressed in echidna, suggesting that they are the remnants of the evolutionary history of the ancestral venom gland. Gene ontology terms associated with the top 100 most highly expressed genes in echidna, showed functional terms associated with steroidal and fatty acid production, suggesting that echidna “venom” may play a role in scent communication during the breeding season. The loss of the ability to erect the spur and other unknown evolutionary forces acting in the echidna lineage resulted in the gradual decay of venom components and the evolution of a new role for the crural gland. PMID:24265746

  13. Moments of action provide insight into critical times for advection-diffusion-reaction processes

    NASA Astrophysics Data System (ADS)

    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.BIOJAU0006-349510.1016/j.bpj.2010.07.045 99, L59 (2010); Phys. Rev. EPLEEE81539-375510.1103/PhysRevE.83.051906 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.IJAMDM0272-496010.1093/imamat/47.2.193 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.

  14. Temporal-Spatial Transcriptome Analyses Provide Insights into the Development of Petaloid Androecium in Canna indica

    PubMed Central

    Tian, Xueyi; Yu, Qianxia; Liu, Huanfang; Liao, Jingping

    2016-01-01

    Canna indica (Zingiberales) is one of the most important ornamental species characterized with beautiful petaloid staminodes, which are considered to evolve from stamens. However, the genetic basis for the development of petaloid staminodes remains unclear largely because the genomic sequences are not available. By using RNA-Seq, we sequenced the transcripts in the flower of C. indica, and quantified the temporal gene expressions in flower primordium and differentiated flower, as well as the spatial gene expressions in petal and petaloid staminode. In total, 118,869 unigenes were assembled, among which 67,299 unigenes were annotated. Quantification analysis identified the differentially expressed genes in the temporal and spatial two comparisons, based on which, Gene Ontology enrichment analysis highlighted the representative terms in each sample, such as specification of organ number in flower primordium, growth in differentiated flower, secondary cell wall biogenesis in petal and cell division in petaloid staminode. Among the 51 analyzed MADS-box unigenes, 37 were up-regulated in differentiated flower compared with those in flower primordium. A-class unigenes were expressed higher in petal than in petaloid staminode, and C-class unigenes were expressed oppositely, whereas B-class unigenes demonstrated close expression levels in these two organs, indicating that petaloid staminode retains stamen identity to some degree. In situ hybridization provided more detailed expression patterns of these unigenes, and revealed the extended expression of B-class to the carpel at later stages when the style turned flat. These results constitute a preliminary basis for the study of flower development in C. indica and can be applied in further study of the evolution of Zingiberales. PMID:27582744

  15. Wing shape of four new bee fossils (Hymenoptera: Anthophila) provides insights to bee evolution.

    PubMed

    Dehon, Manuel; Michez, Denis; Nel, André; Engel, Michael S; De Meulemeester, Thibaut

    2014-01-01

    Bees (Anthophila) are one of the major groups of angiosperm-pollinating insects and accordingly are widely studied in both basic and applied research, for which it is essential to have a clear understanding of their phylogeny, and evolutionary history. Direct evidence of bee evolutionary history has been hindered by a dearth of available fossils needed to determine the timing and tempo of their diversification, as well as episodes of extinction. Here we describe four new compression fossils of bees from three different deposits (Miocene of la Cerdanya, Spain; Oligocene of Céreste, France; and Eocene of the Green River Formation, U.S.A.). We assess the similarity of the forewing shape of the new fossils with extant and fossil taxa using geometric morphometrics analyses. Predictive discriminant analyses show that three fossils share similar forewing shapes with the Apidae [one of uncertain tribal placement and perhaps near Euglossini, one definitive bumble bee (Bombini), and one digger bee (Anthophorini)], while one fossil is more similar to the Andrenidae. The corbiculate fossils are described as Euglossopteryx biesmeijeri De Meulemeester, Michez, & Engel, gen. nov. sp. nov. (type species of Euglossopteryx Dehon & Engel, n. gen.) and Bombus cerdanyensis Dehon, De Meulemeester, & Engel, sp. nov. They provide new information on the distribution and timing of particular corbiculate groups, most notably the extension into North America of possible Eocene-Oligocene cooling-induced extinctions. Protohabropoda pauli De Meulemeester & Michez, gen. nov. sp. nov. (type species of Protohabropoda Dehon & Engel, n. gen.) reinforces previous hypotheses of anthophorine evolution in terms of ecological shifts by the Oligocene from tropical to mesic or xeric habitats. Lastly, a new fossil of the Andreninae, Andrena antoinei Michez & De Meulemeester, sp. nov., further documents the presence of the today widespread genus Andrena Fabricius in the Late Oligocene of France. PMID

  16. Tectonic subsidence provides insight into possible coral reef futures under rapid sea-level rise

    NASA Astrophysics Data System (ADS)

    Saunders, Megan I.; Albert, Simon; Roelfsema, Chris M.; Leon, Javier X.; Woodroffe, Colin D.; Phinn, Stuart R.; Mumby, Peter J.

    2016-03-01

    Sea-level rise will change environmental conditions on coral reef flats, which comprise extensive habitats in shallow tropical seas and support a wealth of ecosystem services. Rapid relative sea-level rise of 0.6 m over a relatively pristine coral reef in Solomon Islands, caused by a subduction earthquake in April 2007, generated a unique opportunity to examine in situ coral reef response to relative sea-level rise of the magnitude (but not the rate) anticipated by 2100. Extent of live coral was measured from satellite imagery in 2003, 2006, 2009 and 2012. Ecological data were obtained from microatolls and ecological surveys in May 2013. The reef was sampled at 12 locations where dense live hard coral remained absent, remained present or changed from absent to present following subsidence. Ecological data (substratum depth, live coral canopy depth, coral canopy height, substratum suitability, recruitment, diversity and Acropora presence) were measured at each location to identify factors associated with coral response to relative sea-level rise. Vertical and horizontal proliferation of coral occurred following subsidence. Lateral expansion of live coral, accomplished primarily by branching Acropora spp., resulted in lower diversity in regions which changed composition from pavement to dense live coral following subsidence. Of the ecological factors measured, biotic factors were more influential than abiotic factors; species identity was the most important factor in determining which regions of the reef responded to rapid sea-level rise. On relatively pristine reef flats under present climatic conditions, rapid relative sea-level rise generated an opportunity for hard coral to proliferate. However, the species assemb