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991.
992.
Shoko Shinya Takayuki Ohnuma Reina Yamashiro Hisashi Kimoto Hideo Kusaoke Padmanabhan Anbazhagan André H. Juffer Tamo Fukamizo 《The Journal of biological chemistry》2013,288(42):30042-30053
Two carbohydrate binding modules (DD1 and DD2) belonging to CBM32 are located at the C terminus of a chitosanase from Paenibacillus sp. IK-5. We produced three proteins, DD1, DD2, and tandem DD1/DD2 (DD1+DD2), and characterized their binding ability. Transition temperature of thermal unfolding (Tm) of each protein was elevated by the addition of cello-, laminari-, chitin-, or chitosan-hexamer (GlcN)6. The Tm elevation (ΔTm) in DD1 was the highest (10.3 °C) upon the addition of (GlcN)6 and was markedly higher than that in DD2 (1.0 °C). A synergistic effect was observed (ΔTm = 13.6 °C), when (GlcN)6 was added to DD1+DD2. From isothermal titration calorimetry experiments, affinities to DD1 were not clearly dependent upon chain length of (GlcN)n; ΔGr° values were −7.8 (n = 6), −7.6 (n = 5), −7.6 (n = 4), −7.6 (n = 3), and −7.1 (n = 2) kcal/mol, and the value was not obtained for GlcN due to the lowest affinity. DD2 bound (GlcN)n with the lower affinities (ΔGr° = −5.0 (n = 3) ∼ −5.2 (n = 6) kcal/mol). Isothermal titration calorimetry profiles obtained for DD1+DD2 exhibited a better fit when the two-site model was used for analysis and provided greater affinities to (GlcN)6 for individual DD1 and DD2 sites (ΔGr° = −8.6 and −6.4 kcal/mol, respectively). From NMR titration experiments, (GlcN)n (n = 2∼6) were found to bind to loops extruded from the core β-sandwich of individual DD1 and DD2, and the interaction sites were similar to each other. Taken together, DD1+DD2 is specific to chitosan, and individual modules synergistically interact with at least two GlcN units, facilitating chitosan hydrolysis. 相似文献
993.
José M. Pastor Vicente Bernal Manuel Salvador Montserrat Argando?a Carmen Vargas Laszlo Csonka ángel Sevilla José L. Iborra Joaquín J. Nieto Manuel Cánovas 《The Journal of biological chemistry》2013,288(24):17769-17781
Bacterial osmoadaptation involves the cytoplasmic accumulation of compatible solutes to counteract extracellular osmolarity. The halophilic and highly halotolerant bacterium Chromohalobacter salexigens is able to grow up to 3 m NaCl in a minimal medium due to the de novo synthesis of ectoines. This is an osmoregulated pathway that burdens central metabolic routes by quantitatively drawing off TCA cycle intermediaries. Consequently, metabolism in C. salexigens has adapted to support this biosynthetic route. Metabolism of C. salexigens is more efficient at high salinity than at low salinity, as reflected by lower glucose consumption, lower metabolite overflow, and higher biomass yield. At low salinity, by-products (mainly gluconate, pyruvate, and acetate) accumulate extracellularly. Using [1-13C]-, [2-13C]-, [6-13C]-, and [U-13C6]glucose as carbon sources, we were able to determine the main central metabolic pathways involved in ectoines biosynthesis from glucose. C. salexigens uses the Entner-Doudoroff pathway rather than the standard glycolytic pathway for glucose catabolism, and anaplerotic activity is high to replenish the TCA cycle with the intermediaries withdrawn for ectoines biosynthesis. Metabolic flux ratios at low and high salinity were similar, revealing a certain metabolic rigidity, probably due to its specialization to support high biosynthetic fluxes and partially explaining why metabolic yields are so highly affected by salinity. This work represents an important contribution to the elucidation of specific metabolic adaptations in compatible solute-accumulating halophilic bacteria. 相似文献
994.
Joakim Lundqvist Ilka Braumann Marzena Kurowska André H. Müller Mats Hansson 《The Journal of biological chemistry》2013,288(33):24012-24019
The ATP-dependent insertion of Mg2+ into protoporphyrin IX is the first committed step in the chlorophyll biosynthetic pathway. The reaction is catalyzed by magnesium chelatase, which consists of three gene products: BchI, BchD, and BchH. The BchI and BchD subunits belong to the family of AAA+ proteins (ATPases associated with various cellular activities) and form a two-ring complex with six BchI subunits in one layer and six BchD subunits in the other layer. This BchID complex is a two-layered trimer of dimers with the ATP binding site located at the interface between two neighboring BchI subunits. ATP hydrolysis by the BchID motor unit fuels the insertion of Mg2+ into the porphyrin by the BchH subunit. In the present study, we explored mutations that were originally identified in semidominant barley (Hordeum vulgare L.) mutants. The resulting recombinant BchI proteins have marginal ATPase activity and cannot contribute to magnesium chelatase activity although they apparently form structurally correct complexes with BchD. Mixing experiments with modified and wild-type BchI in various combinations showed that an exchange of BchI subunits in magnesium chelatase occurs during the catalytic cycle, which indicates that dissociation of the complex may be part of the reaction mechanism related to product release. Mixing experiments also showed that more than three functional interfaces in the BchI ring structure are required for magnesium chelatase activity. 相似文献
995.
Hugh Kim Hervé Falet Karin M. Hoffmeister John H. Hartwig 《The Journal of biological chemistry》2013,288(48):34352-34363
Platelets are immunologically competent cells containing cytokines such as TGF-β1 that regulate cell-mediated immunity. However, the mechanisms underlying cytokine secretion from platelets are undefined. The Wiskott-Aldrich syndrome protein (WASp) regulates actin polymerization in nucleated hematopoietic cells but has other role(s) in platelets. WASp-null (WASp−/−) platelets stimulated with a PAR-4 receptor agonist had increased TGF-β1 release compared with WT platelets; inhibiting WASp function with wiskostatin augmented TRAP-induced TGF-β1 release in human platelets. TGF-β1 release is dissociated from α-granule secretion (P-selectin up-regulation) and occurs more gradually, with ∼10–15% released after 30–60 min. Blockade of Src family kinase-mediated WASp Tyr-291/Tyr-293 phosphorylation increased TGF-β1 release, with no additive effect in WASp−/− platelets, signifying that phosphorylation is critical for WASp-limited TGF-β1 secretion. Inhibiting F-actin assembly with cytochalasin D enhanced secretion in WT platelets and further increased TGF-β1 release in WASp−/− platelets, indicating that WASp and actin assembly independently regulate TGF-β1 release. A permeabilized platelet model was used to test the role of upstream small GTPases in TGF-β1 release. N17Cdc42, but not Rac1 mutants, increased TGF-β1 secretion and abrogated WASp phosphorylation. We conclude that WASp function restricts TGF-β1 secretion in a Cdc42- and Src family kinase-dependent manner and independently of actin assembly. 相似文献
996.
Ana Casa?al Ulrich Zander Cristina Mu?oz Florine Dupeux Irene Luque Miguel Angel Botella Wilfried Schwab Victoriano Valpuesta José A. Marquez 《The Journal of biological chemistry》2013,288(49):35322-35332
Pathogenesis-related 10 (PR-10) proteins are involved in many aspects of plant biology but their molecular function is still unclear. They are related by sequence and structural homology to mammalian lipid transport and plant abscisic acid receptor proteins and are predicted to have cavities for ligand binding. Recently, three new members of the PR-10 family, the Fra a proteins, have been identified in strawberry, where they are required for the activity of the flavonoid biosynthesis pathway, which is essential for the development of color and flavor in fruits. Here, we show that Fra a proteins bind natural flavonoids with different selectivity and affinities in the low μm range. The structural analysis of Fra a 1 E and a Fra a 3-catechin complex indicates that loops L3, L5, and L7 surrounding the ligand-binding cavity show significant flexibility in the apo forms but close over the ligand in the Fra a 3-catechin complex. Our findings provide mechanistic insight on the function of Fra a proteins and suggest that PR-10 proteins, which are widespread in plants, may play a role in the control of secondary metabolic pathways by binding to metabolic intermediates. 相似文献
997.
Michela Campagna Laura Marcos-Villar Francesca Arnoldi Carlos F. de la Cruz-Herrera Pedro Gallego José González-Santamaría Dolores González Fernando Lopitz-Otsoa Manuel S. Rodriguez Oscar R. Burrone Carmen Rivas 《Journal of virology》2013,87(2):807-817
Posttranslational modification by SUMO provides functional flexibility to target proteins. Viruses interact extensively with the cellular SUMO modification system in order to improve their replication, and there are numerous examples of viral proteins that are SUMOylated. However, thus far the relevance of SUMOylation for rotavirus replication remains unexplored. In this study, we report that SUMOylation positively regulates rotavirus replication and viral protein production. We show that SUMO can be covalently conjugated to the viroplasm proteins VP1, VP2, NSP2, VP6, and NSP5. In addition, VP1, VP2, and NSP2 can also interact with SUMO in a noncovalent manner. We observed that an NSP5 SUMOylation mutant protein retains most of its activities, such as its interaction with VP1 and NSP2, the formation of viroplasm-like structures after the coexpression with NSP2, and the ability to complement in trans the lack of NSP5 in infected cells. However, this mutant is characterized by a high degree of phosphorylation and is impaired in the formation of viroplasm-like structures when coexpressed with VP2. These results reveal for the first time a positive role for SUMO modification in rotavirus replication, describe the SUMOylation of several viroplasm resident rotavirus proteins, and demonstrate a requirement for NSP5 SUMOylation in the production of viroplasm-like structures. 相似文献
998.
Adriano Cavalleri André R. de Souza Fábio Prezoto Laurence A. Mound 《Biological journal of the Linnean Society. Linnean Society of London》2013,109(2):332-341
The insects known as thrips are commonly thought of as flower‐living and pestiferous organisms, but we report here a novel interaction between a phlaeothripine thrips species, Mirothrips arbiter gen. et sp. nov. and three species of social paper wasps in Brazil. This thrips species breeds inside the wasp colonies, and larval and adult thrips feed on wasp eggs, which become severely damaged. Infested nests can contain up to 300 M. arbiter gen. et sp. nov. individuals. The closest relatives of M. arbiter are two presumably predaceous species: Mirothrips bicolor sp. nov. , which inhabits abandoned Cecidomyiidae galls, and Mirothrips analis comb. nov. , described from individuals collected in the silken bags of the caterpillars of Psychidae moths. The behaviour exhibited by M. arbiter represents one of the most evolutionarily advanced lifestyles known among Thysanoptera, and we predict that other polistine species serve as hosts for this thrips in Brazil. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 332–341. 相似文献
999.
Szilvia Kusza Katalin Priskin Ante Ivankovic Bogumila Jedrzejewska Tomasz Podgorski András Jávor Sándor Mihók 《Biological journal of the Linnean Society. Linnean Society of London》2013,109(1):54-65
The aim of this work was to gather information about the origin and genetic characterization of the Central European Hucul horse based on 71 horses using 17 microsatellites and the D‐loop region of mtDNA. Their genetic relationship to the Polish Konik (N = 7), German (N = 4) and Hungarian wild Przewalski horses (N = 4) and 200 horse sequences from GenBank was also analysed. Both microsatellite and mtDNA analysis showed a high genetic variation in the Hucul. A total of 130 alleles were detected, the mean number of observed alleles per microsatellite was 7.647, and the number of effective alleles was 4.401. The average observed and expected heterozygosity were 0.706 and 0.747, respectively. The high heterozygosity values and Wright's fixation index (FIS) (?0.128) indicated a low level of inbreeding, low or no selection pressure, and large number of alleles. mtDNA analysis revealed 18 haplotypes for the Hucul population with a total of 23 variable sites. Haplotype and nucleotide diversities were 0.935 ± 0.011 and 0.022 ± 0.012, respectively. Neutrality tests (Tajima's D and Fu's Fs) were non‐significant, and mismatch distribution was ragged, indicating that the Hucul population is in genetic equilibrium. The most frequent mtDNA D‐loop region belonged to haplogroup A (48%), which was also present in Przewalski Wild horse samples, while Polish Konik samples belonged to three haplotypes and C, F, and G haplogroups. Large and significant pairwise ΦST values along with a small number of common haplotypes indicated a low level of gene flow and lack of genetic structure among the three studied breeds (Hucul, Konik, and Przewalski Wild horse). The present work contributes to our knowledge of the genetic diversity of the Hucul horse and helps to define its genetic conservation. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 54–65. 相似文献
1000.
Roberto Zenteno-Cuevas Francisco X Silva-Hernández Fabiola Mendoza-Damián Maria Dolores Ramírez-Hernández Karen Vázquez-Medina Lorena Widrobo-García Aremy Cuellar-Sanchez Raquel Mu?íz-Salazar Leonor Enciso-Moreno Lucia Monserrat Pérez-Navarro José Antonio Enciso-Moreno 《Memórias do Instituto Oswaldo Cruz》2013,108(6):718-723
Tuberculosis (TB) is an infectocontagious respiratory disease caused by members
of the Mycobacterium tuberculosis complex. A 7 base pair (bp)
deletion in the locus polyketide synthase
(pks)15/1 is described as polymorphic among members of the
M. tuberculosis complex, enabling the identification of
Euro-American, Indo-Oceanic and Asian lineages. The aim of this study was to
characterise this locus in TB isolates from Mexico. One hundred
twenty clinical isolates were recovered from the states of Veracruz and Estado
de Mexico. We determined the nucleotide sequence of a ± 400 bp fragment of the
locus pks15/1, while genotypic characterisation was
performed by spoligotyping. One hundred and fifty isolates contained the 7 bp
deletion, while five had the wild type locus. Lineages X (22%),
LAM (18%) and T (17%) were the most frequent; only three (2%) of the isolates
were identified as Beijing and two (1%) EAI-Manila. The wild type
pks15/1 locus was observed in all Asian lineage isolates
tested. Our results confirm the utility of locus pks15/1 as a
molecular marker for identifying Asian lineages of the M.
tuberculosis complex. This marker could be of great value in the
epidemiological surveillance of TB, especially in countries like Mexico, where
the prevalence of such lineages is unknown. 相似文献