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31.
Sidi AO Babah KO Brimer N Nominé Y Romier C Kieffer B Pol SV Travé G Zanier K 《Protein expression and purification》2011,80(1):8-16
E6 is a small oncoprotein involved in tumorigenesis induced by papillomaviruses (PVs). E6 often recognizes its cellular targets by binding to short motifs presenting the consensus LXXLL. E6 proteins have long resisted structural analysis. We found that bovine papillomavirus type 1 (BPV1) E6 binds the N-terminal LXXLL motif of the cellular protein paxillin with significantly higher affinity as compared to other E6/peptide interactions. Although recombinant BPV1 E6 was poorly soluble in the free state, provision of the paxillin LXXLL peptide during BPV1 E6 biosynthesis greatly enhanced the protein's solubility. Expression of BPV1 E6/LXXLL peptide complexes was carried out in bacteria in the form of triple fusion constructs comprising, from N- to C-terminus, the soluble carrier protein maltose binding protein (MBP), the LXXLL motif and the E6 protein. A TEV protease cleavage site was placed either between MBP and LXXLL motif or between LXXLL motif and E6. These constructs allowed us to produce highly concentrated samples of BPV1 E6, either covalently fused to the C-terminus of the LXXLL motif (intra-molecular complex) or non-covalently bound to it (inter-molecular complex). Heteronuclear NMR measurements were performed and showed that the E6 protein was folded with similar conformations in both covalent and non-covalent complexes. These data open the way to novel structural and functional studies of the BPV1 E6 in complex with its preferential target motif. 相似文献
32.
Experiments were conducted to determine whether low-speed swimming during recovery from exhaustive exercise improved both metabolic recovery and performance during a swimming challenge. For these experiments, brook trout were allowed to recover from exhaustive exercise for 2 h while swimming at 0, 0.5, 1.0, or 1.5 body length (BL) s(-1) or allowed to recover from exhaustive exercise for 1, 2, or 3 h while swimming at 1.0 BL s(-1). At the appropriate interval, either (i) muscle and blood samples were removed from the fish or (ii) fish were assessed for performance (i.e., fatigue time) during a fixed-interval swimming test. Low-speed swimming during recovery from exhaustive exercise resulted in significantly longer fatigue times compared with fish recovering in still water (i.e., 0 BL s(-1)). However, swimming during recovery did not expedite recovery of muscle lactate or blood variables (e.g., lactate, osmolarity, glucose). These observations suggest that metabolic recovery and subsequent swimming performance may not be directly linked and that other factors play a role in swimming recovery in brook trout. 相似文献
33.
34.
Under iron-deficient conditions, the Gram-negative bacterium Pseudomonas aeruginosa ATCC 15692 secretes a peptidic siderophore, pyoverdin PaA, composed of an aromatic chromophore derived from 2,3-diamino-6,7-dihydroxyquinoline and a partially cyclized octapeptide, D-Ser-L-Arg-D-Ser-L-FoOHOrn-(L-Lys-L-FoOHOrn-L-Thr-L-Thr) (FoOHOrn: delta N-formyl-delta N-hydroxyornithine), in which the C-terminal carboxyl group forms a peptidic bond with the primary amine of the L-Lys side chain. Ferric iron is chelated by the catechol group on the chromophore and the two hydroxyornithine side chains. In aqueous solution, the (1)H-NMR spectrum of pyoverdin PaA-Ga(III), in which Ga(III) is used instead of Fe(III) for spectroscopic purposes, showed clear evidence of exchange broadening, preventing further structural characterization. The use of cryo-solvents allowed measurements to be made at temperatures as low as 253 K where two distinct conformations with roughly equivalent populations could be observed. (13)C and (15)N labeling of pyoverdin PaA enabled complete assignment of both forms of pyoverdin PaA-Ga(III) at 253 and 267 K, using triple-resonance multidimensional NMR experiments commonly applied to doubly labeled proteins. 相似文献
35.
Brian C. Brajcich Andrew L. Iarocci Lindsey A. G. Johnstone Rory K. Morgan Zachary T. Lonjers Matthew J. Hotchko Jordan D. Muhs Amanda Kieffer Bree J. Reynolds Sarah M. Mandel Beth N. Marbois Catherine F. Clarke Jennifer N. Shepherd 《Journal of bacteriology》2010,192(2):436-445
Rhodoquinone (RQ) is an important cofactor used in the anaerobic energy metabolism of Rhodospirillum rubrum. RQ is structurally similar to ubiquinone (coenzyme Q or Q), a polyprenylated benzoquinone used in the aerobic respiratory chain. RQ is also found in several eukaryotic species that utilize a fumarate reductase pathway for anaerobic respiration, an important example being the parasitic helminths. RQ is not found in humans or other mammals, and therefore inhibition of its biosynthesis may provide a parasite-specific drug target. In this report, we describe several in vivo feeding experiments with R. rubrum used for the identification of RQ biosynthetic intermediates. Cultures of R. rubrum were grown in the presence of synthetic analogs of ubiquinone and the known Q biosynthetic precursors demethylubiquinone, demethoxyubiquinone, and demethyldemethoxyubiquinone, and assays were monitored for the formation of RQ3. Data from time course experiments and S-adenosyl-l-methionine-dependent O-methyltransferase inhibition studies are discussed. Based on the results presented, we have demonstrated that Q is a required intermediate for the biosynthesis of RQ in R. rubrum.Rhodospirillum rubrum is a well-characterized and metabolically diverse member of the family of purple nonsulfur bacteria (29, 61). R. rubrum is typically found in aquatic environments and can adapt to a variety of growth conditions by using photosynthesis, respiration, or fermentation pathways (28, 70). In the light, R. rubrum exhibits photoheterotrophic growth using organic substrates or photoautotrophic growth using CO2 and H2 (15, 70). In the dark, R. rubrum can utilize either aerobic respiration (70, 73) or anaerobic respiration with a fumarate reduction pathway or with nonfermentable substrates in the presence of oxidants such as dimethyl sulfoxide (DMSO) or trimethylamine oxide (15, 58, 73). R. rubrum can also grow anaerobically in the dark by fermentation of sugars in the presence of bicarbonate (58). The focus of this work was the biosynthesis of quinones used by R. rubrum for aerobic and anaerobic respiration.Rhodoquinone (RQ; compound 1 in Fig. Fig.1)1) is an aminoquinone structurally similar to ubiquinone (coenzyme Q or Q [compound 2]) (44); however, the two differ considerably in redox potential (that of RQ is −63 mV, and that of Q is +100 mV) (2). Both RQ and Q have a fully substituted benzoquinone ring and a polyisoprenoid side chain that varies in length (depending on the species; see Fig. Fig.11 for examples). The only difference between the structures is that RQ has an amino substituent (NH2) instead of a methoxy substituent (OCH3) on the quinone ring. While Q is a ubiquitous lipid component involved in aerobic respiratory electron transport (9, 36, 60), RQ functions in anaerobic respiration in R. rubrum (19) and in several other phototrophic purple bacteria (21, 22, 41) and is also present in a few aerobic chemotrophic bacteria, including Brachymonas denitrificans and Zoogloea ramigera (23). In these varied species of bacteria, RQ has been proposed to function in fumarate reduction to maintain NAD+/NADH redox balance, either during photosynthetic anaerobic metabolism (12, 15-18, 64) or in chemotrophic metabolism when the availability of oxygen as a terminal oxidant is limiting (23). Another recent finding is that RQH2 is capable of inducing Q-cycle bypass reactions in the cytochrome bc1 complex in Saccharomyces cerevisiae, resulting in superoxide formation (7). If RQ/RQH2 coexists in the cytoplasmic membrane with Q/QH2 in R. rubrum, it might serve as both a substrate for and an inhibitor of the bc1 complex (47).Open in a separate windowFIG. 1.Proposed pathways for RQ biosynthesis. The number of isoprene units (n) varies by species (in S. cerevisiae, n = 6; in E. coli, n = 8; in C. elegans, n = 9; in helminth parasites, n = 9 or 10; in R. rubrum, n = 10; in humans, n = 10). RQ is not found in S. cerevisiae, E. coli, or humans. Known Coq (from S. cerevisiae) and Ubi (from E. coli) gene products required for the biosynthesis of ubiquinone (Q, compound 2) are labeled. A polyisoprenyl diphosphate (compound 5) is assembled from dimethylallyl disphosphate (compound 3) and isopentyl diphosphate (compound 4). Coupling of compound 5 with p-hydroxybenzoic acid (compound 6) yields 3-polyprenyl-4-hydroxybenzoic acid (compound 7). The next three steps differ between S. cerevisiae and E. coli. However, they merge at the common intermediate (compound 8), which is oxidized to demethyldemethoxyubiquinone (DDMQn, compound 9). RQ (compound 1) has been proposed to arise from compound 9, demethoxyubiquinone (DMQn; compound 10), demethylubiquinone (DMeQn; compound 11), or compound 2 (by pathway A, B, C, or D). Results presented in this work support pathway D as the favored route for RQ biosynthesis in R. rubrum.RQ is also found in the mitochondrial membrane of eukaryotic species capable of fumarate reduction, such as the flagellate Euglena gracilis (25, 53), the free-living nematode Caenorhabditis elegans (62), and the parasitic helminths (65, 66, 68, 72). Similar to R. rubrum, these species can adapt their metabolism to both aerobic and anaerobic conditions throughout their life cycle. For example, most adult parasitic species (e.g., Ascaris suum, Fasciola hepatica, and Haemonchus contortus) rely heavily on fumarate reduction for their energy generation while inside a host organism, where the oxygen tension is very low (30, 65, 72). Under these conditions, the biosynthesis of RQ is upregulated; however, during free-living stages of their life cycle, the helminth parasites use primarily aerobic respiration, which requires Q (30, 65, 72). The anaerobic energy metabolism of the helminthes has been reviewed (63, 67). Humans and other mammalian hosts use Q for aerobic energy metabolism but do not produce or require RQ; therefore, selective inhibition of RQ biosynthesis may lead to highly specific antihelminthic drugs that do not have a toxic effect on the host (35, 48).R. rubrum is an excellent facultative model system for the study of RQ biosynthesis. The complete genome of R. rubrum has recently been sequenced by the Department of Energy Joint Genome Institute, finished by the Los Alamos Finishing Group, and further validated by optical mapping (57). The 16S rRNA sequence of R. rubrum is highly homologous to cognate eukaryotic mitochondrial sequences (46). Due to the similarities in structure, the biosynthetic pathways of RQ and Q have been proposed to diverge from a common precursor (67). Proposed pathways for RQ biosynthesis (A to D), in conjunction with the known steps in Q biosynthesis, are outlined in Fig. Fig.11 (31, 34, 60). Parson and Rudney previously showed that when R. rubrum was grown anaerobically in the light in the presence of [U-14C]p-hydroxybenzoate, 14C was incorporated into both Q10 and RQ10 (50). In their growth experiments, the specific activity of Q10 was measured at its maximal value 15 h after inoculation and then began to decrease. However, the specific activity of RQ10 continued to increase for 40 h before declining. These results suggested that Q10 was a biosynthetic precursor of RQ10, although this was not directly demonstrated using radiolabeled Q10; hence, the possibility remained that the labeled RQ10 was derived from another radiolabeled lipid species. We have done this feeding experiment with a synthetic analog of Q where n = 3 (Q3) and monitored for the production of RQ3. The synthesis and use of farnesylated quinone and aromatic intermediates for characterization of the Q biosynthetic pathway in S. cerevisiae and Escherichia coli has been well documented (4, 5, 38, 52, 59). The other proposed precursors of RQ shown in Fig. Fig.11 were also fed to R. rubrum, and the lipid extracts from these assays were analyzed for the presence of RQ3, i.e., demethyldemethoxyubiquinone-3 (DDMQ3; compound 9), demethoxyubiquinone-3 (DMQ3; compound 10), and demethylubiquinone-3 (DMeQ3; compound 11).In S. cerevisiae and E. coli, the last O-methylation step in Q biosynthesis is catalyzed by the S-adenosyl-l-methionine (SAM)-dependent methyltransferases Coq3 and UbiG, respectively (26, 52); this final methylation step converts DMeQ to Q. Using the NCBI Basic Local Alignment Search Tool, an O-methyltransferase (GeneID no. 3834724 Rru_A0742) that had 41% and 59% sequence identity with Coq3 and UbiG, respectively, was identified in R. rubrum. S-Adenosyl-l-homocysteine (SAH) is a well-known inhibitor of SAM-dependent methyltransferases (13, 24). Because SAH is the transmethylation by-product of SAM-dependent methyltransferases, it is not readily taken up by cells and must be generated in vivo (24). SAH can be produced in vivo from S-adenosine and l-homocysteine thiolactone by endogenous SAH hydrolase (SAHH) (37, 71). A search of the R. rubrum genome also confirmed the presence of a gene encoding SAHH (GeneID no. 3836896 Rru_A3444). It was proposed that if DMeQ is the immediate precursor of RQ, then SAH inhibition of the methyltransferase required for Q biosynthesis should have little effect on RQ production. Conversely, if Q is required for RQ synthesis, then inhibition of Q biosynthesis should have a significant effect on RQ production. Assays were designed to quantify the levels of RQ3 produced from DMeQ3 and Q3 in R. rubrum cultures at various concentrations of SAH. 相似文献
36.
37.
In contrast to animals, organogenesis in plants is continuous, allowing development in response to intrinsic and extrinsic signals. Organs arise from primordia formed on the flanks of meristems. The apical meristem produces primordia that acquire leaf identity, while floral meristems form primordia which develop into four organ types: sepals, petals, stamens and carpels. The production of mature organs involves two distinct processes, the initiation of organ primordia and the establishment of meristem, primordia and cell identities. Here we concentrate on floral organogenesis in Arabidopsis and examine the extent to which these processes utilize similar control mechanisms and regulatory molecules. 相似文献
38.
Rezaï X Faget L Bednarek E Schwab Y Kieffer BL Massotte D 《Cellular and molecular neurobiology》2012,32(4):509-516
Delta opioid receptors participate in the control of chronic pain and emotional responses. Recent data have also identified
their implication in drug-context associations pointing to a modulatory role on hippocampal activity. We used fluorescent
knock-in mice that express a functional delta opioid receptor fused at its carboxy terminus with the green fluorescent protein
in place of the native receptor to investigate the receptor neuroanatomical distribution in this structure. Fine mapping of
the pyramidal layer was performed in hippocampal acute brain slices and organotypic cultures using fluorescence confocal imaging,
co-localization with pre- and postsynaptic markers and correlative light-electron microscopy. The different approaches concurred
to identify delta opioid receptors on presynaptic afferents to glutamatergic principal cells. In the latter, only scarce receptors
were detected that were confined within the Golgi or vesicular intracellular compartments with no receptor present at the
cell surface. In the mouse hippocampus, expression of functional delta opioid receptors is therefore mostly associated with
interneurons emphasizing a presynaptic modulatory effect on the pyramidal cell firing rate. 相似文献
39.
STYLIANOS MICHAIL SIMAIAKIS EVEN TJØRVE GABRIELE GENTILE ALESSANDRO MINELLI MOISIS MYLONAS 《Biological journal of the Linnean Society. Linnean Society of London》2012,105(1):146-159
The present study article examines the shapes of centipede species–area relationships (SARs) in the Mediterranean islands, compares the results of the linear form of the power model between archipelagos, discusses biological significance of the power model parameters with other taxa on the Aegean archipelago, and tests for a significant small‐island effect (SIE). We used 11 models to test the SARs and we compared the quality‐of‐fit of all candidate models. The power function ranked first and Z‐values was in the range 0.106–0.334. We assessed the presence of SIEs by fitting both a continuous and discontinuous breakpoint regression model. The continuous breakpoint regression functions never performed much better than the closest discontinuous model as a predictor of centipede species richness. We suggest that the relatively low Z‐values in our data partly reflect better dispersal abilities in centipedes than in other soil invertebrate taxa. Longer periods of isolation and more recent island formation may explain the somewhat lower constant c in the western Mediterranean islands compared to the Aegean islands. Higher breakpoint values in the western Mediterranean may also be a result of larger distance to the mainland and longer separation times. Despite the differences in the geological history and the idiosyncratic features of the main island groups considered, the overall results are quite similar and this could be assigned to the ability of centipedes to disperse across isolation barriers. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 146–159. 相似文献
40.
Kieffer TL De Meyer S Bartels DJ Sullivan JC Zhang EZ Tigges A Dierynck I Spanks J Dorrian J Jiang M Adiwijaya B Ghys A Beumont M Kauffman RS Adda N Jacobson IM Sherman KE Zeuzem S Kwong AD Picchio G 《PloS one》2012,7(4):e34372