21 research outputs found
Formation of a Trimeric Xpo1-Ran[GTP]-Ded1 Exportin Complex Modulates ATPase and Helicase Activities of Ded1.
No full textThe DEAD-box RNA helicase Ded1, which is essential in yeast and known as DDX3 in humans, shuttles between the nucleus and cytoplasm and takes part in several basic processes including RNA processing and translation. A key interacting partner of Ded1 is the exportin Xpo1, which together with the GTP-bound state of the small GTPase Ran, facilitates unidirectional transport of Ded1 out of the nucleus. Here we demonstrate that Xpo1 and Ran[GTP] together reduce the RNA-stimulated ATPase and helicase activities of Ded1. Binding and inhibition of Ded1 by Xpo1 depend on the affinity of the Ded1 nuclear export sequence (NES) for Xpo1 and the presence of Ran[GTP]. Association with Xpo1/Ran[GTP] reduces RNA-stimulated ATPase activity of Ded1 by increasing the apparent KM for the RNA substrate. Despite the increased KM, the Ded1:Xpo1:Ran[GTP] ternary complex retains the ability to bind single stranded RNA, suggesting that Xpo1/Ran[GTP] may modulate the substrate specificity of Ded1. These results demonstrate that, in addition to transport, exportins such as Xpo1 also have the capability to alter enzymatic activities of their cargo
The NES of Ded1ΔC is critical for a Ran[GTP]-dependent interaction with Xpo1.
No full text<p><b>(a)</b> The three protein components yield unique profiles by size exclusion chromatography. The profiles for each component applied individually on a Superdex 200 10/300 column are overlaid for comparison: Xpo1 (dark gray), Ded1ΔC (medium gray), and Ran[GTP]<sub>Q71L</sub> (light gray). The second, larger peak in the Ran[GTP]<sub>Q71L</sub> profile is excess GTP included in the injected sample. Analysis of each experiment is also shown by SDS-PAGE, illustrating the distribution of peak fractions of each protein in isolation. <b>(b)</b> Xpo1, Ded1ΔC and Ran[GTP]<sub>Q71L</sub> form a complex. The elution profile for Xpo1, Ded1ΔC and Ran[GTP]<sub>Q71L</sub> co-incubated together before injection is shown as a black trace. The three solid gray peaks of the individual components, as shown in (a), are given for comparison. As shown in the chromatogram and by SDS-PAGE, all three proteins elute in earlier fractions when co-incubated together, consistent with a higher molecular weight complex. <b>(c)</b> Ded1ΔC and Xpo1 do not form a stable complex without Ran[GTP]<sub>Q71L</sub>. The elution profile for Ded1ΔC and Xpo1 co-incubated together before injection is shown as a black trace. The three solid gray peaks of the individual components, as shown in (a), are given for comparison. As shown in the chromatogram and by SDS-PAGE, Ded1ΔC and Xpo1 elute at similar volumes as when they are injected individually, suggesting that a stable complex is not formed. <b>(d)</b> Ded1ΔC<sub>I13A</sub> does not form a complex with Xpo1 and Ran[GTP]<sub>Q71L</sub>. The elution profile for Xpo1, Ded1ΔC<sub>I13A</sub> and Ran[GTP]<sub>Q71L</sub> co-incubated together before injection is shown as a black trace. The three solid gray peaks of the individual components, as shown in (a), are given for comparison. As shown in the chromatogram and by SDS-PAGE, all three proteins elute at similar volumes as when they are injected individually, indicating that the I13A disruption of the NES interferes with formation of a stable complex. All chromatograms and elution profiles are representative of three or more experiments.</p
Ded1ΔC binds to the exterior of the Xpo1 toroid.
No full text<p><b>(a)</b> Raw scattering data for a purified Xpo1:Ran[GTP]<sub>Q71L</sub>:Ded1ΔC complex. <b>(b)</b> Gunier plot for the raw data in (a). The black squares indicate the linear Gunier region. <b>(c)</b> P(r) curve for the raw data in (a). <b>(d)</b> R<sub>g</sub> and D<sub>max</sub> values calculated for Xpo1 alone and in the presence of Ran[GTP]<sub>Q71L</sub> and Ded1ΔC. Published values are taken from ref. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131690#pone.0131690.ref033" target="_blank">33</a>]. <b>(e)</b> A putative docking of the crystal structure of snurportin 1 bound to Xpo1 and Ran[GTP] (3GJX; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131690#pone.0131690.ref013" target="_blank">13</a>]) with an averaged, filtered bead model calculated for the Xpo1: Ded1ΔC:Ran[GTP]<sub>Q71L</sub> complex.</p
Xpo1 and Ran[GTP] reduce but do not prevent ATP-dependent single-stranded RNA binding activity of Ded1.
No full text<p><b>(a)</b> FAM-labeled 25mer RNA oligomers were incubated in the presence and absence of Ded1 variants (900 nM), Xpo1 (990 nM), and Ran[GTP]<sub>Q71L</sub> (1.17 μM), and resolved by native PAGE after addition of excess unlabeled RNA. Reactions additionally incubated in the presence of the non-hydrolyzable ATP analog AMP-PNP showed super-shifted species, suggestive of protein-RNA complexes. Open triangles indicate shifted RNA species present when Ded1 is incubated with ATP and RNA alone; closed triangles indicate super-shifted RNA species present only in reactions containing Ded1:Xpo1:Ran[GTP] complexes. <b>(b)</b> Quantification of the fraction of super-shifted RNA from reactions containing AMP-PNP, full length Ded1 variants, and the presence or absence of Ran[GTP]<sub>Q71L</sub> and Xpo1. Averages and standard deviations shown are from three independent experiments.</p
Xpo1 and Ran[GTP] reduce the ATPase activity of Ded1 in an NES-dependent manner.
No full text<p><b>(a)</b> The RNA-dependent ATPase activity of Ded1 is reduced in the presence of Xpo1/Ran[GTP]. The rates of ATP hydrolysis for either wildtype (+) or an ATPase-dead variant (Ded1<sub>D306N</sub>) of Ded1 (500 nM) in the presence or absence of yeast whole-cell RNA (320 μg/mL), Xpo1 and/or Ran[GTP]<sub>Q71L</sub>, as indicated. The approximate molar ratios of Xpo1/Ran[GTP]<sub>Q71L</sub> relative to Ded1 are indicated, corresponding to Xpo1/Ran[GTP]<sub>Q71L</sub> concentrations of 0.55/0.65 μM (1x), 1.1/1.3 μM (2x), 2.75/3.25 μM (5x), 5.5/6.5 μM (10x), and 11/13 μM (20x). The reaction shown in column 13 additionally contained Ded1(1–368)<sub>sNES</sub> (12.5 μM), the truncated and ATPase-dead version of Ded1, which relieved inhibition by Xpo1 and Ran[GTP]. <b>(b)</b> A tight-binding NES reduced the amount of Xpo1 and Ran[GTP] required for maximal inhibition of Ded1 ATPase activity. RNA-stimulated ATPase activities of Ded1<sub>sNES</sub> are shown in the absence (column 15) and presence (columns 16–20) of Xpo1 and Ran[GTP]<sub>Q71L</sub>. The concentrations of Xpo1 and Ran[GTP]<sub>Q71L</sub> are the same as those shown in columns 8–12 of (a). <b>(c)</b> Disrupting the NES of Ded1 with the I13A substitution prevents inhibition by Xpo1 and Ran[GTP]. RNA-stimulated ATPase activities of Ded1<sub>I13A</sub> are shown in the absence (column 21) and presence (columns 22–26) of Xpo1 and Ran[GTP]<sub>Q71L</sub>. The concentrations of Xpo1 and Ran[GTP]<sub>Q71L</sub> are the same as those shown in columns 8–12 of (a). **P<0.0005; n.s., not significant.</p
Xpo1 and Ran[GTP] reduce the RNA unwinding activity of Ded1.
No full text<p><b>(a)</b> RNA duplexes consisting of a FAM-labeled 13mer annealed to an unlabeled 41mer were incubated with Ded1 variants (50 nM) under unwinding conditions in the presence and absence of Xpo1 (550 nM) and Ran[GTP]<sub>Q71L</sub> (650 nM). The time points used for each experiment were 0.33, 0.66, 1, 1.33. 2, 10, 20, and 90 min. The reactions were monitored by native PAGE, with a representative gel shown from three independent experiments. <b>(b)</b> Quantification of RNA unwinding experiments. Each set of points represents the average of three separate experiments. Single exponential fits are shown for Ded1 variants alone (solid lines) and in the presence of Xpo1/Ran[GTP]<sub>Q71L</sub> (dotted lines). <b>(c)</b> Values for k<sub>unwind</sub> for data plotted in (b). The reductions in helicase activity upon addition of Xpo1 and Ran[GTP], given as fold-inhibition, are the ratios of k<sub>unwind</sub> values in the absence and presence of Xpo1 and Ran[GTP].</p
Synthesis and antimicrobial evaluation of new nitric oxide‐donating fluoroquinolone/oxime hybrids
No full textA new series of nitric oxide‐donating fluoroquinolone/oximes was prepared in this study. The nitric oxide release from the prepared compounds was measured using a modified Griess colorimetric method. The antitubercular evaluation of the synthesized compounds indicated that ketone derivatives 2b and 2e and oximes 3b and 3d exhibited somewhat higher activity than their respective parent fluoroquinolones. Mycobacterial DNA cleavage studies and molecular modeling of Mycobacterium tuberculosis DNA gyrase were pursued to explain the observed bioactivity. More important, antibacterial evaluation showed that oximes 3c–e are highly potent against Klebsiella pneumoniae, with minimum inhibitory concentration (MIC) values of 0.06, 0.08, and 0.034 µM, respectively, whereas ketone 2c and oxime 4c are more active against Staphylococcus aureus than ciprofloxacin (MIC values: 0.7, 0.38, and 1.6 µM, respectively). Notably, the antipseudomonal activities of compounds 2a and 4c were much higher than those of their respective parent fluoroquinolones
New fluoroquinolones/nitric oxide donor hybrids: design, synthesis and antitubercular activity
No full textNew nitric oxide (NO) donating fluoroquinolones/nitrate ester hybrids were prepared and their structures were characterizedby various spectroscopic and analytical tools. The release of NO from the prepared nitrate esters was measured using the modified Griess colorimetric method. Evaluation of antitubercular activity showed that most of tested compounds exhibited comparable or higher activity than the parent fluoroquinolones. Compounds 2b, 3a, 4a, 5a, and 2d showed better activity than ciprofloxacin. Nevertheless, none of the new compounds were superior to the parent fluoroquinolones in terms of DNA cleavage stimulation in mycobacteria. The additional growth inhibition effect that is distinct from gyrase poisoning may be due to release of NO or enhancement of lipophilicity. These data are augmented by docking results where the docked compounds did not exert additional significant bindings over the parent fluoroquinolones
Design, synthesis and molecular docking of new N-4-piperazinyl ciprofloxacin-triazole hybrids with potential antimicrobial activity
No full textNew N-4-piperazinyl ciprofloxacin-triazole hybrids 6a-o were prepared and characterized. The in vitro antimycobacterial activity revealed that compound 6a experienced promising antimycobacterial activity against Mycobactrium smegmatis compared with the reference isoniazide (INH). Additionally, compound 6a exhibited broad spectrum antibacterial activity against all the tested strains either Gram-positive or Gram-negative bacteria compared with the reference ciprofloxacin. Also, compounds 6g and 6i displayed considerable antifungal activity compared with the reference ketoconazole. DNA cleavage assay of the highly active compounds 6c and 6h showed a good correlation between the Mycobactrium cleaved DNA gyrase assay and their in vitro antimycobactrial activity. Moreover, molecular modeling studies were done for the designed ciprofloxacin derivatives to predict their binding modes towards Topoisomerase II enzyme (PDB: 5bs8)
Recommended from our members
PRE-SHOT SIMULATIONS OF NEAR-FIELD AND FAR-FIELD GROUND MOTION FOR THE SOURCE PHYSICS EXPERIMENT (SPE) EXPLOSIONS AT THE CLIMAX STOCK, NEVADA NATIONAL SECURITY SITE: SPE3
No full text
