Characterization of the ATPase and unwinding activities of the yeast DEAD-box protein Has1p and the analysis of the roles of the conserved motifs

The yeast DEAD-box protein Has1p is required for the maturation of 18S rRNA, the biogenesis of 40S r-subunits and for the processing of 27S pre-rRNAs during 60S r-subunit biogenesis. We purified recombinant Has1p and characterized its biochemical activities. We show that Has1p is an RNA-dependent ATPase in vitro and that it is able to unwind RNA/DNA duplexes in an ATP-dependent manner. We also report a mutational analysis of the conserved residues in motif I (86AKTGSGKT93), motif III (228SAT230) and motif VI (375HRVGRTARG383). The in vivo lethal K92A substitution in motif I abolishes ATPase activity in vitro. The mutations S228A and T230A partially dissociate ATPase and helicase activities, and they have cold-sensitive and lethal growth phenotypes, respectively. The H375E substitution in motif VI significantly decreased helicase but not ATPase activity and was lethal in vivo. These results suggest that both ATPase and unwinding activities are required in vivo. Has1p possesses a Walker A-like motif downstream of motif VI (383GTKGKGKS390). K389A substitution in this motif significantly increases the Has1p activity in vitro, which indicates it potentially plays a role as a negative regulator. Finally, rRNAs and poly(A) RNA serve as the best stimulators of the ATPase activity of Has1p among the tested RNA

Characterization of the ATPase and unwinding activities of the yeast DEAD-box protein Has1p and the analysis of the roles of the conserved motifs

The yeast DEAD-box protein Has1p is required for the maturation of 18S rRNA, the biogenesis of 40S r-subunits and for the processing of 27S pre-rRNAs during 60S r-subunit biogenesis. We purified recombinant Has1p and characterized its biochemical activities. We show that Has1p is an RNA-dependent ATPase in vitro and that it is able to unwind RNA/DNA duplexes in an ATP-dependent manner. We also report a mutational analysis of the conserved residues in motif I ((86)AKTGSGKT(93)), motif III ((228)SAT(230)) and motif VI ((375)HRVGRTARG(383)). The in vivo lethal K92A substitution in motif I abolishes ATPase activity in vitro. The mutations S228A and T230A partially dissociate ATPase and helicase activities, and they have cold-sensitive and lethal growth phenotypes, respectively. The H375E substitution in motif VI significantly decreased helicase but not ATPase activity and was lethal in vivo. These results suggest that both ATPase and unwinding activities are required in vivo. Has1p possesses a Walker A-like motif downstream of motif VI ((383)GTKGKGKS(390)). K389A substitution in this motif significantly increases the Has1p activity in vitro, which indicates it potentially plays a role as a negative regulator. Finally, rRNAs and poly(A) RNA serve as the best stimulators of the ATPase activity of Has1p among the tested RNAs

Točnost sinteze seril-tRNA

The high level of translational fidelity is ensured by various types of quality control mechanisms, which are adapted to prevent or correct naturally occurring mistakes. Accurate aminoacyl-tRNA synthesis is mostly dependent on the specificity of the aminoacyl-tRNA synthetases (aaRS), i.e. their ability to choose among competing structurally similar substrates. Our studies have revealed that accurate seryl-tRNA synthesis in yeast and plants is accomplished via tRNA-assisted optimization of amino acid binding to the active site of seryl-tRNA synthetase (SerRS). Based on our recent kinetic data, a mechanism is proposed by which transient protein : RNA complex activates the cognate amino acid more efficiently and more specifically than the apoenzyme alone. This may proceed via a tRNA induced conformational change in the enzyme’s active site. The influence of tRNASer, on the activation of serine by SerRS variants mutated in the active site, is much less pronounced. Although SerRS misactivates structurally similar threonine in vitro, the formation of such erroneous threonyl-adenylate is reduced in the presence of nonchargeable tRNASer analog. Thus, the sequence-specific tRNA : SerRS interactions enhance the accuracy of amino acid recognition. Another type of quality control mechanism in tRNA serylation is assumed to be based on the complex formation between SerRS and a nonsynthetase protein. Using in vivo interaction screen, yeast peroxin Pex21p was identified as SerRS interacting protein. This was confirmed by an in vitro binding assay. Kinetic experiments performed in the presence of Pex21p revealed that this peroxin acts as an activator of seryl-tRNA synthetase in the aminoacylation reaction.Točnost biosinteze proteina nadziru različiti kontrolni mehanizmi koji sprečavaju ili ispravljaju gre{ke u translaciji. Specifičnost aminoacil tRNA-sintetaza (aaRS) pri izboru i kovalentnom povezivanju pripadnih aminokiselina i tRNA ključna je u ovom procesu. Istraživanja u našem laboratoriju pokazala su da se specifičnost i učinkovitost sinteze seril-tRNA u kvascu i biljkama povećavaju tRNA-ovisnim prilagođavanjem veznog mjesta za serin u aktivnom mjestu seril tRNA-sintetaze (SerRS). Dakle, makromolekularni kompleksi tRNA i enzima imaju bolja katalitička svojstva od apoenzima. Naši rezultati kinetike pokazuju da se vezanjem tRNA bitno mijenja konformacija veznog mjesta za serin u enzimu divljeg tipa, dok je konformacijska promjena slabija kod enzima s mutacijama u aktivnom mjestu. Iako SerRS može aktivirati i serinu sličan treonin, stvaranje treonil adenilata smanjeno je u prisutnosti aminoacilacijski inaktivnog analoga tRNA. Time je pokazano da su interakcije između pripadne tRNA i SerRS bitne za točan izbor aminokiseline. Djelotvornost serilacije povećava se i interakcijom SerRS s nesintetaznim proteinom, peroksinom Pex21p. Ta neočekivana interakcija uočena je prvo in vivo, pretragom kvaščeve biblioteke u sustavu dvaju hibrida sa SerRS kao interakcijskim proteinom. Interakcija je potvrđena in vitro. Kinetički eksperimenti pokazali su da Pex21p djeluje kao aktivator SerRS, što ovu neobičnu interakciju čini biološki značajnom jer povećava učinkovitost aminoaciliranja

Subcutaneous Interferon Beta-1a inPediatric Multiple Sclerosis: A Retrospective Study

To expand current knowledge, we examined the safety and tolerability of subcutaneous interferon b-1a in patients with pediatriconset multiple sclerosis. Records from 307 patients who had received at least 1 injection of subcutaneous interferon b-1a for demyelinating events when aged younger than 18 years were reviewed. Overall, 168 (54.7%) patients had at least 1 prespecified medical event related to or under close monitoring with subcutaneous interferon b-1a or specific to pediatric patients, 184 (59.9%) had nonserious medical events related to treatment or of unknown causality, and 12 (3.9%) had serious medical events irrespective of causality. The most common laboratory abnormalities were increased alanine (74/195; 37.9%) and aspartate aminotransferase levels (59/194; 30.4%). Annualized relapse rates were 1.79 before treatment and 0.47 during treatment. In conclusion, adult doses of subcutaneous interferon b-1a (44 and 22 mg, 3 times weekly) were well tolerated in pediatric patients and were associated with reduced relapse rates

The Accuracy of Seryl-tRNA Synthesis

The high level of translational fidelity is ensured by various types of quality control mechanisms, which are adapted to prevent or correct naturally occurring mistakes. Accurate aminoacyl-tRNA synthesis is mostly dependent on the specificity of the aminoacyl-tRNA synthetases (aaRS), i.e. their ability to choose among competing structurally similar substrates. Our studies have revealed that accurate seryl-tRNA synthesis in yeast and plants is accomplished via tRNA-assisted optimization of amino acid binding to the active site of seryl-tRNA synthetase (SerRS). Based on our recent kinetic data, a mechanism is proposed by which transient protein : RNA complex activates the cognate amino acid more efficiently and more specifically than the apoenzyme alone. This may proceed via a tRNA induced conformational change in the enzyme’s active site. The influence of tRNASer, on the activation of serine by SerRS variants mutated in the active site, is much less pronounced. Although SerRS misactivates structurally similar threonine in vitro, the formation of such erroneous threonyl-adenylate is reduced in the presence of nonchargeable tRNASer analog. Thus, the sequence-specific tRNA : SerRS interactions enhance the accuracy of amino acid recognition. Another type of quality control mechanism in tRNA serylation is assumed to be based on the complex formation between SerRS and a nonsynthetase protein. Using in vivo interaction screen, yeast peroxin Pex21p was identified as SerRS interacting protein. This was confirmed by an in vitro binding assay. Kinetic experiments performed in the presence of Pex21p revealed that this peroxin acts as an activator of seryl-tRNA synthetase in the aminoacylation reaction

Aliquots (1 μg) of purified wild-type (Has1p) and mutant proteins were analyzed by SDS–PAGE, and the polypeptides were visualized by staining with Coomassie blue

<p><b>Copyright information:</b></p><p>Taken from "Characterization of the ATPase and unwinding activities of the yeast DEAD-box protein Has1p and the analysis of the roles of the conserved motifs"</p><p>Nucleic Acids Research 2005;33(3):999-1009.</p><p>Published online 17 Feb 2005</p><p>PMCID:PMC549409.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> The positions of marker proteins (in kDa) are indicated at the left. Proteins K92A–K389A and R376A are not shown; they were purified to the comparable purity and homogeneity

Ziritaxestat, a Novel Autotaxin Inhibitor, and Lung Function in Idiopathic Pulmonary Fibrosis:The ISABELA 1 and 2 Randomized Clinical Trials

There is a major need for effective, well-tolerated treatments for idiopathic pulmonary fibrosis (IPF). To assess the efficacy and safety of the autotaxin inhibitor ziritaxestat in patients with IPF. The 2 identically designed, phase 3, randomized clinical trials, ISABELA 1 and ISABELA 2, were conducted in Africa, Asia-Pacific region, Europe, Latin America, the Middle East, and North America (26 countries). A total of 1306 patients with IPF were randomized (525 patients at 106 sites in ISABELA 1 and 781 patients at 121 sites in ISABELA 2). Enrollment began in November 2018 in both trials and follow-up was completed early due to study termination on April 12, 2021, for ISABELA 1 and on March 30, 2021, for ISABELA 2. Patients were randomized 1:1:1 to receive 600 mg of oral ziritaxestat, 200 mg of ziritaxestat, or placebo once daily in addition to local standard of care (pirfenidone, nintedanib, or neither) for at least 52 weeks. The primary outcome was the annual rate of decline for forced vital capacity (FVC) at week 52. The key secondary outcomes were disease progression, time to first respiratory-related hospitalization, and change from baseline in St George's Respiratory Questionnaire total score (range, 0 to 100; higher scores indicate poorer health-related quality of life). At the time of study termination, 525 patients were randomized in ISABELA 1 and 781 patients in ISABELA 2 (mean age: 70.0 [SD, 7.2] years in ISABELA 1 and 69.8 [SD, 7.1] years in ISABELA 2; male: 82.4% and 81.2%, respectively). The trials were terminated early after an independent data and safety monitoring committee concluded that the benefit to risk profile of ziritaxestat no longer supported their continuation. Ziritaxestat did not improve the annual rate of FVC decline vs placebo in either study. In ISABELA 1, the least-squares mean annual rate of FVC decline was -124.6 mL (95% CI, -178.0 to -71.2 mL) with 600 mg of ziritaxestat vs -147.3 mL (95% CI, -199.8 to -94.7 mL) with placebo (between-group difference, 22.7 mL [95% CI, -52.3 to 97.6 mL]), and -173.9 mL (95% CI, -225.7 to -122.2 mL) with 200 mg of ziritaxestat (between-group difference vs placebo, -26.7 mL [95% CI, -100.5 to 47.1 mL]). In ISABELA 2, the least-squares mean annual rate of FVC decline was -173.8 mL (95% CI, -209.2 to -138.4 mL) with 600 mg of ziritaxestat vs -176.6 mL (95% CI, -211.4 to -141.8 mL) with placebo (between-group difference, 2.8 mL [95% CI, -46.9 to 52.4 mL]) and -174.9 mL (95% CI, -209.5 to -140.2 mL) with 200 mg of ziritaxestat (between-group difference vs placebo, 1.7 mL [95% CI, -47.4 to 50.8 mL]). There was no benefit with ziritaxestat vs placebo for the key secondary outcomes. In ISABELA 1, all-cause mortality was 8.0% with 600 mg of ziritaxestat, 4.6% with 200 mg of ziritaxestat, and 6.3% with placebo; in ISABELA 2, it was 9.3% with 600 mg of ziritaxestat, 8.5% with 200 mg of ziritaxestat, and 4.7% with placebo. Ziritaxestat did not improve clinical outcomes compared with placebo in patients with IPF receiving standard of care treatment with pirfenidone or nintedanib or in those not receiving standard of care treatment. ClinicalTrials.gov Identifiers: NCT03711162 and NCT03733444