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

Richtlijnen voor de participatie van bewoners van sociale huurwoningen in de energietransitie.:Eindrapportage SAL AURORA

Klimaatverandering en de opwarming van onze aarde zijn duurzaamheidsuitdagingen die de politieke en maatschappelijke gemoederen momenteel flink bezighouden. Nederland heeft zich in dit kader gecommitteerd aan het drastisch terugdringen van broeikasgassen: in 2050 moet de uitstoot van broeikasgassen met 95% verminderd zijn ten opzichte van 1990. Dit zijn ambitieuze doelstellingen, die deels behaald kunnen worden via verduurzaming van ons energieverbruik.Er wordt vaak vanuit gegaan dat woningeigenaren zélf initiatief nemen en zélf een energietransitie in gang zetten. In de praktijk blijkt dit echter lastig, vooral wanneer we te maken hebben met kwetsbare groepen in de samenleving, of mensen met een kleinere beurs. Strategisch gezien kan het daarom handig zijn om ook te kijken naar mogelijkheden voor verduurzaming van huurwoningen van woningcorporaties. Woningcorporaties hebben een grote woningvoorraad en kunnen bijdragen aan het behalen van de doelstellingen. Daarnaast hebben de Limburgse woningcorporaties zich gecommitteerd aan de ‘prestatie afspraken’ welke inhouden dat hun gehele woningvoorraad een gemiddeld energielabel B heeft in 2028. Om deze afspraken te behalen, zijn verduurzamende maatregelen nodig.Investeren in duurzamere woningen heeft echter impact op de bewoners van deze woningen. Vervanging van de kozijnen en ramen gaat bijvoorbeeld gepaard met flinke bouwwerken en het betekent dat er mensen over de vloer komen om oude kozijnen weg te halen, en nieuwe te plaatsen. Niet alle huurders zitten hierop te wachten. Toch moet wettelijk gezien 70% van de bewoners akkoord zijn met de te nemen maatregelen om deze doorgang te kunnen verlenen. Het ‘meekrijgen’ van de bewoners van huurwoningen is dus cruciaal om verduurzamende maatregelen te kunnen nemen en de prestatieafspraken te kunnen behalen.De richtlijnen die ten grondslag liggen aan dit rapport beogen dan ook antwoord te geven op de volgende vraag:Hoe kunnen bewoners van (sociale) huurwoningen worden meegenomen in de energietransitie

HUBUNGAN ANTARA KONTROL DIRI DENGAN PERILAKU BELANJA ONLINE PADA MAHASISWA DI MASA PANDEMI COVID-19

Penelitian ini bertujuan untuk mengetahui apakah ada hubungan antara kontrol diri dengan perilaku belanja online pada mahasiswa di masa pandemi COVID-19. Hipotesis yang diajukan adalah “Ada hubungan negatif antara kontrol diri dengan perilaku belanja online pada mahasiswa di masa pandemi COVID-19. Artinya, semakin tinggi kontrol diri maka semakin rendah perilaku belanja online, dan begitu pula sebaliknya”. Subjek penelitian ini adalah mahasiswa S1 dengan kriteria memiliki akun e-commerce untuk berbelanja dan pernah melakukan transaksi belanja secara online serta pernah menerima barang belanja yang dibeli secara online. Jumlah keseluruhan subjek penelitian adalah 140 mahasiswa. Alat ukur yang digunakan Skala Kontrol Diri dan Skala Perilaku Belanja Online pada Mahasiswa di Masa Pandemi COVID-19. Alat ukur yang digunakan sudah teruji validitas dan reliabilitasnya. Teknik analisis data yang digunakan adalah korelasi Product Moment. Hasil penelitian menunjukkan korelasi negatif yang signifikan (rxy = -0,170; p=0,022). Dengan demikian hipotesis yang diajukan diterima, yaitu ada hubungan negatif yang signifikan antara kontrol diri dengan perilaku belanja online pada mahasiswa di masa pandemi COVID-19

Peroxin Pex21p Interacts with C-terminal Noncatalytic Domain of Yeast Seryl-tRNA Synthetase and Forms a Specific Ternary Complex with tRNA\u3csup\u3eSer\u3c/sup\u3e

The seryl‐tRNA synthetase from Saccharomyces cerevisiae interacts with the peroxisome biogenesis‐related factor Pex21p. Several deletion mutants of seryl‐tRNA synthetase were constructed and inspected for their ability to interact with Pex21p in a yeast two‐hybrid assay, allowing mapping of the synthetase domain required for complex assembly. Deletion of the 13 C‐terminal amino acids abolished Pex21p binding to seryl‐tRNA synthetase. The catalytic parameters of purified truncated seryl‐tRNA synthetase, determined in the serylation reaction, were found to be almost identical to those of the native enzyme. In vivo loss of interaction with Pex21p was confirmed in vitro by coaffinity purification. These data indicate that the C‐terminally appended domain of yeast seryl‐tRNA synthetase does not participate in substrate binding, but instead is required for association with Pex21p. We further determined that Pex21p does not directly bind tRNA, and nor does it possess a tRNA‐binding motif, but it instead participates in the formation of a specific ternary complex with seryl‐tRNA synthetase and tRNASer, strengthening the interaction of seryl‐tRNA synthetase with its cognate tRNASer

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

A conformational change in the helicase core is necessary but not sufficient for RNA unwinding by the DEAD box helicase YxiN

Cooperative binding of ATP and RNA to DEAD-box helicases induces the closed conformation of their helicase core, with extensive interactions across the domain interface. The bound RNA is bent, and its distortion may constitute the first step towards RNA unwinding. To dissect the role of the conformational change in the helicase core for RNA unwinding, we characterized the RNA-stimulated ATPase activity, RNA unwinding and the propensity to form the closed conformer for mutants of the DEAD box helicase YxiN. The ATPase-deficient K52Q mutant forms a closed conformer upon binding of ATP and RNA, but is deficient in RNA unwinding. A mutation in motif III slows down the catalytic cycle, but neither affects the propensity for the closed conformer nor its global conformation. Hence, the closure of the cleft in the helicase core is necessary but not sufficient for RNA unwinding. In contrast, the G303A mutation in motif V prevents a complete closure of the inter-domain cleft, affecting ATP binding and hydrolysis and is detrimental to unwinding. Possibly, the K52Q and motif III mutants still introduce a kink into the backbone of bound RNA, whereas G303A fails to kink the RNA substrate

Expression of the RNA helicase DDX3 and the hypoxia response in breast cancer

<p>Aims: DDX3 is an RNA helicase that has antiapoptotic properties, and promotes proliferation and transformation. In addition, DDX3 was shown to be a direct downstream target of HIF-1α (the master regulatory of the hypoxia response) in breast cancer cell lines. However, the relation between DDX3 and hypoxia has not been addressed in human tumors. In this paper, we studied the relation between DDX3 and the hypoxic responsive proteins in human breast cancer.</p> <p>Methods and Results: DDX3 expression was investigated by immunohistochemistry in breast cancer in comparison with hypoxia related proteins HIF-1α, GLUT1, CAIX, EGFR, HER2, Akt1, FOXO4, p53, ERα, COMMD1, FER kinase, PIN1, E-cadherin, p21, p27, Transferrin receptor, FOXO3A, c-Met and Notch1. DDX3 was overexpressed in 127 of 366 breast cancer patients, and was correlated with overexpression of HIF-1α and its downstream genes CAIX and GLUT1. Moreover, DDX3 expression correlated with hypoxia-related proteins EGFR, HER2, FOXO4, ERα and c-Met in a HIF-1α dependent fashion, and with COMMD1, FER kinase, Akt1, E-cadherin, TfR and FOXO3A independent of HIF-1α.</p> <p>Conclusions: In invasive breast cancer, expression of DDX3 was correlated with overexpression of HIF-1α and many other hypoxia related proteins, pointing to a distinct role for DDX3 under hypoxic conditions and supporting the oncogenic role of DDX3 which could have clinical implication for current development of DDX3 inhibitors.</p&gt