A sejtmagi fehérje-import Importin-alfa2 faktorának citoplazmatikus funkciói = Cytoplasmic functions of Importin-alpha2, a factor of nuclear protein import

Az importin-alfa2D14 + / + importin-ßKetelRE34 Drosophila nőstényektől származó embriók letálisak. Jellemeztük a maternális interakciót. Főbb eredményeink: - embrió letalitás a szinciciális blasztoderma állapotban a metafázisban blokkolt osztódási orsó hibái miatt. Konfokális mikroszkópiával jellemeztük a mutáns fenotípust. - importin-alfa1, -alfa2, -alfa3 transzgének közül csak az i-alfa2 menekítette az embrió letalitást (paralóg-specifikus szerep) - Importin-alfa2 (I-alfa2) fehérje az NLS-kötő domainjén keresztül köti az ISWI, CP190 és laminDm0 mitotikus faktorokat (ko-immunprecipitáció). Genetikai interakcióval azonosítottuk még a Rpl7 és Nup358 kölcsönható fehérjéket. - DNS-szekvenálás: az importin-ßKetelRE34 (RE34) egy D725N szuppresszor mutációt is hordoz. - a RE34 mutáns fehérje nagyobb affinitással köti a RanGDP/GTP-t, mint a vad típus (GST pull-down). Számítógépes modellezés szerint ennek valószínű oka a RE34-nek a vad típusnál nyitottabb konformációja. Modellünk: A Drosophila petében a mitótikus orsót felépítő fehérjéket NLS-szekvenciájuknál fogva az Importin-alfa2/ Importin-ß komplex köti és tartja inaktív állapotban. A komplex RanGTP hatására szétesik, az orsófehérjék felszabadulnak, és megindul az osztódási orsók képződése. A maternális letalitás valószínű oka az, hogy az Importin-alfa2/ Importin-ß KetelRE34 /NLS komplex a RE34 fokozott RanGTP kötése már alacsony RanGTP koncentrációnál szétesik, és a mitótikus fehérjék abnormális mennyiségben szabadulnak fel. | importin-alpha2D14 + / + importin-ßKetelRE34 heterozygous Drosophila females give rise to lethal embryos. We characterized this maternal interaction, the main results are as follow: - the embryos die in the syncytial blastoderm stage because of abnormal mitotic spindles blocked in metaphase. We characterized the mutant phenotype by confocal microscopy. - of the importin-alpha1, -alpha2, -alpha3 transgenes, only i-alpha2 rescued embryo lethality (paralog-specific role of i-2) I-alpha2 protein binds the ISWI, CP190 and laminDm0 mitotic factors through its NLS-binding domain (co-immunoprecipitation). By genetic interaction, we identified the Rpl7 and Nup358 interactor proteins. - DNA sequencing revealed a second-site D725N mutation in importin-ß-KetelRE34 probably suppressing the dominant female sterile phenotype of KetelD. - Importin-ß-KetelRE34 protein (RE34) binds RanGDP/GTP with higher affinity than wild type (GST pull-down). It is likely explained by the more open than normal conformation of RE34 (computer modeling). Our model: in the Drosophila egg the I-alpha2/ I-ß complex binds inactive the mitotic proteins through their NLS sequences. RanGTP dissociates the complex liberating constituents of the mitotic spindle, and spindle formation begins. The maternal lethality of mutant embryos is likely caused by the higher than normal affinity to RanGTP of Importin-ß liberating abnormal amounts of spindle proteins

FMRFamid-rokon neuropeptidek és receptoraik hatásmechanizmusának genetikai analízise Drosophila melanogaster-ben = Insect FMRFamide-related neuropeptides (FaRPs) and receptors: genetic analysis of their mechanism of action in Drosophila melanogaster

Genetikai módszerekkel tanulmányoztuk ecetmuslica (Drosophila melanogaster) modellben az FMRF-rokon (FARP) és azok receptorait. (1) Intragénikus deléciós mutánsokat izoláltunk a DMS-R1 and FMRFa génekben. (2) Kettős-szálú RNS (RNAi) segítségével csendesítettük a peptid- és receptor géneket. (3) Gal4 driver-eket készítettünk az FMRFa gén 5’ régiójának fragmentjeivel, majd FMRFa peptiderg neuronokat elimináltunk. Az RNAi tesztben először az Act5C-Gal4 drivert alkalmaztuk, amely az FMRFa, DMS, DMS-R1, DSK-R1 és DSK-R2 gének esetében letalitást eredményezett. A pánneurális elav-Gal4 driverrel viszont minden kombináció életképes volt. Homozigóta életképesek az FMRFa és DMS-R1 deléciós mutánsok is. Az eredmények magyarázata az „off-target” hatás.. A mutáns kombinációknak az adult viselkedésre gyakorolt hatását stressz-indukált mozgási tesztben vizsgáltuk. A mozgási aktivitást szignifikánsan csökkentették a deléciós mutánsok és az RNAi kombinációk, valamint bizonyos FMRF-specifikus neuronok ablációja. | We accomplished a genetic analysis in Drosophila melanogaster of the FMRF-related group of peptides (FARPs) comprising (DMS the dFMRFamide, dromyosuppressin) and drosulfakinin (DSK) peptides, and their specific receptors (FP, DMS-R1 and -R2, DSK-R1 and –R2). We used three approaches: (i) isolating intragenic deletions by P transposon remobilization in the DMS-R1 and FMRFa genes, (ii) gene silencing by double-stranded inhibitory RNA (RNAi) and (iii) constructing new Gal4 drivers (RS- 8, -11 ans -17) with 5’ regulatory fragments of the FMRFa gene, and using them to ablate specific FMRFa peptidergic neurons. In the RNAi tests, when using the Act5C-Gal4 driver expressing continuously in all the tissues, silencing the FMRFa, DMS, DMS-R1, DSK-R1 and DSK-R2 genes resulted in lethality, while with the pan-neuronal elav-Gal4 driver all the combinations remained alive. Similarly, the FMRFa and DMS-R1 deletion mutants are homozygous viable. The RNAi-induced lethality is likely explained by off-target effect, i.e. silencing non-specific genes which have sections in their DNA sequence similar to that of the target gene. In order to compare the effects of the above mutant combinations on adult behavior, we examined the startle-induced locomotor activity of flies by measuring the mean velocity of movement (MVM) following repeated air-puffs. In general, the MVM was decreased by the deletion mutants, the elav-Gal4-induced RNAi knockdown and the ablation of some FMRF-specific neurons

Deletion of proteasomal subunit S5a/Rpn10/p54 causes lethality, multiple mitotic defects and overexpression of proteasomal genes in Drosophila melanogaster

The regulatory complex of the 26S proteasome is responsible for the selective recognition and binding of multiubiquitinated proteins. It was earlier shown that the subunit S5a/Rpn10/p54 of the regulatory complex is the only cellular protein capable of binding multiubiquitin chains in an in vitro overlay assay. The role of this subunit in substrate selection, however, is a subject of debate, following the observation that its deletion in Saccharomyces cerevisiae is not lethal and instead causes only a mild phenotype. To study the function of this subunit in higher eukaryotes, a mutant Drosophila strain was constructed by deleting the single copy gene encoding subunit S5a/Rpn10/p54. This deletion caused larval-pupal polyphasic lethality, multiple mitotic defects, the accumulation of higher multimers of ubiquitinated proteins and a huge accumulation of defective 26S proteasome particles. Deletion of the subunit S5a/Rpn10/p54 does not destabilise the regulatory complex and does not disturb the assembly of the regulatory complex and the catalytic core. The pupal lethality is a consequence of the depletion of the maternally provided 26S proteasome during the larval stages and a sudden increase in the proteasomal activity demands during the first few hours of pupal development. The huge accumulation of the fully assembled 26S proteasome in the deletion mutant and the lack of free subunits or partially assembled particles indicate that there is a highly coordinated accumulation of all the subunits of the 26S proteasome. This suggests that in higher eukaryotes, as with yeast, a feedback circuit coordinately regulates the expression of the proteasomal genes, and this adjusts the actual proteasome concentration in the cells according to the temporal and/or spatial proteolytic demand

Identification and functional characterization of the pheromone biosynthesis activating neuropeptide receptor isoforms from Mamestra brassicae

Communication : 28th Conference of European Comparative Endocrinologists ; Organisme : European Society for Comparative Endocrinology (ESCE), Louvain, Belgique ; date de début de l'événement : 2016-08-21, date de fin de l'évenement : 2016-08-25International audienceIn most moth species, including Mamestra brassicae, pheromone biosynthesis activating neuropeptide (PBAN) regulates pheromone production. Generally, PBAN acts directly on the pheromone gland (PG) cells via its specific G protein-coupled receptor (i.e. PBANR) with Ca2+ as a second messenger. In this study, we identified cDNAs encoding three variants (A, B and C) of the M. brassicae PBANR (Mambr-PBANR). The full-length coding sequences were transiently expressed in cultured Trichoplusia ni cells and Sf9 cells for functional characterization. All three isoforms dose-dependently mobilized extracellular Ca2+ in response to PBAN analogs with Mambr-PBANR-C exhibiting the greatest sensitivity. Fluorescent confocal microscopy imaging studies demonstrated binding of a rhodamine red-labeled ligand (RR10CPBAN) to all three Mambr-PBANR isoforms. RR10CPBAN binding did not trigger ligand-induced internalization in cells expressing PBANR-A, but did in cells expressing the PBANR-B and-C isoforms. Furthermore, activation of the PBANR-B and-C isoforms with the 18 amino acid Mambr-pheromonotropin resulted in co-localization with a Drosophila melanogaster arrestin homolog (Kurtz), whereas stimulation with an unrelated peptide had no effect. PCR-based profiling of the three transcripts revealed a basal level of expression throughout development with a dramatic increase in PG transcripts from the day of adult emergence with PBANR-C being the most abundant

The degradation of p53 and its major E3 ligase Mdm2 is differentially dependent on the proteasomal ubiquitin receptor S5a.

p53 and its major E3 ligase Mdm2 are both ubiquitinated and targeted to the proteasome for degradation. Despite the importance of this in regulating the p53 pathway, little is known about the mechanisms of proteasomal recognition of ubiquitinated p53 and Mdm2. In this study, we show that knockdown of the proteasomal ubiquitin receptor S5a/PSMD4/Rpn10 inhibits p53 protein degradation and results in the accumulation of ubiquitinated p53. Overexpression of a dominant-negative deletion of S5a lacking its ubiquitin-interacting motifs (UIM)s, but which can be incorporated into the proteasome, also causes the stabilization of p53. Furthermore, small-interferring RNA (siRNA) rescue experiments confirm that the UIMs of S5a are required for the maintenance of low p53 levels. These observations indicate that S5a participates in the recognition of ubiquitinated p53 by the proteasome. In contrast, targeting S5a has no effect on the rate of degradation of Mdm2, indicating that proteasomal recognition of Mdm2 can be mediated by an S5a-independent pathway. S5a knockdown results in an increase in the transcriptional activity of p53. The selective stabilization of p53 and not Mdm2 provides a mechanism for p53 activation. Depletion of S5a causes a p53-dependent decrease in cell proliferation, demonstrating that p53 can have a dominant role in the response to targeting S5a. This study provides evidence for alternative pathways of proteasomal recognition of p53 and Mdm2. Differences in recognition by the proteasome could provide a means to modulate the relative stability of p53 and Mdm2 in response to cellular signals. In addition, they could be exploited for p53-activating therapies. This work shows that the degradation of proteins by the proteasome can be selectively dependent on S5a in human cells, and that this selectivity can extend to an E3 ubiquitin ligase and its substrate

A szavak befolyása Donald Trump kommunikációjában

Szakdolgozatom a politikai kommunikáció témakörében járja körül Donald Trumpot, és azt, hogy mennyire tudja manipulálni a közvéleményt, tömeget stílusával, szóhasználatával. Mindezt a Twitteren közvetített üzenetei által vizsgáltam meg. A téma többek között azért is releváns, mert az újonnan megjelenő kommunikációs formák sok lehetőséget, azonban veszélyeket is rejtenek. Az elemzésem alapján arra a konklúzióra jutottam, hogyan lehet kiaknázni ezeket a modern kommunikációban lévő lehetőségeket