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Inhibition of the Ubiquitin Proteasome System Enhances Long-Term Depression in Rat Hippocampal SlicesLouie, LeeAnn N 01 January 2013 (has links)
The ubiquitin proteasome system (UPS) depends on three enzymes called E1, E2, and E3 to ubiquitinate proteins and several isopeptidases to de-ubiquitinate them. Ubiquitination serves as a post-translational modification that either tags proteins for degradation by the proteasome or serves to modulate their function. This dynamic system plays a role in synaptic plasticity and dysfunction of the UPS is associated a variety of neurodegenerative diseases. In this study, three inhibitors the UPS, ziram, clasto-lactacystin β-lactone (lactacystin) and G5 were employed to illuminate involvement of the UPS in long-term and short term plasticity in area CA1 of rat hippocampal slices. Ziram, lactacystin and G5 inhibits the E1 ubiquitin-activating enzyme, the proteasome and isopeptidases, respectively. It was found that UPS inhibition enhanced long-term plasticity, by specifically increasing the magnitude of long-term depression (LTD) and altered short term plasticity, measured with paired pulse facilitation (PPF), to varying degrees. These findings establish that the UPS may play a regulatory role in LTD and PPF, and the changes in PPF further indicate that the UPS may be acting presynaptically. Overall, the results suggest ubiquitination and proteasome-mediated proteolysis are important in both long-term and short-term plasticity.
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In vitro and in vivo characterization of the E3 ubiquitin ligase RNF157 in the brainLee, Shih-Ju 01 December 2014 (has links)
No description available.
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Schistosoma mansoni: caracterização do perfil de resposta aos estresses oxidativo, térmico e químico / Schistosoma mansoni: Caracterização do perfil de resposta aos estresses oxidativo, térmico e químicoRenato Graciano de Paula 15 February 2013 (has links)
A esquistossomose mansônica é a segunda maior endemia parasitária do mundo em termos de extensão das áreas endêmicas e do número de pessoas infectadas com 200 milhões de pessoas acometidas. Esta doença é causada pelo parasito trematódeo Schistosoma mansoni, o qual apresenta adequados mecanismos de resposta ao estresse envolvendo a regulação da expressão gênica e proteica, reparo ou substituição de moléculas danificadas, recuperação do balanço redox, controle do ciclo celular e apoptose. O sistema ubiquitina- proteassoma é importante para manter a homeostase proteica durante o estresse celular. Inibidores do proteassoma podem interferir em processos como crescimento, progressão do ciclo celular e replicação, e os seus efeitos vem sendo caracterizados em muitos parasitos. Nosso laboratório demonstrou que MG132 reduz o número de esquistossômulos, a carga parasitária e a ovoposição em camundongos infectados com S. mansoni. Neste trabalho, são descritos os efeitos in vitro do estresse oxidativo, choque térmico e estresse químico em vermes adultos de S. mansoni. Observou-se alteração no perfil de expressão proteica durante estresse oxidativo e térmico, sendo identificadas dezoito proteínas upreguladas nestas condições. Estas proteínas estão envolvidas em muitas vias intracelulares como dobramento de proteínas, proteólise, ligação a íons cálcio, regulação de proteínas e resposta a estresse. Além disso, o estresse oxidativo gerou mudanças em vermes adultos de S. mansoni em processos como produção de ovos, motilidade, morfologia do tegumento, viabilidade e pareamento dos vermes. O estresse químico induzido com Curcumina, IBMX e MG132 aumentou a produção de ROS intracelular e alterou o perfil de expressão de enzimas antioxidantes em S. mansoni. As enzimas SmGPx1 e SmPGx2 tiveram a expressão aumentada no estresse com Curcumina e IBMX, enquanto que SmSOD e SmTGR foram induzidas no estresse com Curcumina. As enzimas do proteassoma SmHul5 e SmUbp6 tiveram a expressão modulada durante o estresse oxidativo, choque térmico e estresse químico. Em adição, a análise de expressão no ciclo de vida de S. mansoni revelou que estes genes apresentam um nível alto de expressão em esporocistos, esquistossômulos e miracídios. Estes resultados sugerem que estas proteínas acessórias do proteassoma participam da resposta ao estresse e desenvolvimento do parasito. O nível de expressão de SmHul5 e SmUbp6 foi cerca 9 e 16 vezes menor em relação ao controle no estresse químico induzido com IBMX, respectivamente, sugerindo a desmontagem do proteassoma. Por outro lado, Curcumina, MG132, estresse oxidativo e choque térmico aumentaram o nível de expressão de SmHul5 e SmUbp6. Além disso, o nível de expressão da proteína de maturação do proteassoma (SmPOMP) aumentou no estresse com Curcumina, MG132 e estresse oxidativo, sugerindo a síntese de novas populações de proteassoma. Em relação ao estresse oxidativo, nós demonstramos o aumento no nível proteico de proteassoma 20S e da subunidade alfa-3 do proteassoma sugerindo que em S. mansoni as proteínas oxidadas são degradadas pelo proteassoma 20S. Além do mais, nós observamos que vermes adultos de S. mansoni parecem utilizar mecanismos de resposta similares para diferentes estresses. Nossos resultados demonstraram que o estresse oxidativo, choque térmico e estresse químico modificam o perfil de expressão de genes relacionados ao sistema ubiquitina-proteassoma e sugerem que o proteassoma é importante para as respostas celulares ao estresse neste parasito. / Schistosomiasis is a neglected tropical disease caused by blood flukes (genus Schistosoma) and affecting 200 million people worldwide. This disease continues to rank, following malaria, at the second position of the world\'s parasitic diseases in terms of the extent of endemic areas and the number of infected people. There are different types of stress and the organisms have many mechanisms to respond to these stressor agents. The responses involve the regulation of gene and protein expression and consist in events such as repair or substitution of damaged molecules, recovery of redox balance, cell cycle control and apoptosis. The proteasomal system is important to support the protein homeostasis during the cellular stress. Effect of proteasome inhibitors has been described in many protozoans, either inhibiting growth or cell cycle progression, or blocking replication. Our laboratory\'s results have shown that MG132 reduces the number of lung stage schistosomula, the worm burden and consequently decreases oviposition in S. mansoni-infected mice. Here, we describe the in vitro effects of oxidative stress, heat shock and chemical stress in S. mansoni adult worms. We report that the oxidative stress and heat shock cause drastic changes in the protein profile of S. mansoni adult worms, and we identified a total of eighteen upregulated proteins in these conditions. These proteins are involved with many intracellular pathways as protein folding, proteolysis, calcium ion binding, regulator proteins and stress response. In addition, oxidative stress induced with H2O2 generated significative changes in the adult worms concerning process such as egg production, motor activity, tegument morphology, viability and pairing of worms. Chemical stress induced with Curcumin, IBMX and MG132 increases ROS production and changes the gene expression profile of antioxidant enzymes of S. mansoni adult worms. The enzymes SmGPx1 and SmGPx2 were upregulated in Curcumin and IBMXinduced chemical stress, and both SmSOD and SmTGR were upregulated- Curcumin. The proteasomal enzymes SmHul5 and SmUbp6 had their gene expression modified during oxidative stress, heat shock and chemical stress. Besides of, expression analyses in the S. mansoni life cycle indicate that genes are different express in sporocyst, schistosomula and miracidia. These results suggest these accessory proteins proteasome participates of stress response and parasite development. The expression level of SmHul5 and SmUbp6 were 16 and 9 times less than the control in chemical stress induced by IBMX, and we suggest that these results are due to the proteasome disassembling. On the other hand, Curcumin, MG132, oxidative stress e heat shock increases the expression of SmHul5 and SmUbp6. Furthermore, the expression level of maturation proteasome protein (SmPOMP) increases in stress induced by Curcumin, MG132 and oxidative stress suggesting new proteasome synthesis. In addition, we demonstrate increase the both 20S level and alpha-3 subunit proteasome in the oxidative stress, suggesting that in S. mansoni oxidized protein formed due to oxidative damage are degrade by proteasome 20S. We observed that S. mansoni adult worms utilize similar mechanisms to respond different stresses. Ours results demonstrate that oxidative stress, heat shock and chemical stress modified the expression profile of genes related with the ubiquitinproteasome system and suggest that the proteasome is important to responses the cellular stresses in the parasite.
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Envolvimento de microRNAs na interação Carica papaya L. e Papaya meleira virus com potencial biotecnológicoAbreu, Paolla Mendes do Vale de, Abreu, Paolla Mendes do Vale de 26 February 2015 (has links)
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Tese_defesa_Paolla M. V. Abreu_para capa dura.pdf: 9884169 bytes, checksum: 4a899fd021d8b6129d63eb15679acdaf (MD5) / CAPES / O mamoeiro (Carica papaya L.) é uma das fruteiras mais cultivadas e o mamão um
dos frutos mais consumidos nas regiões tropicais e subtropicais do mundo. O Brasil
é um dos maiores produtores mundiais de mamão e está dentre os principais países
exportadores. As doenças, no entanto, constituem os principais fatores limitantes da
produção. A meleira do mamoeiro, causada pelo Papaya meleira virus (PMeV) é
uma doença importante na produção de mamão capaz de causar a perda completa
da produção. Apesar disso, pouco se conhece sobre os mecanismos de interação e
de resposta do mamoeiro contra o PMeV e ainda não existe no mercado cultivares
resistentes a este vírus. Sabe-se que a expressão de proteínas como a subunidade
20S do proteasoma é maior durante a infecção, sugerindo que a proteólise seja um
mecanismo importante de resposta de defesa. Atualmente 10.598 microRNAs de
plantas estão depositados no banco de dados Plant miRNAs Database, mas
somente dois, miR162 e miR403 são codificados especificamente por C. papaya.
Neste estudo, sequências conhecidas de microRNAs provenientes de diferentes
espécies de plantas foram utilizadas na predição in silico de microRNAs no genoma
de mamoeiro. Um total de 462 microRNAs foram preditos, representando 72 famílias
de miRNAs conhecidas. A expressão de 11 microRNAs, cujos alvos estão
envolvidos na degradação via proteasoma 20S e 26S e em outras vias de resposta a
estresse, foi comparada por qRT-PCR em amostras de folhas de plantas sadias e
infectadas com PMeV. A expressão de microRNAs envolvidos na degradação de
proteínas via proteasoma aumentou em resposta a um baixo acúmulo relativo de
PMeV e diminuiu com o aumento do acúmulo relativo viral. Por outro lado, a
expressão de microRNAs envolvidos na resposta da planta ao estresse biótico
diminuiu em resposta ao baixo acúmulo relativo viral e aumentou com o aumento do
acúmulo relativo de PMeV. Os genes descritos como alvos de alguns dos miRNAs
tiveram sua expressão modulada de uma maneira dependente. Diante dos
resultados, alguns miRNAs foram apontados como importantes para a aplicação
biotecnológica. Este estudo representa uma compreensiva predição de microRNAs
em mamoeiro. A expressão diferencial de microRNAs específicos e a modulação de
seus genes alvos é de grande ajuda para entender a interação particular entre o
mamoeiro e o PMeV, responsável pelo desenvolvimento da meleira. / Carica papaya L. is one of the most cultivated and consumed fruits in tropical and
subtropical regions worldwide. Brazil is among the largest producers and exporters of
papaya fruit. The pre-harvest diseases of papaya plants are the main limitation for
fruit production. Papaya sticky disease is caused by the Papaya meleira virus
(PMeV). It is a commercially important pathology in papaya culture potentially
causing the complete loss of fruit production. Despite of this, little is known about the
papaya interaction and response mechanisms against PMeV and there is not a
papaya variety resistant to the virus. It is known that papaya 20S proteasome subunit
levels of increase during PMeV infection, suggesting that proteolysis is an important
feature of the plant defense response mechanisms. To date, 10,598 plant microRNAs
have been identified in the Plant miRNAs Database (name of the DB), but only two
microRNAs, miR162 and miR403, are from papaya. In this study, plant microRNA
sequences were used to search for putative microRNAs in the papaya genome. A
total of 462 microRNAs, representing 72 microRNA families, were predicted to occur
in papaya. Out of these, the expression of 11 microRNAs, whose targets are known
to be involved in 20S and 26S proteasomal degradation and in other stress response
pathways, was estimated using real-time PCR, comparing healthy and infected
papaya leaf tissues. The expression of miRNAs involved in proteasomal degradation
increased in response to very low levels of PMeV titre and decreased as the viral titre
increased. In contrast, biotic stress-related miRNAs levels decreased in papaya
tissues infected with low virus titre and increased at high PMeV levels. Corroborating
this results, analysed target genes for this miRNAs had their expression modulated in
a dependent manner. With the results, some miRNAs were identified as relevant to
the biotechnological application. This study represents a comprehensive prediction of
miRNAs in papaya. The data presented here might help to complement the available
molecular and genomic tools for the study of papaya. The differential expression of
specific miRNAs and the modulation of their target genes will be helpful for
understanding the particular interaction of PMeV and papaya responsible of disease
development
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SEL1Lの分解中間体はポリグルタミンタンパク質の細胞質での凝集を促進する服部, 徳哉 24 January 2022 (has links)
京都大学 / 新制・課程博士 / 博士(理学) / 甲第23600号 / 理博第4762号 / 新制||理||1683(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)准教授 細川 暢子, 教授 杤尾 豪人, 教授 森 和俊 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM
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Regulation of the RNA/DNA helicase Sen1 by proteasome-mediated degradationAleman Alvarado, Marjorie Andrea 04 1900 (has links)
Sen1 est une hélicase ARN/ADN impliquée dans la protection du génome de la levure en résolvant les hybrides ARN/ADN et dans la terminaison de la transcription de courts ARN non codants. Malgré la demande cellulaire généralisée pour l'action Sen1, son abondance cellulaire est très faible, ce qui suggère que des mécanismes régulent les niveaux de protéine Sen1 dans la cellule. Nous avons confirmé que Sen1 est dégradé via une voie dépendante du protéasome. Ce mécanisme dépend de l’activité catalytique de Glc7, une protéine phosphatase dont il a été précédemment démontré qu’elle déphosphoryle Sen1 in vitro et qu’elle interagit avec Sen1 via un motif RVxF selon des expériences à deux hybrides.
Notre hypothèse de travail est que Glc7 contrôle les niveaux de protéine Sen1 via la déphosphorylation d'un phospho-dégron. Fait intéressant, un site potentiel dans la région N-terminale de Sen1 (sérine 863) qui peut fonctionner comme un phospho-dégron a été identifié dans une analyse à l'échelle du protéome de la co-occurrence de phosphorylation et d'ubiquitylation. Afin d'identifier les sites de phosphorylation Sen1 qui sont enrichis en l'absence de Glc7, nous avons réalisé une immunoprécipitation de Sen1 suivie d'une spectrométrie de masse. Cette analyse a identifié un site de phosphorylation dans Sen1 à la sérine 1505 qui pourrait agir comme un site de dégron potentiel. A noter que ce site, a également été signalé dans les études antérieures phosphoprotéomiques sur la levure. De plus, l'interaction entre Sen1 et Glc7 et l’importance du motif RVxF (par la mutation du résidu F2003) pour cette interaction ont été confirmée par co-immunoprécipitation. De manière surprenante, la prévention de cette interaction n'affecte pas la stabilité de Sen1 ou la croissance cellulaire.
Dans l'ensemble, nous avons identifié un petit groupe de sites de phosphorylation Sen1 avec une pertinence biologique potentielle. Nos résultats confirment également que la mutation du motif RVxF de Sen1 altère l'interaction avec Glc7 in vivo. Ces données approfondissent notre compréhension de la régulation de la protéine Sen1 par Glc7 dans les cellules de levure qui peuvent fournir des indices sur le rôle de la sénataxine, orthologue humaine, dans les troubles neurodégénératifs. / Sen1 is an RNA/DNA helicase involved in protecting the yeast genome by resolving RNA/DNA hybrids and in the transcription termination of short non-coding RNAs. Despite the widespread cellular demand for Sen1 action, its cellular abundance is very low, suggesting that mechanisms regulate Sen1 protein levels in the cell. We have confirmed that Sen1 is degraded via a proteasome-dependent pathway. This mechanism depends on the catalytic activity of Glc7, a protein phosphatase that was previously shown to dephosphorylate Sen1 in vitro, and to interact with Sen1 through an ‘RVxF’ motif.
Our working hypothesis is that Glc7 controls Sen1 protein levels via dephosphorylation of a phospho-degron. Interestingly, a potential site in the N-terminal region of Sen1 (serine 863) that may work as a phospho-degron has been identified in a proteome-wide analysis of phosphorylation and ubiquitylation cross-talk. In order to identify Sen1 phosphorylation sites enriched in the absence of Glc7, we conducted immunoprecipitation of Sen1 followed by mass spectrometry. This analysis identified one phosphorylation site within Sen1 at serine 1505 that could act as a potential degron site. Note that this site has also been reported in previous phosphoproteomic studies on yeast. Furthermore, the interaction between Sen1 and Glc7 and the importance of the RVxF motif (by mutation of residue F2003) for this interaction was confirmed by co-immunoprecipitation. Surprisingly, prevention of this interaction does not affect the stability of Sen1 or cell growth.
Overall, we have identified a small group of Sen1 phosphorylation sites with potential biological relevance. Our findings also confirm that mutating the RVxF motif of Sen1 impairs the interaction with Glc7 in vivo. These data further our understanding of Sen1 protein regulation by Glc7 in yeast cells that may provide clues to the role of senataxin, human orthologue, in neurodegenerative disorders.
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Regulation of Hsp70 function by nucleotide-exchange factorsGowda, Naveen Kumar Chandappa January 2016 (has links)
Protein folding is the process in which polypeptides in their non-native states attain the unique folds of their native states. Adverse environmental conditions and genetic predisposition challenge the folding process and accelerate the production of proteotoxic misfolded proteins. Misfolded proteins are selectively recognized and removed from the cell by processes of protein quality control (PQC). In PQC molecular chaperones of the Heat shock protein 70 kDa (Hsp70) family play important roles by recognizing and facilitating the removal of misfolded proteins. Hsp70 function is dependent on cofactors that regulate the intrinsic ATPase activity of the chaperone. In this thesis I have used yeast genetic, cell biological and biochemical experiments to gain insight into the regulation of Hsp70 function in PQC by nucleotide-exchange factors (NEFs). Study I shows that the NEF Fes1 is a key factor essential for cytosolic PQC. A reverse genetics approach demonstrated that Fes1 NEF activity is required for the degradation of misfolded proteins associated with Hsp70 by the ubiquitin-proteasome system. Specifically, Fes1 association with Hsp70-substrate complexes promotes interaction of the substrate with downstream ubiquitin E3 ligase Ubr1. The consequences of genetic removal of FES1 (fes1Δ) are the failure to degrade misfolded proteins, the accumulation of protein aggregates and constitutive induction of the heat-shock response. Taken the experimental data together, Fes1 targets misfolded proteins for degradation by releasing them from Hsp70. Study II describes an unusual example of alternative splicing of FES1 transcripts that leads to the expression of the two alternative splice isoforms Fes1S and Fes1L. Both isoforms are functional NEFs but localize to different compartments. Fes1S is localized to the cytosol and is required for the efficient degradation of Hsp70-associated misfolded proteins. In contrast, Fes1L is targeted to the nucleus and represents the first identified nuclear NEF in yeast. The identification of distinctly localized Fes1 isoforms have implications for the understanding of the mechanisms underlying nucleo-cytoplasmic PQC. Study III reports on the mechanism that Fes1 employs to regulate Hsp70 function. Specifically Fes1 carries an N-terminal domain (NTD) that is conserved throughout the fungal kingdom. The NTD is flexible, modular and is required for the cellular function of Fes1. Importantly, the NTD forms ATP-sensitive complexes with Hsp70 suggesting that it competes substrates of the chaperone during Fes1-Hsp70 interactions. Study IV reports on methodological development for the efficient assembly of bacterial protein-expression plasmids using yeast homologous recombination cloning and the novel vector pSUMO-YHRC. The findings support the notion that Fes1 plays a key role in determining the fate of Hsp70-associated misfolded substrates and thereby target them for proteasomal degradation. From a broader perspective, the findings provide information essential to develop models that describe how Hsp70 function is regulated by different NEFs to participate in protein folding and degradation. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.</p>
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Modulation des mécanismes de Contrôle Qualité des Protéines dans la dystrophie musculaire de Duchenne / Modulation of Protein Quality Control mechanisms in Duchenne Muscular DystrophyWattin, Marion 21 December 2017 (has links)
De nombreuses études ont mis en évidence l’importance du contrôle qualité des protéines, c’est à dire des mécanismes de reconformation (chaperons moléculaires) et de dégradation (autophagie, proteasome) des protéines dans différentes pathologies musculaires telles que la dystrophie musculaire d’Ullrich (UCMD), de Duchenne (DMD) ou d’Emery-Dreifuss (EDMD) ; cependant, à l’heure actuelle, aucune n’a été menée sur l’ensemble de ces mécanismes dans un seul et même modèle et sur des cellules musculaires avant leur différenciation en muscles. Nous nous sommes donc intéressés à la fonctionnalité des mécanismes de Contrôle Qualité des Protéines et à leurs interconnexions dans des myoblastes immortalisés de donneurs sains ou de patients atteints de DMD. Nous avons observé une augmentation de l’agrégation protéique dans les cellules DMD. Ce phénomène s’accompagne d’une dérégulation des mécanismes de séquestration par les chaperons moléculaires, conséquence d’une modulation de l’expression des protéines HSPB5 et HSPB8. Les mécanismes de dégradation sont également dérégulés; en effet, nous avons observé d’une part, une diminution de l’activité enzymatique du protéasome ainsi que des molécules d’adressage des protéines multiubiquitinées au protéasome et d’autre part, une augmentation de l’activité du facteur de transcription NF?B, de l’expression de protéines intervenant dans l’autophagie et des complexes BAG3/HspB8 conduisant à une augmentation du flux autophagique. L’ensemble de ces dérégulations reflète l’existence d’un stress d’agrégation protéique dans les myoblastes issus de patients DMD. Dans ce contexte, la modulation pharmacologique du PQC dans ces cellules pourrait représenter une nouvelle stratégie thérapeutique pour la Dystrophie Musculaire de Duchenne / Various studies have highlighted the importance of Protein Quality Control (PQC), including protein refolding (molecular chaperones) and degradation (autophagy, proteasome) mechanisms in inherited muscle disorders such as Ullrich Congenital Muscular Dystrophy (UCMD), Duchenne Muscular Dystrophy (DMD) or Emery-Dreifuss Muscular Dystrophy (EDMD); however, to date, no extensive study has been conducted on these mechanisms in a same model, in muscle cells before muscle differentiation. Thus, we were interested in PQC mechanisms functionality and their interconnection in human immortalized myoblasts from healthy donors or patients suffering from DMD. We observed an increase of protein aggregation in DMD cells. This phenomenon is accompanied by a deregulation of sequestration mechanisms by molecular chaperones, reflected by the modulation of HSPB5 and HSPB8 expression. Degradation mechanisms are also deregulated; indeed, we observed on one hand a decrease of proteasome enzymatic activity and multiubiquitinated proteins UPS-adressing molecules and on the other hand, an increase of NF?B transcription factor’s activity, involved in autophagy, and of BAG3/HSPB8 complexes, leading to an increase of the autophagic flux. These PQC defects reflect the existence of a protein aggregation stress in myoblasts coming from DMD patients. In this context, pharmacological modulation of PQC in these cells could represent a new therapeutic strategy for Duchenne Muscular Dystrophy
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Delineating the interplay between the PB2 protein of influenza A viruses and the host Ubiquitin Proteasome System / Analyse comparative des interactions entre l'ARN polymérase des virus influenza A et le système ubiquitine-protéasome de la cellule hôteBiquand, Elise 31 October 2017 (has links)
On estime que 10%-20% de la population mondiale est infectée chaque année par des virus influenza A (IAV) saisonniers, causant 250 à 500 000 morts. De plus ces virus présentent des risques de pandémie, et sont à ce titre un problème de santé publique majeur. Le cycle viral est dépendant de la capacité du virus à manipuler le protéome cellulaire. Par ailleurs, le système ubiquitine-protéasome (SUP) cellulaire est impliqué dans de nombreux processus de régulation cellulaires par l'induction de la dégradation de protéines, ou par la modification de leur activation ou de leur localisation sub-cellulaire. Le SUP est une cible privilégiée des virus lors de l'infection. Des études récentes indiquent qu'un réseau d'interactions entre les protéines virales des IAV et les protéines du SUP pourrait contribuer à la réplication virale et l’échappement du virus face au système immunitaire. Cependant ces interactions restent encore mal connues. Nous avons construit une banque contenant 570 facteurs du SUP, ce qui représente environ 60% des facteurs SUP humains connus. Puis nous avons mis au point une méthodologie permettant de réaliser un crible comparatif des interactions entre cette banque SUP et cinq PB2 provenant de souches de virus influenza A de virulence différentes chez l’homme : deux souches saisonnières circulant actuellement dans la population humaine (H1N1pdm09 et H3N2), deux souches hautement pathogènes chez l’homme (H7N9 et H1N1-1918) et une souche de laboratoire (H1N1-WSN). Cette première phase de cartographie a permis de sélectionner 42 facteurs du SUP interagissant avec au moins une des protéines PB2 étudiées. Par ailleurs, l’analyse des similarités de profils d’interaction PB2/UPS des souches étudiées a permis de mettre en évidence une corrélation avec le temps de circulation de chaque souche dans la population humaine. Nous avons ensuite caractérisé le rôle fonctionnel des partenaires de PB2 dans le cycle viral par des expériences de déplétion transitoire de l’expression des facteurs cellulaires par siARN, et validé 36 des 42 facteurs testés. La très grande quantité de facteurs identifiés impliqués dans le cycle viral démontre la qualité de la méthodologie développée pour l’identification de ces interacteurs. Parmi ces facteurs, nous avons étudié plus en détail le rôle de trois deubiquitinases (DUBs) dans l’infection. Nous avons montré que les DUBs sont impliquées dans les phases précoces et tardives du cycle viral. De plus, avec des collègues de Hong Kong nous avons mis en évidence que la DUB OTUB1 est impliquée dans la réponse cellulaire à l’infection produisant des cytokines, et probablement dans l’assemblage des nouveaux virions. Nous avons identifié que la DUB OTUD6A est également impliquée dans les phases tardives du cycle viral. A l’inverse PAN2 qui fait partie des complexes de poly-d’adénylation est impliqué dans les phases précoces. Nous poursuivons nos études afin d’élucider le rôle de ces DUBs dans l’infection par IAV. / An estimated 10%-20% of the world's population is affected each year by seasonal epidemic influenza, causing about 250,000 to 500,000 fatal cases. The pandemic risk reinforces the trait of influenza A virus (IAV) infection as a public health issue. The virus life cycle critically relies on its ability to manipulate the host proteome. Besides, the ubiquitin-proteasome system (UPS) is involved in many regulatory processes in mammalian cells by inducing protein degradation, mediating protein activation or shaping their sub-cellular localisation. Therefore, UPS is a prime target hijacked by viruses. Recent evidence indicates that an intricate regulatory network involving viral proteins and the cellular UPS is likely to contribute to viral replication and immune evasion of influenza A viruses. However, usurpation of the host UPS by IAV is far from being comprehensively deciphered. To gain better understanding, we assessed the interplay between the human UPS and the PB2 subunit of the influenza A virus polymerase through a global proteomic profiling approach. For that purpose, an UPS-dedicated library of 590 human cDNAs, comprising 63% of the whole human UPS, was constituted and characterised. In an initial screen, UPS factors were challenged using a high-throughput split luciferase assay for interaction with the PB2 protein from 5 influenza A strains of different pathogenicity in human. A total of 80 UPS factors emerged as potential PB2 partners, of which 42 were validated as high-confidence PB2 partners for at least one of the strains. Further comparison of interaction profiles of the 5 PB2 with the UPS by hierarchical clustering revealed an interaction dendrogram fitting with the circulation time in the human population.Functional importance of interactors was tested by siRNA-mediated knock down experiments using luciferase tagged recombinant IAV viruses. Depletion of 36 out of the 42 tested UPS factors showed an effect on the infection with all or a subset of IAV strains, underlying the strong functional output of the developed methodology. Among these factors three deubiquitinases (DUBs) were further studied to decipher their involvement in IAV viral cycle. We have shown that they are involved in early and late stage of the infection and began to draw their function in viral cycle. We demonstrated with our colleagues in Hong-Kong that OTUB1 is involved in the host cytokine response and most probably in virus assembly. OTUD6A was also shown to be implicated in late stages of the infection but we still don't know its exact role. Contrariwise, the inactive DUB PAN2, which is part of poly-deadenylation complexes, is implicated in early phase of IAV infection, but surprisingly apparently not through viral mRNA regulation. More work is on-going to precise by which mechanisms these DUBs are implicated in IAV infection.
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Application Of Surface-enhanced Raman Scattering (sers) Method For Genetic AnalysesKarabicak, Seher 01 March 2011 (has links) (PDF)
Raman spectroscopy offers much better spectral selectivity but its usage has been limited by its poor sensitivity. The discovery of surface-enhanced Raman scattering (SERS) effect, which results in increased sensitivities of up to 108-fold for some compounds, has eliminated this drawback.
A new SERS active substrate was developed in this study. Silver nanoparticle-doped polyvinyl alcohol (PVA) coated SERS substrate prepared through chemical and electrochemical reduction of silver particles dispersed in the polymer matrix. Performances of the substrates were evaluated with some biologically important compounds.
The specific detection of DNA has gained significance in recent years since increasingly DNA sequences of different organisms are being assigned. Such sequence knowledge can be employed for identification of the genes of microorganisms or diseases. In this study, specific proteasome gene sequences were detected both label free spectrophotometric detection and SERS detection. In label free spectrophotometic detection, proteasome gene probe and complementary target gene sequence were attached to the gold nanoparticles separately. Then, the target and probe oligonucleotide-modified gold solutions were mixed for hybridization and the shift in the surface plasmon absorption band of gold nanoparticles were followed.
SERS detection of specific nucleic acid sequences are mainly based on hybridization of DNA targets to complementary probe sequences, which are labelled with SERS active dyes. In this study, to show correlation between circulating proteasome levels and disease state we suggest a Raman spectroscopic technique that uses SERGen probes. This novel approach deals with specific detection of elevated or decreased levels of proteasome genes&rsquo / transcription in patients as an alternative to available enzyme activity measurement methods. First, SERGen probes were prepared using SERS active labels and specific proteasome gene sequences. Then DNA targets to complementary SERGen probe sequences were hybridized and SERS active label peak was followed.
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