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1
Schorova, Lenka. "Étude des mécanismes de régulation synaptique de la balance sumoylation/désumoylation." Thesis, Université Côte d'Azur (ComUE), 2018. http://www.theses.fr/2018AZUR4021.
Full textAbstract:
La SUMOylation est une modification post-traductionnelle essentielle pour toutes les cellules eucaryotes. C’est un processus enzymatique qui permet la liaison covalente du polypeptide SUMO sur des résidus lysine de protéines cibles. La SUMOylation est un processus réversible sous l’action de désumoylases appelées SENP. Il est critique de maintenir un équilibre entre forme modifiée et non modifiée d’un substrat donné. En effet, la dérégulation de la balance SUMOylation/déSUMOylation a été mise en évidence dans plusieurs pathologies cérébrales. La synapse est le point de contact entre les neurones où s’effectue la communication synaptique. Ce sont des structures très denses où le processus de SUMOylation régule l’interaction et la fonction de multiples protéines. Durant ma thèse, j'ai combiné l’utilisation de l'imagerie en temps réel sur cellules vivantes avec des approches biochimiques et pharmacologiques pour identifier les mécanismes de régulation du transport de SENP1. J'ai ainsi démontré que l'activation neuronale augmente les niveaux synaptiques de SENP1. Cette augmentation synaptique résulte de la modification de la vitesse de diffusion de l’enzyme SENP1 entre les dendrites et les synapses d’une part, et d’autre part, de l’augmentation importante du temps de rétention synaptique de l’enzyme. Je rapporte également que ce mécanisme de régulation dynamique de SENP1 implique l'activation des récepteurs métabotropiques du glutamate. De plus, je suggère la participation du processus de phosphorylation dans cette régulation synapto-dendritique de SENP1 mettant ainsi en lumière un nouveau mécanisme de régulation de la balance neuronale entre SUMOylation et déSUMOylationSumoylation is a vital eukaryotic posttranslational modification. Sumoylation occurs as an enzymatic cycle that conjugates SUMO proteins to target proteins. SUMO proteases (SENP) deconjugate SUMO from modified proteins and thus maintain balanced levels of SUMOylated and un-SUMOylated proteins required for physiological homeostasis. Neuronal synapses are protein-rich structures that underlie synaptic transmission and plasticity. Strong evidence exists that sumoylation occurs in synapses and regulates the function of synaptic proteins. Indeed, distortion of the SUMO balance has been linked to several pathologies of the synapse. Gaining a deeper understanding into the molecular mechanisms regulating the SUMO balance is a prerequisite to envisaging the development of novel therapies. In my PhD work, I used a combination of live-cell confocal imaging, protein biochemistry and pharmacological approaches to identify SENP1 regulatory mechanisms at synapses. I provided evidence that synaptic activation increases SENP1 protein levels at synapses. I showed that the increase in synaptic SENP1 upon synaptic activation is a result of two processes: Although (a) fewer SENP1 proteins enter into spines at low diffusion speed (b) a significant proportion of SENP1 becomes immobile and is retained in spines. I demonstrate that the regulatory mechanisms of SENP1 dynamics involve a direct activation of mGlu1/5 receptors. Moreover, I suggest that phosphorylation may play an important regulatory role in SENP1 synapto-dendritic diffusion. Altogether, I propose a novel mechanism driving for the SUMO balance at synapses
2
Reichhold, Christian [Verfasser]. "The role of SENP1 in B cell development and differentiation / Christian Reichhold." Mainz : Universitätsbibliothek Mainz, 2014. http://d-nb.info/1048654141/34.
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3
Haindl, Markus. "Charakterisierung der SUMO-Isopeptidasen SENP3 und SENP6." Diss., lmu, 2008. http://nbn-resolving.de/urn:nbn:de:bvb:19-150775.
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4
Era, Saho. "The SUMO protease SENP1 is required for cohesion maintenance and mitotic arrest following spindle poison treatment." Kyoto University, 2013. http://hdl.handle.net/2433/174794.
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5
Rimsa, Vadim. "Targeting an E3 ubiquitin ligase Siah1 and a cysteine protease SENP1 using SPR and DSF-based fragment screening." Thesis, University of Dundee, 2013. https://discovery.dundee.ac.uk/en/studentTheses/0dfaa23f-8048-423a-b3c0-560ac40de2a4.
Full textAbstract:
This dissertation presents fragment screening studies against two human proteins Siah1 and SENP1, which function in post-translational modification pathways. Siah1 is an E3 ubiquitin ligase that functions as a scaffold to transfer ubiquitin bound to an E2 ubiquitin-conjugating enzyme to a substrate as part of the ubiquitination pathway. SENP1 is a cysteine protease that catalyses two essential reactions in the SUMO pathway. It processes pre-SUMO proteins to their mature form and removes SUMO from the target proteins. Siah1 interactions with other proteins involve large surface areas, while SENP1 has a small active site, making it hard to identify ligands for these proteins. The fragment-based approach has emerged as a complementary method to high-throughput screening of finding novel small molecules. The main aim of the study was to examine whether fragment screening would identify any ligands against these targets. Chapter 1 introduces post-translational modifications and presents fragment-based approach used in drug discovery. Chapter 2 describes the experimental methods used. The results from fragment screening against Siah1 using SPR and DSF are reported in chapter 3. The chapter also presents the structure of Siah1 refined to 1.95 Å that displays new parts of the structure, previously missing due to the absence of reliable electron density. Chapter 4 contains results from the fragment screens against SENP1 using DSF and NMR. The crystal structure of SENP1 was determined with a number of improvements made over earlier structures. Besides performing fragment screening, the binding between Siah1 interacting proteins reported in the literature and Siah1 was investigated. A number of Siah1 binding partners were successfully expressed and purified as described in chapter 5. One of those, SIP showed a clear interaction with Siah1, as observed by the shift on a size exclusion column of the complex relative to the individual protein species. Siah1 was reported to collaborate with PEG3 in the regulation of ß-catenin degradation. A SCAN domain, located at the N-terminus of PEG3, was tested for binding using gel filtration chromatography and NMR, but no interaction was observed. PEG3 was used in the crystallographic studies and a structure of its SCAN domain was solved using molecular replacement and refined to 1.95 Å. The structure of PEG3-SCAN domain revealed a stable homodimer with an extensive dimerization interface. The structure of a zinc-dependent cytosolic carboxypeptidase from Burkholderiacenocepacia was determined and is reported in chapter 6. This work was a side project assessing a new refinement strategy, which involved the use of the automated protocols embedded in the PDB_REDO server. The structure revealed that carboxypeptidase is a tetramer and provides details of its active site, whose spatial conformation of residues supports the notion that the protein might function as a deglutamylase.
6
Alegre, Kamela Olivya. "Structural and Fumctional Analysis of the SUMO Proteases SENP6 and SENP7." Doctoral thesis, Universitat Autònoma de Barcelona, 2012. http://hdl.handle.net/10803/121596.
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Intercambiar la especifidad de las isoformas de SUMO1 y SUMO2/3 para SENP6/SENP7
SENP6 y SENP7 son los miembros más divergentes de la familia de proteasas SENP y los únicos miembros que llevan cuatro inserciónes o “loops” localizadas en su dominio catalítico. Al sobreponer el dominio catalítico de SENP7 sobre el complejo SENP2-SUMO podemos ver una posible interfaz entre el Loop1 de SENP7 y SUMO. También identificamos diferentes residuos de SUMO1 y SUMO2 que podrían formar parte de la interfaz. Diseñamos una serie de mutantes de SUMO1 y SUMO2 donde se intercambian entre ellos los residuos que forman parte de la interfaz. De esta manera fuimos capaces de intercambiar la actividad proteolítica de SENP6 y SENP7 hacia estos substratos.
El Loop1 de SENP6 y SENP7 es responsable de la especificidad de la interfaz entre los SUMOs y las SENP6/7
Diseñamos una serie de mutantes del Loop1 de SENP7 dentro de la posible interfaz entre el Loop1 de SENP7 y SUMO para determinar los papeles estructurales y funcionales de los residuos que están dentro de esta región. Los mutantes de SENP6/7 sin el Loop1 perdían gran parte de su actividad, esta se podía recuperar un poco al reemplazar las cuatro prolinas del Loop1 por glicinas. De esta manera probamos que el Loop1 tiene un papel por lo menos estructural en el reconocimiento de SUMO. También identificamos el residuo Lys691 del Loop1 de SENP7 como elemento indispensable en la actividad de la enzima ya que al mutar este residuo por un aspártico disminuye la actividad para sustratos con SUMO2. Por otra parte el residuo Asp71 de SUMO2 es uno de los residuos propuestos para el reconocimiento de SUMO2/3. Cuando mutamos este residuo a lisina, vemos una bajada en la actividad de SENP7. Este hecho sugiere que Asp71 de SUMO2 está interaccionando con Loop1 y quizas con la Lys691, y que la bajada en la actividad es causada por una repulsión de cargas.
Por otro lado con el objectivo de estudiar el efecto de Loop1 en la desconjugación de especies SUMOyladas, insertamos los ocho residuos de SENP6 Loop en el dominio catalítico de SENP2. Esta inserción provoca un aumento de la actividad sustancial de SENP2 contra diSUMO en comparación con la forma wild type.
Complejos con substratos
Para ver si SENP6 es capaz de formar algún complejo estable, producimos cantidades en miligramos de los mutantes inactivos de Δ3SENP6C1030S y de Δ2Δ3SENP6C1030S. Incubamos cada proteasa con los precursors de SUMO, con RanGAP1-SUMO2 y con diSUMO2. De todos los substratos que probamos, diSUMO2 fue el único que podía formar un complejo estable con Δ3SENP6CS y Δ2Δ3SENP6CS por copurificación en una columna de gel fitración. Con Δ2SENP6CS no pudimos formar ningún complejo. Con esta información llegamos a la conclusión que el Loop3 de SENP6 reduce, quizá por razones de entropía, la capacidad de SENP6 de formar un complejo estable con diSUMO2.
Caracterización del Loop3 de SENP6
Para descifrar el papel que este loop tiene en el contexto de la proteasa, producimos y purificamos los 185 residuos de Loop3 de SENP6. Análysis 1-H 1-D RMN mostraba una falta de estructura terciaria dentro del loop, pero proteólisis limitada y espectroscopia de masas mostraban un fragmento estable de cerca de 11kDa. Dicroísmo circular y FTIR tambien sugieren la presencia de algunos elementos de estructura secundaria. Globalmente, la caracterización biofísica del Loop3 de SENP6 muestra que el loop es una proteína desestructurada.Swapping the SUMO Isoform Specificity of SENP6/7 SENP6 and SENP7 are the most divergent members in the SENP family of proteases and they are the only members that bear four loop insertions dispersed throughout their catalytic domains. The superposition of the SENP7 catalytic domain with the SENP2-SUMO complex revealed a tentative SENP7 Loop1-SUMO interface and upon further inspection, distinct residues on SUMO1 and SUMO2 were identified at the interface. A series of mutants were constructed bearing characteristics of both SUMOs and by swapping the residues from SUMO1 to SUMO2 and vice versus we were able to both decrease and increase the activity of the SENP6 and SENP7 toward these substrates. Loop1 SENP6/7 Is Responsible For SUMO Interface Specificity In addition to mutations on SUMO we constructed a series of mutations on SENP7-Loop1 within the tentative SENP7-Loop1-SUMO interface to determine the structural and functional roles of the residues that reside within this region. We were able to recover some of the activity lost by the removal of Loop1 by replacing the four prolines of Loop1 with glycines proving that Loop1 plays at least a structural role in SUMO recognition. We also identified Lys691 of Loop1 in SENP7 as indispensible to the activity of the enzyme. D71 is one of the residues proposed to confer SUMO2/3 specificity in the previous swapping experiments. We mutated this residue in diSUMO2 and saw a decrease in activity of SENP7. This could be explained by our theory that this region on SUMO is interacting with Loop1, more specifically K691, and the decrease in activity was caused by a charge clash between SUMO2 and SENP7 Loop1. To further show the utility of Loop1 in deconjugation of multi-SUMOylated species we inserted the eight residues of SENP6 Loop1 into SENP2. We saw an overall increase in activity of SENP2 against diSUMO2 but not against any other substrate tested. Complexes With Substrates In order to see if SENP6 was able to form any stable complexes in solution, we produced milligram amounts of Δ3SENP6CS and Δ2Δ3SENP6CS (the two constructs of the protein that showed both good yields in protein production and high performance in activity assay). We incubated each protease with SUMO precursors, RanGAP1-SUMO2 and diSUMO2 substrates. Of all the substrates tested, only diSUMO was able to form a stable complex with Δ3SENP6CS and Δ2Δ3SENP6CS. Δ2SENP6CS was also tested but there was no indication of any complex formation, leading to the hypothesis that SENP6 Loop3 was impeding, perhaps entropically, the ability of SENP6 to form a stable complex with diSUMO2. SENP6 Loop3 Characterization Loop3 takes up roughly 40% and 20% of the catalytic domains of SENP6 and SENP7 respectively. In our loop deletion experiments we saw an overall increase in the activity when Loop3 was not present and removal of Loop3 proved vital to the ability of the enzyme to form a stable complex with diSUMO2. In order to try to decipher what role this loop plays in the context of the protease, we isolated the 184 insert from SENP6 and produced and purified the protein. 1-H 1-D NMR pointed to an overall lack of tertiary structure within the loop but limited proteolysis and mass spectrometry analyses showed a stable fragment of around 11kDa. Further circular dichroism and Fourier Transform Infrared Spectroscopy suggested the presence of some secondary structural elements but overall characterization of SENP6 Loop3 showed a mainly unstructured loop.
7
Haindl, Markus [Verfasser], and Stefan [Akademischer Betreuer] Jentsch. "Charakterisierung der SUMO-Isopeptidasen SENP3 und SENP6 / Markus Haindl. Betreuer: Stefan Jentsch." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2008. http://d-nb.info/1028490453/34.
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8
Schorova, Lenka. "Étude des mécanismes de régulation synaptique de la balance sumoylation/désumoylation." Electronic Thesis or Diss., Université Côte d'Azur (ComUE), 2018. http://theses.univ-cotedazur.fr/2018AZUR4021.
Full textAbstract:
La SUMOylation est une modification post-traductionnelle essentielle pour toutes les cellules eucaryotes. C’est un processus enzymatique qui permet la liaison covalente du polypeptide SUMO sur des résidus lysine de protéines cibles. La SUMOylation est un processus réversible sous l’action de désumoylases appelées SENP. Il est critique de maintenir un équilibre entre forme modifiée et non modifiée d’un substrat donné. En effet, la dérégulation de la balance SUMOylation/déSUMOylation a été mise en évidence dans plusieurs pathologies cérébrales. La synapse est le point de contact entre les neurones où s’effectue la communication synaptique. Ce sont des structures très denses où le processus de SUMOylation régule l’interaction et la fonction de multiples protéines. Durant ma thèse, j'ai combiné l’utilisation de l'imagerie en temps réel sur cellules vivantes avec des approches biochimiques et pharmacologiques pour identifier les mécanismes de régulation du transport de SENP1. J'ai ainsi démontré que l'activation neuronale augmente les niveaux synaptiques de SENP1. Cette augmentation synaptique résulte de la modification de la vitesse de diffusion de l’enzyme SENP1 entre les dendrites et les synapses d’une part, et d’autre part, de l’augmentation importante du temps de rétention synaptique de l’enzyme. Je rapporte également que ce mécanisme de régulation dynamique de SENP1 implique l'activation des récepteurs métabotropiques du glutamate. De plus, je suggère la participation du processus de phosphorylation dans cette régulation synapto-dendritique de SENP1 mettant ainsi en lumière un nouveau mécanisme de régulation de la balance neuronale entre SUMOylation et déSUMOylationSumoylation is a vital eukaryotic posttranslational modification. Sumoylation occurs as an enzymatic cycle that conjugates SUMO proteins to target proteins. SUMO proteases (SENP) deconjugate SUMO from modified proteins and thus maintain balanced levels of SUMOylated and un-SUMOylated proteins required for physiological homeostasis. Neuronal synapses are protein-rich structures that underlie synaptic transmission and plasticity. Strong evidence exists that sumoylation occurs in synapses and regulates the function of synaptic proteins. Indeed, distortion of the SUMO balance has been linked to several pathologies of the synapse. Gaining a deeper understanding into the molecular mechanisms regulating the SUMO balance is a prerequisite to envisaging the development of novel therapies. In my PhD work, I used a combination of live-cell confocal imaging, protein biochemistry and pharmacological approaches to identify SENP1 regulatory mechanisms at synapses. I provided evidence that synaptic activation increases SENP1 protein levels at synapses. I showed that the increase in synaptic SENP1 upon synaptic activation is a result of two processes: Although (a) fewer SENP1 proteins enter into spines at low diffusion speed (b) a significant proportion of SENP1 becomes immobile and is retained in spines. I demonstrate that the regulatory mechanisms of SENP1 dynamics involve a direct activation of mGlu1/5 receptors. Moreover, I suggest that phosphorylation may play an important regulatory role in SENP1 synapto-dendritic diffusion. Altogether, I propose a novel mechanism driving for the SUMO balance at synapses
9
Martin, Sarah Friede. "Fluorescence resonance energy transfer studies of protein interactions." Thesis, St Andrews, 2008. http://hdl.handle.net/10023/537.
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10
Hattersley, Neil. "Characterisation of the SUMO protease SenP6." Thesis, University of Dundee, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.521677.
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Diaz, Nuñez Indira Mariaeugenia. "Resolución N° 021-2014-OEFA-TFA-SEP1." Bachelor's thesis, Pontificia Universidad Católica del Perú, 2021. http://hdl.handle.net/20.500.12404/19942.
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El objetivo general del trabajo es identificar los vacíos normativos que
identificamos en procedimientos administrativos sancionadores, y como ante
dicho contexto, la Administración Pública puede incurrir en vicios que vulneren
los derechos a los administrados sujetos a su competencia. Pero, este análisis no
solo tiene por objetivo identificar dichas falencias y vacíos normativos, sino
también plantear soluciones que permiten contribuir de mejor manera en la
actuación de la Administración Pública.
Los instrumentos que se ha utilizado en presente trabajo es el marco normativo
que aplica al sector ambiental dentro un procedimiento administrativo
sancionador, la jurisprudencia que aborda los principios del procedimiento
administrativo y la doctrina que aborda temas no solo de principios sino también
temas como procedente vinculantes en materia administrativa. En ese sentido,
entre las principales conclusiones en que llega en el análisis de un caso surgido
entre la Empresa Administradora Chungar y el Organismo de Evaluación y
Fiscalización Ambiental, es que, se ha vulnerado claramente los principios que
rigen el procedimiento administrativo sancionador, y que, por ende, correspondía
a uno de los órganos que se interviene dentro del procedimiento que declare la
nulidad de lo actuado, pero al no pasar esto, tiene por resultado emitir un
precedente vinculante que contraviene el marco normativo aplicable.The general objective of the work is to identify the regulatory gaps that we identify in administrative sanctioning procedures, and as in this context, the Public Administration may incur in vices that violate the rights of the administrations subject to its competence. However, this analysis is not only intended to identify these gaps and regulatory gaps, but also to propose solutions that allow a better contribution to the performance of the Public Administration. The instruments that have been used in this work are the normative framework that applies to the environmental sector within an administrative sanctioning procedure, the jurisprudence that addresses the principles of the administrative procedure and the doctrine that addresses issues not only of principles but also issues such as binding in administrative matter. In this sense, among the main conclusions reached in the analysis of a case that arose between the Chungar Administrative Company and the Environmental Assessment and Enforcement Agency, is that the principles that govern the administrative sanctioning procedure have been clearly violated, and that Therefore, it corresponded to one of the bodies that intervenes within the procedure to declare the nullity of the action, but when this does not happen, the result is to issue a binding precedent that contravenes the applicable regulatory framework.
12
Eckers, Jaimee Claire. "SEPP1 and FoxM1 regulate oxidative stress-mediated radiation response." Diss., University of Iowa, 2013. https://ir.uiowa.edu/etd/1449.
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Radiation is a common mode of cancer therapy that is well known to generate reactive oxygen species leading to cell damage and death. However, there are many limitations to radiation therapy including normal tissue toxicity and the presence of quiescent cancer cells that are radio-resistant. There are many factors that regulate normal and cancer cell radiation response including the cellular redox environment which includes a complex network of antioxidants. In this study, two specific objectives will be explored: (A) SEPP1 regulation of normal cell toxicity; and (B) FoxM1 regulation of quiescence-associated radiation resistance in human oral squamous carcinoma cells. Results from DHE-oxidation analysis show that in irradiated proliferating normal cells there is a late ROS accumulation that occurs independent of cell cycle checkpoint activation and precedes cell death. Additionally, Q-RT-PCR and immunoblot analysis show an increase in Selenoprotein P (SEPP1) expression following radiation. SEPP1 is an extracellular glycoprotein with proposed selenium transport and antioxidant functions. However, pretreatment of normal cells with sodium selenite or overexpression of sepp1 is able to mitigate radiation-induced normal cell toxicity.
It is well-accepted that quiescent populations exist in most solid tumors and are often the reason for tumor recurrence. In this study, we see that quiescent head and neck cancer cells that are resistant to radiation have low basal expression of Forkhead box M1 (FoxM1) compared to proliferating cancer cells. FoxM1 is a transcription factor that has recently been implicated in the cellular response to oxidative stress. Results indicate that although basal expression is low in quiescent cells, following irradiation FoxM1 is increased in quiescent cancer cells but not in proliferating cancer cells. Additionally, pharmacological and genetic knockdown of FoxM1 led to sensitization of quiescent cancer cells indicating that FoxM1 inhibitors could be useful radiation sensitizers. Together, these objectives will help to identify possible treatment options to use in addition to radiation therapy to better target quiescence-associated resistant tumors and induce less normal cell toxicity.
13
Chinchay, Habich Fiorella Alexandra. "Informe sobre la Resolución N° 004-2014-OEFA/TFA-SEP1." Bachelor's thesis, Pontificia Universidad Católica del Perú, 2020. http://hdl.handle.net/20.500.12404/18346.
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En este informe se analiza la Resolución Nº 004-2014-OEFA/TFA-SEP1, emitida por el
Tribunal de Fiscalización Ambiental del Organismo de Evaluación y Fiscalización
Ambiental. La justificación radica en los constantes cambios normativos
administrativos, el alto nivel de especialidad y el enfoque interdisciplinario
para su resolución. Se parte de la hipótesis que la Resolución no respeta principios y
normas del ordenamiento administrativo, ambiental y minero, todo lo cual nos aleja
de la verdad material -reconocida como un valor que orienta la actividad del proceso
sancionador- y de los fines del derecho administrativo -entre ellos, conocer si en el
caso particular existió o no daño ambiental y cómo debe diferenciarse el daño
potencial y el daño efectivamente causado-. Para ello se analiza la legislación
vigente al momento de la Resolución, así como doctrina autorizada nacional y
comparada y jurisprudencia. En este trabajo se concluye que la Resolución no
cumple con los plazos previstos por el ordenamiento para su emisión, transgrede el
principio de tipicidad -tano en el aspecto relacionado a las normas sustanciales como
en lo relacionado a la norma tipificadora-. En consecuencia, la Resolución no es
correcta, ya que no se analizaron aspectos relevantes que hubieran cambiado el
sentido del pronunciamiento.
14
Han, Zhong. "Characterization of the mechanisms of transcription termination by the helicase Sen1." Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLS202/document.
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La transcription cachée est un phénomène répandu aussi bien chez les eucaryotes que chez les procaryotes. Elle se caractérise par une production massive d’ARNs non-codants au niveau de régions non-annotées du génome et est potentiellement dangereuse pour la cellule car elle peut interférer avec l’expression normale des gènes. Chez S. cerevisiae, l’hélicase Sen1 induit la terminaison précoce de la transcription non-codante et joue ainsi un rôle clé dans le contrôle de la transcription cachée. Sen1 est très conservée et des mutations dans son homologue humain, senataxin (SETX), ont été associées à des maladies neurodégénératives. Malgré de nombreuses recherches menées sur ces protéines, leurs propriétés biochimiques ainsi que leurs mécanismes d’action restent peu connus. Durant ma thèse, j’ai étudié le mécanisme de terminaison par Sen1.Premièrement, j’ai caractérisé les activités biochimiques de Sen1 et analysé comment elles permettent d’induire la terminaison. Pour cela, j’ai utilisé un ensemble de techniques in vitro, notamment un système de transcription-terminaison qui contient uniquement des composants purifiés : Sen1, l’ARN polymérase II (Pol II) et les ADN matrices. Ce système permet de modifier les différents éléments de façon contrôlée afin de comprendre leur rôle précis dans la terminaison. J’ai tout d’abord analysé la fonction des différents domaines de Sen1 dans la terminaison. Sen1 est une protéine de taille importante qui possède un domaine central catalytique flanqué par deux domaines impliqués dans l’interaction avec d’autres facteurs. J’ai montré que le domaine hélicase est suffisant pour déclencher la terminaison de la transcription in vitro. Ensuite, j’ai montré que Sen1 utilise l’énergie de l’hydrolyse de l’ATP pour se déplacer sur des acides nucléiques simple bras (ARN et ADN) dans le sens 5’ vers 3’. J’ai alors étudié le rôle des différents acides nucléiques du système dans la terminaison par Sen1 et j’ai montré que l’interaction de Sen1 avec l’ADN n’est pas nécessaire; en revanche Sen1 doit s’associer à l’ARN naissant et se déplacer vers la polymérase. J’ai aussi montré qu’une fois que Sen1 entre en collision avec la Pol II, elle y exerce une action mécanique qui conduit à la terminaison uniquement quand la Pol II marque une pause. Cela indique que la terminaison est fortement dépendante de la pause transcriptionnelle. Deuxièmement, en collaboration avec le groupe d’E. Conti, nous avons réalisé une analyse structure-fonction du domaine hélicase de Sen1. Nous avons observé que Sen1 présente une organisation similaire à celle d’autres hélicases proches avec un core composé de deux domaines de type RecA avec plusieurs domaines auxiliaires. En général, le core est très conservé au sein des hélicases proches, alors que les domaines accessoires ont des caractéristiques distinctes qui confèrent des propriétés spécifiques aux différentes hélicases. En effet, nous avons identifié un sous-domaine spécifique à Sen1 mais conservé au cours de l’évolution que nous avons appelé le “brace”. Nous avons également détecté des différences notables au niveau d’un autre domaine accessoire que nous avons nommé le “prong”. Nous avons pu montrer que le “prong” est essentiel pour la terminaison par Sen1. Nos données suggèrent que les caractéristiques structurales spécifiques de Sen1 que nous avons révélées sont des déterminants majeurs de son activité dans la terminaison de la transcription. Finalement, nous avons utilisé Sen1 comme modèle pour étudier des mutations dans SETX qui sont associées à des maladies neurodégénératives. Nous avons introduit chez Sen1 une partie des mutations liées à des maladies et nous avons réalisé une caractérisation biochimique complète de chaque mutant. Nous avons ainsi montré que toutes les mutations sont fortement délétères pour la terminaison de la transcription. En conclusion, nos résultats ont permis d’améliorer la compréhension de l’origine des maladies provoquées par des mutations dans SETXPervasive transcription is a common phenomenon both in eukaryotes and prokaryotes that consists in the massive production of non-coding RNAs from non-annotated regions of the genome. Pervasive transcription poses a risk that needs to be controlled since it can interfere with normal transcription of canonical genes. In S.cerevisiae, the helicase Sen1 plays a key role in restricting pervasive transcription by eliciting early termination of non-coding transcription. Sen1 is highly conserved across species and mutations in the human Sen1 orthologue, senataxin (SETX), are associated with two neurological disorders. Despite the major biological relevance of Sen1 proteins, little is known about their biochemical properties and precise mechanisms of action. During my PhD I have studied in detail the mechanisms of termination by Sen1.In a first project, I have characterized the biochemical activities of Sen1 and investigated how these activities partake in termination. To this end I have employed a variety of in vitro approaches, including a minimal transcription-termination system containing only purified Sen1, RNA polymerase II (RNAPII) and DNA transcription templates that allows modifying the different elements of the system in a controlled manner to understand their role in termination. First, we have analysed the function of the different domains of Sen1 in termination. Sen1 is a large protein composed of a central catalytic domain flanked by additional domains with proposed roles in protein-protein interactions. We have demonstrated that the central helicase domain is sufficient to elicit transcription termination in vitro. Next, we have shown that Sen1 can translocate along single-stranded nucleic acids (both RNA and DNA) from 5’ to 3’. Then, we have analysed the role of the different nucleic acid components of the elongation complex (i.e. nascent RNA and DNA transcription templates) in termination. Our results indicate that termination does not involve the interaction of Sen1 with the DNA but requires Sen1 translocation on the nascent RNA towards the RNAPII. Importantly, we show that upon encountering RNAPII, Sen1 can apply a mechanical force on the polymerase that results in transcription termination when RNAPII is paused under certain conditions. This indicates that RNAPII pausing is a strict requirement for Sen1-mediated termination. In a second project, in collaboration with the group of E. Conti we have performed a structure-function analysis of the helicase domain of Sen1. Comparison of Sen1 structure with that of other related helicases has revealed an overall similar organization consisting in two tandem RecA-like domains from which additional accessory subdomains protrude. In general, the core RecA-like domains are very well conserved among related helicases and most variation is found in the accessory subdomains, that often confer specific characteristics to different helicases. Indeed, we have found that Sen1 contains a unique but evolutionary conserved structural feature that we have dubbed the “brace”. In addition, Sen1 is different from other helicases in an auxiliary subdomain that we have named the “prong”. Importantly, we have shown that the integrity of this subdomain is critical transcription termination by Sen1. We propose that the specific features identified in our structural analyses are important determinants of the transcription termination activity of Sen1. Finally, we have used Sen1 as a model to investigate the molecular effect of SETX mutations linked to neurodegenerative diseases. We have introduced disease-associated mutations in Sen1 and performed a complete biochemical characterization of the different mutants in vitro. Importantly, we found that all mutants were severely affected in transcription termination. Taken together, our results elucidate the key structural determinants of the function of Sen1 and shed light on the molecular origin of the diseases associated with SETX mutations
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Nait, Achour Thiziri. "Les rôles de la SUMO protéase SENP2 et du corépresseur LCoR dans la signalisation œstrogénique." Thesis, Montpellier 2, 2011. http://www.theses.fr/2011MON20206.
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Les œstrogènes sont impliqués dans la prolifération des cellules épithéliales du sein normal et l'exposition prolongée à ces hormones s'accompagne d'une augmentation du risque de développement de cancer du sein. Les œstrogènes exercent leurs effets via les récepteurs des œstrogènes (REs). L'activité de ces récepteurs est finement régulée par un grand nombre de cofacteurs transcriptionnels, mais également par les modifications post-traductionnelles. Mon travail de thèse a eu pour objectif la compréhension de l'impact de ces deux niveaux de régulation sur la signalisation œstrogénique. Il a été récemment décrit que la sumoylation affectait de manière drastique l'activité du RE. La sumoylation est une modification dont le caractère réversible est assuré par des isopeptidases appelé SENPs (SENtrin Proteases). Dans une première étude nous avons montré que SENP2 pouvait fortement réprimer l'activité transcriptionnelle dépendante des œstrogènes ainsi que la prolifération cellulaire. Dans une seconde étude, nous nous sommes attelés à mieux caractériser les mécanismes d'action à l'origine du caractère répresseur du cofacteur transcriptionnel LCoR (Ligand-dependent Corepressor). Nous nous sommes plus précisément intéressés aux relations existant entre LCoR et un autre cofacteur du RE, RIP140 (Receptor Interacting Protein of 140 kDa) répresseur majeur de l'activité œstrogénique. Nous avons pu caractériser, outre les modes de recrutement des deux protéines, les modulations d'expression exercées par les deux cofacteurs. L'ensemble de nos travaux identifie de nouveaux cofacteurs des REs et contribue à une meilleure compréhension de la signalisation œstrogéniqueEstrogens are involved in the proliferation of normal breast epithelial cells. The prolonged exposure to these hormones comes along with an increase of the risk of breast cancer.development. Estrogen receptors (ERs) mediate the effects of estrogens. The activity of these receptors is finely tuned by a large number of transcriptional cofactors, but also by post-translational modifications. This work aimed at understanding the impact of these regulations on estrogenic signalling. It was recently described that sumoylation could strongly affect ER-dependent activity. SUMO conjugation is a dynamic process which is reversed by SUMO specific proteases also known as SENtrin Proteases (SENPs). In a first study, we investigated the role of SENP2, in ER-dependent transcriptional activity. We showed that SENP2 could acts as a transcriptional cofactor independently of its catalytic activity by strongly repressing ER-dependent transcriptional activity. We also provided evidence for a role in in breast cancer cell line proliferation. In a second part of the work we investigated the mechanism of action of the transcriptional cofactor LCoR (Ligand-dependent Corepressor) with a specific emphasis on the relationship between LCoR and another ER cofactor, RIP140 (Receptor Interacting Protein of 140 kDa). We characterized a crossed expression modulation of the two transcription cofactors. We also depicted an interaction between these two corepressors and a regulation of LCoR activity by RIP140. Our work provides new insights in identifying new coregulators of ER and contributes to a better understanding of both LCoR and RIP140 mechanism of action, and therefore of estrogenic signalling
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Rivosecchi, Julieta. "Sen1-mediated RNAPIII transcription termination controls the positioning of condensin on mitotic chromosomes." Thesis, Lyon, 2019. http://www.theses.fr/2019LYSEN041/document.
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Le complexe condensine est le moteur de la condensation mitotique des chromosomes, un processus essentiel à la stabilité du génome au cours de la division cellulaire. De nombreuses données publiées indiquent qu’il existe des liens fonctionnels étroits entre le processus de transcription des gènes et le processus d’organisation des chromosomes par condensine. Ces données sont toutefois souvent contradictoires et aucun modèle ne fait actuellement consensus pour expliquer les liens entre transcription et condensine. Au cours de cette thèse, nous avons montré chez la levure Schizosaccharomyces pombe qu’en l’absence de l’hélicase à ADN/ARN Sen1, condensine s’accumule spécifiquement à proximité des gènes transcrits par l’ARN Polymérase III. Nous avons utilisé ces observations pour mieux comprendre les liens entre transcription par l’ARN polymérase III et le positionnement de condensine. Nos données montrent que Sen1 est un cofacteur de l’ARN Polymérase III impliqué dans la terminaison de la transcription. Ce résultat est important car il démontre que les modèles existants qui affirment que l’ARN polymérase III termine de transcrire de façon autonome sont erronés. Nous avons ensuite démontré que les défauts de terminaison de l’ARN polymérase III observés en l’absence de Sen1 suffisent entièrement à expliquer l’accumulation de condensine en ces sites. Cette observation importante démontre que le contrôle de qualité de la transcription est directement impliqué dans le positionnement de condensine sur les chromosomes en mitose. Nos résultats nous permettent de proposer qu’au-delà d’un certain seuil, la densité en ARN polymérases est un obstacle à la translocation de condensine sur les chromosomesThe condensin complex is a key driver of chromosome condensation in mitosis. The condensin-dependent assembly of highly compacted chromosomes is essential for the faithful transmission of the genome during cell division. Many independent studies have established that gene transcription impacts the association of condensin with chromosomes, but the molecular mechanisms involved are still unclear. This is especially true as a number of sometimes contradictory mechanisms have been proposed so far. Here, we show in Schizosaccharomyces pombe that condensin accumulates specifically in the vicinity of a subset of RNA polymerase III-transcribed genes in the absence of the conserved DNA/RNA helicase Sen1. We demonstrate that Sen1 is a cofactor of RNA polymerase III (RNAPIII) required for efficient transcription termination. These results are important because they fundamentally challenge the pre-existing view that RNAPIII terminates transcription autonomously. Strikingly, we show that the RNAPIII transcription termination defects are directly responsible for the accumulation of condensin in the absence of Sen1. This indicates that the quality control of transcription impacts the distribution of condensin on mitotic chromosomes. We propose that above a certain density threshold, the accumulation of RNAPIII constitutes a barrier for the translocation of condensin on chromosomes
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Concnetino, Eralda Luiza Castro. "Estudo de mutações no gene SEPN1 em pacientes brasileiros portadores de miopatias e distrofias musculares congênitas." Universidade Federal de Minas Gerais, 2011. http://hdl.handle.net/1843/BUOS-8M4H48.
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Introduction: different kinds of myopathy are related to the selenoprotein N gene, among which: congenital muscular dystrophy with rigid spine (RSMD) multiminicore myopathy (MmD), desminopathy with Mallory body and congenital fiber-type disproportion myopathy (CFTD). Until the present moment there aren´t national studies that evaluated mutations in the gene SEPN1 in brazilian patients with myopathy related to selenoprotein N. This study is a dissertation and will be presented in the form of two papers: the first, a literature review of myopathies related to selenoprotein N and the second shows the results of the study of SEPN1 gene in Brazilian patients with such condictions. Objective: to evaluate the presence of mutations in the SEPN1 gene in Brazilian patients with myophathies related to selenoprotein N and to correlate the clinical, histopathological and genetic findings. Methodology: patients with myopathies related to SEPN1 gene were submited to: clinical evaluation, measurement of creatinephosphokinase (CPK), electromyography (ENMG) and muscle biopsy with histochemistry study because of diagnosis. We conducted a research protocol, through the medical record and clinical data of patients. We reviewed the muscle histopathology of patients included in the research and molecular study of patients, some family members and normal controls, by technique of polymerase chain reaction (PCR) and sequencing of the gene SEPN1. Results: Were included eighteen patients from sixteen different families. This patients ten were classified with, seven patients were classified with MmD and one patient with CFTD. The molecular analysis revealed mutations in six families. Family 1: A patient with classic phenotype of MmD with compound heterozygous mutation (cG1010T/cT1384G). Family 2 and 3: Two patients unrelated with RSMD phenotype with insertion of a base out of phase in heterozygous (c713- 714insA). Família 4: One patient with CFTD showed insertion of 12 bases in phase (c316-317 Ins.12bp). Family 5: One patient with MmD prenatal form with arthrogryposis showed a missense mutation in heterozigose (cG583A) in which the complementar change was not identified. Family 6: Three affected siblings with myopathy and stiff neck which was identified one insertion in phase of three base pairs in heterozygous (c438-439ins3bp). Conclusion: Myopathies related to selenoprotein N have a wide clinical phenotype and histology. Among the six mutations found two (C713-714insA and cT1384G) had previously been described in the literature as pathogenic in patients with DMER and MmD. The others four changes were not found descriptions in the literature. The mutation cG1010T was found in compound heterozygous with the pathogenic mutation cT1384G in a patient with MmD, being considered due to theircharacteristics and absence in normal controls, a new pathogenic mutation. The mutation c438-439ins3bp, is a polymorphism because it was found homozygous in a parent asymptomatic and in 2% of normal controls.The other two mutations cG583A and c316-317Ins12bp require further studies for better elucidation as to its pathogenicity. The molecular analysis can help confirm the diagnosis, as well as genetic counseling of familiesIntrodução: diferentes formas de miopatias estão relacionadas ao gene da selenoproteína N, entre as quais a distrofia muscular congênita tipo espinha rígida (DMER), miopatia multiminicore (DMm), desmiopatia com mallory body e miopatia com desproporção congênita de fibras (MDCF). Até o momento, não há estudos nacionais sobre mutações no gene SEPN1 em pacientes brasileiros com miopatias relacionadas à selenoproteína N. O presente estudo é uma dissertação de mestrado apresentada na forma de dois artigos científicos: o primeiro, uma revisão da literatura sobre miopatias relacionadas à selenoproteína N; e o segundo mostra os resultados do estudo do gene SEPN1 em pacientes brasileiros com tais afecções. Objetivo: avaliar a presença de mutações no gene SEPN1 em paciente brasileiros com miopatias relacionadas à selenoproteína N e correlacionar os achados clínicos, histopatológicos e genéticos. Metodologia: pacientes portadores de miopatias relacionadas ao gene SEPN1 foram submetidos a: avaliação clínica, dosagem de creatinofosfoquinase (CPK), eletroneuromiografia (ENMG) e biópsia muscular com estudo histoquímico por ocasião do diagnóstico. Realizou-se protocolo de pesquisa, no prontuário, dos dados clínicos dos pacientes. Fez-se revisão da histopatologia muscular dos pacientes incluídos na pesquisa bem como estudo molecular dos pacientes, de alguns familiares e de controles normais, pela técnica de reação em cadeia da polimerase (PCR) e sequenciamento do gene SEPN1. Resultados: incluíram-se 18 pacientes de 16 famílias diferentes. Desses, 10 foram classificados como portadores de DMER, sete com DMm e um com MDCF. O estudo molecular revelou mutações em seis famílias. Família 1: um paciente com fenótipo clássico de DMm, com mutação em heterozigose composta (cG1010T/cT1384G). Famílias 2 e 3: dois pacientes não aparentados, com fenótipo de DMER, com inserção de uma base fora de fase em heterozigose (c713- 714insA). Família 4: um paciente com MDCF com inserção de 12 bases em fase (c316-317Ins12bp). Família 5: um paciente com DMm forma pré-natal com artrogripose e mutação com sentido trocado em heterozigose (cG583A), cuja alteração complementar não foi identificada. Família 6: três irmãos afetados com quadro de miopatia e rigidez cervical, nos quais foi identificada uma inserção em fase, de três pares de base, em heterozigose (c438-439ins3bp). Conclusão: as miopatias relacionadas à selenoproteína N apresentaram amplo fenótipo clínico e histopatológico. Das seis mutações encontradas, duas (c713-714insA e cT1384G) estavam descritas na literatura como patogênicas, em pacientes com DMER e DMm. Das outras quatro não houve registros. A mutação cG1010T foi detectada em heterozigose composta com a mutação patogênica cT1384G em um paciente com DMm, sendo considerada, devido às suas características e ausência em controles normais, mutação nova patogênica. A mutação c438-439ins3bp é umpolimorfismo, pois foi encontrada em homozigose em um dos pais assintomáticos e em 2% dos controles normais. As outras duas mutações cG583A e c316- 317Ins12bp requerem mais estudos para melhor esclarecimento de sua patogenicidade. O estudo molecular pode auxiliar na confirmação do diagnóstico, bem como no aconselhamento genético familiar
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Romeo, Kelly. "Régulation de l'enrichissement en protéines HP1 par la SUMO-protéase SENP7 à l'hétérochromatine péricentrique de souris." Paris 6, 2013. http://www.theses.fr/2013PA066179.
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L'organisation en chromatine permet de compacter l’ADN génomique et de réguler finement le métabolisme de l’ADN. La chromatine peut s’organiser en domaines nucléaires caractérisés par un enrichissement en facteurs spécifiques. Un enjeu majeur est de comprendre les mécanismes qui contrôlent l’établissement et le maintien de ces domaines au cours des divisions cellulaires. Au cours de ma thèse, je me suis intéressée aux domaines péricentriques. Il s’agit de l’hétérochromatine constitutive du centromère caractérisée épigénétiquement par l’enrichissement en protéines HP1, qui confèrent à ces régions une fonction critique pour la ségrégation des chromosomes. Les mécanismes de régulation de la localisation de HP1 à ces régions sont donc à considérer. Mes travaux de thèse ont mis en évidence un nouveau composant enrichi à l’hétérochromatine péricentrique de souris, la SUMO-protéase SENP7. SENP7 interagit directement avec HP1 grâce à deux motifs conservés dits PxVxL. La fonction de SENP7 à ces régions est particulièrement critique car la perte de SENP7 induit une perte d’enrichissement en protéines HP1α aux domaines péricentriques. Ce mécanisme ne dépend que des deux motifs PxVxL de SENP7. Notre hypothèse est que SENP7 verrouillerait via ces motifs PxVxL les molécules de HP1 à l’hétérochromatine péricentrique, afin de stabiliser HP1 en restreignant sa dynamique. Ces résultats ouvrent la porte à de nombreuses perspectives de recherche visant à identifier les mécanismes de stabilisation des protéines HP1 aux domaines péricentriques, et au-delà de HP1, à caractériser des mécanismes impliqués dans le maintien de l’architecture nucléaire et ainsi dans la stabilité du génomeIn the nucleus of all eukaryotic organisms, genomic DNA is organized in chromatin. This organization contributes to different degrees of DNA compaction, which define functional nuclear domains characterized by the enrichment of specific factors. A central question is to understand how such functional chromatin domains are established and maintained throughout multiple cellular divisions. During my PhD, I focused on pericentric domains, the constitutive heterochromatin of the centromere. This heterochromatin is characterized epigenetically by an enrichment of HP1 proteins that confer a critical function in chromosome segregation. It is thus crucial to maintain HP1 enrichment at pericentric domains for genome stability and integrity. During my PhD, I identified a new component of pericentric domains, the SUMO-protease SENP7. SENP7 is enriched at pericentric domains and interacts with HP1 with two HP1-interaction motifs, the so-called PxVxL motifs. SENP7 function is particularly important at pericentric heterochromatin as it is required for HP1 enrichment at these regions. Notably, the SENP7 desumoylation activity is not involved in this function. Rather, I showed that the two HP1-interaction motifs are critical for HP1 enrichment. We propose that HP1 interactors with two HP1-interaction motifs could lock nucleosome-bound HP1 and act as key contributors for maintaining HP1 enrichment at heterochromatin domains. These results open up new avenues to explore the maintenance mechanisms of HP1 enrichment that would stabilize HP1 proteins, but also mechanisms associated to other proteins and functional nuclear domains that could impact nuclear architecture and genome stability
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Zhen, Yang. "Structural and functional studies on the regulation of the USP28 de-ubiquitinase and the SENP5 de-SUMOylase." Doctoral thesis, Universitat Autònoma de Barcelona, 2014. http://hdl.handle.net/10803/285770.
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USP28 és un enzim de-ubiquitinasa (DUBs), homologa a USP25, que pertany a la família USPs. USP28 s’ha involucrat en apoptosis induïda per IR i en l’estabilitat de vari regulador de DDR (Reparació de Dany al DNA). D’altra banda, SENP5 és una proteasa especifica per Sentrin/SUMO de la família de SENP, que s’ha descrit que està involucrada en mitosi i citocinesi. En aquesta tesi, el principal objectiu ha estat la resolució de les seves estructures cristal·lines i la caracterització de la regulació de la seva activitat proteolítica.
Respecte a la part de USP28, hem aconseguit produir diferent constructes del seu Domini Catalític (CD) i de la regió N-terminal (NT), i hem caracteritzat les seves activitats sobre diferent substrats poli-Ubiquitina. També hem identificat el lloc de SUMOilització primari de USP28 per Espectrometria de Masses (K99). També hem aconseguit obtenir cristalls del NT de USP28 i del CD de USP25. Desafortunadament, la difracció d’aquests cristalls és dèbil i la seva estructura no s’ha pogut resoldre. També hem identificat l’especificitat de cadena di-Ubiquitin en USP28 (K11, K48 i K63 cadenes). Els nostres assajos in vitro indiquen que l’activitat proteolítica de USP28 es pot regular per modificació covalent per SUMO a la regió N-terminal. De totes formes, la presencia de la regió N-terminal no és estrictament necessària per l’activitat USP28. Si considerem que tenim un domini SIM, un domini UBA i dos dominis UIMs a la regió N-terminal, fóra interessant estudiar aquest aspecte amb més detall en el futur.
Respecte a la part de SENP, hem posat molts esforços en la producció d’una construcció soluble del Domini Catalític (CD) de SENP3 i SENP5. Finalment, hem aconseguit produir en grans quantitats un constructe soluble de SENP5-CD en E.coli. Hem caracteritzat in vitro les reaccions de processament de SUMO i de deconjugació de SUMO. També ham format complexes entre un mutant inactiu de SENP5-CD-C712S amb el precursor de SUMO2 (Sp5-S2p) i amb el substrat RanGAP1-SUMO2. Només hem aconseguit obtenir cristalls de Sp5-S2p, malauradament la difracció era tan dèbil que no hem pogut resoldre l’estructura a alta resolució.USP28 is a member of a family of deubiquitinating enzymes (DUBs), homologous to USP25, belonging to the USP family. USP28 has been involved in IR-induced apoptosis and in the stability of numerous DDR regulators. On the other hand, SENP5 is a member of the Sentrin/SUMO-specific proteases (SENP) in humans, which is reported to be involved in mitosis and/or cytokinesis. In this thesis, the main goal has been to elucidate their crystal structures and to characterize the regulation of their proteolytic activities. With respect to the USP28, we have produced different constructs of their Catalytic Domain (CD) and N-terminal región (NT), and have characterized their activities against different poly-Ubiquitin substrates. We have also identified the USP28 SUMOylation primary site by Mass Spectrometry (K99). We have been able to obtain nice crystals of the NT of USP28 and of the CD of USP25. Unfortunately, the diffractions of these crystals were weak and the structure could not be solved. We have also identified the di-Ubiquitin chain specificity of USP28 (K11, K48 and K63 linkages). Our in vitro analysis indicates that the USP28 proteolytic activity can be regulated by covalent SUMO modification at the N-terminal region; however, the presence of this N-terminal region is not strictly necessary for the USP28 activity. Considering there are one SIM, one UBA and two UIMs in the N-terminal region, this issue would be interesting to be explored deeply in the future. Regarding to the SENP part, we have put many efforts on the protein expression of different constructs of the Catalytic Domains (CD) of SENP3 and SENP5. Finally we have been able to produce in high yields a soluble construct of SENP5-CD in E.coli. We have characterized the SUMO processing and SUMO deconjugation reactions of SENP5-CD. We also have formed complexes between the inactive mutant of the SENP5-CD-C712S with SUMO2 precursor (Sp5-S2p) and with RanGAP1-SUMO2 substrates. We could only get crystals of Sp5-S2p, however the diffraction was so weak that we could not solve the structure at high resolution.
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Dudhal, Swati. "Selenoprotein N as a novel regulator of the muscle progenitor’s cell fate decision process : balancing differentiation and self-renewal." Thesis, Sorbonne Paris Cité, 2018. http://www.theses.fr/2018USPCC288.
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Les mutations du gène codant la sélénoprotéine N (SEPN1) provoquent une myopathie congénitale nommée SEPN1-related myopathy (SEPN1-RM), caractérisée par une faiblesse et une amyotrophie majeures des muscles du cou et du tronc, une scoliose et une insuffisance respiratoire potentiellement létale. SEPN1-RM a été associée avec un stress oxydant, une diminution de la population de cellules souches musculaires (cellules satellites) et des défauts de la régénération musculaire. Avec l’objectif de rechercher les mécanismes impliqués dans ces défauts, et en particulier un rôle potentiel de SEPN1 dans la régulation de l’équilibre entre le renouvellement et la différentiation du pool de cellules satellites, j’ai étudié des cellules satellites primaires de souris knocked-out pour Sepn1et la lignée musculaire murine C2C12 knocked-down pour Sepn1à différents stades de différentiation (cellules quiescentes, myoblastes etmyotubes). Utilisant un système de suspension pour générer une quiescence synchronisée des C2C12, j’ai trouvé que l’absence de SEPN1 dans les cellules en G0 n’est pas incompatible avec la sortie et le retour dans le cycle cellulaire, mais entraîne une moindre sous-régulation de l’expression de deux facteurs clé de la différentiation myogénique (augmentation des transcrits de MYOD1etMYOG par rapport aux contrôles) et une augmentation des niveaux de Cycline D1 (mRNAdeCCND1) en conditions de quiescence. Des études de microarrayet deqRT-PCR ont montré que la déplétion de SEPN1 dans des C2C12 prolifératives est associée à une augmentation significative de l’expression des facteurs de transcription myogéniques MYOG and MYOD1. En parallèle, des études d’immunoblot ont confirmé un niveau augmenté des protéines régulatrices du cycle cellulaire p21 and Cyclin D3 en conditions de prolifération. De plus, des cellules satellites primaires isolées à partir des muscles gastrocnemiusetplantarisde souris KO Sepn1ont montré une fusion accélérée des myoblastes au cours de la différentiation myogénique initiale. Par la suite, j’ai explore les voies mécanistiques impliquées dans ce phénotype cellulaire par western blot et/ou qRT-PCR utilisant des cellulesC2C12 knocked-down pour Sepn1. J’ai pu montrer l’absence de changements nets des voies de l’AMPK et p38, ainsi que du taux d’expression des marqueurs de stress du réticulum endoplasmique GRP78 oucalnexine. Par contre, nos données suggèrent que les voies HDAC5 etmTOR pourraient être impliquées dans le phénotype de différentiation musculaire accélérée. En conclusion, la déplétion de SEPN1 entraîne une quiescence incomplète et une différentiation myogénique accélérée. Par conséquent, ce travail identifie SEPN1 comme un nouveau régulateur du processus de décision du destin cellulaire des progéniteurs musculaires, l’absence de SEPN1 favorisant la différentiation au détriment du renouvellement cellulaire. Ces résultats peuvent contribuer à expliquer la déplétion de la population de cellules satellites et les défauts de régénération observés dans les modèles de SEPN1-RM, et aider à identifier de nouveaux biomarqueurs cellulaires qui seront utiles à l’avenir pour évaluer des approches thérapeutiquesMutations of Selenoprotein N (SEPN1) cause a congenital myopathy, SEPN1-related myopathy (SEPN1-RM), characterized by severe weakness and wasting of neck and trunk muscles, scoliosis and lethal respiratory failure. SEPN1-RM has been associated with oxidative stress, reduced satellite cell population and defective muscle regeneration. To investigate the underlying mechanisms, particularly a potential role of SEPN1 in regulating the balance between self-renewal and differentiation of the satellite cell pool, I used Sepn1 KO mice primary satellite cells and C2C12 cells knocked down for Sepn1, at different stages of differentiation (quiescent cells, myoblasts and myotubes). Using a suspension system to generate synchronized quiescence on C2C12, I found that Sepn1 absence in G0 cells does not prevent cell cycle exiting and re-entering but prevents normal downregulation of two key myogenic factors (MYOD1 and MYOG mRNAs) and leads to higher Cyclin D1 levels (CCND1 mRNA) in quiescence conditions. Microarray and qRT-PCR studies showed that Sepn1 depletion in proliferative C2C12 cells leads to significant increase in the levels of the transcription factors MYOG and MYOD1. In parallel, immunoblot analysis showed an increased expression of the cell cycle regulator proteins p21 and Cyclin D3. Moreover, primary murine satellite cells isolated from gastrocnemius and plantaris muscles from the Sepn1 KO mice showed increased myoblast fusion during early myogenic differentiation. Next, I explored the mechanistic pathways leading to this cell phenotype by western blots and/or qRT-PCR using Sepn1 knockdown C2C12 cells. I found no clear-cut abnormalities of the AMPK or the p38 mediated pathways, and no consistent changes in the expression of the ER stress markers GRP78 or calnexin. In contrast, our data suggest that HDAC5 and mTOR could be involved in the accelerated differentiation phenotype. Other mechanistic studies are in the progress. In conclusion, lack of SEPN1 leads to incomplete quiescence and accelerated myogenic differentiation. Thus, we identify SEPN1 as a novel regulator of the muscle progenitor’s cell fate decision process and SEPN1 depletion favors differentiation over self-renewal. These results potentially explain the depletion of the satellite cell population and the regeneration defect in SEPN1-RM models, and identify novel biomarkers useful to assess potential therapeutic interventions
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Leonaitė-Pittelkov, Bronislava Verfasser], and Elena [Akademischer Betreuer] [Conti. "Structural and biochemical studies of the S. cerevisiae DNA/RNA helicase Sen1 / Bronislava Leonaitė-Pittelkov ; Betreuer: Elena Conti." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2018. http://d-nb.info/1156852005/34.
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Haidara, Noûhou El Moctar. "Analyse du rôle de la terminaison de la transcription des ARN non-codants dans la régulation de l’expression des gènes Modulated termination of non-coding transcription partakes in the regulation of gene expression Intrinsic and extrinsic mechanisms cooperate to ensure efficient termination of RNAPIII transcription Termination of non-coding transcription in yeast relies on both an RNA Pol II CTD interaction domain and a CTD-mimicking region in Sen1." Thesis, université Paris-Saclay, 2021. http://www.theses.fr/2021UPASL010.
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La transcription pervasive est un phénomène universel et constitue une source importante d’ARN non-codants. Elle peut s’s’interférer avec l’expression normale des gènes. Cependant,la transcription pervasive peut aussi jouer un rôle dans la régulation de l’expression des gènes en favorisant la répression des gènes spécifiques par un mécanisme d’interférence transcriptionnelle. L’efficacité de la terminaison de la transcription non-codante peut fortement influencer sa capacité de réguler l’expression des gènes voisins. Cependant, à ce jour, il n’est pas clair s’il existe des mécanismes qui modulent l’efficacité de la terminaison non-codante en réponse aux signaux environnementaux afin de réguler l’expression des gènes. Durant ma thèse, j’ai abordé cette question en me focalisant sur la régulation de l’hélicase Sen1, un facteur clé de la terminaison de la transcription non-codante chez S. cerevisiae.Dans un premier temps, nous avons identifié une phosphorylation sur une thréonine conservée située dans le domaine catalytique de Sen1 et nous avons montré que cette phosphorylation réduit la capacité de liaison de Sen1 à l’ARN et par conséquent affecte la terminaison de la transcription non-codante. Par la suite, nous avons réalisé des analyses transcriptomiques à haute résolution et montré que cette phosphorylation conduit à la répression de l’expression de ZAP1 codant pour le régulateur central de la réponse au zinc par interférence transcriptionnelle via un ARN non-codant que nous avons baptisé ZRN1(Zap1 Repressor Non-coding gene 1). En plus, de nombreux gènes supplémentaires présentent un profil d’expression imitant les conditions d’excès de zinc où l’expression de ZAP1 est naturellement réprimée, suggérant que la phosphorylation de Sen1 pourrait participer à la régulation de l’expression des gènes impliqués dans l’homéostasie du zinc. Dans un second temps, en collaboration avec le laboratoire de F. Posas (IRB, Barcelone, Espagne), nous avons identifié des résidus de Sen1 qui peuvent être phosphorylés in vitropar la MAP kinase Hog1, un régulateur clé de la réponse au stress osmotique. Nous avons réalisé des analyses transcriptomiques et montré que certains gènes non-codants cibles de Sen1 sont affectés dans la terminaison de la transcription en condition de stress osmotique. Ces défauts de terminaison sont parfois accompagnés de la répression des gènes en aval ouen antisens et donc, pourraient potentiellement être impliqués dans la régulation de ces gènes en réponse au stress. Nos analyses protéomiques ont indiqué une diminution de l’interaction de Sen1 avec le complexe « Mediator » dans la même condition. Ces résultats nous permettent d’imaginer que les défauts de terminaison en condition de stress osmotique pourraient être provoqués par une diminution de l’efficacité de recrutement de Sen1 par le complexe médiateur. Ces résultats bien que préliminaires, pourraient suggérer l’implication de Sen1 dans la régulation de l’expression des gènes dans la réponse au stress osmotique. Enfin, nous avons analysé le rôle de l’interaction Sen1 avec la phosphatase Glc7, une protéine impliquée dans la terminaison de la transcription de certains gènes non-codants. Nous avons délété le motif d’interaction de Glc7 dans la protéine Sen1 et montré que cette interaction semble être importante pour la terminaison d’un nombre très restreint d’ARN non-codants. Nous avons ensuite montré que la perte de l’interaction de Sen1 avec Glc7 est associée à une augmentation de son interaction avec Nrd1 et Nab3, deux protéines qui interagissent aussi bien avec Sen1 qu’avec les ARN cibles de Sen1. Ces données suggèrent que Glc7 pourrait participer au relâchement de Sen1 de Nrd1 et Nab3 au niveau de certainsPervasive transcription is a universal phenomenon that leads to the production of amultitude of non-coding RNAs. If left uncontrolled, pervasive transcription can bepotentially deleterious for normal gene expression. However, non-coding transcription canalso play important regulatory roles, for instance by promoting the repression of specificgenes by a mechanism of transcriptional interference. The efficiency of transcriptiontermination can strongly influence the regulatory capacity of non-coding transcriptionevents, yet very little is known about the mechanisms modulating the termination of non-coding transcription in response to environmental cues.During my PhD I have addressed this question by investigating the mechanisms thatregulate the activity of the main actor in termination of non-coding transcription in S.cerevisiae, the helicase Sen1. We have identified a phosphorylation at a conservedthreonine of the catalytic domain of Sen1 and we have shown that this phosphorylationreduces the efficiency of Sen1-mediated termination by interfering with Sen1 interactionwith the RNA. Interestingly, we have found that this phosphorylation impairs terminationat an unannotated non-coding gene just upstream of the gene encoding the masterregulator of Zn homeostasis, Zap1, and thus, repression of ZAP1 expression bytranscriptional interference. We have named this non-coding gene ZRN1 for Zap1Repressor Non-coding gene 1. Furthermore, we have found that many additional genesexhibit an expression pattern mimicking conditions of Zn excess, where ZAP1 is naturallyrepressed. Taken together, our results support the idea that Sen1 phosphorylation couldbe involved in the regulation of the expression of genes involved in zinc homeostasis. In parallel, in collaboration with the laboratory of F. Posas (IRB, Barcelona, Spain), wehave identified several residues that can be phosphorylated in vitro by the MPK Hog1, themaster regulator of the osmotic stress response. We have performed high-resolutiontranscriptomic analyses and we have shown that certain Sen1-dependent non-codinggenes exhibit impaired transcription termination under osmotic stress. Interestingly, insome cases these termination defects occur concomitantly with the repression ofdownstream or antisense protein-coding genes, suggesting a possible implication in theregulation of these genes in response to stress. Our proteomic analyses indicated adecrease in the interaction of Sen1 with the mediator complex in the same conditions.These results suggest that the termination defects observed under osmotic stress mightbe due to a decrease in the recruitment of Sen1 by the mediator complex. Thesepreliminary data suggest a possible role for Sen1 in the regulation of gene expression inthe response to osmotic stress. Finally, we analyzed the role of the interaction of Sen1 with the Glc7 phosphatase, an essential protein that has previously been implicated in transcription termination at a subset of non-coding genes. We have deleted the Glc7 interaction motif in Sen1 and we have shown that this interaction is important for transcription termination at a small subset of non-coding genes. In addition, we have shown that the loss of Sen1 interaction with Glc7 is associated with an increase in the interaction with of Sen1 with Nrd1 and Nab3, two proteins that interact with both Sen1 and Sen1 target RNAs. These data suggest that Glc7 may modestly participate in the release of Sen1 from Nrd1 and Nab3 tothe RNA, which might allow more efficient transcription termination by Sen1 in some cases
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Castets, Perrine. "Etude des pathologies liées à SEPN1 : rôles de la sélénoprotéine N au cours du développement et de la regénération du tissu musculaire." Paris 6, 2009. http://www.theses.fr/2009PA066376.
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La sélénoprotéine N (SelN) est impliquée dans plusieurs pathologies neuromusculaires regroupées sous le terme SEPN1-Related Myopathies. L’expression prédominante de la protéine dans les tissus humains fœtaux et le phénotype musculaire précoce développé par les poissons zèbre invalidés, suggèrent que SelN pourrait être impliquée dans la myogenèse. Nous avons montré que le gène est exprimé précocement dans les précurseurs musculaires chez la souris et que l'expression protéique chute brutalement au cours de la période périnatale. Le modèle murin déficient en SelN ne présente pas de phénotype évident. En revanche, ces souris développent des défauts marqués lorsqu'elles sont soumises à un test de nage forcée. En parallèle, des défauts modérés de régénération musculaire ont été observés chez ces souris après injection de cardiotoxine. Une réduction drastique de la population de cellules satellites a en outre été mise en évidence et confirmée par le fait qu’une seconde régénération efficace n’est pas possible chez ces souris. Ces résultats ouvrent des perspectives nouvelles quant à la compréhension des pathologies associées à SelN.
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Tudek, Agnieszka. "Role of Nrd1p and Ctk1p in transcription termination and the metabolism of non-coding RNAs in Saccharomyces cerevisiae." Thesis, Paris 11, 2014. http://www.theses.fr/2014PA112045.
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L’ARN polymérase II (ARNPII) synthétise des ARNs codants et des ARNs non-codants (ARNnc) tels que les petits ARNs nucléaire/nucléolaire (sn/snoRNAs) et les CUTs (Cryptic Unstable Transcripts). Les CUTs sont des transcrits ubiquitaire souvent produits dans des régions codants dont la transcription peut interférer avec l’expression des gènes. Le contrôle de l’expression des ARNnc est essentiel et se fait aux niveaux de la terminaison de la transcription et la dégradation de l’ARN. Chez la levure Saccharomyces cerevisiae la terminaison de la transcription des gènes codants est effectuée par le Facteur de Clivage et de Polyadénylation (CPF), tandis que les ARNnc courts sont terminés par le complexe Nrd1p-Nab3p-Sen1p (NNS). La terminaison de la transcription est régulée par la phosphorylation du domaine C-terminal (CTD) de l’ARNPII composé de répétitions du motif Y1S2P3T4S5P6S7. Un niveau élevé de phosphorylation des résidus Ser5 près du promoteur permet l’activité du complexe NNS. La phosphorylation des résidus Ser2 est catalysée durant la transcription par la kinase Ctk1p et ces résidus sont reconnus par des éléments de la voie CPF. Mon travail de thèse a porté sur le mécanisme de terminaison de la transcription par le complexe NNS. La terminaison NNS dépend de la liaison de Nrd1p et Nab3p à des motifs dans l’ARN naissant et l’activité hélicase de Sen1p qui provoque le relarguage de la polymérase. La sous-unité Nrd1p interagit avec le domaine CTD de l’ARNPII phosphorylé sur Ser5 à travers son domaine CID (CTD-interaction domain). Le rôle du CID dans la terminaison à été proposé mais pas encore clairement démontré. En collaboration avec le groupe de P. Cramer (Université Louis-et-Maximilien de Munich Allemagne) nous avons mis en évidence que le CID est requis pour une terminaison efficace par la voie NNS et qu’il est important pour le recrutement de Nrd1p sur l’ARNPII. Le CID est aussi impliqué de manière directe ou indirecte dans l’interaction de Sen1p avec Nrd1p et Nab3p. En parallèle, avec le groupe de F. Holstege (Université Centre Médicale de Utrecht, Pays-Bas) nous avons montré que la phosphorylation en Ser2 du domaine CTD est requise pour une terminaison efficace par la voie NNS. De manière surprenante, ce résidu joue un rôle mineur dans la terminaison des ARNs codants effectuée par le complexe CPF. Les ARNs naissant terminés par le complexe NNS sont rapidement ciblés par le complexe nucléase exosome/Rrp6p et son cofacteur TRAMP ce qui mène a la maturation des sn/snoRNAs et la destruction des CUTs. Le complexe NNS interagit in vivo avec l’exosome et le complexe TRAMP, ce qui facilite la dégradation. Cependant les détails moléculaires de cette interaction restent inconnus. Nous avons montré que le domaine CID est requis et suffisant in vivo et in vitro pour l’interaction de Nrd1p avec la partie C-terminale de la sous-unité Trf4p du complexe TRAMP, que nous avons appelé NIM (Nrd1p-Interaction Motif). En collaboration avec le groupe de R. Stefl (Université Masaryk, République Tchèque) nous avons étudié par spectroscopie RMN la structure de ce motif NIM lié au CID. Nous avons mis en évidence que le CID lie le NIM et le CTD de façon similaire, et que ces interactions sont mutuellement exclusives. Le NIM se lie au CID environ 100 fois plus fortement qu’au CTD. Nous proposons que ces interactions alternatives de Nrd1p définissent des formes différentes du complexe NNS, une qui fonctionne dans la terminaison de la transcription, l’autre qui est active dans la dégradation. In vitro l’interaction du NIM avec le CID stimule l’activité poly(A)-polymérase de Trf4p ce qui suggère une fonction importante de cette interaction dans la dégradation. Nous montrons aussi que Rrp6p interagit directement avec Trf4p et cette liaison in vivo sert à recruter le complexe TRAMP à l’exosome Nous proposons que ce jeu serré d’interactions entre les complexes NNS, TRAMP et l’exosome/Rrp6p contribue à augmenter l’efficacité de dégradation de l’ARN in vivoThe RNA polymerase II (RNAPII) synthesizes protein-coding RNAs and many non-coding RNAs (ncRNAs) such as small nuclear/nucleolar (sn-/snoRNAs) and Cryptic Unstable Transcripts (CUTs). CUTs are ubiquitously transcribed including overlapping and antisense to genes, which can interfere with gene expression. Control of ncRNA expression is vital and also operates at the level of transcription termination and RNA degradation.In yeast Saccharomyces cerevisiae transcription of protein-coding genes is terminated by the Cleavage and Polyadenylation Factor (CPF), while short ncRNAs are generated by transcription termination dependent from the Nrd1p-Nab3p-Sen1p (NNS) complex. Transcription termination is regulated by phosphorylation of the carboxy-terminal domain (CTD) of the Rpb1p subunit of RNAPII, composed of repeats of the Y1S2P3T4S5P6S7 motif. Promoter-proximal high levels of serine 5 phosphorylated (Ser5P) CTD favors the function of the NNS pathway while the Ser2 phosphorylated mark (Ser2P), which is gradually introduced during transcription by Ctk1p, is recognized by components of the CPF pathway. The study of the mechanism of action of the NNS complex was the subject of my PhD work.NNS-dependent transcription termination is driven by the recognition of four nucleotide motifs in the nascent RNA by Nrd1p and Nab3p and the release of the RNAPII by the Sen1p helicase. Nrd1p interacts with the CTD-Ser5P via its CTD-interaction domain (CID). Thus a role of the CID in termination was anticipated but not demonstrated. In collaboration with the group of P. Cramer (Ludwig Maximilian University of Munich, Germany), we have shown that the Nrd1p CID domain is required for efficient transcription termination at most NNS-target genes and that it is important for the recruitment of Nrd1p to the RNAPII. This domain is also involved, directly or indirectly, in the interaction of the Sen1p helicase with Nrd1p and Nab3p. In the second project, in collaboration with F. Holstege group (University Medical Center Utrecht, Netherlands), we have shown that the CTD-Ser2P mark is important for efficient transcription termination by the NNS pathway but, surprisingly, it appears to play a minor role in termination of mRNA-coding genes by the CPF-complex.Shortly after NNS-dependent termination, the released ncRNAs are targeted by the nuclear exosome/Rrp6p nuclease complex and its cofactor the TRAMP which results in trimming of sn-/snoRNAs to a mature form and complete degradation of CUTs. The NNS complex co-purifies in vivo with the TRAMP/exosome, which is believed to facilitate subsequent degradation and processing. However, the molecular details of this interaction are unknown. We show that the CID is required and sufficient in vivo and in vitro for the interaction of Nrd1p with a motif present in the C-terminal region of Trf4p, which we called NIM (for Nrd1p-Interaction Motif). In collaboration with the group of R. Stefl (Masaryk University, Czech Republic), we obtained the NMR structure of the CID bound to the NIM and demonstrated that the CID binds in a similar manner to the CTD and the NIM. The CID interacts with the CTD and the NIM in a mutually exclusive manner and the former interaction is roughly 100 times stronger than the first. We propose that these alternative interactions represent two forms of the NNS complex, one functioning in termination and the other in degradation. Importantly, the NIM-CID interaction is likely to be functionally relevant since in vitro it results in the stimulation of the polyA polymerase activity of the Trf4p. We further show that Trf4p interacts directly with Rrp6p, which in vivo serves to recruit the TRAMP to the core exosome complex. This tight interplay between the NNS, TRAMP and exosome/Rrp6p complexes most likely accounts for the efficiency of RNA degradation in vivo
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Michaelis, Marten. "Einfluss von Selenoprotein P auf die intestinale Tumorigenese im Mausmodell." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2009. http://dx.doi.org/10.18452/15874.
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Das essentielle Spurenelement Selen (Se) wird als einziges Spurenelement über den genetischen Code als Bestandteil der 21. proteinogene Aminosäure Selenocystein (SeCys) in eine kleine Gruppe von Proteinen eingebaut. Als Bestandteil des aktiven Zentrums dieser Selenoproteine ist Se bzw. SeCys essentiell für deren Funktion. Die Biosynthese der Selenoproteine ist durch eine Reihe von Besonderheiten gekennzeichnet, so z.B. durch eine hierarchisch abgestimmte Versorgung der unterschiedlichen Organe mit dem limitierenden Spurenelement bzw. durch eine hierarchische Versorgung der einzelnen Selenoproteine während ihrer Biosynthese. Für die biologische Verwertung und Verteilung ist das Selenoprotein SePP von zentraler Bedeutung. Transkriptomanalysen haben aufgezeigt, dass gerade in Tumoren die Expression von SePP stark erniedrigt ist. In dieser Arbeit wurde untersucht, inwieweit der Verlust von SePP einen kausalen Einfluss auf die Tumorigenese nimmt. Hierzu wurden zwei transgene Mausmodelle verkreuzt: zum einen Mäuse mit einem partiellen bzw. kompletten genetischen Verlust der SePP-Expression und zum anderen Mäuse mit einer Mutation im APC-Tumorsuppressorgen, welche multiple intestinale Neoplasien (Min) auslöst und als Paradigma in der experimentellen Darmkrebsforschung dient. Der komplette Verlust des SePP-Gens bewirkte eine stark erhöhte Tumorrate im Dünndarm der APCmin-Mäuse. Hierdurch konnte SePP als neuer wichtiger Modulator der APC-abhängigen Tumorigenese etabliert werden. Interessanterweise genügte bereits der Verlust eines einzelnen SePP-Allels, um mehr, größere und weniger differenzierte Adenome im Dünndarm entstehen zu lassen. Diese Beobachtung deutet auf einen entscheidenden Gen-Dosis-Effekt von SePP für die intestinale Tumorigenese hin und könnte damit als weiteres sinnvolles Kriterium zur Feindiagnostik von Darmkrebs dienen. Die molekularbiologischen Untersuchungen der Tumore lassen eine Aktivierung von Zellzyklus-, Angiogenese- und Akutphaseprozessen vermuten. Hierdurch und über die erhöhte Produktion von Wachstumsfaktoren kann die vermehrte Tumorigenese bei SePP-Mangel erklärt werden. Weitergehend konnte auch der Darm, ungeachtet seiner primären Rolle bei der Selenaufnahme, als abhängig von einer regulären SePP-Expression erkannt werden. Somit stellt sich SePP als zentraler Vermittler des Selenmetabolismus dar, und könnte auf lange Sicht als funktioneller Biomarker des Selenstatus für die individuelle Risikoabschätzung etabliert werden.Selenium (Se) is the only trace element which is encoded in the genome as the 21st proteinogenic amino acid selenocystein (Sec). Se is essential for the catalytic activity of the small group of Sec-containing selenoproteins. The biosynthesis of this group of extraordinary proteins is characterized by several specialities, e.g. the distribution of Se differs between the organs giving rise to a hierarchical biosynthesis of the selenoproteins and there is an intracellular hierarchy of selenoprotein biosynthesis in times of Se depletion. One particular selenoprotein is of central importance for the organification and trafficking of Se within the organism, i.e., Selenoprotein P (SePP). From transcriptome analyses it was deduced that this Se transport protein is markedly reduced in tumours of several origins. The aim of this thesis was to elucidate whether SePP has a causal impact on the tumourigenesis within the intestinal tract. For this purpose, the SePP-KO mouse model with a genetically impaired SePP expression was crossed with the well-established APCmin intestinal tumour model. A stop mutation in the APC tumour suppressor gene causes multiple intestinal neoplasias (Min) in these mice. The combined deletion of SePP caused a sharp increase in tumour incidence in the small intestines of APCmin mice. Interestingly, even the inactivation of only one SePP allele was sufficient to induce more and less well differentiated adenomas in the small intestine. These results indicate that SePP acts as an important modulator of APC dependent tumorigenesis in a gene dose dependent manner. In the long run, SePP might turn out as another valuable biomarker to estimate the individual cancer risk. From a mechanistic point of view, the transcriptome analyses indicate that an impaired SePP expression favors cell cycle progression, angiogenesis and acute phase response. In addition, an elevated production of growth factors in response to SePP deficiency might contribute to the phenotype of bigger and more undifferentiated tumours. Additional analyses of the intestines revealed that the intestinal tract is dependent on a regular SePP expression in order to synthesise its regular set of selenoproteins even so it represents the prime organ of Se absorption. Therefore, SePP represents a central Se transport and storage protein also within the intestinal tract, highlighting its essential role to preserve health and regular Se metabolism.
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NISKA, JOANNA. "TERMINATING REPLICATION AT TERS AT EUKARYOTIC CHROMOSOMES." Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/234148.
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Faithful transmission of genetic material is challenged by the presence of natural impediments affecting replication fork progression that jeopardize genome integrity. Transcription, which competes with DNA replication for the same template, is a common barrier to replication in both prokaryotes and higher eukaryotes. Multiple mechanisms minimize the consequences of DNA replication and transcription collisions in order to prevent torsional stress accumulation that occurs when replication fork encounters the transcription machinery. Defects in resolving topological problems during chromosome replication lead to fork reversal, R loop formation and recombination-induced genome rearrangements. Our interest is focused on the processes that coordinate replication with transcription at TERs (termination sites) and on the molecular pathways involved in termination of DNA replication. We investigated the roles of Rrm3, a DNA helicase that assists replication fork progression, and of Sen1, a DNA/RNA helicase that resolves the conflicts between replication and transcription. We found that Rrm3 and Sen1 mediate replication termination at specific TERs, preventing aberrant events ultimately leading to chromosome fragility. Our results contribute to the elucidation of mechanisms coordinating replication and transcription at TER zones in eukaryotes.
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Chen, Sung-Yuan, and 陳松遠. "miR-1236 regulates epithelial-mesenchymal transition and metastatic activity through repressing sentrin-specific protease1(SENP1) and HDAC3." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/tda32k.
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博士國立陽明大學
生化暨分子生物研究所
105
Intratumoral hypoxia induces epithelial–mesenchymal transition and promotes cancer metastasis. MicroRNAs (miRNAs) are endogenous, single-strand RNA molecules that regulate gene expression. MiRNAs control cell growth, proliferation, differentiation and cell death and may function as oncogenes or tumor suppressors. HDAC3 and SENP1 are two molecules involved in hypoxia-induced EMT and HIF-1α stability, respectively. In this proposal, we show that miR-1236 plays a critical role in hypoxia-induced EMT and metastasis. MiRNA prediction programs TargetScan and miRanda show that miR-1236 may target HDAC3 and SENP1. MiR-1236 represses the luciferase activity of reporter constructs containing 3’UTR of HDAC3 and SENP1 as well as the expression levels of HDAC3 and SENP1. MiR-1236 abolishes hypoxia-induced EMT and inhibits migration and invasion activity of tumor cells. Hypoxia represses miR-1236 expression. The promoter region of miR-1236 is identified as the NELFE promoter. Twist1, an EMT regulator activated by hypoxia/HIF-1α, is shown to repress the reporter construct driven by the NELFE promoter. The binding site of Twist1 in the NELFE promoter is identified and chromatin immunoprecipitation assays show the direct binding of Twist1 to this site. Overexpression or knockdown of Twist1 in stable cell lines shows the inverse correlation between Twist1 and miR-1236 expression. These results identify a miRNA that regulates hypoxia-induced EMT and metastasis through repressing HDAC3 and SENP1 expression and present a regulatory network that involves many key players in hypoxia-induced EMT.
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Wu, Yu-Chih, and 吳友志. "The Role of SENP1 in OCT4 Protein Stability and Chemotherapeutic Sensitivity of Testicular Embryonal Carcinoma Cells Under Hypoxic Conditions." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/69551670324971150982.
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博士國防醫學院
生命科學研究所
101
Testicular germ cell tumors (TGCTs) generally respond well to chemotherapy, but tumors that express low levels of the transcription factor OCT4 are associated with chemoresistance and poor prognosis. Hypoxia is known to induce drug resistance in TGCTs, however, the mechanistic basis for reduced expression of OCT4 and drug resistance is unclear. Here we demonstrate that hypoxia reduces OCT4 levels and increases the resistance of embryonal carcinoma (EC) cells to cisplatin and bleomycin. Furthermore, we show that the loss of OCT4 expression under hypoxia can be triggered by sumoylation, which was regulated by SUMO1 and the SUMO1 peptidase SENP1. Under hypoxic conditions, overexpression of SUMO1gg (the active form of SUMO1) not only increased the level of sumoylated OCT4 (Su-OCT4), but also decreased the stability of OCT4 protein. Additionally, overexpression of SENP1 reduced the Su-OCT4 level induced by SUMO1gg overexpression, thereby maintaining OCT4 levels and enhancing chemosensitivity. Mechanistic investigations revealed that OCT4 sumoylation occurred at K123, as overexpression of an OCT4-K123R mutant effectively reduced the level of Su-OCT4 under hypoxic conditions. Taken together, our results demonstrated that hypoxia reduces OCT4 expression levels in ECs to increase drug resistance, and that these effects could be countered to ablate the suppressive effects of hypoxia on chemosensitivity. Our findings also highlight SENP1 as a potential therapeutic target for drug-resistant TGCTs.
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Finkel, Jonathan Sewell. "Dissecting the multiple phenotypes of the yeast Sen1p." 2008. http://www.library.wisc.edu/databases/connect/dissertations.html.
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Rasmussen, Theodore P. "SnoRNA processing in yeast requiring the action of Sen1p." 1997. http://catalog.hathitrust.org/api/volumes/oclc/39824689.html.
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Thesis (Ph. D.)--University of Wisconsin--Madison, 1997.Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 129-143).
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Zhang, Yinglu. "Structural studies of the yeast transcription termination complex Nrd1-Nab3-Sen1." Thesis, 2019. https://doi.org/10.7916/d8-n4dw-1684.
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The Nrd1-Nab3-Sen1 (NNS) complex carries out the RNA polymerase II (Pol II) transcription termination of non-coding RNAs (ncRNAs) in yeast, although the detailed interactions among its subunits remain obscure. In this dissertation, we have identified three sequence motifs in Sen1 that mediate direct interactions with the Pol II CTD interaction domain (CID) of Nrd1, determined the crystal structures of these Nrd1 interaction motifs (NIMs) bound to the CID, which elucidated the molecular basis for their recognition by Nrd1 CID, and characterized the interactions in vitro and in yeast. Although the Sen1 NIMs are not essential for supporting viability from the in vivo studies, termination defects were observed from NIM deletions in a reporter assay. In addition, the conservation of Sen1 NIMs suggests these interactions are very likely to promote NNS function.
This dissertation also describes the structural studies of the flowering time control protein FPA in plants, which regulates the alternative 3’-end processing of the FLOWERING LOCUS C (FLC) antisense RNA. FPA belongs to the split ends (SPEN) family of proteins, which contain N-terminal RNA recognition motifs (RRMs) and a SPEN paralog and ortholog C-terminal (SPOC) domain. The SPOC domain is highly conserved among FPA homologs in plants, but the conservation with the domain in other SPEN proteins is much lower. We have determined the crystal structure of Arabidopsis thaliana FPA SPOC domain at 2.7 Å resolution. Structural and sequence analyses identify a surface patch that is conserved among plant FPA homologs. Mutations of two residues in this surface patch did not disrupt FPA functions, suggesting that either the SPOC domain is not required for the role of FPA in regulating RNA 3’-end formation or the functions of the FPA SPOC domain cannot be disrupted by this combination of mutations.
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Mei, Yan (Angela). "Analysis of DOCK2 and SENP2 mutations on the immune system and CD8 T cell survival." Phd thesis, 2018. http://hdl.handle.net/1885/165368.
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The discovery of genes essential for normal T cell numbers and T cell-mediated immune responses provides valuable insights into the adaptive immune system and targets for modulating disorders of immunity in cancer, transplantation, infection and autoimmunity. The studies presented in this thesis investigate two novel gene mutations that diminish the number of circulating T cells. The mutations were discovered by genome-wide ENU-mutagenesis in mice by flow cytometric screening of lymphocyte subsets in splenocytes and in peripheral blood. Genetic mapping and sequencing identified the two mutations. The first was a premature stop-gain mutation in Dock2, encoding a GTP exchange factor
for the Rac family of cytoplasmic small G proteins, which has previously been found to be critical for T cells and immunity in mice and humans. The second mutation was a synonymous exonic nucleotide substitution in Senp2, encoding a specific protease for cleaving the ubiquitin-like SUMO protein from its conjugates with other proteins, whose critical role in T cells had not previously been shown.
Mice homozygous for the ‘dockland’ E775X mutation in DOCK2 (doc/doc) had 80% fewer naïve (CD44low) CD8 and CD4 T cells, 70% fewer NKT cells, 50% fewer B cells in the blood and 80% fewer B cells in the spleen, and absence of splenic marginal zone B cells. Mutant T cells nevertheless divided normally to anti-CD3 in vitro, consistent with intact TCR signaling, and peripheral CD44high activated memory T cells were present in normal or increased numbers consistent with homeostatic proliferation. Thymocytes showed evidence of developmental defects, including 50% decreased CD4+CD8+ double positive (DP) cells with
lower CD5 expression and greatly decreased NKT cells. The percentage of CD4+ or CD8+ single positive (SP) thymocytes lacking CD69 was increased, consistent with delayed egress of fully mature cells. In competitive bone marrow transplantation experiments, mutant hematopoietic stem cells engrafted efficiently based on their contribution to granulocytes, but failed to contribute to peripheral T cells due to progressive under-representation at the DP and SP thymocyte stages
of development. Mutant B cells contributed poorly to the follicular and germinal centre subsets, and were completely absent from the marginal zone subset. These results establish that DOCK2 is essential within T and B lymphocytes.
Despite decreased circulating naïve T cells, Dock2 mutant doc/doc mice exposed sequentially to H3N2 and H1N1 influenza viruses formed normal numbers of memory CD8 T cells binding H2-Db MHC tetramers bearing the dominant influenza NP peptide epitope, NP366-374 (NP+), in contrast to Dock8 mutant mice analysed in parallel which had greatly diminished memory T cells. Dock2 mutant mice nevertheless formed fewer NP-binding effector CD8 cells than wildtype at the peak of the primary and recall response. During acute lung infection with PR8-strain H1N1 influenza virus, Dock2 mutant mice were protected from
weight loss and morbidity occurring between days 5 and 7 in wild-type mice.
Mice with combined homozygous mutations in Dock2 and Dock8 tended to have
fewer circulating naïve T cells and NKT cells than either single mutant, although
this did not achieve statistical significance in the cohort available. Collectively,
these results extend earlier published findings in Dock2 knockout mice, and
provide new insights into the effects of DOCK2 deficiency on the anti-viral
immune response that are relevant to the fatal infections and T cell lymphopenia
in DOCK2-deficient children.
In the second mutant strain, duan, a 70% decrease selectively in circulating naïve
CD8 T cells mapped as a recessive trait to a synonymous A>G substitution at
Ch16:22,036,478 in exon 11 of Senp2. The mutation was +9 nucleotides from the
exon 11 splice acceptor and diminished splicing of exon 10 to this acceptor. Of
spliced mRNA that was produced, 55% skipped exon 11 to create an in-frame
deletion of 15 amino acids. Upon expression of GFP-tagged SENP2, the internally
deleted protein nevertheless exhibited normal intracellular localization to the
nuclear membrane. While Senp2-/- null mutation in mice is homozygous lethal
prior to blastocyst implantation, a complementation cross with the duan point
mutant revealed that compound heterozygous Senp2dua/- mice were viable and
recapitulated the CD8 T cell deficiency of Senp2dua/dua homozygotes, confirming
that partial deficiency of SENP2 causes a selective T cell deficit.
Thymic T cells subsets were unaffected in Senp2dua/dua mice, and the basis for the
decreased number of naïve CD8 T cells was shown to be diminished survival in
the periphery. This was most clearly shown by comparing the persistence of
ovalbumin-specific OT-I CD8 T cells with normal or mutant Senp2 after the T
cells were “parked” in wild-type mice without antigenic stimulation. In this
setting, mutant OT-I T cells transduced with a retroviral vector expressing wildtype
SENP2 preferentially survived, further confirming that SENP2 deficiency
was responsible. Competitive bone marrow transplantation also showed a cell
autonomous requirement for normal splicing of Senp2 to maintain peripheral CD8
T cell numbers but not for CD4 T cells or B cells. Upon sequential exposure to
recombinant H3N2 and H1N1 influenza viruses expressing ovalbumin peptide,
OT-I T cells bearing the Senp2dua/dua mutation formed an equally large population
of effector CD8 T cells 7 days after primary or recall challenge, but persistence of
memory CD8 T cells was decreased more than 90%. Senp2 mutant CD8 T cells
responded normally to CD3 stimulation in vitro as measured by cell division.
They also responded normally to IL-7 in vitro as measured by STAT5
phosphorylation, despite a cell autonomous 25% decrease in IL7Ra/CD127 on
their cell surface. Collectively, these results define a key role for controlling
SUMO conjugation by SENP2 in the survival of naïve and memory CD8 T cells,
apparently involving a pathway independent of the established TCR and IL-7
signalling mechanisms.
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Chen, Nien-Ming, and 陳年明. "Study of the Self-polymerization of SUMO1 and Characterization of the PolySUMO Chain Degradation Catalyzed by SENPs." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/79780442723922019089.
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碩士國立臺灣大學
生化科技學系
99
The conjugation of SUMO moiety by SUMO conjugation system and the removal of the moiety by SENPs are important aspects of protein modification in cells. Analogous to ubiquitination, sumoylation regulates many physiological functions of proteins. It has been demonstrated that ubiquitin can form various polymeric chains via specific lysines to execute distinct functions. Although certain types of polymeric SUMO chains have been described, their roles in biological processes and the mechanism of formation remain unclear. In this study, I transformed the enzymes required for sumoylation into Escherichia coli BL21 (DE3) to establish a sumoylation system, which lacks SENP-mediated desumoylation for analyzing the formation of polymeric SUMO chains. The results suggest that SUMO1, which has been considered unable to form polymers, is able to form polymeric chains mainly through self-conjugation at specific lysine residues nearby the flexible N terminus, indicating that the steric hindrance is a key factor modulating polySUMO chains formation. To investigate the character of polySUMO degradation catalyzed by SENPs, I examined the degradation of polySUMO chains by conducting the in vitro SENP activity analysis. The data showed that the catalytic domains of all SENPs, except SENP5, are able to deconjugate both polySUMO1 chains and polySUMO2 chains. Moreover, the catalytic core of SENP1 and SENP2 present similar preference toward polySUMO1 and polySUMO2 chains. I also found that the catalytic cores of SENP6 and SENP7 exhibit an exquisite substrate selectivity that polySUMO2 chains are deconjugated more efficiently than polySUMO1 chains.
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Wang, Pin-Ya, and 王品雅. "Characterization of the specialized gene expression during the onset of primary spermatocytes: using senp2 as a key paradigm." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/16390202286685559080.
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碩士東海大學
畜產與生物科技學系
93
The quality of semen relies immensely on spermatogenesis. In this study, we intended to understand the differential gene expression patterns between spermatogonia and primary spermatocytes. We prepared total RNA from the 8-day-old (containing only Sertoli cells and spermatogonia) and the 10-day-old (primary spermatocytes starting to appear) mouse testes, respectively, for the DD RT-PCR analysis to study the differential gene expression between these two cell populations. We recovered differentially expressed cDNA fragments, and cloned them by TA Cloning for DNA sequence analysis. The cloned sequences were compared with the nucleotide sequences deposited in the GenBank database. Most of the clones contain unknown sequences. Three clones contain gene sequences known as activating transcription factor 1 (ATF-1), PHD finger protein and SUMO-1 protease 2 (SENP2). Among them, we studied gene expression of senp2 during spermatogenesis in details. By reverse-transcription PCR we found that senp2 differentially expressed between spermatogonia and primary spermatocytes. Expression of senp2 increased in the primary spermatocytes, and was highest in secondary spermatocytes. Western blotting analysis indicated that SENP2 expression pattern was consistent with its RNA expression in the testis during prepubertal stages. In addition, the reverse-transcription PCR results indicated that senp2 transcript was also expressed in MA-10 Leydig tumor cell line. Furthermore, by immuno-fluorescent staining assay, we found that the SENP2 expression was high in germ cells, mainly the secondary spermatocytes in a form of perinucleus, but was low in somatic cells, e.g., the Leydig cells. We conclude that senp2 expressed in all populations of germ cells in a stage-dependent manner. Its expression was predominantly elevated in the secondary spermatocytes, and reduced to barely detectable levels in mature spermatozoa.
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Jaafari, N., F. A. Konopacki, T. F. Owen, Sriharsha Kantamneni, P. Rubin, T. J. Craig, K. A. Wilkinson, and J. M. Henley. "SUMOylation Is Required for Glycine-Induced Increases in AMPA Receptor Surface Expression (ChemLTP) in Hippocampal Neurons." 2012. http://hdl.handle.net/10454/8124.
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yesMultiple pathways participate in the AMPA receptor trafficking that underlies long-term potentiation (LTP) of synaptic transmission. Here we demonstrate that protein SUMOylation is required for insertion of the GluA1 AMPAR subunit following transient glycine-evoked increase in AMPA receptor surface expression (ChemLTP) in dispersed neuronal cultures. ChemLTP increases co-localisation of SUMO-1 and the SUMO conjugating enzyme Ubc9 and with PSD95 consistent with the recruitment of SUMOylated proteins to dendritic spines. In addition, we show that ChemLTP increases dendritic levels of SUMO-1 and Ubc9 mRNA. Consistent with activity dependent translocation of these mRNAs to sites near synapses, levels of the mRNA binding and dendritic transport protein CPEB are also increased by ChemLTP. Importantly, reducing the extent of substrate protein SUMOylation by overexpressing the deSUMOylating enzyme SENP-1 or inhibiting SUMOylation by expressing dominant negative Ubc9 prevent the ChemLTP-induced increase in both AMPAR surface expression and dendritic SUMO-1 mRNA. Taken together these data demonstrate that SUMOylation of synaptic protein(s) involved in AMPA receptor trafficking is necessary for activity-dependent increases in AMPAR surface expression.
Medical Research Council, the European Research Council and the Wellcome Trust
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Wohlmuth, Jan. "Možnosti využití nanočástic na bázi selenu v ochraně rostlin vůči bakteriálním patogenům." Master's thesis, 2019. http://www.nusl.cz/ntk/nusl-426622.
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The work is focused on the observation of the effect of selenium nanoparticles [Se] on seedlings of head cabbage, whose seeds were inoculated with Xanthomonas campestris pv campestris (Pammel) Dowson. The paper summarizes protocols of nanoparticle synthesis using various methods. A special part was devoted to methods of synthesis of selenium [Se] nanoparticles. Methods of conventional synthesis and methods of biological synthesis have been described. When using a preparation containing nanoparticles, the effect on bacteria inoculated on seed was clearly seen. Increasing concentration of the product visibly appeared to be stronger. The use of SelenBact appears to be very promising when applied to seed contaminated with bacteria Xanthomonas campestris pv campestris (Pammel) Dowson.
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Greta, Baggio. "Investigation on Bacterial Selenite Reduction to Elemental Selenium by Bacillus mycoides SeITE01 and Stenotrophomonas maltophilia SeITE02 through Spectroscopic and Metabolomics Analyses, with Characterization of Biogenic Selenium Nanoparticles (Bio-SeNPs)." Doctoral thesis, 2020. http://hdl.handle.net/11562/1015752.
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Bacillus mycoides SeITE01 and Stenotrophomonas maltophilia SeITE02 are environmental bacterial isolates that rely on detoxification processes to transform selenite (SeO32-), a highly toxic and bioavailable chemical species of selenium, into insoluble and virtually nontoxic elemental selenium (Se0) with the formation of biogenic selenium nanoparticles (Bio-SeNPs). In the last decade, Bio-SeNPs have attracted attention for their interesting applications in the nanotechnology, industrial and medical fields not only due to their special physico-chemical features, but also for their attractive antimicrobial activities and anticancer properties. These worthwhile biotechnological traits are related to the presence on Bio-SeNPs of an external organic coating, whose composition and role are mostly unknown and currently under investigation. In the first part of this thesis, FTIR (Fourier Transform Infrared) spectroscopy was applied to study the SeO32- bio-reduction process analysing the bio-molecular composition of both SeITE01 and SeITE02 cells. The analysis was conducted during the diverse cellular growth phases and in different conditions, namely untreated (growth without the presence in the medium of sodium selenite Na2SeO3) and Se-treated (exposure to the stress factor SeO32-). Moreover, along with FTIR spectroscopic analyses, the biogenic intracellular and extracellular SeNPs bio-produced by the bacterial strains and collected with two extraction methods (vacuum filtration and pelleting processing) were examined also using DLS (Dynamic Light Scattering) measurements and TEM (Transmission Electron Microscopy) imaging. In the second part of this research, metabolomics was used to investigate the biological reduction and effect of Na2SeO3 on SeITE01 and SeITE02 cells by a LC-MS (Liquid Chromatography Mass-Spectrometry) approach. Both intracellular and extracellular metabolites and their concentration fluxes during a defined time course and in response to the exposure of bacterial cells to SeO32- were studied. From the results obtained with all the investigation techniques it was possible to observe and underline two distinct behaviors and trends assumed by the bacterial strains. SeITE01 cells showed substantial changes when exposed to SeO32-, activating a series of macromolecular responses and biochemical pathways to defend the cells against the toxic action of both the oxyanion and the Se nanostructures. Moreover, different FTIR spectral trends were acquired with regard to the organic coatings present on the surfaces of the biogenic SeNPs synthesized by this bacterium, and the most marked differences were recorded according to their different localization (intracellular or extracellular). The Bio-SeNPs production in this Gram-positive microorganism was also described by the data collected with the DLS and the zeta-potential measurements. The analyzed Bio-SeNPs showed an average dimension between 637 and 393 nm for the intracellular Bio-SeNPs, and 147 nm for the extracellular ones. Low value of negative potentials suggest a lower stability of these nanostructures in solution. On the contrary, the Gram-negative bacterium SeITE02 did not present drastic changes in the macromolecular composition after SeO32- exposure, and the variations recorded were mainly due to the maintenance of vital cellular functions. DLS data revealed an average size of 315 nm for the intracellular Bio-SeNPs and 160 nm for the extracellular ones, whilst negative potential values at or below -30 mV were recorded, indicating a remarkable stability of these biogenic Se nanostructures and a lower tendency to form aggregates and thus to precipitate. Thus, the results obtained in the course of this investigation revealed two distinct attitudes and responses on the part of the two microorganisms SeITE01 and SeITE02 to the SeO32- exposure and Bio-SeNPs production.
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Collin, Pierre. "Caractérisation du domaine C-terminal de l'ARN polymérase II et de la phosphatase Glc7 dans la terminaison transcriptionnelle chez Saccharomyces cerevisiae." Thèse, 2018. http://hdl.handle.net/1866/21788.
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