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1
Mokas, Sophie. "Mécanismes d'assemblage des granules de stress." Thesis, Université Laval, 2012. http://www.theses.ulaval.ca/2012/28583/28583.pdf.
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Les granules de stress (GS) sont des sites de régulation de la traduction qui permettent aux cellules cancéreuses de survivre au stress. Elles apparaissent sous différentes conditions de stress et disparaissent une fois remises de celles-ci. Elles se forment selon deux voies, l’une dépendante de la phosphorylation du facteur eIF2α et l’autre indépendante. Les mécanismes d’assemblage des GS par cette dernière voie restent méconnus. Afin de définir ces mécanismes, l’objectif principal de ma maîtrise était de caractériser les étapes critiques de la formation de GS. En utilisant des approches pharmacologiques et d’interférence à l’ARN, nous démontrons que l’inactivation de plusieurs facteurs d’initiation de la traduction provoque la formation de GS indépendamment de la phosphorylation d’eIF2α. Par contre, l’inactivation du facteur eIF4E, ainsi que ceux permettant l’association du 60S à l’ARNm, n’induit pas de GS. De nouvelles stratégies anti-cancer inhibant la traduction et bloquant la formation de GS serait alors possible.
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Fung, Gabriel. "Interplay between stress granules, cellular stress response, and coxsackievirus B3 infection." Thesis, University of British Columbia, 2016. http://hdl.handle.net/2429/58510.
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Viral infection affects a multitude of cellular processes to facilitate successful replication. Such responses include the formation of stress granules (SGs) and the activation of autophagy. SGs are stalled translational complexes and function to restore cellular homeostasis after stress. Autophagy is a cellular process that recycles misfolded proteins and damaged organelles and plays an important role in various stress responses. We previously demonstrated that infection with Coxsackievirus B3 (CVB3), a common human pathogen for viral myocarditis, disrupts the autophagic process to support effective viral replication. However, the interplay between CVB3 and SGs, and the ability of SGs to regulate autophagy have not been investigated. Here we showed that SGs are formed early and actively disassembled late during CVB3 infection due to viral protease 3Cpro-mediated cleavage of Ras-GAP SH3 domain binding protein 1 (G3BP1), a key nucleating protein of SGs. Overexpression of G3BP1 inhibits CVB3 replication, indicating an anti-viral function of SGs. We further demonstrated that the C-terminal product of G3BP1 has a toxic gain-of-function that further inhibits SG formation. We also examined the interaction between CVB3 and the transactive response DNA-binding protein-43 (TDP-43), an RNA binding protein that mislocates to SGs under cellular stress. We found that TDP-43 is translocated from the nucleus to SGs upon infection through the activity of viral protease 2Apro, followed by cleavage by protease 3Cpro. The C-terminal product of TDP-43 is quickly degraded by the proteasome, whereas the N-terminal truncate acts as a dominant-negative mutant that inhibits the function of native TDP-43 in alternative RNA splicing. Knockdown of TDP-43 results in an increase in viral titres, suggesting a protective role for TDP-43 in CVB3 infection. Lastly, we explored the possible role of G3BP1-SGs in regulating autophagy. We showed that G3BP1 inhibits autophagic flux, likely by binding to cytoplasmic signal transducer and activator of transcription 3 (STAT3). Taken together, our results reveal that the host SGs and associated proteins, including G3BP1 and TDP-43, are utilized and modified during CVB3 infection to promote efficient viral replication and induce viral pathogenesis. Moreover, we propose a novel mechanism by which G3BP1 binds cytoplasmic STAT3 to inhibit autophagy.Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
Graduate
3
Fanous, Alaa. "Elucidating the Functional Role of TDRD3 in Stress Granules." Thèse, Université d'Ottawa / University of Ottawa, 2014. http://hdl.handle.net/10393/31019.
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Tudor domain-containing protein 3, TDRD3, was first identified in a proteomic survey of the spliceosome machinery. Although its function remains elusive, elevated TDRD3 gene expression is associated with poor prognosis of estrogen receptor-negative breast cancer. The Tudor domain of TDRD3 is highly similar to the Tudor domain of the survival of motor neuron (SMN) and accordingly, it has been shown to bind dimethylated arginine residues. Our lab has previously demonstrated the association of TDRD3 with the translation machinery and most importantly, its localization to stress granules (SG) upon various cellular stresses. In this study, it was revealed that TDRD3 knockdown facilitates and accelerates SG assembly and consequently accelerates SG disassembly. Moreover, we showed that wildtype TDRD3 rescued this defect while a mutation in the Tudor domain of TDRD3, E691K, was not able to do so. Taken together, these findings allude to a prominent role for TDRD3, via its Tudor domain, in the proper formation of SGs.
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Riemschoß, Katrin [Verfasser]. "Similarities of stress granules and cytosolic prions / Katrin Riemschoß." Bonn : Universitäts- und Landesbibliothek Bonn, 2019. http://d-nb.info/1206246170/34.
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Bounedjah, Ouissame. "Mécanismes d'assemblage des granules de stress dans des conditions de stress oxydatif et osmotique." Thesis, Evry-Val d'Essonne, 2014. http://www.theses.fr/2014EVRY0017/document.
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Les granules de stress (GSs) sont des entités cytoplasmiques très dynamiques et dépourvues de membranes, ils apparaissent suite à des conditions de stress. En raison du fait que les GSs sont instables et dépourvus de membranes, leur isolement biochimique n'a pas été accompli. En effet, toutes les fonctions qui sont attribuées aux GSs se basent principalement sur l'observation par microscopie optique de quelques protéines régulatrices des ARNm. A travers cette étude, nous avons déterminé la composition des GSs à l'échelle nanométrique dans deux conditions de stress différentes (stress osmotique et stress oxydatif). Nous avons d'abord cartographié les GSs par microscopie électronique puis ces mêmes granule sont été analysés par microscopie ionique. Grâce au marquage isotopique de l'ARN, nous avons montré que ces structures sont très riche sen ARN, par rapport au reste du cytoplasme et ceci dans les deux conditions de stress. Le deuxième volet de notre étude nous a permis de mettre en évidence un rôle fonctionnel des GSs dans la réponse au stress osmotique. En effet, l'augmentation de la force ionique et de l'encombrement macromoléculaire (deux paramètres qui sont accentués dans les conditions de stress osmotique) permet la dissociation des polysomes et l'assemblage des GSs. Néanmoins, quelques heures après, l'accumulation des osmolytes compatibles dans le cytoplasme par les transporteurs spécifiques réduit la force ionique et l'encombrement macromoléculaire permettant ainsi la dissociation des GSs et le retour progressif de la traduction. Nous avons démontré également que le préconditionnement des cellules avec des osmolytes compatibles avant leur exposition à un stress osmotique sévère bloque la formation des GSs et augmente le taux de survie des cellules. L'ensemble de ces résultats prouve que les osmolytes compatibles favorisent la survie cellulaire et l'adaptation des cellules aux conditions de stress osmotique partiellement via la dissociation des GSs et la reprise de la traductionStress granules (SGs) are highly dynamical cytoplasmic bodies laking encapsuling membarnes which appear in reponse to a wide variety of stresses. Due to their lack of membranes and their instability, their biochemical isolation from cells has not yet been accomplished. All functions attributed to SGs are mostly based on optical microscopy observations of key proteins involved in mRNA processing. In the first part of our study, we explored the RNA composition SGs at a nanometric scale and their biophysical properties in two different conditions (osmotic and oxydative stresses). To do so, we imaged and identified the SGs by electron microscopy and analyzed the distribution of N15-uridine labeled-RNA via ionic microscopy. We show that the SGs are enriched in RNA compared to rest of cytoplasm in the two stress conditions. The second part of our study, we tackled the functional role of the SGs in response to osmotic stress. The increase of ionic strength and macromolecular crowding which are the hallmark of osmotic stress lead to SGs assembly in cells after polysome disassembly. However, several hours after the onset of stress, the compatible osmolyte accumulation in the cell by specific transporters reduces the ionic strength and macromolecular crowding thus allowing the diassembly of SGs and the progressive return of translation. In line with this, celle preconditioning with compatible osmolytes before their exposition to severe osmotoc stress prevents the assembly of SGs and increases the rate of cell survival. Together, these results show that compatible osmolytes favors cell survival and adaptation to osmotoc stress via the disassembly of SGs ans recovery of translation
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Gasparinho, Goncalves A. C. "Understanding the role of stress granules in the inner ear." Thesis, University College London (University of London), 2017. http://discovery.ucl.ac.uk/1553331/.
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The human ear undergoes stress constantly. Exposure to noise, drugs or ageing contribute to the irreversible loss of hair cells, resulting in hearing loss. To understand why we go deaf, it is important to understand how the ear responds to stress. Stress granules (SGs) are aggregates of mRNA and proteins that are formed during stress. The SG-pathway has been implicated in the cochlea’s response to aminoglycoside antibiotics, suggesting that SGs play an important role during ototoxicity. Dysregulation of SG-formation has also been linked to neurodegeneration, supporting the hypothesis that SGs play a critical role in cell survival. Here, the formation and regulation of SGs have been investigated in an inner ear context using a combination of inner ear-derived UB/OC-2 cells, cochlear explants and the in-vivo mouse cochlea. Cells were labelled for two SG-proteins, TIA-1 and Caprin-1, and polyA+ mRNA was detected within SGs using RNA-immuno-FISH. A novel quantification method was developed to characterise in detail the number and size of SGs upon two stress paradigms, heat shock and arsenite. PolyA+ mRNA was observed to aggregate within SGs following different types of stress, suggesting that SGs are involved in post transcriptional regulation of gene expression in the cochlea. Experiments in cochlear explants suggest that pharmacological induction of SGs promotes outer hair cell survival during aminoglycoside exposure. In addition, SG formation was observed in the in-vivo C57BL/6 cochlea during ageing, suggesting that SGs may be related to cochlear degeneration. Hsp70, previously shown to promote hair cell survival following ototoxicity has been associated with SGs in other systems. Here, Hsp70 expression was evaluated in OC 2 cells following different stressors and evidence suggests it to be a key regulator of SGs. Taken together, these data implicate the SG pathway with maintenance of auditory function as a potential therapeutic target for further investigation.
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CONI, PAOLA. "Ruolo della TDP-43 nella formazione dei granuli da stress nella Sclerosi Laterale Amiotrofica." Doctoral thesis, Università degli Studi di Cagliari, 2016. http://hdl.handle.net/11584/266642.
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Amyotrophic Lateral Sclerosis (ALS) is a late-onset neurodegenerative disease characterized by the selective loss of upper and lower motor neurons; most ALS cases are sporadic, and only 5-10% are familial. About 4% of familial cases, are due to mutations in TARDBP, the gene encoding TDP-43, that is an ubiquitous nuclear protein that regulates mRNA functions and metabolism.
Recent studies suggest that TDP-43 may regulates stress granules dynamics that are cytoplasmic structures composed of non-translating messenger ribonucleoproteins (mRNPs) that rapidly aggregate in cells exposed to adverse environmental conditions. Stress granules function in part to triage RNA and sequester transcripts not needed for coping with the stress.
We evaluated stress granule dynamics in primary fibroblast cultures from skin of ALS patients carriying TARDBPA382T mutation, ALS patients without any TARDBP mutation and healthy controls. After treatment with sodium arsenite (0,5 mM), for 30 and 60 minutes, we observed a significantly higher number of cells exhibiting stress granules, identified by immunostaining for specific markers (TIA-1 and HuR), in fibroblasts from healthy controls compare with those from ALS patients carriying TARDBPA382T mutation. Moreover, fibroblasts from healthy controls showed more stress granules per cell compare with those from ALS patients, while no differences were observed in stress granule size between groups. Fibroblasts from ALS patients without any TARDBP mutation, showed the same ability to form stress granules as cells from healthy controls, confirming that the decrease was associated to TARDBPA382T mutation rather than other factors attributable to ALS.
In all samples analyzed TDP-43 immunostaining was always observed into the nucleus of all the cells and even after sodium arsenite treatment TDP-43 was never localized in stress granules.
The involvement of TDP-43 in stress granule assembly was confirmed by silencing TARDBP gene in fibroblasts from healthy controls. After sodium arsenite treatment, fibroblasts in wich TARDBP gene was silenced, showed a significantly lower number of cells exhibiting stress granules compare with unsilenced controls.
Following stress stimuli, we observed, using MTT assay, a significant higher cytotoxicity in fibroblasts from patients carriying TARDBPA382T mutation compare with healthy controls. Expression of G3BP, a core stress granule component, was significantly lower after sodium arsenite treatment in fibroblasts from patients carriying TARDBPA382T mutation compare with healthy controls.
We can conclude that TARDBPA382T mutation caused a reduction in the ability of human fibroblasts to respond to stress through loss of TDP-43 function in stress granule nucleation. The pathogenetic action revealed in our study model does not seem to be mediated by changes in the localization of the TDP-43 protein, but we found that this protein contributes to stress granule formation through a regulatory effect on the G3BP core protein.
These data demonstrate that TDP-43 may modulate stress granule formation contributing to the cellular response to acute stress and suggest that TARDBPA382T mutation may compromise the cellular stress response, contributing to neuronal vulnerability in ALS.
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Bahri, Alia. "Rôle des condensats préexistants dans la modulation de l'agrégation induite par le stress." Electronic Thesis or Diss., Université Côte d'Azur, 2024. http://www.theses.fr/2024COAZ6048.
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Les condensats de ribonucléoprotéines (RNP sont des organelles sans membrane concentrant des protéines et ARN spécifiques, et organisent le transcriptome et le protéome cellulaires. Une mauvaise régulation de ces condensats pourrait entraîner des maladies, lorsque ces condensats « physiologiques », normalement fluides et réversibles, deviennent des agrégats rigides et irréversibles, dits « pathologiques ». Bien que plusieurs recherches aient étudié le rôle du mauvais repliement des protéines dans cette transition, celui de l'ARN est moins exploré. Mon travail comble cette lacune en explorant la contribution de l'ARN aux transitions entre condensats physiologiques et agrégats pathologiques. J'ai utilisé les granules induits par le stress (SiG) et les P-bodies (PB) dans les ovocytes de C. elegans comme modèles. Les PBs, constitutifs, représentent les condensats « physiologiques », tandis que les SiG, formés en réponse au stress, sont plus enclins aux transitions pathologiques.Pour répondre à cette question, j'ai analysé trois critères : (1) la densité de l'ARNm dans les condensats ou les agrégats, mesurée par smiFISH ; (2) la persistance des condensats ARN, observée par imagerie des protéines de granule fusionnées à la GFP ; et (3) la létalité cellulaire, mesurée via la létalité embryonnaire. Chez C. elegans sans PBs préexistants, un stress de chaleur prolongé accroit la densité d'ARNm dans les condensats, accompagnée d'une persistance des condensats devenus insolubles et de mort cellulaire, suggérant qu'un stress prolongé entraîne un enchevêtrement d'ARN responsable de la persistance des condensats ARN et de la mort cellulaire. Cependant, chez les C. elegans avec des PBs préexistants, ces effets étaient réduits, suggérant que les PBs préexistants limitent l'agrégation pathologique des ARNm induite par le stress. Pour évaluer les changements transcriptomiques dans les PB sous stress, j'ai séquencé leur transcriptome par FAPS-seq sous stress de chaleur et de froid. Nos résultats montrent que le transcriptome des PBs est modifié de manière spécifique au type de stress, suggérant qu'une condensation sélective des ARNm participe à l'adaptation de la traduction aux conditions environnementalesRibonucleoprotein (RNP) condensates are membrane-less organelles that concentrate specific proteins and RNA and organize the cellular transcriptome and proteome. Dysregulation of these condensates could lead to diseases when these "physiological" condensates, normally fluid and reversible, become rigid and irreversible aggregates, known as "pathological" aggregates. While several studies have examined the role of protein misfolding in this transition, the role of RNA is less explored. My work fills this gap by investigating the contribution of RNA to the transitions between physiological condensates and pathological aggregates. I used stress-induced granules (SiG) and P-bodies (PB) in C. elegans oocytes as models. The constitutive PBs represent "physiological" condensates, while the SiG, formed in response to stress, are more prone to pathological transitions.To address this question, I analyzed three criteria: (1) mRNA density in condensates or aggregates, measured by smiFISH; (2) the persistence of RNA condensates, observed through imaging of GFP-tagged granule proteins; and (3) cell lethality, measured via embryonic lethality. In C. elegans without pre-existing PBs, prolonged heat stress increases mRNA density in the condensates, accompanied by the persistence of insoluble condensates and cell death, suggesting that prolonged stress leads to RNA entanglement responsible for the persistence of RNA condensates and cell death. However, in C. elegans with pre-existing PBs, these effects were reduced, suggesting that pre-existing PBs limit stress-induced pathological aggregation of mRNAs. To assess transcriptomic changes in PBs under stress, I sequenced their transcriptome using FAPS-seq under heat and cold stress. Our results show that the PB transcriptome is specifically modified according to the type of stress, suggesting that selective condensation of mRNAs contributes to the adaptation of translation to environmental conditions
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Moutaoufik, Mohamed Taha. "Granules de stress cytoplasmiques à ARN induits par le rayonnement ultraviolet (UV)." Thesis, Université Laval, 2012. http://www.theses.ulaval.ca/2012/28900/28900.pdf.
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Chez les eucaryotes, différents types de granules à ARN sont des acteurs importants dans les mécanismes de la régulation post-transcriptionnelle de l'expression des gènes.
L’irradiation aux UV induit la formation des petits granules cytoplasmiques (GUV) qui ne sont pas des processing bodies et qui semblent être une nouvelle sous classe de granules de stress. Ces granules n’ont pas la même cinétique de formation et de disparition ainsi que la taille, le nombre et la capacité de fusion que les granules de stress classiques. D’autre part, la formation de ces granules UV ne semble pas affecter le niveau de traduction, ni d’induire la réponse au stress. Toutefois, nous avons observé que l’apparition des granules coïncide avec l’arrêt de la prolifération cellulaire. En effet, dans les conditions expérimentales utilisées, la prolifération est décalée de 24 à 48 h selon la dose d’irradiation.
L’ensemble de ces observations suggère fortement l'existence, d'une nouvelle sous classe de granules de stress induit par les UV, dont le rôle semble être la répression de la traduction des ARNm codant pour des facteurs importants de prolifération cellulaire.
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Ng, Siew Kit. "Investigating the localization of ADAR1 to cytoplasmic stress granules in mammalian cells." Thesis, University of Cambridge, 2014. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708396.
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Aznar, Nicolas. "L’histone déacétylase HDAC6, un nouvel effecteur du suppresseur de tumeur LKB1." Thesis, Lyon 1, 2011. http://www.theses.fr/2011LYO10043.
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Le gène suppresseur de tumeur LKB1 code une sérine/thréonine kinase qui régule le métabolisme énergétique et la polarité cellulaire. Son action biologique s'exerce en partie via la protéine kinase activée par l'AMP (AMPK), substrat de LKB1 dont la phosphorylation stimule l'activité catalytique. Nous avons récemment mis en évidence une interaction entre LKB1 et la déacétylase HDAC6. HDAC6 régule principalement l'état d'acétylation de protéines localisées dans le cytoplasme telles que la molécule chaperon HSP90, la tubuline α, et la cortactine. HDAC6 contrôle la stabilité des protéines liées à HSP90 mais agit aussi sur la polarité et l'adhérence des cellules. De plus, HDAC6 répond à différentes situations de stress cellulaire en favorisant le transport des protéines polyubiquitinées vers les aggrésomes, où celles ci sont dégradées, et en promouvant la formation des granules de stress, complexes ribonucléoprotéiques participant au stockage des ARNm et au blocage de la traduction. Mon projet de recherche a porté sur les conséquences fonctionnelles de l'interaction entre LKB1 et HDAC6. J'ai ainsi pu montrer que la formation de ce complexe est renforcée en condition de stress oxydatif et thermique. Dans cette situation biologique, LKB1 interfère avec la capacité de HDAC6 à fixer les protéines ubiquitinylées, et par conséquent prévient la formation des aggrésomes et des granules de stress. A l'inverse, LKB1 stimule l'activité déacétylase de HDAC6, et cette action de LKB1 est requise pour la migration orientée des cellules ainsi que pour la polarisation apico-basale dans un modèle de culture d'entérocytes. Ce travail nous a ainsi permis d'identifier un nouvel effecteur de LKB1 qui intervient dans la réponse au stress et dans la polarisation cellulaire. Il s'agit aussi de la première mise en évidence d'une régulation de l'activité de liaison à l'ubiquitine de HDAC6. Ces données suggèrent que LKB1, via son effet sur HDAC6, pourrait limiter la réponse adaptative des cellules soumises à des stress exogènes et endogènes, comme ceux que les cellules en voie de transformation rencontrent dans leur microenvironnement, une propriété qui pourrait s'avérer essentielle pour son activité de suppresseur de tumeurThe tumor suppressor LKB1 is a serine-threonine kinase that acts as a critical regulator of energy homeostasis and cell polarity 1,2. LKB1 relays its intracellular signal through the AMP-activated protein kinase (AMPK) as well as twelve additional members of the AMPK sub-family 3-5. However, despite the identification of these LKB1 effectors, the mechanisms that underlie LKB1-mediated biological effects remain incompletely understood. We now report that LKB1 interacts with and phosphorylates HDAC6, a deacetylase that protects cells against extrinsic insults through its ability to ligate polyubiquinated misfolded proteins and to dynamically associate with both the microtubule and the actin cytoskeleton networks 6. We further found that the formation of the LKB1-HDAC6 complex was promoted in response to diverse stressful stimuli. As a consequence, HDAC6 ubiquitin-binding activity was inhibited, thus impeding the formation of aggresomes and stress granules, two transient cellular structures that, respectively, prevent the accumulation of aggregated proteins 7 and remodel messenger ribonucleoprotein complexes following stresses that block translation 8. Collectively, these data identify HDAC6 as a key downstream component of the LKB1 signalling pathway. Our findings further suggest that LKB1, via its inhibitory effect on HDAC6 ubiquitin-binding activity, limits the cellular adaptive response to a protracted stress, a distinctive biological property that is likely to contribute to its tumor-suppressive function
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Christen, Kimberley Estelle. "Detection and Characterisation of Compounds Inhibiting Stress Granule Formation in Cancer Cells." Thesis, Griffith University, 2019. http://hdl.handle.net/10072/386552.
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Cancer is one of the leading causes of death worldwide and despite significant improvements to treatment and prevention, cancer cases remain on the rise. Chemotherapy is used to treat patients with cancer, however these do not only kill the cancer cells but also kill normal, healthy cells in the patient. Furthermore, cancer cells have the capacity to become resistant to chemotherapeutic treatment. Therefore, new treatments need to be developed to overcome this problem. One avenue that is being researched is the use of natural products as chemotherapeutic drugs. Over 60% of anticancer drugs used today are either natural products, or their synthetic derivatives and new research is being performed to screen plant and animal secondary metabolites to discover new compounds with anti-cancer therapeutic potential. The research reported in this thesis uses two compounds purified from natural products to explore a novel approach for cancer treatment.
Stress granules (SGs) are messenger ribonucleoprotein particles that are produced in the cytoplasm of the cell in response to stress. Stress granules have been linked to the inhibition of apoptosis and development of multiple drug resistance and it has been suggested that cancer cells can hijack stress granules and use their biological activities to enhance cancer cell survival. In a study by Fournier et al the inhibition of stress granules in bortezomib resistant cancer cells allowed these cells to become sensitive to bortezomib treatment and resulted in an increase in cell death from 15% to 75% (Fournier et al., 2010). This suggests that the inhibition of stress granule formation may restore chemo-sensitivity to the cancer cells, however, the full effect has not been explored beyond the cell based experiments described by Fournier et al.
This research project was based on the research by Fournier et al, suggesting that SG inhibition can increase the efficacy of bortezomib. The aims of this project were to discover natural products that inhibited SG formation and use these natural products in combination with the chemotherapeutics bortezomib and sorafenib to increase their efficacies.
Chapter 3 describes the optimisation and characterisation of SG formation in HEK293, MCF7, T47D, Vero, HeLa, MDAMB231 and MCF7MDR cells for the development of a SG inhibition assay. Chapter 4 describes the screening of 36 compounds from the Davis Open Access Compound Library from which 2 compounds, RAD112 and psammaplysin F were discovered; having activity inhibiting SG formation in the in vitro SG inhibition assay. In chapter 5, the mechanism of action of RAD112 and psammaplysin F were explored. RAD112 belongs to the chalcone class and this class of compound is known for the disruption of microtubules. A microtubule assay was performed analysing the effect of RAD112 against a known microtubule inhibitor, nocodazole and it was confirmed that RAD112 was disrupting microtubules. Psammaplysin F did not cause the disruption of microtubules, therefore, the most common pathway of SG formation, phosphorylation of eIF2α (p-eIF2α) was analysed. It was discovered that psammaplysin F was reducing the amount of p-eIF2α in HEK293, MCF7, Vero and MCF7MDR cells. The mechanism of action studies of both compounds show promising results that warrant further evaluation.
Combinational therapies have many advantages over single chemotherapy regimes in breast cancer as it has been shown that it can increase the patients disease free survival rate and reduce the risk of reoccurrence. In chapter 6 RAD112 and psammaplysin F were used in combination with bortezomib or sorafenib and the interaction between RAD112 with bortezomib or sorafenib and psammaplysin F with bortezomib or sorafenib was determined by cell viability assays and analysed using Compusyn software. The increase in efficacy of bortezomib or sorafenib when combined with RAD112 and psammaplysin F was also examined in the in vitro combinational assay. All combinations of the compounds with the drugs resulted in a synergistic interaction in most cell lines. However, psammaplysin F and sorafenib had the strongest synergistic interaction in MCF7MDR cells, with a combination index (CI) value of <0.4. The IC50 of sorafenib was decreased 4 fold in MCF7MDR cells and 7 fold in MCF7 cells when combined with psammaplysin F. The efficacy of bortezomib and sorafenib was increased after treatment with RAD112 and psammaplysin F suggesting that psammaplysin F and bortezomib have the potential to be used in combination with known chemotherapeutics to restore drug efficacy.
Altogether, this thesis has identified two compounds that inhibit SG formation, RAD112 and psammaplysin F. The mechanism of action studies has revealed two different mechanisms of action for SG inhibition, microtubule inhibition and inhibition of p-eIF2α for RAD112 and psammaplysin F respectively. Combinational studies has resulted in synergistic interactions between RAD112 and bortezomib or sorafenib and psammaplysin F and bortezomib and sorafenib and increased efficacy of bortezomib and sorafenib. These findings show promise as a new strategy in the battle against cancer and further studies involving the complete mechanism of action of these compounds have to be carried out before they can move onto pre-clinical evaluation.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Environment and Sc
Science, Environment, Engineering and Technology
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Singla, Ashima. "PHOSHOLIPASE Cβ INTERACTS WITH ARGONAUTE 2 IN STRESS GRANULES TO CHANGE THE MICRORNAs POPULATION IN RESPONSE TO OSMOTIC STRESS." Digital WPI, 2017. https://digitalcommons.wpi.edu/etd-theses/1248.
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"When cells are exposed to environmental stress, they respond by compartmentalizing mRNA and translation proteins in stress granulates to protect mRNA. However, the mechanism through which external stress is communicated into the cell to form stress granules is unknown. Phospholipase Cβ (PLCβ) is activated by Gq on the plasma membrane in response to sensory stimuli to initiate calcium signals resulting in a variety of cellular responses. Here, we show that PLCβ binds to major proteins that organize stress granules as well as the main component of the RNA-induced silencing machinery, Argonaute-2 (Ago2). Under stress, PLCβ moves from the plasma membrane to the cytosol to escort Ago2 into stress granules and potentially inhibit mRNA degradation by regulating microRNAs (miRs) expression. Using a model muscle cell line functionally adapted to handle stress, we find that upon osmotic stress, the movement of PLCβ into the cytosol to move Ago2 into stress granules changes the population and distribution of miRs, and in particular, members of the let family. The impact of changes in let is to acutely affect glucose metabolism allowing cells to adapt to stress conditions. Our studies present a model in which PLCβ relays information about external stress to promote stress granule formation and protect mRNAs."
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ICHIKAWA, KENJI, TAKANORI NAKAMURA, YUJI KUBOTA, and MUTSUHIRO TAKEKAWA. "REGULATION OF STRESS-ACTIVATED MAP KINASE PATHWAYS DURING CELL FATE DECISIONS." Nagoya University School of Medicine, 2011. http://hdl.handle.net/2237/14910.
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Lian, Xian Jin 1968. "Delineating the role of stress granules in senescent cells exposed to external assaults." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=112534.
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As we age, our ability to cope with a variety of stresses significantly decreases. One of the features of an ageing organism is the dramatic increase in the number of cells arrested in the G1 phase, a process known as senescence. It is well established that the senescence phenotype leads to a change in the way cells respond to stress. However, the molecular mechanisms by which these cells cope and/or respond to a variety of environmental challenges remain unknown. In general, cells respond to stress by engaging a variety of mechanisms; one of them is the assembly of cytoplasmic foci known as stress granules (SGs). These entities are considered as part of the survival pathways that are activated at the beginning of any stress to protect key cellular elements which allow a quick recovery if the stress is rapidly removed. However, we do not know whether SGs formation is activated during senescence. In this study, we investigated the formation and the role of SGs in senescent cells exposed to various stresses. We demonstrated that while SGs can assemble in response to oxidative stress (OS) during all the steps leading to senescence activation, their number significantly increases at late stage of senescence. This increase correlates with a rapid decrease in the expression of the cyclin kinase inhibitor p21, one of the main players in the activation of the senescence phenotype. Although the OS-induced recruitment of p21 mRNA to SGs correlates with a significant increase in its half-life, this translocation interferes with p21 translation only at late senescence. This translation inhibition could be explained by the co-recruitment of CUGBP1, a known translation activator during senescence of p21, and p21 mRNA to SGs. Therefore, our data suggest that SGs formation and the reduction in p21 protein levels represent two main events through which senescent cells respond to stress conditions.
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Cid, Samper Fernando 1991. "Computational approaches to characterize RNP granules." Doctoral thesis, Universitat Pompeu Fabra, 2020. http://hdl.handle.net/10803/668449.
Full textAbstract:
Ribonucleoprotein granules (RNP granules) are liquid-liquid phase separated
complexes composed mainly by proteins and RNA. They are responsible of many
processes involved in RNA regulation. Alterations in the dynamics of these proteinRNA complexes are associated with the appearance of several neurodegenerative
disorders such as Amyotrophic Lateral Sclerosis ALS or Fragile X Tremor Ataxia
Syndrome FXTAS. Yet, many aspects of their organization as well as the specific
roles of the RNA on the formation and function of these complexes are still unknown.
In order to study RNP granules structure and formation, we integrated several state of
the art high-throughput datasets. This includes protein and RNA composition obtained
from RNP pull-downs, protein-RNA interaction data from eCLIP experiments and
transcriptome-wide secondary structure information (produced by PARS). We used
network analysis and clustering algorithms to understand the fundamental properties
of granule RNAs. By integrating these properties, we produced a model to identify
scaffolding RNA. Scaffolding RNAs are able to recruit many protein components into
RNP granules. We found that the main protein components of stress granules (a kind
of RNP granules) are connected through protein-RNA interactions. We also analyzed
the contribution of RNA-RNA interactions and RNA post-transcriptional
modifications on the granule internal organization.
We applied these findings to understand the biochemical pathophysiology of FXTAS
disease, employing as well some novel experimental data. In FXTAS, a mutation on
the FMR1 gene produces a 5´microsatellite repetition that enhances its scaffolding
ability. This mutated mRNA is able to sequester some important proteins into nuclear
RNP granules, such as TRA2A (i.e. a splicing factor), impeding their normal function
and therefore producing some symptoms associated with the progress of the disease.
The better understanding of the principles governing granules formation and structure
will enable to develop novel therapies (e.g. aptamers) to mitigate the development of
several neurodegenerative diseases.Los gránulos ribonucleoproteicos (gránulos RNP, por sus siglas en inglés) son complejos producidos mediante separación líquido-líquido y están constituidos principalmente por proteínas y ARN. Son responsables de numerosos procesos involucrados con la regulación del ARN. Alteraciones en la dinámica de estos complejos de proteínas y ARN están asociadas con la aparición de diversas enfermedades neurodegenerativas como el ELA o FXTAS. Sin embargo, todavía se desconocen muchos aspectos relativos a su organización interna así como las contribuciones específicas del RNA en la formación y funcionamiento de estos complejos. A fin de estudiar la estructura y formación de los gránulos RNP, hemos integrado varias bases de datos de alto rendimiento de reciente aparición. Esto incluye datos sobre la composición proteica y en ARN de los RNP, sobre la interacción de proteínas y ARN extraída de experimentos de eCLIP y sobre la estructura secundaria del transcriptoma (producida mediante PARS). Todos estos datos han sido procesados para comprender las propiedades fundamentales de los ARNs que integran los gránulos, mediante el empleo de métodos computacionales como el análisis de redes o algoritmos de agrupamiento. De esta manera, hemos producido un modelo que integra varias de estas propiedades e identifica candidatos denominados ARNs de andamiaje. Definimos ARNs de andamiaje como moléculas de ARN con una alta propensión a formar gránulos y reclutar un gran número de componentes proteicos a los gránulos RNP. También hemos encontrado que las interacciones proteína-ARN conectan los principales componentes proteicos de consenso de los gránulos de estrés (un tipo específico de gránulos RNP). También hemos estudiado la contribución de las interacciones ARN-ARN y las modificaciones post-transcriptionales del RNA en la organización interna del gránulo. Hemos aplicado estos resultados para la comprensión de la fisiopatología molecular de FXTAS, empleando también algunos datos experimentales originales. En FXTAS, una mutación en el gen FMR1 produce una repetición de microsatélite en 5´ que incrementa su capacidad como ARN de andamiaje. Este mARN mutado es capaz de secuestrar algunas proteínas importantes como TRA2A (un factor de ayuste alternativo) en gránulos RNP nucleares, impidiendo su normal funcionamiento y por consiguiente produciendo algunos síntomas asociados con el progreso de la enfermedad. Una mejor comprensión de los principios que gobiernan la formación y estructura de los gránulos puede permitir desarrollar nuevas terapias (ej: aptámeros) para mitigar el desarrollo de diversas enfermedades neurodegenerativas.
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Lavrynenko, Kyrylo. "The interaction of Caprin1 and G3BP1, major proteins in stress granule assembly, promotes the messenger RNA recruitment by G3BP1." Electronic Thesis or Diss., université Paris-Saclay, 2024. http://www.theses.fr/2024UPASL133.
Full textAbstract:
Les protéines de liaison à l'ARN (RBP) jouent un rôle majeur dans la régulation de la traduction de l'ARN messager et l'adaptation du métabolisme cellulaire à divers signaux environnementaux. La présence dans de nombreuses RBP d'une combinaison unique de domaines fonctionnels structurés et de régions intrinsèquement désordonnées, leur permet de générer des condensats riches en ARN par le processus de séparation de phase liquide-liquide. G3BP1 est une RBP centrale dans le processus de protection de l'ARNm lorsque les cellules sont exposées à des stress environnementaux via la formation des granules de stress - des condensats de ribonucléoprotéines qui s'assemblent en réponse au stress. Les granules de stress (SG) pourraient servir de point de contrôle dans la destinée des ARNm : stockage de l'ARNm pour éviter l'expression de protéines inutiles à la survie cellulaire, transfert des transcrits d'ARNm vers les p-bodies pour dégradation ou transfert dans des polysomes pour traduction de protéines essentielles à la survie cellulaire. G3BP1 est un élément central dans l'assemblage des SGs car elle possède des domaines de liaison à l'ARN et une activité hélicase, et interagit spécifiquement avec plusieurs protéines présentes dans les SG comme Caprin1.Le but de cette étude est d'analyser la coopération entre G3BP1 et Caprin1 dans la liaison à l'ARN et la formation de condensats. Des études antérieures mettent en évidence le caractère central de G3BP1 dans l'assemblage de SG, mais G3BP1 ne possède pas de domaine de type prion contrairement aux nombreuses protéines impliquées dans la nucléation des SG et son affinité pour l'ARNm est assez faible. On propose que Caprin1, en tant que partenaire de G3BP1 à travers le domaine NTF2L et également une protéine impliquée dans la formation de stress granules, favorise la liaison de G3BP1 à l'ARNm et son recrutement dans les granules de stress. De plus, on a décortiqué la fonction des différents domaines de G3BP1 dans cette interaction.Pour démontrer l'interaction G3BP1-Caprin1-ARNm, nous avons utilisé plusieurs méthodes de biologie structurale et cellulaire. Nous proposons que Caprin1, qui est à la fois un partenaire connu de G3BP1 via le domaine NTF2-L de G3BP1 et une protéine de liaison à l'ARNm via son domaine de faible complexité riche en RGG, puisse améliorer l'interaction entre ARNm et G3BP1 et favoriser le recrutement de l'ARNm dans les SGs. L'analyse de l'influence des différents domaines de G3BP1 dans cette interaction démontre que l'amélioration du recrutement de l'ARNm via l'interaction avec Caprin1 ne fonctionne que si G3BP1 conserve ses domaines de liaison à l'ARNm. La coopération entre Caprin1 et G3BP1 permet de mieux comprendre la centralité de G3BP1 dans l'assemblage des SGs et ouvre des perspectives sur le recrutement d'ARNm spécifiques lors d'épisodes de stressRNA-binding proteins play major role in regulation of messenger RNA translation and the adaptation of cellular metabolism to various environmental signals. This is accomplished due to RBPs possessing unique combination of structured functional domains and non-structured intrinsically disordered regions, which allows them to undergo liquid-liquid phase separation and form separate condensates with mRNA. G3BP1 is a central protein in a network of RBPs that participate in protection of mRNA from environmental stress by forming stress granules - ribonucleoprotein condensates that assemble in response to stress. Stress granules (SGs) might function as checkpoint for mRNA fate: storage of translationally silent mRNA, transfer of mRNA transcripts to P-bodies for degradation or transfer back into polysomes for translation. G3BP1 possesses RNA-binding domains, helicase activity and recruits several proteins into SGs, with some of them considered central nucleators in SG assembly, Caprin1 among them. The aim of this study is to investigate the cooperation between G3BP1 and Caprin1 in RNA-binding and condensate formation. Previous studies evidence the centrality of G3BP1 in SG assembly but, unlike other SG-nucleating proteins, G3BP1 lacks a prion-like domain and its direct mRNA binding is not clear. We propose that Caprin1, which is a known G3BP1 partner through the NTF2L domain of G3BP1 and a SG protein, may promote the G3BP1 mRNA binding and improve the mRNA recruitment in SG. In addition, we analyzed the function of the different G3BP1 domains in this interaction To demonstrate G3BP1-Caprin1-mRNA interplay, we used several methods of structural and cellular biology. We confirmed that G3BP1 and Caprin1 can co-localize and recruit mRNA in vivo, moreover, NTF2L-domain of G3BP1 is needed for this interaction. The mRNA recruiting capabilities of G3BP1 are improved in presence of Caprin1, and the RNA-binding domains of G3BP1 are of fundamental importance. Similarly, the enhanced mRNA recruitment of G3BP1-Caprin1 complex to SGs is at play only when full length G3BP1 is present. The consequence of G3BP1-Caprin1 interaction explain the centrality of G3BP1 in SG assembly and complement the model in which the shift RNA concentration triggers the conformational switch of G3BP1 at the heart of SG assembly by liquid-liquid phase separation
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Goggin, Kevin. "Altération de la réponse au stress par des agrégats cytoplasmiques de la protéine prion." Mémoire, Université de Sherbrooke, 2008. http://hdl.handle.net/11143/5537.
Full textAbstract:
Les encéphalopathies spongiformes transmissibles (EST) sont des maladies neurodégénératives infectieuses qui résultent de l'agrégation de formes anormales de la protéine prion cellulaire (PrP[indice supérieur C]). La maladie de Creutzfeldt-Jakob est la plus répandue chez les humains alors que chez les animaux, la maladie de la vache folle est celle qui a l'impact économique le plus important et est la seule EST animale clairement transmissible à l'homme. Des études suggèrent que des agrégats cytoplasmiques de la protéine prion (CyPrP) pourraient être responsables de la neurodégénérescence observée lors des EST, toutefois, le mécanisme de toxicité de ces agrégats est encore inconnu. Nous avons émis l'hypothèse que la production d'agrégats cytoplasmiques de la protéine prion entraîne un stress considérable et possiblement létal pour les cellules neuronales. Nous avons analysé la capacité de ces agrégats d'activer ou d'inhiber certaines composantes de la réponse au stress intégrée. Cette réponse a pour but de limiter les dommages cellulaires en conditions de stress et consiste en l'arrêt de la synthèse protéique, l'assemblage de granules de stress et l'induction de chaperonnes moléculaires. Nos résultats démontrent que les agrégats cytoplasmiques de la protéine prion induisent un stress pour la cellule qui résulte en l'activation de la kinase du stress PKR, la phosphorylation du facteur d'initiation de la traduction eIF2[alpha] et une diminution d'environ 80% de la synthèse protéique. De façon surprenante, la formation des granules de stress est inhibée dans les cellules qui produisent des agrégats cytoplasmiques de PrP. L'hybridation in situ et la chromatographie d'affinité sur résine de cellulose-oligo(dT) nous ont permis de démontrer que les ARNm étaient séquestrés en grande partie au sein des agrégats de CyPrP. Nous avons aussi démontré que l'induction de Hsp70 était inhibée suite à un stress dans les cellules qui produisent des agrégats et que ces cellules sont beaucoup plus sensibles à un stress oxydatif. Nous proposons que l'activation d'une réponse au stress inadéquate par les agrégats cytoplasmiques de la protéine prion altère considérablement la capacité des neurones de résister à de nombreux stress physiologiques. Nous suggérons que ces événements pourraient contribuer à la toxicité et à la neurodégénérescence observée au cours des maladies à prions.
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Chiacchiera, Fulvio. "Characterization of a novel p63/p73 interacting protein." Doctoral thesis, Università degli studi di Trieste, 2008. http://hdl.handle.net/10077/2627.
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2006/2007I tumori sono tra le maggiori cause di morte nelle popolazioni occidentali. D'altra parte anomalie congenite nello sviluppo nonostante non siano ugualmente frequenti richiedono uno sforzo notevole in termini di assistenza e cure da parte delle istituzioni e delle famiglie coinvolte. La comprensione dei processi molecolari alla base di queste patologie è quindi di fondamentale importanza per la medicina. Diverse evidenze sperimentali dimostrano come geni coinvolti nello sviluppo embrionale e nel differenziamento sono spesso coinvolto nella genesi dei tumori. In particolare membri della famiglia di p53 rivestono un ruolo fondamentale nell'omeostasi della cellula e le loro funzioni risultano spesso alterate nei tumori ed in alcune malattie genetiche. a livello molecolare l'attività di queste proteine è finemente regolata tramite una serie di modificazioni post-trascrizionali ed interazioni proteiche. Ogni singolo interattore risulta quindi un possibile bersaglio per nuove strategie farmacologiche. In questo lavoro presentiamo la caratterizzazione del prodotto del gene umano c16orf35, un nuovo interattore di p63e p73 isolato da uno screening volto a cercare nuovi interattori di p53 di Drosophila melanogaster. C16orf35 è una proteina nucleare evolutivamente molto conservata ed espressa ubiquitariamente. è in grado di associare con compartimenti cellulari specifici definiti "stress granules" e "p-bodies" in cui gli RNA subiscono diversi tipi di modificazioni strutturali. L'aumento forzato dei livelli cellulari di c16orf35 induce la formazione degli stress granules ed inibisce la proliferazione di cellule tumorali in coltura. Ciò suggerisce un possibile ruolo di questa proteina nelle vie che regolano la crescita cellulare.
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Giordano, Tiziana. "Insulin and chromogranin B secretory granules in β cell lines under physiological and stress conditions." Thesis, Open University, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.439344.
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Liu-Yesucevitz, LiQun. "The biology of TDP-43 stress granules: novel insights about protein aggregation in neurodegenerative diseases." Thesis, Boston University, 2012. https://hdl.handle.net/2144/12483.
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Thesis (Ph.D.)--Boston University
PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.TDP-43 is the principal protein component in the neuronal inclusion bodies of patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTLD). In afflicted CNS areas, TDP-43 mislocalizes to cytoplasm and forms inclusion bodies. Emerging evidence identifies TDP-43 as an RNA-binding protein that governs RNA metabolism, trafficking and protein translation. Studies in this thesis demonstrate that TDP-43 is a bona fide stress granule (SG)-associated protein, participating in stress granule pathways. Expressing C-terminal fragments of TDP-43, which mimic pathological TDP-43 recovered from diseased-human CNS tissues, are sufficient to elicit SG formation and cytotoxicity, suggesting that pathological TDP-43 interacts with the SG pathway and could impair protein translation and RNA metabolism in neurons. Colocalization of TDP-43 and SG markers in spinal cords of ALS donors supports the hypothesis that pathological TDP-43 associates with SGs to form neuronal inclusions in ALS. We also discovered that ALS-linked mutations in TDP-43 increase its tendency to become insoluble, aggregate, and form SGs. ALS-Iinked mutations in TDP-43 increase its direct interaction with TIA-1, a key molecular organizer and component of SG, suggesting a mechanism by which mutations in TDP-43 could modify the formation and composition of RNA granules generally and SGs specifically. In neurons, proteins are locally translated at dendrites to fulfill the demands of synaptic plasticity in response to neuronal activity changes. Imaging studies performed using primary hippocampal neurons indicate that TDP-43 localizes in both transport RNA granules and SGs at dendrites, but not P-bodies. Importantly the distribution of TDP-43 RNA granules along dendrites is disturbed by ALS-Iinked TDP-43 mutation. Live cell imaging provides us a delicate tool for tracking granule motility. We discovered that disease-linked mutations in TDP-43 dampen the mobility of TDP-43 in RNA granules, and also inhibit the movement of TDP-43-enriched RNA granules. The movement impairment of TDP-43 RNA granules raises the possibility that protein translation and RNA metabolism might be dysregulated locally in dendrites. Overall, our studies on TDP-43 RNA granules provide a new model for protein aggregation in neurodegenerative diseases that is based on the SG pathway, and suggest mechanisms by which mutations in TDP-43 might contribute to disease.
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Shah, Khyati H. "REGULATION, COMPOSITION AND FUNCTIONS OF RNP GRANULES IN QUIESCENT CELLS OF SACCHAROMYCES CEREVISIAE." The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1417541239.
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Budkina, Karina. "The role of an mRNA-binding protein YB-1 in formation of stress granules and translation." Thesis, université Paris-Saclay, 2021. http://www.theses.fr/2021UPASL006.
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Au cours de la vie de l'ARNm dans la cellule, l'ARNm existe en complexe avec des protéines et n'est jamais libre. Dans le cytoplasme, l'ARNm actif est associé aux ribosomes pour former les polyribosomes tandis que les ARNm réprimés s’associent avec certaines protéines de liaison à l'ARN (RBP) pour former des mRNP. Les mRNP réprimés sont généralement isolés dans le cytoplasme mais ils peuvent également être trouvés dans des compartiments appelés granules d’ARN, notamment lors d'un stress cellulaire. Ces granules d’ARN sont des organelles non membranaires contenant principalement de l'ARNm inactif et coexistent avec des polysomes. Selon les conditions environnementales, il y a un changement dans le ratio des ARNms trouvés dans les granules d’ARN ou dans les polysomes. De plus, il existe des différences dans la teneur en ARNm des différents types de ces organelles en fonction de leur localisation et de leurs fonctions. Actuellement, les granules de stress présentent un grand intérêt pour les chercheurs en raison de leur relation avec certaines maladies neurologiques. Les mutations trouvées dans certaines protéines de liaison à l'ARN telles que TDP43 et FUS sont directement liées à certaines maladies neurodégénératives telles que la sclérose latérale amyotrophique (SLA), la démence frontotemporale (FTLD) et la maladie d'Alzheimer (MA). Dans les neurones affectés, TDP-43 et FUS forment des agrégats cytoplasmiques alors que ces protéines se trouvent généralement dans le noyau dans des conditions physiologiques. Comme elles ont également été trouvées dans les granules de stress cytoplasmiques, les granules de stress peuvent servir d'intermédiaires pour la formation d'agrégats de FUS et TDP-43. En outre, FUS et TDP-43 contiennent des régions intrinsèquement désordonnées (IDR) qui contribuent à leur agrégation.La formation de granules de stress est stimulée par l'exposition à différents facteurs internes et / ou externes. Les granules de stress servent de lieu de stabilisation des ARNm et à les maintenir inactifs jusqu'à ce que les facteurs de stress disparaissent. On considère que les structures secondaires de l'ARNm jouent un rôle important dans l'assemblage des granules de stress. De telles structures servent aussi de sites de liaison pour les RBP, qui les stabilisent davantage (par exemple G3BP). La protéine de liaison Y-box 1 (YB-1) a également été identifiée comme un marqueur pour les granules de stress. YB-1 est une protéine de liaison à l'ARN qui accompagne l'ARNm dès sa synthèse dans le noyau jusqu’à sa dégradation dans le cytoplasme. YB-1 contient un domaine de choc froid (CSD) avec deux motifs de reconnaissance d'ARN (RNP-1 et RNP-2), ainsi qu'un domaine CTD non structuré similaire aux IDR. Pour la plupart des protéines impliquées dans la formation des granules de stress, leur activité stimulante de l'IDR dans ce processus a été démontrée. Dans le même temps, il existe quelques controverses concernant le rôle de YB-1 dans l'assemblage des granules de stress. Selon certains modèles, il y a lieu de le considérer comme un régulateur négatif dans la formation des granules de stress. Selon d'autres, YB-1 présente les propriétés d'un agent favorisant de l'assemblage de granules de stress. Par ailleurs, peu de travaux ont n'a été faits pour déchiffrer l'action de la protéine sur la traduction sous stress oxydatif. Ici, notre objectif était de démêler les mécanismes structuraux par lesquels YB-1 peut réguler négativement la formation de granules de stress et de clarifier son influence sur la traduction dans des conditions de stressDuring mRNA life in cell mRNA exists in complex with proteins and is never free. In the cytoplasm, active mRNA is associated with ribosomes to form polyribosomes while repressed mRNAs in association with RNA-binding proteins forms mRNPs. Repressed mRNPs are generally isolated in the cytoplasm but they can also be found in compartments called mRNP granules, notably during cellular stress. Such mRNP granules are non-membrane organelles contains mostly translationally inactive mRNA and coexist with polysomes. Depending on the environmental conditions, there is a change in the ratio of mRNA found in these types of granules or in polysomes. In addition, there are differences in the mRNA content of the different types of such organelles depending on their localization and functions. Currently, stress granules are of great interest to researchers due to their relation to some neurological diseases. Mutations of some RNA-binding proteins such asTDP43 and FUS are directly linked to some neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTLD), and Alzheimer's disease (AD). In the affected neurons, TDP-43 and FUS form cytoplasmic aggregates while these proteins are generally found in the nucleus under physiological conditions. As they were also found in cytoplasmic stress granules, stress granules may serve as intermediates for the formation of FUS and TDP-43 aggregates. In addition, FUS and TDP-43 contain intrinsically disordered regions (IDRs) which contribute to their aggregation. The formation of stress granules is stimulated by exposure to different internal and/or external factors. Stress granules serve as a place for mRNA stabilization and keeping it inactive until stress factors disappear. It is considered that secondary structures of mRNA play a significant role in the assembly of stress granules. Such structures serve as binding sites for RBPs, which further stabilize them (e.g. G3BP). The Y-box binding protein 1 (YB-1) was also identified as a marker for stress granules. YB-1 is an RNA-binding protein that accompanies mRNA from its synthesis in the nucleus to degradation in the cytoplasm. YB-1 contains a cold shock domain (CSD) with two RNA-recognition motifs (RNP-1 and RNP-2), as well as an unstructured CTD domain similar to IDRs. For most of the proteins involved in the formation of stress granules, their stimulating activity of IDR in this process has been shown. At the same time, there are some controversies regarding the role of YB-1 in the assembly of granules. According to some sources, there is reason to consider it as a negative regulator. According to others, YB-1 exhibits the properties of an inducer during the assembly of stress granules. At the same time, no attempts were made to decipher the mechanism of action of the protein under oxidative stress.Here our aim was to unravel the structural mechanisms by which YB-1 can negatively regulate the formation of stress granules and to clarify its influence on translation in stress conditions
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Courtney, Sean C. "Functional Analysis of Host Cell Proteins and Stress Responses that Inhibit West Nile Virus Infection." Digital Archive @ GSU, 2011. http://digitalarchive.gsu.edu/biology_diss/101.
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Resistance to flavivirus-induced disease is conferred by a single gene that encodes oligoadenylate synthetase (Oas) 1b (Oas1b). Oas1b is not a functional synthetase suggesting its anti-flavivirus mechanism is RNase L-independent and that it may be mediated by interactions with other host cell protein(s). A yeast two-hybrid screen was used to identify host cell binding partners of Oas1b. Candidate partners were confirmed by yeast co-transformation and co-immunoprecipitation analyses. Oxysterol binding protein-related 1L (ORP1L) and ATP binding cassette subfamily F 3 (ABCF3) were found to interact with Oas1b. RNAi knockdown studies suggested that ORP1L and ABCF3 form a tripartite complex with Oas1b that is critical for the flavivirus-induced disease resistance mechanism.
Stresses including oxidation, nutrient starvation, and viral infections often induce the formation of stress granules (SGs) in eukaryotic cells. In response to stress, eIF2α kinases phosphorylate eIF2α leading to stalled 48S pre-initiation complexes and SG formation. West Nile virus (WNV) Eg101 infections were previously shown not to induce the formation of SGs. Infections with viruses of other natural WNV strains, as well as a WNV lineage 1/2-based infectious clone (W956IC) were analyzed and only W956IC infections were found to induce SGs. eIF2α kinase knockout MEFs were used to show that the W956IC-induced SGs were PKR-dependent. WNV chimeras were made by inserting Eg101 genes into the W956IC backbone. Chimeras replacing NS5 or NS1 and NS5 or NS1 and NS3 and NS4a reduced SG formation as well as early viral RNA synthesis similar to Eg101 infections. W956IC infections but not Eg101 infections were shown to produce exposed viral dsRNA at early times after infection. The data suggest that natural WNV infections evade the cell SG response by suppressing the amplification of viral RNA until cytoplasmic membranes have been remodeled to protect replication complexes from detection.
It was previously reported that WNV Eg101 infections inhibited the formation of arsenite-induced SGs. The ability of other natural WNV strain infections to inhibit SG formation by arsenite (HRI), DTT (PERK), W956IC co-infection (PKR), and heat shock treatments was assessed. WNV infections only inhibited arsenite-induced SG formation suggesting that WNV infections specifically suppress the response to oxidative intermediates.
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Ferrier, Emilie. "Rôle et mode d'action de l'UTP : RNA Uridylyltransférase URT1 dans l'uridylation et la dégradation des ARNm chez Aradopsis thaliana." Thesis, Strasbourg, 2013. http://www.theses.fr/2013STRAJ053/document.
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La dégradation des ARN est un mécanisme essentiel à la régulation de l’expression des génomes. L’importance de l’uridylation dans les mécanismes de dégradation des ARN commence juste à être appréciée. Cette thèse présente l’étudede l’UTP :RNA Uridylyltransferase 1 (URT1) et de son rôle dans la dégradation des ARN chez Arabidopsis thaliana. L’étude des propriétés catalytiques de URT1 montre que cette uridylyltransférase est intrinsèquement spécifique des UTP et distributive pour les premières uridines ajoutées. URT1 est responsable in vivo de l’uridylation des ARNm après une étape de déadénylation, protégeant leur extrémité 3’ et polarisant la dégradation de 5’ en 3’. URT1 est localisée dans le cytosol au niveau des granules de stress et des processing bodies. Le mécanisme d’adressage de URT1 dans les processing bodies implique une partie de la région N terminale prédite comme intrinsèquement désorganisée, alors que le domainenucléotidyltransférase C terminal semble suffisant pour permettre l’adressage de URT1 au niveau des processing bodies et granules de stress en réponse à un stress thermique. Ces travaux de thèse ont permis de mieux comprendre les mécanismes et les rôles de l’uridylation dans la dégradation des ARNm chez Arabidopsis. Ils ouvrent des perspectives dans l’étude d’autres fonctions de l’uridylation comme l’inhibition de la traductionRNA degradation is an essential mechanism for the regulation of genome expression. The importance of uridylation for RNA degradation is just emerging. This thesis presents the study of URT1 (UTP :RNA Uridylyltransferase 1) and its role in RNA degradation in Arabidopsis thaliana. URT1 is an uridylyltransferase intrinsically and strictly specific for UTP and is distributive for the first nucleotides added. URT1 uridylates mRNA in vivo after a deadenylation step. This uridylation protects mRNA’s3’ end from further attacks and polarise degradation in the 5’ to 3’ direction. This protection of 3’ ends by uridylation and its conferred polarity of 5’ to 3’ degradation are also detected in polysomes. Uridylation is therefore likely important in case of cotranslational degradation of mRNAs. A region in URT1’s N terminal region predicted to be intrinsically disorganised is required for addressing URT1 to processing bodies. However, following heat shock, the nucleotidyltransferase domain present in the C terminal region of URT1 is sufficient to address URT1 to both processing bodies and stress granules, This work contributes to a better understanding of the mechanisms and roles of uridylation in RNA degradation in Arabidopsis thaliana. These results also open perspectives for studying other functions of uridylation such as translation inhibition
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Swisher, Kylie. "Assembly of mRNP Complexes During Stress and Nonsense-Mediated mRNA Decay Quality Control in Saccharomyces cerevisiae." Diss., The University of Arizona, 2011. http://hdl.handle.net/10150/204068.
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In eukaryotes, mRNA is in constant flux between an actively translating state and translationally repressed states. Specifically, mRNA degradation and repression factors compete with translation factors to direct mRNAs out of translation for storage or decay. This process often leads to formation of cytoplasmic aggregates. P-bodies are granules that contain mRNA and degradation factors, suggesting they are sites of mRNA decay or storage. Stress granules form in response to stress conditions and contain mRNAs and translation factors.P-bodies and stress granules consist of mRNPs of different compositions, believed to mature and transition between the states. It is proposed that mRNAs transition between the two granules. In the work described below, we use
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Seng, Ng Chen. "INHIBITION OF HOST INNATE IMMUNE RESPONSES THROUGH THE MODULATION OF CYTOPLASMIC STRESS GRANULES BY ENCEPHALOMYOCARDITIS VIRUS PROTEASE." 京都大学 (Kyoto University), 2014. http://hdl.handle.net/2433/192227.
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Haghandish, Nasim. "Characterizing the Role of Protein Arginine Methyltransferase 7 (PRMT7) in Breast Cancer." Thesis, Université d'Ottawa / University of Ottawa, 2019. http://hdl.handle.net/10393/38672.
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The development of more efficient therapeutic strategies in the treatment of breast cancer relies on understanding the biological events that promote its progression. Protein arginine methyltransferases (PRMTs) are enzymes that catalyze the methylation of arginine residues within proteins resulting in changes in several biological processes. PRMTs have been shown to be aberrantly expressed in many cancers and promote tumourigenesis and cancer progression. Specifically, PRMT7 mRNA expression correlates with breast cancer aggressiveness and invasiveness. Thus, we sought to determine whether PRMT7 promotes breast cancer progression/tumourigenesis and to further identify the functional mechanisms through which this is possible. We have shown that PRMT7 is upregulated in both breast cancer tissues and cell lines. Moreover, we have shown both in vitro and in vivo that PRMT7 enhances breast cancer cell invasion and metastasis. Using biochemical experimentation, we demonstrated that PRMT7 induces the expression of matrix metalloproteinase 9 to promote invasion and subsequent metastasis. Furthermore, using proteomic experiments, we discovered many novel PRMT7-interacting proteins. Further biochemical experimentation identified eukaryotic translation initiation factor eIF2α as an interacting protein and substrate of PRMT7. We demonstrated a regulatory interplay between eIF2α methylation and phosphorylation upon cellular stress: methylation is required for S51 phosphorylation. Accordingly, we have shown that stress granule formation, in the face of cellular stresses, was significantly diminished in PRMT7-knockdown cells. We additionally found that PRMT7 plays a regulatory role in protein translation. Overall, these findings suggest that PRMT7 plays a critical role in promoting breast cancer cell invasion, metastasis, stress regulation, and protein translation.
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Lui, Jennifer. "Investigation into the localisation of mRNA into cytoplasmic granules following glucose starvation in Saccharomyces cerevisiae." Thesis, University of Manchester, 2012. https://www.research.manchester.ac.uk/portal/en/theses/investigation-into-the-localisation-of-mrna-into-cytoplasmic-granules-followingglucose-starvation-in-saccharomyces-cerevisiae(bf2a8964-fbc9-4cbe-ad0d-3e456b577eae).html.
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Cytoplasmic mRNA-containing granules in eukaryotic cells play key roles inthe storage, localisation and degradation of mRNA. In yeast, depletion of glucoseleads to the rapid inhibition of translation initiation and consequent appearance of Pbodiesand EGP-bodies. P-bodies contain factors of the mRNA decay pathway andtherefore, are likely to be sites in which mRNAs targeted for degradation arelocalised. In contrast, EGP-bodies lack decay components and contain onlytranslation initiation factors and RNA binding proteins. Thus EGP-bodies have beensuggested to be storage repositories for mRNAs that need to be rapidly translatedfollowing glucose readdition. In this study we utilised the m-TAG system to investigate the localisation ofendogenous MS2-tagged mRNAs with P-bodies and EGP-bodies. A triplefluorescent labelled system developed show that a class of unregulated mRNAslocalised into P-bodies following glucose starvation. It was also observed that thesespecific abundant classes of mRNAs can be found in aggregates prior to any cellularstress and upon glucose starvation these aggregates coalesce into larger granules thatcolocalise with P-body components. This coalescence of mRNA aggregatesfollowing glucose starvation does not rely upon the recruitment of mRNA decayfactors and appears to precede this event. Indeed mRNAs in mutants deficient in Pbodyformation still develop large aggregates following glucose stress. In unstressedcells it appears that the mRNA granules are implicated in high-level translation ofthese specific abundant mRNAs. Following the inhibition of translation initiation inresponse to stress, these granules nucleate P-body formation via aggregation and therecruitment of mRNA decay factors.
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De, Leeuw Frédéric. "Etude de la protéine CIRP et sa fonction dans le métabolisme des ARN messagers." Doctoral thesis, Universite Libre de Bruxelles, 2008. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/210577.
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La protéine CIRP (Cold Induced RNA binding Protein) est une petite protéine de liaison à l’ARN de 172 acides aminés, qui est constituée du côté amino-terminal d’un domaine de liaison à l’ARN de type RRM (RNA recognition motif), et d’une partie carboxy-terminale riche en glycine et arginine qui comprend plusieurs motifs RGG. Elle a été identifiée comme étant inductible par hypothermie mais aussi par irradiation aux UV et par hypoxie. Nous avons analysé son expression et sa localisation en réponse à différents stress cellulaires. Nous avons montré qu’un traitement à l’arsénite qui induit un stress oxydant n’altère pas l’expression de CIRP provoque sa localisation dans les granules de stress (SG). Les SG sont des structures ribonucléoprotéiques cytoplasmiques contenant des complexes de pré-initiation incompétents pour la traduction, et qui s’accumulent dans les cellules exposées à un stress. Ces structures constituent des sites de triages des ARNm, dans lesquels les ARNm sont soit stockés en attente d’une réinitiation de la traduction une fois le stress surmonté, soit destinés à être dégradés. La protéine CIRP se localise dans les SG que ce soit suite à un stress cytoplasmique ou du réticulum endoplasmique. Nous avons montré également que la localisation de la protéine CIRP dans les SG se déroule indépendamment de la présence de la protéine TIA-1 qui a été décrite comme responsable de l’assemblage des SG. De plus la surexpression de la protéine CIRP conduit à la formation de SG. Nous suggérons donc qu’il existe plusieurs voies qui mènent à l’assemblage de ces structures. En outre, nous avons analysé la localisation de mutants par délétion de la protéine CIRP et avons montré que le domaine RRM et le domaine RGG peuvent indépendamment localiser la protéine dans les SG. Par contre, la méthylation des résidus arginine du domaine RGG est une modification nécessaire à la localisation de CIRP dans les SG. Ensuite, nous avons étudié la fonction de la protéine CIRP dans le métabolisme des ARN messagers. Nous avons montré par une méthode d’adressage, que CIRP est un répresseur de la traduction des ARNm et que le domaine carboxy-terminal est nécessaire et suffisant à cette fonction.Doctorat en Sciences
info:eu-repo/semantics/nonPublished
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Qin, Qingsong. "Characterization of mammalian orthoreovirus (MRV) induced stress granules (SGs) and implications of eIF2[alpha] phosphorylation on viral translation." [Ames, Iowa : Iowa State University], 2010. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3403826.
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Emara, Mohamed Maged. "Analysis of the Cellular Proteins, TIA-1 and TIAR, and their Interaction with the West Nile Virus (WNV) 3' SL Minus-Strand RNA." Digital Archive @ GSU, 2007. http://digitalarchive.gsu.edu/biology_diss/70.
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The 3' terminal stem loop of the WNV minus-strand [WNV3'(-) SL] RNA was previously shown to bind the cell protein, T-cell intracellular antigen-1 (TIA-1), and the related protein, TIAR. These two proteins are known to bind AU-rich sequences in the 3' UTRs of some cellular mRNAs. AU stretches are located in three single-stranded loops (L1, L2, and L3) of the WNV3'(-) SL RNA. The RNA binding activity of both proteins was reduced when L1 or L2, but not L3, AU sequences were deleted or substituted with Cs. Deletion or substitution with Cs of the entire AU-rich sequence in either L1 or L2 in a WNV infectious clone was lethal for the virus while mutation of some of these nt decreased the efficiency of virus replication. Mutant viral RNAs with small plaque or lethal phenotypes had similar translational efficiencies to wildtype RNA, but showed decreased levels of plus-strand RNA synthesis. These results correlated well with the efficiency of TIA-1 and/or TIAR binding in in vitro assays. In normal cells, TIA-1 and TIAR are evenly distributed in the cytoplasm and nucleus. Between 6 and 24 hr after WNV infection, TIAR concentrated in the perinuclear region and TIA-1 localization to this region began by 24 hr. Similar observations were made in DV2 infected cells but at later times after infection. In infected cells, both proteins colocalized with dsRNA, a marker for viral replication complexes, and with viral non-structural proteins. Anti-TIAR or anti-TIA-1 antibody coimmunoprecipitated viral NS3 and possibly other viral nonstructural proteins. In response to different types stress, TIA-1 and TIAR recruit cell mRNA poly(A)+ into cytoplasmic stress granules (SG) leading to general translational arrest in these cells. SG were not induced by flavivirus infection and cells became increasingly resistant to arsenite induction of SG with time after infection. Processing Body (PB) assembly was also decreased beginning at 24 hr. These data suggest that the sequestration of first TIAR and then TIA-1 via their interaction with viral components in flavivirus infected cells inhibits SG formation and prevents the shutoff of host translation.
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Martin, Sophie. "Le composant des granules de stress G3BP : caractérisation phénotypique de souris KO, et identification de son interactome ribonucléoprotéique dans le cerveau de souris." Thesis, Montpellier 2, 2012. http://www.theses.fr/2012MON20247.
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Les protéines capables de lier des ARNs sont essentielles pour les différentes étapes de maturation de l'ARN messager (ARNm), en dirigeant leur localisation et leur devenir dans la cellule, et en formant avec les ARNs des particules ribonucléoprotéiques (mRNPs). Les mRNPS peuvent former des structures cellulaires dynamiques qui sont adressées vers des fonctions spécifiques. Ces granules, tels que les granules de stress formés suite à un stress cellulaire, contiennent des ARNm dont la traduction est inhibée et qui sont stockés transitoirement. Ma thèse a consisté en la caractérisation fonctionnelle de G3BP (RasGAP SH3 binding protein), une RBP exprimée de façon ubiquitaire chez l'homme et la souris, et impliquée dans l'assemblage des granules de stress. Par recombinaison homologue classique, des souris knock-out pour G3BP ont été générées. Ces souris ont une espérance de vie faible et des défauts du comportement associés au Système Nerveux Central, en particulier un phénotype de type ataxie. Des expériences d'électrophysiologie ont aussi montré une altération de la plasticité synaptique dans l'hippocampe des souris KO. J'ai donc réalisé des expériences d'immunoprécipitation après cross-link (Cross-Linking and Immunoprecipitation, CLIP) pour purifier à partir de cerveau de souris un complexe stable contenant G3BP, et les ARNs associés ont été identifiés par séquençage haut débit (High-Throughput Sequencing, HITS-CLIP). De façon surprenante, la plupart des cibles de G3BP correspondent à des transcrits codants mais qui contiennent des séquences introniques, et des ARNs non codants. De plus, mes résultats ont montré que l'absence de G3BP1 affecte la stabilité de ces transcrits pré-matures spécifiquement dans le cervelet, ce qui peut être corrélé au phénotype d'ataxie des souris KO G3BP1. Cela suggère un nouveau mécanisme de régulation qui passe par la stabilisation de transcrits pré-matures, qui pourraient être convertis en transcrits matures par exemple lors d'un stress et de la séquestration de G3BP dans les granulesRNA binding proteins (RBPs) are essential in the different steps of processing of the messenger RNAs (mRNAs), directing their localization and fate within the cell, and forming with them the ribonucleoprotein particles (mRNPs). mRNPs can assemble into dynamic cellular structures in which they are routed towards specific functions. RNA granules such as stress granules (SGs) contain translationally silenced mRNPs storing transiently repressed mRNAs.My thesis work consisted in the functional characterization of G3BP (RasGAP SH3 binding protein), an RBP that is expressed ubiquitously in both humans and mice and is involved in the assembly of SGs. Using classical homozygous recombination, viable G3BP1 knock out mice were generated that demonstrated short lifespan.and behavioral defects linked to the Central Nervous System (CNS), notably an ataxia phenotype. Electrophysiology experiments showed an alteration of synaptic plasticity in the hippocampus of KO mice. Therefore, I used Cross-Linking and Immunoprecipitation (CLIP) to purify from mouse brain a stable complex containing G3BP, and performed High-Throughput Sequencing (HITS-CLIP) to identify associated RNAs. Strikingly, most of the G3BP targets correspond to intron sequence-retaining transcripts and non-coding RNAs. My results also showed that G3BP1 depletion influences the stability of these premature transcripts in the cerebellum, which can be correlated to the ataxia phenotype of the G3BP1 KO mice. This comprehensive analysis suggests a new mechanism of gene regulation based on stabilization of silenced premature transcripts which might be converted to mature transcripts under stress condition and sequestration of G3BP in SGs
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Di, Marco Barbara. "Modulation of Stress Granules formation: Role of mGlu5 receptor and FMRP and implications for pathophysiology of Fragile X Syndrome." Doctoral thesis, Università di Catania, 2015. http://hdl.handle.net/10761/3838.
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Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and autism. The genetic defect in FXS is a CGG trinucleotide repeat expansion (>200) in the promoter region of the FMR1 (fragile X mental retardation 1) gene; this amplification causes the absence of the encoded protein FMRP (Fragile X Mental Retardation Protein). FMRP is an RNA-binding protein involved in mRNA transport and translation. Despite numerous studies, the available treatments are only symptomatic. There is no cure to replace FMRP expression, yet. FMRP can interact with RNA-binding proteins such as FXR1P, FXR2P, NUFIP and 82-FIP, and with proteins that do not bind RNA, like CYFIP1 and CYFIP2. The interaction with these different proteins may modulate FMRP functions and its RNA affinity. A new role of FMRP in mRNA metabolism as component of stress granules (SGs) has been identified. FMRP seems to lead mRNAs in SGs upon cellular stress, during which protein synthesis is blocked. SGs are ribonucleoproteic aggregates containing translation initiation components and RNA binding proteins, like eIF2a and FMRP. Several data also indicate that some of the FXS symptoms are a consequence of a defect in group-I metabotropic glutamate receptor, namely mGlu5; pharmacological blockade of mGlu5 receptors provide a therapeutic target in FXS. mGlu5 receptor, like FMRP, regulates protein synthesis but in a functionally opponent manner: mGlu5 receptor activates protein synthesis, FMRP suppresses it. In the absence of FMRP, mGlu5-dependent protein synthesis is unchecked, with consequent excessive translation. Activation of mGlu5 receptors stimulates FMRP-mediated mRNA transport and protein synthesis, but its role in SGs formation is unknown.The aim of this PhD thesis was to better investigate FMRP function studying the relationship of FMRP with its interacting proteins and the role of FMRP in stress response under activation of mGlu5 receptor. In Paper I, we analyzed the expression pattern of FMRP and its interacting proteins in different brain areas, at different ages in wild type (WT) mice to better define the interplay between FMRP and its interacting proteins during development. FMRP was strongly expressed at P3, peaked at P7-P14 and gradually decreases thereafter. The analysis of expression pattern of several proteins carried out, indicate that FMRP and its interacting proteins have distinct developmental patterns of expression and suggest that FMRP may be preferentially associated to certain proteins in early and late developmental stages. We found that the RNA binding and cytoskeleton remodeling functions of FMRP may be differently modulated during development. In Paper II we studied FMRP under stress condition using WT and Fmr1 knockout (KO) astrocytes. We have demonstrated that the lack of FMRP impairs SGs formation and furthermore that activation of mGlu5 receptor affects SGs formation through a FMRP-mediated mechanism in WT. Interestingly, the mGlu5 receptor blockade restores SGs formation in Fmr1 KO. Also, mGlu5 receptor activation before stress reduced FMRP recruitment in SGs and phosphorylation of eIF2a and FMRP. In contrast, mGlu5 receptor activation did not affect SGs formation in Fmr1 KO astrocytes. Since phosphorylation of eIF2a and FMRP are two crucial key events in SGs formation and modulation of protein synthesis, mGlu5 receptors may act by shifting the balance from inhibition to activation of protein synthesis during stress. These findings suggest a potential novel role for mGlu receptors in SGs formation. We suggest that FMRP may have a positive role in stress response, facilitating and enhancing SGs formation to prevent stress damages. This process is useful to understand what happens in FXS, in which can occur abnormal modulation of different proteins during development with consequent abnormal response during adversal conditions, like oxidative stress that represent a frequent component in FXS and neurodegenerative disorders.
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Jain, Saumya. "The Analysis of mRNP Granule Composition and Structure in Saccharomyces cerevisiae." Diss., The University of Arizona, 2015. http://hdl.handle.net/10150/556224.
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A recurring theme in biology is the aggregation of mRNA-protein complexes (mRNPs) into higher order assemblies. Often these complexes play important roles in the regulation of gene expression, but the function of the conserved cytoplasmic mRNP assemblies - P bodies and stress granules, is not known. It is believed that the misregulation of granule assembly is related to disorders like Amyotrophic Lateral Sclerosis and Frontotemporal Lobe Degeneration. Determining the complete composition of these granules may hold the key to understanding the function and mechanism of assembly of these granules. This work describes multiple approaches taken to identify new protein and mRNA components of P bodies and stress granules. New members of the P body and stress granule proteome reveal a role for these granules in diverse cellular processes including signal transduction, transcription and metabolism. Additionally, a new stress granule resident complex - the CCT complex, was also identified as a novel regulator of granule disassembly. This work also describes the first purification scheme for stress granules and presents a new system for in vitro study of stress granules. Together, the findings shed new light on the composition, function, structure and regulation of P bodies and stress granules in yeast.
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Coudert, Laetitia. "La formation des granules de stress : un possible mécanisme général de la réponse des cellules cancéreuses aux drogues anti-cancers." Thesis, Université Laval, 2014. http://www.theses.ulaval.ca/2014/30404/30404.pdf.
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Le réflexe naturel d’une cellule eucaryote, soumise à un stress (ex : radiations, drogues anti-cancers…), est d’activer des mécanismes de défense afin de s’adapter aux conditions extrêmes imposées, leur permettant de survivre. Un des mécanismes activé en condition de stress est l’inhibition de l’initiation de la traduction menant à la formation de granules de stress (GS). Les GS sont des corps cytoplasmiques dynamiques renfermant des facteurs d’initiation de la traduction, des ARNms, des protéines de liaison à l’ARN ainsi que des molécules de signalisation impliquées dans les voies de mort cellulaire. La formation des GS fut identifiée comme un évènement clé inactivant les voies de mort cellulaire, constituant donc un mécanisme majeur de survie, qui dans le cas du cancer peut engendrer une chimiorésistance. Nous avons précédemment conduit un criblage des facteurs d’initiations de la traduction impliqués dans la formation des GS. Ces travaux (Mazroui et al, 2006 ; Mokas et al, 2009) ont permis d’identifier plusieurs facteurs dont l’inactivation induit la formation des GS. Par contre, l’inactivation du facteur eIF4E, qui est responsable de la reconnaissance des ARNms lors de l’initiation de la traduction, n’induit pas la formation des GS. Mon travail de thèse a permis de mettre en évidence un nouveau rôle du facteur d’initiation de la traduction eIF4E ainsi que son partenaire eIF4GI dans la formation des GS induites par le traitement chimiothérapeutique Bortezomib. Ce rôle est stimulé par la voie oncogénique mTORC1, qui est la voie de signalisation responsable de l’interaction eIF4E-eIF4GI. De plus, notre étude a démontré que l’inhibition spécifique d’eIF4E, d’eIF4GI ou l’inactivation de mTORC1 empêche l’activation des voies anti-apoptotiques associées au GS, sensibilisant ainsi les cellules cancéreuses aux traitements chimiothérapeutiques. Néanmoins, la formation des GS n’est pas restreinte au Bortezomib. En effet, notre criblage des drogues chimiothérapeutiques a identifié le Sorafenib (Nevaxar®) et le Lapatinib (Tykerb/Tyverb®) comme deux puissants inducteurs des GS au sein des cellules cancéreuses. En conclusion, ces travaux ont mis en lumière un nouveau mécanisme de formation des GS ainsi que deux potentiels inducteurs d’assemblage de ces granules.The natural reflex of a eukaryotic cell under stress (e.g.: radiation, anti-cancer drugs, thermal or oxidative stress) is to activate defense mechanisms to adapt to extreme conditions imposed, allowing them to survive. One mechanism activated under stress conditions is the inhibition of translation initiation leading to the formation of stress granules (SG). SG are dynamic cytoplasmic body containing translation initiation factors, mRNAs, RNA binding proteins and signaling molecules involved in cell death pathways. SG formation was identified as a key event inactivating cell death pathways, thus establishing a major survival mechanism, which in the case of cancer can lead to drug resistance. We previously conducted a screening of the translation initiation factors involved in the SG formation. These works (Mazroui et al, 2006; Mochas et al, 2009) have identified several factors that inactivation induces the formation of GS. For cons, the inactivation of factor eIF4E, which is responsible for the recognition of mRNAs during translation initiation, does not induce the formation of SG. My thesis has highlighted a new role for the translation initiation factors eIF4E and its partner eIF4GI in the SG formation induced by chemotherapeutic drug Bortezomib. This role is stimulated by oncogenic mTORC1 pathway, which is the key regulator of the eIF4E-eIF4GI interaction. In addition, our study demonstrated that specific inhibition of eIF4E, eIF4GI or the inactivation of mTORC1 prevents anti-apoptotic pathways associated with SG and sensitizing cancer cells to chemotherapeutic treatments. The SG formation is not restricted to Bortezomib. Indeed, our screening of chemotherapeutic drugs has identified Sorafenib (Nevaxar ®) and Lapatinib (Tykerb / Tyverb ®) as two potent inducers of SG in cancer cells. Our results indicate that the mechanism of action of these two drugs appears to be similar to Bortezomib and they induce the formation of SG by inhibiting translation initiation. In addition, the formation of SG induced by Sorafenib or Lapatinib also seems to depend on the eIF4E-eIF4GI complex formation. Therefore, my work provides a general role of eIF4E-eIF4GI interaction in the assembly of SG and the cancer cells resistance to chemotherapy.
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Wu, Yuhong. "Structural studies of Human Caprin Protein." OpenSIUC, 2019. https://opensiuc.lib.siu.edu/dissertations/1652.
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Human Caprin-1 and Caprin-2 are prototypic members of the caprin (cytoplasmic activation/proliferation-associated protein) protein family. Vertebrate caprin proteins contain two highly conserved homologous regions (HR1 and HR2) and C-terminal RGG motifs. Drosophila caprin (dCaprin) shares HR1 and RGG motifs but lacks HR2. Caprin-1 and Caprin-2 have important and non-redundant functions. The detailed molecular mechanisms of their actions remain largely unknown.
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Beaudoin, Simon. "Caractérisation du rôle de la protéine prion cellulaire et de ses formes pathologiques dans la régulation des ARNm et de la réponse au stress cellulaire." Thèse, Université de Sherbrooke, 2013. http://hdl.handle.net/11143/6662.
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Certaines maladies neurodégénératives sont associées au mauvais repliement de la protéine prion (PrP[indice supérieur c]) et sont connues sous le nom de maladies à prion ou les encéphalopathies spongiformes transmissibles (ESTs). Récemment, il a été démontré que PrP[indice supérieur c] augmente l'efficacité des micro-ARN (miARN) via son interaction directe avec la protéine Argonaute 2 (Ago2) au niveau de la membrane des corps multivésiculaires (multivesicular bodies (MVBs)). Ago2 est une des protéines centrales du complexe RISC (RNA-induced silencing complex) qui est responsable de l'efficacité des ARNi. Certaines formes pathologiques de PrP[indice supérieur c] induisent l'activation de la réponse au stress eiF2a dépendante, une étape déterminante pour le développement de la pathologie. Cependant, l'activation de la réponse au stress eiF2a dépendante, favorise normalement la survie cellulaire via la formation de deux types de granules d'ARN, les granules de stress (GSs) et les P-Bodies. Aucune étude n'a investigué le rôle de ces deux types de granules d'ARN et de la régulation de l'efficacité des miARN par PrP[indice supérieur c] dans la neurotoxicité associée aux ESTs. Mon premier objectif est d'approfondir nos connaissances sur le nouveau rôle de PrP[indice supérieur c] dans la régulation du système miARN et de l'implication des miARN et du système endosomal dans les ESTs génétiques. Mon second objectif est d'investiguer le rôle des GSs et des P-Bodies, dans la neurotoxicité reliée aux ESTs et établir un lien entre la dérégulation de la réponse au stress et du système ARNi. PrP[indice supérieur c] augmente l'efficacité des miARN via l'interaction de sa région octapeptidique répété (OR, octapeptide repeat region) avec la protéine Ago2. L'effet d'un mutant cytoplasmique artificiel nommé CyPrP (connu comme agent neurotoxique potentiel) et de cinq mutants familiaux de PrP[indice supérieur c] sur le système endosomal et miARN a été caractérisé. Les mutants de PrP[indice supérieur c] affectent la maturation des MVBs et, conséquemment, induisent une délocalisation de GW182, une diminution l'efficacité des miARN et de la production des exosomes. Nous proposons que cette dérégulation de la voie endosomale et miARN par les mutants familiaux de PrP[indice supérieur c] contribue à la neurodégénérescence observée dans les ESTs. Les mutants de PrP[indice supérieur c] induisent également l'expression de la protéine PACT qui est responsable de l'activation de la kinase de stress PKR et de la phosphorylation d'eiF2a. Cependant, malgré la phosphorylation d'eiF2a, les mutants familiaux de PrP[indice supérieur c] et PrP[indice supérieur Sc] inhibent la formation des GSs et des P-Bodies augmentant la susceptibilité des cellules aux différents stress. L'inhibition des P-bodies par les mutants confirment également que les mutants de PrP[indice supérieur c] induisent une diminution de l'efficacité des miARN. Je propose que le maintien de l'activation de la voie PACT-PKR-eiF2a et l'inhibition des GSs et des P-Bodies contribuent à la dérégulation du système d'ARNi, à la susceptibilité des neurones et à la neurodégénérescence observée dans les ESTs. Une meilleure compréhension des mécanismes de neurotoxicité plus particulièrement de l'inhibition du système ARNi et de la réponse au stress par les formes mal repliées de PrP[indice supérieur c] peut mener au développement de médicaments afin de contrer l'évolution de la pathologie. [symboles non conformes]
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Melhado, Elise Spencer. "Characterizing Novel Pathways for Regulation and Function of Ataxin-2." BYU ScholarsArchive, 2019. https://scholarsarchive.byu.edu/etd/8574.
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Ataxin-2 is an RNA-binding protein that is involved in many crucial cellular processes such as R-loop regulation, mRNA stability, TOR signaling regulation, and stress granule formation. Ataxin-2 is highly conserved, found in organisms ranging from Saccharomyces cerevisiae to Caenorhabditis elegans and Homo sapiens. Recently, ataxin-2 has been linked to the neurodegenerative disease Amyotrophic Lateral Sclerosis (ALS). ALS is a fatal disease that causes loss of motor neurons. In addition to ataxin-2 interacting with known ALS risk factor proteins, research into the relationship between ataxin-2 and ALS shows that polyglutamine expansions in ataxin-2 are gain-of-function mutations that lead to overactivity of ataxin-2 and probable neurodegeneration. In fact, targeting ataxin-2 using gene silencing techniques dramatically slows the progression of ALS in both mice and man.The Grose laboratory has characterized a serine-threonine kinase, PAS kinase as upstream kinase and putative activator of ataxin-2. We hypothesize that knockdown of PAS kinase could, therefore, have similar effects to directly downregulating ataxin-2 and its cellular functions. Characterization of Ataxin-2 has revealed that its gain or loss of function lead to distinct cellular phenotypes. One study concluded that lowering ataxin-2 levels reduced the size and number of stress granules in mammalian cells, which was observed through microscopy. Another study found that activation and overexpression of ataxin-2 lead to reduced mTOR levels because of its sequestration to stress granules. Lastly, preliminary data obtained by the Grose laboratory suggests that yeast deficient in Pbp1 (the yeast homologue of ataxin-2) have altered cell cycles.This project describes the cellular readouts used to determine if PAS kinase downregulation confers the same cellular phenotypes as ataxin-2 downregulation. First, we found that PAS kinase does influence ataxin-2 abundance in mammalian cells. Using yeast as a model, we found that Pbp1 influences the cell cycle through its binding partners, causing a reduction in the percentage of cells in the G2 phase compared to the G1 phase. PAS kinase conferred an opposite change, most likely due to the activity of other PAS kinase substrates. Additionally, we found that Pbp1 deficiency is synthetically lethal when in conjunction with deficiency of any one of its cell cycle-related binding partners. The cellular changes cause by Pbp1 deficiency highlight not only the importance of ataxin-2 in the cell, but also the importance of understanding the effects of downregulation of ataxin-2.
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Bogamuwa, Srimathi Priyadarshani. "FUNCTIONAL CHARACTERIZATION OF THREE SEED-SPECIFIC TANDEM CCCH ZINC FINGER PROTEINS IN Arabidopsis thaliana." The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1417514831.
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Kaushansky, Laura J. "Investigating the Effects of Mutant FUS on Stress Response in Amyotrophic Lateral Sclerosis: A Thesis." eScholarship@UMMS, 2015. https://escholarship.umassmed.edu/gsbs_diss/792.
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During stress, eukaryotes regulate protein synthesis in part through formation of cytoplasmic, non-membrane-bound complexes called stress granules (SGs). SGs transiently store signaling proteins and stalled translational complexes in response to stress stimuli (e.g. oxidative insult, DNA damage, temperature shifts and ER dysfunction). The functional outcome of SGs is proper translational regulation and signaling, allowing cells to overcome stress.
The fatal motor neuron disease Amyotrophic Lateral Sclerosis (ALS) develops in an age-related manner and is marked by progressive neuronal death, with cytoplasmic protein aggregation, excitotoxicity and increased oxidative stress as major hallmarks. Fused in Sarcoma/Translocated in Liposarcoma (FUS) is an RNA-binding protein mutated in ALS with roles in RNA and DNA processing. Most ALS-associated FUS mutations cause FUS to aberrantly localize in the cytoplasm due to a disruption in the nuclear localization sequence. Intriguingly, pathological inclusions in human FUSALS cases contain aggregated FUS as well as several SG-associated proteins. Further, cytoplasmic mutant FUS incorporates into SGs, which increases SG volume and number, delays SG assembly, accelerates SG disassembly, and alters SG dynamics.
I posit that mutant FUS association with stress granules is a toxic gain-of-function in ALS that alters the function of SGs by interaction with SG components. Here, I show that mutant FUS incorporates in to SGs via its Cterminal RGG motifs, the methylation of which is not required for this localization. Further, I identify protein interactions specific to full-length mutant FUS under stress conditions that are potentially capable of interacting with FUS in SGs. Finally, I demonstrate a potential change in the protein composition of SGs upon incorporation of mutant FUS. These findings advance the field of ALS and SG biology, thereby providing groundwork for future investigation.
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Kaushansky, Laura J. "Investigating the Effects of Mutant FUS on Stress Response in Amyotrophic Lateral Sclerosis: A Thesis." eScholarship@UMMS, 2008. http://escholarship.umassmed.edu/gsbs_diss/792.
Full textAbstract:
During stress, eukaryotes regulate protein synthesis in part through formation of cytoplasmic, non-membrane-bound complexes called stress granules (SGs). SGs transiently store signaling proteins and stalled translational complexes in response to stress stimuli (e.g. oxidative insult, DNA damage, temperature shifts and ER dysfunction). The functional outcome of SGs is proper translational regulation and signaling, allowing cells to overcome stress.
The fatal motor neuron disease Amyotrophic Lateral Sclerosis (ALS) develops in an age-related manner and is marked by progressive neuronal death, with cytoplasmic protein aggregation, excitotoxicity and increased oxidative stress as major hallmarks. Fused in Sarcoma/Translocated in Liposarcoma (FUS) is an RNA-binding protein mutated in ALS with roles in RNA and DNA processing. Most ALS-associated FUS mutations cause FUS to aberrantly localize in the cytoplasm due to a disruption in the nuclear localization sequence. Intriguingly, pathological inclusions in human FUSALS cases contain aggregated FUS as well as several SG-associated proteins. Further, cytoplasmic mutant FUS incorporates into SGs, which increases SG volume and number, delays SG assembly, accelerates SG disassembly, and alters SG dynamics.
I posit that mutant FUS association with stress granules is a toxic gain-of-function in ALS that alters the function of SGs by interaction with SG components. Here, I show that mutant FUS incorporates in to SGs via its Cterminal RGG motifs, the methylation of which is not required for this localization. Further, I identify protein interactions specific to full-length mutant FUS under stress conditions that are potentially capable of interacting with FUS in SGs. Finally, I demonstrate a potential change in the protein composition of SGs upon incorporation of mutant FUS. These findings advance the field of ALS and SG biology, thereby providing groundwork for future investigation.
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Emara, Mohamed Maged. "Analysis of the Cellular Proteins, TIA-1 and TIAR, and their Interaction with the West Nile Virus (WNV) 3' SL Minus-Strand RNA." Digital Archive @ GSU, 2008. http://digitalarchive.gsu.edu/biology_diss/38.
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The 3' terminal stem loop of the WNV minus-strand [WNV3'(-) SL] RNA was previously shown to bind the cell protein, T-cell intracellular antigen-1 (TIA-1), and the related protein, TIAR. These two proteins are known to bind AU-rich sequences in the 3' UTRs of some cellular mRNAs. AU stretches are located in three single-stranded loops (L1, L2, and L3) of the WNV3'(-) SL RNA. The RNA binding activity of both proteins was reduced when L1 or L2, but not L3, AU sequences were deleted or substituted with Cs. Deletion or substitution with Cs of the entire AU-rich sequence in either L1 or L2 in a WNV infectious clone was lethal for the virus while mutation of some of these nt decreased the efficiency of virus replication. Mutant viral RNAs with small plaque or lethal phenotypes had similar translational efficiencies to wildtype RNA, but showed decreased levels of plus-strand RNA synthesis. These results correlated well with the efficiency of TIA-1 and/or TIAR binding in in vitro assays. In normal cells, TIA-1 and TIAR are evenly distributed in the cytoplasm and nucleus. Between 6 and 24 hr after WNV infection, TIAR concentrated in the perinuclear region and TIA-1 localization to this region began by 24 hr. Similar observations were made in DV2 infected cells but at later times after infection. In infected cells, both proteins colocalized with dsRNA, a marker for viral replication complexes, and with viral non-structural proteins. Anti-TIAR or anti-TIA-1 antibody coimmunoprecipitated viral NS3 and possibly other viral nonstructural proteins. In response to different types stress, TIA-1 and TIAR recruit cell mRNA poly(A)+ into cytoplasmic stress granules (SG) leading to general translational arrest in these cells. SG were not induced by flavivirus infection and cells became increasingly resistant to arsenite induction of SG with time after infection. Processing Body (PB) assembly was also decreased beginning at 24 hr. These data suggest that the sequestration of first TIAR and then TIA-1 via their interaction with viral components in flavivirus infected cells inhibits SG formation and prevents the shutoff of host translation.
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Palud, Amandine. "Liquid-liquid phase separation mediated by low complexity sequence domains promotes stress granule assembly and drives pathological fibrillization." Thesis, Paris 6, 2015. http://www.theses.fr/2015PA066560/document.
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Il a été observé que l’altération des fonctions des granules de stress, entités cytoplasmiques non-membranaires composées d’ARN et de protéines liant l’ARN (RBPs), peut conduire au développement de maladies telles que la sclérose latérale amyotrophique, la démence fronto-temporale, la myopathie à inclusions et la maladie de Paget des os. Ces pathologies sont caractérisées par un dépôt cytoplasmique d’inclusions solides enrichies en RBPs et comprenant des fibrilles. Une connexion génétique a été suggérée entre la persistance des granules de stress et l’accumulation de ces inclusions pathologiques dans le cytoplasme des patients. Dans mon manuscrit de thèse, il est mis en évidence le fait que la protéine hnRNPA1, dont les mutations entrainent les maladies mentionnées plus haut, subit une séparation de phases entre deux liquides connue également sous l’appellation « Séparation de Phases Liquide-Liquide » (LLPS) dans des gouttelettes enrichies en protéines. Bien que le domaine composé d’une séquence à faible complexité (Low Complexity sequence Domains ou LCD) soit suffisant pour obtenir cette séparation de phases, les domaines de liaison à l’ARN y contribuent également en présence d’ARN. Cela a permis d’envisager l’existence de plusieurs mécanismes intervenant dans la régulation de l’assemblage de ces granules. Un autre résultat a mis en exergue le fait que la formation de fibrilles n’est pas une obligation pour permettre la séparation de phases mais que les gouttelettes, enrichies en protéines, entrainent, par ailleurs, une augmentation de la formation de ces fibrilles. La séparation de phases liquide-liquide induite par le domaine composé d’une séquence à faible complexité semble contribuer à l’assemblage des granules de stress et à leurs propriétés liquides. Finalement, cette étude propose d’établir une réelle corrélation entre la formation des granules de stress qui deviennent persistants et l’accumulation d’inclusions pathologiques dans le cytoplasme des patientsStress granules are membrane-less organelles composed of RNA-binding proteins (RBPs) and RNA. Functional impairment of stress granules has been implicated in amyotrophic lateral sclerosis, inclusion body myopathy, Paget’s disease of bone and frontotemporal dementia; these diseases are characterized by solid, fibrillar, cytoplasmic inclusions that are rich in RNA binding proteins (RBPs). Genetic evidence suggests a link between persistent stress granules and the accumulation of pathological inclusions. In this thesis manuscript, I demonstrate that the disease-related RBP hnRNPA1 undergoes liquid-liquid phase separation (LLPS) into protein-rich droplets mediated by a low complexity sequence domain (LCD). While the LCD of hnRNPA1 is sufficient to mediate LLPS, the folded RNA recognition motifs contribute to LLPS in the presence of RNA, potentially giving rise to several mechanisms for regulating assembly of stress granules. Importantly, while not required for LLPS, fibrillization is enhanced in protein-rich droplets. I suggest that LCD-mediated LLPS contributes to the assembly of stress granules and their liquid properties, and provides a mechanistic link between persistent stress granules and fibrillar protein pathology in disease
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Gareau, Cristina. "La surexpression de p21 WAF1/CIP1 via CUGP1 et les Granules de Stress procurent une résistance aux cellules cancéreuses face à l'apoptose médiée par le Bortézomib." Doctoral thesis, Université Laval, 2016. http://hdl.handle.net/20.500.11794/26707.
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Raisonnement: Les mécanismes post-transcriptionnels occupent une place importante au sein de la régulation de l’expression génique. L’expression génique est cruciale au bon développement de la cellule, mais également à sa survie. L’altération des mécanismes post-transcriptionnels fait maintenant l'objet de nombreuses études sur la cause ou la conséquence de différentes pathologies humaines telles que le cancer. Récemment, les Granules de Stress (GS) ont été trouvées à agir comme un nouveau mécanisme post-transcriptionnel, qui permet à la cellule de survivre en conditions de stress. Résultats: Notre étude démontre pour la première fois, la formation de GS dans les cellules cancéreuses traitées avec un agent chimiothérapeutique. De cela, nous avons élucidé un sentier spécifique de formation des GS en réponse au Bortézomib (Bz). Nous avons montré que cet inhibiteur de protéasome réduit l’initiation de la traduction via la phosphorylation du facteur de l’initiation de la traduction (eIF2). Cette phosphorylation d’eIF2 se produit via l’activation de la kinase de stress HRI (Heme-Regulated kinase). La suppression de la voie de phosphorylation d’eIF2-GS via la déplétion de HRI favorise une mort cellulaire accrue chez les cellules cancéreuses traitées au Bz. Ces données révèlent donc un rôle important pour HRI dans la résistance des cellules cancéreuses face au Bz, en partie par l’entremise de sa capacité à réguler la formation de GS. Pour faire suite à cette étude, nous décrivons que le facteur anti-apoptotique p21 est séquestré à l’intérieur des GS qui sont induites par le Bz. L’emprisonnement de l’ARNm p21, hautement instable, à l’intérieur des GS permet à cet ARNm d’être protégé, stabilisé et donc accumulé. Nous démontrons que la protéine de liaison à l’ARN, CUGBP1, est responsable de la localisation de l’ARNm p21 à l’intérieur des GS en réponse au Bz. Après un traitement prolongé de Bz, les GS sont désassemblées et ainsi relâchent une grande quantité d’ARNm p21 qui devient alors disponible pour être traduite. Cette traduction massive de p21 apporte alors un élan anti-apoptotique à la cellule cancéreuse ce qui lui permet de survivre au traitement chimiothérapeutique de Bz. Conclusions et perspectives: En somme, ces études décrivent une nouvelle voie spécifique de survie cellulaire qui implique un rôle potentiel pour les GS dans le cancer et qui pourrait être ciblée en thérapie. En perspective, des tumeurs xénogreffes chez la souris seront utilisées pour tester si (i) la suppression des GS via l’inactivation de HRI, et (ii) l’inactivation de la voie CUGBP1-p21, qui est régulée par les GS, sensibiliseraient les tumeurs au Bz, validant ainsi notre modèle in vivo. Ces études apporteraient des preuves de concept pour le développement de nouvelles stratégies ciblant les voies associées au GS et qui pourraient être utilisées en thérapie combinatoire pour diminuer le risque de résistance face au traitement de Bz.Rationale: Post-transcriptional mechanisms play an important role in the regulation of gene expression. Gene expression is crucial for the proper development of the cell but also for its survival. The alteration of post-transcriptional mechanisms is now the subject of numerous studies on the cause, or on the consequence, of various human diseases such as cancer. Recently, Stress Granules (SG) have been found to act as a new post-transcriptional mechanism, which allows the cell to survive in stress conditions. Results: Our study demonstrates for the first time, the formation of SGs in cancerous cells, in response to a chemotherapeutic agent. From this we have elucidated a specific pathway of SG formation in response to Bortezomib (Bz). We demonstrate herein that this proteasome inhibitor reduces translation initiation via the phosphorylation of the initiation factor (eIF2). This phosphorylation of eIF2 is controlled through the activation of the heme-regulated kinase (HRI). The alteration of the pathway phospho-eIf2-SG, through depletion of HRI, causes massive cellular death in Bz treated cancerous cells. These data thus reveal a crucial role for HRI in the resistance of cancerous cells against Bz, in part via its capacity to regulate SG formation. Furthermore, we describe the anti-apoptotic factor p21 to be trapped inside Bz-SGs. This sheltering of the highly unstable p21 mRNA allows this one to be protected from degradation, which can be stabilized and accumulated. We also demonstrate, herein, that the RNA-binding protein CUGBP1 acts as a factor responsible for the localization of the p21 mRNA inside Bz-SGs. After prolonged treatment of Bz, SGs disassemble and release a high dose of p21 mRNA that becomes available for translation. This massive translation of anti-apoptotic p21 gives a boost to the cell that allows it to survive the stress. Perspectives and Conclusion: In sum, our studies describe a new specific pathway of cell survival that implies a potential role for SGs in cancer, which could be targeted in therapy. In perspective, xenograft tumors in mice will be used to test if (i) the inhibition of SG formation via the inactivation of HRI, and (ii) the inactivation of the CUGBP1-p21 pathway that is regulated by SGs, can both sensitize tumors to Bz treatment thus validating our model in vivo. These studies will provide us with a proof of principle for the development of new strategies targeting SG-associated pathways. Combinatorial therapies implicating the termination of such pathways could be developped in order to reduce the risk of recurrence against Bz.
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Catanzaro, Nicholas Jr. "Molecular Mechanisms of Host Responses to Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Infection." Diss., Virginia Tech, 2020. http://hdl.handle.net/10919/97907.
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Porcine reproductive and respiratory syndrome virus (PRRSV) is arguably the most economically devastating pathogen affecting the global swine industry. Since the emergence of the virus in the late 1980s, vaccination strategies aimed to control the virus have not been very effective. Current commercial vaccines are generally protective against homologous or closely-related strains but ineffective at conferring heterologous protection against genetically-diverse strains of the virus. Consequently, emergence of variant and sometime more pathogenic strains of PRRSV continues in global swine herds. As such, there is a need for better understanding of the molecular mechanisms involved in the replication of the virus.
In order to better understand the molecular mechanisms of host responses to PRRSV replication, we first sought to evaluate the ability of the virus to induce stress granules (SGs) during PRRSV infection. SGs are intracellular, cytoplasmic aggregates of RNA-binding proteins (RBPs) and mRNA. Formation of SGs is observed upon cellular stress and ultimately function to arrest cellular translation to promote cellular survival until the stress has been remedied. Indeed, several viruses have been shown to modulate the SG pathways to facilitate viral replication and even suppress the host's immune response. However, it is currently unknown whether PRRSV modulates the SG response. First, we used confocal microscopy and fluorescent in situ hybridization (FISH) to determine the distribution of known SG marker proteins and cellular mRNAs. Our findings revealed that PRRSV induces a potent SG response at late time points post-infection, and that SGs were closely associated with viral replication complexes (VRCs). Subsequently, we demonstrated that SGs are dispensable for viral replication, as short hairpin RNA (shRNA)-mediated knockdown of critical SG components (G3BP1 and G3BP2) did not affect viral replication. Interestingly, we found that the PRRSV-induced SGs are formed in a PERK-dependent manner. PERK is an important sensor of ER stress and activator of the unfolded protein response (UPR). Further investigation into the PERK signaling pathway revealed that PRRSV induces a significant amount of ER stress upon the cell during viral infection, and that exogenous stress significantly impaired the ability of the virus to replicate in MARC145 cells. We also showed that PRRSV potently induces all three signaling branches of the UPR, including PERK. While PERK knockdown had no effect on cell viability or viral replication, it significantly upregulated the mRNA expression of interferon-β and interferon stimulated genes (ISGs). The results from our studies suggest a critical role for PERK in regulating the host innate immune response to PRRSV infection. Only with a better understanding of the underlying molecular mechanisms of PRRSV replication will we be able to rationally design more effective vaccines against the virus.Doctor of Philosophy
Porcine reproductive and respiratory syndrome virus (PRRSV) causes an economically-devastating disease in the global swine industry. Annually, PRRSV is estimated to cause more than $600 million in economic losses to the swine industry in the United States alone. Current commercial vaccines against the virus are not effective against the diverse field strains largely due to the extreme heterogeneity of the virus. PRRSV is also able to potently suppress several aspects of the host's immune response and therefore establish a persistent infection. The underlying mechanisms of PRRSV-mediated immune suppression are not well understood. Therefore, in this dissertation we decided to investigate the molecular mechanisms of host responses to PRRSV infection. We first investigated the ability of the virus to induce stress granules (SGs). SGs are important intracellular regulatory components that modulate many aspects of the host's cellular processes, and have even been shown to play roles in regulating viral replication and controlling immune responses to viral infection. We demonstrate that PRRSV not only induces SGs, but that the PRRSV-induced SGs are closely associated with viral replication complexes (VRCs) within infected cells. The PRRSV-induced SGs were dispensable for viral replication. PRRSV-induced SGs were previously shown to form in a PERK dependent manner. Therefore, in the second part of this dissertation research, we decided to investigate the PERK signaling pathway during PRRSV infection. PERK is an important sensor of ER stress and activator of the unfolded protein response (UPR). Our results showed that PRRSV potently induces ER stress and all three signaling branches of the UPR, including PERK. Furthermore, we revealed that PERK may play an important role in regulating the type I interferon response to PRRSV infection. The results from our studies will aid in understanding the underlying molecular mechanism of PRRSV replication which will help rationally design the next generation of more effective vaccines against this devastating swine pathogen.
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Pape, Jenny Adele. "Characterizing the Function of PAS kinase in Cellular Metabolism and Neurodegenerative Disease." BYU ScholarsArchive, 2019. https://scholarsarchive.byu.edu/etd/8552.
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The second identified substrate of PAS kinase discussed is Pbp1. The human homolog of Pbp1 is ataxin-2, mutations in which are a known risk factor for amyotrophic lateral sclerosis (ALS). As diet and sex have been shown to be important factors regarding PAS kinase function, they also are strong contributing factors to ALS and are extensively reviewed herein. Pbp1 is known to be sequestered by PAS kinase under glucose depravation, and it can sequester additional proteins along with it to regulate different cellular pathways. To shed light on the pathways affected by Pbp1, we performed a yeast two-hybrid assay and mass spectrometry, identifying 32 novel interacting partners of Pbp1 (ataxin-2). We provide further analysis of the direct binding partner Ptc6, measuring mitophagy, mitochondrial content, colocalization, and respiration. This work elucidates novel molecular mechanisms behind the function of PAS kinase and yields valuable insights into the role of PAS kinase in disease.
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Mollet, Stéphanie. "Analyse dynamique des P-Bodies et des granules de stress, deux structures cytoplasmiques impliquées dans le stockage et la dégradation des ARNm, dans les cellules de mammifères." Paris 11, 2010. http://www.theses.fr/2010PA112200.
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Les P-Bodies (PB) et les granules de stress (GS) sont deux structures cytoplasmiques, dépourvues de membrane, impliquées dans le stockage et la dégradation des ARNm. Dans les PB, il y a accumulation de la machinerie de dégradation des ARNm. Dans les GS, induits par un stress, il y a accumulation d'une partie des ARNm non traduits. Une fois le stress levé, ils disparaissent, suggérant que les ARNm qu'ils contiennent reprennent leur traduction. PB et GS sont mobiles et nous avons caractérisé leur mobilité ainsi que les fréquences, les vitesses et les distances parcourues par les PB. Nous avons aussi déterminé le(s) réseau(x) du cytosquelette impliqués dans ces mouvements. Par ailleurs, nous avons montré que les GS se forment à proximité des PB et que les protéines des PB s'accumulant dans les GS, lors de certains stress, ne proviennent pas des PB mais du cytoplasme. Par ailleurs, la majorité des ARNm réprimés sont en dehors des GS, ce qui va à l'encontre d'un rôle direct des GS dans le stockage des ARNm (Mollet et al. , 2008). Par microscopie électronique, nous avons déterminé l'ultra-structure des GS. Ils sont peu compacts et fibrillo-granulaires tandis que les PB sont plus denses et fibrillaires (Soucjuere et al. , 2009). Nous avons montré que les PB interagissent avec les mitochondries de façon dynamique. La déplétion des PB ne semble pas affecter les mitochondries et l'altération de la fonction mitochondriale ne modifie pas les contacts PB/mitochondries. Cependant, l'inactivation des mitochondries entraîne une diminution de l'efficacité de l'interférence ARN, qui est accompagné d'une délocalisation d'Ago2 des PB vers le cytoplasme (Huan et al. , 2010, manuscrit en révision)P-Bodies (PB) and stress granules (SG) are two cytoplasmic granules, not delineated by a membrane, involved in mRNA degradation and storage. Ln PB, there is accumulation of the mRNA degradation machinery. Ln SG, induced by stress, there is accumulation of a part of the nontranslated mRNA. After stress, SG disappear, suggesting that the mRNA which they contain resume their translation. PB are mobile structures and we were able to characterize their mobility by measuring the frequencies, the speeds and the distances covered by PB. We determined the cytoskeleton network involved in these movements. Besides, we showed that the SG form near P-Bodies and that the proteins of PB accumulating in the SG, during sorne stress, do not originate from adjacent PB but from cytoplasm. Furthermore, most arrested mRNAs are not in the SG. These data do not support a direct role of SG to mRNA storage (Mollet et al. , 2008). By electronic microscopy, we determined the ultra structure of SG. They are loose and fibrillo-granular structures whereas PB are denser and fibrillar (Souquere et al. , 2009). We also showed that PB interact with mitochondria in a dynamic way. The depletion of PB does not seem to affect mitochondria and the change of mitochondrial activity does not modify the contacts between PB end mitochondria. However, the inactivation of mitochondria leads a decrease of thé RNA interference efficiency, who is accompanied by a delocalization of the Ago2 protein out of the PB (Huang et al. , 2010, Manuscript in revision)
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Pradhan, Tejaswini [Verfasser], Bernd [Akademischer Betreuer] Reif, Bernd [Gutachter] Reif, and Johannes [Gutachter] Buchner. "NMR Investigations of Antibody Light Chains Involved in AL-Amyloidosis and the Stress granules forming protein TIA-1 / Tejaswini Pradhan ; Gutachter: Bernd Reif, Johannes Buchner ; Betreuer: Bernd Reif." München : Universitätsbibliothek der TU München, 2019. http://d-nb.info/120707490X/34.
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Singh, Mamata. "Insights into the Renal Protective Mechanisms of mRNA Binding Protein HuR." The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1300995188.
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