Mitogen-activated protein kinases (MAPKs) belong to the most conserved signaling pathways and are found in all eukaryotes, including humans where they play important roles in various diseases and cancer. Stimulation of this signal transduction pathway by microbe-associated molecular patterns (MAMP) results in a multitude of events to regulate innate immune responses in Arabidopsis thaliana stimulating large-scale changes in gene expression. Starting from a phosphoproteomic screen in Arabidopsis thaliana wild type and mpk3, mpk4 and mpk6 mutants following microbe-associated molecular pattern (MAMP) treatment, several novel chromatin-associated proteins were identified that are differentially phosphorylated by stress-induced protein kinases. Arabidopsis Ras GTPase-activating protein SH3-domain-binding protein (AtG3BP-1) is a downstream putative substrate of the MAMP-stimulated MAPK pathway that is phosphorylated by MPK3, 4 and 6 in in vitro kinase assays. AtG3BP1 belongs to a highly conserved family of RNA-binding proteins in eukaryotes that link kinase receptormediated signaling to RNA metabolism. Here, we report the characterization of the Arabidopsis homolog of human G3BP1 in plant innate immunity. AtG3BP1 negatively regulates plant immunity and defense immune responses. Atg3bp1 mutant lines show constitutive stomata closure, expression of a number of key defense marker genes, and accumulate salicylic acid but not jasmonic acid. Furthermore, Atg3bp1 plants exhibit enhanced resistance to the biotrophic pathogen Pseudomonas syringae pv. tomato. Pathogen resistance was mediated by stomatal and apoplastic immunity in Atg3bp1. More generally, our data reinforce that AtG3BP1 is a key mediator of plant defense responses and transient expression of AtG3BP1 delivered striking disease resistance in the absence of yield penalty, highlighting a potential application of this gene in crop protection.

Type 2 diabetes (T2D) is a prevalent metabolic disorder affecting approximately 529 million people globally, with an estimated increase to 1.27 billion by 2050. This condition is characterized by chronic, uncontrolled hyperglycemia, resulting from insufficient insulin secretion by pancreatic beta cells in relationship to metabolic needs due to elevated insulin resistance. Accordingly, a reduction of the functional beta cell mass is a key aspect of T2D pathogenesis. Recently, our transcriptomic analyses of laser-captured micro-dissected pancreatic islets from human living donors, identified G3BP1, which codes for a RNA-binding protein, to be among the most significantly downregulated genes in donors with T2D compared to normoglycemic individuals. This finding prompted us to investigate the role of G3BP1 in pancreatic beta cell function. Beta cells are specialized in maintaining glucose homeostasis, secreting insulin into the bloodstream in response to elevated blood glucose levels. These cells synthesize insulin at an impressive rate, exceeding 3,000 molecules per minute per cell. Given that insulin transcription is not immediate post-glucose stimulation, the regulation of insulin mRNA translation relies predominantly on post-transcriptional mechanisms. Several RNA-binding proteins (RBPs) have been identified to regulate insulin mRNA translation, notably PTBP1, which binds to the 3’ and 5’ UTRs, enhancing translation and stabilizing the transcript. Building on this, our research focused on evaluating the potential function of the RBP G3BP1 in beta cell function. G3BP1 is prominently known for its role in the formation of stress granules, membraneless phase-separated bodies comprising various mRNAs, small ribosomal subunits (40S), and RBPs. Their primary function is to halt translation, conserving energy during stress. However, our previous findings indicated the presence of G3BP1+ condensates under resting glucose concentrations, suggesting a non-stress induced response. The aim of this thesis was to investigate whether the presence of these G3BP1+ condensates signify a physiological response and to determine the specific function of G3BP1 in beta cells. Initially, in MIN6-K8 insulinoma cells G3BP1 and its paralog G3BP2 were observed in condensates that colocalize with Ins1/2 mRNA at resting glucose concentrations. Significantly, these G3BP1/2+ condensates dissolved upon shifting to stimulating glucose concentrations, as assessed by immunohistochemistry coupled with RNA FISH. Subsequently, a stress response could be excluded upon discovering that eIF2 activation was substantially lower under glucose-only conditions compared to glucose plus an oxidative stress agent, sodium arsenate. Additionally, it was found that under glucose-only conditions, signaling predominantly involved AMPK- activation. Following this, G3BP1 and G3BP2 single knockout cell lines were created using CRISPR-Cas9 genomic editing. In these cell lines, G3BP1, in contrast to G3BP2, proved essential for maintaining appropriate levels of insulin transcript variants and pro-insulin levels, as measured by qPCR and ELISA. Moreover, G3BP1 was crucial for facilitating polysome peak enrichment after glucose stimulation, as made evident through polysomal profiling. Interestingly, unlike insulin, other secretory granule proteins did neither exhibit similar patterns in transcript localization to G3BP1+ condensates, nor showed comparable transcript/protein level changes in G3BP1 knockout cells. Additionally, other insulin secretagogues such as Exendin-4 and palmitic acid induced a reduction in G3BP1+ condensates under resting glucose conditions, though not as pronounced as with stimulating glucose levels. In contrast, KCl led to an increase in G3BP1+ condensates under resting conditions, suggesting it may act as a stressor for beta cells. Importantly, the observed phenotype of G3BP1/2+ condensate presence co-localizing with Ins1/2 mRNA in resting glucose conditions and their subsequent dissolution in stimulating conditions were validated in mouse and human primary beta cells. This was particularly evident in mouse dispersed pancreatic islet cells and fresh human pancreatic islets, using immunohistochemistry with RNA FISH or RNAscope, respectively. Moreover, INS mRNA+ granular structures that partially co-localized with G3BP1+ signal were identified in frozen human pancreatic tissue sections from non-diabetic living donors, an important finding as these tissues were immediately snap-frozen post-extraction. The novelty of these findings lies in identifying a process typically associated with stress conditions that also occurs in physiological conditions, likely utilizing similar machinery to the stress related response. This discovery unveils new molecular mechanisms for investigating the pathogenesis of T2D, thereby paving the way for more focused research and the identification of novel therapeutic targets for its treatment. / Typ-2-Diabetes (T2D) ist eine weit verbreitete Stoffwechselerkrankung, von der weltweit etwa 529 Millionen Menschen betroffen sind, mit einer geschätzten Zunahme auf 1,27 Milliarden bis 2050. Diese Erkrankung ist gekennzeichnet durch chronische, unkontrollierte Hyperglykämie, die auf unzureichende Insulinsekretion durch pankreatische Betazellen im Verhältnis zu den metabolischen Bedürfnissen des Organismus aufgrund erhöhter Insulinresistenz peripheren Gewebes zurückzuführen ist. Entsprechend ist eine Reduktion der funktionellen Betazellmasse ein Schlüsselaspekt der T2D-Pathogenese. Kürzlich identifizierten unsere transkriptomischen Analysen aus Laser mikrodissezierten Pankreasinseln von lebenden menschlichen Spendern G3BP1, eine Gensequenz welche ein RNA-bindendes Protein kodiert, als eines der am stärksten herunterregulierten Gene bei Spendern mit T2D im Vergleich zu normoglykämischen Individuen. Dieser Befund veranlasste uns, die Rolle von G3BP1 in der Funktion von Betazellen zu untersuchen. Betazellen sind darauf spezialisiert, die Glukosehomöostase aufrechtzuerhalten, indem sie Insulin in den Blutkreislauf abgeben, wenn der Blutzuckerspiegel erhöht ist. Diese Zellen synthetisieren Insulin mit einer beeindruckenden Rate, die mehr als 3.000 Moleküle pro Minute pro Zelle übersteigt. Da die Insulintranskription nach Glukosestimulation nicht sofort eintritt, beruht die Regulation der Insulin-mRNA-Übersetzung hauptsächlich auf posttranskriptionellen Mechanismen. Mehrere RNA-bindende Proteine (RBPs) wurden identifiziert, welche die Insulin-mRNA-Übersetzung regulieren. Unteranderem PTBP1, das an die 3’- und 5’-UTRs bindet, und so die Translation verstärkt und das Transkript stabilisiert. Aufbauend darauf konzentrierte sich unsere Forschung auf die Bewertung der potenziellen Funktion des RBP G3BP1 in Betazellen. G3BP1 ist vor allem für seine Rolle bei der Bildung von Stressgranula bekannt. Diese sind membranlose, phasenseparierte Kondensate, die aus verschiedenen mRNAs, kleinen ribosomalen Untereinheiten (40S) und RBPs bestehen. Ihre Hauptfunktion ist es, die Translation zu stoppen und Energie während Zellstresses zu konservieren. Unsere früheren Ergebnisse wiesen jedoch auf das Vorhandensein von phasenseparierten G3BP1+ Kondensaten unter Ruheglukosekonzentrationen hin, was auf eine physiologische statt einer stressbedingten Reaktion hinweisen könnte. Das Ziel dieser Arbeit war es, zu untersuchen, ob das Vorhandensein dieser G3BP1+ Kondensate eine physiologische Reaktion darstellt, und die spezifische Funktion von G3BP1 in Betazellen zu bestimmen. Zunächst wurden in MIN6-K8 Insulinomzellen G3BP1 und sein Paralog G3BP2 in Kondensaten beobachtet, die mit Insulin-mRNA bei Ruheglukosekonzentrationen kolokalisierten. Auffällig war, dass sich diese G3BP1/2+ Kondensate bei dem Wechsel zu stimulierenden Glukosekonzentrationen auflösten, wie durch Immunhistochemie in Kombination mit RNA FISH beurteilt wurde. Anschließend wurde eine Stressreaktion ausgeschlossen, nachdem beobachtet wurde,, dass die eIF2-Aktivierung unter Glukosebedingungen wesentlich geringer war als unter der gleichzeitigen Zugabe von Glukose und einem oxidativen Stressmittel, Natriumarsenat. Außerdem wurde festgestellt, dass unter Glukosebedingungen die Signalgebung hauptsächlich auf einer AMPK--Aktivierung beruht. Daraufhin wurden G3BP1- und G3BP2-Einzelknockout-Zelllinien mit CRISPR-Cas9-Genomeditierung erstellt. In diesen Zelllinien erwies sich G3BP1 im Gegensatz zu G3BP2 als unerlässlich für die Aufrechterhaltung angemessener Spiegel von Insulin-Transkriptvarianten und Pro-insulinspiegeln, gemessen durch qPCR und ELISA. Darüber hinaus war G3BP1 entscheidend für die Erleichterung der Polysomenpeakanreicherung nach Glukosestimulation, was durch polysomales Profiling deutlich wurde. Interessanterweise zeigten im Gegensatz zu Insulin andere sekretorische Granulproteine weder ähnliche Muster in der Transkriptlokalisierung zu G3BP1+ Kondensaten, noch vergleichbare Transkript-/Proteinleveländerungen in G3BP1-Knockout-Zellen. Außerdem induzierten andere Insulinsekretagoge wie Exendin-4 und Palmitinsäure eine Reduktion von G3BP1+ Kondensaten unter Ruheglukosebedingungen, allerdings nicht so ausgeprägt wie bei stimulierende Glukosespiegel. Im Gegensatz dazu führte KCl zu einer Zunahme von G3BP1+ Kondensaten unter Ruhebedingungen, was darauf hindeutet, dass es als Stressor für Betazellen wirken könnte. Wichtig ist, dass der beobachtete Phänotyp des Vorhandenseins von G3BP1/2+ Kondensaten, die mit Insulin-mRNA unter Ruheglukosebedingungen kolokalisieren und sich unter stimulierenden Bedingungen auflösen, in primären Maus- und menschlichen Betazellen validiert wurde. Dies war besonders deutlich in der Immunhistochemie mit RNA FISH bzw. RNAscope von disseminiert Maus-Pankreasinselzellen und frischen menschlichen Pankreasinseln., Darüber hinaus wurden in gefrorenen menschlichen Pankreasgewebeschnitten von nicht-diabetischen, lebenden Spendern INS-mRNA+ granuläre Strukturen identifiziert, die teilweise mit G3BP1+ Kondensaten kolokalisierten.Das ist ein wichtiger Befund, da diese Gewebe unmittelbar nach der Entnahme schockgefroren wurden, was darauf hindeutet, dass diese Strukturen unter physiologischen Bedingungen vorhanden sind. Die Neuheit dieser Erkenntnisse liegt darin, einen Prozess zu identifizieren, der typischerweise mit Stressbedingungen assoziiert ist, der aber auch unter physiologischen Bedingungen auftritt und wahrscheinlich eine ähnliche Maschinerie verwendet, jedoch durch physiologische statt stressbedingte Antworten ausgelöst wird. Diese Entdeckung enthüllt neue molekulare Mechanismen für die Untersuchung der Pathogenese von T2D und ebnet damit den Weg für gezieltere Forschung und die Identifizierung neuer therapeutischer Ziele für deren Behandlung.

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.

G3BP1 and G3BP2 are members of a highly conserved family of multi-functional RNA binding proteins, which appear to co-ordinate signal transduction and post-transcriptional gene regulation. Both proteins are over-expressed in cancer, and G3BP1 promotes cell proliferation and survival. Aberrant expression of various RNA binding proteins is common in cancer, and several of these proteins influence tumorigenesis. Therefore, detailed examination of RNA binding proteins, such as G3BPs, may provide insights into the post-transcriptional mechanisms underlying tumorigenesis. Tumours arise as a consequence of genetic mutation or alteration, which often result from stress-induced DNA damage. Cancer progression is facilitated by various epigenetic stress adaptation mechanisms. Stressful stimuli induce transitory translational shut-off, mediated by phosphorylation of eukaryotic initiation factor alpha;(eIF2alpha;). This phosphorylation event leads to formation of discrete cytoplasmic foci known as stress granules (SGs), which are translationally-silent sites of mRNA sorting. It was initially thought that an RNA-binding protein, T-cell internal antigen 1 (TIA-1), was instrumental in both the formation and functioning of SGs, because over-expression of TIA-1 induces spontaneous SGs and concomitantly causes a decrease in reporter gene expression. It is now clear that SG content can change depending on the type of stress, and that various proteins, including G3BP1, can induce spontaneous SGs. In vitro evidence previously implicated both G3BP1 and G3BP2 as endoribonucleases, so it was suggested that G3BPs act to target mRNA for decay at the SG. This project sought to further investigate this proposal, and in this way gain insight into the specific function of G3BPs in post-transcriptional regulation during tumorigenesis. Characterisation of G3BP1 and G3BP2 expression and localisation patterns in human cells and cancer was necessary before functional analyses in human cell systems could be undertaken. Both proteins were found to be over-expressed in breast cancer, irrespective of cancer stage or grade. G3BP1 and G3BP2 were also expressed in all human cell lines tested, despite previously observed tissue-specific expression. These results support the notion that G3BP expression is switched on in parallel with cell proliferation, and as such, may influence tumorigenesis. The results of further analyses suggested that the diverse functions attributed to G3BP1 and G3BP2 may be facilitated by isoform-specific expression, various post-translational modifications and sub-cellular localisation. Despite the absence of a canonical endoribonuclease domain, it was previously reported that site-specific phosphorylation of G3BP1 enables the protein to degrade a synthetic c-myc RNA substrate in vitro. This finding implicated G3BP in the specific regulation of a proto-oncogene. Tailored reporter assays were thus designed in order to address the in vivo consequences of G3BP's putative endoribonuclease activity. Contrary to expectations, all G3BP family members increased or maintained the expression of a range of reporters, at both the mRNA and protein level, irrespective of the presence of any particular cis-acting element, coding sequence or promoter. These results support the emerging notion that G3BPs positively affect the expression of at least some of their target mRNAs, and may also indirectly promote transcription. In contrast to the theory that G3BPs degrade proto-oncogenic mRNA/s, these findings are consistent with a role for G3BP in promoting cell proliferation and survival. Further analyses showed that G3BP1 and G3BP2 simultaneously increased reporter gene expression and induced SG formation. These findings highlighted the fact that SGs are dynamic sorting stations for mRNAs, and not merely sites of stalled translation. This result also supports the notion that a variety of proteins may be recruited to the SG to facilitate a multitude of mRNA fates. Although the precise role of the SG in stress adapation is not known, it is clear that an appropriate integrated stress response (ISR) is required for cells to survive in sub-optimal conditions. It was found that specific G3BP1 knockdown inhibited SG formation and cell survival, and this appeared to occur downstream of eIF2alpha; phosphorylation. The phosphorylation of eIFalpha; is the only factor known to be necessary for SG formation and cell survival. This data is the first to implicate SG formation itself, downstream of eIF2alpha; phosphorylation, in the survival phase of the ISR. The results also suggest that G3BP1 plays a pivotal role in the post-transcriptional mechanisms underlying stress adaptation. To facilitate future analysis of G3BP roles in the regulation of specific transcripts and in SG biology, a pilot study to identify G3BP RNA ligands was undertaken. Immunoprecipitation of epitope-tagged G3BP1 from stable cell lines facilitated purification and isolation of RNA in association with G3BP1. Specific RNA transcripts were subsequently detected and identified by microarray. Many genes were enriched in the G3BP1 immunoprecipitate. Transcript enrichment in the control immunoprecipitate was comparatively weak and seemingly random, suggesting that several replicates will enable generation of a reliable target list. This work forms a promising basis for further investigations into G3BP functionality, and also provides a platform for broader and more large-scale analyses of the mechanisms of post-transcriptional gene regulation. The work presented in this thesis addressed the potential post-transcriptional mechanisms by which the G3BP family of proteins mediate cell proliferation and survival. Both G3BP1 and G3BP2 were shown to be over-expressed in tumours and each appeared to promote reporter gene expression. G3BP1 was also found to play a pivotal role in stress adaptation. A technique to identify novel RNA ligands was assessed, and it was found that G3BP1 may interact with various mRNA transcripts. It is hypothesised that the G3BP family of proteins, and in particular G3BP1, function to determine the fate of specific RNAs in response to cellular stress and other stimuli. In this way, G3BP proteins may facilitate appropriate responses to extra-cellular stimuli which allow for cell proliferation and survival.

Les troubles neurodégénératifs, tels que la sclérose latérale amyotrophique (SLA) et la démence frontotemporale (DFT), ont été associés aux protéines de liaison à l'ARN (RBP). Les principales caractéristiques de la SLA sont l'agrégation d'une protéine de liaison à l'ARN appelée protéine de liaison TAR (TDP-43). Il a été démontré que TDP-43 se lie à G3BP1, un facteur de nucléation pour l'assemblage des granules de stress, pour le stabiliser. Les granules de stress sont des structures séparées par phases qui se forment dans des conditions stressantes et favorisent la survie cellulaire. Une altération de l’assemblage des granules de stress et une réduction du G3BP1 sont signalées dans la SLA. Cette réduction est due à un défaut dans les transcriptions codantes pour G3BP1 stabilisant TDP-43. Par conséquent, une réponse défaillante des granules de stress pourrait jouer un rôle majeur dans la maladie. Ainsi, ce projet de recherche se concentre sur la restauration de G3BP1, dont la déplétion est liée à la perte de fonction de TDP-43. En utilisant des composés de petites molécules identifiés lors d'une campagne de dépistage de médicaments, nous cherchons à augmenter l'expression de G3BP1, rétablissant ainsi le mécanisme SG endogène et favorisant la survie neuronale. La découverte de candidats principaux (NPX-047, NPX-000-115 et NPX-001-280) qui sauvent efficacement l'expression et la fonction de G3BP1 est prometteuse pour des thérapies potentielles contre la SLA. Ces composés ont été testés sur des cellules SHSY5Y traitées avec du si-TDP, mais aucune récupération de l'ARNm de G3BP1 n'a été observée malgré des niveaux plus élevés de signaux de luciférase. Ainsi, une enquête approfondie sur les divergences dans nos résultats constitue notre prochaine étape, ce qui n’a pas été possible pendant la durée limitée de cette mémoire. De plus, les cibles non ciblées de ces composés seront étudiées à l’aide du séquençage Bru Chase. Dans l’ensemble, cette étude explore de nouvelles stratégies pour restaurer l’expression de G3BP1, offrant ainsi une voie potentielle d’intervention thérapeutique dans la SLA. / Neurodegenerative disorders, such as Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD), have been associated with RNA-binding proteins (RBPs). Major hallmark of ALS is aggregation of an RNA-binding protein called TAR binding protein (TDP-43). TDP-43 has shown to bind to G3BP1, a nucleating factor for stress granule assembly, to stabilize it. Stress granules (SGs) are phase separated structures that form under stressful conditions and promote cell survival. Impaired stress granules assembly and reduced G3BP1 is reported in ALS. This reduction is due to a defect in TDP-43 stabilizing G3BP1 encoding transcripts; thus, a failed stress granule response could have a major role in the disease. Thus, this research focuses on restoring G3BP1, whose depletion is linked to TDP-43 loss of function. By utilizing small-molecule compounds identified through a drug screening campaign, we seek to increase G3BP1 expression, consequently reinstating the endogenous SG mechanism and promoting neuronal survival. The discovery of lead candidates (NPX-047, NPX-000-115, and NPX-001-280) that effectively rescue G3BP1 expression and function offers promise for potential ALS therapies. These compounds were tested on SH-SY5Y cells treated with si-TDP however no rescue of G3BP1 mRNA was observed despite higher levels of luciferase signals. Thus, in-depth investigation of discrepancies in our results is our next step which was not possible during the limited timeline of this thesis. In addition, off-targets of these compounds will be investigated using BruChase-sequencing. Overall, this study explores novel strategies to restore G3BP1 expression, providing a potential avenue for therapeutic intervention in ALS.

No description available.

La sclérose latérale amyotrophique (SLA) et la démence fronto-temporale (DFT) sont des maladies neurodégénératives fatales, actuellement sans traitement. Ces maladies entrainent la dégénérescence des neurones moteurs et corticaux, engendrant des troubles moteurs et cognitifs et ultimement menant à la mort des patients souvent par détresse respiratoire trois à cinq ans après l’apparition des premiers symptômes. À l’échelle d’une vie, le risque de développer ces pathologies est de 1 pour 300-400 pour la SLA et 1 pour 742 pour la DFT, faisant de ces pathologies un risque majeur. Avec le vieillissement de la population que nous connaissons actuellement, il est évident que l’incidence de ces maladies deviendra de plus en plus élevée. Ainsi il est essentiel de comprendre les mécanismes moléculaires sous-jacents à ces pathologies dans le but de développer des thérapies effectives et prévenir l’impact de ces pathologies dans notre société. À ce jour, l’étiologie de la SLA-DFT est encore débattue, cependant la communauté scientifique s’accorde sur le fait que l’interaction entre la génétique et l’environnement joue un rôle essentiel dans le développement de ces maladies. La caractéristique moléculaire principale de ces pathologies est la localisation cytoplasmique de la protéine, normalement, nucléaire TDP-43. TDP-43 est un régulateur clef de l’homéostasie des ARNs. Parmi ces nombreuses fonctions, TDP-43 régule la formation des granules de stress, en régulant leur protéine régulatrice G3BP1. Ces granules formés d’ARN et de protéines se forment pour protéger les cellules durant une période de stress. Récemment, ces granules ont fait l’objet de nombreuses études et leurs dysfonctions ont été associées à la SLA-DFT. Dans cette thèse, nous avons approfondi l’étude de la régulation de TDP-43 sur G3BP1. Nous avons défini que TDP-43 stabilise les transcrits de G3BP1 de par une liaison forte à une séquence conservée à travers l’évolution se situant dans le 3’UTR de G3BP1. La perte de localisation nucléaire, la présence de mutations ou de TDP-35, une isoforme pathologique de TDP-43, sont associées à une diminution des niveaux de G3BP1. Également, d’un point de vue histopathologique, dans le cortex orbitofrontal des patients atteints de SLA-DFT, les neurones présentant une localisation cytoplasmique de TDP-43 ont une perte des niveaux transcriptionnels de G3BP1, associant alors directement G3BP1 à la maladie. Par la suite, nous avons défini que la perte de fonction en tant que stabilisateur, permet la liaison de microARNs sur les transcrits de G3BP1, engendrant leur dégradation. Le blocage de la liaison de microARNs sur G3BP1 empêche la dégradation des transcrits et restaure les fonctions de la protéine. Ainsi, nous avons déterminé un moyen de contrer la perte de fonction de TDP-43 sur G3BP1. De façon intéressante, en plus de la formation des granules de stress, G3BP1 est essentielle pour l’homéostasie neuronale et la survie neuronale post-stress. Par conséquent, la restauration de la protéine est potentiellement une avenue thérapeutique multi-approche pour le traitement de ces maladies. / Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two fatal neurodegenerative diseases, currently without cure. These diseases lead to the degeneration of motor and cortical neurons, causing motor and cognitive disorders and ultimately leading to death, often from respiratory distress three to five years after the onset. Over a lifetime, the risk of developing these conditions is 1 in 300-400 for ALS and 1 in 742 for FTD, making these conditions a major risk. With the current aging of the population, it is evident that the incidence of these diseases will become increasingly high. It is therefore essential to understand the molecular mechanisms underlying these pathologies in order to develop effective therapies. To this day, the etiology of ALS-FTD is still debated. However, the scientific community agrees that the interaction between genetics and the environment play an essential role in the development of these diseases. The main molecular characteristic of these pathologies is the cytoplasmic localization of the normally nuclear protein TDP-43. TDP-43 is a key regulator of RNA homoeostasis. Among these many functions, TDP-43 regulates the formation of stress granules, by regulating their nucleator protein G3BP1. These granules of RNA and protein form to protect cells during times of stress. Recently these granules have been the subject of several studies and their dysfunction has been associated with ALS-FTD. In this thesis, we have deepened the study of the regulation of TDP-43 on G3BP1. We have defined that TDP-43 stabilizes G3BP1 transcripts by strong binding to a sequence conserved through evolution located in the 3'UTR of G3BP1. Loss of nuclear localization, the presence of mutations or of TDP-35, a pathological isoform of TDP-43, are associated with decreased levels of G3BP1. Also, histopathologically, in the orbitofrontal cortex of patients with ALS-DFT, neurons with cytoplasmic localization of TDP-43 have a loss of transcriptional levels of G3BP1, directly associating G3BP1 with the disease. Subsequently, we defined that TDP-43 loss of function as a stabilizer allows the binding of two microRNAs on the G3BP1 transcripts, causing their degradation. Blocking the binding of these microRNAs to G3BP1 prevents the degradation of the transcripts and restores the functions of the protein. Thus, we have determined a way to counter the loss of function of TDP-43 on G3BP1. Interestingly, in addition to the formation of stress granules, G3BP1 is essential for neuronal homoeostasis and post-stress neuronal survival. Therefore, the restoration of the protein is potentially a multi-approach therapeutic avenue for the treatment of these diseases.

Ubiquitinbindande protein 2-liknande (UBAP2L) och Ras GTPas-aktiverande proteinsbindande protein 1 (G3BP1) är två kärn-RNA-bindande proteiner (RBPs) som är involverade i bildandet av SGs. Nyligen genomförda studier föreslog att UBAP2L kan fungera som ett upströmsprotein till G3BP1/2 med förmågan att främja SG-bildning oberoende. NTF2-domänen dimeriserar G3BP1 och bildar en plattform för protein-protein-interaktioner. Dess interaktion med UBAP2L-DUF-regionen var avgörande för bildandet av mogna SG. Detta projekt syftade till att avslöja protein-protein-interaktionen mellan G3BP1 och UBAP2L in vitro och karakterisera den kända interaktionen mellan G3BP1-NTF2 och UBAP2L-DUF-regionen. I detta projekt renades homodimeren av G3BP1-NTF2-domänen och komplexades med en syntetiserad peptid som motsvarar rester 505–534 av UBAP2L (UBAP2L 505-534). Låga diffraktionskristallträffar erhölls genom att co-kristallisera G3BP1-NTF2 med peptiden. Ytterligare optimeringar krävs fortfarande. Tre konstruktioner av UBAP2L (N, C och L) designades och utsattes för reningsförsök. Fullängds-G3BP1 renades för att identifiera domäninteraktionen inom UBAP2L-konstruktionerna. Pull-down-testet med His-G3BP och samuttrycket av G3BP1/UBAP2L-N föreslog en mycket svag interaktion mellan G3BP1 och UBAP2L-N. Med tanke på de tillgängliga resultaten från vår studie och co-IP-tester från andra publicerade artiklar bör vi fokusera mer på studien av G3BP2, som föreslogs spela en mer direkt roll i att binda UBAP2L. / Ubiquitin Binding Protein 2-like (UBAP2L) and Ras GTPase-activating protein-binding protein 1 (G3BP1) are two core RNA-binding proteins (RBPs) involved in the assembly of SGs. Recent studies suggested that UBAP2L might serve as an upstream protein of G3BP1/2 with the ability to promote SG assembly independently. The NTF2 domain dimerizes G3BP1 and forms a platform for protein-protein interactions. Its interaction with the UBAP2L-DUF region was critical for mature SG formations. This project aimed to reveal the protein-protein interaction between G3BP1 and UBAP2L in vitro and characterize the known interaction between G3BP1-NTF2 and UBAP2L-DUF region. In this project, the G3BP1-NTF2 domain homodimer was purified and complexed UBAP2L-DUF derived peptide. Low diffraction crystal hits were obtained by co-crystallizing G3BP1-NTF2 with a synthesized peptide corresponding to residues 505 – 534 of UBAP2L (UBAP2L 505-534). While further optimizations are still required. Three constructs of UBAP2L (N, C, and L) were designed and subjected to purification trials.  Full-length G3BP1 was purified to identify the domain interaction within UBAP2L constructs. The his-G3BP pull-down assay and co-expression of G3BP1/UBAP2L-N suggested a very weak interaction between G3BP1 and UBAP2L-N. Considering the available results from our study and co-IP assays from other published papers, we should focus more on the study of G3BP2 which was suggested to play a more direct role in binding UBAP2L.

Les Granule de Stress (GS) sont des inclusions cytoplasmiques contenant des protéines et des ARNm qui s’assemblent en réponse à l’exposition à un stress. Leur formation fait partie intégrante de la réponse cellulaire au stress et est considérée comme une étape déterminante pour la résistance au stress et la survie cellulaire. Actuellement, les GS sont reliés à divers pathologies allant des infections virales aux maladies neurovégétatives. L’une d’entre elle, la Sclérose Latérale Amyotrophique (SLA) est particulièrement agressive, caractérisée par une perte des neurones moteurs aboutissant à la paralysie et à la mort du patient en cinq ans en moyenne. Les mécanismes de déclenchement de la pathologie restent encore à déterminer. TDP-43 (TAR DNA binding protein 43) et FUS (Fused in liposarcoma) sont deux protéines reliées à la pathologie qui présentent des similarités de structure et de fonction, suggérant un mécanisme commun de toxicité. TDP-43 et FUS sont toutes les deux recrutées au niveau des GS en condition de stress. Nous avons démontré pour la première fois que la fonction des GS est de protéger les ARNm de la dégradation induite par l’exposition au stress. Cette fonction n’était que suspectée jusqu’alors. De plus nous avons mis en évidence que G3BP1 (Ras GTPase-activating protein-binding protein 1) est l’effectrice de cette fonction via son implication dans la dynamique de formation des GS. TDP-43 étant un régulateur de G3BP1, nous prouvons ainsi que la perte de fonction de TDP-43/G3BP1 aboutit à un défaut de réponse au stress aboutissant à une vulnérabilisation cellulaire. Le mécanisme de toxicité emprunter par FUS diffère de celui de TDP-43 et ne semble pas passer par une perte de fonction dans le cadre de la réponse au stress. / Stress Granule (SGs) are cytoplasmic inclusions sequestering proteins and mRNAs following a stress exposure. Their assembly is part of the cell stress response and is considered an important step for stress resistance and cell survival. SG are currently linked to different pathogenesis from viral infection to neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS).ALS is an aggressive disease, characterized by neuronal death leading to paralysis and death within five years. Pathogenesis mechanisms are still not fully understood. TDP-43 (TAR DNA binding protein 43) and FUS (Fused in liposarcoma) are two proteins linked to the disease that share many structural features and functions suggesting a common toxicity mechanism. TDP-43 and FUS are both recruited to SGs in stress conditions. We demonstrate for the first time that SGs function to protect mRNA from degradation induced after stress exposure, a function that was only suspected until now. We also prove that G3BP1 (Ras GTPase-activating protein-binding protein 1) is the effector of this function via it’s implication in the dynamics of SG formation. As TDP-43 is a regulator of G3BP1, we prove that loss of TDP-43/G3BP1 function leads to a stress response defect yielding increased cellular vulnerability. Furthermore, we have discovered that the mechanism of toxicity for FUS is different from TDP-43, and does not implicate a loss of function mechanism during the cell stress response.

No description available.