No description available.

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.

Poly(ADP-ribose) polymerases belong to a family of proteins with 17 members in human beings. PARP1, the founding member of the family is a protein that synthesizes linear or branched polymers of ADP-ribose on itself or on target proteins. Different members of this family, that do not all possess ADP-ribosyl polymerase activity, are involved in the regulation of various cellular mechanisms. Some members of the family are particularly involved in the positive or negative control of the immune response. PARP1 is a key player in the regulation of inflammation, through its positive control of cell death and of proinflammatory cytokine production. On the other hand, the tankyrases (PARP5a and PARP5b) and PARP14 seem to regulate inflammatory responses in a negative fashion. PARP12 is a poorly characterized member of the family, whose expression is greatly increase following stimulation with type-I interferons, cytokines mainly involved in antiviral defences.<p>PARP12 is a protein that possesses three main domains: A putative RNA binding N-terminal domain composed of tandem CCCH zinc-fingers, a central WWE domain and a C-terminal PARP catalytic domain. In this work, we have shown that the expression of PARP12 is strictly-dependent on type-I interferons, that it possesses ADP-ribosyl transferase activity and that in can regulate the translation of messenger RNA into proteins. PARP12 can be found in stress granules, sites of storage of untranslated mRNAs, and is capable of directly inhibiting the translation of a reporter mRNA when tethered to it, in a manner dependent on its catalytic activity. Furthermore overexpression of wild-type PARP12, in contrast to overexpression of a mutant with no detectable catalytic activity (PARP12-G575W), leads to a general arrest of most cellular translation.<p>On the other hand, we have shown that PARP12 can activate the transcription of genes under the control of an NFκB-dependent promoter, especially when its zinc-fingers are deleted or mutated (PARP12ΔZnF). PARP12ΔZnF is located in structures that can enclose TRIF, RIP1, NEMO, p62/SQSTM1 and ubiquitin. These proteins have all possess an important role in the activation of NFκB signalling cascades. Moreover, we have shown that endogenous PARP12 is situated in ALIS (Aggresome-Like Induced Structures) in LPS-stimulated macrophages. These structures have a possible role in the presentation of antigens on class I major histocompatibility complexes, implying that PARP12 may be involved in the regulation of antigen presentation. <p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

No description available.

ALBA proteins were discovered in Archaea more than 30 years ago. They were gradually identified to be well conserved in Eucaryotes as well. A functional dimeric form of these proteins with DNA and RNA-binding capability was claimed in both mentioned domains of organisms. However, their roles diversified during evolution and vary in between organisms. In Archaea, ALBAs are involved in the genome organization and RNA-protein interactions. In Eukaryotes, there are presented two different subfamilies of ALBA proteins - Rpp20 and Rpp25 subfamily. A sole protein from each subfamily was identified in some organisms though they were multiplied in plants, respectively. These proteins can interact with each other and participate in ontogenetic development and stress responses. According to several studies, ALBA proteins were found to be involved in DNA stability maintenance or pre-rRNA splicing in the nucleus of Arabidopsis thaliana. However, they have been shown to play a role in the cellular metabolism and stress responses in cytoplasm. Six ALBA proteins were identified in the genome of A. thaliana, three from each subfamily. In this study, all heterodimeric protein- protein interactions were investigated by the bimolecular fluorescence complementation (BiFC) assay which revealed positive results in...

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.

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.

The protein Ataxin-2 is a known positive regulator of stress granules in humans, mice and yeast (known as yeast PBP1). Due to the role that stress granules play in diseases including Amyotrophic Lateral Sclerosis (ALS) and cancer, this thesis investigates the role of Ataxin-2 and its protein binding partners in stress granule development and its effects on various metabolic phenotypes of the cell. PAS kinase is a sensory protein kinase, conserved from yeast to man, which regulates respiration and lipid biosynthesis. Our lab discovered that PAS kinase phosphorylates and activates Ataxin-2 in yeast, and that PAS kinase overexpression enhances localization of Ataxin-2 to stress granules. Our preliminary results from yeast show that PAS kinase positively regulates stress granule formation in response to metabolic stress. Ataxin-2 normally functions to promote stress granule formation and it has been specifically shown to sequester and inhibit mammalian target of rapamycin complex I (mTORC1), a major player in the regulation of cell growth, to stress granules in both yeast and mammalian cells. To build upon this knowledge we performed a large-scale yeast interactome to identify Pbp1 binding partners through yeast-two hybrid and mass spectrometry. We identified 32 novel putative binding partners. A protein of note was Ptc6, a known regulator of mitophagy with human homolog PPM1K, which is not known to be involved in stress granules. Through colocalization with Ppb1 we determined that Ptc6 is sequestered to stress granules under glucose depravation. Under Pbp1 overexpression, Ptc6 was shown to increase localization to a stress granule marker, Pab1, showing that Pbp1 may be actively promoting Ptc6 to stress granules. We investigated the effects of eliminating Pbp1 and Ptc6 in yeast cells, including on mitophagy, mitochondrial quantification, whole cell respiration and mitochondrial reactive oxidative species. In a separate project, due to the outbreak of a worldwide pandemic and early concerns that currency could be a potential SARS-CoV-2 fomite, we investigated whether the virus could survive on varying types of currency. We conducted environmental studies and found no viable virus on bank notes or money cards. In vitro studies with live virus suggested SARS-CoV-2 was highly unstable on banknotes, however SARS-CoV-2 displayed increased stability on money cards with live virus detected after 48 hours.

No description available.

Because it can expand the range of a protein’s interactions or modulate its activity, post-translational methylation of arginine residues in proteins must be duly coordinated and ‘decoded’ to ensure appropriate cellular interpretation of this biological cue. This can be achieved through modulation of the enzymatic activity/specificity of the protein arginine methyltransferases (PRMTs) and proper recognition of the methylation ‘mark’ by a subset of proteins containing ‘methyl-sensing’ protein modules known as ‘Tudor’ domains. In order to gain a better understanding of these regulatory mechanisms, we undertook a detailed biochemical characterization of the predominant member of the PRMT family, PRMT1, and of the novel Tudor domain-containing protein 3 (TDRD3). First, we found that PRMT1 function can be modulated by 1) the expression of up to seven PRMT1 isoforms (v1-7), each with a unique N-terminal region that confers distinct substrate specificity, and by 2) differential subcellular localization, as revealed by the presence of a nuclear export sequence unique to PRMT1v2. Second, our findings suggest that TDRD3 is recruited to cytoplasmic stress granules (SGs) in response to environmental stress potentially by engaging in methyl-dependent protein-protein interactions with proteins involved in the control of gene expression. We also found that arginine methylation may serve as a general regulator of overall SG dynamics. Finally, we uncovered that alteration of PRMT1, TDRD3, and global arginine methylation levels in breast cancer cells may be closely associated with disease progression and poor prognosis. Therefore, further studies into the pathophysiological consequences ensuing from misregulation of arginine methylation will likely lead to the development of novel strategies for the prevention and treatment of breast cancer.