RelA/p65 is a main subunit of nuclear factor-kappaB (NF-kappaB) that regulates expression of genes involved in cell growth and survival, stress response, and inflammation, but its oncogenic or tumor-suppressive function in tumorigenesis has been highly controversial. Hundreds of NF-kappaB inhibitors have been developed for targeted cancer therapy, but fail to achieve anticipated anticancer efficacy. Complexity of posttranslational modifications may contribute to tumorigenic diversity of RelA/p65, but mechanisms of action remain unclear. Here we show that phosphorylation of RelA/p65 at Ser536 functions as a tumor suppressor. In normal human colon mucosa, RelA/p65 phosphorylation at Ser536 is gradually increased with the maturation and apoptotic shedding of cryptic cells, but the phosphorylation is significantly decreased in colon tumors. In RelA/p65-silenced breast and colon cancer cells, reconstitution of a constitutively active RelA/p65, whose Ser536 was replaced by aspartic acid (named p65/S536D), triggered dramatic apoptosis and autophagy or senescence upon the cell context, by affecting the expression of a range of cell death/survival genes. Reconstitution with a phosphorylation-deficient RelA/p65 whose Ser536 was replaced by alanine (called p65/S536A) had no effect on cell growth and survival. Intratumoral delivery of p65/S536D effectively suppressed tumor growth in nude mice and was marked with apoptotic cell death. Together our data suggest that it is the phosphorylation at Ser536 that confers NF-kappaB RelA/p65 a tumor-suppressive role. In addition to driving cell death, the phosphorylation of RelA/p65 on Ser536 is also beneficial for chemosensitivity in cancer therapy. Literature reports that NF-kappaB activation contributes to chemoresistance by regulating oncogenic pathways and multi-drug resistance genes, but recent studies on senescence and chemotherapy have suggested that NF-kappaB activation is essential for chemotherapy induced senescence and tumor regression. The mechanisms underlying the contradictory observations are elusive. Here we show that site-differential phosphorylation of NF-kappaB RelA/p65 modulates chemosensitivity and results in distinct outcomes in cancer cells. Phospohrylation of RelA/p65 on Ser276, Ser536 and Ser468 were detected in RITA (reactivation of p53 and induction of tumor cell apoptosis) treated cells and RITA-resistant sublines, and they showed distinct dynamics. Ectopic expression of p65/S536D dramatically enhanced RITA sensitivity while the constitutively phosphorylated form of S276 (S276D) compromised RITA effects in treated cells. The constitutive phosphorylation of Ser468 (S468D) showed very mild effects on RITA sensitivity. In addition, p65/S536D resensitized RITA-resistant sublines to RITA treatment. P65/S536D also enhanced the doxorubicin sensitivity, but not paclitaxel in these multi-drug resistant sublines. ATP-binding cassette (ABC) transporters are critical multidrug resistant factors. We found that in the RITA-resistant sublines, multiple ABC transporter genes were upregulated, particularly the ABCC6. Further studies dissected that ABCC6 was involved in site-specific phosphorylation- related chemoresistance. P65/S536D decreased ABCC6 expression and sensitized cells to doxorubicin and RITA, but not paclitaxel that is not the substrate of ABCC6 transporter. Conversely, p65/S276D enhanced chemoresistance by upregulating ABCC6. The p65/S536D enhanced RITA chemosensitivity is also confirmed in in vivo study using tumor xenografts in nude mice. Taken together, phosphorylation of p65 on Ser536 contributes to chemosensitivity by targeting ABCC6 while Ser276 leads to chemoresistance. These findings solve the controversial issues of NF-kappaB RelA/p65 in tumorigenesis and in chemosensitivity, and are important in developing NF-kappaB targeted cancer therapy, such as inhibitors. This study also highlights that better understanding of distinct active sites on NF-kappaB RelA/p65 is necessary for discussing the tumorigenic roles of NF-kappaB and developing efficient NF-kappaB targeted therapies.

No description available.

Macroautophagy (hereafter referred to as autophagy) is a highly conserved cellular process that promotes cytoplasmic homeostasis via lysosomal degradation of proteins and organelles. Dysfunctional autophagy occurs in numerous human pathologies, including neurodegeneration and cancer. Vinexin (encoded by SORBS3) is a physiologically important adaptor protein for two main reasons: 1. SORSB3 mRNA expression increases in normal human brain ageing, 2. SORBS3 is a candidate tumour suppressor in hepatocellular carcinoma (HCC). This dissertation builds on published data from an siRNA screen for autophagy regulations under basal conditions, which indicates vinexin knockdown upregulates autophagy. I replicate this finding in multiple cell lines, before characterising the impact of siSORBS3 treatment on autophagy; autophagosome biogenesis is increased, while flux through the autophagy pathway remains intact. Having excluded several possible mechanisms suggested by the literature, I focus on the transcriptional coactivators YAP and TAZ. The rationale here is: 1. YAP/TAZ activity is implicated in autophagy, 2. YAP/TAZ and vinexin are both linked to HCC. My data show that YAP/TAZ transcriptional activity is upregulated upon vinexin depletion. Moreover, increased autophagy following siSORBS3 treatment requires YAP and TAZ. A key focus of this dissertation is the mechanism by which vinexin knockdown upregulates YAP/TAZ and hence, autophagy. This centres on altered actin cytoskeleton dynamics; an increase in F-actin structures appears to compete with YAP/TAZ for binding to angiomotins, established sequesterers of YAP/TAZ in the cytosol. In this way, siSORBS3 treatment facilitates YAP/TAZ nuclear localisation and consequent transcriptional activity. Angiomotin overexpression therefore ameliorates the increase in autophagy caused by vinexin depletion. Published RNA sequencing data is used to confirm that SORBS3 mRNA expression increases in normal brain ageing, not only in the frontal cortex (as previously published), but also in the hippocampus. This sits alongside decreased expression of core autophagy genes in both tissues. Accordingly, vinexin could contribute to the decline in autophagic potential thought to occur in normal brain ageing. With regards to SORBS3 as a candidate tumour suppressor in HCC, I show that stably re-expressing vinexin in a HCC cell line downregulates YAP/TAZ and hence, autophagy. These cells also show reduced clonogenicity. My data therefore support the hypothesis that SORBS3 is a tumour suppressor in HCC; YAP and TAZ are well-known to increase proliferation and resistance to apoptosis, while autophagy can enable tumour cells to survive stressors such as nutrient starvation. The conclusions of this dissertation are that vinexin depletion upregulates autophagy in a YAP/TAZ-dependent manner and that this has physiologically important implications, especially with regards to HCC.

Orientador: Marciane Milanski Ferreira / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Aplicadas / Made available in DSpace on 2018-08-23T21:13:22Z (GMT). No. of bitstreams: 1 Araujo_MarianaPortovedodeOliveira_M.pdf: 2272542 bytes, checksum: 478a6f7d3c1173dce497f2be39d260ba (MD5) Previous issue date: 2013 / Resumo: O hipotálamo mediobasal é conhecido como o local primário que coordena o balanço entre a ingestão alimentar e o gasto energético. Nesse contexto, dietas com alto conteúdo de ácidos graxos saturados são apontadas como fatores dietéticos importantes responsáveis pelo desenvolvimento da obesidade. Dados recentes demonstraram que a autofagia, um processo que regula a homeostase celular por degradar organelas e proteínas disfuncionais, é crucial para a manutenção da funcionalidade de neurônios hipotalâmicos AgRP e POMC, que por sua vez, são responsáveis por coordenar o balanço energético corporal. Nesse trabalho, avaliamos a distribuição e o conteúdo de proteínas da maquinaria da autofagia em um modelo animal de obesidade induzida por dieta hiperlipídica com 8 ou 16 semanas de dieta e também em resposta a injeções intracerebroventriculares de ácido graxo esteárico. Demonstramos que a exposição crônica à dieta hiperlipídica levou ao aumento da expressão de marcadores inflamatórios e diminuição de autofagia. Além disso, o tratamento intracerebroventricular com ácido esteárico parece ser capaz de diminuir a autofagia em hipotálamo, sugerindo que ácidos graxos saturados podem ser os responsáveis pela diminuição da autofagia encontrada nos animais obesos. Por fim, a indução de autofagia em animais obesos com rapamicina foi capaz de melhorar a homeostase da glicose e reverter parâmetros inflamatórios e metabólicos, sem alterar o peso e a ingestão alimentar dos animais. Entender os mecanismos celulares e moleculares destes processos é crucial para identificar novos alvos terapêuticos para a obesidade / Abstract: The mediobasal hypothalamus is known as the primary site which coordinates the balance between food ingestion and energy expenditure. In this context, diets with high content of saturated fatty acids are pointed as the main diet factor responsible for the development of obesity. Recent data have shown that autophagy, a process which regulates cellular homeostasis by degrading dysfunctional proteins and organelles, is crucial to maintain the functionality of hypothalamic AgRP and POMC neurons, which in turn are responsible for coordinate body energy homeostasis. In this study we evaluated the hypothalamic distribution and content of autophagy's machinery proteins in an animal model of diet-induced obesity at 8 or 16 weeks of high-fat diet and in response to intracerebroventricular injections of a saturated fatty acid. We demonstrate that chronic exposure to a high-fat diet can lead to an increased expression of inflammatory markers and downregulation of autophagy. Also, intracerebroventricular treatment with stearic acid appears to contribute to the decrease of hypothalamic autophagy, suggesting that saturated fatty acids can contribute to the downregulation of autophagy found in obese mice. Finally, induction of autophagy in obese mice with rapamycin was able to improved glucose homeostasis and reverse inflammatory and apoptosis markers, the main mechanisms involving dysregulation of energy balance related to hypothalamic neurons, while no weight loss was observed during the treatment / Mestrado / Metabolismo e Biologia Molecular / Mestra em Ciências da Nutrição e do Esporte e Metabolismo

La macro-autophagie est un processus de dégradation intracellulaire qui impliquela formation d'une vésicule à double membranes, l'autophagosome, permettantla séquestration des molécules à dégrader. La formation de l'autophagosome dé-bute par la nucléation de membranes isolées dans le cytosol. Elle se poursuit parune phase de croissance de la membrane en forme de coupe. Dans l'étape _nale,l'autophagosome se referme en fusionnant par ses bords pour former la vésicule.La morphologie de l'autophagosome évolue donc durant sa formation. Certainesétudes montrent que cela est dû à l'implication de machineries protéiques. Indé-pendamment, d'autres recherches mettent en avant les rôles joués par les propriétésphysiques de la membrane.Dans mon travail de thèse, j'ai cherché à montrer un couplage entre l'activité d'uneprotéine impliquée dans l'autophagie et les caractéristiques physiques de la membrane.Cette protéine, nommée GABARAP, est considérée comme le marqueurde l'autophagosome car elle est la seule protéine, avec ses homologues, à s'ancrerspécifiquement sur sa membrane lors de sa formation. En incorporant GABARAPdans des vésicules géantes unilamellaires micromanipulées, j'ai pu montrer unein_uence de la composition et de la courbure de la membrane sur la distribution,l'oligomérisation et la dynamique de la protéine. Ensuite, j'ai mesuré une diminutiondu module de courbure de la membrane lorsque la protéine y était ancrée.Cette dernière étude m'a amenée à développer un modèle permettant de prédire ladistribution de la protéine sur une membrane possédant deux régions de courburesdiérentes. Enfin, j'ai déterminé la nature de l'interaction en trans de GABARAP. / Macro-autophagy is an intracellular degradation process that involves a doublemembrane vesicle, the autophagosome, to engulf a cargo. Its formation starts withthe nucleation of isolated membrane in the cytosol. Then the membrane growsas a cup-shape around the cargo to finally fuse at its edge and enclose the molecules to be degraded. Thus, the autophagosome morphology evolves during itsformation. Studies show that protein machineries support such shape changes. Independently, other researches point membrane physical properties roles during itsrearrangement.During my PhD, I investigated the coupling between the activity of one autophagyprotein and membranes physical characteristics. This protein, GABARAP,is considered as the autophagosome marker, because, with its homologs, it is theonly protein to be specifically anchored to its membrane during all its formation.The reconstitution of GABARAP in micromanipulated giant unilamellar vesicles(GUVs) allowed me to study the interplay between membrane characteristics andprotein behaviours. In a first part, I showed that membrane composition and curvature trigger specific distribution, oligomerization and dynamic of GABARAP.Then I measured a decrease of the membrane bending modulus when the proteinwas anchored. This last result led me to propose a model that predicts proteinsdistribution on membranes with two regions of diferent curvatures. Finally, I determined the nature of GABARAP / GABARAP trans interaction.

No description available.

L'autophagie est l'une des voies de signalisation qui maintiennent l'homéostasie cellulaire en condition basale, mais aussi en réponse à un stress. Son rôle est essentiel pour assurer plusieurs fonctions physiologiques, et son altération est associée à de nombreuses maladies, parmi lesquelles le cancer, les maladies immunitaires et les maladies neurodégénératives. Un nombre croissant d'études a établi que la voie autophagique est finement contrôlée. Cependant, très peu est connu sur les mécanismes moléculaires assurant sa régulation mais la famille des E3-ligases joue un rôle primordiale. La Gigaxonine est un adaptateur de la famille des E3 ligases CUL3, qui spécifie les substrats pour leur ubiquitination et leur successive dégradation. Des mutations «perte de fonction» de la Gigaxonine causent la Neuropathie à Axones Géants (NAG), une maladie neurodégénérative sévère et fatale, qui impacte tout le système nerveux et provoque une agrégation anormale des Filaments Intermédiaires (FI) dans l'organisme entier. Grâce à la modélisation de la pathologie dans les cellules de patients et chez la souris, le laboratoire a pu mettre en avant le rôle crucial de la Gigaxonine dans la dégradation de la famille des FIs, à travers son activité d'ubiquitination.Au cours de ma thèse, j'ai étudié les mécanismes de neurodégénerescence de la NAG, et la possible altération de la voie autophagique.Pour cela, j'ai développé un nouveau modèle neuronal de la maladie, à partir de notre modèle murin NAG, qui reproduit la mort neuronale et l'agrégation des FIs retrouvées chez les patients. Pour étudier l'implication de l'autophagie dans la neurodégénérescence, j'ai évalué l'effet de la déplétion de la Gigaxonine sur la formation des autophagosomes, le flux autophagique, la fusion avec le lysosome et la dégradation. J’ai ainsi révélé un défaut dans la dynamique autophagique dans les neurones NAG -/-. Pour déchiffrer les mécanismes moléculaires sous-jacents, j'ai étudié l'effet de l'absence de la Gigaxonine sur différentes régulateurs de la voie. En utilisant des techniques complémentaires, j'ai montré que la Gigaxonine est essentielle pour le turn-over d’un interrupteur autophagique, à travers son activité d’E3-ligase.En conclusion, nous avons identifié un nouveau mécanisme moléculaire impliqué dans le contrôle des premières phases de l'autophagie. Non seulement ces résultats présentent une avancée significative dans le domaine de l'autophagie, ils contribuent également à la compréhension de son dysfonctionnement dans les maladies neurodégénératives, et pourraient générer une nouvelle cible pour une intervention thérapeutique chez l'homme. / The autophagic route is one of the signaling pathways that sustain cellular homeostasis in basal condition, but also in response to stress. It has been shown to be crucial for several physiological functions and its impairment is associated with many diseases, including cancer, immune and neurodegenerative diseases. While an expanding number of studies have shown that autophagic route is finely controlled, little is known about the molecular mechanisms ensuring its function, but a fundamental role is sustained by the family of E3 ligases. Gigaxonin is an adaptor of a Cul3-E3 ligase, which specifies the substrates for their ubiquitination and their subsequent degradation. “Loss of function” mutations in Gigaxonin cause Giant Axonal Neuropathy (GAN), a severe and fatal neurodegenerative disorder that impacts broadly the nervous system and cause an abnormal aggregation of Intermediate Filaments (IFs) through the body. Modeling the disease in patient’s cells and in mouse, the laboratory has demonstrated the crucial role of Gigaxonin in degrading the entire family of IFs through its ubiquitination activity.During my PhD, I studied the neurodegenerative mechanisms in GAN disease, and the possible impairment of autophagy pathway.For that purpose, I developed a new neuronal model of the disease from our GAN mouse, which reproduced the neurodegeneration and the IF aggregation found in patients. To investigate the involvement of autophagy in neurodegeneration, I evaluated the effect of Gigaxonin depletion on autophagosome formation, autophagic flux, lysosome fusion and degradation, and I revealed a defect in autophagy dynamics. To decipher the molecular mechanism of autophagosome impairment, I investigated the effect of Gigaxonin depletion on different autophagy regulators. Using complementary techniques, I showed that Gigaxonin is essential for the turn-over of a specific molecular switch, through its E3 ligase activity.Altogether, we identified a new exciting molecular mechanism in the control of autophagy. Not only these findings present a significant advance in the comprehension of the fundamental field of autophagy, but it also contribute in the understanding of its dysfunction in neurodegenerative diseases, and may generate a new target for therapeutic intervention in humans.

ULK1 (UNC-51 like kinase 1) is a serine/threonine protein kinase that has been shown to play a crucial role in autophagy, a process of self digestion implicated in maintaining cellular homeostasis and in mediating type II programmed cell death. However, the exact mechanism by which ULK1 controls autophagy remains elusive, mostly because none of the known ULK1 targets have been directly linked to autophagy. To address this issue, I have employed a protein microarray screening approach to identify novel ULK1 substrates. I found five putative targets: MERTK (proto-oncogene tyrosine-protein kinase MER), B-RAF (v-raf murine sarcoma viral oncogene homologue B1), NOL4 (nucleolar protein 4), TBC1D22B (TBC1 domain family member 22B) and ACVRL1 (activin A receptor type II-like 1). My preliminary experiments have not confirmed that MERTK or B-RAF can be phosphorylated by ULK1 in vitro. However, further investigation will be required to firmly rule out MERTK and B-RAF as downstream targets of ULK1 and to test the ability of ULK1 to phosphorylate the other candidates. In addition, I have identified by in-gel kinase assay a ULK1 kinase at 34-kDa whose ability to phosphorylate the kinase domain of ULK1 was increased upon starvation. Using the genome information, I predicted this upstream kinase to be Pim1 (Proto-oncogene serine/threonine-protein kinase pim-1). I confirmed that Pim1 phosphorylated ULK1 in vitro at S147 and S224. Results of site directed mutagenesis suggest that phosphorylation at S224 correlates with increased ULK1 activity. This is consistent with observation that Pim1 is capable of activating ULK1 in vitro. Furthermore, I present preliminary data suggesting that Pim1 promotes autophagy in HeLa cells.

Francisella tularensis is the causative agent of tularemia and is categorized by the CDC as a Tier 1 select agent. This gram-negative, facultative-intracellular bacterium infects macrophages by escaping the phagosome and replicating with high efficacy in the cytosol. Multiple virulence factors, including capsule and lipopolysaccharide (LPS), are expressed by F. tularensis. Biosynthesis of capsule and LPS O-antigen requires the same O-antigen biosynthesis gene cluster and, together, expression of capsule and O-antigen confer serum resistance. Mutations in the O-antigen biosynthesis gene cluster not only result in serum sensitivity, but also attenuate the ability to cause disease in vivo. In addition to changes in F. tularensis virulence, individual capsule and O-antigen mutants appear to have distinct intracellular phenotypes in macrophages. As previously shown by Lindemann et al. (2011), the capsule and O-antigen mutants FTT1236, FTT1237, and FTT1238 all replicated in human monocyte derived macrophages (MDMs) up to 16 hr and then ceased to replicate after that. This is hypothesized to be due to MDM cytotoxicity. In contrast, Raynaud et al. (2007) showed that the capsule and O-antigen mutant wbtA completely lacked replication in J774 macrophages, the reason for which has not been identified. A potential explanation for the loss of F. tularensis capsule and O-antigen mutant replication is capture and degradation by the host cell's autophagy pathway. Capture and degradation by autophagy is an accepted innate immune response to many intracellular pathogens. When small subpopulations of bacteria that normally replicate in membrane-bound vacuoles become cytosolic, such as Mycobacterium tuberculosis and Salmonella enterica serovar Typhimurium, they are targeted to forming autophagosomes through ubiquitination and binding of autophagy receptors. Pathogens have also developed methods to circumvent recognition and degradation by autophagy. Since F. tularensis replicates in the cytosol, it stands to reason that it has a means of evading detection by autophagy. We propose that expression of capsule and O-antigen acts as a mechanism used by F. tularensis to protect itself in an extracellular environment, as well as during intracellular infection. In this thesis we characterized nine different capsule and O-antigen mutants, and found different replication phenotypes in MDMs and varying degrees of MDM cytotoxicity. Also, only a subset of the mutants was detected by the autophagy marker, ubiquitin, supporting our hypothesis that different capsule and O-antigen mutants have diverse fates in MDMs. We also show that LVS and Schu S4 wbtA mutants had similar phenotypes. Upon further evaluation, we found that LVS wbtA more readily colocalized with ubiquitin, autophagy receptors, and the autophagy membrane protein LC3B, but not Beclin-1 or LAMP-1. This supports our hypothesis that capsule and O-antigen mutants are more susceptible to recognition by autophagy. Yet, because we did not observe LAMP-1 colocalization, there may be defects in the maturation of autophagosomes to degradative autolysosomes. Finally, we found that the fate of LVS wbtA in MDMs is dissimilar from J774 macrophages, suggesting macrophage species affect mutant fate. This thesis shows that different capsule and O-antigen mutants have multiple fates in MDMs, and suggest that F. tularensis capsule and O-antigen act as protective virulence factors that limit detection by autophagy.

archives@tulane.edu / Alcoholic and nonalcoholic fatty liver disease is projected to be the most common cause of liver disease in developing countries. The main significant risk factors are obesity, diabetes mellitus type 2, cardiovascular disease, and dyslipidemia. Louisiana is ranked seventh in liver cancer diagnoses and ranked sixth in the leading cause of death. Recent findings indicated that multifaceted stress response due to the accumulation of fatty acids from the diet is the driving force of disease progression. We sought to study multifaceted integrated stress response (ISR) in liver cells cultured with saturated fatty acids. Understanding the process that ISR takes to either induce or inhibit autophagy, self-eating machinery, in strongly permissive HUH 7.5 cells is vital when treating liver abnormalities. The major protein kinase, P-EIF2 alpha, was the targeted factor contributing the most to autophagy due to its functional link to the endoplasmic reticulum, mitochondria, and cellular membrane by further assessment using the inductive drug, Sephin 1. HUH, 7.5 liver cells are treated with increasing amounts of palmitic acid for 24 hours in DMEM with 10% FBS. ISR activated after substantial cellular damage leading to autophagy impairment. The cell culture was assessed for lipid accumulation, and the expression of PKR, IRE1 alpha, PERK, ATF6, P-EIF2 alpha, HRI, MTORC1, GCN2, P62, and LC3B was achieved by immunoblot analysis. Membrane fluidity PKR, lysosomal MTORC1, and protein synthesis GCN2 activated to elicit an integral response to the ISR pathway. Endoplasmic reticulum protein kinases induced in response to UPR activation lead to an integration of the P-EIF2 alpha pathway. Mitochondrial stress heme regulated inhibitor proliferated to provoke an activation in the significant protein kinase leading to autophagy impairment. The P-EIF2 alpha kinase invoked autophagic deficiency even when dephosphorylation was prevented by Sephin 1 drug treatment. ISR constrained autophagy in the liver-derived cell line due to the accumulation of the toxic saturated fatty acid. Keywords: palmitate, autophagy, fatty liver disease, integrated stress response, Sephin 1 / 1 / Glory Ogunyinka