Tumor cells often hijack endoplasmic reticulum (ER) mediated signaling to facilitate tumor progression by adapting to the cellular stress evoked by oncogene overexpression and adverse microenvironment. Despite the prevalence of MYC-driven cancers, how the MYC oncoprotein regulates ER stress response pathways during tumorigenesis remains incompletely understood. Here we show that MYC drives continuous upregulation of ubiquitin fusion degradation 1 (UFD1) during T-cell acute lymphoblastic leukemia (T-ALL) development. As the E2 component of an ER-associated degradation (ERAD) complex, UFD1 facilitates the elimination of misfolded/unfolded proteins from the ER. We found that genetic and pharmacological disruption of UFD1 function exacerbates ER stress and activates the unfolded protein response (UPR). Specifically, UFD1 knockdown in human T-ALL cells impairs ERAD and promotes the proapoptotic UPR through the PERK-CHOP-BCL2 axis. This effect is demonstrated by an upregulation of PERK, phospho-PERK and its downstream effector CHOP, as well as a downregulation of BCL2 and BCLxL. Indeed, CHOP inactivation or BCL2 overexpression is sufficient to rescue tumor-cell apoptosis induced by UFD1 knockdown. Allelic loss of ufd1 in zebrafish similarly induces tumor-cell apoptosis and impairs MYC-driven T-ALL progression without affecting general animal health. These studies establish the UFD1-mediated ER stress response as an important mediator of MYC-driven tumor progression and suggest strategies for targeted therapy in T-ALL, and perhaps other MYC-driven cancers. Although UFD1-specific inhibitors have yet to be developed, inhibitors that target the p97 co-factor in UFD1-mediated ERAD are readily available. Importantly, we show that treatment with CB-5083, a selective and oral bioactive inhibitor of p97, can effectively kill human MYC-overexpressing T-ALL patient cells ex vivo and inhibits tumor progression in zebrafish models of MYC-driven T-ALL. Thus, CB-5083 treatment may represent an effective targeted therapy for T-ALL, especially relapsed/refractory ones with gain-of-function NOTCH1 mutations and thus MYC-overexpression.

A imunodeficiência comum variável (CVID) é caracterizada por hipogamaglobulinemia. Anteriormente identificou-se uma paciente com CVID que apresenta nível aumentado de estresse de retículo endoplasmático (ER), secundário a desregulação da via UPR. No presente trabalho, estendemos esta análise para outros pacientes e avaliamos o perfil de maturação de seus linfócitos B. Métodos: Western-blot, RT-PCR, Q-PCR, Citometria de Fluxo e cultura de células B ex vivo e imortalizadas. Resultados: A análise de 16 pacientes com CVID e 9 indivíduos saudáveis revelou três pacientes com porcentagens aumentadas de linfócitos B imaturos no sangue periférico. A análise da expressão de RNAm para BiP e XBP-1 em linfócitos B destes pacientes, após estímulo com LPS in vitro, identificou que os linfócitos B de um deles apresenta estresse de RE. Conclusão: Identificamos um subgrupo de pacientes com CVID que apresentam linfócitos B imaturos no sangue periférico. Um membro deste subgrupo apresenta estresse aumentado de ER. / Common Variable Immunodeficiency (CVID) is characterized by hypogammaglobulinemia. Previously a CVID patient was identified with increased levels of Endoplasmic Reticulum (ER) stress due to dysregulation of the UPR. In the present study these analyses were performed in other patients and healthy donors. Maturation markers of B lymphocytes were also characterized in these individuals. Methods: Western-blot, RT-PCR, Q-PCR, Flow cytometry and culturing of ex vivo and immortalized B cells. Results: The analysis of 16 CVID patients and 9 healthy donors revealed three patients that present higher percentage of immature B cells in peripheral blood. Analysis of expression of BiP and XBP1 induced by LPS treatment of B lymphocytes from these patients revealed that one patient present increased levels of ER stress.

Les bactéries intracellulaires Wolbachia ont développé une vaste gamme d’interactions symbiotiques, du parasitisme reproductif au mutualisme chez les arthropodes terrestres et les nématodes filaires, devenant ainsi les endosymbiotes les plus répandus sur terre. Bien qu’elles se développent lentement dans les cultures cellulaires d’insectes pour lesquelles les marqueurs sont limités et qu’elles ne sont génétiquement pas manipulables, il existe un intéret croissant de déchiffrer leur mode de vie intracellulaire pour 2 raisons. Premièrement, Wolbachia intervient dans le développement et la transmission des arbovirus et deuxièmement, les filarioses lymphatiques sont traitables grâce à la susceptibilité des Wolbachia qui infectent les nématodes filaires aux antibiotiques. Au début de ce projet, j’ai infecté 2 lignées cellulaires de Drosophila melanogaster qui sont transcriptomiquement divergentes par une même souche de Wolbachia pouvant naturellement infecter Drosophila melanogaster. J’ai utilisé ces 2 lignées cellulaires qui sont différentiellement permissive à l’infection pour explorer l’interaction de Wolbachia avec le réticulum endoplasmique. Les observations par microscopie à fluorescence en temps réel et par microscopie électronique prouvent que cet organite est une source de membranes pour Wolbachia et possiblement, une source de nutriments. Pourtant, les analyses d’expression génique et les approches d’immunofluorescence démontrent que Wolbachia n’induit ni un stress au niveau du réticulum endoplasmique ni une protéolyse via la voie de signalisation ERAD suggérant dès lors, que Wolbachia subvertissent d’autres mécanismes pour assurer leur besoin en acides aminés. Au cours de ce projet, j’ai commencé à mettre en place une technique pour transformer Wolbachia par biolistique. La validation de cette technique de transformation a ouvert la voie vers l’optimisation de la procédure de sélection des transformants pour enfin pouvoir génétiquement manipuler Wolbachia. / The intracellular bacteria Wolbachia have developed a wide range of symbiotic interactions, from being opportunistic reproductive parasites to mutualists with terrestrial arthropods and filarial nematode species, making them the most common endosymbionts on earth. The discovery that they interfere with arboviruses development and transmission by mosquito vectors and that filarial diseases can be cured by targeting Wolbachia, have created a strong interest in deciphering the mechanisms underlying their intracellular lifestyle. However, being obligate intracellular endosymbionts, Wolbachia remain genetically intractable. They grow slowly in insect cell cultures, for which markers are limited. Despite these obstacles, and to limit cell line-specific phenotypes, I chose to infect 2 Drosophila melanogaster cell lines presenting different sets of expressed genes, with a unique Wolbachia strain, naturally hosted by Drosophila melanogaster. Using these 2 cell lines that are differently permissive to the infection, I explored the interaction of Wolbachia with the endoplasmic reticulum (ER). Through fluorescence time-lapse confocal and electron microscopy observations, I provide strong evidence that this organelle is the source of membrane for Wolbachia, and possibly a source of nutrients. However, gene expression analyses and immunofluorescence approaches demonstrate that Wolbachia do not induce ER stress nor an increased ERAD- induced proteolysis, suggesting; unlike previously reported, that Wolbachia salvage amino acids by other subversion mechanisms. Additionally, I pioneered biolistic bombardement of Wolbachia-infected cells and the validation of this transformation technique has paved the way towards optimization of transformant selection steps and ultimately to the genetic engineering of Wolbachia.

Organelles sequester biological phenomena within the cell, and allow an additional layer of complexity to life. The presence and maintenance of these organelles is crucial for cellular function. Two of the most expansive and complex organelles are the mitochondria and endoplasmic reticulum. These organelles contribute energy, protein folding and secretion, lipids, calcium regulation, and various other metabolites to the biology of the cell. Importantly, these organelles accumulate damage and cannot be derived de novo, therefore must be inherited and maintained in a functioning state. The study of these organelle quality control processes serves as the basis for my thesis. We use the budding yeast as a model organism to uncover conserved pathways affecting organelle, and ultimately cellular homeostasis. In yeast we find mitochondrial inheritance is critical for cell survival. Furthermore, not only is inheritance critical, but inheritance of a certain threshold of functional mitochondria appears critical in maintaining normal lifespan in yeast, identifying mitochondria as an aging determinant. By examining mutants that negatively affect mitochondrial inheritance in yeast, we established a role for phosphatidylcholine biosynthesis in organelle maintenance and inheritance. Glycerophospholipid biosynthesis plays a clear role not only in mitochondrial inheritance but also in that of the endoplasmic reticulum. We use insights gained from yeast to guide research into a human disease caused by similar glycerophospholipid biosynthetic deficiency.

In eukaryotic cells, proteins continuously flux through the organelles of the secretory pathway in an essential cellular process called protein secretion. This dynamic process originates at the endoplasmic reticulum (ER), where translating ribosomes push linear peptides into the ER membrane and lumen. ER chaperones assist in folding nascent peptides into three-dimensional conformations and proteins are concentrated into membrane-encapsulated vesicles bound for the Golgi apparatus. ER to Golgi transport is mediated by a set of cytosolic coat proteins called COPII. The COPII coat polymerizes into a lattice on the ER membrane that is able to bend the membrane around secretory cargo and bud off a spherical vesicle. Protein secretion is subject to rapid changes as a cell responds to its environment and requirements for viability alter. In addition to accommodating short-term demands, such as translational up-regulation, evolved complexity of secretory proteins over time, has also required that secretory components adapt. In both cases changes in secretory demands require that the COPII proteins have an inherent flexibility to navigate these changes without disrupting secretory flux. In this work I have examined a family of quintessential secretory cargo, p24 proteins, that challenge protein secretion. This family of proteins forms a hetero-tetrameric complex that cycles between the ER and the Golgi and mediates transport of glycosylphosphatidylinositol-anchored proteins (GPI-APs). Here I present evidence that suggests, when present in vesicles, both p24 proteins and their GPI-AP cargo present a challenge to vesicle formation. I posit that three attributes of these proteins present a local barrier to membrane bending: Lumenal asymmetric distribution across the membrane, high cellular abundance and affinity for ceramide rich membranes. I have also elucidated mechanisms that the coat has evolved to accommodate troublesome cargo such as p24 proteins, which enhance structural scaffolding and increase average vesicle size. Finally I present preliminary findings indicating that p24s also contribute to ER homeostasis by preventing aberrant incorporation of proteins into vesicles. Comprehensively, these findings have shed light on the role of p24 proteins in vesicles. Traditionally thought to be canonical ER cargo receptors, these proteins also appear capable of contributing to the composition of the vesicles in which they reside, and impacting trafficking efficiency in two ways: First by directly mediating transport of GPI-APs and second by uniformly packing vesicles to avoid wasteful secretion. My work has contributed to a growing notion in the field that secretory cargo are not inert passengers but active participants in vesicle mediated secretion.

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The unfolded protein response (UPR) is a cellular mechanism whose primary functions are to sense perturbations in the protein-folding capacity of the endoplasmic reticulum and to take corrective steps to restore homeostasis. Although the UPR is conserved across all eukaryotic cells, it is considerably more complex in mammalian cells, due to the presence of three interconnected pathways triggered by separate sensor proteins, a translation attenuation mechanism, and a negative feedback loop. The mechanisms of these interacting biochemical pathways in the mammalian UPR allow for a better fine-tuning of the response than in the case of lower eukaryotes, such as yeasts. The present thesis develops a quantitative mathematical model for the dynamics of the UPR in mammalian cells, which incorporates all the proteins and interactions between them that are known to play a role in this response. This model can be used to provide quantitative information about the levels of its components throughout the response, and to analyze the ramifications of perturbations of the UPR. The model uses a system of ordinary nonlinear differential equations based on biochemical rate equations to describe the dynamics of the UPR as a network of interacting proteins and mRNAs. An early model is presented as a first attempt to investigate the UPR network and construct an inclusive wiring diagram, as well as suggesting a framework to model the differential equations. Then, a refined, quantitative model is designed based on experimental data collected on Mouse Embryonic Fibroblasts treated with Thapsigargin to induce stress and trigger the UPR. The model defines the differential equations and determines the unknown kinetic parameters by optimizing the fit of the system's solution to the experimental data. It includes the UPR's intrinsic feedback loops and allows for the integration of various forms of external stress signals. To the best of our knowledge, it is the first, data-validated, quantitative model in the literature for the UPR in mammalian cells. The last chapters of the thesis address, from a modeling point of view, two important questions for the UPR: (1) cell survival versus apoptosis; and (2) incompleteness of the biological wiring diagram. Recent experimental results show that the UPR is capable of producing qualitatively different results leading to cell survival or death depending on the nature, strength, and persistence of the inducing stress. This thesis proposes several approaches by which the equations can be modified to model the transition from adaptation to apoptosis as a dynamic switch, while taking into account the various hypotheses of cell death mechanisms. Finally, we use recently-developed computational algebra techniques to infer an optimal structure of the UPR network, based solely on the experimental data; the resulting wiring diagram provides insights on elements of the structure of the model that may have been overlooked during the classical (mechanistic) approach to our original data-based model.

No description available.

Thesis (M. Phil.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

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