Thesis (Ph. D.)--Indiana University, 2008. / Title from screen (viewed June 24, 2009). Department of Biochemistry and Molecular Biology, Indiana University-Purdue University Indianapolis (IUPUI). Advisor(s): Maureen Harrington. Includes vita. Non-Latin script record. Includes bibliographical references (leaves 126-132).

Le trafic intracellulaire est un processus fondamental qui maintient l'homéostasie cellulaire. Les RAB GTPases sont des régulateurs clés du trafic intracellulaire. RAB6 est la RAB résidente la plus abondante du Golgi. RAB6 est un régulateur clé de l'homéostasie Golgienne. Mon projet de thèse s'est intéressé à l'étude de la fonction de RAB6 dans la sécrétion post-Golgienne. Des études précédentes ont montré que la déplétion de RAB6 inhibe l'arrivée à la membrane plasmique de différents cargos : dans des cellules HeLa, NPY et VSV-G, et TNFα dans les macrophages. Nous avons donc émis l'hypothèse que RAB6 pourrait être un régulateur général de la sécrétion post-Golgienne. A l'aide de cellules MEFs RAB6 KO, nous avons d'abord montré que la sécrétion de toutes les protéines nouvellement synthétisées est inhibée. Pour comprendre les mécanismes entraînant cet effet, nous avons étudié le rôle de RAB6 dans le transport post-Golgien de trois types différents de cargos : GPI-APs (PLAP et CD59), collagen X, une protéine soluble, et une protéine transmembranaire TNFα. Afin de synchroniser le transport de cargos, nous avons utilisé le système RUSH. Ainsi, nous avons montré que RAB6 est présent sur les vésicules post-Golgiennes contenant les 3 types de cargos et que la déplétion de RAB6 affecte leur sécrétion. Les effecteurs de RAB6 sont aussi impliqués: Myosine II dans leur fission du Golgi, KIF5B dans leur transport vers la périphérie cellulaire, ELKS dans leur arrimage à la membrane plasmique. Finalement, nous avons pu montrer que les 3 cargos sont présents dans les mêmes vésicules post-Golgiennes avec RAB6. Ces résultats montrent que RAB6 régule la sécrétion de différents cargos. / Intracellular trafficking is a fundamental process which ensures cell homeostasis. RAB GTPases are key regulators of intracellular trafficking. RAB6 is the most abundant Golgi resident RAB and is a key regulator of Golgi homeostasis. My Ph.D project focused on understanding the function of RAB6 in post-Golgi secretion.Previous reports have shown that RAB6 depletion impairs the arrival at the plasma membrane of different cargoes: in HeLa cells, NPY and VSV-G and TNFα in macrophages. We thus hypothesized that RAB6 could be a general regulator of post-Golgi transport steps. Using MEF cells from RAB6 KO mice, we first showed that the secretion of all newly synthesized proteins is affected. To decipher the mechanisms leading to this inhibition, we have then investigated the role of RAB6 in the post-Golgi transport of three different classes of proteins, GPI-anchored proteins (such as Placental Alkaline phosphatase or PLAP and CD59), collagen X, a soluble protein, and the transmembrane protein TNFα. In order to synchronize transport of newly-synthetized cargoes along the secretory pathway, we used the RUSH system. Here, we show that RAB6 is present on post Golgi vesicles containing the three types of cargo and that RAB6 depletion affects their secretion to the plasma membrane. RAB6 effectors are also implicated: Myosin II for their fission from the Golgi, KIF5B for their transport to the cell periphery, ELKS/RAB2IP2 for their docking with the plasma membrane. Finally, we could show that these three cargoes are present in the same post-Golgi transport carriers with RAB6. Altogether, these results show that RAB6 regulates the secretion of a wide number of cargo proteins.

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Indiana University-Purdue University Indianapolis (IUPUI) / The innate and adaptive immune responses are critical for host survival. The TNFα/NF-κB signaling pathway is a major regulator of the immune response. The TNFα/NF-κB signaling pathway has also been proposed to play a role in the regulation of hematopoiesis. In the TNFα signaling pathway, full induction of NF-κB (specifically the p65 subunit) dependent transcription is regulated by a co-activator SIMPL. The biological significance of SIMPL in TNFα dependent responses is poorly understood. To study SIMPL in vitro and in vivo in mammalian cells, a knockdown system utilizing shRNA (short hairpin RNA) was used. Analysis of hematopoietic progenitor cells infected with a retrovirus encoding the SIMPL shRNA was used to study the role of SIMPL in hematopoiesis. The ability of progenitor cells lacking SIMPL to grow and differentiate was not compromised. In contrast in the progenitors cells lacking SIMPL, TNFα mediated inhibition of colony formation was significantly enhanced. These growth inhibitory effects of SIMPL were not due to an increase in apoptosis. The enhanced inhibitory affects were specific for TNFα and not found in other common hematopoietic inhibitors (TGF-β1 and IFNγ). Results of this work reveal that SIMPL is a component of the hematopoiesis that is required for TNFα dependent effects upon myeloid progenitors.

Adipose tissue (AT) expansion and remodeling that maintains healthy function relies on stromal preadipocytes capable of differentiating into new adipocytes (adipogenesis). During chronic positive energy balance, a relative deficit in adipogenesis, from either a decrease in preadipocyte number or their capacity to differentiate, leads to excessive adipocyte hypertrophy and AT dysfunction. AT contains macrophages whose number and activation state is dynamically regulated with changes in AT mass. This study aims to investigate the effect of macrophage-secreted factors on preadipocyte survival. To assess the effect of macrophage-secreted factors on preadipocytes, murine 3T3-L1 preadipocytes or human primary preadipocytes were incubated with macrophage-conditioned medium (MacCM), prepared from either murine (J774A.1, RAW264.7, bone marrow-derived) or human (THP-1, monocyte-derived) macrophage models, respectively. MacCM inhibited preadipocyte apoptosis and activated pro-survival signaling in both preadipocyte models. Inhibition of PDGFR, Akt, or ERK1/2 reduced the pro-survival effect of MacCM in 3T3-L1 preadipocytes. Inhibition of reactive oxygen species (ROS) generation, or enhancement of ROS clearance, reduced MacCM-dependent 3T3-L1 preadipocyte survival. Whereas anti-inflammatory activated macrophages retained the ability to prevent preadipocyte apoptosis, pro-inflammatory activated macrophages did not. TNF-α immunoneutralization restored the survival activity of pro-inflammatory MacCM on 3T3-L1 preadipocytes. These studies reveal a novel pro-survival effect of MacCM on preadipocytes, and identify signaling molecules (PDGF, Akt, ERK1/2, and ROS) that underlie this action. Macrophage activation was found to regulate the pro-survival activity of MacCM. These in vitro cell culture studies are consistent with a model in which the extent of preadipocyte apoptosis in vivo may determine preadipocyte number and the ability of AT to expand while maintaining healthy function during chronic positive energy balance.

Adipose tissue (AT) expansion and remodeling that maintains healthy function relies on stromal preadipocytes capable of differentiating into new adipocytes (adipogenesis). During chronic positive energy balance, a relative deficit in adipogenesis, from either a decrease in preadipocyte number or their capacity to differentiate, leads to excessive adipocyte hypertrophy and AT dysfunction. AT contains macrophages whose number and activation state is dynamically regulated with changes in AT mass. This study aims to investigate the effect of macrophage-secreted factors on preadipocyte survival. To assess the effect of macrophage-secreted factors on preadipocytes, murine 3T3-L1 preadipocytes or human primary preadipocytes were incubated with macrophage-conditioned medium (MacCM), prepared from either murine (J774A.1, RAW264.7, bone marrow-derived) or human (THP-1, monocyte-derived) macrophage models, respectively. MacCM inhibited preadipocyte apoptosis and activated pro-survival signaling in both preadipocyte models. Inhibition of PDGFR, Akt, or ERK1/2 reduced the pro-survival effect of MacCM in 3T3-L1 preadipocytes. Inhibition of reactive oxygen species (ROS) generation, or enhancement of ROS clearance, reduced MacCM-dependent 3T3-L1 preadipocyte survival. Whereas anti-inflammatory activated macrophages retained the ability to prevent preadipocyte apoptosis, pro-inflammatory activated macrophages did not. TNF-α immunoneutralization restored the survival activity of pro-inflammatory MacCM on 3T3-L1 preadipocytes. These studies reveal a novel pro-survival effect of MacCM on preadipocytes, and identify signaling molecules (PDGF, Akt, ERK1/2, and ROS) that underlie this action. Macrophage activation was found to regulate the pro-survival activity of MacCM. These in vitro cell culture studies are consistent with a model in which the extent of preadipocyte apoptosis in vivo may determine preadipocyte number and the ability of AT to expand while maintaining healthy function during chronic positive energy balance.

Adipose tissue (AT) expansion and remodeling that maintains healthy function relies on stromal preadipocytes capable of differentiating into new adipocytes (adipogenesis). During chronic positive energy balance, a relative deficit in adipogenesis, from either a decrease in preadipocyte number or their capacity to differentiate, leads to excessive adipocyte hypertrophy and AT dysfunction. AT contains macrophages whose number and activation state is dynamically regulated with changes in AT mass. This study aims to investigate the effect of macrophage-secreted factors on preadipocyte survival. To assess the effect of macrophage-secreted factors on preadipocytes, murine 3T3-L1 preadipocytes or human primary preadipocytes were incubated with macrophage-conditioned medium (MacCM), prepared from either murine (J774A.1, RAW264.7, bone marrow-derived) or human (THP-1, monocyte-derived) macrophage models, respectively. MacCM inhibited preadipocyte apoptosis and activated pro-survival signaling in both preadipocyte models. Inhibition of PDGFR, Akt, or ERK1/2 reduced the pro-survival effect of MacCM in 3T3-L1 preadipocytes. Inhibition of reactive oxygen species (ROS) generation, or enhancement of ROS clearance, reduced MacCM-dependent 3T3-L1 preadipocyte survival. Whereas anti-inflammatory activated macrophages retained the ability to prevent preadipocyte apoptosis, pro-inflammatory activated macrophages did not. TNF-α immunoneutralization restored the survival activity of pro-inflammatory MacCM on 3T3-L1 preadipocytes. These studies reveal a novel pro-survival effect of MacCM on preadipocytes, and identify signaling molecules (PDGF, Akt, ERK1/2, and ROS) that underlie this action. Macrophage activation was found to regulate the pro-survival activity of MacCM. These in vitro cell culture studies are consistent with a model in which the extent of preadipocyte apoptosis in vivo may determine preadipocyte number and the ability of AT to expand while maintaining healthy function during chronic positive energy balance.

The tumor necrosis factor (TNF) receptor-associated factors (TRAFs) were initially discovered as proteins that inducibly interact with the intracellular region of TNF receptors (TNFRs). Because the TNFRs lack intrinsic catalytic activity, the TRAFs are hypothesized to orchestrate signaling activation downstream of the TNFR superfamily, however their mechanism of activation remains unclear (Inoue et al., 2000; Bishop, 2004). Originally, the TRAFs were compared to the signal transducers and activators of transcription (STAT) protein family, due to their sequence homology, and the presence of multiple RING- and zinc-finger domains, suggesting that their function may be to regulate transcriptional activity (Rothe et al., 1994; Hu et al., 1994; Sato et al. 1995; Cheng et al., 1995). However, subsequent research focused predominantly on their cytoplasmic functions, and more recently on their function as E3 ubiquitin ligases (Pineda et al., 2007). In my research, I analyzed the subcellular localizations of the TRAFs following CD40 ligand (CD40L)-stimulation, and found that TRAF2 and 3 rapidly translocate into the nucleus of primary neurons and Neuro2a cells. Interestingly, similar analysis conducted in pre-B lymphocytes (Daudi cells) revealed a different response to CD40L-stimulation, with TRAF2 and 3 being rapidly degraded within 5-minutes of stimulation. These findings are significant because they demonstrate for the first time that the TRAFs translocate into the nucleus and suggest that they may function within the nucleus in a cell-specific manner. I next analyzed the ability of TRAF2 and 3 to bind to DNA, and found that they both bind to chromatin and the NF-kappaB consensus element in Neuro2a cells, following CD40L-stimulation. Similar analyses of the chromatin binding of TRAF2 and 3 in Daudi cells revealed that they were rapidly degraded, similar to the results from my analysis of their subcellular localization. These findings show for the first time that the TRAFs interact with DNA, and therefore support the hypothesis that the TRAFs may function within the nucleus as transcriptional regulators. Finally, I analyzed the ability of the TRAFs to regulate transcriptional activity by luciferase assay. Previous studies showed that overexpression of TRAF2 and 6 could induce NF-kappaB transcriptional activity; however researchers have not been able to determine the mechanism by which they do so. In my studies, I found that every TRAF can directly regulate transcriptional activity either as co-activators or co-repressors of transcription, in a cell- and target protein-specific manner. Additionally, I found that TRAF2 can act as a transcriptional activator, and that its ability to regulate transcription is largely dependent upon the presence of its RING-finger domain. In conclusion, these studies have revealed an entirely novel function for the TRAFs as immediate-early transcriptional regulators. Future research into the genes that are regulated by the specific TRAF complexes will further elucidate how the TRAFs regulate TNFR signaling, as well as whether dysfunctions in TRAF signaling may be associated with known disorders. If specific TRAF complexes are found to regulate specific genes, then pharmacological targeting of the individual TRAF complexes may allow for the highly specific inhibition of signaling events downstream of the TNFRs, without compromising overall receptor signaling, transcription factor pathways, or cellular systems.

Das Proteasomensystem der Zelle ist für die Degradation von Proteinen verantwortlich und spielt eine zentrale Rolle bei der Generierung von Epitopen, die auf MHC-Klasse-I Molekülen den cytotoxischen T-Lymphozyten (CTLs) präsentiert werden. Die Stimulation von Zellen mit Interferon-gamma (IFNgamma) führt zu der Bildung von Immunoproteasomen, die im Vergleich zu den konstitutiven Proteasomen eine verbesserte Generierung vieler MHC-Klasse-I Epitope aufweisen. In gesunden Mäusen werden Immunoproteasomen vorwiegend in den lymphatischen Geweben exprimiert, wohingegen nicht-lymphatische Gewebe hauptsächlich konstitutive Proteasomen enthalten. In der vorliegenden Arbeit wurde der Einfluss der Listeria monocytogenes Infektion auf die aus der Leber, der Milz, dem Dünndarm und dem Colon stammenden murinen 20S Proteasomen untersucht. Die Struktur der isolierten 20S Proteasomen wurde mittels zweidimensionaler Gelelektrophorese und Westernblot ermittelt, während die Funktion durch in vitro Prozessierung von drei oligomeren Peptidsubstraten analysiert wurde. Die Prozessierungsprodukte wurden mittels HPLC-ESI-Ionenfalle massenspektrometrisch identifiziert sowie quantifiziert. Die vorliegende Arbeit zeigt zum ersten Mal, dass nach einer Infektion die aus den nicht-lymphatischen Organen und Zellen isolierten 20S Proteasomen eine strukturelle und funktionelle Plastizität aufweisen: Nach der Infektion wurde die Bildung von Immunoproteasomen induziert, was mit der gesteigerten Generierung der immunrelevanten Fragmente korreliert werden konnte. Dies verlief unabhängig von der direkten Präsenz von Listeria monocytogenes in den Organen und wurde ausschließlich durch das Cytokin IFNgamma reguliert. Es konnte außerdem eine Zunahme der posttranslationalen Modifikation von Leberproteasomen mit dem Monosaccharid N-Acetylglucosamin nach der Infektion nachgewiesen werden. Des Weiteren wurde eine detaillierte Analyse der massenspektrometrischen Daten hinsichtlich des Schnittverhaltens der konstitutiven und Immunoproteasomen etabliert. Die Auswertung ergab, dass die Immunoproteasomen nach der Infektion durch schnellere und veränderte Nutzung bestehender Spaltstellen an der verbesserten Epitoppräsentation beteiligt sind. / The proteasome system of the cell is responsible for the degradation of proteins and plays a central role in the generation of epitopes which are presented to cytotoxic T-lymphocytes (CTLs) on MHC-class-I molecules. The stimulation of cells by interferon-gamma (IFNgamma) leads to the formation of immunoproteasomes that show an improved generation of many MHC-class-I epitopes compared to constitutive proteasomes. In healthy mice, immunoproteasomes are mainly expressed in the lymphatic tissues, whereas the non-lymphatic organs predominantly contain constitutive proteasomes. In this project the effect of Listeria monocytogenes infection on murine 20S proteasomes derived from the liver, spleen, small intestine and colon were investigated. The structure of the isolated proteasomes was analyzed by two-dimensional gel electrophoresis and western blots while the function was studied by in vitro processing of three oligomeric peptide substrates. Identification and quantification of the processing products was performed by HPLC-ESI-ion trap mass spectrometry. The project showed for the first time, that after infection 20S proteasomes isolated from non-lymphatic organs as well as from non-lymphatic cells displayed structural and functional plasticity: immunoproteasomes were induced post infection which could be correlated with the enhanced generation of immuno-relevant fragments. This was independent of the direct presence of Listeria monocytogenes in the organs and solely controlled by the cytokine IFNgamma. In addition, an increased posttranslational modification with the monosaccharide N-acetylglucosamine could be detected in liver-derived proteasomes after infection. Furthermore, a detailed analysis of the mass spectrometry data was established according to the cleavage site usage of constitutive and immunoproteasomes. The result was that immunoproteasomes are involved in improved generation of the immuno-relevant fragments by the faster cleavage and the changed usage of existing cleavage sites after infection.