Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 116-125).

Thesis (Ph. D.)--University of Virginia, 2009. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Thesis (Ph. D.)--Cornell University, May, 2008. / Vita. Includes bibliographical references (leaves 148-168).

No description available.

The small GTP-binding protein Rad (RRAD, Ras associated with diabetes) is the founding member of the RGK (Rad, Rem, Rem2, and Gem/Kir) family that regulates voltage-dependent calcium channel function. Given its expression in both excitable and non-excitable cell types, the control mechanisms for Rad regulation and the potential for novel functions for Rad beyond calcium channel modulation are open questions. Here we report a novel interaction between Rad and Enigma, a scaffolding protein that also binds to the E3 ubiquitin ligase Smad ubiquitin regulatory factor 1 (Smurf1). Overexpression of Smurf1, but not of a catalytically inactive mutant enzyme, results in ubiquitination of Rad and down regulation of Rad protein levels. The Smurf1-mediated decrease in Rad levels is sensitive to proteasome inhibition and requires the ubiquitination site Lys204, suggesting that Smurf1 targets Rad for degradation. Rad protein levels, but notably not mRNA levels, are increased in the hearts of Enigma-/- mice, leading to the hypothesis that Enigma may function as a scaffold to enhance Smurf1 regulation of Rad. In addition to ubiquitination, phosphorylation of RGK proteins represents another potential means of regulation. Indeed, Rem phosphorylation has been shown to abolish calcium channel inhibition. We demonstrate that b-adrenergic signaling promotes Rad phosphorylation at Ser39. Rad Ser39 phosphorylation is correlated with a decrease in the interaction between Rad and the CaVb subunit of the calcium channel and an increase in Rad binding to 14-3-3. Interestingly, Enigma overexpression promotes an increase in Rad Ser39 phosphorylation as well. Despite an interaction between Enigma and the CaV1.2 calcium channel subunit, overexpression of Enigma had no effect on Rad-mediated channel inhibition. Thus, Rad Ser39 phosphorylation alters its association with the calcium channel, but its impact on calcium channel regulation has yet to be determined. Finally, we report a novel function for Rad in the regulation of bone homeostasis. Rad deletion in mice results in a significant decrease in bone mass. Dynamic histomorphometry in vivo and primary calvarial osteoblast assays in vitro demonstrate that bone formation and osteoblast mineralization rates are depressed in the absence of Rad. Microarray analysis revealed that canonical osteogenic gene expression is not altered in Rad-/- osteoblasts; instead robust up-regulation of matrix Gla protein (MGP, +11-fold), an inhibitor of mineralization and a protein secreted during adipocyte differentiation, was observed. Strikingly, Rad deficiency also resulted in significantly higher bone marrow adipose tissue (BMAT) levels in vivo and promoted spontaneous in vitro adipogenesis of primary calvarial osteoblasts. Adipogenic differentiation of WT osteoblasts resulted in the loss of endogenous Rad protein, further supporting a role for Rad in the control of BMAT levels. These findings reveal a novel in vivo function for Rad signaling in the complex physiological control of skeletal homeostasis and bone marrow adiposity. In summary, this dissertation expands our understanding of Rad regulation through identification of a novel binding partner and characterization of post-translational regulatory mechanisms for Rad function. This work also defines a new role for Rad that may not depend upon its calcium channel regulatory properties: regulation of the bone-fat balance. These findings suggest that the regulation of Rad GTPase is likely more complex than guanine nucleotide cycling and that functions of Rad in non-excitable tissues warrant further study.

Pancreatic cancer is one of the deadliest cancers with a five-year survival rate of 6%. Pancreatic cancer is resistant to conventional chemotherapy and is usually diagnosed at late stages. Current treatment options have minimal effects in extending patients' lives beyond 10 months. One significant limitation in developing treatments to combat pancreatic cancer is its immunosuppressive microenvironment. Pancreatic cancer secretes factors that activate immunosuppressive cells, such as regulatory T cells (Tregs). These Tregs suppress effector CD4+ and CD8+ T cell anti-tumor immune responses. Therefore, novel treatment options to reduce Treg-mediated immune suppression and increase the numbers and functions of CD4+ and CD8+ T cells are paramount to enhance anti-tumor immunity in pancreatic cancer tumor-bearing (TB) hosts. The alternatively spliced transcription factor Ikaros is essential for lymphocyte development and is considered a tumor suppressor in T cells. Ikaros' protein stability and function are regulated by its phosphorylation and dephosphorylation by protein kinase CK2 and phosphatase 1 (PP1), respectively. Mutations and functional inactivation of Ikaros have mainly been investigated in T cell leukemias and lymphomas. In this dissertation, we investigated the role of Ikaros in regulating T cell homeostasis in murine pancreatic cancer. In this study, we report that Ikaros proteins are degraded by the ubiquitin-proteasome pathway in response to factors produced by murine pancreatic cancer cells. Our results further suggest that an increase in CK2 activity leads to Ikaros' degradation and disrupts its localization to pericentromeric heterochromatin in our murine pancreatic TB model. This loss of Ikaros expression is accompanied by an imbalance in T cell homeostasis. More specifically, we observe a significant decrease in effector CD4+ and CD8+ T cells but an increase in Treg percentages in TB and spontaneous pancreatic cancer models. T-cell specific defects in Ikaros protein expression were also observed in TB CD3+ T cells. Apigenin, a natural plant flavonoid and CK2 inhibitor, restored expression of some Ikaros isoforms in our TB model. Apigenin also displayed immunological benefits evident by enhanced anti-tumor immunity in TB mice. These data provide mechanistic and functional evidence that pharmacological inhibition of CK2 can regulate Ikaros expression and identifies the possible involvement of Ikaros in regulating T cell immune responses in murine pancreatic cancer.

Themis est une protéine de signalisation des récepteurs des lymphocytes T (TCR) essentielle pour la sélection positive des cellules T. La fonction moléculaire de Themis a été controversée mais de récentes études suggèrent qu'il est un régulateur positif des voies de signalisation des TCR. Nous avons montré dans une étude préliminaire que Themis interagit avec des déubiquitinases et qu'il est ubiquitiné dans les thymocytes. L'objectif de ma thèse était de caractériser les mécanismes moléculaires qui régulent l'ubiquitination de Themis et de déterminer si ces processus affectent la fonction de Themis durant le développement des lymphocytes T. Nous avons montré que si l'expression des ARNm codant pour Themis diminue dans les stades précoces de la sélection positive, son expression protéique est parallèlement augmentée, suggérant une stabilisation de Themis par des modifications post-traductionnelles durant cette étape. Nous avons montré que la déubiquitinase USP9X déubiquitine Themis pour stabiliser son expression durant la stimulation des TCR. L'ensemble de nos résultats proposent qu'USP9X soit activé durant la stimulation des TCR grâce à son recrutement dans les complexes proximaux des TCR par l'intermédiaire de l'adaptateur Grb2 et Themis, entrainant la stabilisation de l'expression de Themis. Nous pensons que ce mécanisme est important pour maintenir l'expression de Themis durant la sélection positive afin de favoriser l'induction d'un signal des TCR soutenu, requis pour l'efficacité de ce processus. / The protein Themis is a new actor of the T cell receptor (TCR) signaling essential for the positive selection of T cells. The molecular function of Themis has been controversial but recent findings suggest that it acts as positive regulator of TCR signaling. We demonstrated in an initial research that Themis interacts with deubiquitylases and is covalently associated to ubiquitin chains in thymocytes. The aim of my PhD project was to characterize the molecular process that regulates the ubiquitination of Themis and to investigate how these post-translational modifications affect Themis function during T cell development. We demonstrated that Themis mRNA expression is progressively decreased after positive selection whereas Themis protein expression is enhanced at the early stages of positive selection, suggesting that Themis is stabilized by post-translational modifications during positive selection. We demonstrated that USP9X allows the deubiquitination of Themis and its stabilization following TCR engagement. Ours results suggest that USP9X is activated during TCR engagement following its recruitment to proximal signaling complexes through Grb2 and Themis, leading to the deubiquitination and stabilization of Themis expression. We believe that this mechanism is important to sustain Themis expression during positive selection and to promote durable TCR signals required for the efficiency of this process.

La régulation des protéines par les modifications post-traductionnelles (PTMs) est un événement clé dans le maintien des fonctions biologiques de la cellule. Parmi elles, on retrouve les modifications causées par une famille de molécules appelées Ubiquitin Like Modifiers (UBls), incluant l’ubiquitination, la neddylation ou encore la SUMOylation. Au contraire des modifications classiques faisant intervenir des petits groupements chimiques, telles que la phosphorylation ou l’acétylation, les UBls sont eux-mêmes des protéines se greffant sur le groupement amine en position e des lysines des protéines ciblées, générant des protéines ramifiées. Alors que la principale fonction de l’ubiquitination est la dégradation des protéines par le protéasome, les autres UBls sont encore mal caractérisées. Dans ce contexte, le but de cette thèse était de développer de nouvelles approches protéomiques afin de définir le rôle de la SUMOylation dans des cellules humaines. En effet, l’identification des sites de SUMOylation par spectrométrie de masse (MS) est un défi. Ceci s’explique par la très faible abondance des protéines SUMOylées dans la cellule ainsi que par la longue chaine de 19 à 34 acides aminés laissés sur la protéine ciblée après digestion à la trypsine. Afin de pallier à ces deux problèmes, un mutant de la protéine SUMO a été généré au sein du laboratoire. La première altération sur ce mutant est l’insertion d’une séquence 6xHis à l’extrémité N-terminale de la protéine afin de faciliter l’enrichissement des protéines SUMOylés. La seconde altération de la protéine SUMO est la mutation d’une glutamine en arginine en position 6 à partir du C-terminal. Cette mutation a pour effet de libérer des peptides trypsiques ramifiés contenant seulement 5 acides aminés provenant de SUMO sur le peptide ciblé. Le premier but de cette thèse était de développer une méthode permettant de cibler spécifiquement les peptides SUMOylés lors d’une analyse par LC-MS. Cette méthode repose sur le patron de fragmentation propre de la chaine de 5 acides aminés commune à tous les peptides SUMOylés et utilise la technologie Sequential Window Acquisition of all THeoretical Mass Spectra (SWATH). Lors d’une telle analyse, l’échantillon est injecté une première fois en fragmentant de larges fenêtres de masses. Ceci permet d’obtenir des spectres MS/MS pour tous les peptides présents dans l’échantillon. Un algorithme est ensuite utilisé afin de détecter les fenêtres de masses contenant des peptides SUMOylés et de recalculer le rapport masse sur charge des peptides candidats. Les injections subséquentes permettent ensuite de fragmenter uniquement les peptides candidats. Cette méthode s’est avérée être complémentaire aux méthodes conventionnelles et a permis l’identification d’un total de 54 peptides SUMOylés à partir d’extraits protéiques enrichis sur billes NiNTA. La seconde approche envisagée était d’ajouter une étape d’enrichissement supplémentaire au niveau peptidique. Pour cela, un anticorps reconnaissant la chaine de 5 acides aminés laissée après digestion tryptique a été produit. Cette étape d’immuno-purification supplémentaire a permis l’identification d’un total de 954 sites de SUMOylation dans des cellules humaines lors d’une analyse à grande échelle. Afin de valider les nouvelles cibles identifiées, une étude fonctionnelle de la SUMOylation de la protéine CDC73 a été réalisée. Cette étude a montré que la SUMOylation de CDC73 était requise pour sa rétention nucléaire, confirmant ainsi un rôle important pour la SUMOylation de cette protéine. Cependant, le principal défaut de la précédente approche était la nécessité de cultiver 500 millions de cellules par condition étudiée. Cette approche a donc été optimisée afin de pouvoir réduire le nombre de cellules utilisées dans une analyse. L’optimisation de chacun des paramètres analytiques nous a permis de réduire ce nombre de 50 fois, permettant ainsi d’identifier plus de 1000 sites de SUMOylation à partir de seulement 10 millions de cellules. De plus, nous avons montré que cette approche permet l’identification concomitante des sites de SUMOylation et d’ubiquitination dans un seul échantillon biologique. Ceci a permis d’identifier un nouveau mécanisme de régulation des deubiquitinases par les UBls, ainsi que d’élucider les mécanismes de translocation du protéasome dans la cellule. Dans l’ensemble, nous avons développé des méthodes permettant de mieux caractériser la SUMOylation des protéines et avons prouvé que ces méthodes sont applicables à l’étude de plusieurs UBls en parallèle. Nous sommes certains que l’approche par immuno-purification permettra à l’avenir d’identifier la SUMOylation à un niveau endogène. / Protein regulation by post-translational modification (PTMs) is a key event in regulating cellular function. These modifications include a group termed Ubiquitin-Like modifiers (UBLs) that contain, but is not limited to, ubiquitylation, neddylation and SUMOylation. While conventional modifications, such as phosphorylation or acetylation, involve a small chemical group, UBLs are proteins attached from their C-terminus to the epsilon amine group of a lysine contained in the targeted protein, thus generating branched proteins. While the main function of ubiquitylation is protein degradation by the proteasome, other UBLs remain mostly unexplored. In this context, the aim of this thesis was to develop new proteomics strategies to characterize SUMOylation in human cells. Indeed, identification of SUMOylation sites by mass spectrometry (MS) is a challenge. This is due to the low abundance of SUMOylated proteins in the cells as well as the long 19 to 34 amino acid SUMO remnant left of the target after trypsin digestion. In this context, our research group has developed a mutant of SUMO containing two mutations. The first mutation consists of a 6xHis tag at the N-terminus of SUMO in order to facilitate SUMOylated substrates enrichment at the protein level. A second mutation was also introduced at the 6th position from the C-terminus and consists in a glutamine to arginine substitution in order to release shorter SUMOylated peptides after trypsin digestion. The first goal of this thesis was to develop a targeted approach to specifically fragment SUMOylated peptides during an LC-MS run. This was enabled by the common fragmentation pattern of all SUMOylated peptides arising from the five amino acid SUMO remnant. Digested peptides were first analyzed using SequentialWindow Acquisition of all THeoretical Mass Spectra (SWATH). In this experiment, large mass windows are fragmented. A custom algorithm is then used that detects mass windows in which candidates are located and determine their intact mass. In subsequent injections these peptides were then specifically targeted. This method was complementary to data dependent acquisition and enabled the identification of 54 SUMOylated peptides. In a second approach, we wanted to enrich for SUMOylated substrates at the peptide level. An antibody was raised against the five amino acid SUMO remnant and used for immunopurification of SUMOylated peptides. In total, we identified 954 SUMOylation sites in human cells. Moreover, functional analysis of the newly identified substrate CDC73 revealed that SUMOylation on K136 is required for its nuclear retention, thus showing a new role for the SUMOylation of this protein. Although this approach gave new insights into the characterization of SUMOylated substrates, high amounts of material were still required to obtain such results. The last goal of this thesis was to optimize the previously developed immunopurification. Systematic optimization of every analytical parameter was done and enabled the reduction of the number of cells required by a factor of 50, without affecting the number of SUMOylation sites identified. Moreover, we used this approach to profile for SUMOylation and ubiquitylation dynamics in human cells upon proteasomal inhibition with MG132. This revealed an unexpected regulation mechanism of deubiquitinating enzymes by UBLs and unraveled translocation mechanisms of the proteasome in the cell. Our SUMO proteomic approach demonstrates capability for the concomitant analysis of SUMOylation and ubiquitylation. In the future, we hope to extend this approach to endogenous SUMOylation.

Philosophiae Doctor - PhD / Retinoblastoma-binding protein 6 (RBBP6) is a multi-domain human protein known to play a role in mRNA splicing, cell cycle control and apoptosis. The protein interacts with tumour suppressor proteins p53 and pRb and recent studies have shown that it plays a role in the ubiquitination of p53 by interacting with Hdm2, the human homologue of mouse double minute protein 2 (Mdm2), in which the RING finger domain plays an essential role. Recently, RBBP6 has been shown to ubiquitinate the mRNA-associated proteinYB-1 through its RING finger domain, causing it to be degraded in the proteasome.RING (Really Interesting New Gene) fingers are small commonly-occurring domains of approximately 70 amino acids in length which coordinate two zinc ions in a cross-brace fashion.They are characterized by a conserved pattern of eight Cysteine or Histidine residues which are involved in coordinating the zinc ions. In terms of this conserved consensus, the RING finger from RBBP6 is expected to coordinate the zinc ions through eight Cysteine residues, making it a “C4C4” RING finger similar to those identified in transcription-associated proteins CNOT4(CCR4-NOT transcription complex, subunit 4) and p44 (interferon-induced protein 44). The amino acid sequence of the domain also shares many similarities with the U-box family of domains, which have an identical three-dimensional structure despite not requiring zinc ions in order to fold. This thesis reports the bacterial expression of a fragment containing the RING finger domain from human RBBP6, and determination of its structure using heteronuclear Nuclear Magnetic Resonance (NMR) spectroscopy. Preliminary NMR analysis of the fragment revealed that the domain was folded, but that it was preceded by an unstructured region at the N-terminus. A shortened fragment was therefore expressed and used for structural studies. Isotope-enriched protein samples were generated by growing bacteria in minimal media supplemented with 15NNH4Cl and 13C-glucose and purified using a combination of glutathione agarose affinity chromatography, anion exchange and size exclusion chromatography. A complete set of heteronuclear NMR data was collected at 600 MHz from which almost complete assignment of the backbone, side-chain and aromatic resonances was achieved. By exchange of Zn2+ with 113Cd2+ we managed to confirm that the domain binds two Zn2+ ions, and confirm that they are coordinated in the expected cross-brace manner. Structural data in the form of 2-Dimensional Nuclear Overhauser Enhancement Spectroscopy (2D-NOESY), 15N-separated NOESY and 13Cseparated NOESY spectra were recorded and used to determine the structure using restrained molecular dynamics on the Combined Assignment and Dynamics Algorithm for NMR Applications (CYANA) platform.As expected, the structure contains a triple-stranded β-sheet packing against an α-helix and two zinc-stabilized loops as found in all RING fingers. However, it also contains a C-terminal helix which packs against an N-terminal loop which is similar to that found in many U-box domains.A search using the DALI server revealed that the structure is most similar to the U-box from CHIP (C-terminus of Hsp70-interacting protein), an E3 ligase that cooperates with Hsp70 to degrade unfolded proteins that cannot be refolded. Using NMR we showed that the domain dimerizes with a KD of approximately 200 Μm, which means that it is dimeric at the concentrations used for NMR structure determination. Chemical shift analysis showed the dimerization interface to be very similar to that identified in U-box domains found in C-terminus of Hsp70 interacting proteins (CHIP).The structural similarities reported here between the RING finger from RBBP6 and the U-box family lead us to conclude that RBBP6 may, like CHIP, play a role in protein quality control.

Philosophiae Doctor - PhD / Retinoblastoma Binding Protein 6 (RBBP6) is a 250 kDa multi-domain protein that has been implicated in diverse cellular processes including apoptosis, mRNA processing and cell cycle regulation. Many of these functions are likely to be related to its interaction with tumour suppressor proteins p53 and the Retinoblastoma protein (pRb), and the oncogenic Y-Box Binding Protein-1 (YB-1). RBBP6 inhibits the binding of p53 to DNA and enhances the HDM2-mediated ubiquitination and proteasomal degradation of p53. Disruption of RBBP6 leads to an embryonic lethal phenotype in mice as a result of widespread p53-mediated apoptosis. RBBP6 promotes ubiquitination and degradation of YB-1, leading to its proteasomal degradation in vivo.The first part of this thesis describes in vitro investigations of the interaction betweenbacterially-expressed human p53 and fragments of human RBBP6 previously identified as interacting with p53, in an attempt to further localise the region of interaction on both proteins. GST-pull down assays and immunoprecipitation assays confirmed the interaction, and localised it to the core DNA binding domain of p53 and a region corresponding to residues 1422-1668 of RBBP6. However in Nuclear Magnetic Resonance (NMR) chemical shift perturbation assays no evidence was found for the interaction. NMR showed the relevant region of RBBP6 to be unfolded,and no evidence was found for interaction-induced folding. The R273H mutant of the p53 core domain did not abolish the interaction, in contrast to reports that the corresponding murine mutation (R270C) did abolish the interaction.The second part of this thesis describes in vitro investigations of the ubiquitination of YB-1 by RBBP6. A fragment corresponding to the first 335 residues of RBBP6,denoted R3, was expressed in bacteria and found to be soluble. Contrary to expectation, in a fully in vitro assay R3 was not able to ubiquitinate YB-1. However,following addition of human cell lysate, YB-1 was degraded in an R3-dependent and proteasome-dependent manner, indicating that R3 is required for ubiquitination and proteasomal degradation of YB-1. However R3 is not sufficient, with one or more factors being supplied by the cell lysate. In view of the pro-tumourigenic effects of YB-1 in many human cancers, these results lay the foundation for an understanding of the regulatory effect of RBBP6 on YB-1 and its potential role in anti-tumour therapy.