The Epstein-Barr virus (EBV) infects over 90% of the human population and is associated with several human malignancies. The EBNA1 protein of EBV binds recognition sites in the latent origin of replication (oriP) and is important for the replication and segregation of EBV genomes in latently-infected cells. EBNA1 is also directly implicated in malignant transformation and immortalization of the host cell. EBNA1 does not have any known enzymatic activity and it employs cellular proteins to mediate its functions. One such protein is the ubiquitin specific protease, USP7, which is a key regulator of the p53 tumor suppressor. The aim of this thesis was to functionally characterize the interaction between EBNA1 and USP7. Here I show that USP7 promotes the DNA-binding activity of EBNA1 and is recruited along with an accessory protein, GMPS, to the oriP. The USP7-GMPS complex can deubiquitinate histone H2B and may enable epigenetic regulation of latent viral infection. Additionally, I present evidence for a direct role of EBNA1 in EBV-mediated carcinogenesis. EBNA1 prevents stabilization of p53 by USP7 and abrogates p53 activation by disrupting promyelocytic leukemia nuclear bodies (PML-NBs) that acetylate p53. This interferes with p53-activated gene expression and inhibits apoptosis. EBNA1-expressing cells also have impaired ability to repair DNA, but survive as well as or better than control cells. Thus EBNA1 creates a cellular environment conducive to transformation and immortalization. These studies have also allowed me to learn more about and expand on the known functions of USP7. I provide biochemical evidence suggesting that a P/A/ExxS motif is a preferred sequence for binding the USP7 N-terminal domain. Furthermore, I show USP7 is a negative regulator of PML proteins and PML-NBs and promotes p53 DNA-binding activity. Surprisingly, neither function required the deubiquitinase activity of USP7.

Tie2 is a receptor tyrosine kinase (RTK) involved in numerous aspects of both normal and pathological angiogenesis. Proper functioning of this receptor is essential for normal development of the vasculature in the embryo as well as vessel maintenance and at sites of active angiogenesis in the adult. A growing list of pathological states has been attributed to a disruption of the angiogenic ‘balance’ including psoriasis, arthritis, atherosclerosis and diabetic retinopathy. Elucidating the molecular mechanisms behind this important biological process will provide insight into the various molecules involved as well as provide potential targets for novel angiogenic therapies. In an attempt to better understand the signaling pathways downstream of the Tie2 receptor we have studied tyrosine residues on the receptor believed to play an important role in Tie2 function. Of these, we have identified Y1111 as a negative regulatory site on Tie2. Mutation of this site affects receptor phosphorylation and kinase activity. Furthermore, protease digestion studies indicate that mutation of Y1111 may alter receptor conformation and potentially relieve negative inhibition imparted by the C-tail of Tie2. As well, we examined potential Tie2 downstream binding partners, specifically the novel Grb7 family of proteins. This work describes for the first time tyrosine phosphorylation of Grb14, an adaptor molecule previously shown to bind Tie2 in vitro. Moreover, our data suggests a role for this adaptor in Tie2 signal transduction involving two tyrosine residues in the receptor C-terminal tail; Y1100 and Y1106. These studies provide important insight into both signal transduction downstream of Tie2 as well as help us understand some of the molecular mechanisms behind the intrinsic ability of this RTK to regulate its own activity.

The role of antioxidants in cancer has been controversial for a long time. Although the public’s belief is that antioxidants prevent and/or inhibit cancer, there is increasing evidence to suggest the opposite: that cancer cells require antioxidants to survive. We wanted to interrogate the role of antioxidants in cancer by investigating both upstream regulators and downstream effectors of antioxidant signaling. We have identified protein tyrosine phosphatase non-receptor type 12 (PTPN12) as a novel regulator of antioxidant signaling in cancer. PTPN12 reduces reactive oxygen species (ROS) levels by promoting activity of the forkhead box O (FOXO) family of antioxidant transcription factors. We have also elucidated the impact of glutathione (GSH), the most abundant antioxidant in the cell, on tumorigenesis. We have found that GSH is required for cancer initiation, yet dispensable once transformation has occurred due to compensation provided by the thioredoxin (TXN) antioxidant pathway. Together, these studies expand our knowledge of the role of antioxidants in cancer and provide numerous avenues of research for the future.

Integral membrane proteins are targeted to discrete compartments through the action of a number of transport pathways. The post-translational modification of cargo with ubiquitin is a key regulator of protein sorting. Ubiquitinated cargo are bound by specific cargo sorting machinery and directed towards the appropriate destination. Therefore, the identification and characterization of the proteins involved in cargo ubiquitination is critical to understanding the regulation of protein sorting. In the work presented here, we characterize the role of the E3 ubiquitin ligases, RNF126 and Rabring7, in two distinct membrane trafficking pathways. First, we show that RNF126 and Rabring7 are involved in the ligand induced downregulation of cell surface receptors. RNF126 and Rabring7 associate with the EGFR, amongst other RTKs, and promotes its ubiquitination. RNF126 and Rabring7 are required for the efficient sorting of the EGFR through the late endocytic compartment. We also show that the depletion of Rabring7 attenuates the degradation of MET and that both RNF126 and Rabring7 regulate the sorting of CXCR4 from an early endocytic compartment. In addition, the depletion of RNF126 or Rabring7 destabilizes ESCRT-II and reduces the number of multivesicular bodies formed after EGF stimulation. Second, we found that RNF126 regulates the sorting of the CI-MPR. In cells transiently depleted of RNF126, the CI-MPR is dispersed into a transferrin receptor positive endocytic compartment. This effect is specific to the CI-MPR as other cargos that are sorted between the endosome at the Golgi remain unaffected. We found that RNF126 physically associates with the clathrin adaptor GGA3 and promotes its ubiquitination, suggesting that RNF126 regulates GGA3 mediated CI-MPR sorting. Together, this work furthers our understanding regarding the role of ubiquitin in membrane traffic.

Constitutive secretion is responsible for the targeting of transmembrane proteins to the plasma membrane, and for the secretion of extracellular matrix proteins, hormones, and other cellular products. The basic steps of secretion are well understood – proteins synthesized in the endoplasmic reticulum are transported in lipid-bound intermediates to the Golgi, and from the Golgi to the plasma membrane or cell exterior. Dysfunction of the secretory pathway – either constitutive or regulated – is involved in many disease states. One such state is diabetic nephropathy (DN). DN is characterized by renal hypertrophy and fibrosis, and is the leading cause of renal failure worldwide. Our lab had previously shown that munc13 is both upregulated and activated in the diabetic kidney, and that munc13 is an effector of rab34. Study of rab34 in HeLa cells revealed that rab34 is localized to the Golgi, and that it is required for the secretion of the Vesicular Stomatitis Virus glycoprotein. Colocalization experiments, as well as the use of Brefeldin A, localized the effect of rab34 to intra-Golgi transport. Further experiments indicated that glucose-induced upregulation of munc13 in rat mesangial cells increased the rate of constitutive secretion to the plasma membrane, and that this effect depended on its interaction with rab34. Finally, munc13 and rab34 were found to be required for the high glucose-mediated stimulation of Transforming Growth Factor-β secretion from mesangial cells, placing these two proteins at a key point in a pathway of physiological significance in the pathology of DN.

In recent years, research in the area of cancer stem cells has spiked tremendously. Numerous investigators have found that several types of cancers contain a subpopulation of tumor cells that display many defining characteristics of normal tissue stem cells, including multipotent differentiation potential, long-term self-renewal capacity, and expression of molecular markers of stemness. Most importantly, these cancer stem cells (CSCs) have very high tumor initiating potential, a finding that has led to the development of the cancer stem cell model for tumor progression. This model suggests that tumors are organized in a developmental hierarchy (similar to healthy tissue), with long-term tumor progression being driven by self-renewing CSCs at the top of the hierarchy. The CSC model represents a significant shift in our understanding of tumor progression, and as such, it may be possible to expand our knowledge of other aspects of tumor biology by re-examining them in the context of the CSC model. My work focuses on investigating interactions between CSCs and the tumor vasculature. Previous work has demonstrated heterogeneity in the proangiogenic potential of cells in a tumor. Considering the possibility that angiogenesis may be driven by specific subsets of tumor cells, I investigated the contribution of the CSC fraction to tumor angiogenesis. Comparing tumors with low or high CSC fractions, I have found that CSCs contribute to tumor vascular development through promotion of endothelial cell activity and recruitment of bone marrow-derived proangiogenic cells, mediated in part by vascular endothelial growth factor (VEGF) and stromal-derived factor 1 (SDF1). Since some tissue stem cells are known to reside in a vascular niche, I investigated the possibility that CSCs may also be supported by blood vessels in the tumor microenvironment, and that consequently CSCs may be targeted by disruption of tumor vasculature with antiangiogenic therapy. By testing multiple antiangiogenic therapeutic strategies, I have found that antiangiogenic therapy sensitizes CSCs to the effects of cytotoxic chemotherapy. Taken together, my work demonstrates a bi-directional relationship in which CSCs promote tumor vascular development, and tumor vasculature supports and protects CSCs. This work has implications for our understanding of CSC biology, tumor angiogenesis and antiangiogenic therapy, and provides insight into strategies for targeting the critical CSC population.

Anemia occurs in 60% of patients with Shwachman Diamond Syndrome (SDS). Although bi-allelic mutations in SBDS cause SDS, it is unclear whether SBDS is critical for erythropoiesis and what the pathogenesis of anemia is in SDS. I hypothesize that SBDS protects early erythroid progenitors from p53 family member mediated apoptosis by promoting ribosome biosynthesis and translation. SBDS deficiency by vector-based shRNA led to impaired cell expansion of differentiating K562 cells due to accelerated apoptosis and reduced proliferation. Furthermore, the cells showed general reduction of 40S, 60S, 80S ribosomal subunits, loss of polysomes and impaired global translation during differentiation. An upregulation of the pro-apoptotic p53 family member, TAp73, was found in resting SBDS deficient cells; however, not in differentiating cells. These results demonstrate SBDS plays a critical role in erythroid expansion by promoting survival of early erythroid progenitors and in maintaining ribosome biogenesis during erythroid maturation independently of p53 family members.

Microvilli are actin-based protrusions at the apical surface of epithelial cells and have diverse functions. My bioinformatic analysis suggests that human Cadherin 23, which is critical for normal microvillus development, has three paralogous homologues in Drosophila, Cad74A, Cad87A, and Cad88C. All three fly cadherins are present in follicle cell microvilli in late stages of oogenesis. The combined loss of Cad74A, Cad87A, and Cad88C did not produce any obvious defects in follicle cell microvilli or egg morphology. However, in a Cad74A Cad88C double mutant, Cad87A is strongly reduced at the apical surface of follicle cells. Furthermore, females overexpressing Cad74A produced abnormal eggs. This phenotype was rescued by increasing or reducing Cad87A expression. Together, my data suggest genetic interactions between the three cadherins, and that Cad74A and Cad87A may be involved in eggshell formation.

The course of pathogenic bacterial infection is dependent on the interactions between the host immune response and the bacterial virulence mechanisms. Our lab previously discovered that the Survival of Motor Neuron (SMN) protein complex undergoes a change in subcellular localization during infection with invasive Shigella bacteria, forming novel cytoplasmic aggregates called "U bodies". Similar results were obtained with other intracellular bacterial pathogens suggesting that these U bodies are a fundamental entity in microbial pathogenesis. Notably, the SMN complex normally plays a key role in the assembly of the spliceosomal U snRNA. We have shown during infection that there are changes in U snRNA maturation and splicing patterns. Importantly, we have found that U bodies are downstream of a stress pathway involving the stress-inducible ATF3 protein. Altogether, intracellular bacterial infection induces novel cellular stress pathways that disrupt normal SMN complex function and leads to changes in U snRNA associated functions.

Microvilli are actin-based protrusions at the apical surface of epithelial cells and have diverse functions. My bioinformatic analysis suggests that human Cadherin 23, which is critical for normal microvillus development, has three paralogous homologues in Drosophila, Cad74A, Cad87A, and Cad88C. All three fly cadherins are present in follicle cell microvilli in late stages of oogenesis. The combined loss of Cad74A, Cad87A, and Cad88C did not produce any obvious defects in follicle cell microvilli or egg morphology. However, in a Cad74A Cad88C double mutant, Cad87A is strongly reduced at the apical surface of follicle cells. Furthermore, females overexpressing Cad74A produced abnormal eggs. This phenotype was rescued by increasing or reducing Cad87A expression. Together, my data suggest genetic interactions between the three cadherins, and that Cad74A and Cad87A may be involved in eggshell formation.