The pathogenic subgroup C feline leukemia virus (C virus) arises in infected cats by mutations in the envelope gene (env) of subgroup A FeLV (A virus) that switches the host receptor used for infection. To better understand C virus emergence and potential FeLV variants that may arise, I characterized FeLV Env sequence isolated from the primary FY981 FeLV isolate derived from an anemic cat. I show that the FY981 pseudotype virus is capable of using both the A virus receptor THTR1, and the C virus receptor FLVCR1 for infection, consistent with the FY981 Env being a hybrid A virus/C virus Env. Furthermore, I propose that pathogenic C virus arise in infected cats through FeLV variants or intermediates that are multi-tropic in their receptor use.

Autophagy, a cellular degradative pathway, plays a key role in protecting the cytosol from bacterial colonization, but the mechanisms of bacterial recognition by this pathway are unclear. Autophagy is also known to degrade cargo tagged by ubiquitinated proteins, including aggregates of misfolded proteins, and peroxisomes. Autophagy of ubiquitinated cargo requires p62, an adaptor protein with multiple protein-protein interaction domains. Previous studies demonstrated that the intracellular bacterial pathogen S. typhimurium is targeted by autophagy during infection of host cells. Here I show that p62 is recruited to S. typhimurium targeted by autophagy, and that the recruitment of p62 is associated with ubiquitinated proteins localized to the bacteria. Expression of p62 is required for efficient autophagy of bacteria, and restriction of their intracellular replication. My study demonstrates that the surveillance of misfolded proteins and bacteria occurs via a conserved pathway and reveals a novel function of p62 in innate immunity.

Metabolic channelling occurs when two enzymes that act on a common substrate pass that intermediate directly from one active site to the next without allowing it to diffuse into the surrounding aqueous medium. In this study, properties of channelling are investigated through the use of computational models and cell simulation tools. The effects of enzyme kinetics and thermodynamics on channelling are explored with the emphasis on validating the hypothesized roles of metabolic channelling in living cells. These simulations identify situations in which channelling can induce acceleration of reaction velocities and reduction in the free concentration of intermediate metabolites. Databases of biological information, including metabolic, thermodynamic, toxicity, inhibitory, gene fusion and physical protein interaction data are used to predict examples of potentially channelled enzyme pairs. The predictions are used both to support the hypothesized evolutionary motivations for channelling, and to propose potential enzyme interactions that may be worthy of future investigation.

Insulin resistance associated with obesity has been suggested to contribute to an increased risk and poor prognosis for breast cancer. In this study, a HER2/Neu transgenic mouse model of breast cancer was used to assess how obesity-induced insulin resistance and hyperinsulinemia can influence the development and progression of breast cancer. We investigated the effect of a high-fat diet and found a tumor-promoting effect in the absence of overt insulin resistance. In contrast, a high-fat combined with fructose diet induced significant hyperinsulinemia but no tumor promoting or growth effect was observed. Treatment with the anti-diabetic, insulin-lowering agent metformin led to a delay in tumor onset in mice on control diet, but this effect was abrogated by the high-fat fructose diet. These data indicate that the effects and potential interactions of insulin, nutrition and drugs on breast cancer development and progression are complex and require further study.

Reversible protein phosphorylation is an essential regulatory mechanism used by eukaryotes to coordinate the biochemical processes of the cell. The PP2A phosphatase functions to dephosphorylate specific proteins originally targeted by stimulus-activated protein kinases. The identification of a sub-network surrounding the human PP2A-Striatin holoenzyme, termed STRIPAK, provides insight into novel mechanisms for PP2A function and regulation. I reveal that STRIPAK participates in at least two mutually-exclusive sub-complexes, one of which contains the putative cortactin-binding protein, CTTNBP2NL. I show that CTTNBP2NL is enriched at the actin cytoskeleton, likely in a STRIPAK-independent manner. This study also reveals that STRIPAK interacts with a subunit of the dynein motor, at least partially, through CTTNBP2NL. This work will serve as a platform for the structural and functional characterization of STRIPAK and will ultimately assist in defining novel mechanisms of regulation and function for the human PP2A phosphatase.

Drosophila cadherin Cad99C is a critical regulator of follicle cell microvilli during Drosophila oogenesis. I extended the functional analysis of Cad99C by identifying Drosophila Myosin VIIA, and Cad74A and Cad87A, which are homologues of human Myosin VIIA and Cadherin 23, respectively, as components of the Cad99C protein complex and by studying their interactions in follicle cell microvilli. My co-immunoprecipitation data show that the cytoplasmic tail of Cad99C interacts with Myosin VIIA, and the extracellular cadherin repeats of Cad99C interact with Cad87A and Cad74A, independent of the cytoplasmic tail of Cad99C. Genetic studies indicate that 1) Cad99C and Cad74A/Cad87A localize independently to microvilli, although their amount is affected by changes in the ratio of their concentrations. 2) All cadherins affect microvillus morphology when overexpressed although the phenotypes are different. The overexpression effect of Cad74A depends on Cad99C but not vice versa. These data point to complex interactions between the microvillus cadherins.

The Saccharomyces cerevisiae origin recognition complex (ORC) binds to replication origins at the ARS consensus sequence (ACS), serving as a scaffold for the assembly of replication complexes needed for the initiation of DNA synthesis. I generated a genome-wide map of nucleosome positions surrounding replication origins because the precise locations of nucleosomes may influence replication. My map revealed a nucleosome-free region surrounding the ACS that is bordered by two well-positioned nucleosomes. I was able to explain differences in origin properties by clustering nucleosome profiles. I found an association between the replication time and nucleosome profile for a given origin cluster. An ORC depletion mutant nucleosome map indicated a shift in nucleosomes towards the ACS. I present the first genome-wide view of origin nucleosome architecture, indicate a relationship between chromatin structure and replication timing, and suggest a model whereby the interplay between DNA sequence and ORC binding defines the nucleosome occupancy pattern.

The erythroblastoma (ErbB) receptor family consists of four members that are implicated in many human cancers. To gain insight into their biological function, we investigated their interacting partners to derive a comprehensive protein interaction network. Using the membrane yeast two-hybrid (MYTH) system we probed the ErbB2, ErbB3, and ErbB4 interaction space and validated a subset of interacting partners using the luminescence-based mammalian interactome mapping (LUMIER) system. The integrated use of these two complementary protein interaction technologies generated high confidence data and identified many novel ErbB binding partners, a subset of which was supported by co-immunoprecipitation in the breast adenocarcinoma cell line Sk-Br-3 including the GPCR GPRC5B, the cysteine protease CAPN1, and WIF1, a secreted protein containing 5 EGF domains that may represent a novel ErbB ligand. Our systematic approach offers an unbiased systems level view that may identify novel drug targets and contribute to therapeutic research.

Neuroblastoma (NB) is the most common extra-cranial solid tumor in children. Recently discovered neuroblastoma tumor-initiating cells (NB-TICs) have many properties of cancer stem cells and form tumors with as few as 10 cells. To elucidate the signaling pathways driving NB- TIC survival and proliferation, we surveyed the phospho-tyrosine containing subset of the NB- TIC proteome. Over 300 phosphorylated proteins were identified, including 21 tyrosine kinases of which several belong to the Src kinase family. Using bioinformatics tools, several hematopoietic signaling pathways were identified, including the B cell receptor (BCR) pathway. Further proteomic approaches substantiated molecular hematopoietic features in NB-TICs. Inhibitors of BCR proximal kinases SYK and SFKs were cytotoxic to NB-TICs. Clinically utilized inhibitors of SFKs induce apoptosis in NB-TICs. Targeting hematopoietic survival pathways in NB-TICs from the bone marrow, which have thus far not been predicted to play a role in this neural malignancy, may provide new drug therapies for NB.

The pediatric sarcoma alveolar rhabdomyosarcoma (ARMS) is highly aggressive with a poor prognosis for diagnosed patients. Here, we demonstrate that targeting the unique oncogene integrin-linked kinase (ILK) in ARMS cells in conjunction with the common chemotherapy agent vincristine, a synergistic effect is found in the reduction of cell viability in vitro. This result was achieved by both RNAi-mediated depletion of ILK and using a small molecule kinase inhibitor specific for ILK. Both techniques were found to disrupt important protein interactions at the site of the centrosome. Combination ILK disruption and vincristine treatment of cells induced the expression of apoptotic markers and arrested cells in the G2/M stage of the cell cycle. Interestingly, protein levels of JNK and its target c-Jun were regulated with combined treatment. Altogether, these findings indicate that the use of molecular targets like ILK may further improve the clinical treatment of ARMS.