Ovarian cancer is the leading cause of death among all gynecological disorders. Aberrant glycosylation, or more specifically, increased sialylation of proteins has been observed in this malignancy. Several sialyltransferase genes have been shown to be up-regulated at both mRNA and and protein levels in a number of cancers, including that of the ovary. In the present study, we have analyzed the glycosylation patterns of kallikrein 6 in the context of ovarian cancer. We have discovered that the carbohydrate structures found at the single N-glycosylation site of kallikrein 6 derived from ovarian cancer cells found in the ascites fluid of ovarian cancer patients is enriched in sialic acid moieties and has an increased branching pattern when compared to controls. We have also developed a reliable anion-exchange HPLC-based methodology capable of quantifying different glycoform subpopulations of kallikrein 6 in serum and other biological fluids, which was capable of differentiating between samples from ovarian cancer patients and healthy controls. A variety of classic molecular biology and mass spectrometry based techniques were utilized in these experiments. Based on the results of the analysis of kallikrein 6 glycosylation and other literature reports showing upregulated sialylation of proteins in ovarian cancer, we have also identified sialylated glycoproteins from ovarian cancer proximal fluids and conditioned media of ovarian cancer cell lines. Sialylated proteins were enriched utilizing lectin affinity or hydrazide chemistry. In total, 333 sialylated glycoproteins and 579 glycosylation sites were identified. A list of 21 potential candidate ovarian cancer biomarkers was produced from proteins that were identified solely in ovarian cancer proximal fluids, which could form the basis for any future studies.

When prions are transmitted between species, there can be a delay in pathogenesis due to a phenomenon referred to as the transmission barrier. Some species also show very low susceptibility to prion disease. In this study I hypothesized that the susceptibility of species to prion disease is proportional to the tendency of their endogenous prion protein, PrP, to adopt the β-state, an oligomeric form of misfolded recombinant PrP that is rich in β sheet. Using a novel method of two-wavelength CD analysis, it could be shown that recombinant PrP from prion-susceptible species have a higher propensity to refold to the β-state than resistant species. The crystal structure of rabbit PrPC 121-230 revealed a helix-cap motif at the N-terminus of helix-2 that contributes to the reduced β-state propensity of rabbit PrP. Single amino acid changes in the sequence of PrP can lead to a transmission barrier and/or resistance in species. Mutating single residues in rabbit PrPC to those found in corresponding positions in hamster PrPC, ablated the helix-cap observed in the wild-type and caused an increase in the β-state propensity of rabbit PrP. Conversely, a decrease in β-state propensity was observed when rabbit mutations were introduced into PrP of hamster, a susceptible species. A dimeric association is hypothesized to be involved in the function of PrP and/or the conversion mechanism to infectious prion. In the structures of the wild-type and mutant rabbit PrPCs a dimeric arrangement was observed in the asymmetric unit of the crystals. Using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), a dimer of rabbit and hamster PrPC was crosslinked in solution. The dimer crosslink was specific and dependent on the tertiary structure of PrPC. Crosslinking of the β-state octamer with EDC showed that similar contacts may be present in this oligomeric form. Together these data provide strong evidence that species susceptibility is linked to β-state propensity.

TTumor hypoxia (poor oxygenation) adversely affects patient prognosis by promoting therapeutic resistance and an aggressive tumor phenotype. We aimed to understand how urokinase plasminogen activator receptor (uPAR), a cysteine-rich protein implicated in the malignant phenotype and poor patient prognosis, matures in hypoxia. We hypothesized that secretion of uPAR during hypoxia is conferred by a superior ability to form disulfide bonds without oxygen. A model and assay was established to monitor the oxygen-dependency of suPAR (a soluble secreted isoform of uPAR) folding and secretion. We found that suPAR maturation involves disulfide formation and N-linked glycosylation in normoxia. In anoxia, suPAR disulfide formation was impaired, but suPAR was nevertheless secreted. We propose that suPAR has low dependency on disulfide formation for efficient secretion in comparison to other disulfide-containing proteins. Mechanisms supporting protein expression during hypoxia may potentially be targeted to mitigate the adverse effects of tumor hypoxia and ultimately improve cancer therapy.

EmrE from Escherichia coli is a member of the small multidrug resistance protein family that oligomerizes to export hydrophobic cationic antimicrobials by utilizing the proton motive force. We studied the helix-helix interactions of the four transmembrane (TM) segments of EmrE to determine how this protein might assemble into its oligomeric forms. Using a combination of biochemical and biophysical techniques, we assessed the oligomerization propensities of Lys-tagged EmrE TM peptides in membrane-mimetic environments. Our results established that each of the TMs of EmrE display detergent-sensitive self-association, but in particular, TM2 had the greatest dimerization capability that was not completely abolished even by scrambling the native sequence. Mutations made to TM2 in full-length EmrE also revealed that efflux-defective mutations are located on one face of the helix. These findings reveal another potential oligomerization site for EmrE - and perhaps SMRs - and may provide a target for development of novel efflux-inhibitors.

The BTB domain is a well-conserved protein-protein interaction motif. There are 43 BTB-ZF transcription factors in the human proteome. Many of these transcription factors play crucial roles in cancer and developmental processes. The purpose of this project is to identify lists of interactors of the BTB domains of six BTB-ZF proteins with high confidence using a mass spectrometry based approach. The BTB domains BCL6, PLZF, Kaiso, LRF, FAZF, and Miz1 were studied. This study was able to identify 142 putative interactors. The list of putative interactor proteins participates in a wide array of biological functions. Selected putative interactors of the BCL6BTB were also validated using biochemical techniques. In conclusion, this project was able to provide an analysis of the protein-protein interactions mediated by the BTB domains of six BTB-ZF transcription factors. The information generated is valuable to guide future functional and structural studies of the BTB domains.

The BTB domain is a well-conserved protein-protein interaction motif. There are 43 BTB-ZF transcription factors in the human proteome. Many of these transcription factors play crucial roles in cancer and developmental processes. The purpose of this project is to identify lists of interactors of the BTB domains of six BTB-ZF proteins with high confidence using a mass spectrometry based approach. The BTB domains BCL6, PLZF, Kaiso, LRF, FAZF, and Miz1 were studied. This study was able to identify 142 putative interactors. The list of putative interactor proteins participates in a wide array of biological functions. Selected putative interactors of the BCL6BTB were also validated using biochemical techniques. In conclusion, this project was able to provide an analysis of the protein-protein interactions mediated by the BTB domains of six BTB-ZF transcription factors. The information generated is valuable to guide future functional and structural studies of the BTB domains.

Ovarian cancer is the leading cause of death among all gynecological disorders. Aberrant glycosylation, or more specifically, increased sialylation of proteins has been observed in this malignancy. Several sialyltransferase genes have been shown to be up-regulated at both mRNA and and protein levels in a number of cancers, including that of the ovary. In the present study, we have analyzed the glycosylation patterns of kallikrein 6 in the context of ovarian cancer. We have discovered that the carbohydrate structures found at the single N-glycosylation site of kallikrein 6 derived from ovarian cancer cells found in the ascites fluid of ovarian cancer patients is enriched in sialic acid moieties and has an increased branching pattern when compared to controls. We have also developed a reliable anion-exchange HPLC-based methodology capable of quantifying different glycoform subpopulations of kallikrein 6 in serum and other biological fluids, which was capable of differentiating between samples from ovarian cancer patients and healthy controls. A variety of classic molecular biology and mass spectrometry based techniques were utilized in these experiments. Based on the results of the analysis of kallikrein 6 glycosylation and other literature reports showing upregulated sialylation of proteins in ovarian cancer, we have also identified sialylated glycoproteins from ovarian cancer proximal fluids and conditioned media of ovarian cancer cell lines. Sialylated proteins were enriched utilizing lectin affinity or hydrazide chemistry. In total, 333 sialylated glycoproteins and 579 glycosylation sites were identified. A list of 21 potential candidate ovarian cancer biomarkers was produced from proteins that were identified solely in ovarian cancer proximal fluids, which could form the basis for any future studies.

Systematic identification of protein-protein interactions (PPIs) on a genome scale has become an important focus of biology, as the majority of cellular functions are mediated by these interactions. Several high throughput experimental techniques have emerged as effective tools for querying the protein-protein interactome and can be broadly categorized into those that detect direct, physical protein-protein interactions and those that yield information on the composition of protein complexes. Tandem affinity purification followed by mass spectrometry (TAP/MS) is an example of the latter that identifies proteins that co-purify with a given tagged query (bait) protein. Though TAP/MS enables these co-complexed associations to be identified on a proteome scale, the amount of data generated by the systematic querying of thousands of proteins can be extremely large. Data from multiple purifications are combined to form a very large network of proteins linked by edges whenever the corresponding pairs might form an association. Only a fraction of these pairwise associations correspond to physical interactions, however, and further computational analysis is necessary to filter out non-specific associations. This thesis examines how differing computational procedures for the analysis of TAP/MS data can affect the final PPI network, and outlines a procedure to accurately identify protein complexes from data consolidated from multiple proteome-scale TAP/MS experiments in the budding yeast \textit{Saccharomyces cerevisiae}. In collaboration with the Greenblatt and Emili laboratories at the University of Toronto, this methodology was extended to yeast membrane proteins to derive a comprehensive network of 13,343 PPIs and 720 protein complexes spanning both membrane and non-membrane proteins.

TTumor hypoxia (poor oxygenation) adversely affects patient prognosis by promoting therapeutic resistance and an aggressive tumor phenotype. We aimed to understand how urokinase plasminogen activator receptor (uPAR), a cysteine-rich protein implicated in the malignant phenotype and poor patient prognosis, matures in hypoxia. We hypothesized that secretion of uPAR during hypoxia is conferred by a superior ability to form disulfide bonds without oxygen. A model and assay was established to monitor the oxygen-dependency of suPAR (a soluble secreted isoform of uPAR) folding and secretion. We found that suPAR maturation involves disulfide formation and N-linked glycosylation in normoxia. In anoxia, suPAR disulfide formation was impaired, but suPAR was nevertheless secreted. We propose that suPAR has low dependency on disulfide formation for efficient secretion in comparison to other disulfide-containing proteins. Mechanisms supporting protein expression during hypoxia may potentially be targeted to mitigate the adverse effects of tumor hypoxia and ultimately improve cancer therapy.

EmrE from Escherichia coli is a member of the small multidrug resistance protein family that oligomerizes to export hydrophobic cationic antimicrobials by utilizing the proton motive force. We studied the helix-helix interactions of the four transmembrane (TM) segments of EmrE to determine how this protein might assemble into its oligomeric forms. Using a combination of biochemical and biophysical techniques, we assessed the oligomerization propensities of Lys-tagged EmrE TM peptides in membrane-mimetic environments. Our results established that each of the TMs of EmrE display detergent-sensitive self-association, but in particular, TM2 had the greatest dimerization capability that was not completely abolished even by scrambling the native sequence. Mutations made to TM2 in full-length EmrE also revealed that efflux-defective mutations are located on one face of the helix. These findings reveal another potential oligomerization site for EmrE - and perhaps SMRs - and may provide a target for development of novel efflux-inhibitors.