This thesis describes studies that are designed to investigates the hypothesis that mitochondrial production of reactive oxygen species (O2*- and H2O2) cause oxidative stress during PCB exposure and this increased production of ROS contributes to the biological effects of PCBs on cell proliferation in human breast and prostate epithelial cells. Exponentially growing non-malignant human breast epithelial cells (MCF-10A) and non-malignant human prostate epithelial cells (RWPE-1) were treated with selected PCBs and their metabolites (PCB3, 77, 153, Aroclor and 4ClBQ). Results showed that PCBs and their metabolites could significantly inhibit MCF-10A and RWPE-1 cell growth as well as inducing clonogenic cell killing. These PCBs were also found to increase steady-state levels of mitochondrial O2*- and H2O2. Furthermore, the same PCBs were also found to induce alterations in SOD activities in MCF-10A and RWPE-1 cells. Finally, treatment with either N-acetyl-cysteine (NAC), or the combination of polyethylene glycol (PEG) conjugated CuZnSOD and PEG-catalase added 1 hour after PCBs, significantly protected MCF-10A and RWPE-1 cells from PCB-induced toxicity even when added following PCB exposure. Similar experiments were also accomplished using airborne PCBs treated RWPE-1 cells. 4-OH-PCB11, a metabolite of airborne PCB 11 is shown to lead to steady-state increases in superoxide and hydroperoxides in exponentially growing RWPE-1 human nonmalignant prostate epithelial cells. This increased level of ROS was accompanied by the inhibition of cell growth and clonogenic cell killing. Furthermore treatment of cells with antioxidants one hour following exposure to 4-OH-PCB11 was able to significantly diminish the toxicity in human prostate epithelial cells. These results strongly supported the hypothesis that exposure to PCBs or their metabolites can induce the cytotoxicity and alterations in cellular proliferation as well as causing oxidative stress in exponentially growing human breast and prostate epithelial cells. More importantly, the data also provide clear evidence that antioxidant manipulations after PCB exposure are capable of protecting human cells against PCB-induced cytotoxicity. Based on these observations, the long term goal of this work is to develop a mechanism based biochemical rationale for the development of pharmaceutical manipulations to protect humans from PCB intoxication.

Adeno-associated Virus (AAV) Rep proteins are multifunctional proteins that carry out various DNA transactions required for the life cycle of AAV. The Rep proteins have been found to be important for genome replication, gene regulation, site-specific integration and play an essential role in genome packaging. There are two main groups of Rep proteins: large and small Reps; both groups are SF3 helicase family members. During DNA packaging, studies have shown that the small Rep proteins are critical to produce fully packed particles. Using stopped-flow kinetic analysis, we show a significant difference in helicase activity between the small and large Rep proteins that support the notion that the small Rep proteins are the primary motor to package DNA due to more efficient motor activity. That leaves the large Rep proteins to serve a different role during packaging. In previous studies, we have shown that the large Rep proteins have the ability to change their oligomeric state depending on the nature of the DNA substrate. We can observe double octameric rings with single-stranded DNA (ssDNA) and heptameric complex with double-stranded DNA (dsDNA). To understand Rep protein structural plasticity, we solved a 6.96 Å cryo-EM structure of Rep68*/ssDNA complex illustrating that the formation of Rep octamer rings is dominated by interactions between their N-terminal origin-binding domain (OBD) using the same interface utilized to recognize dsDNA specifically. Our analysis of the structural data suggests that the double octameric ring structure is stabilized by ssDNA that bridges octameric rings together. The structure shows that the helicase domains are highly flexible and that ssDNA is present at the center of the ring. In addition, we have solved a preliminary 12 Å model of Rep68*/dsDNA complex showing a heptameric ring encircling a DNA molecule. Our structural and functional data offer insights to the various Rep-DNA scaffolds that can perform diverse functions during the AAV life cycle.

<p>Bacteria of the genus <em>Deinococcus</em> are perhaps the most resilient life forms ever discovered, demonstrating extreme resistance to ionizing radiation, ultraviolet radiation, desiccation, and a variety of mutagenic chemical agents. The most studied member of this genus, <em>D. radiodurans</em>, has been observed to rapidly reassemble its genome following severe fragmentation by hundreds of γ-radiation induced double-strand DNA breaks. Amongst the numerous factors contributing to DNA repair, a single-stranded annealing protein, DdrB, is believed to play an important role during the initial phases of recovery. The work described in this thesis represents the first structural characterization of DdrB, revealing a novel fold for single-stranded DNA binding. Together with biochemical data delineating the DNA-binding interface, two crystal structures of the DdrB/ssDNA complex were also solved, providing a comprehensive illustration of this interaction. Quaternary assemblies observed in these crystal structures also informed on the potential contribution of higher-order nucleoprotein complexes to the function of DdrB in single-stranded annealing. Most significantly, a face-to-face assembly of DdrB/ssDNA complexes provided insight into the mechanism by which DdrB mediates annealing of DNA, which may represent a common mechanism shared by other single-stranded annealing proteins.<strong></strong></p> / Doctor of Philosophy (PhD)

The twin-arginine protein transport (Tat) system is one of two general protein transport systems in the cytoplasmic membranes of bacteria, and is conserved in the thylakoid membranes of plants. The Tat system transports fully folded proteins of varying sizes across the membrane. This feat is achieved by the use of just three conserved integral membrane proteins, TatA, TatB and TatC. Passenger proteins are designated for transport by the system by the use of an N-terminal extension, termed a signal peptide which contains an invariant twin arginine motif. The TatC component of the transport system is responsible for recognising the signal peptide, as well as binding to TatB and the TatA components. TatC therefore emerges as the functional and organisational ‘core’ of the Tat system. There is a wealth of functional data relating to the TatC protein, but no high resolution structural information on TatC was available to interpret this data. In order to gain an understanding of the Tat system at the molecular level, a structure was required of TatC. Preliminary expression screening using green fluorescent protein had previously identified the TatC protein from the thermophilic bacterium Aquifex aeolicus as being amenable for structural work. This study purified the A. aeolicus TatC construct in a variety of detergents, used native mass spectrometry and light scattering techniques to assay the sample homogeneity and set up extensive crystallisation trials. Following optimisation of a selenomethionine minimal media growth protocol, crystals grown from selenomethionine-substituted TatC purified in lauryl maltose neopentyl glycol diffracted to 3.5Å resolution, and allowed the structure of TatC to be solved by single wavelength anomalous dispersion. Analysis of the novel structure of TatC, in combination with an isothermal titration calorimetric binding assay, gave information on how TatC binds to signal peptides. Further structural analysis combined with additional experimental data from collaborators allowed a model to be proposed for how TatC binds to the two additional integral membrane components of the system, TatB and TatA, as part of the transport mechanism. Subsequent work was undertaken in this study to attempt to gain a co-crystal structure of TatC with the signal peptide. Construct and detergent screening was carried out in order to aim towards high resolution structural characterisation of the TatBC complex.

The primary function of the vascular system is the maintenance of oxygen homeostasis for all metazoan tissue. Angiogenesis, the remodeling and maintenance of new blood vessels from an existing vessel, is primarily controlled through the endothelial specific receptor tyrosine kinase Tie2, and the orphan receptor tyrosine kinase, Tie1. Although these receptors share highly conserved, genetic and biochemical analysis has shown these receptors have distinct and essential roles in angiogenesis. Tie2 activation typically results in vessel stability and quiescences and has further been shown to interact with all four sub-types of the angiopoietin signaling factors, Ang1-4. Conversely, Tie1 is involved in vascular remodeling and has no known ligands. The aim of this study is to resolve the molecular mechanism in which Tie1 modulates Angiopoietin-induced Tie2 signaling. Using biophysical, structural, and biochemical assays we show Tie1 directly interacts with Tie2 via electrostatic interactions housed within the extracellular domains. The binding of Tie1 to Tie2 attenuates Tie2 phosphorylation. We further show the constitutive agonist of Tie2, Ang-1, is capable of excluding Tie1 initiating Tie2 activation. Whereas the antagonist, Ang-2, is in incapable of excluding Tie1. Finally, we identify a region within the angiopoietin receptor-binding domain that is capable of including or excluding Tie1 from Tie2. Based upon the available data, we provide a model for Angiopoietin-induced Tie2 signaling.

Sortases have been known to be essential in Gram-positive bacteria for attaching proteins onto the peptidoglycan layer of the bacterium. Sortase A has been found to be useful as a “molecular stapler”, although; in vivo, the enzyme is responsible for attaching proteins to the peptidoglycan layer of Gram-positive bacteria. It accomplishes both of these tasks by joining two proteins together via an LPXTG sorting sequence. The enzyme has been proven to be very useful in attaching any two proteins together without worrying about recombinant techniques to generate the fusion protein. The problem with this enzyme is that the catalytic diad, which is composed of Cys-184 and His-120, has to be in a certain form that exists .2% of the time at pH 7.0. There is also a hydrolytic shunt that the enzyme can undergo instead of the productive transpeptidase reaction. These issues lead to groups attempting to place S.aureus SrtA through directed evolution in order to increase the catalytic efficiency of the enzyme. Although mutants have been generated that increase the catalytic efficiency 13-fold and 130-fold, the structural basis behind this increase is poorly understood. Using crystallography, we will attempt to discover the structural basis behind the rate enhancement as well as understand more about different species of SrtA. We also will attempt to kinetically characterize the S.aureus SrtA enzyme, its mutants, and different strains of SrtA. Thus far G.moribillorum SrtA has been crystallized and its structure shows that there is a distinction in the β6/β7 loop which has been implied to be important to catalysis. Furthermore, the pentaglycine kinetics shone some light on how the different mutants interact with the pentaglycine substrate of S.aureus SrtA.

The nucleosome remodeling and deacetylase (NuRD) complex is an abundant deacetylase complex, which couples histone deacetylation and chromatin remodeling ATPase activities, and has a broad cellular and tissue distribution. Although the working model of how this complex forms and functions is not well known, we have demonstrated that the coiled-coil interaction between two proteins (MBD2 and p66α) is critical for DNA methylation dependent gene silencing in vivo. Chapter one: ‘Unique features of the anti-parallel, heterodimeric coiled-coil interaction between methyl-cytosine binding domain 2 (MBD2) homologues and p66α dictate high affinity binding’ describes this unique coiled coil interaction. Coiled-coils were studied using a variety of biophysical techniques including analytical ultracentrifugation (AUC), isothermal titration calorimetry (ITC) and circular dichroism (CD). Results were compared across homologues and mutation studies were carried out to test our hypotheses. The studies reported in this chapter add to our understanding of coiled-coil interaction and thereby facilitate development of small peptide based drugs which target such interactions in nature.A number of proteins have been identified in humans that specifically bind to methylated CpG via a methyl binding domain (MBD). The human genome encodes at least five MBD proteins: MeCP2 and MBD1 through MBD4, which are homologous in their methyl binding domains but not many similarities are seen outside the MBD. Out of the five MBDs, MBD4 has a c-terminal glycosylase domain through which it recognizes mCpG.TpG mismatch and is important for base excision repair system. Chapter two: ‘Dynamic behavior of MBD4 in methylated DNA recognition’ focuses on MBD4 and its preference for DNA methylation mark. Techniques of surface plasmon resonance (SPR), nuclear magnetic resonance (NMR) spectroscopy are used to study binding affinity for variations of methylated DNA mark. Chemical exchange studies are used to demonstrate how MBD4 scans for methylation mark and these studies have added a new dimension to our understanding of how MBD proteins ‘read’ DNA methylation marks. Chapter three: ‘Solving the solution structure of MBD domain of MBD4 on methylated DNA by NMR’ describes a process of structure determination using NMR spectroscopy. The focus of this chapter is not on developing a new technique but rather on using current resources to solve a protein structure, which can be used to further understand our biological system. Here, I have discussed the workflow used to determine a final three-dimensional structure starting from sample preparation, data collection, data analysis to structure calculation.

Lysophosphatidic acid (LPA), a naturally occurring phospholipid present in serum and malignant effusions, elicits diverse biological functions through binding and activating specific cell surface G-protein coupled receptors. In addition to the conventional LPA1/Edg2, LPA2/Edg4 and LPA3/Edg7 receptors of the endothelial differentiation gene (Edg) family, LPA4/p2y9/GPR23 of the purinergic receptor family and the related LPA5/GPR92 and LPA6/p2y5 have been identified as novel LPA receptors. These newly identified LPA receptors are structurally distant from the Edg LPA1-3 receptors and couple to Gq, G12/13 and probably Gs subunits. However, the roles of the LPA4-6 receptors in LPA signal transduction and physiology are poorly understood. This project has used biochemical and genetic approaches to study biological functions of LPA4. In the first part of the study, we confirmed that LPA4 is indeed a functional LPA receptor mediating some cellular and biochemical responses to LPA including stimulation of neurite retraction, protein tyrosine phosphorylation. LPA4 also physically binds to LPA when ectopically expressed in cell lines. Mammalian cells usually express multiple LPA receptor subtypes and respond to LPA, making it difficult to link LPA receptors to specific responses. Targeted deletion has become a necessary approach to probe functions of individual LPA receptors. We therefore disrupted LPA4-encoding gene (lpa4/p2y9/gpr23) in mice. LPA4-deficient mice were born at the expected frequency and displayed no apparent abnormalities at least at early ages, indicating that LPA4 is not required for fertility, embryonic development or normal physiology. This is similar to knockouts of other LPA receptors. The backup and/or redundant receptor subtypes of LPA may suffice to compensate for the loss of individual LPA receptors in vivo. Alternatively, LPA may not be the only or rate-limiting mediator physiologically required in vivo. LPA signaling may be more critical in pathophysiological conditions when levels of the lipid mediator are locally and temporally altered. The availability of LPA4-null mice provides a valued model to analyze the roles of LPA4 in pathophysiological processes. Despite the lack of apparent phenotypes in mice, we took advantage of the LPA4- negative mouse embryonic fibroblasts (MEFs) to evaluate the effects of lpa4 deletion on cellular responses to LPA. Strikingly, LPA4-deficient MEFs were hypersensitive to LPA induced migration. Consistent with negative modulation of the phosphatidylinositol 3 kinase (PI3K) pathway by LPA4, LPA4 deficiency potentiated AKT and Rac but decreased Rho activation induced by LPA. Reconstitution of LPA4 converted LPA4-negative cells into a less motile phenotype. In support of the biological relevance of these observations, ectopic expression of LPA4 strongly inhibited migration and invasion of human cancer cells. When coexpressed with LPA1 in B103 neuroblastoma cells devoid of endogenous LPA receptors, LPA4 attenuated LPA1-driven migration and invasion, indicating functional antagonism between the two subtypes of LPA receptors. These results provide genetic and biochemical evidence that LPA4 is a suppressor of LPA-dependent cell migration and invasion. LPA4 may thus play a role in negative regulation of LPA signal transduction and specific cellular responses.

Lysophosphatidic acid (LPA), a naturally-occurring, simple phospholipid, is present at elevated levels in the blood and ascites of ovarian cancer patients. LPA is a ligand of seven cell surface G protein-coupled receptors. It has been known as an oncogenic growth factor in ovarian cancer and other types of human malignancies. However, the precise biological functions of LPA in ovarian oncogenesis remain to be fully elucidated. Our laboratory is interested in studying the potential role of LPA, as a tumor microenvironment factor, in regulation of cancer cell metabolism. A fundamental change associated with most cancer is the switch of glucose metabolism from mitochondrial oxidative phosphorylation to aerobic glycolysis, a phenomenon described by Otto Warburg nearly a century ago. This seems to be necessary to meet bioenergetic and biosynthetic demands of rapidly dividing tumor cells. However, the mechanism underlying the switch from aerobic respiration to aerobic glycolysis in cancer cells remains poorly understood. In this thesis project, my goal was to explore the effect of LPA on glycolysis and to compare LPA with other important growth factors in their capability to promote the glycolytic pathway in ovarian cancer cells. We demonstrated that LPA stimulated aerobic glycolysis as well as cell proliferation in ovarian cancer cell lines. The two parallel responses were LPA dose dependent. To determine whether LPA is unique in driving glycolysis, we compared the effect of LPA with other growth factors, including EGF, insulin and IGF-1 which are all involved in pathogenesis of ovarian cancer. While doses of these growth factors could be adjusted to achieve similar levels of cell proliferation, LPA and EGF were much more potent than insulin and IGF-1 in stimulation of glycolytic flux and lactate production. Therefore, we identified LPA and EGF as highly glycolytic factors relevant to the development and maintenance of the glycolytic phenotype of ovarian cancer cells. The next part of my study was focused on the molecular mechanism for the differential effects of LPA, EGF, insulin and IGF-1 on glycolytic metabolism. We used the glucose metabolism RT-PCR array to profile expression of glycolytic genes. The most remarkable change induced by LPA and EGF was the robust induction of hexokinase 2 (HK2) that stimulates irreversible entry of glucose to the glycolytic pathway. However, insulin and IGF-1 only weakly induced HK2 expression. Further experimental evidence using HK2 inhibitors indicated that HK2 up-regulation was the critical mediator of LPA-induced glycolysis. Further, the cells grown in LPA and EGF-stimulated conditions appear to show larger volume compared to insulin and IGF-1-treated cells, consistent with the hypothesis that active glycolysis contributes to biosynthetic processes to maintain cell sizes. Taken together, these findings of the current study revealed high glycolytic effects of LPA and EGF in ovarian cancer and the underlying HK2-mediated mechanism that distinguishes LPA and EGF from other growth factors such as insulin and IGF-1.

Overexpression of the DNA repair protein, X-ray repair cross-complementing protein 3 (XRCC3), has been shown to increase breast cell invasiveness. This raises the question of whether XRCC3 is a positive modulator of invasion or whether overexpressed XRCC3 is trapped in non-functional complexes, resulting in a dominant negative effect. To address this we transiently downregulated XRCC3 in a triple-negative breast cell line model of pre-invasive to invasive transition. Downregulation of XRCC3 in invasive T4-2 cells enhances cell invasiveness and increases FAK activation and signaling. This effect is abolished by EGF or TGFβ suggesting that XRCC3 is upstream of these factors. Conditioned media (CM) from XRCC3-downregulated T4-2 cells enhances the invasion of naïve pre-invasive and invasive cells. Mass spectrometric analysis reveals that secreted cyclophilin A (CYPA) is upregulated in the CM by XRCC3-siRNAs. The addition of recombinant CYPA to naïve T4-2 cells enhances their invasiveness, suggesting that CYPA is sufficient to induce neighboring cell invasiveness. Inhibiting the secreted CYPA by CYPA-inhibitor Cyclosporine-A (CsA) in CMs from low-XRCC3 T4-2 cells renders the CM incapable of increasing invasiveness, suggesting that CYPA is downstream of XRCC3. Furthermore, intracellular CYPA levels increase in progression from S1 to invasive T4-2 cells, consistent with previous observations of overexpression of CYPA in cancers. Overexpressing XRCC3 in S1 cells has no effect on invasion, whereas overexpressing XRCC3 in T4-2 cells increases invasion, but also viability. Overexpressing XRCC3 T241M mutation in S1 cells significantly increases their invasiveness, but in T4-2 cells it moderately increases invasion, suggesting an early role of this polymorphism in breast cancer invasion. Therefore, XRCC3 is an invasion suppressor, upstream of adhesion, growth factors, and paracrine effectors.