The kinetic characterization of cytochrome P450s that catalyze multi-step, sequential reactions is the focus of this work. Two novel substrates were identified for P450 1A2, one of which showed a high degree of homotropic positive cooperativity. Structural modeling was used to explain why cooperativity was substrate dependent. Pre-steady-state kinetics were used to characterize substrate binding, and fitting of these and the sigmoidal rate vs. substrate concentration plots yielded a kinetic model for the cooperative, sequential reaction. <p> A robust heterologous expression and purification strategy for P450 19A1 was developed. Steady-state and pre-steady-state kinetic parameters were measured for the substrate, intermediates, and product. Unlike many other P450s that catalyze multi-step reactions, P450 19A1 was shown to be a distributive enzyme in that the intermediates freely dissociated during the course of the reaction. Global fitting of kinetic experiments resulted in a kinetic model of the three-step reaction catalyzed by P450 19A1.

Replication Protein A (RPA) is the primary eukaryotic ssDNA binding protein utilized in preventing secondary structure formation and re-annealing of unwound DNA strands, thereby controlling access to DNA templates in diverse DNA transactions in the cell. Importantly, RPA serves as a scaffold for the assembly/disassembly of DNA processing machinery during normal cell cycle conditions and when DNA damage is encountered. The question of how RPA recognizes replication versus repair proteins, or those of other pathways remains obscure. However it is known that hyperphosphorylation of RPA signals recognition of DNA damage. RPA is a heterotrimer composed of three subunits (RPA70, RPA32, RPA14) that contain seven globular domains (70N, 70A, 70B, 70C, 32D, 32C) and one unstructured domain (32N) harboring all phosphorylation sites observed in DNA damage recognition. The domains are connected by flexible linkers that enable substantial inter-domain motion essential to RPA function. Small angle X-ray scattering (SAXS) experiments on two multi-domain constructs from the N-terminus of the large subunit (RPA70) were used to examine the structural dynamics of these domains and their response to the binding of ssDNA. The SAXS data combined with molecular dynamics simulations reveal substantial interdomain flexibility for both RPA70AB (the tandem high affinity ssDNA binding domains A and B connected by a 10-residue linker) and RPA70NAB (RPA70AB extended by a 70-residue linker to the RPA70N protein interaction domain). Binding of ssDNA to RPA70NAB reduces the interdomain flexibility between the A and B domains, but has no effect on RPA70N. These studies provide the first direct measurements of changes in orientation of these three RPA domains upon binding ssDNA. The results support a model in which RPA70N remains structurally independent of RPA70AB in the DNA bound state and therefore freely available to serve as a protein recruitment module. Nuclear magnetic resonance (NMR) experiments on RPA32N and an RPA32N phospho-mimic mutant (RPA32N-D8) were used to systematically examine the effect of hyperphosphorylation on interactions of RPA32N with other RPA domains. Multiple weak interactions between RPA32N-D8 and the 70N, 70A and 70B domains were observed. Control experiments demonstrated there were no interactions with the 32D, 32C and 14 domains. The data were interpreted within a framework in which RPA becomes more compact upon hyperphosphorylation. Such a change in the structural dynamics of RPA would be expected to influence its protein interaction network as part of the switch in activity upon damage of DNA.

The mechanism controlling the switch between gene activation and repression is critically important for understanding the process of transcriptional regulation. Gene expression is highly controlled through a dynamic exchange between co-activators and co-repressors from DNA bound transcription factors. TBL1 is an essential multi-domain scaffolding protein that appears to mediate the switch between transcriptional activation and repression of β-catenin and nuclear hormone receptors (NHRs). The mechanism of TBL1 as a transcriptional regulator has been the main focus of this dissertation. The role of TBL1 was first investigated in the context of the SCF(TBL1) complex in the poly-ubiquitination and proteasomal degradation of β-catenin under UV-induced genotoxic stress. Over-expression and purification protocols were developed for each of the SCF(TBL1) proteins, enabling a systematic analysis of β-catenin ubiquitination using an in vitro ubiquitination assay. This study revealed that Siah-1 alone was able to poly-ubiquitinate β-catenin. Moreover, TBL1 was found to protect β-catenin from Siah-1 ubiquitination in vitro and from Siah-1-targeted proteasomal degradation in cells. Both Siah-1 and TBL1 bind to the armadillo repeat domain of β-catenin, suggesting poly-ubiquitination of β-catenin is regulated by competition between Siah-1 and TBL1. To gain insight into the function of TBL1 within multi-protein complexes that regulate the transcriptional activity of β-catenin and NHRs, this work pursued the structural and biochemical characterization of TBL1. Expression, purification and analysis of full length TBL1 and different domain constructs revealed that it forms a stable tetramer through the N-terminal LisH domain. The structural architecture of full length TBL1 and the spatial organization of the domains were characterized by small angle x-ray scattering (SAXS) and analytical ultracentrifugation (AUC). An ab initio model of TBL1 was generated, revealing an extended anti-parallel dimer of dimers. The structure of TBL1 has a large surface area that can accommodate multiple binding partners, suggesting a role for TBL1 tetramerization in facilitating the formation of multi-protein assemblies. TBL1 mutants that form only dimers were designed and validated to enable future functional studies of the mechanism of TBL1 in regulating the transcriptional activity of β-catenin and NHRs.

Analysis of cellular signaling networks typically involves targeted measurements of phosphorylated protein intermediates. However, phosphoproteomic analyses usually require affinity enrichment of phosphopeptides and can be complicated by artifactual changes in phosphorylation caused by uncontrolled preanalytical variables, particularly in the analysis of tissue specimens. I hypothesized that changes in protein expression, which are more stable and easily analyzed, could reflect network stimulation and inhibition. This approach was employed to analyze stimulation and inhibition of the epidermal growth factor receptor (EGFR) by EGF and selective EGFR inhibitors. Shotgun analysis of proteomes from proliferating A431 cells, EGF-stimulated cells and cells co-treated with the EGFR inhibitors cetuximab or gefitinib identified groups of differentially expressed proteins. Comparisons of these protein groups identified 13 proteins whose EGF-induced expression changes were reversed by both EGFR inhibitors. Targeted multiple- reaction-monitoring (MRM) analysis verified differential expression of 12 of these proteins, which comprise a candidate EGFR inhibition signature. I then tested these 12 proteins by MRM analysis in 3 other models: 1) a comparison of DiFi (EGFR inhibitor-sensitive) and HCT116 (EGFR-insensitive) cell lines, 2) in formalin-fixed, paraffin-embedded (FFPE) mouse xenograft DiFi and HCT116 tumors, and 3) in tissue biopsies from a patient with the gastric hyperproliferative disorder Ménétriers disease, who was treated with cetuximab. Of the proteins in the candidate signature, a core group, including c-Jun, jagged-1, and claudin 4 were decreased by EGFR inhibitors in all three models. Although the goal of these studies was not to validate a clinically-useful EGFR inhibition signature, the results confirm the hypothesis and outline a prototypical approach to derive and test protein expression signatures for drug action on signaling networks. A secondary goal of this research was to apply a new method to quantify protein modification changes to EGFR using internal reference peptides (IRP). The major focus of this work was to assess the performance of this newly developed MS-based quantitation method to detect phosphorylation changes on EGFR by comparing the performance characteristics to stable isotope dilution (SID) methods. Initial studies are presented along with suggestions for future studies using overall findings in this dissertation.

Human topoisomerase I and topoisomerase IIα and IIβ play critical roles in regulating the topological state of DNA and are targets for some of the most widely prescribed anticancer drugs currently in clinical use. These drugs act by stabilizing covalent-topoisomerase cleavage complexes that are requisite intermediates in the enzyme catalytic cycles. It is the accumulation of these cleavage complexes ahead of DNA tracking systems, on overwound DNA, that ultimately leads to cell death. Therefore, work in this dissertation more fully characterizes the effects that DNA interactions have on the activities of topoisomerase I and topoisomerase II and drugs that target these enzymes. Results show that all clusters of positively charged residues located in the C-terminal domain of topoisomerases IIα are necessary for the preferential relaxation of overwound substrates. Experiments also show that topoisomerase I preferentially relaxes and cleaves overwound substrates in the absence or presence of anticancer drugs. Additionally, DNA intercalating compounds were found to act as topological poisons of topoisomerase I, enhancing cleavage activity on underwound substrates and in cells by changing the apparent topological state of the DNA. Finally, F14512, an etoposide derivative that contains a spermine moiety in place of the C4 carbohydrate, was shown to be a more potent poison of human topoisomerase II than the parent compound. The drug shows similar contacts as etoposide in the enzyme-drug binary complex but, unlike etoposide, interacts with DNA in the absence of enzyme. The enhanced activity of the drug correlates with a stronger interaction of the compound in the ternary enzyme-drug-DNA complex, and the covalent linkage of the spermine moiety to the drug core is necessary for this enhanced activity. Together, data presented in this dissertation increase our understanding of the role that topoisomerase-DNA interactions play in enzyme regulation of DNA topology and highlight the importance of enzyme-DNA-drug interactions when designing new topoisomerase-targeted compounds with potentially enhanced therapeutic potential.

Histone lysine (K) methylation is a dynamic process that plays an important role in regulating chromatin structure and gene expression. This study has identified Jhd2, a JmjC domain-containing protein, as an H3K4-specific demethylase in budding yeast. In addition, I show that Jhd2 has intrinsic activity to remove all three states of H3K4 methylation in vivo, and can dynamically associate with chromatin to modulate H3K4 methylation levels on both active and repressed genes and in the subtelomeric regions. I also provide evidence that the interaction between the JmjN and JmjC domains in Jhd2 is important for its protein stability, and Not4 (an E3 ubiquitin ligase) monitors the structural integrity of this inter-domain interaction to maintain the overall protein levels of Jhd2. Moreover, I find that the PHD finger of Jhd2 is important for its chromatin association in vivo by binding to the N-terminal region of H2A, suggesting a novel docking site on chromatin for Jhd2. In summary, this study has revealed important insights into the function and regulation of the H3K4 demethylase Jhd2.

Axonal regeneration in the central nervous system is prevented, in part, by inhibitory proteins expressed by myelin, including Myelin-associated glycoprotein (MAG). Although injury to the corticospinal tract can result in permanent disability, little is known regarding the mechanisms by which MAG affects cortical neurons. Here, we demonstrate that cortical neurons plated on MAG expressing CHO cells, exhibit a striking reduction in process outgrowth. Interestingly, none of the receptors previously implicated in MAG signaling, including the p75 neurotrophin receptor or gangliosides, contributed significantly to MAG-mediated inhibition. However, blocking the small GTPase Rho or its downstream effector kinase, ROCK, partially reversed the effects of MAG on the neurons. In addition, we identified the lipid phosphatase PTEN as a mediator of MAGs inhibitory effects on neurite outgrowth. Knockdown or gene deletion of PTEN or over expression of activated AKT in cortical neurons resulted in significant, although partial, rescue of neurite outgrowth on MAG-CHO cells. PTEN knockout and Rho inactivation resulted in an additive effect on reversal of neurite outgrowth inhibition by MAG, suggesting that both molecules contribute to inhibition of cortical neurite outgrowth by MAG. Moreover, MAG decreased the levels of phospho-Akt, suggesting that it activates PTEN in the neurons. Taken together, these results suggest a novel pathway activated by MAG in cortical neurons involving the PTEN/PI3K/AKT axis.

Histone deacetylases (HDACs) are enzymes that regulate chromatin structure through removal of acetyl groups from lysine residues on histones. HDAC3 is a unique member of class 1 HDACs that is a critical component of the SMRT/N-CoR co-repressor complex. Interestingly, Hdac3 deletion in immortalized NIH 3T3 cells results in decreased rates of cellular proliferation. Therefore, structure function studies were carried out in order to understand the mechanism by which Hdac3 regulates cell growth. A homology model and crystal structure were used to provide insight into the surface of HDAC3 and potential protein interaction regions. With this information in hand, mutations in HDAC3 were generated to disrupt HDAC3 protein interactions, eliminate HDAC3 deacetylase activity, and remove the ability of HDAC3 to undergo phosphorylation. HDAC3 mutants generated were expressed in Hdac3-/- NIH 3T3 cells, and their ability to complement the observed growth defect was monitored. While HDAC3-mediated changes in proliferation were not dependent upon phosphorylation, RelA binding, or protein interactions with hydrophobic regions, catalytic activity was required for normal growth rates. Furthermore, HDAC3 catalytic activity was dependent on its interaction with the deacetylase activating domain of SMRT/N-CoR mediated through Ins(1,4,5,6)P4. Therefore, HDAC3-mediated effects on cell growth are dependent on functional deacetylase activity and interaction with SMRT/N-CoR.

DETERMINANTS OF CYCLOOXYGENASE-2-MEDIATED OXIDATIVE METABOLISM OF THE ENDOCANNABINOID, 2-ARACHIDONOYL GLYCEROL, IN VITRO AND EX VIVO Joel Musee Dissertation under the direction of Professor Lawrence J. Marnett PGHSs catalyze the oxygenation of fatty acyl substrates (FAH) such as arachidonic acid (AA). This is the committed step in the generation of prostaglandin H2 (PGH2). PGH2 is the substrate for five downstream isomerases that lead to the generation of prostaglandins. Prostaglandins mediate a variety of physiological effects, such as pain, inflammation, fever, vascular homeostasis, and parturition, by their actions at several prostaglandin-specific G-protein coupled receptors (GPCRs). The endogenous ligands for the cannabinoid receptors, arachidonoyl ethanolamine (AEA) and 2-arachidonoyl glycerol (2-AG) are substrates for the second isoform of PGHS, PGHS-2. Their oxygenation leads to the generation of AEA and 2-AG derived prostaglandins (PG-EAs and PG-Gs). PG-Gs have been shown to have unique actions at yet to be identified GPCRs including, mobilizing Ca2+ at the picomolar level in cell culture, and causing a concentration dependent hyperalgesia (exaggerated pain) and allodynia (pain in response to non-pain evoking stimuli). To determine the biochemical determinants of the oxygenation of 2-AG by PGHS-2, I compared the abilities of AA derived hydroperoxides to 2-AG derived peroxides as substrates and activators of the oxygenase function of PGHS. I demonstrated that PGG2 and PGG2-G were both equivalent substrates for PGHS-2. Interestingly, the oxygenation of 2-AG demonstrated and increased need for the concentrations of peroxide required to activate its oxygenation. In the presence of increased peroxide scavenging by glutathione peroxide (GPx), the oxygenation of 2-AG was almost abrogated compared to relatively unaffected AA oxygenation. Specifically, I demonstrated that the depletion of the membrane associated GPx (GPx4), leads to increased oxidant stress and peroxide tone, and significantly increased oxygenation of 2-AG in murine derived cells. These results led us to conclude that the oxygenation of 2-AG is exquisitely sensitive to the concentration of oxygenase activating peroxide. Chemical ligands specifically designed to target the peroxidase active site of PGHS have to date not been reported. I designed a chemical mimic of PGHS-2 substrate PGG2 and developed a chemical ligand that inhibited the turnover of peroxide at the peroxidase active site of PGHS. This lead compound forms a new class of molecule that can specifically and potently inhibit the oxygenation of 2-AG by PGHS-2, while having no impact on AA oxygenation. It could form a useful tool for the dissection of the physiological role of PGHS-2 mediated 2-AG oxygenation.

All living organisms encode at least one type II topoisomerase. These enzymes help to regulate the superhelical density of the bacterial chromosome and remove knots and tangles from the double helix. Previous studies indicate that eukaryotic type II enzymes utilize a novel variant of the canonical two-metal-ion mechanism to catalyze DNA scission. However, the role of these metal ions in the reaction mediated by bacterial type II enzymes has been controversial. To resolve this issue, we characterized the cleavage reaction of Escherichia coli topoisomerase IV. We utilized a series of divalent metal ions with varying thiophilicities along with oligonucleotides that replaced bridging and non-bridging oxygen atoms at the scissile bond with sulfur atoms. DNA scission was enhanced when thiophilic metal ions were used with sulfur-containing substrates. The metal ion dependence of DNA cleavage was sigmoidal in nature. Furthermore, rates and levels of DNA cleavage increased when metal ion mixtures were used in reactions. Based on these findings, we propose that topoisomerase IV cleaves DNA using a two-metal-ion mechanism in which one of the metal ions makes a critical interaction with the 3-bridging atom of the scissile phosphate and facilitates DNA scission. Etoposide is a widely prescribed anticancer drug that stabilizes covalent topoisomerase II-cleaved DNA complexes. The drug contains a polycyclic ring system (rings AD), a glycosidic moiety at C4, and a pendant ring (Ering) at C1. Interactions between human topoisomerase IIα and etoposide in the enzyme-drug complex appear to be mediated by substituents on the A-, B-, and E-rings, but not the D-ring or C4 glycosidic moiety. To address the contributions of the D-ring to the activity of etoposide, drug derivatives were characterized. D-ring alterations diminished the ability of etoposide to enhance DNA cleavage mediated by human topoisomerase IIα in vitro and in cultured cells. They also decreased etoposide binding in the ternary enzyme-drug-DNA complex and altered sites of enzyme-mediated DNA cleavage. Based on these findings, we propose that the D-ring of etoposide has important interactions with DNA in the ternary topoisomerase II cleavage complex.