<p> The Cytotoxic Effect of Methylglyoxal on Yeast Cell Growth Methylglyoxal (MG) is a highly reactive, cytotoxic dicarbonyl compound, mainly formed as a by-product of glycolysis. It is one of the most potent glycating agents and readily reacts with proteins, lipids and nucleic acids to form advanced glycation end products (AGEs). However, the molecular targets of MG are largely unknown. Glucose is the preferred carbon source of yeast <i>Saccharomyces cerevisiae</i> which it can sense and utilize efficiently over a broad range of concentrations. It prefers to ferment rather than oxidize glucose, even when oxygen is abundant. The yeast cell-surface glucose sensors Rgt2 and Snf3 function as glucose receptors that sense extracellular glucose and generate a signal for induction of genes encoding glucose transporters (Hxts). Using molecular and cell biology approaches, including Western blotting, qRT-PCR analysis and fluorescence microscopy, I have provided evidence that MG inhibits expression of the Hxts (Hxt1 and Hxt3) by inactivating the low-affinity yeast glucose sensor Rgt2. MG inhibits the growth of glucose-fermenting yeast cells by inducing endocytosis and degradation of the glucose sensor. However, the glucose sensor with mutations at their putative ubiquitin-acceptor lysine residues is resistant to MG-induced degradation. The results of this study suggest that the low-affinity glucose sensor Rgt2 is inactivated through ubiquitin-mediated endocytosis and degraded in the presence of MG. Under physiological conditions, MG is detoxified by the glyoxalase system into D-lactate, with glyoxalase 1 (Glo1) as the key enzyme in the anti-glycation defense. This study further indicates that the inhibitory effect of MG on the glucose sensor is greatly enhanced in the cells lacking Glo1. Thus, the stability of this glucose sensor seems to be critically regulated by intracellular MG levels. Taken together, these findings suggest that MG attenuates glycolysis by promoting degradation of the cell surface glucose sensor and thus identify MG as a potential glycolytic inhibitor.</p>

<p> Apolipoprotein E3 (apoE3) is a 299 residue exchangeable apolipoprotein that has the ability to exist in lipid-free and lipoprotein-bound state. It plays a crucial role as an anti-atherogenic agent by removing cholesterol and triglycerides from circulation via the low-density lipoprotein receptor family of proteins. It also functions in reverse cholesterol transport in atherosclerosis by promoting cholesterol efflux from macrophages, a process that leads to the initial formation of nascent discoidal high-density lipoproteins (nHDL) composed of a bilayer of lipids surrounded by apoE3. </p><p> The objective of this study is to examine the molecular organization of apoE3 in HDL. Reconstituted HDL (rHDL) was prepared using single Cys variants of apoE3 (1-299) and synthetic phospholipids of physiological relevance. Pyrene fluorescence spectroscopy and Cys specific cross-linking studies were carried out to determine the spatial proximity of specified sites on neighboring apoE3 molecules in rHDL. Our results indicate that regardless of the location of the Cys residue, there was a decrease in excimer emission and absence of cross linker-mediated dimer in rHDL suggesting, that two apoE3 molecules are aligned in an anti- parallel fashion with respect to each other around the phospholipid bilayer. Our study offers new insights into the conformation of lipid-associated apoE3 in nascent HDL particles generated by macrophages, a critical step in atherosclerosis and cardiovascular disease.</p>

<p> Aurein is a cationic antimicrobial peptide, rich in phenylalanine residues. Although the peptide has been extensively studied, its mechanism of action is not fully understood and has not been established. This project is focused on studying the interactions of aurein with model biological membranes and antimalarials using Fourier Transform Infrared (FTIR), fluorescence, dynamic light scattering (DLS), atomic force microscopy (AFM), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC) techniques. FTIR data revealed conformational changes to the secondary structure of the peptide in the presence of the model membranes. The strongest interactions of aurein were found with DOPC and lipid raft systems. Fluorescence data revealed some differences in the mechanism of interaction between aurein and lipid rafts. Topographical analysis was performed using Atomic force microscopy (AFM). AFM images of the peptide with its lipid rafts showed a change in surface roughness suggesting a different mechanism of interaction. DLS data in agreement with FTIR confirmed that aurein interacts differently with the lipid rafts. The results gathered from this study provided new insights on the interaction of aurein. On the other hand, drug-drug interaction issues continue to present a major dilemma for the clinician caring for complex patients such as those infected with infectious disease. This study has examined the interaction of aurein with quinine, primaquine, and chloroquine. Significant interactions between aurein and antimalarials occured at a higher concentration of antimalarials. Interactions between aurein and antimalarials reveal a strong interaction between aurein and primaquine. Interactions between aurein and quinine or chloroquine were found to be weak and negligible. FTIR, TGA, and DSC may be used in a complementary way to gain insights into the possible drug-drug interactions involving aurein. These studies are needed to initiate in vivo controlled interaction studies between antibiotics and antimalarials.</p>

<p> As a continuation of the research on the pigment formation from catecholamines, we studied the polysaccharide-mediated oxidation of serotonin and other 5-hydroxy indoles into pigmented substances. As for catecholamines, we observed that many polysaccharides promote the oxidation of such compounds, particularly in the presence of Cu (II). The same polysaccharides, e.g., carrageenan or fucoidan, which strongly promoted the oxidation of catecholamines, strongly promoted the oxidation of serotonin, leading to the formation of darkly colored pigments. The reactions were evaluated using RP-HPLC and size exclusion chromatography (SEC) as the main analytical techniques. SEC proved particularly informative as these analyses allowed us to monitor (1) the decline in the substrate, (2) the formation of low-molecular mass oxidation products, (3) the formation of polysaccharide-associated pigments, and (4) the formation of potential pigment-based nanoparticles. We observed that increased amounts of polysaccharide or Cu (II) increased the amount of pigment generated. However, other cations like Co(II), Ni(II), Mn(II), or Fe(II) had no or very little effect on the reactivity. Apart from serotonin, 5-hydroxy indole could serve as a substrate to generate polysaccharide-associated pigments. However, reactions with the related substrate, 5-hydroxy indole-3-acetic acid yielded a low molecular mass chromophore, but not any polysaccharide-associated pigments. Large-scale reactions were set up in an attempt to isolate and characterize any pigments that were generated. The reaction mixtures could readily be dialyzed and lyophilized to obtain polysaccharide-associated pigments. Pigments were evaluated using UV-Vis spectroscopy, SEC analysis, FT_IR spectroscopy, and atomic absorption spectroscopy to evaluate the amount of Cu remaining in the materials.</p>

Genomic replication is a highly challenging task. The DNA replication machinery must precisely duplicate billions of base pairs while tolerating a multitude of obstacles including damaged DNA, collisions with transcriptional machineries, unusual DNA structures and other difficult to replicate sequences. Many of these obstacles stall replication forks and activate replication stress responses that stabilize and restart persistently stalled forks. These mechanisms include fork remodeling to regress replication forks into a chicken foot DNA structure. Fork regression may facilitate DNA repair or template switching to bypass the obstruction. Several members of the SNF2 family of DNA-dependent ATPases including SMARCAL1 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A-like protein 1), HLTF (Helicase like transcription factor) and ZRANB3 (Zinc finger Ran-binding protein 2- type containing 3) are replication stress response proteins that catalyze fork remodeling including fork regression. The enzymatic activities of SMARCAL1 and HLTF are dependent on a SNF2 ATPase motor domain and a substrate recognition domain (SRD) that is thought to mediate binding to specific structures at stalled replication forks. The SRD of SMARCAL1 is its HARP2 domain, which is required for SMARCAL1 binding to branched DNA structures as well as its DNA-dependent ATPase and fork regression activities. The SRD in HLTF is its HIRAN domain, which is unrelated in sequence and structure to the HARP domain and interacts with the exposed 3â end of small DNA flaps. The HIRAN domain is also important for HLTF mediated fork regression activity. Interestingly, unlike SMARCAL1 and HLTF, ZRANB3 contains a highly conserved ATP-dependent, substrate specific HNH endonuclease domain and catalyzes nuclease activity to branched DNA substrates. How ZRANB3 catalyzes fork remodeling and endonuclease activities is unknown. This work identified a substrate recognition domain within ZRANB3 that is needed for it to recognize and bind forked DNA structures, hydrolyze ATP, and catalyze fork remodeling and endonuclease activities. Importantly, this work provides a mechanistic understanding of how these enzymes operate within the replication stress response to restart stalled replication forks.

All cells attempt to maintain optimal levels of essential metal nutrients while eschewing toxic metal concentrations. To achieve this balance, metal homeostasis mechanisms have evolved to maintain appropriate levels (1). Of the many proteins which play a crucial role in metal homeostasis, the P-type ATPase family of transmembrane transporters is involved in the active transport of metal ions across the membrane using ATP hydrolysis as an energy source (2-5). P(1B) ATPases are found in all life kingdoms, from extremophilic archaea to mammals, and are a subgroup of P-type ATPases which are specific for heavy metal transport (4, 6). These transporters confer metal tolerance to archaea and bacteria and aid in metal efflux from the cytoplasm, thus conferring survival under conditions of high levels of metal in the environment. In humans defects in metal transport can result in severe hepatic and neurological disorders such as Menkes' and Wilson's disease, and thus understanding the molecular function of copper transporters is of paramount importance in human health (7). CopB, the 690 amino acid P(1B-3)-ATPase from Archaeoglobus fulgidus, is the central focus of this dissertation. CopB has a multiple cytosolic domains (ATP binding domain, actuator domain, and metal binding domain) in addition to 8 transmembrane helices (8).The transporter has a distinctive histidine rich N-terminal metal binding domain (MBD), which has been postulated to have a regulatory role in the transport process (9, 10). Although the MBD lacks sequence similarity to cytosolic metal binding domains belonging to homologous P-type ATPases, the preponderance of histidine residues suggests one or more metal coordination sites. Although membrane extracts of A. fulgidus CopB were shown to transport Cu(II), the metal binding affinity of the MBD, any potential metal dependent interactions with the ATP binding domain (ATPBD), and the structures of the cytosolic domains themselves remained unresolved when this research was initiated. Since then, the purpose of this study had been tripartite. First, I probed the relationship of the metal binding to the function of the N-terminal metal binding domain by investigating the metal binding properties of the MBD and the impact on its structure and inter domain interactions with the ATPBD and the A-domains, which has been seen in other homologous ATPases and shown to play an important regulatory role. The second focus was to determine the structure and characterize the ligand-induced conformational changes of the ATPBD. Finally I investigated the effects of some prominent Wilson's disease mutations which are conserved on the ATPBD of CopB, thereby using CopB as a model system. These studies have helped in understanding how heavy metal ATPases with distinct His-rich metal binding domains work in maintaining critical levels of metal ions across the cell membrane.

During development of the nervous system, approximately 50% of the neurons generated undergo apoptosis as part of a normal pruning process. The neuronal corpses must be efficiently cleared to prevent an immune system response. We demonstrated that satellite glial precursor cells are the main phagocytes in the developing dorsal root ganglia (DRG), and identified a novel engulfment receptor necessary for this engulfment, Jedi-1. Jedi-1 contains immunoreceptor tyrosine based activation motifs (ITAMs) that bind to the tyrosine kinase Syk, which is required for phagocytosis. Jedi-1 also contains an NPXY motif that is required for interaction with the adapter protein GULP, which was also necessary for engulfment. Jedi-1 associates with GULP and recruits clathrin heavy chain (CHC), which promotes actin rearrangement required for engulfment. To determine the role of Jedi-1 in vivo we generated jedi-1 -/- mice. Apoptotic neurons accumulate in the developing DRG of jedi-1 -/- mice, and the mice develop autoimmune disease including the production of autoantibodies and kidney dysfunction. The jedi-1 null mice also exhibit excessive itching which results in skin lesions. Our data suggests that this itch phenotype may be due to activation of satellite glial cells in response to defective clearance of apoptotic neurons.

The work to be reported in this thesis has been restricted to the study of carbohydrate absorption. Since a number of aspects of the problem have received attention, these aspects have been dealt with in different chapters. It was thought that the subject could be more easily followed if the literature concerning each aspect was reviewed in the opening paragraphs of the separate chapters. For this reason the General Introduction will be limited to a brief description of the most common methods used in the past for the study of the intestinal absorption of carbohydrates, and following this to a consideration of the evidence quoted by Verzar in support of a phosphorylative mechanism of absorption. The plausibility of the phosphorylation theory has gained for it wider acceptance than the proof advanced would seem to warrant, and subsequent researches have been greatly influenced by this concept.

The topological state of DNA has a dramatic effect on nucleic acid processes in cells. Type II topoisomerases are necessary enzymes that help regulate the topological state of the genome, but their activity requires the creation of transient DNA breaks. This potential to induce DNA damage is exploited by topoisomerase II poisons, which stabilize the covalent enzyme-cleaved DNA complex and eventually overwhelm the cell with DNA breaks. Although several topoisomerase II poisons are highly successful chemotherapeutic drugs or antibacterials, much about the interactions between topoisomerase II poisons, type II topoisomerases, and their DNA substrates has remained elusive. The first part of this dissertation examines interactions between human topoisomerase IIα and covalent poisons. Thymoquinone, a natural product from black seed, acts as a covalent poison even in its complex herbal form. This suggests that its chemopreventive properties may be related to its effects on topoisomerases. Covalent poisons cannot enhance cleavage mediated by the catalytic core of topoisomerase IIα, but can still inhibit the enzyme if incubated with it prior to the addition of DNA. Therefore, covalent poisons appear to have multiple interaction sites within the enzyme, although not every interaction stabilizes cleavage complexes. The second part of this dissertation describes the recognition of DNA geometry by bacterial type II topoisomerases. Bacillus anthracis gyrase relaxes overwound DNA quickly and processively while maintaining low levels of cleavage complexes, indicating that it is well suited to work on overwound DNA ahead of replication forks and tracking systems. Conversely, topoisomerase IV relaxes overwound DNA faster than underwound molecules but is unable to distinguish supercoil geometry during cleavage. Based on results with different constructs of Mycobacterium tuberculosis gyrase, it appears that the ability to distinguish supercoil geometry during cleavage is embedded within the N-terminal domain. Finally, the last part of this dissertation describes the biochemical basis of interactions between quinolone antibacterials and B. anthracis gyrase. Results indicate that the primary interaction occurs through a conserved water-metal ion bridge. However, altering quinolone substituents can allow for new interactions and overcome resistance.

Human cytochrome P450 (P450) 17A1 is an essential enzyme in the steroid biosynthesis pathway that mediates a critical branch point which leads to either glucocorticoid or sex hormone production. P450 17A1 is a bifunctional enzyme that catalyzes 17α-hydroxylation of pregnenolone and progesterone and subsequently a 17,20-lyase reaction that generates dehydroepiandrosterone and androstenedione, respectively. While the 17α-hydroxy steroids are substrates for the androgen generating cleavage reaction, they are also precursors to glucocorticoids. Selective inhibition of the 17,20-lyase reaction to reduce androgen levels, without disrupting glucocorticoid production, is a current goal in prostate cancer therapy. The objective of this investigation was to evaluate the chemical and kinetic mechanisms of human P450 17A1 that enable catalysis of two distinct reactions, with the purpose of contributing greater insight for the development of cleavage-selective inhibitors. The chemical mechanism of the 17,20-lyase reaction was assessed and the results provide compelling support for a Compound I-mediated mechanism but have not ruled out a ferric peroxide mechanism. Novel hydroxylation products were identified in the course of these studies. The majority of the new products were 16-hydroxy steroids and, remarkably, hydroxylation of a B-ring carbon was also observed (6β,16α,17α-trihydroxyprogesterone). One critical factor for reaction-selective inhibition of P450 17A1 is the processivity in catalyzing the sequential reactions. The kinetic mechanism of human P450 17A1 was evaluated using steady-state and pre-steady-state methods, and a minimal kinetic model was developed using KinTek Explorer® software. Human P450 17A1 was primarily distributive in catalyzing the two reactions but was more processive with pregnenolone than with progesterone. Furthermore, cytochrome b5 (b5) enhanced the processivity and the results indicate that it functions as a shunt towards sex hormone production. A theoretical model was proposed that includes multiple enzyme conformations, including a more efficient, processive conformation that may be stabilized by b5. As such, selective inhibition of the 17,20-lyase reaction is complicated by the propensity of human P450 17A1 to catalyze the sequential reactions processively when b5 is present, and it is possible that it may only be accomplished by disrupting the interactions between the two proteins.