The ocular lens is a transparent optical element that focuses light onto the retina for clear vision. The bulk of the lens is composed of elongated, anuclear fiber cells, which are continuously synthesized throughout life and added to older, mature fiber cells in the center of the lens. Mature fiber cells experience no cellular turnover, and the lens contains very long-lived proteins that become modified over time. Changes in protein abundance, solubility, post-translational modifications (PTMs), and protein-protein interactions can modulate protein function during lens development, differentiation, and aging, especially in regions of the lens where no new protein can be synthesized. By spatially characterizing proteins in different regions of the lens, we can gain insight into programmed and age-related modifications that may modulate protein function and therefore affect lens health or cataract development. One critical lens protein is Aquaporin-0 (AQP0), a water channel essential for lens transparency. Here, we used MALDI IMS to characterize several AQP0 PTMs across a wide age range of human lenses. Our results shed light on the recently-discovered fatty acid acylation of AQP0, confirming it is an irreversible modification that remains on the protein during age-related C-terminal truncation. This lipid modification was detected in 2-month and 4-month human lenses, suggesting fatty acid acylation is programmed in young fiber cells. We performed in situ digestion and MALDI IMS to generate AQP0 C-terminal peptide fragments, enabling imaging of deamidated peptide forms for the first time. These unprecedented images revealed rapid deamidation of AQP0 in very young fiber cells, followed by truncation at labile asparagine residues. In a separate study, we investigated changes to a very narrow region of the human lens outer cortex that contains the morphologically-distinct remodeling zone. Using laser capture microdissection (LCM) and quantitative proteomics, our results revealed significant changes between the remodeling zone and surrounding regions. Notably, the vimentin intermediate filament to beaded filament switch occurs at the remodeling zone. Changes to other intermediate filament interacting proteins (IFAPs) were also observed. Together, these experiments advance the lens field by providing additional information on the spatial distribution of lens proteins correlated with development and age.

The pivotal role of the transcription factor p53 in tumor suppression remains unchallenged; however, the role of its family member, p73, in normal cellular function and tumorigenesis is far from certain. The goal of this dissertation is to better understand the mechanisms that regulate p73 function as well as develop significant insight into the biological systems that p73 controls through its transcriptional activity. To elucidate these goals we investigated biological systems in which p73 activity is essential as well as identified putative protein-protein interactions that regulate p73. ¬¬¬ In this dissertation, we discovered that that p73 is a necessary transcriptional regulator of the process of ciliary biogenesis within multiciliated cells (MCCs). Loss of MCCs provided a unifying mechanism for many phenotypes observed in p73 knockout mice including hydrocephalus, hippocampal dysgenesis, sterility and chronic inflammation/infection of lung, middle ear and sinus. We found p73 is expressed in MCCs as well as co-expressed with p63 in a subset of basal, tracheal epithelial cells, suggesting that p73 âmarksâ these cells for MCC differentiation. Furthermore, through in situ p63/p73 ChIP-seq of the murine trachea, we identified genomic binding sites in proximity to genes that regulate MCC differentiation, from cell cycle arrest (Cdkn1a) and amplification of centrioles (Myb) to apical docking of centrioles with components that make up the axoneme [Foxj1 and Traf3ip1]. By combining our ChIP-seq data with RNA-seq of tracheal epithelial cells, we found evidence for p73-dependent, direct and indirect transcriptional regulation of a broad network of cilia-associated genes. In sum, p73 is essential for MCC differentiation, functions as a critical regulator of a transcriptome required for MCC differentiation and, like p63, has an essential role in development of tissues. Research results presented in this dissertation are not only significant value to the understanding of p73 but also to the understanding of MCC differentiation. This work lays essential groundwork for future studies investigating roles and regulation of p73 within the correct biological contexts.

Clostridium difficile is the leading cause of hospital-acquired diarrhea in the United States. The two main virulence factors of C. difficile are the large toxins, TcdA and TcdB, which enter colonic epithelial cells and cause inflammation, fluid secretion, and cell death. Using a gene trap insertional mutagenesis screen in the human colonic epithelial cell line Caco-2, we identified 45 host genes necessary for TcdB-mediated cytotoxicity. We performed detailed follow up on one of these hits, Poliovirus Receptor Like-3 (PVRL3). Disruption of PVRL3 protein expression using gene-trap mutagenesis, shRNA, or CRISPR-Cas9 mutagenesis resulted in resistance of cells to TcdB. Complementation of the gene-trap and CRISPR mutants resulted in full restoration of TcdB-mediated cell death. Purified receptor ectodomain bound TcdB directly by pull-down outside of the CROPS domain, which had previously been hypothesized to be the receptor binding domain. Pretreatment of cells with a monoclonal antibody specific for PVRL3 or pre-binding TcdB with purified receptor ectodomain also inhibited cytotoxicity. The receptor is highly expressed on the surface epithelium of the human colon and was observed to colocalize with TcdB in both an explant model and in tissue from a patient with pseudomembranous colitis. These data suggest that PVRL3 can serve as a new target for the prevention of TcdB-induced necrosis in C. difficile infection.

Apoptosis signal-regulating kinase 1 (ASK1) is a critical sensor of cellular stress that is capable of integrating several chemically distinct signals into a single response pathway and is believed to play a role in several human pathologies. ASK1 is believed to be regulated both by protein-protein interactions (as a multiprotein complex termed the signalosome) and phosphorylation of several key residues. These two mechanisms have predominantly been studied after activation of ASK1 by H2O2, which represents only one of the several stressors that is known to trigger signaling through the ASK1 pathway. In this dissertation, I examined both regulatory mechanisms of ASK1 in response to activation with 4-hydroxy-2-nonenal (HNE), which is chemically and mechanistically distinct from H2O2. I tested the hypothesis that there is a consensus two-state signalosome that dynamically assembles around ASK1 in response to activation using targeted mass spectrometry assays. I performed absolute quantitation assays on known ASK1-interacting proteins and reported the first stoichiometric estimate for the ASK1 signalosome composition. My data suggests that this complex is stably composed of ASK1, ASK2, and 14-3-3 proteins in a 2:2:1 ratio. Fourteen other protein-protein associations with ASK1 were detected as dynamic in response to HNE treatment, but appear to be transient in nature. Thus it is likely that the ASK1 signalosome is actually composed of a stable core component that transiently associates with other proteins as needed, resulting in the concurrent presence of many different signalosomes in the cell as opposed to two distinct multiprotein complexes. In order to determine if ASK1 senses different stressors by the same mechanism, I treated ASK1 cells with HNE and H2O2 and monitored the dynamic changes in the phosphorylation state of ASK1. I detected unique phosphosites for each of the stressors that exhibited concentration-dependent responses, indicating that ASK1 senses these stressors by different mechanisms. I also detected a core set of phosphorylations on ASK1 that were consistent between both treatments, which likely represent the residues necessary for general regulation of activity. The use of these approaches to monitor protein interaction and phosphorylation state dynamics can be extended to study most other multiprotein complexes with a higher degree of confidence than methods currently commonly employed.

Topoisomerase II plays many essential roles in genome maintenance. To carry out its physiological functions, the enzyme generates transient double-stranded breaks in the DNA to help resolve topological problems that occur naturally. Thus, while essential to cell survival, topoisomerase II has the potential to fragment the genome. Beyond its critical cellular functions, human topoisomerase II is the target for a number of widely prescribed anticancer drugs that are used in the treatment of solid tumors and hematological malignancies. Drugs that target topoisomerase II act by increasing levels of enzyme-mediated DNA strand breaks. Many of these âtopoisomerase II poisonsâ are derived from natural sources. Identification and characterization of topoisomerase II poisons from natural sources can potentially provide novel drug scaffolds or chemopreventative agents. The goals of my research were to identify naturally derived compounds that alter human topoisomerase II? activity and characterize their mechanism of action. I examined compounds that were originally derived from natural sources or synthesized based on a natural parent compound for activity against topoisomerase II?. Naturally occurring polyphenols from the olive plant, such as oleuropein, hydroxytyrosol, and verbascoside, and the soil fungi Septofusidium berolinense, 3,6-dihydroxy-2-propylbenzaldehyde (GE-1) and 2-hydroxymethyl-3-propylcyclohexa-2,5-diene-1,4-dione (GE-2), enhanced human topoisomerase IIalpha-mediated DNA cleavage. The presence of an oxidant increased the potency of catechol- and hydroquinone-based covalent poisons, and was able to convert a reduced quinone that inhibited topoisomerase II activity into a topoisomerase poison. The olive and fungal metabolites were covalent topoisomerase IIalpha poisons and appeared to act at sites outside of the catalytic core. Ellipticine, a natural product first isolated from the Australian evergreen tree, is an antineoplastic agent that intercalates into DNA and alters topoisomerase II activity. Two novel C5-demethylated derivatives, ET-1 and ET-2, were catalytic inhibitors of human topoisomerase II?. The potency of ET-1 and ET-2 appeared to be related to their ability to intercalate into the double helix. The results of these studies may provide a cellular target for some chemopreventative therapies and a platform for developing new anticancer drugs.

Every cell divisions cycle, over 6.8 billion base pairs of DNA must be accurately replicated. To further complicate this process, the DNA is damaged at a rate of ~10,000 lesions per cell per day. To overcome these obstacles, the kinase ATR (Ataxia telangiectasia mutated and RAD3 related) activates every S-phase. ATR maintains genomic integrity through regulation of origin firing, promotion of DNA repair, stabilization of stalled replication forks, modulation of the cell cycle, and regulation of senescence and apoptosis. The activation of ATR is a multistep process and results in phosphorylation of hundreds of downstream substrates. The consequences of which remain mostly undefined. Additionally, many common DNA-damaging chemotherapies activate ATR. As such, ATR is a promising new cancer therapeutic target. Recently developed ATR inhibitors are currently entering phase II clinical trials, however which patients should be treated with these compounds remains unknown. In my dissertation work, I completed several projects, which revolved around modulation of ATR kinase activity. I examined the consequences of ATR inhibition on the replication fork on a single molecule level. ATR inhibition results in rapid slowing of the replication fork and increased origin firing, and in the presence of replication stress and ATR inhibition, replication forks collapse. Further work examining potential ATR autophosphorylation sites identified point mutations dramatically altering the kinase activity. These point mutations suggest the conformational changes undergone within ATR during activation of the kinase involve the HEAT repeats. Last, a whole genome siRNA screen was conducted in combination with the ATR inhibitor to identify new ATR pathway genes as well as identify clinically actionable synthetic lethal relationships. We identified the ATR pathway and DNA replication as the top pathways when lost that result in increased sensitivity to the ATR inhibitor. Our work indicates patients with cancers containing ATR pathway defects will have increased sensitivity to the ATR inhibitor alone or in combination with cisplatin.

Prostaglandin E2 (PGE2), a cyclooxygenase metabolite that generally acts as a systemic vasodepressor, has been shown to have vasopressor effects under certain physiologic conditions. Previous studies have demonstrated that PGE2 receptor signaling modulates angiotensin II (Ang II)-induced hypertension, but the interaction of these two systems in the regulation of vascular reactivity is incompletely characterized. We hypothesized that Ang II, a principal effector of the renin-angiotensin-aldosterone system, potentiates PGE2-mediated vasoconstriction. Here we demonstrate that pre-treatment of arterial rings with Ang II potentiated PGE2-evoked constriction in a concentration dependent manner. Using genetic deletion models and pharmacological antagonists, we demonstrate that this potentiation effect is mediated via concurrent signaling between the angiotensin II receptor 1 (AT1) and the PGE2 E-prostanoid receptor 3 (EP3) in the mouse femoral artery. EP3 receptor-mediated vasoconstriction is shown to be dependent on extracellular calcium in combination with proline-rich tyrosine kinase 2 (Pyk2) and Rho-kinase. Thus, our findings reveal a novel mechanism through which Ang II and PGE2 regulate peripheral vascular reactivity.

Chemotaxis is the directional movement of cells in response to an extracellular chemical gradient. It is thought to be involved in cancer cell metastasis by recognizing chemokines and growth factors; therefore, deregulation of chemotactic pathways can result in increased tumor cell metastasis. The Target of Rapamycin Complex 2 (TORC2) regulates chemotaxis downstream of these signaling molecules, or chemoattractants. Examining the role of TORC2 in chemotaxis of cancer cells could provide insight into the deregulation of signals leading to cancer cell metastasis. The non-tumorgenic cell line MCF10A and the two breast cancer cell lines MCF7 and MDA-MB-231 were employed in this study. Epidermal Growth Factor (EGF) and Insulin-like Growth Factor 1 (IGF-1) showed potential as chemoattractants by stimulating TORC2. Wound healing assays were performed on MCF10A, MCF7, and MDA-MB-231 cells exposed to TOR inhibitors, as well as MCF10A cells with Rictor knocked down. Cells with Rictor knocked down and MCF10A cells exposed to Torin 2 showed a decrease in cell migration. Ibidiμ-Slide chemotaxis slides were used to perform a chemotaxis assay with MCF10A cells in response the EGF. Cells showed greater directionality toward EGF in the experimental well as compared to the control with EGF on both sides of the cell chamber. Future examination of other potential chemoattractants as well as chemotaxis assays with other chemoattractants will give more insight into the goals of this research.

<p> Nutritional iron acquisition by bacteria is well described, but almost nothing is known about bacterial iron export. Here, we show that <i> Bradyrhizobium japonicum</i> MbfA (Blr7895) is an inner membrane protein expressed in cells specifically under high iron conditions. An mbfA deletion mutant is severely defective in iron export activity, contains >2-fold more intracellular iron than the parent strain, and displays an aberrant iron-dependent gene expression phenotype. The findings suggest that iron export plays an important role in bacterial iron homeostasis, and MbfA is responsible for the iron export activity of <i>B. japonicum.</i> The N-terminal Ferritin like domain (FLD) of MbfA is localized to the cytoplasmic side of the inner membrane and is required for export activity. Purified FLD is a dimer in solution implying that MbfA functions as a dimer. </p><p> An <i>mbfA</i> mutant is sensitive to short term exposure to high levels iron or H₂O₂ but not when grown in elevated iron media, suggesting a stress response adaptation. The bfr gene encodes the iron storage protein bacterioferritin. An <i>mbfA bfr</i> double mutant showed a loss of stress adaptation, and had a severe growth phenotype in high iron media. The double mutant exhibits elevated intracellular iron content than the wild type, and displays aberrant gene expression even when grown in relatively low iron media. These results suggest that MbfA and Bfr work in concert to manage iron and oxidative stresses. In addition, the need for iron detoxification is not limited to extreme environments, but is also required for normal cellular function.</p><p> <i>B. japonicum</i> cannot make siderophores for acquisition of iron in aerobic environments. The mechanism of iron uptake in the absence of xenosiderophores is unknown. Exploiting the synthetic lethal phenotype of the <i>mbfA bfr</i> double mutant, we identified suppressor strains that can grow in high iron concentrations. The suppressor strains harbor loss of function mutations in the <i>feoAB</i> operon, which is a ferrous iron transport system. Interestingly, FeoAB system is required for ferric iron utilization and is required for high affinity uptake of both ferric and ferrous iron by <i>B. japonicum. feoB</i> and <i>feoA</i> incited small, poorly developed, non-nitrogen fixing nodules on soybean plants suggesting the requirement of FeoAB system for establishment of symbiosis. A suppressor strain harboring a Glu-40 to Lys mutation in FeoA (<i>feoAE40K </i>) has diminished but measurable iron uptake activity in free living cells. It elicited nitrogen fixing nodules on soybean but the bacteroids in the nodules displayed lower iron uptake activity compared to wildtype bacteroids. This strongly suggests that the FeoAB transport system is involved in iron uptake within symbiotic bacteroids. Thus our results indicate that <i>B. japonicum</i> employs a single iron transporter to adapt to diverse environmental conditions.</p>

<p>The outer-leaflet of the outer membrane of Gram-negative bacteria is composed of lipopolysaccharide (LPS), which is attached to the membrane via a hexa-acylated saccharolipid called lipid A. The fourth step of lipid A biosynthesis involves the cleavage of the pyrophosphate group of UDP-2,3-diacyl-GlcN to form lipid X; this step is carried out by LpxH in E. coli and the majority of Gamma- and Beta-Proteobacteria. LpxH has been previously characterized, however sample impurity and non-optimized assay conditions hindered meaningful conclusions. The enzyme was suggested to contain signature motifs found in the calcineurin-like phosphoesterase (CLP) family of metalloenzymes, however the extent of biochemical data fails to demonstrate a significant level of metal activation in LpxH assays. We report cloning, purification, and detailed enzymatic characterization with a highly purified sample of H. influenzae LpxH (HiLpxH). HiLpxH shows over 600-fold stimulation of activity in the presence of Mn2+. Furthermore, EPR studies reveal the presence of a Mn2+ cluster in LpxH. Finally, point mutants of residues in the conserved metal-binding motifs of the CLP family greatly inhibit HiLpxH activity, highlighting their importance in enzyme function. Overall, through optimized purification and assay methods, our work unambiguously establishes LpxH as a membrane-associating CLP containing a Mn2+ cluster coordinated by conserved residues. These results set the scene for further structural investigation of the enzyme and for design of novel antibiotics targeting lipid A biosynthesis.</p><p>Several species of Gram-negative bacteria lack LpxH orthologs, yet retain other lipid A biosynthetic enzymes and still produce lipid A. An unrelated protein, LpxI, is responsible for UDP-DAGn hydrolysis is several such organisms. Interestingly, some bacteria, such as the human pathogen Chlamydia trachomatis, have neither LpxH nor LpxI orthologs, suggesting the presence of a third UDP-DAGn hydrolase. Through implantation of a novel complementation screen that used a C. trachmatis genomic library and a conditional-lethal lpxH mutant E. coli strain, we were able to identify an open reading frame encoding an new enzyme capable of lipid X production. Due to its ability to complement UDP-DAGn hydrolase function in vivo and catalyze the formation of lipid X in vitro, we have designated the enzyme LpxG. Further biochemical analysis with purified LpxG revealed it facilitates hydrolysis through attack on the alpha phosphate of its substrate and is activated by Mn2+ in vitro. LpxG is in the same CLP superfamily as LpxH, however it shows very little homology to LpxH or LpxI. Identification of LpxG improves our understanding of the lipid A biosynthetic pathway in C. trachomatis. More broadly, as limited genetic tools are available for the study of the prevalent pathogen, it provides an advantageous method for the functional screening of other C. trachomatis genes.</p> / Dissertation