<p> Histidine kinase CheA is central to signaling in bacterial chemotaxis. This kinase is responsible for the phosphorylation of two response regulators, CheY and CheB. CheY controls flagellar rotation and thus motility. CheB is crucial for sensory adaptation. CheA is dramatically activated, up to 1000-fold, and put under the control of chemoreceptors by formation of the signaling complex. As measured by phosphorylation of CheY, this control modulates the activity of CheA in a range as wide as two orders of magnitude. This change in the activity of CheA is the essence of chemotactic signaling. However, the enzymatic properties altered by kinase incorporation into signaling complexes, chemoreceptor ligand binding or receptor adaptational modification are largely undefined. This dissertation describes the kinetic analysis of kinase CheA. Data are fit to the Michaelis-Menten equation, from which important parameters K_M and k_cat are obtained. Based on these parameters for CheA at different signaling conditions, important observations and conclusions are made to contribute to better understanding of the control of the activity of kinase CheA, thus the bacterial chemotaxis signaling system. </p><p>

<p> Head and neck cancers (HNCs) account for a small percentage of all cancers in the United States, but their localization complicates surgical treatment. Therefore, chemotherapy is the most viable treatment option. In this study, the hypopharyngeal carcinoma cell line FaDu was treated with collagen/cell-penetrating hybrid peptides that have a potential to be effective drug-carriers. Confocal microscopy shows that one of the hybrid peptides, FL8V1, is able to function as an effective drug carrier: it is internalized by FaDu via endosomal uptake and manages to escape from the endosome before it matures into a lysosome. </p><p> In addition, it was observed an unusual localization of hybrid peptides at the cell surface, that was not observed in previous experiments involving different malignant and healthy cells. Initially, it was hypothesized that the studied peptides interact with the tight junctions of the FaDu cell membrane. However, colocalization experiments performed with one of the hybrid peptides and the tight junction protein JAM-A in FaDu cells showed that they did not colocalize. Subsequently, the salt wash of FaDu cells was performed to determine if the interaction of peptide with the FaDu cell surface was specific or non-specific. These experiments showed that the interactions were non-specific. Compared to other cell lines studied in the past, FaDu is the only cell line that exhibits this behavior, therefore it is speculated that the FaDu cell membrane is uniquely polarized in comparison to other malignant cells. More studies are required to assess the reason for the hybrid peptide preconcentration at the FaDu cell surface. The differences in the behavior between FaDu and other malignant cells may lead to clinical applications in the treatment of HNCs in the future.</p><p>

<p> A comprehensive understanding of the design of proteins puts heavy emphasis on certain key residues. These key residues can often be identified by the level of conservation in nature, which acts as a reliable witness mark in order to study the pressures that select for residues which play a critical role in the function of the protein. In the case of triosephosphate isomerase (TIM), the fifth enzyme in glycolysis, the second-shell residue Glu97 has been found to be fully conserved across all known TIM sequences. Its proximity to the active site as well as several previous studies has pointed to a possible direct role in catalysis. However, the present study shows when Glu97 is mutated to Ala, Gln, and Asp in <i>Trypanosoma brucei brucei</i> (tbb) that the resulting effects on <i>k</i>_cat are small. Previous results from other studies that have observed larger mutational effects may be due to nearby non-conserved residues that are specific to the TIM homolog in which these studies are performed. The structural studies detailed here suggest that instead, Glu97 is involved in the structural stability of the enzyme, as well as participating in dimer formation. Size-exclusion chromatography analysis suggests that several <i>tbb</i>TIM mutants may in fact be monomeric.</p><p>

SNARE proteins are the engines that drive membrane fusion throughout the cell. They provide this energy by zippering up into a parallel four helix bundle in a thermodynamically favored process. Because the zippering of SNAREs is spontaneous, fusion events occur immediately upon a vesicle interacting with its target membrane. But, in certain circumstances, such as in synaptic vesicles, spontaneous fusion is not desired, so a clamp protein is necessary to prevent this fusion until signaled to do otherwise. In synapses, this protein is called Complexin and a second protein, called Synaptotagmin, releases the clamp upon a rapid influx of calcium, the hallmark of an action potential. How Complexin clamps is a subject of great interest in the field, and an area of active research. What is known is that a so-called Accessory helix (residues 28-47) is responsible for clamping, while another, Central Helix (reisudes 48-70) is responsible for physically binding to the helix. A recently solved crystal structure revealed how CPX might behave before the SNAREs fully zipper, namely that the accessory helix extends away from the SNAREs at a 45° angle. But, because of the packing of the crystal, it is entirely possible that the crystal is an artifact of packing, and/or truncationIn this thesis, my work first validates the crystal structure, using a FRET pair I developed for this purpose. I establish that the angled-out positioning of the accessory helix does, in fact, occur in solution, and is not due to crystal packing or the truncation of the VAMP2 (the neuronal vesicle-associated SNARE), but rather is due to the fact that its C-terminus is not present. I describe a mechanism by which Complexin can clamp. Further, I demonstrate that the residues in VAMP2 which are responsible for the switch from the "open" to the "closed" conformation are a patch of asparatates in VAMP2 (residues 64, 65, an 68). I also establish that these three aspartates are responsible for the release of the clamp and that without them, Complexin cannot be brought into the angled-in configuration. I propose a model for how the clamp might be released by Synaptotagmin.

Contemporary interpretations of Crick's original Adaptor Hypothesis view both the amino acid and the tRNA body as passive participants during aminoacyl-tRNA (aa-tRNA) selection by the ribosome. Recent experimental evidence investigating tRNA mutants that miscode due to mutations that lie outside the anticodon as well as data from the unnatural amino acid mutagenesis field that shows that different unnatural amino acids, when esterified to the same suppressor tRNA, produce different amounts of protein, suggest that aa-tRNA selection is considerably more complex than originally envisioned in Crick's Adaptor Hypothesis and that aa-tRNA selection may extend to the amino acid itself. Here, using unnatural amino acids as substrates on natural, fully modified tRNAs and a highly purified in vitro translation system, we investigate the substrate specificity of the ribosome with respect to the amino acid.

Structural changes are critical to the ability of proteins, particularly enzymes, to carry out their biological function. However, flexibility also leaves proteins vulnerable to denaturation and degradation; thus a balance must be struck between the dynamics required for function and the rigidity required for maintaining a globular protein's characteristic folded structure. These relationships have been studied in detail through comparison of homologous proteins from organisms adapted to varying properties of the bulk environment. In particular, organisms adapted to temperature extremes offer fruitful platforms for the investigation of adaptive changes in protein stability as a function of environmental pressures. Thermostable proteins are widely reported to be more rigid than their homologs from mesophilic organisms, and those from psychrophiles more flexible; this suggests the possibility of evolutionary conservation of the balance between dynamics and stability. Thus specifically functional aspects of protein dynamics may be isolable through the comparative analysis of members of protein families from organisms adapted to different thermal environments. The best experimental tool for characterizing internal conformational dynamics of proteins on a range of timescales and at site-specific resolution is nuclear magnetic resonance (NMR) spectroscopy, which has found widespread use in the study of protein flexibility and dynamics. However, it is often difficult to provide a detailed structural interpretation of NMR observations. This gap can be bridged using molecular dynamics (MD) simulations, which can directly simulate motional processes that have been observed experimentally. The potential for deep synergy between these two complementary tools has been recognized since MD methods were first applied to biological macromolecules, and recent technological developments have reinforced the mutually beneficial relationship between the two techniques. Ribonuclease HI (RNase H), an 18 kD globular protein that hydrolyzes the RNA strand of RNA:DNA hybrid substrates, has been extensively studied by NMR to characterize the differences in dynamics between homologs from the mesophilic organism \textit{E. coli} and the thermophilic organism \textit{T. thermophilus}. However, these dynamic differences are subtle and difficult to interpret structurally. The series of studies described in the present work was conceived in the pursuit of an improved understanding of the complex relationships between protein dynamics, activity, and thermostability in the RNase H protein family. The organizing principle of the work presented herein has been the close coupling between molecular dynamics simulations and NMR observations, permitting both validation of the MD trajectories by rigorous comparison to experiment and improved interpretation of the dynamics observed by NMR. Previous NMR observations of E. coli and T. thermophilus are integrated into an interpretive framework derived from simulations of the larger RNase H family. First, comparative analysis of molecular dynamics simulations of a total of five homologous RNase H families from organisms of varying preferred growth temperature reveals systematic differences in the conformational dynamics of the handle region, a loop previously identified as contributing to substrate binding. Second, analysis of the effects of activating mutations on the dynamics of ttRNH identifies rotamer dynamics whose contributions to increased catalytic activity can be rationalized in the context of observed differences in sidechain orientation in the wild-type ecRNH and ttRNH simulations. Third, a combined MD-NMR study finds that the active site residues of ecRNH, and likely of the entire RNase H family, are rigid on the ps-ns timescale while undergoing substantial conformational exchange upon Mg2+ binding; this suggests that the active site is electrostatically preorganized for binding the first metal ion, which in turn induces dynamic reorganization at longer timescales. Finally, long-timescale simulations of the RNase H family, despite unexpected local unfolding for some family members, identify handle-loop and rotamer preferences for the C. tepidum RNase H (ctRNH) homolog that unexpectedly differ from those observed for ecRNH and ttRNH, and which can be experimentally tested by NMR spectroscopy of this recently characterized and less well-studied example of an RNase H homolog from a thermophilic organism.

The development of orally bioavailable small molecule drugs targeting protein-protein interactions (PPIs) has been challenging1. Unlike conventional targets, PPIs’ extended, open surface makes it difficult for small molecules to bind. In order to achieve strong binding, it is frequently necessary to use larger molecules, which traditionally is considered to disfavor druglikeness2. However, PPIs possess great therapeutic potential due to their abundance and regulatory roles in cells3. More extensive studies are needed to identify larger chemotypes that retain good druglike properties and therefore might have utility against PPI targets. NF-κB Essential Modulator (NEMO), interacting with IκB Kinase subunit β (IKKβ), is an important PPI target because of its regulatory role in NF-κB signaling4. Literature suggests that the N-terminal domain of NEMO is intrinsically disordered in the absence of bound ligand5. To test this hypothesis, I developed variants of the NEMO N-terminal domain, and studied their secondary structure, stability, and affinity for IKKβ, showing that the N-terminal domain of NEMO is intrinsically structured (Chapter Two). I also characterized partially peptidic NEMO inhibitors from our collaborator, Carmot Therapeutics. We tested the binding of these compounds and their peptidic fragments to full-length NEMO using fluorescence anisotropy (FA)6 and surface plasmon resonance (SPR). The results provided information about hit validity, binding affinity and kinetics (Chapter Three). Macrocycles are of interest for inhibiting PPIs partly because of their proposed good membrane permeability7. To evaluate this hypothesis, I implemented a membrane permeability assay, tested the permeability of a set of macrocyclic compounds, and used the results to develop a multiple linear regression model to predict permeability from macrocycles’ physicochemical properties. The model suggests that hydrophobicity correlates positively with good permeability, while high polarity or high aromatic ring count renders macrocycles less permeable (Chapter Four). Finally, in a separate project, to elucidate the origins of protein-ligand binding energy between interleukin-2 (IL-2) and its known small molecule inhibitors8, I developed a SPR based binding assay, and validated it by showing that the KD value of known inhibitor Ro26-45508 agrees with the literature value (Chapter Five). The assay will be useful in future studies of IL-2 inhibitors and their fragments.

Epithelial-derived cancers such as breast cancer consist of transformed epithelial cells surrounded by a stroma that contains numerous cell types. Understanding the kinetics of stromal activation and characterizing both the cell types involved in the cancer stroma as well as their secreted proteins may lead to future therapies to stop breast cancer progression. It is unknown how stromal cells communicate with transformed epithelial cells to promote tumor growth and metastasis. The transforming growth factor β (TGFβ) pathway promotes epithelial to mesenchymal transition (EMT). Our lab has identified a secreted protein, aortic carboxypeptidase-like protein (ACLP) that is expressed in the breast cancer stroma and can exert effects by activating the TGFβ pathway. The role of ACLP in EMT during cancer progression is not known. We hypothesize that changes in the breast cancer stroma drive cancer progression and correlate with increased expression of α-smooth muscle actin (α-SMA) and collagen I (Col I) in stromal fibroblasts and that ACLP promotes EMT in the cancerous mammary epithelium. The first goal of this project was to develop an in vivo mouse model to first track and then isolate activated cells in the breast cancer stroma. The second goal was to test whether ACLP induces gene expression changes consistent with EMT. To identify changes in the breast cancer stroma we crossed mice expressing the Her2/neu receptor, which is part of the epidermal growth factor receptor family and occurs in 20-30% of all human breast cancers, with transgenic mice harboring α-SMA (SMA-mCherry) and collagen (Col I-Tpz) promoters driving red and green variants of green fluorescent protein (GFP) respectively. Initial experiments characterized the expression of these reporters at baseline and in tumors. Both the tumor and mammary gland tissue were isolated and a new method to detect fluorescent reporter activity was developed. Whole mount and sections of the tumors and the surrounding stroma defined at least two distinct cell populations: those that express SMA and those that express Col I, and a possibility of a third population expressing both. In contrast to the stroma of a normal mammary duct, which contained dispersed Col I+ cells and not cells of the other two populations, the stroma in the tumor sections contained a population of SMA+ cells and an increase in Col I+ cells dispersed on the periphery of the tumor. Aligned streaks of Col I+ cells were identified. Normal murine mammary gland (NMuMG) cells treated with ACLP showed a decrease in E-cadherin mRNA levels and increase in vimentin mRNA levels. Immunofluorescence staining revealed decreased E-cadherin on the cell borders after ACLP treatment, and, when also treated with TGFβ results in increased detected intracellular vimentin. In summary, we developed a new model to detect changes in the stroma associated with breast cancer development as well as a procedure for preserving fluorescence in mouse tissue for analysis. We also found evidence to support the idea that ACLP promotes EMT. Ongoing work will characterize the stromal cells by flow cytometry and expression profiling and develop strategies to target stroma-derived proteins to treat breast cancer. / 2019-06-30T00:00:00Z

Tumorigenesis is a complex process that begins with the accumulation of several aberrant gene mutations that often favor cell proliferation while antagonizing anti-proliferative signaling pathways. Tumor cells acquire additional genetic alterations, which allow them to change their cellular metabolism, promote angiogenesis, invade tissue, and metastasize. Together, the genetic changes that abrogate the function of tumor suppressors and promote oncogenesis are key to cellular transformation and tumorigenesis. A novel Ig domain-containing adhesion molecule (Ig-CAM), Transmembrane and Immunoglobulin (Ig) Domain-containing 1 (TMIGD1), was recently identified in our laboratory. TMIGD1 is expressed in renal epithelial cells and plays a protective role against oxidative cell injury. TMIGD1 expression is downregulated in human renal cancers. However, the mechanisms of downregulation of TMIGD1 in renal cancer have yet to be defined. In this study, we investigated the transcriptional regulation of TMIGD1 using a green fluorescent protein (GFP) reporter assay. The proximal promoter of human TMIGD1 contains multiple CCAAT box sequences with the GGCCAATCT consensus, which are putative binding sites for the transcription factor CCAAT/Enhancer-binding protein (C/EBPβ). This study demonstrates that the transcriptionally active C/EBPβ/LAP activates the TMIGD1 promoter, while the transcriptionally inactive C/EBPβ/LIP inhibits TMIGD1 promoter activity. When the putative CCAAT box sequences are deleted from the TMIGD1 promoter, C/EBPβ/LAP no longer has a marked effect on the promoter activity. Through additional analysis, we show that several transcription factors and proteins that are commonly implicated in renal cancers, including pVHL, HIF1α, HIF2α, β-catenin, and APC, do not seem to have a noticeable effect on TMIGD1 promoter activity. Collectively, the present study identifies C/EBPβ as an important transcription factor in the transcriptional regulation of TMIGD1. Furthermore, the study suggests a possible role for C/EBPβ in downregulation of TMIGD1 in renal cancer. / 2018-07-11T00:00:00Z

Angiogenesis, the formation of new vessels, is a highly regulated and complex cellular process, which plays a crucial role in physiological processes such as embryological development and wound healing. Aberrant angiogenesis is a key feature of common human pathologies, including cancer and inflammation. Neurogenic locus notch homology protein 2 (NOTCH2) signaling is an evolutionarily conserved pathway and a major player in regulating angiogenesis. Despite its fundamental involvement in both embryonic development and human diseases, the processes through which the NOTCH pathway modulates angiogenesis are not fully elucidated. We have identified Major Intrinsically disordered NOTCH2-Associated Receptor (MINAR) as a novel ligand for NOTCH2. The main objectives of this project were to demonstrate the mechanism of association between MINAR with NOTCH2, and its biological importance in angiogenesis. Our findings reveal that MINAR is an intrinsically disordered cell surface receptor, which is highly expressed in endothelial cells and other tissues of human vasculature. The physical association between MINAR and NOTCH2 increases its order and stability, and also reduces the degradation of MINAR. Moreover, we demonstrate that MINAR regulates NOTCH2 activation to inhibit angiogenesis. Taken together, the data suggest that MINAR is a novel ligand of NOTCH2 and a key regulator of angiogenesis. / 2018-07-11T00:00:00Z