Browsing by Subject "Biochemistry"
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Item Open Access A Comprehensive Study of Guanosine-5'-triphosphate Hydrolysis by the Bacterial Cell Division Protein FtsZ(2018) Salsburg, AndrewThe bacterial protein FtsZ plays a vital role in cytokinesis in prokaryotes as it polymerizes to form an FtsZ ring (Z ring) at the division septum midcell. FtsZ exhibits a GTP hydrolysis activity and attempts have been made to model the kinetics of this process. There is a major discrepancy, however, over the concentration of GTP needed for activity. The dissociation constant (KD) between wild-type FtsZ protein and GTP was measured to be 30 nM using isothermal titration calorimetry. In contrast, several research groups have reported that GTP hydrolysis required GTP concentrations in the millimolar range. They used Michaelis-Menten kinetics to model the GTP concentration dependence and obtained an apparent binding constant (Km) in the range of 300-1,000 µM GTP. Km and KD are not identical measures of binding given that they differ by the kinetic constant governing catalysis, kcat, but we suggest that a five order of magnitude difference between the values is unprecedented and this was a problem that needed investigating.
My overall goal in this work has been to perform a comprehensive in vitro study of the FtsZ GTP hydrolysis activity using an enzyme-coupled regenerating system. With this system rates of GTP hydrolysis by FtsZ are obtained spectrophotometrically. I have confirmed for wild-type FtsZ that GTP hydrolysis rates show little to no dependence on GTP concentrations in the range of 50-3,000 µM, contradicting the high values of Km reported in some previous studies. Since we have failed to reproduce the high Km with three different preparations of FtsZ protein, we cannot propose a definitive mechanism for the previous results.
I also measured the GTP hydrolysis of several mutants of FtsZ: E238A, L169R, and FtsZ84 (G105S). I investigated each of these mutants to see if they had a high apparent Km. FtsZ84 had a low overall hydrolysis rate, but did show a large increase in hydrolysis rates when GTP was increased from 50-3,000 µM. We hypothesize that a lower affinity for GTP is not a Michaelis-Menten Km, but likely a reflection of a weak binding of GTP by FtsZ84, giving KD in the millimolar range.
Item Open Access A Novel DNA Damage Response Network Associated with the CTD of RNA Polymerase II(2012) Winsor, Tiffany SabinSince RNA Polymerase II (RNAPII) transcribes much of the genome, it is well situated to encounter and initiate a response to various types of DNA damage. However, to date very little is known about any response of RNAPII to DNA damage outside of Transcription Coupled Nucleotide Excision Repair (TC-NER). A link between DNA damage response mechanisms and the C-terminal domain of RNAPII (CTD) is suggested by an overlap between proteins that bind the CTD and genes required for resistance to DNA damaging agents. In this thesis, I show that proper deployment of CTD associated proteins is required to respond to DNA damaging agents. Furthermore, I show that a CTD associated protein (Set2) is required for response to DNA damage, but its catalytic activity is not. Finally, I show that the recombinational ability of strains lacking the CTD kinase, Ctk1, is deficient. Based on these lines of evidence, I propose a novel CTD Associated DNA Damage Response (CAR) system of proteins that is required for proper response to DNA damaging agents.
Item Open Access A Novel Vascular Graft Diagnostic and Reversible Aptamers for the Purification of Therapeutic Cells(2017) Nichols, Michael DouglasCreation of novel tools for biomedical applications is critical for the improvement of patient diagnostics and therapeutics. Two particularly important needs lie in (1) improved in vitro testing and increased performance of prosthetic vascular grafts and (2) purification methods for cells that do not compromise their utility. Progress in these areas is urgently needed and would facilitate the availability of higher quality devices and treatments that raise the quality of patient care. This work focused on developing new approaches toward that goal.
A tremendous and immediate need exists for high-performance small-diameter synthetic vascular grafts, as a fifth of the 500,000 annual coronary artery bypass grafting (CABG) patients lack suitable autologous vessels for revascularization. This problem has driven intense research and development of increasingly diverse prosthetics that could be viable alternatives in the years to come. Evaluating these designs in vitro offers high-throughput, low-cost screening for promising graft technologies ahead of more stringent vetting in vivo.
Offering a fresh take on assessing vascular graft thrombogenicity in vitro, the buildup of pressure upstream to a clot was used as a metric to quantify the physical interaction between the graft lumen and a maturing thrombus. A closed tubing system was devised and continuously monitored as clotting solutions of fibrin glue, platelet-rich plasma or whole blood were cured to varying maturities and then purged from small-diameter ePTFE grafts or Tygon graft mimics. This approach provided insight into how blood flow resistance is influenced by a number of clinically relevant factors, such as the level of vessel occlusion and the physical nature of the resident coagulum.
Endothelialization of synthetic vascular grafts yields viable alternatives to native vessels and can be accomplished non-invasively using late-outgrowth endothelial progenitor cells (LO-EPCs) isolated from peripheral blood. However, the time required to amass sufficient cells to prepare a graft with current methods is risky for waiting CABG patients. An ambitious approach conceived to significantly decrease this wait period involved developing affinity ligands selective for LO-EPCs that would enable their capture directly from the circulation to facilitate rapid amassment. An in vitro directed evolution strategy to generate aptamers, the nucleic acid analogs of antibodies, that specifically bind these cells was carried out with initially promising but ultimately unsuccessful results. While the particular strategy executed here did not prevail, the high value and impact LO-EPC aptamers would deliver merit revisiting this work with a revised strategy such as the one proposed in this document.
Purification of autologous and allogenic cells is essential for their use in a variety of therapeutic and basic research applications in addition to augmenting graft performance. However, the antibody stains conventionally used to selectively purify cells are permanent and their continued presence can elicit an immune response in vivo and compromise native cell behavior. To avoid these issues, a cell purification strategy was crafted utilizing aptamers and matched oligonucleotide antidotes that enabled reversible cell staining. The reversible stains were robust enough for cell purification via fluorescence-activated cell sorting (FACS) yet subsequently able to be removed with gentle heat treatment and antidote. Importantly, cell function that was compromised without antidote was rescued to match the native behavior of non-stained cells following purification and antidote treatment.
Item Open Access A Role for Cytoplasmic 3'-Nucleotide Hydrolysis in Liver and Intestine Function(2012) Hudson, BenjaminBisphosphate 3'-nucleotidase (Bpnt1) is a member of a family of small molecule phosphatases whose activities depend on divalent cations and are inhibited by lithium. While the enzymes share many commonalities, they have distinct and non-overlapping substrate pools. Of the seven mammalian members, two enzymes, gPAPP and Bpnt1, hydrolyze the same small molecule 3'-phosphoadenosine 5'-phosphate (PAP) but act in separate subcellular compartments, the Golgi apparatus and cytoplasm respectively. Hydrolysis of PAP, which is a metabolite of the inorganic sulfate incorporation pathway, is highly conserved throughout evolution from bacteria to yeast to humans. Evidence in multiple species has shown that inhibiting PAP hydrolysis leads to cellular toxicity as a result of its accumulation and also that these effects can be ameliorated by modulating the rate of its production. However, despite the abundant evidence of its importance
from studies in lower eukaryotes, the role of the cytoplasmic PAP phosphatase, Bpnt1, in more complicated mammalian physiological remains poorly understood. Here we report for the first time the generation and characterization of mice deficient for Bpnt1. Bpnt1 null mice do not exhibit skeletal defects, but instead develop severe liver
pathologies and deficiencies in intestinal iron absorption. Loss of Bpnt1 leads to tissue-specific elevations of the substrate PAP. To test the hypothesis that a toxic cellular accumulation of PAP accounts for the observed phenotypes, we generated a double mutant mouse that concomitantly down regulates bisphosphorylated nucleotide synthesis in the context of Bpnt1 deficiency. Remarkably, double mutants do not display any detectable physiological defects seen in Bpnt1 null mice. In addition, we have identified and characterized a novel substrate of 3'nucleotidases, 3'-phosphoadenosine 5'-diphosphate (PAPP) that co-accumulates with PAPS and PAP and might play a role in mediating certain aspects of the physiological defects of Bpnt1 null mice. Overall, our study defines a role for Bpnt1 in mammalian physiology and provides mechanistic insights into the importance of cytoplasmic 3'-
nucleotide hydrolysis to normal cellular function.
Item Open Access A Sweet Embrace: Control of Protein-Protein Interactions by O-Linked β-N-Acetylglucosamine.(Biochemistry, 2018-01) Tarbet, Heather J; Toleman, Clifford A; Boyce, MichaelO-Linked β-N-acetylglucosamine (O-GlcNAc) is a critical post-translational modification (PTM) of thousands of intracellular proteins. Reversible O-GlcNAcylation governs many aspects of cell physiology and is dysregulated in numerous human diseases. Despite this broad pathophysiological significance, major aspects of O-GlcNAc signaling remain poorly understood, including the biochemical mechanisms through which O-GlcNAc transduces information. Recent work from many laboratories, including our own, has revealed that O-GlcNAc, like other intracellular PTMs, can control its substrates' functions by inhibiting or inducing protein-protein interactions. This dynamic regulation of multiprotein complexes exerts diverse downstream signaling effects in a range of processes, cell types, and organisms. Here, we review the literature about O-GlcNAc-regulated protein-protein interactions and suggest important questions for future studies in the field.Item Open Access A tale of two metallophosphatases: biochemical and functional characterization of novel substrates of PP1 and MESH1(2017) Rose, Joshua StevenAddition and removal of phosphate is an important post-translational modification involved in cellular signaling. The enzymes responsible for removing this phosphorylation mark, called phosphatases, play a vital role in the cellular decision making processes. In this work we discuss two discoveries, a novel enzyme for a known signaling function involving control of transcription and a novel target for an important cellular stress response enzyme.
In the first project we sought to determine a novel enzyme responsible for dephosphorylating the C-terminal domain of RNA polymerase II. This domain serves as a vital signaling platform for transcription of mammalian genes, with the ability to recruit cofactors that bind to specific patterns of phosphorylation throughout its repeating amino acid sequence. Using a functional assay for phosphatase activity at the Thr4 position we biochemically isolated the unknown enzyme and identified it as PP1 and validated its function in vitro and in vivo.
The second phosphatase studied in this dissertation is MESH1—a mammalian ortholog of the bacterial stringent response protein SpoT that dephosphorylates ppGpp. Because ppGpp is absent in mammalian cells MESH1 lacks a viable target. We established NADPH as a substrate of MESH1 biochemically and corroborated these results by determining the substrate bound structure. Our results reveal a novel regulatory role of MESH1 in a pathway that resembles the bacterial stringent response.
Item Open Access A Theoretical and Experimental Study of DNA Self-assembly(2012) Chandran, HarishThe control of matter and phenomena at the nanoscale is fast becoming one of the most important challenges of the 21st century with wide-ranging applications from energy and health care to computing and material science. Conventional top-down approaches to nanotechnology, having served us well for long, are reaching their inherent limitations. Meanwhile, bottom-up methods such as self-assembly are emerging as viable alternatives for nanoscale fabrication and manipulation.
A particularly successful bottom up technique is DNA self-assembly where a set of carefully designed DNA strands form a nanoscale object as a consequence of specific, local interactions among the different components, without external direction. The final product of the self-assembly process might be a static nanostructure or a dynamic nanodevice that performs a specific function. Over the past two decades, DNA self-assembly has produced stunning nanoscale objects such as 2D and 3D lattices, polyhedra and addressable arbitrary shaped substrates, and a myriad of nanoscale devices such as molecular tweezers, computational circuits, biosensors and molecular assembly lines. In this dissertation we study multiple problems in the theory, simulations and experiments of DNA self-assembly.
We extend the Turing-universal mathematical framework of self-assembly known as the Tile Assembly Model by incorporating randomization during the assembly process. This allows us to reduce the tile complexity of linear assemblies. We develop multiple techniques to build linear assemblies of expected length N using far fewer tile types than previously possible.
We abstract the fundamental properties of DNA and develop a biochemical system, which we call meta-DNA, based entirely on strands of DNA as the only component molecule. We further develop various enzyme-free protocols to manipulate meta-DNA systems and provide strand level details along with abstract notations for these mechanisms.
We simulate DNA circuits by providing detailed designs for local molecular computations that involve spatially contiguous molecules arranged on addressable substrates via enzyme-free DNA hybridization reaction cascades. We use the Visual DSD simulation software in conjunction with localized reaction rates obtained from biophysical modeling to create chemical reaction networks of localized hybridization circuits that are then model checked using the PRISM model checking software.
We develop a DNA detection system employing the triggered self-assembly of a novel DNA dendritic nanostructure. Detection begins when a specific, single-stranded target DNA strand triggers a hybridization chain reaction between two distinct DNA hairpins. Each hairpin opens and hybridizes up to two copies of the other, and hence each layer of the growing dendritic nanostructure can in principle accommodate an exponentially increasing number of cognate molecules, generating a nanostructure with high molecular weight.
We build linear activatable assemblies employing a novel protection/deprotection strategy to strictly enforce the direction of tiling assembly growth to ensure the robustness of the assembly process. Our system consists of two tiles that can form a linear co-polymer. These tiles, which are initially protected such that they do not react with each other, can be activated to form linear co-polymers via the use of a strand displacing enzyme.
Item Open Access Achieving Cell-Specific Delivery of Multiple Oligonucleotide Therapeutics with Aptamer Chimeras(2012) Kotula, Jonathan WCurrent standard cancer treatments such as chemotherapeutics, and radiation therapy are nearly as likely to kill the patient as cure the cancer. Therapies that have such a narrow window of efficacy are necessary for the treatment of aggressive diseases, but safer alternatives must be created. By discovering novel therapeutics that target specific disease processes within specific diseased cells, while leaving healthy cells unaltered, we can improve the lives of millions of cancer sufferers and their families. A therapeutic's window of efficacy can be measured by the therapeutic index. For many anti-cancer therapeutics, the therapeutic index is very small, the dose of treatment that kills cancer cells and shrinks tumors is nearly the dose that causes toxicity. In cancer patients, this toxicity causes many serious conditions such as gastrointestinal distress, organ damage, and death.
Recently, the model of cancer treatment has evolved from non-specific cytotoxic agents to more selective therapeutics that target cellular processes necessary for cancer cell survival. If a therapy can be targeted to selectively bind and internalize targeted cells, its toxicity would only impact the targeted cells and healthy cells in the immediate vicinity, which would greatly reduce the toxic effects on the rest of the body. Targeting cancer cells can be done through cancer biomarkers, which are cell surface proteins, expressed exclusively, or are much more abundant on the surface of cancer cells than on somatic cells.
Advances in antibody and aptamer technology have enabled researchers to design those molecules to bind specifically to cancer cells, and deliver drugs that alter specific cellular processes. An aptamer designed to bind PSMA, a prostate cancer biomarker, only bound to a specific subset of cancer cells, and delivered a therapeutic siRNA that prevented a specific survival process from occurring. While this technology is promising, it is currently limited to targeting small subsets of cancer types. To generate an aptamer therapeutic that would have greater utility and efficacy, I have examined the properties of a nucleolin aptamer-mediated delivery system that targets multiple types of cancer cells, and delivers various oligonucleotide therapeutics.
The nucleolin aptamer targeted cancer cells by binding to membrane–associated nucleolin. Nucleolin, a conserved protein found in all eukaryotes, shuttles from the nucleus, through the cytoplasm to the cell membrane. Cancer cells express a far greater amount of membrane–associated nucleolin than somatic cells, making nucleolin an ideal cancer biomarker. The shuttling, and oligonucleotide binding attributes of the protein enable it to deliver aptamer chimeras from the cell surface to the nucleus. Therefore the nucleolin aptamer has unique access to the nuclei of cancer cells, and can deliver therapeutic oligonucleotide cargoes through nucleolin binding.
The nucleolin aptamer delivered splice–switching oligonucleotides, a form of antisense technology, improving their efficacy, and potentially increasing their therapeutic viability. The ability to deliver antisense oligonucleotides to the nuclei of cancer cells has the potential for other therapeutic possibilities including the inhibition of transcription with antisense triplexes.
The nucleolin aptamer can also deliver therapeutic aptamers. The nucleolin aptamer–β–arrestin aptamer chimera prevented the stem cell renewal phenotype necessary for leukemia progression in human patient tissue samples. The ability to effectively deliver therapeutic aptamers may lead to clinical applications for many of the aptamers that have been selected against intracellular targets including transcriptional activators.
Oligonucleotide research continues to advance our understanding of potentially therapeutic oligonucleotides. Long non–coding RNAs for example, may impact epigenetics, and transcription. Additionally, locked nucleic acids have been developed to improve binding affinity, thus increasing the efficacy of antisense oligonucleotides. In order to bring these discoveries into the clinic, they must be safely and specifically delivered to their target cells.
This work demonstrated that the nucleolin aptamer could deliver oligonucleotide therapeutics to specific cancer cells. Nucleolin aptamer chimeras have the potential to develop into safe and effective cancer therapies, thus improving the treatment options for cancer sufferers.
Item Open Access Advancing DNA-based Nanotechnology Capabilities and Applications(2014) Marchi, Alexandria NicoleBiological systems have inspired interest in developing artificial molecular self-assembly techniques that imitate nature's ability to harness chemical forces to specifically position atoms within intricate assemblies. Of the biomolecules used to mimic nature's abilities, nucleic acids have gained special attention. Specifically, deoxyribonucleic acid is a stable molecule with a readily accessible code that exhibits predictable and programmable intermolecular interactions. These properties are exploited in the revolutionary structural DNA nanotechnology method known as scaffolded DNA origami. For DNA origami to establish itself as a widely used method for creating self-assembling, complex, functional materials, current limitations need to be overcome and new methods need to be established to move forward with developing structures for diverse applications in many fields. The limitations discussed in this dissertation include 1) pushing the scale of well-formed, fully-addressable origami to two and seven times the size of conventional origami, 2) testing cost-effective staple strand synthesis methods for producing pools of oligos for a specified origami, and 3) engineering mechanical properties using non-natural nucleotides in DNA assemblies. After accomplishing the above, we're able to design complex DNA origami structures that incorporate many of the current developments in the field into a useful material with applicability in wide-ranging fields, namely cell biology and photonics.
Item Open Access Analysis and Redesign of Protein-Protein Interactions: A Hotspot-Centric View(2010) Layton, Curtis JamesOne of the most significant discoveries from mutational analysis of protein interfaces is that often a large percentage of interface residues negligibly perturb the binding energy upon mutation, while residues in a few critical "hotspots" drastically reduce affinity when mutated. The organization of protein interfaces into hotspots has a number of important implications. For example, small interfaces can have high affinity, and when multiple binding partners are generated to the same protein, they are predisposed to binding the same regions and often have the same hotspots. Even small molecules that bind to interfaces and disrupt protein-protein interactions (PPIs) tend to bind at hotspots. This suggests that some hotspot-forming sites on protein surfaces are intrinsically more apt to form protein interfaces. These observations paint a hotspot-centric picture of PPI energetics, and present a question of fundamental importance which remains largely unanswered: why are hotspots hot?
In order to gain insight into the nature of hotspots I experimentally examined the small, but high-affinity interface between the synthetically evolved ankyrin repeat protein Off7 with E. coli maltose binding protein by characterization of mutant variants and redesigned interfaces. In order to characterize many mutants, I developed two high-throughput assays to measure protein-protein binding that integrate with existing technology for the high-throughput fabrication of genes. The first is an ELISA-based method using in vitro expressed protein for semi-quantitative analysis of affinity. Starting from DNA encoding protein partners, binding data is obtained in just a few hours; no exogenous purification is required. For the second assay, I develop data fitting methods and thermodynamic framework for determination of binding free energies from binding-induced shifts in protein thermal stability monitored with Sypro Orange.
Analysis of Off7/MBP variants using these methods reveals that conservative mutagenesis or local computational repacking is tolerated for many residues in the interface without drastic loss of affinity, except for a single essential hotspot. This hotspot contains a Tyr-His-Asp hydrogen bonding network reminiscent of a common catalytic motif. Substitution of the tyrosine with phenylalanine shows that a single hydrogen bond across the interface is critical for binding. Analysis of the protein database by structural bioinformatics shows that, although rare, this motif is present in other naturally evolved interfaces. Such a triad was found in the homodimeric interface of PH0642 from Pyrococcus horikoshii, and is conserved between many homologues in the nitrilase superfamily, meeting one of the key criteria by which potential hotspots can be identified. This analysis supports a number of analogies between hotspot residues and catalytic residues in enzyme active sites, and raises the intriguing possibility that hotspots may be associated with other structural motifs that could be used for identification or design of PPIs.
Item Open Access Antineoplastic Cytotoxicity and Immune Adjuvancy of a Recombinant Oncolytic Poliovirus(2016) Brown, Michael ClavonOur group has pioneered the development of a live-attenuated poliovirus, called PVSRIPO, for the purpose of targeting cancer. Despite clinical progress, the cancer selective cytotoxicity and immunotherapeutic potential of PVSRIPO has not yet been mechanistically dissected. Defining such mechanisms may inform its clinical application.
Herein I describe the discovery of a mechanism by which the MAP-Kinase Interacting Kinases (MNKs) regulate PVSRIPO cytotoxicity in cancer. In doing so, I delineate a novel, intricate network connecting the MNK and mTOR signaling pathway that regulates activity of a splicing kinase called the Ser-Arg Rich Protein Kinase (SRPK), and define SRPK as an impediment to IRES mediated translation. Moreover, I demonstrate that MNK regulates mTORC1 associations that determine its substrate proximity and thus, activity. In a collaborative effort, we found that PVSRIPO oncolysis produces antigen specific, cytolytic anti-tumor immunity in an in vitro human system and that much of the observed adjuvancy is due to the direct infection of dendritic cells (DCs) by the virus itself; implicating PVSRIPO as a potent adjuvant. In summary, oncogenic signaling in part through MNK leads to cancer specific cytotoxicity by PVSRIPO that engages an inflammatory environment conducive to DC activation and antigen specific T cell antigen immunity.
Item Open Access Application of the Stability of Proteins from Rates of Oxidation Technique to the Analysis of Mouse Models of Aging and Parkinson's Disease(2017) Roberts, Julia HamiltonRecently, several mass spectrometry-based proteomics techniques have been developed for the large-scale analysis of thermodynamic measurements of protein stability. This has created the possibility of characterizing disease states via differential thermodynamic stability profiles. Described here is the application of the Stability of Proteins from Rates of Oxidation (SPROX) technique to characterize mouse models of disease. The mouse models studied here are of normal aging and two genetically induced Parkinson’s Disease (PD) models.
Thermodynamic stability profiles were generated for 809 proteins in brain cell lysates from C57BL/6 mice at age 6- (n=7) and 18-months (n=9). The biological variability of the protein stability measurements was low, and within the experimental error of the SPROX technique. Remarkably, the large majority of the 83 brain protein hits were destabilized in the old mice, and the hits were enriched in proteins that have slow turnover rates (p<0.07). Furthermore, 70% of the hits have been previously linked to aging or age-related disease.
One of the PD mouse models involved characterizing the protein interactions induced by mutated leuceine-rich repeat kinase 2 (LRRK2) at a pre-symptomatic time point (3 months old). The models used were a control, overexpressed wildtype LRRK2, and overexpressed R1441G mutated LRRK2 (n=2 for all models). Comparative analyses on thermodynamic stability profiles of ~470 proteins revealed relatively few differences. In fact, the observed hit rate in each comparative analysis was close to that associated with the biological variability of the mice. However, four protein hits, dihydropyrimidinase-related protein 2, eukaryotic translation initiation factor 4A2, Rap1 GTP-GDP dissociation stimulator 1 and myelin basic protein, were identified with consistent thermodynamic stability in multiple mice within a biological state and as hits in multiple comparisons suggesting they are the most likely to be true positives.
The second PD mouse model studied was one in which the human α-synuclein protein, containing the known PD mutation A53T, was overexpressed. To characterize the disease progression of PD induced by this mutation, mice were sacrificed at 1 month (n=4), 6 months (n=4) and when they became symptomatic at 10-16 months (n=3). Thermodynamic stability profiles were generated for >850 proteins at each time point. The relative stabilities of these proteins were assayed in a series of comparative analyses involving mice at the different time points and the normally aged mice from above. In total 244 peptides were found to be differentially stabilized during PD progression. A subset of 52 peptide hits was identified to be of particular interest. Of these 52 peptides 22 were identified with early disease progression, 5 peptides showed late disease progression, 5 peptides reported a gradual difference in stability over disease progression and 20 peptides indicated no disease progression trend. More than 90% of the 32 peptides indicating a trend in disease progression showed progression related destabilization.
The results of this thesis help validate the use of thermodynamic stability measurements to capture disease-related proteomic differences in mice. Furthermore, these results establish a new biophysical link between the hit proteins identified and their role in aging, LRRK2 protein interactions, and PD progression.
Item Open Access Aqueous Desolvation and Molecular Recognition: Experimental and Computational Studies of a Novel Host-Guest System Based on Cucurbit[7]uril(2012) Wang, YiMolecular recognition is arguably the most elementary physical process essential for life that arises at the molecular scale. Molecular recognition drives events across virtually all length scales, from the folding of proteins and binding of ligands, to the organization of membranes and the function of muscles. Understanding such events at the molecular level is massively complicated by the unique medium in which life occurs: water. In contrast to recognition in non-aqueous solvents, which are driven largely by attractive interactions between binding partners, binding reactions in water are driven in large measure by the properties of the medium itself. Aqueous binding involves the loss of solute-solvent interactions (desolvation) and the concomitant formation of solute-solute interactions. Despite decades of research, aqueous binding remains poorly understood, a deficit that profoundly limits our ability to design effective pharmaceuticals and new enzymes. Particularly problematic is understanding the energetic consequences of aqueous desolvation, an area the Toone and Beratan groups have considered for many years.
In this dissertation, we embark on a quest to shed new light on aqueous desolvation from two perspectives. In one component of this research, we improve current computational tools to study aqueous desolvation, employing quantum mechanics (QM), molecular dynamics (MD) and Monte Carlo (MC) simulations to better understand the behavior of water near molecular surfaces. In the other, we use a synthetic host, cucurbit[7]uril (CB[7]), in conjunction with a de novo series of ligands to study the structure and thermodynamics of aqueous desolvation in the context of ligand binding with atomic precision, a feat hitherto impossible. A simple and rigid macrocycle, CB[7] alleviates the drawbacks of protein systems for the study of aqueous ligand binding, that arise from conformational heterogeneity and prohibitive computational costs to model.
We first constructed a novel host-guest system that facilitates internalization of the trimethylammonium (methonium) group from bulk water to the hydrophobic cavity of CB[7] with precise (atomic-scale) control over the position of the ligand with respect to the cavity. The process of internalization was investigated energetically using isothermal titration microcalorimetry and structurally by nuclear magnetic resonance (NMR) spectroscopy. We show that the transfer of methonium from bulk water to the CB[7] cavity is accompanied by an unfavorable desolvation enthalpy of just 0.49±0.27 kcal*mol-1, a value significantly less endothermic than those values suggested from previous gas-phase model studies. Our results offer a rationale for the wide distribution of methonium in biology and demonstrate important limitations to computational estimates of binding affinities based on simple solvent-accessible surface area approaches.
To better understand our experimental results, we developed a two-dimensional lattice model of water based on random cluster structures that successfully reproduces the temperature-density anomaly of water with minimum computational cost. Using reported well-characterized ligands of CB[7], we probed water structure within the CB[7] cavity and identified an energetically perturbed cluster of water. We offer both experimental and computational evidence that this unstable water cluster provides a significant portion of the driving force for encapsulation of hydrophobic guests.
The studies reported herein shed important light on the thermodynamic and structural nature of aqueous desolvation, and bring our previous understanding of the hydrophobic effect based on ordered water and buried surface area into question. Our approach provides new tools to quantify the thermodynamics of functional group desolvation in the context of ligand binding, which will be of tremendous value for future research on ligand/drug design.
Item Open Access Auxiliary Wnt3A Signaling in Cell Fate Decisions of C3H10T1/2 Mesenchymal Stem Cells(2011) Rossol-Allison, Jessica K.Activation of Wnt signaling pathways is critical to a variety of developmental events across all animal taxa. These highly evolutionarily conserved pathways are also important in the adult organism for maintaining homeostasis of self-renewing tissues. Because of its role in such important physiological processes, deregulation of Wnt signaling can have severe consequences; indeed, inappropriate activation of this pathway has been implicated in multiple human diseases, including cancer.
Upon binding their cellular receptors, canonical Wnt ligands, like Wnt 3A, stimulate the stabilization, accumulation, and nuclear translocation of a multifunctional cellular protein βcatenin, the consequence of which is induction of βcatenin-dependent transcription. This work describes the identification and characterization of two Wnt3A-stimulated intracellular signaling pathways activated in parallel to βcatenin stabilization: the RhoA pathway and the ERK pathway. These two auxiliary pathways do not affect βcatenin stability, accumulation, or subcellular localization; rather, they modulate βcatenin -dependent transcriptional activity through other mechanisms. As a result of their influence on βcatenin-dependent transcription, these pathways instruct cell fate decisions in C3H10T1/2 mesenchymal stem cells, in particular inhibition of adipogenesis and promotion of osteoblastogenesis.
Expression microarray analysis and biochemical and pharmacological techniques were used to further characterize the two Wnt3A-stimulated auxiliary pathways in C3H10T1/2 cells. Remarkably, each pathway influences βcatenin function via a novel mechanism. In the Wnt3A/RhoA pathway, Wnt3A-stimulated trimeric G proteins activate a RhoA-ROCK-SRF cascade. Activated SRF can cooperate with βcatenin to enhance the induction of Wnt3A target genes, like Ctgf, that also contain SRF binding sites within regulatory elements. In the Wnt3A/ERK pathway, Wnt3A transactivates the EGFR in a concentration-dependent manner, leading ultimately to ERK activation, which interacts with and promotes βcatenin/Tcf4 interaction and enhances induction of βcatenin/Tcf4 target genes.
These data emphasize the complexity of Wnt signaling and have intriguing implications regarding cross-regulation of the pathway, especially in stem cells. Also, since not all cells are capable of responding to Wnt3A by activation of these auxiliary pathways, this work identifies novel mechanisms that could underlie cell type-specific responses to Wnts and provides mechanistic insight into cellular responses to Wnt concentration gradients. Moreover, this work identifies novel transcriptional mechanisms important for promoting osteogenic cell fate specification, which could ultimately provide new therapeutic targets in disease states with bone loss or ineffective bone formation.
Item Open Access Bacterial Extracellular Vesicles and the Plant Immune Response(2021) McMillan, Hannah MaryCells from all levels of life secrete vesicles, which are nanoscale proteoliposomes packaged with a variety of proteins, lipids, and small molecule cargo. Depending on their origin, these extracellular vesicles are termed exosomes, microvesicles, exomeres, and membrane vesicles, to list a few. Vesicles released from Gram-negative bacteria bud from the outer membrane and are, therefore, referred to as outer membrane vesicles (OMVs). In mammalian systems, OMVs facilitate bacterial survival by alleviating membrane stress, serving as a decoy for bacteriophage and antibiotics, and providing a fast membrane remodeling mechanism. OMVs also contribute to virulence by delivering toxins and other soluble and insoluble cargo to the host cell. The role OMVs play in plant systems remains unknown.
Previous studies revealed that plant pathogenic bacterial vesicles contain virulence factors, type III secretion system effectors, plant cell wall-degrading enzymes, and more, suggesting that vesicles may play similar roles to those from mammalian pathogens in host-pathogen interactions. Further, OMVs elicit several markers for pathogen-associated molecular pattern triggered immunity in plants. These responses include increased transcription of defense markers such as FRK1 and production of reactive oxygen species. Building on these findings, here we show that OMVs from the plant pathogen Pseudomonas syringae and the plant beneficial Pseudomonas fluorescens elicit plant immune responses in Arabidopsis thaliana that protect against future pathogen challenge. Intriguingly, protection is independent of salicylic acid plant defense pathways and bacterial type III secretion. OMVs also inhibit seedling growth, another indication of plant immune activation.
Our initial biochemical studies suggested that the immunogenic OMV cargo was larger than 10 kDa and differed between the pathogen and beneficial species despite similar plant immunity outcomes. Interestingly, protective OMV-mediated responses were protein-independent, while the seedling growth inhibition phenotype was entirely protein dependent. Proteomics analysis confirmed that OMV protein cargo differed between P. syringae and P. fluorescens. While media culture conditions did not dramatically impact the immunogenic activity of isolated OMVs from either species, proteomics analysis revealed a significant shift in P. syringae OMV cargo between complete and minimal media conditions. P. fluorescens OMV cargo was largely the same in the two media conditions, with no significantly enriched proteins in minimal or complete media. Further analysis of the proteins enriched in the P. syringae minimal OMV condition identified one set of proteins with the same baseline abundance in P. syringae and P. fluorescens complete OMVs and another set with a lower baseline abundance compared to P. fluorescens OMVs. These two subsets could contribute to virulence and stress tolerance, respectively. Enrichment analysis uncovered particularly interesting protein categories in the subset with the same baseline abundance. Of interest, several lipoprotein and lipid binding categories were enriched, and proteins involved in synthesis of the phytotoxin coronatine were also enriched in this same-baseline subset. These results support our hypothesis that proteins enriched in P. syringae minimal OMVs with the same baseline abundance in P. fluorescens complete OMVs may contribute to OMV-mediated bacterial virulence in plants. Our findings also suggest that our forthcoming OMV metabolomic analyses may reveal non-proteinaceous cargo that is critical for OMV-mediated plant immune activation.
The work presented here lays the groundwork for future exploration of OMV-plant interactions and adds a new layer of complexity to plant-bacteria interactions. Further, these results reveal that OMVs elicit complex plant immune responses that would be difficult for pathogens to adapt to and overcome, supporting a role for bacterial OMVs in agricultural applications to promote durable resistance and revealing a new potential avenue for disease prevention and management.
Item Open Access Biased Signaling at the β2-adrenergic Receptor is established by Receptor-Transducer Interactions(2018) Choi, Minjungβ-Adrenergic receptors (βAR) are one of the key modulators of cardio-pulmonary functions and belong to a large family of membrane proteins, termed as G-protein coupled receptors (GPCRs). β-blockers (βAR antagonists) and βAR agonists are the mainstay treatments for heart failure and asthma respectively, which reflects the significance of βARs as therapeutic targets. The binding of catecholamines (e.g. adrenaline) to βARs activates intracellular transducer proteins such as hetero trimeric GTP binding proteins (G-proteins) or β-arrestins (βarr), which results in the regulation of cardiac output and bronchodilation.
The bifurcated signaling pathways initiated by G-protein and β-arrestin downstream of βAR, as well as other members in the GPCR family can be selectively activated, a phenomenon termed as ‘biased agonism’. Biased ligands, which can pharmacologically separate these pathways, are of major therapeutic interest due to their potential for improving the specificity of drug actions. For βAR, biased agonism towards β-arrestin is expected to render cardo-protective benefits, while selective activation of G proteins is hypothesized to subdue major side effects from current asthma therapy. Therefore, elucidation of how βARs can preferentially interact with their transducers is at the core of developing the next generation therapeutics, beyond conventional β-blockers and agonists.
Thus far, the exact mechanism behind GPCR biased agonism remains obscure. The leading hypothesis in the field is that GPCRs adopt distinct conformations that preferentially couple to G proteins or β-arrestins. In order to test this hypothesis, we developed and established a G protein biased mutant β2AR (Chapter 2), since efficacious biased ligands for this receptor are yet to be found. Subsequent assessment of GPCR kinase (GRK)-mediated phosphorylation states of this mutant receptor and phosphorylation rescue experiments revealed unexpected findings that contradict the initial hypothesis (Chapter 3). Next, we initiated a biophysical characterization of this mutant β2AR (Chapter 4) to comprehend the conformational and structural basis for its apparent biased phenotype. The cumulative insight gained from experiments described in chapters 2-4 highlight the underappreciated role of GRKs in determining GPCR biased agonism – the mutant β2AR is biased towards G protein due to conformational selection against GRKs, rather than β-arrestins. Furthermore, to obtain a comprehensive understanding of biased agonism, we devised a strategy to map the interface between β2AR-β-arrestin, which can also be used to form stable complexes for further biophysical characterizations (Chapter 5). In summary, this dissertation improves the current understanding of the molecular mechanism behind biased agonism at the prototypical GPCR, β2AR.
Item Open Access Biochemical and Genetic Studies of UDP-2,3-Diacylglucosamine Hydrolysis in Lipid A Biosynthesis(2014) Young, Hayley ElizabethThe 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.
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.
Item Open Access Biochemical and structural mechanisms of multidrug efflux pump transcription regulators, Neisseria gonorrhoeae MtrR and Escherichia coli MprA(2021) Beggs, Grace AnneAs bacterial resistance to multiple antibiotics continues to become a growing problem across the globe, the imperativeness for understanding mechanisms of antibiotic and multidrug resistance is increasingly apparent. Currently, the gram-negative bacteria Neisseria gonorrhoeae and Escherichia coli are considered urgent public health threats due to the rise in multidrug resistant strains. Mechanisms by which these bacteria become resistant to antibiotics include the overexpression of multidrug efflux systems. Prior to the introduction of antibiotics, the primary purpose of these multidrug efflux systems was to protect the bacteria from cytotoxins in the environment including innate host defense molecules or toxic molecules produced by the bacteria. Overtime, the bacteria have adapted these efflux systems to protect against clinically relevant antibiotics used to clear these bacterial infections. These multidrug efflux systems are energetically expensive to synthesize; thus, they are often tightly regulated at the transcription level by transcription repressors or activators. Many multidrug efflux systems are regulated by transcriptional regulators with proximal genes that specifically regulate a single multidrug efflux system. However, the expression of a few multidrug efflux systems is controlled by unique repressors that act as global regulators, which have a larger role in regulating complex virulence and stress response systems. Specifically, examples of multidrug efflux regulators in N. gonorrhoeae and E. coli that act as global regulators within their respective genomes include N. gonorrhoeae MtrR and E. coli MprA. Understanding the global regulatory activities of these two transcription regulators will broaden our understanding of the regulatory mechanisms that enable bacterial survival during host infection, the mechanisms that contribute to antibiotic resistance, as well as the fundamentals of bacterial transcription regulation. To provide novel insight into the global regulatory activities and function of N. gonorrhoeae MtrR, this dissertation expounds a series of original structural, biochemical, and in vivo studies identifying cytotoxin and DNA recognition mechanisms of MtrR. Previous work showed that MtrR represses directly the mtrCDE efflux transporter genes by binding an operator between the mtrR and mtrC genes; additionally, MtrR represses directly the rpoH oxidative stress response sigma factor. MtrR-mediated repression of the mtrCDE genes had been shown to be relieved upon exposure of gonococci to toxic hydrophobic agents and detergents (i. e. MtrR is “induced” by these toxic molecules). However, physiologically relevant innate host molecules recognized by MtrR had not been identified. In this work, we identify bile salts present at extra-urogenital gonococcal infection sites that MtrR directly binds, to result in derepression of the mtrCDE genes in vitro and in vivo. Furthermore, we use x-ray crystallography to solve structures of MtrR in its induced form and bound to the mtrCDE and rpoH operators. With these structures, we determined the structural mechanism of induction of MtrR. In addition, the MtrR-operator structures reveal a degenerate consensus sequence to which MtrR binds within the mtrCDE and rpoH operators. Mechanisms for cytotoxin and DNA recognition were confirmed by structure-guided site-directed mutagenesis studies and a combination of biochemical binding assays utilizing isothermal titration calorimetry (ITC) or fluorescence polarization (FP). Importantly, this structural and biochemical work also reveals the mechanisms by which common mutations in multidrug resistant strains of N. gonorrhoeae confer resistance. To elucidate the function of E. coli MprA and realize its potential as a drug target, this dissertation also includes research describing the ligand-binding mechanisms of MprA. MprA (formerly EmrR) represses directly the EmrAB efflux pump in E. coli. Previously published work identified MprA as the molecular target of a small molecule inhibitor (DU011) of the biosynthesis of an important virulence factor in E. coli, the polysaccharide capsule. This lead molecule has the potential for optimization for drug development and reveals a novel function of MprA as a regulator of polysaccharide capsule synthesis. We characterized the interaction between MprA and DU011 and compared this to the binding between MprA and other previously identified ligands including salicylate and 2,4-dinitrophenol (DNP) utilizing ITC assays. Through these studies, we revealed a novel binding mode for MprA and laid the groundwork for future structural studies and drug optimization. Collectively, this work provides important insight into the breadth of regulatory functions of N. gonorrhoeae MtrR and E. coli MprA, two key global regulators from highly prevalent multidrug resistant pathogens. Specifically, the original research presented here provides a biochemical evaluation of the bacterial stress response mechanisms controlled by MtrR and MprA and their contribution to antibiotic resistance. Indeed, the biochemical and structural characterization of these two regulators will inform future work to combat multidrug resistance.
Item Open Access Biochemical and Structural Studies on PrfA, the Transcriptional Regulator of Virulence in Listeria monocytogenes(2016) Hamilton, KeriAbstract
Listeria monocytogenes is a gram-positive soil saprophytic bacterium that is capable of causing fatal infection in humans. The main virulence regulator PrfA, a member of the Crp/FNR family of transcriptional regulators, activates the expression of essential proteins required for host cell invasion and cell-to-cell spread. The mechanism of PrfA activation and the identity of its small molecule coactivator have remained a mystery for more than 20 years, but it is hypothesized that PrfA shares mechanistic similarity to the E. coli cAMP binding protein, Crp. Crp activates gene expression by binding cAMP, increasing the DNA binding affinity of the protein and causing a significant DNA bend that facilitates RNA polymerase binding and downstream gene activation. Our data suggests PrfA activates virulence protein expression through a mechanism distinct from the canonical Crp activation mechanism that involves a combination of cysteine residue reduction and glutathione (GSH) binding.
Listeria lacking glutathione synthase (ΔgshF) is avirulent in mice; however virulence is rescued when the bacterium expresses the constitutively active PrfA mutant G145S. Interestingly, Listeria expressing a PrfA mutant in which its four cysteines are mutated to alanine (Quad PrfA), demonstrate a 30-fold decrease in virulence. The Quad and ΔgshF double mutant strains are avirulent. DNA-binding affinity, measured through fluorescence polarization assays, indicate reduction of the cysteine side chains is sufficient to allow PrfA to binds its physiological promoters Phly and PactA with low nanomolar affinity. Oxidized PrfA binds the promoters poorly.
Unexpectedly, Quad also binds promoter DNA with nanomolar affinity, suggesting that the cysteines play a role in transcription efficiency in addition to DNA binding. Both PrfA and Quad bind GSH at physiologically relevant and comparable affinities, however GSH did not affect DNA binding in either case. Thermal denaturation assays suggest that Quad and wild-type PrfA differ structurally upon binding GSH, which supports the in vivo difference in infection between the regulator and its mutant.
Structures of PrfA in complex with cognate DNA, determined through X-ray crystallography, further support the disparity between PrfA and Crp activation mechanisms as two structures of reduced PrfA bound to Phly (PrfA-Phly30 and PrfA-Phly24) suggest the DNA adopts a less bent DNA conformation when compared to Crp-cAMP- DNA. The structure of Quad-Phly30 confirms the DNA-binding data as the protein-DNA complex adopts the same overall conformation as PrfA-Phly.
From these results, we hypothesize a two-step activation mechanism wherein PrfA, oxidized upon cell entry and unable to bind DNA, is reduced upon its intracellular release and binds DNA, causing a slight bend in the promoter and small increase in transcription of PrfA-regulated genes. The structures of PrfA-Phly30 and PrfA-Phly24 likely visualize this intermediate complex. Increasing concentrations of GSH shift the protein to a (PrfA-GSH)-DNA complex which is fully active transcriptionally and is hypothesized to resemble closely the transcriptionally active structure of the cAMP-(Crp)-DNA complex. Thermal denaturation results suggest Quad PrfA is deficient in this second step, which explains the decrease in virulence and implicates the cysteine residues as critical for transcription efficiency. Further structural and biochemical studies are on-going to clarify this mechanism of activation.
Item Embargo Biochemical Characterization of an Atypical Polyketide Synthase (PKS) from the Apicomplexan Parasite Toxoplasma gondii(2023) Keeler, AaronThe phylum Apicomplexa encompasses multiple obligate intracellular parasites that pose significant burdens to human health including the causative agents of malaria, toxoplasmosis, and cryptosporidiosis which infect millions of humans and cause hundreds of thousands of deaths each year. During their complex life cycles, apicomplexan parasites coordinate the function of specific proteins to both evade the host immune system and thrive under stressful conditions. Notably, Toxoplasma gondii has been found to harbour multiple polyketide synthase (PKS) genes by bioinformatic analysis, suggesting they can produce secondary metabolite polyketides. While secondary metabolite biosynthetic gene clusters (BGCs) have been known in Apicomplexa for over two decades, limited studies on these enzymes have been completed to date and there have been no characterized products, leaving a void in our understanding of the role of these enzymes in parasite biology. Therefore, characterization of these proteins may aid in our ability to target these biosynthetic enzymes as sources of potential therapeutic candidates in Apicomplexa.While protists are underexplored for biosynthetic potential, research points to this kingdom as an untapped potential for new chemical space. T. gondii for instance possesses multiple putative PKS biosynthetic gene clusters (BGCs) however there have been no secondary metabolite products elucidated thus far. Therefore, our work explores a T. gondii PKS, TgPKS2, and investigates the architecture, predicted structures, and activity of multiple domains within this synthase. Subsequently, Chapters 2 and 3 describes our initial studies on TgPKS2 including hydrolysis activities of acyltransferase (AT) domains, mutagenesis studies, and a first of its kind self-acylation activity of acyl carrier protein (ACP) domains in a modular type I PKS. Chapter 4 further emphasizes the unique attributes of TgPKS2, delving into a never before characterized chain release mechanism, while Chapter 5 compares TgPKS2 transacylation activity to well-characterized bacterial and fungal systems. Combined, these chapters describe our work to biochemically explore TgPKS2, discover the role it plays within the T. gondii life cycle, and further our work to elucidate the metabolite(s) produced by this synthase. Altogether, this research lays the ground work for exploring other apicomplexan and eukaryotic polyketide synthases and significantly increases our knowledge of the biochemical properties of these unique proteins.