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Publication Mitotic SUMOylation: Unraveling the role of DNA Topoisomerase IIα SUMOylation and PIASy SUMO E3 ligase in mitosis(University of Kansas, 2019-12-31) Pandey, Nootan; Azuma, Yoshiaki; Gamblin, Truman Christopher; Lundquist, Erik; Neufeld, Kristi; Blumenstiel, JustinA post-translational modification with SUMO (SUMOylation) can regulate various cellular events such as DNA replication, repair, transcription and cell cycle regulation. Many studies have indicated that SUMOylation is crucial for proper cell cycle progression. With three important enzymes, E1 activating enzyme, E2 conjugating enzyme, and E3 ligase, SUMOylation is mechanistically very similar to ubiquitination. Though, SUMOylation can affect a substrates’ cellular localization, enzymatic activity, or can mediate protein-protein interaction. Using Xenopus egg extracts (XEEs) we have shown that disruption of mitotic SUMOylation causes chromosome segregation defects. Our group has identified DNA topoisomerase IIα (Topo IIα) as one of the important mitotic proteins for SUMOylation. SUMOylated Topo IIα C-terminus (CTD) interacts with Haspin kinase and recruits chromosome passenger complex (CPC) to the mitotic centromeres. In yeast and mammalian cells, the catalytic disruption of Topo IIα is reported to induce a delay in mitosis. However, the molecular mechanism for this mitotic delay is not well understood. In this dissertation, I have provided a molecular insight for this mitotic delay. An additional study had provided important evidence that blockage of Topo IIα enzymatic activity results in the hyper SUMOylation of Topo IIα. We have determined that Topo IIα CTD SUMOylation behaves like a signal transducer to induce a mitotic delay when Topo IIα is catalytically disrupted. Mutation in CTD SUMOylation sites has abrogated the mitotic delay. Further, we have shown that disruption of Topo II strand passage reaction (SPR) results in increased Topo IIα SUMOylation and Aurora B mobilization on chromosome arms. This is a conserved mechanism in XEEs and mammalian cells. Aurora B is a catalytic component of CPC and its precise centromeric recruitment is essential for timely metaphase to anaphase transition. Aurora B mislocalization on chromosomes utilizes the Haspin-H3T3P pathway and is a key factor for the mitotic delay. Further, to understand the role of SUMOylation more clearly in human cells we have targeted one of the important SUMOylation enzymes PIASy SUMO E3 ligase. Earlier in XEEs, we found that PIASy is an essentially important E3 ligase for mitotic SUMOylation. Next, we sought to examine if PIASy has a conserved role in human cells. To address this question, we have established Tet-ON inducible ectopic expression of PIASy and SUMO interacting motif (SIM) mutants in human cells. Our results suggest that PIASy is an important E3 ligase that mediates mitotic SUMOylation in human cells. Altogether, this dissertation research expands our understanding of the significance of SUMOylation during mitosis.Publication Polycyclic aromatic hydrocarbons influence naive CD4+ T cell differentiation(University of Kansas, 2019-12-31) Dunbar, Amanda Jo; Benedict, Stephen H; Egan, Susan; Davido, DavidCD4+ T cells are essential for the effective functioning and appropriate regulation of the immune system. Antigen-naïve T cells require two distinct costimulatory signals to activate and differentiate into effector and memory T cells which can respond to antigen and direct the immune response. The first signal is through cognate antigen presented in major histocompatibility class II on an antigen-presenting cell binding to the T cell receptor. The second signal is the interaction of a costimulatory receptor on the T cell with a counter-receptor expressed on the antigen-presenting cell. This work focuses on the costimulatory receptors CD28, the classic T cell costimulatory molecule, and intercellular adhesion molecule-1 (ICAM-1). ICAM-1 functions in adhesion and extravasation during inflammation, and our lab has previously published that ICAM-1 can function as a T cell second signal for activation. Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants which have been shown by many other investigators to have diverse effects on human health including the immune system. In the present work, we examined the consequences of exposure to two PAHs during naïve T cell costimulation. Bisphenol A, a widely used plasticizer and component of epoxy resins, was found to weakly promote naïve T cell proliferation during ICAM-1 costimulation but not in CD28 costimulation and did not perturb effector and memory cell differentiation in either costimulation. Pyrene, a product of incomplete combustion of organic materials, enhanced proliferation of naïve T cells in both ICAM-1 and CD28 costimulations and inhibited effector and memory cell differentiation in both costimulations. We also compared the differentiation of naïve T cells isolated using StemSep and EasySep naïve CD4+ T cell isolation kits upon being informed by StemCell, the manufacturer of the kits, that StemSep was being discontinued in favor of EasySep. In contrast to StemSep naïve T cells, with which we have published previously, EasySep-purified naïve T cells did not differentiate in response to costimulation through ICAM-1 or CD28 without addition of exogenous cytokines and had dramatically increased cell death. We showed that inclusion of CD25 antibody in the EasySep negative selection antibody cocktail was partially responsible for the observed loss of differentiation and viability. Likewise, the EasySep magnetic beads were partially at fault for observed failure to differentiate and loss of viability in EasySep naïve T cells. This result also suggested an important role for the CD25lo population of naïve T cells in the ability of the cells to differentiate, which is in keeping with the published literature.Publication THE ROAD LESS TRAVELLED: UTILIZATION OF FORMATE IN TWO BIOCHEMICAL REACTIONS IN GRAM-NEGATIVE BACTERIA(University of Kansas, 2019-08-31) Kenjić, Nikola; Lamb, Audrey L; Neufeld, Kristi; Slusky, Joanna; Richter, Mark; Jackson, TimothyFormate is an important intermediate in a number of metabolic reactions. Most of the formate pool in cells is generated from the degradation of primary metabolites (glucose, pyruvate, amino acids) and further degraded into CO2 and CH4. Remaining formate is recycled and utilized in one-carbon metabolism where a one-carbon formyl group is integrated into nucleotide metabolism, protein synthesis, and generation of secondary metabolites such as siderophores. Here I provide two examples of distinct and unique enzymes involved in formate generation and metabolism: PvdF from Pseudomonas aeruginosa and RibB from Vibrio Cholerae. PvdF is one of two enzymes involved in generation of the formyl-hydroxyornithine (fOHOrn) moiety responsible for iron chelation in pyoverdin, a siderophore in Pseudomonas aeruginosa. Biochemical and structural studies suggest that PvdF is a unique new class of transformylase enzyme. PvdF catalyzes the movement of the formyl group from an N10 formyl-THF analogue to the substrate following the bireactant random substrate binding model. Structurally, PvdF has a transformylase fold with secondary structural element insertion so far unprecedented in the literature. RibB is involved in the biosynthesis of riboflavin, vitamin B2. This is a magnesium dependent enzyme that catalyzes the conversion of the sugar ribulose 5-phosphate (Ru5P), a product of the pentose phosphate pathway, into 3,4-dihydroxy-2-butanone 4-phosphate (DHBP). The reaction catalyzed by RibB is an unusual deformylation reaction in which the fourth carbon of the five-carbon sugar is removed as formate. According to the literature, RibB catalyzes this reaction in the presence of di-metal Mg2+ centers following 1,2-methyl shift, called a skeletal rearrangement mechanism. Our evidence, both biochemical and structural, suggests that RibB requires only one Mg2+ for catalysis. Furthermore, NMR and X-ray crystallography data point toward the formation of a 2-phosphoglycolic acid intermediate during RibB catalyzed reaction. These data suggest that a fragmentation mechanism, not a skeletal rearrangement, is the preferred mechanism of RibB catalysis.Publication The Biosynthesis of Opine Metallophores(University of Kansas, 2019-08-31) McFarlane, Jeffrey S; Lamb, Audrey L; Richter, Mark L; Egan, Susan M; Chandler, Josephine; Jackson, TimothyMetal acquisition is a necessity for all cells as metals are essential for the function of numerous metalloproteins and metalloenzymes. Bacterial pathogens employ several strategies for obtaining necessary metal including the direct import of metal ions, the import of metal-containing heme groups, and the secretion of biosynthesized small molecule metallophores to capture metals from the surrounding environment. Metallophore pathways are virulence factors that allow effective competition within the human host and the establishment of infection. They represent viable targets for the development of new antibiotic therapies and an understanding of the production and use of each metallophore contributes to a broader understanding of bacterial pathogenesis. In 2015, Gi and Choi proposed the existence of a novel nicotianamine-like metallophore produced by two enzymes encoded in a four gene operon in Pseudomonas aeruginosa. Nicotianamines are plant-derived metallophores and P. aeruginosa biosynthesizes two well-studied metallophores, pyoverdin and pyochelin, making this hypothesis surprising. In 2016, Ghssein et. al. described a homologous system in Staphylococcus aureus producing a metallophore they named staphylopine biosynthesized by two primary enzymes, a nicotianamine synthase and an opine dehydrogenase. Following heterologous expression of these two orthologous enzymes from the P. aeruginosa operon, a plate-reader screening assay was used to determine the substrates. With substrates known, the predicted structure of pseudopaline was confirmed by mass spectrometry. Pseudopaline is uses L-histidine and -ketoglutarate as substrates in constrast to staphylopine which incorporates D-histidine and pyruvate. Steady-state kinetic parameters for the nicotianamine synthase in S. aureus and P. aeruginosa demonstrated slow kcat values of 1.79 0.02 and 1.07 0.02 min-1 and Km values of 13.0 0.7 and 5.4 0.4 µM respectively. Steady-state parameters had not been previously established for any nicotianamine synthase. This work is detailed in chapter two. The recognition of the enzymatic determinants of opine metallophore production allowed targeted bioinformatics analyses that revealed the presence of like operons in disparate bacterial species living in diverse environments. These analyses are described in chapter one. Opine dehydrogenase enzymes had been structurally characterized from two species (the soil bacterium Arthrobacter sp. 1C and the marine scallop Pecten maximus) prior to this work. Yersinia pestis has a homologous opine metallophore-producing operon. Heterologous expression of the Y. pestis opine dehydrogenase revealed a specificity for L-histidine and pyruvate, although expression of the Y. pestis nicotianamine synthase was unsuccessful and the structure of yersinopine remains presumptive. Y. pestis opine dehydrogenase enriched with selenomethionine crystallized and single wavelength anomalous dispersion (SAD) X-ray diffraction data were collected allowing structure solution. The Y. pestis opine dehydrogenase structure was then used as a molecular replacement model in the structure solution for data sets collected from S. aureus and P. aeruginosa opine dehydrogenase crystals, expanding the number of determined opine dehydrogenase structures in the Protein Data Bank from two to five. A steady-state comparison of these three opine dehydrogenases revealed differences in substrate specificity. S. aureus and Y. pestis use pyruvate and NADPH while P. aeruginosa uses -ketoglutarate and can use either NADH or NADPH. These analyses are described in chapter three. Opine dehydrogenases perform condensation and reduction steps on the prochiral -carbon of an -ketoacid and specifically generate one stereocenter, (R) or (S), depending on their structural family. Opine metallophore-producing opine dehydrogenases are in the family that produce (R) stereocenters. We demonstrated that the S. aureus and P. aeruginosa opine dehydrogenase reactions are reversible and catalyze the reverse reaction only with the (R) diastereomer of staphylopine and pseudopaline respectively. No catalysis was seen in the presence of the (S) diastereomer. In the previously published opine dehydrogenase structures, NADP+ was bound with at least partial electron density, but a structure with bound substrates had not been solved. Structures of P. aeruginosa opine dehydrogenase were solved with both the (R) and (S) diastereomers of pseudopaline. Interestingly, while complete density of (S)-pseudopaline was visualized at 1.64 Å, (R)-pseudopaline was progressively hydrolyzed by the cystal demonstrating reverse catalysis in the X-ray structures. Transient state kinetic analysis of S. aureus opine dehydrogenase revealed that product release was the rate-limiting step of catalysis. These data are described in chapter four. Opine metallophores represent a new class of bacterial metallophore produced by a nicotianamine synthase and an opine dehydrogenase. Nicotianamine synthases remain poorly characterized and future work on the structure and kinetics of this enzyme family will be necessary to provide a mechanistic understanding of function. Our analysis of opine dehydrogenases from S. aureus and P. aeruginosa provides an excellent structure/function analysis and is the foundation for advanced kinetic analyses. These data, along with the expanding analysis of the functional roles of opine metallophore pathways in vivo, are leading toward a global model describing the function of these systems.Publication Gene silencing in the nucleus: mechanisms and new phenomena(University of Kansas, 2019-08-31) NAGARAJAN, VAISHNAVI; Timmons, Lisa; Oakley, Berl; Davido, David; Xu, Liang; Jensen, KirstenSilencing of homologous genes by exogenously introduced dsRNA was first observed in C. elegans. Endogenous small RNAs (siRNAs, piRNAs, miRNAs) mediate regulation of expression of genes post-transcriptionally or at the level of transcription, when argonaute proteins complex with small RNAs to target genomic loci for chromatin modifications in a sequence-specific manner, in the nucleus. There have been previous reports of regulation of expression by targeting mRNA for silencing in the cytoplasm. We identified a region in the genome, flp-17 locus that is amenable to nuclear silencing mechanisms in a wildtype animal. C. elegans exhibits strong anti-foreign genome silencing in their germline as defense against invading viral or transposon DNA. This activity is extended to transgene DNA, resulting in its silencing in the germline. Mos1 is a foreign element, a transposon from Drosophila that is heterologously inserted in C. elegans genome. Trangenes are integrated in the chromosomal DNA by Mos1 based Single Copy insertion technology where homologous regions flanking the gene of interest promotes recombination and thus its integration at a specific mos site on the worm genome. In our experiments we observed robust silencing in the somatic cells of transgenes that were intended to be integrated in ttTi5605 mos site on C. elegans genome by homologous recombination. The silencing phenomenon involves epigenetic mechanisms. We hypothesize that over several generations, the worm has “learned” that mos is a foreign element and when transgene is integrated at that site, it is amenable to silencing by the epigenetic machinery. Furthermore, we identified a previously undescribed mutation (yy14) in eri-6 gene and show evidences pointing to a role in silencing of transgenes in somatic tissues. We observed an increase in trans-spliced mRNA from eri- 6/7 genes in yy14 mutants. Our model reasons out that increase in trans-spliced mRNA results will lead to upregulation of small RNAs (26G RNAs) that efficiently function in the silencing of our transgene, which needs further verification. In addition, ABC transporters in C. elegans have been previously shown to be required for efficient RNAi. haf-2, haf-6 and haf-9 mutants also exhibit defects in transposon mobilization. Chromatin modification by epigenetic machinery prevents mobilization of transposable elements. This includes RNAi effectors like siRNAs, Dicer and argonaute proteins. Thus, a previous study in the lab, highlights a link between ABC transporters and RNAi mechanisms in C. elegans. It is important to characterize the dimerization pattern of half ABC transporter proteins to gain insights on their functions and precise roles in RNAi.Publication The Bacteriophage(University of Kansas, 1933-08-31) Wells, Alvin Y.Publication Studies in anaphylaxis(University of Kansas, 1933-05-31) Kabler, Paul W.Publication A further study of the antigenic properties of Euglena gracilis, Klebs, with related phenomena(University of Kansas, 1933-05-31) Elmore, Mary ElizabethPublication Serological studies of the Reptilia(University of Kansas, 1938-05-31) Bond, Glenn CarlPublication Designing Small Molecule Inhibitors of RNA-Binding Protein Musashi Using New Biochemical and Computational Approaches(University of Kansas, 2019-12-31) Bai, Nan; Xu, Liang; Karanicolas, John; Rafferty, Michael F; De Guzman, Roberto N; Slusky, Joanna; Picking, WilliamABSTRACT RNA-binding proteins (RBPs) are key regulators of post-transcriptional gene expression, and underlie many disease-relevant processes. However, they have historically been challenging to target with drug-like compounds. Inspired by the “anchor residues” of protein-protein interactions, we developed a computational approach for rationally designing small-molecule inhibitors of RBPs. In this dissertation, we first selected Musashi-1 and Musashi-2 to apply our “RNA mimicry” approach. Both Musashi proteins are well-studied RBPs, known principally as stem‑cell markers that are upregulated in many cancers. In the future, we hope our “RNA mimicry” approach can be generally applied to inhibitor design of diverse target RBPs. To design inhibitors of Musashi proteins, we applied our strategy by mimicking the three-dimensional interactions in the protein-RNA complex. As described in Chapter II, by using pharmacophoric screening, we searched for drug-like compounds that can present the same geometric arrangement of functional groups as the RNA in the complex. We hypothesized that such ligands would engage Musashi in a similar manner as the RNA binds to Musashi. Since the interaction geometries can be quite distinct from one another for different RBPs, we anticipated that this strategy would lead to inhibitors that were selective for Musashi. To facilitate characterization of these candidate Musashi inhibitors, I developed the “isothermal analysis” approach. As described in Chapter III, this method allows us to calculate quantitative binding constants by using differential scanning fluorimetry (DSF) data. The method requires only the protein unfolding information at a given temperature as a function of ligand concentration, and thus no thermodynamic parameters are included in the calculation. Finally, I describe the use of computational docking to better understand the basis for PROTAC-mediated degradation of target proteins. PROteolysis TArgeting Chimeras (PROTACs) are heterobifunctional small molecules which can induce target protein degradation through cell ubiquitination process. Rational design of PROTACs is still challenging, however, because of the limited structural understanding of their mechanism. In Chapter IV, I seek to predict the formation of the ternary structure complex by including both effects of the protein-protein interaction and effects of the chemical linker. Looking ahead, I hope to use these ternary structure models to explain the activity and selectivity of the given PROTAC molecules, and ultimately to use our designed Musashi inhibitors as a starting point for building new PROTACs to degrade Musashi. The text of Chapter II is a manuscript that is in preparation for publication as: Bai N‡, Adeshina Y‡, Lan L, Makhov PB, Xia Y, Gowthaman R, Miller SA, Johnson DK, Boumber Y, Xu L, Karanicolas J. Rationally designing inhibitors of the Musashi protein-RNA interaction by hotspot mimicry. ‡equally contributing co-authors The supporting information for this chapter is included as Appendix A.1. The text of Chapter III is a reprint of the material from: Bai N, Roder H, Dickson A, Karanicolas J. Isothermal analysis of ThermoFluor data can readily provide quantitative binding affinities. Sci. Rep. 9, p. 2650 (2019). Note: the software disseminated with this paper has accumulated 1000 downloads in the 9 months since publication The supporting information for this chapter is included as Appendix A.2. The text of Chapter IV is a manuscript that is in preparation for publication as: Bai N, Karanicolas J. Predicting PROTAC-mediated ternary complex formation using Rosetta. The supporting information for this chapter is included as Appendix A.3.Publication Activation and Inhibition of Biological Function through Design of Novel Protein-Ligand Interactions(University of Kansas, 2019-05-31) Budiardjo, Sandi; Slusky, Joanna; Karanicolas, John; Deeds, Eric J; Ray, Christian; Miao, Yinglong; Richter, MarkVirtually every process within a cell involves a protein. They serve as cellular workhorses carrying out functions such as catalysis of essential metabolites, to regulating which genes get turned on or off, to forming the structural scaffolding to retain rigidity of a cell. Proteins form the link between the genetic information encoded in DNA to the observable phenotype of an organism. The way proteins communicate is by direct physical contact with another molecule that alters its shape and dynamics to carry out a particular function. For example, G protein-coupled receptors are membrane imbedded proteins that bind to a small molecule or peptide in the extracellular environment and translate the binding event into an internal signal to regulate processes such as heart rate and even mood. The ability to selectively modulate such fundamental systems offers huge potential with broad applications from the ability to interrogate unknown cellular mechanisms to developing therapeutics when these interactions become aberrant. The scope of this dissertation encompasses determining what properties dictate protein-ligand interactions and the application of these principles to the design of new ones. In particular, chapter 1 covers the design of a molecular switch that is turned on by small molecules. I follow this up in chapter 2 by investigating how to turn off protein function with small molecules in aberrant disease states. In chapter 3 we expand from the world of small molecule ligands to design a protein to turn off function of a protein involved in bacterial pathogenesis.Publication Predicting the Most Tractable Protein Surfaces in the Human Proteome for Developing New Therapeutics(University of Kansas, 2019-05-31) Malhotra, Shipra; Karanicolas, John; Vakser, Ilya; Ray, Christian; Slusky, Joanna; Miao, Yinglong; De Guzman, Roberto; Rafferty, MichaelA critical step in the target identification phase of drug discovery is evaluating druggability, i.e., whether a protein can be targeted with high affinity using drug-like ligands. The overarching goal of my PhD thesis is to build a machine learning model that predicts the binding affinity that can be attained when addressing a given protein surface. I begin by examining the lead optimization phase of drug development, where I find that in a test set of 297 examples, 41 of these (14%) change binding mode when a ligand is elaborated. My analysis shows that while certain ligand physiochemical properties predispose changes in binding mode, particularly those properties that define fragments, simple structure-based modeling proves far more effective for identifying substitutions that alter the binding mode. My proposed measure of RMAC (rmsd after minimization of the aligned complex) can help determine whether a given ligand can be reliably elaborated without changing binding mode, thus enabling straightforward interpretation of the resulting structure-activity relationships. Moving forward, I next noted that a very popular machine learning algorithm for regression tasks, random forest, has a systematic bias in the predictions it generates; this bias is present in both real-world datasets and synthetic datasets. To address this, I define a numerical transformation that can be applied to the output of random forest models. This transformation fully removes the bias in the resulting predictions, and yields improved predictions across all datasets. Finally, taking advantage of this improved machine learning approach, I describe a model that predicts the “attainable binding affinity” for a given binding pocket on a protein surface. This model uses 13 physiochemical and structural features calculated from the protein structure, without any information about the ligand. While details of the ligand must (of course) contribute somewhat to the binding affinity, I find that this model still recapitulates the binding affinity for 848 different protein-ligand complexes (across 230 different proteins) with correlation coefficient 0.57. I further find that this model is not limited to “traditional” drug targets, but rather that it works just as well for emerging “non-traditional” drug targets such as inhibitors of protein-protein interactions. Collectively, I anticipate that the tools and insights generated in the course of my PhD research will play an important role in facilitating the key target selection phase of drug discovery projects.Publication Using Chemical Biology to Modulate Antibody Activity(University of Kansas, 2019-05-31) Khowsathit, Jittasak; Richter, Mark; Slusky, Joanna; Hefty, Scott; Kuczera, Krzysztof; Fischer, ChrisMonoclonal antibodies have shown promising results as therapeutic agents, and yet they can also be associated with adverse side effects due to activity outside the disease site. Aiming to reduce these side effects, we have explored the possibility of a tunable antibody, whose activity can be manipulated via the addition of a small molecule. Previously, we incorporated a single cavity-forming mutation (tryptophan to glycine) into an antibody, and observed reduced antigen-binding activity that could be restored by addition of a complementary ligand (indole) — albeit with binding affinity too low for potential therapeutic applications. Here, I describe a novel computational strategy for enumerating larger cavities in a fluorescein-binding single-chain variable fragment (scFv), leading to a designed variant with three large-to-small mutations (triple mutant) at the domain-domain interface with reduced antigen-binding. Through a complementary virtual screen, we identified a rescuing small molecule (JK43) that enhances binding affinity for antigen. Thorough characterization of this system shows that the loss of activity upon mutation was due to loss of stability and domain dissociation; conversely, addition of JK43 restores stability of the antibody fragment, induces domain re-association, and rescues antigen binding. Beyond this initial model system, I will also describe the transferability of this design (triple mutant and JK43) from the fluorescein-binding scFv onto an unrelated scFv that shares the same three residues used in this design. We hypothesize that this design will also prove transferable onto the many therapeutic antibodies that also share these three residues, including Ipilimumab (anti-CTLA-4), Atezolimumab (anti-PD-L1), Nivolumab (anti-PD-1) and Adalimumab (anti-TNF-α).Publication Cell cycle functions of gamma-tubulin, cyclins, and E3 ubiquitin ligases in Aspergillus nidulans(University of Kansas, 2018-07-31) Paolillo, Vitoria Kate; Oakley, Berl R; Azuma, Mizuki; Azuma, Yoshiaki; Gamblin, Truman C; Neufeld, Kristi; Ward, JoyIn addition to its well-established role in nucleating microtubules at microtubule-organizing centers, γ-tubulin has essential, microtubule-independent functions that are incompletely understood [reviewed in (Oakley et al., 2015)]. Experiments in our lab with the cold-sensitive γ-tubulin mutant mipAD159 in the filamentous fungus Aspergillus nidulans revealed that γ-tubulin has a role in inactivating the anaphase promoting complex/cyclosome (APC/C) resulting in continuous destruction of cyclin B and failure of nuclei to progress through the cell cycle (Nayak et al., 2010). Deletion of the APC/C co-activator CdhA (the A. nidulans Cdh1 homolog) restores cyclin B accumulation and these and other data demonstrate that γ-tubulin plays an important role in inactivating APC/C-CdhA at the G1/S boundary. However, cdhAΔ, mipAD159 strains are as cold sensitive as the mipAD159 parent, indicating that the cold sensitivity is not due to continuous APC/C-CdhA activity (Edgerton-Morgan and Oakley, 2012). Although the underlying molecular mechanism(s) by which γ-tubulin regulates CdhA are not yet known, our data do not support a direct interaction between γ-tubulin and CdhA. Instead, we hypothesize that γ-tubulin acts through regulators of CdhA. Proteins involved in Cdh1 inhibition and inactivation have been identified in other organisms but not in A. nidulans prior to my work. Thus, the first part of my main project consisted of identifying and characterizing regulators of CdhA. As filamentous fungi are hugely important medically, agriculturally and commercially [reviewed in (Meyer et al., 2016)], it is vital that we understand the cell biology of filamentous fungi to be able to combat fungal pathogens and to maximize their growth for production of desirable products. The second part of my main project was aimed at determining whether these CdhA regulators are candidates through which γ-tubulin acts to regulate CdhA at the G1/S transition. In many organisms, initial Cdh1 inactivation at G1/S occurs via phosphorylation by cyclin/CDK complexes which then triggers Cdh1 ubiquitination by the Skp1-Cullin1-F-box (SCF) complex. However, cyclins have not been well studied in members of aspergilli, including A. nidulans. In chapter 3 of this work, I report my identification of all cyclin domain-containing proteins in A. nidulans and establish that this cyclin repertoire is well-conserved in closely and distantly related filamentous ascomycetes. This is significant as the complement of cyclins found in model yeasts (Saccharomyces cerevisiae, Schizosaccharomyces pombe and Candida albicans) differs considerably from one another and from the great majority of filamentous ascomycetes. Thus, A. nidulans is a much better model than these yeasts for studying cyclin function and cell cycle regulation in filamentous fungi. My phylogenetic analyses reveal there are three A. nidulans cyclins that likely carry out cell cycle-related functions (NimECyclin B, PucA, and ClbA). In Chapter 4 of this work, I focus on cyclins PucA and ClbA as they had not been characterized previously. My results reveal that ClbA is not essential, but its destruction is required for mitotic exit. ClbA also appears to function at the G2/M transition. My experiments further demonstrate that both NimECyclin B and ClbA play critical roles in chromosomal disjunction. My results also reveal that PucA is the essential cyclin required for CdhA inactivation at the G1/S transition and that there are no other redundant mechanisms for CdhA inactivation in A. nidulans. Finally, my data indicate that PucA function is required for some of the growth limiting effects of two mipA mutants, including mipAD159, although the mechanism of this interaction is not yet understood. I have also determined that the SCF complex plays a role in regulating CdhA in A. nidulans. In Chapter 5 of this work, I focus on two essential components of the SCF complex, Cullin A (CulA) and SkpA. I have determined their terminal phenotypes via the heterokaryon rescue technique, and I have studied their in vivo localization patterns using fluorescent protein fusions I have generated. Interestingly, CulA-GFP strains in a wild-type background are slightly cold sensitive, and CulA-GFP causes strong synthetic growth reduction with mipAD159. The strong, synthetic genetic interaction between mipAD159 and culA-GFP indicates that γ-tubulin and CulA are involved in a common function that is required for growth. Additionally, I have found that the SCF complex in A. nidulans has a crucial role in suppressing septation near the hyphal tip, which is essential for rapid tip extension in filamentous fungi. My study of cell cycle-related cyclins and SCF components in A. nidulans provides new insights into the regulation of the cell cycle and growth of filamentous fungi. My phylogenetic cyclin analyses also indicate that A. nidulans is a well-suited model compared to popular model yeasts for studying cyclin function and cell cycle regulation in aspergilli and other filamentous fungi. Finally, my data also indicate that PucA and CulA are strong candidates through which γ-tubulin acts to control APC/C-CdhA activity and are worthy of follow-up studies.Publication NEW INSIGHTS INTO THE ROLE OF UNC-6/NETRIN IN GROWTH CONE PROTRUSION, POLARITY AND CYTOSKELETAL ORGANIZATION(University of Kansas, 2018-08-31) Gujar, Mahekta R; Lundquist, Erik A; Ackley, Brian D; Macdonald, Stuart J; Oakley, Berl R; Blumenstiel, Justin PThe formation of complex neuronal circuits is crucial for the proper development of the central nervous system. The wiring structure of the nervous system underlies its function in sensation and movement, and higher order functions such as learning, memory, and cognition. Disruption of this wiring leads to a number of neurodevelopmental disorders such as developmental disability syndromes, autism, and schizophrenia. Axon guidance is an important aspect in this process of development, as neurons are not born with axons but must actively extend these wires in the developing nervous system to reach their appropriate synaptic targets. The developing axons are led by growth cones, dynamic actin-based structures that sense and respond to extracellular guidance cues that drive the forward motion of the axon. Growth cones contain a dynamic lamellipodial body ringed by filopodial protrusions, both important in guiding the axon to its target destination. Motility and guidance behaviors of the growth cone are regulated by its actin and microtubule (MT) cytoskeleton through the modulatory influence of axon guidance cues, such as UNC-6/Netrin and its guidance receptors UNC-40/DCC and UNC-5 that are present at the leading edge of the growth cone. Netrin is a secreted guidance cue that acts as both an axon attractant and repellant in different receptor contexts. Though the role of Netrin and its receptors in axon pathfinding has been extensively studied, very little is known about how Netrin regulates growth cone behavior and morphology in vivo. Caenorhabditis elegans is a useful system to study axon pathfinding and growth cone development in vivo due to its simple, well-characterized nervous system, transparency and fully sequenced genome. The VD and DD motor neurons reside along the ventral nerve cord of the animal, and their axons normally extend straight dorsally to the dorsal nerve cord to form commissures. Though the DD axons develop during embryogenesis, the VD neurons develop post-embryonically in a well-described and stereotypical manner, making them great candidates to study in vivo growth cone development. After a brief introduction to axon guidance and growth cone morphology in chapter I, chapter II describes a novel role for the C. elegans flavin-containing monooxygenase (FMOs) genes in Netrin-mediated axon guidance and growth cone protrusion. We show that the FMO genes are required for VD/DD motor axon guidance and to restrict growth cone filopodial protrusions downstream of the Netrin receptors UNC-40 and UNC-5 and the Rac GTPases CED-10 and MIG-2 in Netrin-mediated growth cone repulsion. In chapter III we present new roles for UNC-6/Netrin in regulating the polarity and extent of growth cone protrusion through its receptors UNC-40 and UNC-5. We demonstrate that UNC-5 signaling regulates three aspects of growth cone morphology during growth away from UNC-6: (i) inhibition of growth cone protrusion (ii) dorsal leading-edge polarization of F-actin and (ii) restriction of MT entry into the growth cone, possibly via the cytoskeletal interacting protein UNC-33/CRMP. We also find that UNC-6 and UNC-40 can stimulate VD growth cone protrusion at the dorsal leading-edge, independent of dorsal F-actin polarity and growth cone MT plus-end accumulation. The characterization of the small GTPases RHO-1 and its guanine nucleotide exchange factor RHGF-1 in modulating growth cone protrusion and microtubule accumulation is detailed in chapter IV. We show that RHO-1 and RHGF-1 are required to prevent MT plus-ends from entering the growth cone as well as limiting excessive filopodial protrusions downstream of UNC-6/Netrin signaling. Chapter V provides new insights into the building blocks of MTs, the tubulins and their importance in MT organization and stability with respect to growth cone morphology. We show that certain missense mutations in the C. elegans alpha and beta-tubulin genes, tba-1 and tbb-1, have different effects on MT stability and in turn growth cone morphology. Mutations that lead to MT destabilitization inhibited growth cone protrusivity, while mutations that lead to the formation of hyperstable MTs caused excess protrusions. This reveals a causal relationship between MT organization and growth cone structure. To address whether other guidance systems cooperate with UNC-6/Netrin to regulate protrusion and directed growth cone migration, chapter VI demonstrates the role of the guidance receptor SAX-3/Robo in dorsal axon guidance and VD growth cone protrusion. We show that sax-3 is required for the guidance of dorsally-directed VD/DD axons and inhibits growth cone protrusion through the Rac GEF UNC-73/Trio. In summary, the results contained here provide new insights into the mechanisms by which Netrin signaling regulates several aspects of growth cone protrusion during the process of axon pathfinding. By studying established and novel genes in repulsion and growth cone inhibition, and by defining their effects on the growth cone cytoskeleton, we are starting to understand how these signaling pathways shape the morphological characteristics of the growth cone during directed migration in vivo.Publication Text Mining for Protein-Protein Docking(University of Kansas, 2018-08-31) Badal, Varsha Dave; Vakser, Ilya A.; Kundrotas, Petras J.; Deeds, Eric J.; Ray, Christian J.; Slusky, Joanna S.G.; Miao, Yinglong; Kuczera, KrzysztofScientific publications are a rich but underutilized source of structural and functional information on proteins and protein interactions. Although scientific literature is intended for human audience, text mining makes it amenable to algorithmic processing. It can focus on extracting information relevant to protein binding modes, providing specific residues that are likely be at the binding site for a given pair of proteins. The knowledge of such residues is a powerful guide for the structural modeling of protein-protein complexes. This work combines and extends two well-established areas of research: the non-structural identification of protein-protein interactors, and structure-based detection of functional (small-ligand) sites on proteins. Text-mining based constraints for protein-protein docking is a unique research direction, which has not been explored prior to this study. Although text mining by itself is unlikely to produce docked models, it is useful in scoring of the docking predictions. Our results show that despite presence of false positives, text mining significantly improves the docking quality. To purge false positives in the mined residues, along with the basic text-mining, this work explores enhanced text mining techniques, using various language processing tools, from simple dictionaries, to WordNet (a generic word ontology), parse trees, word vectors and deep recursive neural networks. The results significantly increase confidence in the generated docking constraints and provide guidelines for the future development of this modeling approach. With the rapid growth of the body of publicly available biomedical literature, and new evolving text-mining methodologies, the approach will become more powerful and adequate to the needs of biomedical community.Publication Current state of tau aggregation inhibitors(University of Kansas, 2018-05-31) Bhattacharya, Reshma; Gamblin, Truman Christopher; Oakley, Berl; Macdonald, StuartTau is an intrinsically disordered, heat stable, and highly soluble protein found primarily in the axons of the central nervous system. It belongs to the family of structural microtubule-associated proteins, the major function of which is promoting and stabilizing microtubule assembly. However, modifications to the protein generate insoluble toxic oligomers of aggregated tau which are amyloid in nature. This modification renders them ineffective in binding to tubulin to stabilize microtubule assembly. Aggregates of tau are pathological hallmarks of many neurodegenerative disorders, collectively known as tauopathies. Some of the most well-known tauopathies are Pick’s disease (PiD), progressive supranuclear palsy (PSP), corticobasal neurodegeneration (CBD), and Alzheimer’s disease (AD). AD is the 6th leading cause of death in the USA and is the most prevalent tauopathy. There are only five FDA approved drugs that can arrest the cognitive decline and other symptoms of AD temporarily, but they do not reverse or inhibit the pathology of the disease. The disease therefore continues to progress, and cognitive functions decline faster when the drugs lose their effectiveness. There is a general consensus that therapies that reduce pathology would be beneficial for treating the disease. AD is characterized by two neuropathological hallmarks – extracellular senile plaques, which are aggregates of amyloid peptide, and intracellular neurofibrillary tangles, which are composed of tau aggregates. Initially, much of the emphasis for drug discovery for AD was focused on inhibiting or reversing the amyloid senile plaque pathology. This focus was due to the observation that familial forms of early onset AD are associated with mutations which enhanced the formation of senile plaques. Additionally, the number of dementia cases involving senile plaques outnumbers dementia cases with pure tauopathies. Recent failures of drugs targeting amyloid accumulation for the treatment of AD and new evidence further strengthening the association of tau pathology with neurodegeneration and AD cognitive impairment have placed importance on the development of tau-based therapeutics. This report discusses the current state of therapies and drugs that are available and are being developed to find an effective cure for AD and other tauopathies.Publication A Step Closer to Precision Oncology: Computational, Biochemical, and Cell-Based Screening to Find Compounds that Stabilize p53(University of Kansas, 2018-05-31) Khar, Karen Rene; Deeds, Eric; Slusky, Joanna; Vakser, Ilya; Ray, Christian; Richter, MarkPersonalized medicine in cancer aims to tailor a treatment plan that takes into account the unique features of a patient's malignancy. One therapeutic target that has a chance to affect a large population of cancer patients is p53. p53 is a tumor suppressor that activates senescence or apoptosis in cells that have accumulated mutations that could lead to cancer. Half of all cancers have mutations in p53, which highlights the importance of its role in disease. A subset of these mutations have been shown to inhibit p53 function by destabilizing p53's core domain. This led to the hypothesis that a personalized drug for patients with this type of destabilized p53 mutation could lead to apoptosis in cancer cells. There has been a lot of evidence supporting this hypothesis. This evidence has inspired many researchers to screen for small molecules that stabilize p53 mutants and rescue function. However, the hits discovered in these screens (with one potential exception) have not been found to be adequate drug leads for several reasons. Many have turned out to rescue function, but not by directly binding p53. Others bind p53, but either lack sufficient binding affinity or cause nonspecific cell responses. All of these are likely to induce side effects if used as part of a cancer therapeutic. This leads to the question: Is there a better way to find a small molecule stabilizer for cancer-associated mutants of p53? Here, I present an alternative approach that focuses on finding a direct binder to p53's core domain in order to avoid off-target effects. Our initial step was a computational approach that uses the crystal structure of p53's core domain in order to virtually screen a set of small molecules for binding. I found a novel pocket on the protein structure that I predicted to be druggable, because the site readily forms pockets during simulations of the core domain. I performed a virtual screen using the DARC, a docking tool from the molecular modeling suite, Rosetta, and selected the 28 best ranked compounds for biochemical testing with purified p53 using two different cancer-associated, destabilizing mutations. Surprisingly, I found that 11 of the 28 compounds stabilized both mutants. Further testing was done in cancer cell lines showing that 7 compounds activated p53 transcription of p21 and PUMA, which are known targets of p53. Using the fluorescent antibody pAb 1620 that binds natively folded p53, we showed that 4 of the compounds lead to a much higher concentration of folded p53 in cells. The excitingly high hit rate was found from a modest sized initial virtual screen of only 64,000 molecules. This suggests that this novel pocket is prone to bind molecules in a manner that rescues structure and function, and should be as a starting point for a larger screen. Also, the compounds from the current screen are intriguing hits that will be further analyzed and optimized to develop new stabilizers of p53.Publication Quorum sensing-controlled genes increase the survival of Chromobacterium violaceum during bacterial interspecies competition(University of Kansas, 2018-05-31) Evans, Kara C; Chandler, Josephine R; Hefty, P. Scott; Hancock, Lynn; Oakley, Berl; Macdonald, Stuart; Blumenstiel, JustinMany Proteobacteria use a cell-cell communication system called quorum sensing (QS) to coordinate gene expression in a cell density-dependent manner. Cell density is detected through the production and diffusion of acyl-homoserine lactones (AHLs). The AHL concentration increases until threshold is reached, and AHLs bind the AHL receptor causing transcription of QS-controlled genes. Many QS-controlled genes include: antimicrobials, biofilm components, and other virulence factors. For this reason, we believe that QS is critical for survival during competition with other bacteria. To test our hypothesis, we have developed a competition model between two soil-saprophytes, Burkholderia thailandensis and Chromobacterium violaceum. Our research demonstrates that QS in C. violaceum and B. thailandensis controls the production of antimicrobials that inhibit the other species’ growth. C. violaceum using QS to increase resistance to bactobolin, a B. thailandensis antibiotic, through a RND efflux pump. This QS-controlled efflux pump is important for limiting QS-deficient mutants in our competition model allowing for the increase in antimicrobial producing cells and increased survival of C. violaceum during competition. Our results demonstrate that interspecies competition can reinforce QS-controlled behaviors by placing constraints on QS-deficient mutants. Additionally, we demonstrate that the AHL receptor, CviR, in C. violaceum Cv017 can bind to N-octanoyl-L-homoserine lactone (C8-HSL) and N-3-hydroxyoctanoyl-L- (3OH-C8)-HSL produced by B. thailandensis. RNA sequencing of transcripts from C. violaceum grown in the presence of C8-HSL or hexanoyl (C6)-HSL revealed an enrichment in the transcriptome for competition associated genes when exposed to C8-HSL. Our transcriptomic and competition results show that the ability of C. violaceum to detect non-cognate AHLs provides an advantage during interspecies competition through the activation of competition associated genes.Publication Structure and Function of Podovirus Sf6 Tail Complex(University of Kansas, 2018-05-31) Liang, Lingfei; Egan, Susan M; De Guzman, Roberto N; Davido, David; Azuma, Mizuki; Fischer, ChrisSf6 is a double-stranded DNA (dsDNA) bacteriophage with a short, non-contractile tail. The tail is a sophisticated molecular machine made of 39 copies of four gene products, including the dodecameric tail adaptor gp7, the hexameric tail nozzle gp8, the trimeric tail needle gp9 and 6 copies of the trimeric tail spike gp14. It has been shown that the tail assembly occurs in a sequential manner. Here we report the high-resolution structure of the Sf6 tail adaptor protein gp7. Comparative structural studies reveal that during tail assembly the gp7 N-terminus undergoes structural rearrangement by repositioning two consecutive repeats of a conserved octad sequence motif, turning the molecule from the preassembly state to the postassembly state, which creates the binding site for the next tail component to attach to. These results provide a structural basis for a mechanism of sequence motifs repositioning by which the adaptor protein mediates the sequential assembly of the phage tail. Tail nozzle gp8 is the following component attached to gp7 in the tail. It is highly conserved between Sf6 and P22, but the structure is not known yet. Here, we did Small-Angle X-ray Scattering (SAXS) analysis on gp8 monomer, showing a brick-shaped, globular protein with a small protrusion. Fitting of the SAXS model into the electron cryo-microscopy (cryoEM) map of the entire tail machine has aided in defining molecular boundaries between gp8 monomers and neighboring subunits of other tail components. One of the important functions of the tail is to deliver viral DNA through host envelope to establish infection. Given the fact that the tail is too short to directly span bacterial envelope, it is assumed that during infection the short tail is extended by three DNA-injection proteins, gp11, gp12, and gp13, to drill through the three-layer envelope of the host cell to inject phage DNA into the host cytoplasm. We achieved the 3D EM reconstruction of gp12 decamer, revealing a tube-like assembly with a constricted channel presumably for dsDNA delivery. We then solved the X-ray structure of the gp12 N-terminal domain (gp12NTD) which, surprisingly, assembles into a undecamer in crystals. This 2.8Å gp12NTD structure represents the first high-resolution structure of tailed virus DNA-injection proteins. The gp12NTD molecule consists of eight α-helices, seven of which forms two helix bundles. Biochemical study suggests that the helix α8 is dispensable for gp12 homo-oligomerization. Analysis on the tertiary structure and the locations of Gly and Pro residues, for the first time, provides experimental foundation for the assumption that internal proteins are partially unfolded when travelling through the narrow tail channel. We also show that P22-gp20 (Sf6-gp12 ortholog) NTD has a highly similar structure and it also assembles to a undecamer. By analyzing the structure characteristics and the conserved features between Sf6-gp12 and P22-gp20, we discussed the possible scenario of gp12/gp20 travelling through tail channel. The gp12CTD is monomeric in solution, and the C-terminal 27 residues are essential for gp12:gp13 interaction. The stoichiometry of gp12 and gp13 is likely to be 1:1. Similarly, gp20 binds with gp16 (counterpart of gp13). Our work sheds light on the roles of the two DNA-injection proteins (Sf6-gp12/P22-gp20 and Sf6-gp13/P22-gp16) in assembly of the extended tail for DNA delivery. A high-resolution X-ray structure of the Non-Structural protein 1 N-terminal domain (NS1N) of Minute Virus of Mice (MVM) is also reported here as an effort to study DNA replication in parvovirus. MVM has a single-stranded DNA (ssDNA) genome with the two ends folding back to from double-stranded hetero-telomeres, providing origin of replication (Ori). NS1N binds to Ori to perform a series of functions including ssDNA nicking. The NS1N structure here shows potential sites for dsDNA binding, ssDNA binding and cleavage on a canonical fold of the histidine-hydrophobic-histidine superfamily of nucleases. Metal derivative crystal structures reveal the nickase active site with an architecture that allows highly versatile metal ligand binding. The structures support a unified mechanism of replication origin recognition for homotelomeric and heterotelomeric parvoviruses, mediated by a basic-residue-rich hairpin and an adjacent helix in the initiator proteins and by tandem tetranucleotide motifs in the replication origins.