Molecular Biosciences Dissertations and Theses
https://hdl.handle.net/1808/8662
2024-03-29T00:15:15ZMitotic SUMOylation: Unraveling the role of DNA Topoisomerase IIα SUMOylation and PIASy SUMO E3 ligase in mitosis
https://hdl.handle.net/1808/31509
Mitotic SUMOylation: Unraveling the role of DNA Topoisomerase IIα SUMOylation and PIASy SUMO E3 ligase in mitosis
Pandey, Nootan
A 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.
2019-12-31T00:00:00ZPolycyclic aromatic hydrocarbons influence naive CD4+ T cell differentiation
https://hdl.handle.net/1808/31485
Polycyclic aromatic hydrocarbons influence naive CD4+ T cell differentiation
Dunbar, Amanda Jo
CD4+ 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.
2019-12-31T00:00:00ZTHE ROAD LESS TRAVELLED: UTILIZATION OF FORMATE IN TWO BIOCHEMICAL REACTIONS IN GRAM-NEGATIVE BACTERIA
https://hdl.handle.net/1808/31360
THE ROAD LESS TRAVELLED: UTILIZATION OF FORMATE IN TWO BIOCHEMICAL REACTIONS IN GRAM-NEGATIVE BACTERIA
Kenjić, Nikola
Formate 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.
2019-08-31T00:00:00ZThe Biosynthesis of Opine Metallophores
https://hdl.handle.net/1808/31356
The Biosynthesis of Opine Metallophores
McFarlane, Jeffrey S
Metal 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.
2019-08-31T00:00:00Z