Pharmacology & Toxicology Department Theses and Dissertations
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The advanced degree programs at the Indiana University of Medicine Department of Pharmacology and Toxicology prepare scientists for careers across the spectrum of biomedical research. The Master of Science (M.S.) degree is a thesis research degree that gives a student the intellectual background to understand and participate in ongoing research projects. The Doctor of Philosophy (Ph.D.) degree is offered for the student who wants to pursue an independent career in research. Students with the Ph.D. degree are prepared for an academic career combining research with teaching or for a career in industrial pharmaceutical research. A combined M.D./Ph.D. degree is open to qualified individuals who ultimately seek to direct biomedical research with a clinical emphasis.
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Browsing Pharmacology & Toxicology Department Theses and Dissertations by Subject "Acetyltransferases"
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Item Functions of the Unique N-terminus of a GCN5 Histone Acetylase in Toxoplasma gondii(2007-05-18T13:14:16Z) Bhatti, Micah M.; Sullivan, William J., Jr.; Chan, Edward M.; Queener, Sherry F.; Safa, Ahmad R.; Sinai, Anthony P.; Vasko, Michael, R.GCN5 is a histone acetyltransferase (HAT) that remodels chromatin by acetylating lysine residues of histones. The GCN5 HAT identified in Toxoplasma gondii (TgGCN5) contains a unique N-terminal “extension” that bears no similarity to known proteins and is devoid of known protein motifs. The hypothesis of this thesis is the N-terminal extension is critical to the function of TgGCN5. Three possible roles of the N-terminus were investigated: nuclear localization, protein-protein interactions, and substrate recognition. Subcellular localization was determined via immunocytochemistry using parasites expressing recombinant forms of TgGCN5 fused to a FLAG tag. Initial studies performed with parasites expressing full length FLAG-TgGCN5 were positive for nuclear localization. Without the N-terminal extension (FLAG-ΔNT-TgGCN5) the protein remains cytoplasmic. Additional studies mapped a six amino acid motif (RKRVKR) as the nuclear localization signal (NLS). When RKRVKR is fused to a cytoplasmic protein, it gains access to the nucleus. Furthermore, we have established the NLS interacts with Toxoplasma importin α, a protein involved in nuclear trafficking. Interaction with importin α provides evidence that the TgGCN5 N-terminal extension is involved in mediating protein-protein interactions. In order to identify additional interacting proteins, FLAG affinity purification was performed on parasites expressing full length FLAG-TgGCN5 and FLAG-ΔNT-TgGCN5. Upon comparing the results of the two purifications, proteins captured with only full length TgGCN5 may be interacting with the N-terminal extension. Full length TgGCN5 affinity purification indicates an interaction with histone proteins, two different homologues of Ada2 (adapter protein reported to interact with GCN5 homologues), and several heat shock proteins. With regard to substrate recognition, the N-terminal extension of TgGCN5 is dispensable for the acetylation of non-nucleosomal histones in vitro. However, the lysine acetylated by TgGCN5 is surprisingly unique. Other GCN5 homologues preferentially acetylate lysine 14 in histone H3, but TgGCN5 exclusively acetylates lysine 18 in histone H3 and has no activity on lysine 14. Taken together, these results argue that the N-terminal extension of TgGCN5 is critical for mediating protein-protein interactions, including those responsible for trafficking the HAT to the parasite nucleus but does not appear to be required for the acetylation of non-nucleosomal histones.Item GCN5-B is a Novel Nuclear Histone Acetyltransferase that is Crucial for Viability in the Protozoan Parasite Toxoplasma gondii(2011-03-16) Dixon, Stacey E.; Sullivan, William J., Jr.; Chan, Rebecca J.; Hocevar, Barbara A.; Queener, Sherry F.; Zhang, Jian-TingInfection with the single-celled parasite Toxoplasma gondii (phylum Apicomplexa) is usually benign in normal healthy individuals, but can cause congenital birth defects, ocular disease, and also life-threatening infection in immunocompromised patients. Acute infection caused by tachyzoites is controlled by a healthy immune response, but the parasite differentiates into a latent cyst form (bradyzoite) leading to permanent infection and chronic disease. Current therapies are effective only against tachyzoites, are highly toxic to the patient, and do not eradicate the encysted bradyzoites, thus highlighting the need for novel therapeutics. Inhibitors of histone deacetylases have been shown to reduce parasite viability in vitro demonstrating that chromatin remodeling enzymes, key mediators in epigenetic regulation, might serve as potential drug targets. Furthermore, epigenetic regulation has been shown to contribute to gene expression and differentiation in Toxoplasma. This dissertation focused on investigating the physiological role of a Toxoplasma GCN5-family histone acetyltransferase (HAT), termed TgGCN5-B. It was hypothesized that TgGCN5-B is an essential HAT that resides within a unique, multi-subunit complex in the parasite nucleus. Studies of TgGCN5-B have revealed that this HAT possesses a unique nuclear localization signal (311RPAENKKRGR320) that is both necessary and sufficient to translocate the protein to the parasite nucleus. Although no other protein motifs have been identified in the N-terminal extension of TgGCN5-B, it is likely that this extension plays a role in protein-protein interactions. All GCN5 homologues function within large multi-subunit complexes, many being conserved among species, but bioinformatic analysis of the Toxoplasma genome revealed a lack of many of these conserved components. Biochemical studies identified several potential TgGCN5-B associating proteins, including several novel apicomplexan transcription factors. Preliminary evidence suggested that TgGCN5-B was essential for tachyzoites; therefore, a dominant-negative approach was utilized to examine the role of TgGCN5-B in the physiology of Toxoplasma. When catalytically inactive TgGCN5-B protein was over-expressed in the parasites, there was a significant decrease in tachyzoite growth and viability, with initial observations suggesting defects in nuclear division and daughter cell budding. These results demonstrate that TgGCN5-B is important for tachyzoite development and indicate that therapeutic targeting of this HAT could be a novel approach to treat toxoplasmosis.Item Identification of TgElp3 as an essential, tail-anchored mitochondrial lysine acetyltransferase in the protozoan pathogen toxoplasma gondii(2014-07-11) Stilger, Krista L.; Nass, Richard M.; Bauer, Margaret E.; Oxford, G. S.; Queener, Sherry F.; Sullivan, William J., Jr.Toxoplasma gondii, a single-celled eukaryotic pathogen, has infected one-third of the world’s population and is the causative agent of toxoplasmosis. The disease primarily affects immunocompromised individuals such as AIDS, cancer, and transplant patients. The parasites can infect any nucleated cell in warm-blooded vertebrates, but because they preferentially target CNS, heart, and ocular tissue, manifestations of infection often include encephalitis, myocarditis, and a host of neurological and ocular disorders. Toxoplasma can also be transmitted congenitally by a mother who becomes infected for the first time during pregnancy, which may result in spontaneous abortion or birth defects in the child. Unfortunately, the therapy currently available for treating toxoplasmosis exhibits serious side effects and can cause severe allergic reactions. Therefore, there is a desperate need to identify novel drug targets for developing more effective, less toxic treatments. The regulation of proteins via lysine acetylation, a reversible post-translational modification, has previously been validated as a promising avenue for drug development. Lysine acetyltransferases (KATs) are responsible for the acetylation of hundreds of proteins throughout prokaryotic and eukaryotic cells. In Toxoplasma, we identified a KAT that exhibits homology to Elongator protein 3 (TgElp3), the catalytic component of a transcriptional elongation complex. TgElp3 contains the highly conserved radical S-adenosylmethionine and KAT domains but also possesses a unique C-terminal transmembrane domain (TMD). Interestingly, we found that the TMD anchors TgElp3 in the outer mitochondrial membrane (OMM) such that the catalytic domains are oriented towards the cytosol. Our results uncovered the first tail-anchored mitochondrial KAT reported for any species to date. We also discovered a shortened form of Elp3 present in mouse mitochondria, suggesting that Elp3 functions beyond transcriptional elongation across eukaryotes. Furthermore, we established that TgElp3 is essential for parasite viability and that its OMM localization is important for its function, highlighting its value as a potential target for future drug development.Item Lysine acetyltransferase Gcn5-B regulates the expression of crucial genes in Toxoplasma and its function is regulated through lysine acetylation(2014-04-02) Wang, Jiachen; Sullivan, William J., Jr.; Queener, Sherry F.; Arrizabalaga, Gustavo; Nass, Richard M.; Lu, TaoHistone acetylation has been linked to developmental changes in gene expression and is a validated drug target of apicomplexan parasites, but little is known about the roles of individual histone modifying enzymes and how they are recruited to target genes. The protozoan parasite Toxoplasma gondii (phylum Apicomplexa) is unusual among invertebrates in possessing two GCN5-family lysine acetyltransferases (KATs). While GCN5a is required for gene expression in response to alkaline stress, this KAT is dispensable for parasite proliferation in normal culture conditions. In contrast, GCN5b cannot be disrupted, suggesting it is essential for Toxoplasma viability. To further explore the function of GCN5b, we generated clonal parasites expressing an inducible HA-tagged form of GCN5b containing a point mutation that ablates enzymatic activity (E703G). Stabilization of this dominant-negative form of GCN5b was mediated through ligand-binding to a destabilization domain (dd) fused to the protein. Induced accumulation of the ddHAGCN5b(E703G) protein led to a rapid arrest in parasite replication. Growth arrest was accompanied by a decrease in histone H3 acetylation at specific lysine residues as well as reduced expression of GCN5b target genes in GCN5b(E703G) parasites, which were identified using chromatin immunoprecipitation coupled with microarray hybridization (ChIP-chip). We also demonstrate that GCN5b interacts with AP2-domain proteins, which are plant-like transcription factors in Apicomplexa. The interactions between GCN5b, AP2IX-7, and AP2X-8 were confirmed by reciprocal co-immunoprecipitation and revealed a “core complex” that includes the co-activator ADA2-A, TFIID subunits, LEO1 polymerase-associated factor (Paf1) subunit, and RRM proteins. The dominant-negative phenotype of ddHAGCN5b(E703G) parasites, considered with the proteomics and ChIP-chip data, indicate that GCN5b plays a central role in transcriptional and chromatin remodeling complexes. We conclude that GCN5b has a non-redundant and indispensable role in regulating gene expression required during the Toxoplasma lytic cycle.