Dictyostelium is a model eukaryote for the study of a multitude of fundamental cellular processes as well as several human diseases. Despite its extensive study relatively little is known about its nucleolus. Only three nucleolar proteins have been identified. The nucleolus in Dictyostelium is different than that of other eukaryotes since it is neither bipartite nor tripartite, possessing no visible subcompartments at the ultrastructural level. Moreover, it exists as two to four patches adjacent to the inner nuclear envelope instead of within the nucleoplasm. The aim of this study was thus to identify and characterize novel nucleolar proteins in Dictyostelium in order to better understand the structure and function of its nucleolus. Previous work had shown that NumA1, a protein linked to cell cycle in Dictyostelium, localizes to similar intranuclear patches suggesting it may be nucleolar. NumA1-binding partners Ca2+-binding protein (CBP) 4a and puromycin-sensitive aminopeptidase A may therefore also reside in the nucleolus. Based on the function of a potential NumA1 homologue in other organisms, BRG1-associated factor 60a homologue Snf12 and checkpoint kinase 2 (Rad53 in yeast) homologue forkhead-associated kinase (Fhk) A were chosen as potential nucleolar proteins in Dictyostelium that may also be involved in cell cycle events. Using a diversity of approaches, this study found that NumA1, CBP4a, Snf12, and FhkA are nucleolar proteins in Dictyostelium while puromycin-sensitive aminopeptidase A is nucleoplasmic. Several nuclear localization signals (NLSs) were identified in these proteins some of which also act as nucleolar localization signals (NoLSs). These NLS/NoLSs (within NumA1 and Snf12) represent the first NoLSs and first NLS/NoLSs identified in Dictyostelium. Treatment with the rDNA transcription inhibitor AM-D led to the budding of nucleolar CBP4a, Snf12, and FhkA from the nucleus to the cytoplasm, a phenomenon not previously observed in any organism. This study also examined for the first time the redistribution of nucleolar proteins during mitosis, a time when the nucleolus disassembles into its component parts. The nuclear envelope was also shown to become permeable at this time. Finally, multiple nucleolar subcompartments were identified suggesting compartmentalization of different functions in the Dictyostelium nucleolus.

Carbon catabolite repression (CCR) is a mechanism by which micro-organisms preferentially utilize more easily metabolizable carbon sources in comparison to less easily metabolizable carbon sources. It prevents the organisms from unnecessary expenditure of energy and enables them to exploit the nutrients in appropriate manner. It represents a complex system of gene regulation. The main aim of this study was to study the intracellular localization of proteins involved in CCR including CreA, CreB, CreC and CreD in A. nidulans in repressing and derepressing conditions. The major regulatory protein involved in CCR in A. nidulans is CreA. It is a DNA-binding repressor, but very little is known about the molecular events that allow CreA function to result in appropriate regulation in response to carbon source. To determine the amount and localization of CreA in different carbon sources, strains were made over-expressing GFP and HA tagged CreA. Western analysis showed that high levels of full length CreA can be present in cells that show normal responses to carbon catabolite repression, whether they are grown in repressing or derepressing media. Hence the amount of CreA is similar in both the conditions and thus degradation of CreA is not a key step in carbon catabolite repression. Fluorescence microscopy studies have shown that CreA is in the nucleus under repressing and derepressing carbon conditions and this is not affected by the absence of CreB or CreD, the other important proteins in A. nidulans. Thus mere localization of CreA in nucleus is not sufficient to cause carbon catabolite repression and there is some modification process involved for CreA to act as a repressor protein in CCR. CreB is a deubiquitinating protein and CreC is a protein containing five WD 40 repeats, a putative nuclear localization signal (NLS) and a proline rich region and both the proteins are present in the cell in a complex. CreB was localized using strains that over-expresses GFP tagged CreB and fluorescence microscopy. CreB is present mainly in the cytoplasm in both repressing and derepressing conditions. Moreover, intracellular localization of CreB is unaffected by the presence or absence of CreD. However, the amount of CreB was higher in a creD+ background as compared to a creD34 mutant background, implying that the presence of CreD affects the amount of CreB in the cell. CreC was localized by using strain that over-expresses YFP tagged CreC and it is also present mainly in the cytoplasm. CreD contains arrestin domains and PY motifs and is highly similar to the Rod1p and Rog3p from S. cerevisiae. CreD is proposed to be involved in ubiquitination process in CCR in A. nidulans. Localization studies have shown that CreD is present throughout the cell in a punctate pattern with more in the cytoplasm than in the nucleus. CreB and CreD co-localize in some regions of the cell whereas in other regions either CreB or CreD is present. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1346526 / Thesis (Ph.D.) - University of Adelaide, School of Molecular and Biomedical Science, 2008

Carbon catabolite repression (CCR) is a mechanism by which micro-organisms preferentially utilize more easily metabolizable carbon sources in comparison to less easily metabolizable carbon sources. It prevents the organisms from unnecessary expenditure of energy and enables them to exploit the nutrients in appropriate manner. It represents a complex system of gene regulation. The main aim of this study was to study the intracellular localization of proteins involved in CCR including CreA, CreB, CreC and CreD in A. nidulans in repressing and derepressing conditions. The major regulatory protein involved in CCR in A. nidulans is CreA. It is a DNA-binding repressor, but very little is known about the molecular events that allow CreA function to result in appropriate regulation in response to carbon source. To determine the amount and localization of CreA in different carbon sources, strains were made over-expressing GFP and HA tagged CreA. Western analysis showed that high levels of full length CreA can be present in cells that show normal responses to carbon catabolite repression, whether they are grown in repressing or derepressing media. Hence the amount of CreA is similar in both the conditions and thus degradation of CreA is not a key step in carbon catabolite repression. Fluorescence microscopy studies have shown that CreA is in the nucleus under repressing and derepressing carbon conditions and this is not affected by the absence of CreB or CreD, the other important proteins in A. nidulans. Thus mere localization of CreA in nucleus is not sufficient to cause carbon catabolite repression and there is some modification process involved for CreA to act as a repressor protein in CCR. CreB is a deubiquitinating protein and CreC is a protein containing five WD 40 repeats, a putative nuclear localization signal (NLS) and a proline rich region and both the proteins are present in the cell in a complex. CreB was localized using strains that over-expresses GFP tagged CreB and fluorescence microscopy. CreB is present mainly in the cytoplasm in both repressing and derepressing conditions. Moreover, intracellular localization of CreB is unaffected by the presence or absence of CreD. However, the amount of CreB was higher in a creD+ background as compared to a creD34 mutant background, implying that the presence of CreD affects the amount of CreB in the cell. CreC was localized by using strain that over-expresses YFP tagged CreC and it is also present mainly in the cytoplasm. CreD contains arrestin domains and PY motifs and is highly similar to the Rod1p and Rog3p from S. cerevisiae. CreD is proposed to be involved in ubiquitination process in CCR in A. nidulans. Localization studies have shown that CreD is present throughout the cell in a punctate pattern with more in the cytoplasm than in the nucleus. CreB and CreD co-localize in some regions of the cell whereas in other regions either CreB or CreD is present. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1346526 / Thesis (Ph.D.) - University of Adelaide, School of Molecular and Biomedical Science, 2008

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed June 23, 2008). Includes bibliographical references.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2004. / Vita. Bibliography: 118-130.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Raymond R. Rowland / Severe acute respiratory syndrome (SARS) is caused by an enveloped, positive-stranded RNA virus, the SARS coronavirus (SARS-CoV). Coronaviruses along with the arteriviruses are placed in the order, Nidovirales. Even though nidovirus replication is restricted to the cytoplasm, the nucleocapsid protein (N) of several coronaviruses and arteriviruses, localize to the nucleolus during infection. Confocal microscopy of N protein localization in Vero cells infected with the SARS-CoV or transfected with the SARS-CoV N gene failed to show presence of N in the nucleoplasm or nucleolus. Recombinant N remained cytoplasmic after the addition of leptomycin B (LMB), a drug that inhibits nuclear export. SARS-CoV N possesses a unique lysine-rich domain, located between amino acids 369-389, which possesses several nuclear localization signal (NLS) and nucleolar localization signal (NoLS) motifs. A chimeric protein composed of the 369-389 peptide substituted for the NLS of equine infectious anemia virus (EIAV) Rev protein (ERev) showed no nuclear localization activity. Three negatively charged amino acids, located at positions 372, 377 and 379 in SARS-CoV N were hypothesized to play a role in the loss of nuclear targeting. Substitution of aspartic acid-372 with alanine restored nuclear localization to the chimeric protein. A full-length recombinant SARS-N protein with the alanine-372 substitution localized to the nucleus. Therefore, the presence of an aspartic acid at position 372 is sufficient to retain N in the cytoplasm The mechanistic basis for how aspartic acid-372 interrupts nuclear transport is unknown, but may lie in the electrostatic repulsion with negatively charged amino acids located within the NLS binding pocket of importin-alpha.

No description available.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Infection 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.

Bone morphogenetic protein 2 (BMP2) is a secreted growth factor that is essential for proper embryonic development and proliferation. Our laboratory discovered a nuclear variant of BMP2 (nBMP2) which is produced when translation is initiated at an alternative start codon within the BMP2 gene. When translation occurs at the downstream start codon, the resulting protein lacks the ER signal peptide, thereby allowing cytoplasmic translation and nuclear localization. Our aim is to distinguish the role of this nuclear localized variant from secreted BMP2. Overexpression of nBMP2 in HEK293 and HT29 cell lines resulted in a higher percentage of cells in proliferative phases of the cell cycle. We determined that nBMP2 does not regulate cell cycle progression by inducing hyperphosphorylation of retinoblastoma protein (Rb), but it may regulate the cell cycle by interacting with ROC1. In order to examine the role of nBMP2 in vivo, we have generated a mouse model in which a mutation of the nuclear localization signal (NLS) disrupts nuclear localization of nBMP2. Aberrant crypts were more abundant in nBmp2NLStm azoxymethane (AOM) treated mice than in wild type mice. Furthermore, H&E staining of colonic tissue showed that mutant mice have increased levels of dysplasia and aberrant crypt foci. This work suggests that nBMP2 is involved in regulating cell cycle progression and proliferation, and therefore may play a role in tumorigenesis.