http://hdl.handle.net/1974/754 Wed, 22 May 2013 11:46:39 GMT 2013-05-22T11:46:39Z http://hdl.handle.net/1974/7776 Title: STRUCTURAL INSIGHTS INTO DICTYOSTELIUM DISCOIDEUM MYOSIN LIGHT CHAIN SPECIFICITY Authors: Liburd, JANINE Abstract: Myosins are molecular motor proteins involved in cell movement, vesicle and organelle transport by moving along the cytoskeletal actin filaments. They include a myosin heavy chain and at least one myosin light chain (LC). The latter are typically bilobal proteins like calmodulin, where each lobe comprises a pair of EF-hand Ca2+-binding motifs. The LCs bind to ~25-residue IQ motifs that loosely conform to an IQXXXRGXXXR consensus sequence, and impart rigidity that is crucial for myosin function. The highly motile amoeba Dictyostelium discoideum expresses seven class I myosins, two of which (MyoD and MyoB) recruit the specific LCs MlcD and MlcB, with MlcB being the first observed single-lobe LC. However, the LCs for the remaining D. discoideum class I myosins are unknown. Identifying and characterizing these LCs is one focus of this thesis, with an overall goal of understanding their role in myosin function and regulation. Nuclear magnetic resonance spectroscopy, site-directed mutagenesis, and computational modeling were used to determine the solution structure of apo-MlcB and identify the MyoB IQ motif-binding site. Apo-MlcB differs from the typical closed conformation of an EF-hand Ca2+-binding protein in the apo-state as helix 1 in its structure is splayed from the remaining helices. The MyoB IQ motif-binding surface is not altered by Ca2+, involves residues from helices 1 and 4, and from residues in the N-terminal canonical EF-hand Ca2+-binding loop, and represents a unique mode of IQ recognition by a myosin LC. Calmodulin was identified as the LC for MyoA and MyoE while another single-lobe LC, MlcC, bound to two of three IQ motifs in MyoC. The solution structure of MlcC was more similar to the C-terminal lobe of apo-calmodulin than to apo-MlcB. Chemical shift perturbation studies suggest that like apo-CaM, MlcC undergoes a global MyoC IQ motif-induced conformational change. Computational modeling of the MlcC-MyoC IQ complex indicates that this is a feasible mode of IQ recognition. The structures of MlcB and MlcC, with their different modes of IQ motif binding, provide novel insights into IQ motif binding specificity and begin to illustrate their role in myosin function and regulation. Description: Thesis (Ph.D, Biochemistry) -- Queen's University, 2013-01-29 11:42:03.428 Tue, 29 Jan 2013 05:00:00 GMT http://hdl.handle.net/1974/7776 2013-01-29T05:00:00Z http://hdl.handle.net/1974/7770 Title: STRUCTURAL INSIGHTS INTO NOVEL MICROBIAL METALLOENZYMES Authors: van Staalduinen, Laura Abstract: Metalloproteins represent a large portion of the total proteome. When bound to a protein a metal ion influences both protein stability and function through structural, catalytic or regulatory roles. Discovery of a metal ion cofactor presents new insight into both the structural and functional aspects of a protein and can be essential for the elucidation of the functional and mechanistic details of a protein of interest. The cupin, 2-oxoglutarate/Fe2+-dependent oxygenases (2OG oxygenases) and the di-iron oxygenase families of metalloproteins exemplify the diversity and catalytic potential of a metal ion cofactor, as well as the conservation of 3-dimensional fold and structural features in proteins with similar functions. The structural and biochemical characterization of three novel microbial metalloenzymes are presented; two Escherichia coli hypothetical proteins of previously unknown function, E. coli cupin sugar isomerase (EcSI) and a 2OG oxygenase, YcfD, and the novel microbial carbon-phosphorus (C-P) bond cleavage enzyme, PhnZ, are presented. In each case the identification of a metal ion cofactor and structure determination led to important functional insights. EcSI is encoded by a gene that is highly conserved among pathogenic bacteria. It has been identified as a sugar isomerase with specificity for the rare sugar D-lyxose as well as D-mannose based on structural homology to the cupin phosphoglucose isomerases, suggesting a role for EcSI in metabolism of alternative carbon sources. Structural homology of YcfD, the second metalloenzyme, to the 2OG oxygenase family, particularily human proteins involved in ribosome assembly, combined with evidence that YcfD interacts with the essential ribosomal protein L-16 provides the first evidence of translational regulation by a 2OG oxygenase in E. coli. The third metalloenzyme, PhnZ, was previously identified as an iron dependent oxygenase. Structural characterization revealed that PhnZ possesses a di-iron cofactor and shows significant structural homology to a di-iron oxygenase, providing structural evidence for its novel mechanism of C-P bond cleavage. Combined, these three structures also highlight several features of metal ion-enzyme interaction and regulation mechanisms employed by metalloenzymes as well as the importance of structure in the elucidation of functional and mechanistic characteristics of a protein. Description: Thesis (Ph.D, Biochemistry) -- Queen's University, 2013-01-24 21:21:36.195 Mon, 28 Jan 2013 05:00:00 GMT http://hdl.handle.net/1974/7770 2013-01-28T05:00:00Z http://hdl.handle.net/1974/7672 Title: THE UNLIGANDED GLUCOCORTICOID RECEPTOR AS A TRANSCRIPTIONAL REGULATOR IN MAMMARY EPITHELIAL CELLS Authors: Ritter, Heather Abstract: This work presents the first evidence of a ligand-independent role for the glucocorticoid receptor (GR) as a positive regulator of gene expression in mammary cells. We have demonstrated that unliganded GR interacts directly with the promoter of the tumour suppressor gene BRCA1, and upregulates its expression. The presence of the stress hormone hydrocortisone (HC) abolished this interaction and resulted in repression of BRCA1. Since low levels of BRCA1 have been implicated in the development of sporadic breast cancer, this may represent a novel mechanism through which prolonged stress signaling increases breast cancer risk. We determined that the interaction between unliganded GR and BRCA1 is mediated through the beta subunit of the Ets transcription factor GABP at the RIBS promoter element. GR and GABPβ were shown to interact in both co-immunoprecipitation and mammalian two-hybrid assays, and this interaction involved the N-terminal to central regions of both proteins. To further characterize the role of unliganded GR in breast cells, we used shRNA to generate mouse mammary cell lines with depleted endogenous GR expression. Loss of GR resulted in an impaired capacity of cells to differentiate into acini, but this effect was rescued by the addition of glucocorticoids, implicating both the liganded and unliganded forms of GR as key regulators of differentiation. We performed expression microarray to identify targets of unliganded GR using the GR-depleted cell lines. This analysis revealed 260 genes negatively regulated and 343 genes positively regulated by unliganded GR. Many of the positively regulated genes were involved in pro-apoptotic networks, and appeared to oppose the activity of liganded GR targets. Validation and further analysis of five candidates of positive regulation by unliganded GR indicated that two of these, Hsd11b1 and Ch25h, were regulated by unliganded GR in a manner similar to Brca1. The Hsd11b1 enzyme regulates intracellular glucocorticoid levels by interconverting cortisol and its inactive metabolite, cortisone. Further investigation of Hsd11b1 expression and regulation indicated that Hsd11b1 activity appears to be unidirectional in breast cells, specifically inactivating cortisol. Overall, this work suggests that gene regulation by unliganded GR represents a mechanism for protecting the breast from tumourigenesis during stress. Description: Thesis (Ph.D, Biochemistry) -- Queen's University, 2012-11-29 11:18:14.596 Mon, 03 Dec 2012 05:00:00 GMT http://hdl.handle.net/1974/7672 2012-12-03T05:00:00Z http://hdl.handle.net/1974/7521 Title: Characterization of Glucocorticoid Receptor Promoter Methylation in Breast Cancer Authors: Nesset, Kirsten A. Abstract: Epidemiological studies have identified psychological stress as a significant risk factor in breast cancer. The stress response is regulated by the HPA axis in the brain and is mediated by glucocorticoid receptor (GR) signalling. It has been found that early life events can affect epigenetic programming of GR, and methylation of the GR promoter has been reported in colorectal tumourigenesis. Decreased GR expression has also been observed in breast cancer. In addition, it has been previously demonstrated that unliganded GR can serve as a direct activator of the BRCA1 promoter in mammary epithelial cells. We propose a model whereby methylation of the GR promoter in the breast significantly lowers GR expression, resulting in insufficient BRCA1 promoter activation and an increased risk of developing cancer. Antibody-based methylated DNA enrichment was followed by qPCR analysis (MeDIP-qPCR) in a novel assay developed to detect locus-specific methylation levels. It was found that 13% of primary breast tumours were hypermethylated at the GR proximal promoter whereas no methylation was detected in normal tissue. RT-PCR and 5’ RACE analysis identified exon 1B as the predominant alternative first exon in the breast. Tumours methylated near exon 1B had decreased GR expression compared to unmethylated samples, suggesting that this region is important for transcriptional regulation of GR. It was also determined that GR and BRCA1 expression was decreased in breast tumour compared to normal tissue. Furthermore, the relative expression of GR and BRCA1 measured by qRT-PCR was correlated in normal tissue but this association was not found in tumour tissue. From this, it appears that lower GR levels with associated decreased BRCA1 expression in tissues may be a predisposing factor for breast cancer. Based on these results we propose a role for GR as a potential tumour suppressor gene in the breast due to its association with BRCA1, also a tumour suppressor gene, as well as its consistently decreased expression in breast tumours and methylation of its proximal promoter in a subset of cancer patients. Description: Thesis (Master, Biochemistry) -- Queen's University, 2012-09-26 18:19:11.006 Wed, 26 Sep 2012 04:00:00 GMT http://hdl.handle.net/1974/7521 2012-09-26T04:00:00Z