Thesis (Ph. D.)--University of Missouri-Columbia, 1996. / Typescript. Vita. Includes bibliographical references (leaves [109]-132. Also available on the Internet.

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The signal transducer and activator of transcription (STAT) and protein inhibitor of STAT(PIAS) system represent an elegant regulatory mechanism of transcriptional control IN mammalian cytokine signalling. Abnormal activation of the system is associated with immune disorders and a large group of diverse tumours. PIAS3 is a multiple domain protein with distinct functions involved in regulation of cytokine-mediated gene activation pathways.Its over-expression significantly inhibits cell growth and renders cancer cells more sensitive to drugs. The objective of this study was to structurally and biochemically characterise the function of the PIAS3 protein using in silico, in vivo and in vitro analysis approaches.The conservation pattern of the PIAS protein family and critical conserved residues in the PINIT (Proline, Isoleucine, Asparagine, Isoleucine, Tyrosine) domain were identified. The PINIT domain model was generated based on the PINIT domain structure of yeast PIAS3 homologue Siz1 and structural determinants in the PIAS3-STAT3 interaction were evaluated.Guided by the in silico findings, in vivo analysis of the localisation of the PIAS3, mutantderivatives of PIAS3 (PIAS3-L97A, PIAS3-R99N, PIAS3-R99Q), PINIT and acidic domain was conducted. PIAS3 was completely localised in the nucleus while PIAS3 mutants appeared to exhibit diffuse cytoplasmic distribution. The PINIT domain was predominantly localised in the nucleus with some apparent perinuclear staining while the acidic domain exhibited a predominantly perinuclear staining pattern. Further analysis of the PINIT domain and the effect of the mutants on PIAS3-STAT3 interaction were assessed by in vitro analysis. Guided by in silico analysis, the PINIT domain and mutant derivatives of PINIT domain (PINIT-L97A, PINIT-R99N, and PINIT-R99Q) were heterologously expressed in Escherichia coli and subsequently purified using a combination of immobilized metal affinity and size exclusion based chromatography. The size and structural elements of the PINIT domain and its mutants were characterised. The 23 kDa PINIT domain was found to exist as a monomer in solution and its secondary structure was shown to consist of 66 % β-sheets by fourier transformed infrared spectroscopy consistent with the generated homology model.Using surface plasmonresonance spectroscopy (SPR) the PINIT domain was shown to bind to STAT3 in a specific concentration dependent manner. Recombinant PINIT-L97A,PINITR99N and PINIT-R99Q mutants, which exhibited similar structural integrity to the wildtype, were found to abrogate binding to STAT3. These findings suggest that these residues form part of a potential binding surface for stat3. In conclusion, this study has provided evidence that the PINIT domain is an important determinant of PIAS3 interaction with STAT3 and that the interaction is mediated by defined conserved residues directly involved in the PINITSTAT3 interaction.

Transcription factors (TFs) are key regulators of gene expression whose failure has been implicated in many diseases, including cancer. They bind at various sites at different specificity depending on the prevailing cellular conditions, disease, development stage or environmental conditions of the cell. TF binding specificity is how well a TF distinguishes functional sites from potential non-functional sites to form a useful regulatory network. Owing to its role in diseases, various techniques have been used to determine TF binding specificity in vitro and in vivo, including chromatin immuno-precipitation followed by massively parallel sequencing (ChIP-seq). ChIP-seq is an in vivo technique that considers how the chromatin landscape affects TF binding. Motif enrichment analysis (MEA) tools are used to identify motifs that are over-represented in ChIP-seq peak regions. One such tool, CentriMo, finds over-represented motifs at the center since peak calling software are biased to declaring binding regions centered at the TF binding site. In this study, we investigate the use of CentriMo and other MEA tools to determine the difference in motif enrichment attributed presence of Chronic Myeloid leukemia (CML)), treatment with Interferon (IFN) and Dexamethasone (DEX) compared to control based on Fisher’s exact test; using uniform peaks ChIP-seq data generated by the ENCODE consortium. CentriMo proved to be capable. We observed differential motif enrichment of TFs with tumor promoter activity: YY1, CEBPA, Egr1, Cmyc family, Gata1 and JunD in K562 while Stat1, Irf1, and Runx1 in Gm12878. Enrichment of CTCF in Gm12878 with YY1 as the immuno-precipitated (ChIP-ed) factor and the presence of significant spacing (SpaMo analysis) of CTCF and YY1 in Gm12878 but not in K562 could show that CTCF, as a repressor, helps in maintaining the required YY1 level in a normal cell line. IFN might reduce Cmyc and the Jun family of TFs binding via the repressive action of CTCF and E2f2. We also show that the concentration of DEX treatment affects motif enrichment with 50nm being an optimum concentration for Gr binding by maintaining open chromatin via AP1 TF. This study has demonstrated the usefulness of CentriMo for TF binding specificity analysis.

The termina1 enzyme in the linoleoyl-CoA desaturase enzyme complex, delta-6-desaturase was implied in the control of cell proliferation in cancer cells. One of the aims of this study was to isolate the terminal enzyme. It was decided that in order to isolate this enzyme it was first necessary to isolate the entire complex and then to enzymatically solubilise the first two components of the complex i e cytochrome b5 reductase and cytochrome b5 from the complex resulting in a pure delta-6-desaturase . The first two components were isolated and purified using simplified and easily reproducible methodologies which could be utilised in the final purification of delta-6- desaturase. The entire enzyme complex, linoleoyl-CoA desaturase was also isolated in a pure form and this pure complex was used to attempt to isolate delta-6-desaturase. The terminal enzyme was isolated with some cytochrome b5 still bound to it. The methods used had proven to be successful and with some modifications should yield a pure enzyme. Zinc and GLA were known to play a role in the inhibition of cancer cell proliferation and zinc was hypothesised to inhibit cell growth by stimulating the activity of the linoleoyl-CoA desaturase enzyme complex which is involved in the regulation of cell proliferation. GLA is the product of the reaction that this enzyme complex catalyses and GLA has been shown to inhibit cancer ce ll growth. The effect of GLA on cell growth and linoleoyl-CoA desaturase activity was thus investigated. Results showed that both zinc and GLA inhibited cell growth and that the combined addition of zinc and GLA generally resulted in the inhibition of cell growth and the activation of linoleoyl-CoA desaturase activity in the BL-6 cells while having a less pronounced effect on the LLCMK cells. The results of this study support the hypothesis that zinc may be a cofactor of linoleoyl-CoA desaturase.

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The concentration of insulin-like growth factor I (IGF-I) in tissue taken from human non-small cell lung carcinoma (NSCLC) is 1.4- to 7-fold higher than the concentration of IGF-I in the surrounding normal lung tissue and therefore IGF-I may be involved in the growth of NSCLC. In this study it was determined that NSCLC cell lines (A549, A427, SK-LU-1) expressed the type I IGF-I receptor protein and IGF-I stimulated the proliferation of low density plated (2000 cells/cm² growth area) carcinoma cells by 1.6- to 3- fold above control after a four day inCUbation period under serum-free conditions (A549, A427) or in the presence of 0.25% serum (SK-LU-1). In addition, when added to detergent-solubilized type I IGF receptors from A549 cells, IGF-I stimulated [1] a dose-dependent increase in the autophosphorylation of the type I IGF receptor, and [2] a dose-dependent increase (1.5- to 4-fold) in the phosphorylation of a tyrosine kinase-specific substrate. These results suggest that the growth promoting activity of IGF-I for the lung carcinoma cells was mediated through the activation of the type I IGF receptor. / Ph. D.

In the present work, the chromatographic behavior and partial characterization of 5 protein phosphatases, 6 paranitrophenyl phosphate (pnpp) phosphatases and 2 protein kinases from <i>Dictyostelium</i> are described. .Two of the protein phosphatases are shown to have subunit structures. All of the protein phosphatase activities described were able to dephosphorylate sites on proteins that had previously been phosphorylated by the cAMP-dependent protein kinase. Therefore it may be that these phosphatases function <i>in vivo</i> to antagonize the action of the cAMP-dependent protein kinase. Various pnpp phosphatase activities, varying in size, cation requirements and pH optima are described. Since many pnpp phosphatases also function with protein substrates it is possible that these activities represent protein phosphatases that function with phosphoproteins that have not been tested. Evidence for the existence of protein kinase C in Dictyostelium is presented. A histone protein kinase was found in <i>Dictyostelium</i> extracts that eluted from DE52 cellulose at the same salt concentration that rabbit brain protein kinase C did. In addition in one experiment clear dependency on calcium and phospholipids is shown. The purification to homogeneity, and characterization of a low molecular weight autophosphorylating protein kinase, pp20, is described. This protein kinase is a major phosphorylated protein and in addition represents at least 0.03% of the total cellular protein. The autophosphorylation reaction can be inhibited by EDTA but not by EGTA. Following inhibition by EDTA, autophosphorylation can be reactivated by the addition of Mn²⁺, Mg²⁺, or Ca²⁺. Western blotting evidence is presented that shows cross reactivity of pp20 and an 85 KDa protein that may possess casein kinase activity. / Ph. D. / incomplete_metadata

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy