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published_or_final_version / abstract / toc / Biochemistry / Master / Master of Philosophy

Chemical inducers of dimerization (CID) represent an important tool that has been implemented in numerous biological applications namely protein functions, protein stability, signal transduction, gene transcription, etc. Most generally CIDs are defined as bivalent molecules capable of inducing proximity between two targeted proteins. This proximity can in turn promote or disfavor a certain biological activity. Cell permeable small molecules in particular represent a very effective method to induce precise temporal and spatial control over a specific biological target. Our lab has devoted much effort in studying and elucidating the activity and functions of protein kinases, which represent a very attractive therapeutic target for the treatment of cancer and many other disorders. Towards this goal we have developed a general CID enabled three-hybrid split-luciferase methodology for the investigation of kinase-inhibitor interactions in vitro. We demonstrate that by modulating the kinase-ligand affinity of the CID we are able to successfully profile many structurally non-related protein kinases. We also investigate the use of weaker affinity kinase ligands to allow competitive displacement of CID by the selected inhibitor. In addition we report the design, synthesis and applications of novel CID's for the profiling of kinase inhibitors in mammalian cells and we demonstrate the feasibility of the assay to be used as a new platform for the discovery of cell permeable kinase inhibitors. Finally, we report a new ligand-gated split-kinase that can be selectively activated by photocleavable inducers of dimerization. We further prove how the activity of split-proteins can be deactivated with temporal control with use of non DNA damaging UV radiation.

Fps/Fes and Fer are members of a distinct subfamily of cytoplasmic protein tyrosine kinases that have recently been implicated in the regulation of innate immunity. Previous studies showed that mice lacking Fps/Fes are hyper-sensitive to systemic lipopolysaccharide (LPS) challenge. This study identifies physiological, cellular and molecular defects that contribute to the hyper-inflammatory phenotype in Fps/Fes-null mice. We showed that plasma tumour necrosis factor (TNF) - levels were elevated in LPS challenged Fps/Fes-null mice as compared to wild type mice, and cultured Fps/Fes-null peritoneal macrophages treated with LPS showed increased TNF- production. Cultured Fps/Fes-null macrophages also displayed prolonged LPS-induced degradation of IB-, increased phosphorylation of the p65 subunit of NF-B, and defective TLR4 internalization. Next, we showed a role for Fps in the regulation of recruitment of inflammatory leukocytes. Using the cremaster muscle intravital microscopy model, we observed increased leukocyte adherence to venules, and increased rates and degrees of transendothelial migration in Fps/Fes-null mice, compared to wild type. There was also increased neutrophil migration into the peritoneal cavity subsequent to thioglycollate challenge. Using flow cytometry, we observed prolonged expression of the selectin ligand PSGL-1 on peripheral blood neutrophils from Fps/Fes-knockout mice stimulated ex-vivo with LPS. Finally, we examined the role of Fps/Fes in regulating apoptosis in response to inflammation. Upon intra-peritoneal challenge with LPS, Fps/Fes-null mice displayed a decreased depletion of macrophages from the peritoneal cavity. In response to ex-vivo TNF- stimulation, macrophages from Fps/Fes-null mice underwent decreased apoptosis and necrosis as assessed by flow cytometry. Immunoblot analysis revealed that Fps/Fes-null macrophages displayed more TNF--induced degradation of IB-α in Fps/Fes-null cells, with corresponding increases in the phosphorylation of the p65 subunit of NF-B. In addition, stimulation of macrophages with TNF-α up-regulated PARP expression in wild-type macrophages; this up-regulation was not observed in Fps/Fes-null macrophages. Finally we observed a decreased recruitment of macrophages to the peritoneal cavities of Fps/Fes-null mice, with a corresponding increase in neutrophil recruitment, 5 days after thioglycollate challenge. Overall, we show that there is a role for Fps/Fes in regulating inflammation at the physiological, cellular, and molecular levels, and that this might be relevant in inflammatory disease. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2007-08-27 11:39:23.393

No description available.

There has been progress towards elucidating the mechanism of Escherichia coli glycerol kinase (EcGK) control by its allosteric effectors fructose-1,6-bisphosphate (FBP) and IIAglc (a member of the phosphoenolpyruvate:glycose phosphotransferase system). Determining the mechanism requires analysis of the interaction between these effectors and the substrates of EcGK. In this study, a structural and kinetic approach was used to determine inhibition by both the effectors. For this work, the use of fluorescence anisotropy to observe ligand binding was investigated. Also, a foundation was laid for future NMR experiments with EcGK. For fluorescence studies, E36C EcGK was labeled with fluorescein and tested for changes in anisotropy in the presence of different ligands. To ensure that E36C was an appropriate representative of wildtype protein, initial velocity, inhibition, and heterotropic coupling assays were performed. Groundwork for future NMR experiments required analyzing substitutions of the native EcGK cysteines by initial velocity and inhibition studies. By comparing wildtype enzyme and E36C (variant of wildtype with an engineered cysteine residue at position 36), it was found that E36C is a suitable substitute and was not drastically affected by labeling with fluorescein. Anisotropy values differed upon binding of different ligands and enabled titrations of the enzyme substrate complexes with both effectors to obtain dissociation constants. This supports using the stopped-flow method to assess the on- and off- rates of substrates and to obtain values for Q coupling. Furthermore, the results for FBP showed that inhibition by FBP is K-type (affects affinity) with respect to ATP and V-type (affects enzyme velocity) with respect to ADP. The findings presented also showed that native cysteine substitutions effect some of the catalytic and allosteric parameters of EcGK and would be powerful reporters for ligand binding in NMR. However, the enzymes are unstable and new protocols for protein isolation will need to be drafted.

This thesis examines Escherichia coli pyruvate kinase type 1 (PK1), a regulatory enzyme core to energy metabolism. Specifically, this thesis characterises a series of mutations in PK1 that were found when populations of E. coli were evolved in a glucose-limited environment for 20,000 generations. The gene pykF, which codes for the PK1 enzyme, was found to have developed nonsynonymous mutations in all replicate populations. Although the mutations at the nucleotide level were not the same (i.e. not parallel), it is not clear whether parallel adaptation exists at the protein structure/function level. This study aimed to address this question by investigating the kinetic and biophysical properties of the wild-type and seven mutant enzymes. The recombinant wild-type PK1 enzyme used in this study was found to have steady state kinetics consistent with those previously reported. Unlike the rabbit kidney PK enzyme, E. coli PK1 was shown to have a very tight tetrameric structure (picomolar range), which was not affected by the enzyme’s substrates (PEP and ADP), or the allosteric effector (FBP), as judged by analytical ultracentrifugation with fluorescence detection. The mutated residues were highly conserved, and found to fall loosely into three groups: those at the active site (P70T, P70Q and D127N); those at the subunit interface (I264F, A301T and A301S); and at the allosteric binding site (G381A). The seven mutated PK1 enzymes were obtained by mutagenesis followed by protein purification. Steady state kinetic analysis showed that the mutated enzymes displayed a variety of functional changes, suggesting that the populations had not evolved in a parallel manner at the enzyme structure/function level. Mutations within the active site (P70T, P70Q and D127N) all showed a decrease in catalytic potency. P70 is located at the hinge connecting the A and B domains, which forms the active site. PK1-P70Q showed strong cooperative binding to PEP, similar to the wild-type enzyme, in the absence of FBP, whereas PK1-P70T had little cooperativity, suggesting changes in the active site. PK1-D127N showed severely attenuated activity, suggesting, for the first time, that this residue is essential for catalysis. Mutations at the subunit interface (I264F, A301T and A301S) all showed altered allosteric regulation, suggesting that this interface is important in the FBP allosteric response. PK1-I264F, which had lower activity, but greater affinity for PEP, displayed a decreased α-helix content (as judged by CD), indicating that a subunit interface helix that includes this residue had altered. Despite still having a similar response to FBP, PK1-G381A showed an increased affinity for PEP, which, together with an increased α-helix content, suggests that this mutation had changed the structure of the FBP binding domain. None of the mutated enzymes showed altered quaternary structure. Although the populations evolved parallel changes with respect to cell physiology, fitness, and gene expression, this study suggests, for the first time, that the populations have not evolved in a parallel way with respect to protein structure and function.

Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 4 uppsatser.

Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Biosciences, 2001? / Copy of author's previously published work inserted. Includes bibliographical references (leaves 160-182).