published_or_final_version / Zoology / Master / Master of Philosophy

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The structure and mechanism of the Protein Tyrosine Phosphatase-like Phytases (PTPLPs) from Selenomonas ruminantium (PhyAsr) and Mitsuokella multacida (PhyAmm) were investigated using a combination of enzyme kinetics, site-directed mutagenesis, and X-ray crystallography. I show that PTPLPs use a classical protein tyrosine phosphatase catalytic mechanism and adopt a core PTP fold. Several unique structural features of PTPLPs confer specificity for inositol phosphates. The effect of ionic strength and oxidation on the kinetics and structure of PTPLPs was investigated. The structural consequences of reversible and irreversible oxidation on PTPLPs and PTPs are compared and discussed. We determine the structural basis of substrate specificity in PTPLPs and propose a novel reaction mechanism for the hydrolysis of inositol polyphosphates by PTPLPs. Finally, the structure and function of a unique tandemly repeated phytase has been determined. We show that the active sites of the tandem repeat possess significantly different specificities for inositol polyphosphate. / xix, 148 leaves : ill. (some col.) ; 29 cm

A novel bacterial protein tyrosine phosphatase (PTP)-like enzyme has recently been isolated that has a PTP-like active site and fold and the ability to dephosphorylate myo-inositol hexakisphosphate. In order to expand our knowledge of this novel class of enzyme, four new representative genes were cloned from 3 different anaerobic bacteria related to clostridia and the recombinant gene products were examined. A combination of site-directed mutagenesis, kinetic, and high-performance ion-pair chromatography studies were used to elucidate the mechanism of hydrolysis, substrate specificity, and pathways of Ins P6 dephosphorylation. The data indicate that these enzymes follow a classical PTP mechanism of hydrolysis and have a general specificity for polyphosphorylated myo-inositol substrates. These enzymes dephosphorylate Ins P6 in a distributive manner, and have the most highly ordered pathways of sequential dephosphorylation of InsP6 characterized to date. Bioinformatic analyses have indicated homologues that are involved in the regulation of cellular function. / x, 138 leaves ; 29 cm.

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The signaling pathways of tyrosine kinase Trk receptors and their downstream biological effects are well known, but the parameters controlling the interactions between the receptors and their natural ligands still remain to be defined. Recent published reports from our laboratory indicate that nerve growth factor (NGF)-induced TrkA receptor activation is dependent on a membrane cellular sialidase. This sialidase activity specifically targets and hydrolyzes sialyl α-2, 3-linked β-galactosyl residues resulting in the desialylation and activation of the receptor. These findings support a novel hypothesis that places mammalian sialidase(s) in a cycle of activation of these receptors by their natural ligand. Taken together, they also predict a prerequisite desialylation of Trk receptors caused by a sialidase on the cell surface enabling the removal of a steric hindrance to receptor dimerization. Until now, the sialidase associated with neurotrophin-treated live Trk-expressing cells has not been identified. The molecular mechanism(s) of sialidase activation by neurotrophin factors binding to their receptors also remains unknown. In this thesis, the novel role of Neu1 sialidase in the activation of ligand-induced TrkA and TrkB receptors has been identified. It has been reported for the first time that Neu1 is already in complex with naïve and ligand-induced Trk receptors. In addition, a membrane sialidase mechanism initiated by NGF binding to TrkA has been indentified. It suggests a potentiation of GPCR-signaling via membrane Gαi subunit proteins and matrix metalloproteinase-9 (MMP-9) activation to induce Neu1 sialidase activation in live TrkA- and TrkB-expressing cells and primary neurons. These results establish a unique mode of regulation of Trk receptors by their natural ligand and define a new function for Neu1 sialidase. Preliminary data indicate that members of the family of tyrosine kinase receptors like epidermal growth factor receptor (EGFR) and insulin receptor are also under the same regulatory control of Neu1 sialidase. Recent reports from the laboratory have indicated that ligand-induced activation of the highly glycosylated Toll-like receptors, TLR-2,-3 and -4 is also dependent on Neu1 sialidase on the cell surface. Taken all together, the findings in this thesis uncover a Neu1 and MMP-9 cross-talk on the cell surface which is critically essential for neurotrophin-induced Trk tyrosine kinase receptor activation and neuron function. / Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2010-04-26 11:44:51.418

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Thesis (Ph. D.)--University of Hawaii at Manoa, 1995. / Includes bibliographical references (leaves 115-129). / Microfiche. / ix, 129 leaves, bound ill. 29 cm

Copy of author's previously published article inserted into back cover pocket. / Bibliography: leaves 157-214. / iv, 214, [131] leaves, [19] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The function of receptor tyrosine kinase, c-Kit is examined in relation to the role of receptor levels in factor dependence and cell transformation and the function of several naturally occurring isoforms of the human c-Kit receptor were analyzed by expressing cDNA encoding these isoforms in murine cells. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1996

"Research conducted at the Department of Haematology, Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science."--T.p. / Includes bibliographical references (leaves 170-214). / xiv, 221 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Elevated levels of receptor tyrosine kinases have been implicated in carcinogenesis. It is possible that high expression of c-Kit by the leukaemic cell provides them with a growth advantage over their normal counterparts in the bone marrow microenvironment. Thus, a means of inhibiting the interaction of c-Kit on these cells with ligand Steel Factor may remove proliferation and survival signals. Main aim of the study was to produce a biological inhibitor of this interaction and evaluate its ability to prevent ligand Steel Factor from binding to c-Kit on live cells. / Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Biosciences, 2001