Phosphorylation/dephosphorylation is likely the most crucial chemical reaction taking place in all living organisms. It is the basis for the regulatory control of many diverse biological events triggered by extracellular effectors. Moreover, it is a ubiquitous element of intracellular signal transduction pathways that regulates a wide range of processes. While protein phosphorylation has been extensively characterized in eukaryotes, far less is known about its emerging counterparts in prokaryotes. This study involved determination of the crystal structures and functional characterization of two protein kinases, YihE and AceK (also a protein phosphatase), and two nucleotide pyrophosphatases, YjjX and YhdE. X-ray crystallographic structure determination combined with bioinformatics analyses, mutageneses and biochemical experiments, both in vitro and in vivo, were utilized for the functional characterization of each protein. YihE was found to be a previously unknown kinase component of a new type of bacterial phospho-relay mechanism, thus adding kinase activity as another response to the Cpx sensing system that functions to maintain cellular homeostasis. AceK, which possesses both kinase and phosphatase activities, modifies isocitrate dehydrogenase (ICDH) to regulate the flux of isocitrate into the glyoxylate cycle. The structures of Acek alone and in complex with its substrate, ICDH, provided us with information to explain the mechanisms underlying its bifunctionality and its molecular switch. Through structural comparison and, particularly, functional characterization, we revealed that YjjX is a novel ITPase/XTPase responsible for the removal of non-canonical nucleotides from the cell during oxidative stress in Escherichia coli. YhdE, identified as a novel dTTPase, was observed to retard cell growth and form a filamentous phenotype when overexpressed in the cell, suggesting that YhdE is involved in the control of cell growth and division by regulating the cell nucleotide pool for DNA synthesis. In summary, this research has made a substantial ii contribution to the investigation of bacterial phosphorylation and dephophorylation systems that respond to various environmental conditions. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2009-05-29 11:41:41.832

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Reversible phosphorylation is a major mechanism for regulating the activity, localization and stability of proteins required for vital cellular processes such as glucose metabolism, gene expression, establishment of polarity, mitosis and cytokinesis. Phospho-regulation is driven by the activities of kinases and phosphatases. Together, these enzymes account for ∼3% of eukaryotic genomes and it is estimated that 30% of the eukaryotic proteome is composed of phospho-proteins. Protein kinases (PKs) have been studied extensively, however relatively little is known regarding the signaling networks of protein phosphatases (PPases). The identification of PPase functional networks has been slow due to the redundant nature of the majority of PPases, the complexity of their substrate recognition in vivo, and the lack of large-scale analyses that would facilitate network analysis. We hypothesized that large-scale analysis of genetic interactions using the Synthetic Genetic Array (SGA) and proteomic analyses using 2D-PAGE Difference Gel Electrophoresis (DiGE) could reveal PPase functional networks. Here, we apply this approach to the essential and conserved PP1 PPase Glc7 as it regulates numerous cellular processes in budding yeast. For this study, we created a glc7 hypomorphic mutant (glc7-E101Q) suited for both SGA and DiGE analyses. SGA analysis of glc7-E101Q revealed a broad network of 147 synthetic sick/lethal (SSL) and 178 synthetic rescue (SR) interactions. DiGE comparison of the glc7-E101Q proteome relative to wild-type at medium-resolution (∼1000 proteins) revealed alterations in 39 proteins that changed as a consequence of both the mutation and growth conditions. One of the proteins identified in this analysis was Eno1, a non-essential enolase that is mis-regulated in the presence of glucose and identified a SR mutation in the glc7-E101Q SGA. Subsequent phenotypic analysis suggests a novel, non-metabolic role for Eno1 in the Glc7 interaction network. Our results reveal that parallel analysis, using SGA and DIGE, can reveal novel functions and networks that a single analysis may not detect.

Thesis (M.S.)--Youngstown State University, 2009. / Includes bibliographical references (leaves 57-66). Also available via the World Wide Web in PDF format.

Thesis (Ph. D.)--University of Texas at Austin, 2005. / Supervisors: Terrence J. Monks and Shawn B. Bratton. Vita. Includes bibliographical references.

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Made available in DSpace on 2014-06-11T19:23:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-02-27Bitstream added on 2014-06-13T19:28:58Z : No. of bitstreams: 1 goncalves_am_me_araiq.pdf: 787474 bytes, checksum: 139bd29c3e4939fc72732e4ef1db4333 (MD5) / Durante a metamorfose dos anuros, além da natação, a cauda tem como função fornecer nutrientes necessários para as transformações morfofisiológicas da fase larval aquática em adultos terrestres, pois os animais não se alimentam durante esse período. A fosfatase alcalina é uma enzima importante na mobilização do fosfato necessário para o anabolismo de biomoléculas essenciais à vida, enquanto a fosfatase ácida é uma marcadora lisossomal. Nesse sentido, o objetivo desse estudo foi avaliar a atividade das fosfatases ácida e alcalina obtidas da cauda de girinos de rã-touro (Lithobates catesbeianus), visando fornecer informações acerca do processo de mobilização de nutrientes durante o desenvolvimento e a metamorfose. Os animais foram mantidos em aquários a 27 °C, e separados por estádios de desenvolvimento. As caudas foram coletadas de animais em cada estádio e homogeneizadas em tampões acetato, TRIS.HCl ou 2-amino-2-metil-1-propanol, contendo MgCl2 2 mM e ZnCl2 1 μM, centrifugadas a 10000 g, durante 10 minutos, a 4°C, congeladas em nitrogênio líquido e armazenadas a -70 °C. A atividade de p-nitrofenilfosfatase foi estável quando as amostras homogeneizadas foram armazenadas em diferentes valores de pH, contudo, a atividade da fosfatase alcalina reduziu aproximadamente 70% após 12 horas de armazenamento no pH 4. O pH ótimo aparente para a hidrólise do p-nitrofenilfosfato pelas fosfatases ácida e alcalina foi 5,0 e 10,5, respectivamente, enquanto a hidrólise do pirofosfato de sódio e do ATP para as atividades de pirofosfatase e ATPase foram de 7,5 e 7,0. A temperatura ótima para a atividade das fosfatases ácida e alcalina foi 45°C por 15 minutos de reação. O estudo da ação de compostos sobre a atividade da fosfatase alcalina revelou que o arsenato, EDTA, metavanadato, teofilina, levamisol, íons fosfato e o p-hidroximercuriobenzoato agem... / During anurans metamorphosis, besides swimming, the tail has the function to supply with nutrients the morphophysiological changes of the larval aquatic stage to terrestrial adults, because the animals do not eat during this period. Alkaline phosphatase is an important enzyme in phosphate mobilization required to the anabolism of essential biomolecules, whereas the acid phosphatase is a lysosomal marker. Thus, the aim of this study was to evaluate the activity of acid and alkaline phosphatase obtained from the tail of bullfrog tadpoles (Lithobates catesbeianus), to provide information about the process of nutrients mobilization during development and metamorphosis. The animals were kept in aquaria at 27 °C, and separated by stages. The tails were collected from animals at each stage and homogenized in acetate buffers, Tris.HCl or 2-amino-2-methyl-1-propanol, containing 2 mM MgCl2 and 1 mM ZnCl2, centrifuged at 10000 g for 10 minutes at 4 °C, frozen in liquid nitrogen and stored at -70 °C. The activity of p-nitrophenylphosphatase was stable when the homogenized samples were stored at different pH values, however, alkaline phosphatase activity decreased about 70% after 12 hours of storage at pH 4. The apparent optimum pH for the hydrolysis of p-nitrophenylphosphate by acid and alkaline phosphatase was 5.0 and 10.5, respectively, while the hydrolysis of sodium pyrophosphate and ATP by the activities of pyrophosphatase and ATPase were 7.5 and 7.0. The optimum temperature for the activity of acid and alkaline phosphatases was 45 ° C for 15 minutes of reaction. The study of the action of compounds on the activity of alkaline phosphatase revealed that arsenate, EDTA, metavanadate, theophylline, levamisole, phosphate ions and p-hydroxymercurybenzoate act as inhibitors, and arsenate showed greater inhibition... (Complete abstract click electronic access below)

Background - The role of protein phosphatases (PPs) during ischemic preconditioning in the rabbit heart was examined. Methods and Results - Fostriecin, a potent inhibitor of PP2A, was administered to isolated rabbit hearts starting either 15 minutes before or 10 minutes after the onset of a 30-minute period of regional ischemia and continuing until the onset of reperfusion. After 2 hours of reperfusion, infarct size was measured with triphenyltetrazolium chloride. In a second study with isolated rabbit cardiomyocytes, the effect of fostriecin pretreatment was assessed by measuring changes in cell osmotic fragility during simulated ischemia. PP1 and PP2A activities of isolated control and ischemically preconditioned cells were also measured. In a third series of experiments, left ventricular biopsies of isolated rabbit hearts were obtained before and at selected times during 60 minutes of global ischemia, and the tissue was assayed for PP1 and PP2A activities. In isolated hearts pretreated with fostriecin, only 8% of the ischemic zone infarcted, significantly less than that in untreated control hearts (33%; P<0.001) but comparable to that in ischemically preconditioned hearts (9%; P<0.001 versus control). Significant protection was also observed in the hearts treated only after the onset of ischemia (18% infarction; P<0.05 versus control). In isolated myocytes, fostriecin also provided protection comparable to that produced by metabolic preconditioning. Preconditioning had no apparent effect on the activity of either PP1 or PP2A in isolated ventricular myocytes or ventricular tissue obtained from heart biopsies. Conclusions - Fostriecin, a potent inhibitor of PP2A, can protect the rabbit heart from infarction even when administered after the onset of ischemia. But inhibition of either PP1 or PP2A does not appear to be the mechanism of protection from ischemic preconditioning.

Tyrosine phosphorylation is an important post-translational modification in cells that modulates key cellular pathways. Tyrosine phosphatases are the class of enzymes that remove this modification from proteins, yet we know relatively little about how they are regulated in various signaling contexts. Activity-based probes that successfully target active sites of tyrosine phosphatases and report on their activities can fill in this gap. We show the assessment of various thiol-reactive groups for their ability to target catalytic cysteine residues with specificity. Then we construct and screen a library of fragment-like compounds for their on-target and off-target reactivities. We also discuss theoretical considerations for screening covalent inhibitors for their kinetic parameters and show this using our experimental data. Lastly, we augment compounds selected from the library to enable click chemistry for reporter group attachment for use on the whole proteome, ultimately through mass spectrometry-based proteomics methods. We show enrichment of target proteins. These target-centric design efforts will yield new insights into the general development processes of activity-based probes or inhibitors.

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