Phosphoryl transfers are essential chemical reactions in key life processes, including energy production, signal transduction and protein synthesis. They are known for having extremely low reaction rates in aqueous solution, reaching the scale of millions of years. In order to make life possible, enzymes that catalyse phosphoryl transfer, phosphoryl transferases, have evolved to be tremendously proficient catalysts, increasing reaction rates to the millisecond timescale. Due to the nature of the electronic structure of phosphorus atoms, understanding how hydrolysis of phosphate esters occurs is a complex task. Experimental studies on the hydrolysis of phosphate monoesters with acidic leaving groups suggest a concerted mechanism with a loose, metaphosphate-like transition state. Theoretical studies have suggested two possible concerted pathways, either with loose or tight transition state geometries, plus the possibility of a stepwise mechanism with the formation of a phosphorane intermediate. Different pathways were shown to be energetically preferable depending on the acidity of the leaving group. Here we performed computational studies to revisit how this mechanistic shift occurs along a series of aryl phosphate monoesters, suggesting possible factors leading to such change. The fact that distinct pathways can occur in solution could mean that the same is possible for an enzyme active site. We performed simulations on the catalytic activity of β-phosphoglucomutase, suggesting that it is possible for two mechanisms to occur at the same time for the phosphoryl transfer. Curiously, several phosphoryl transferases were shown to be able to catalyse not only phosphate ester hydrolysis, but also the cleavage of other compounds. We modeled the catalytic mechanism of two highly promiscuous members of the alkaline phosphatase superfamily. Our model reproduces key experimental observables and shows that these enzymes are electrostatically flexible, employing the same set of residues to enhance the rates of different reactions, with different electrostatic contributions per residue.

Au cours de la division cellulaire, l'information génétique doit être transmise de façon précise et identique de la cellule mère aux cellules filles. Le génome est répliqué au cours de la phase S tandis que la distribution des deux copies entre les cellules filles se fait au cours de la mitose. L'initiation et le maintien de la mitose nécessite un équilibre contrôlé entre les activités des kinases et des phosphatases. La protéine kinase Greatwall est requise pour l'entrée et le maintien de la mitose à travers l'inhibition de la PP2A, la principale phosphatase qui déphosphoryle les substrats du complexe Cdk1-cycline B. Au cours de ce travail, nous avons entrepris l'étude structure/fonction de la protéine kinase Greatwall qui nous a permis de caractériser ses mécanismes d'activation. Nos résultats montrent que Greatwall appartient à la famille des AGC kinases mais qu'elle présente la particularité d'être contrôlée par des mécanismes qui lui sont propres: l'activation de la protéine, qui se fait en deux étapes, est différente de celle décrite pour les autres membres de cette famille de kinases. Par la suite, nous avons identifié deux substrats de la protéine kinase Greatwall, Arpp19 (cAMP-Regulated Phosphoprotein 19) et l'alpha-Endosulfine (ENSA). Nous avons montré qu'une fois phosphorylées par Greatwall, ces deux protéines s'associent à la PP2A et inhibent cette phosphatase. Malgré le fait que ces deux substrats soient capables d'inhiber la PP2A, seul Arpp19 endogène est responsable de l'inhibition de la phosphatase pour promouvoir l'entrée en mitose dans le modèle des extraits d'ovocytes de xénope. Nous nous intéressons à présent à l'étude du rôle d'ENSA. / During cell division, genetic information must be transmitted from the mother cell to the daughter cell in an accurate and identical way. During the S phase the genome is replicated while an equal distribution of two copies of DNA between the daughter cells is made during mitosis. Initiation and maintenance of mitosis require a controlled balance between kinase and phosphatase activities. Greatwall kinase is essential for mitotic entry and maintenance through the inhibition of PP2A, the main phosphatase that dephosphorylates Cdk1/cycline B mitotic substrates. Here we investigate the mechanisms regulating Greatwall. Our results show that Greatwall is a member of the AGC family of kinases that appears to be regulated by a unique two-step mechanism that differs from the other members of this family. Furthermore we identified Arpp19 (cAMP-Regulated Phosphoprotein 19) and alpha-Endosulfine (ENSA) as two substrates of Greatwall that, when phosphorylated by this kinase, associate with and inhibit PP2A. Despite the fact that these two substrates are able to inhibit PP2A, only endogenous Arpp19 is responsible for the phosphatase inhibition at mitotic entry in xenopus egg extratcs. Roles of ENSA are currently under investigation.

Li Mei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 182-202). / Abstracts in English and Chinese. / Abstract --- p.i / 中文摘要 --- p.iii / Acknowledgements --- p.v / List of Abbreviations --- p.vi / CONTENTS --- p.viii / Chapter Chapter 1 --- General introduction --- p.1 / Chapter 1.1 --- Alkaline phosphatase --- p.1 / Chapter 1.1.1 --- Distribution --- p.1 / Chapter 1.1.2 --- Molecular characteristics of alkaline phosphatase --- p.4 / Chapter 1.1.3 --- Properties of alkaline phosphatase --- p.8 / Chapter 1.1.4 --- Role of alkaline phosphatase --- p.10 / Chapter 1.2 --- Mouse embryonic brain development --- p.18 / Chapter 1.2.1 --- General developing process --- p.18 / Chapter 1.2.2 --- The crainal nerve nuclei in the embryonic mouse brainstem --- p.20 / Chapter 1.2.3 --- The process of neurogenesis in central nerve system --- p.22 / Chapter 1.3 --- Alkaline phosphatase expressed in developing neural tube --- p.26 / Chapter 1.4 --- Summary --- p.30 / Chapter 1.5 --- Objectives of study --- p.31 / Chapter Chapter 2 --- AP expression pattern in embryonic mouse brainstem --- p.33 / Chapter 2.1 --- Introduction --- p.33 / Chapter 2.1.1 --- AP expressed in developing neural tube --- p.33 / Chapter 2.1.2 --- Methods for alkaline phosphatase detection --- p.35 / Chapter 2.2 --- Materials and methods --- p.39 / Chapter 2.2.1 --- Animal and procedure --- p.39 / Chapter 2.2.2 --- Preparation of tissue sections and histochemistry --- p.39 / Chapter 2.2.3 --- Electron microscopy study of AP location --- p.41 / Chapter 2.3 --- Results --- p.42 / Chapter 2.3.1 --- Histochemical demonstration of AP --- p.42 / Chapter 2.3.2 --- Stage-specificity and tissue-specificity of AP activity in the neural tube --- p.43 / Chapter 2.3.3 --- Cytochemical localization of AP activity --- p.46 / Chapter 2.3.4 --- Sencitivity to pH of the histochemical staining for AP --- p.46 / Chapter 2.3.5 --- Inactivation of AP activity --- p.47 / Chapter Chapter 3 --- Quantitative studies of AP activity in embryonic mouse brainstem --- p.48 / Chapter 3.1 --- Introduction --- p.48 / Chapter 3.1.1 --- Basic knowledge about enzyme kinetic study --- p.48 / Chapter 3.1.2 --- Enzyme assay for alkaline phosphatase --- p.50 / Chapter 3.2 --- Materials and methods --- p.52 / Chapter 3.2.1 --- Animals and sample preparation --- p.52 / Chapter 3.2.2 --- Measurement of AP activities --- p.53 / Chapter 3.2.3 --- Data analysis --- p.54 / Chapter 3.3 --- Results --- p.54 / Chapter 3.3.1 --- "Determination of reaction duration, initial velocity and Km of AP activity" --- p.54 / Chapter 3.3.2 --- Comparision of AP activity in the brainstem and cortex and at different stages --- p.55 / Chapter 3.3.3 --- Effects of physical and chemical factors on AP activity --- p.55 / Chapter Chapter 4 --- Electrophoresis study of AP activity --- p.57 / Chapter 4.1 --- Introduction --- p.57 / Chapter 4.2 --- Materials and methods --- p.60 / Chapter 4.2.1 --- AP extraction --- p.60 / Chapter 4.2.2 --- Polyacrylamide gel electrophoresis (PAGE) --- p.61 / Chapter 4.2.3 --- Detection of AP activity --- p.61 / Chapter 4.3 --- Results --- p.62 / Chapter 4.3.1 --- Demonstration of AP activity on the gels --- p.62 / Chapter 4.3.2 --- Comparison of AP from the brain at different stages --- p.62 / Chapter 4.3.3 --- "Comparison of AP in the embryonic brainstem with those in the adult mouse placenta, kidney, liver and intestine" --- p.63 / Chapter 4.3.4 --- Effect of heating and chemical factors on AP activity in the embryonic brainstem --- p.63 / Chapter Chapter 5 --- Study of the cell types expressing AP activity --- p.65 / Chapter 5.1 --- Introduction --- p.65 / Chapter 5.2 --- Materials and methods --- p.67 / Chapter 5.2.1 --- Materials --- p.67 / Chapter 5.2.2 --- Immunostaining of AP in the embryonic brainstem --- p.68 / Chapter 5.2.3 --- Double staining for AP and cells markers --- p.70 / Chapter 5.3 --- Results --- p.70 / Chapter 5.3.1 --- Effectiveness of anti-TNAP antibody on the embryonic mouse brain --- p.70 / Chapter 5.3.2 --- Expression pattern of different neural cell markers at E13.5 --- p.71 / Chapter 5.3.3 --- Co-localization of AP and specific cell markers in E13.5 brain --- p.72 / Chapter Chapter 6 --- Discussion --- p.74 / Chapter 6.1 --- Stage-dependence and tissue-specificity of AP expression in the developing mouse brainstem --- p.75 / Chapter 6.2 --- Possible molecular nature of AP expressed in the developing mouse brainstem --- p.80 / Chapter 6.3 --- The possible cell types that express the enzyme activity --- p.83 / "Figures, Tables, Graphs and Legends" --- p.87 / Appendices --- p.165 / Appendix A: Sources of materials --- p.165 / Appendix B: The process of sample for staining --- p.167 / Appendix C: Protocol of histochemical staining for AP --- p.170 / Appendix D: Protocol of electron microscopy study for AP activity --- p.172 / Appendix E: Protocol of enzyme assay for AP activity --- p.174 / Appendix F: Protocol of immunostaining (ABC method) --- p.175 / Appendix G: Protocol of double staining with fluorescent detection --- p.177 / Appendix H: Protocol of electrophoresis analysis for AP --- p.179 / References --- p.182

<p>Although it has been estimated that protein histidine phosphorylation account for about 6 % of the protein phosphorylation in eukaryotic cells; the knowledge of histidine phosphorylation and dephosphorylation is still limited. Lately, studies have appeared of a mammalian 14-kDa phospho- histidine phosphatase, also named protein histidine phosphatase and molecular cloning have provided some information of its physiological role. The object of the present study was to detect the protein expression of protein histidine phosphatase, PHPT1, in mouse tissue, by using immunohistochemistry. Tissue samples from a 4-week-old mouse (heart, liver, kidney, lung, muscle, and spleen), 5-month-old mouse (testis and intestinal), 8-month-old mouse (uterus) and an embryo from 14.5 days old mouse were obtained and processed for light microscopic examination. An absorption test was also made to confirm the specificity of the antibody. The results reveal that PHPT1 is mainly expressed in epithelium, heart- and skeletal muscle. These results provide new evidences for the understanding of the function of eukaryotic histidine phosphorylation and dephosphorylation.</p><p>KEYWORDS</p><p>Phosphohistidine, dephosphorylation, protein histidine phosphatase, phosphohistidine phosphatase, protein phosphorylation</p>

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Heidelberg, Univ., Diss., 2007.

Made available in DSpace on 2014-06-11T19:27:47Z (GMT). No. of bitstreams: 0 Previous issue date: 2005-12-16Bitstream added on 2014-06-13T20:56:53Z : No. of bitstreams: 1 chavesneto_ah_me_araca.pdf: 216462 bytes, checksum: a077ffffb43a7f52121fc844bcf5ba50 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A saliva é coletada através de métodos simples e não invasivos, além de ser facilmente armazenada. A coleta não-traumática é atrativa especialmente para crianças e quando repetidas coletas são requeridas. A desvantagem da saliva como uma ferramenta de diagnóstico é a variabilidade, que ocorre em decorrência dos fatores fisiológicos e do modo de coleta. Numa primeira etapa o trabalho teve por objetivo estudar as atividades das enzimas fosfatase alcalina (FAlc), fosfatase ácida total (FAT) e fosfatase ácida resistente ao tartarato (TRAP) na saliva total não estimulada de crianças, investigando as influências de fatores como sexo, faixa etária e horário de coleta, bem como a correlação das enzimas com a taxa de fluxo salivar. Nesta etapa, 120 crianças saudáveis de ambos os sexos, nas faixas etárias de 1-5 e 6-12 anos de idade tiveram as amostras de saliva coletada entre 8:00-10:00 horas ou entre 14:00-16:00 horas. A segunda parte do trabalho teve por objetivo avaliar a correlação das atividades enzimáticas da FAT, TRAP, proteína tirosina fosfatase de baixa massa molecular relativa (PTP-BMr) e FAlc na saliva e no soro de crianças, além de analisar a influência do sexo e da faixa etária na atividade das enzimas, no soro e na saliva total. Para tanto 32 crianças de ambos os sexos, nas faixas etárias de 1-5 anos e 6-12 anos de idade tiveram as amostras de saliva e sangue coletadas entre 8:00-10:00 horas. Os resultados da primeira etapa sugerem que as atividades da FAT, TRAP e FAlc são influenciadas, de formas distintas, pelos fatores sexo, faixa etária e horário de coleta e que não existe correlação entre as atividades das enzimas e a taxa de fluxo salivar. / Saliva can be collected by simple, non-invasive methods and is easily stored. The non-traumatic collection is specially appealing for children and when repeated collections are required. The main disadvantage of the saliva as a diagnosis tool is the variability that happens due to the physiologic factors and dependence on mode of the collection. In a first stage the work had for objective to study the activities of the enzymes alkaline phosphatase (ALP), total acid phosphatase (TAP) and tartrate-resistant acid phosphatase (TRAP) in the whole unstimulated saliva of children, investigating the influences of factors as sex, age group and time of collection, as well as the correlation of the enzymes with the salivary flow rate. In this stage, 120 healthy children of both sexes, in the age groups of 1-5 and 6-12 years of age had the saliva samples collected between 8:00-10:00 hours or between 14:00-16:00 hours. In a second stage, the work had for objective to evaluate the correlation of the enzymatic activities of TAP, TRAP, low molecular weight protein tyrosine phosphatase (LMW-PTP) and ALP in the saliva and in the children's serum, besides analyzing the influence of the sex and of the age group in the activity of the enzymes, in the serum and in the whole unstimulated saliva. For so much 32 children of both sexes, in the 1-5 year-old age groups and 6-12 years of age had the saliva samples and blood collected between 8:00-10:00 hours. The results of the first stage suggest that the activities of TAP, TRAP and ALP are influenced, in different ways, for the factors sex, age group and time of collection and that correlation doesn't exist between the activities of the enzymes and the salivary flow rate.

Submitted by KETELIN JULIANE DAL PRÁ null (k.dalpra@hotmail.com) on 2017-01-20T02:22:12Z No. of bitstreams: 1 Dissertação de Mestrado Ketelin J. Dal Prá - FOA UNESP.pdf: 1380407 bytes, checksum: aa6eb3e50dc3c4e8a803bd7b396157fa (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-01-24T12:22:26Z (GMT) No. of bitstreams: 1 dalpra_kj_me_araca.pdf: 1380407 bytes, checksum: aa6eb3e50dc3c4e8a803bd7b396157fa (MD5) / Made available in DSpace on 2017-01-24T12:22:26Z (GMT). No. of bitstreams: 1 dalpra_kj_me_araca.pdf: 1380407 bytes, checksum: aa6eb3e50dc3c4e8a803bd7b396157fa (MD5) Previous issue date: 2016-12-16 / O presente estudo teve como objetivo investigar em glândulas submandibulares de ratos tratados com aguardente de cana, a morfologia, atividade funcional das fosfatases e níveis de mucina. 24 ratos machos e adultos foram divididos em 4 grupos (n=6) de acordo com o tipo de bebida fornecida, aguardente de cana (39º GL) ou água, e ao tempo de tratamento de 75 ou 105 dias. Após os períodos de tratamento, os animais foram submetidos à cirurgia para remoção das glândulas submandibulares, seguido da eutanásia. As glândulas submandibulares do lado direito foram processadas para análise histomorfométrica (Image J) dos ductos estriados, ductos granulosos e ácinos. As glândulas do lado esquerdo foram pesadas e armazenadas a -80 °C, para avaliação da atividade funcional da fosfatase ácida total (FAT), fosfatase ácida resistente ao tartarato (FART), fosfatase alcalina (FAL) e determinação dos níveis de mucina. Para isso foram feitos ensaios bioquímicos por métodos espectrofotométricos. Os dados quantitativos foram submetidos à análise estatística (p<0,05). Os pesos absolutos e relativos das glândulas submandibulares apresentam-se reduzidos em relação aos controles (p<0,05). Na análise histomorfométrica, observamos que houve relevante redução da área dos ácinos (p<0,05) e redução não significativa dos ductos estriados (p>0,05). Nos ductos granulosos ocorreu aumento não significativo da área (p>0,05). As atividades de FAT e FART se apresentaram expressivamente diminuídas nos grupos experimentais (p<0,05), enquanto a atividade funcional de FAL diminuiu de forma moderada (p>0,05). Houve redução significativa dos níveis de mucina pelo efeito do alcoolismo crônico (p<0,05). A partir desses dados foi possível concluir que o alcoolismo crônico, por uso de aguardente de cana afeta a funcionalidade bioquímica e a morfologia da glândula submandibular. / The present study aimed to investigate submandibular glands of rats treated with cane brandy, morphology, functional activity of phosphatases and levels of mucin. 24 male and adult rats were divided into 4 groups (n = 6) according to the type of beverage provided, cane brandy (39° GL) or water, and treatment time of 75 or 105 days. After the treatment periods, the animals were submitted to surgery to remove the submandibular glands, followed by euthanasia. The submandibular glands on the right side were processed for histomorphometric analysis (Image J) of the striated ducts, granular ducts and acini. The left glands were weighed and stored at -80 °C for evaluation of the functional activity of total acid phosphatase (TAP), tartrate-resistant acid phosphatase (TRAP), alkaline phosphatase (ALP), and determination of mucin levels. For this, biochemical tests were carried out by spectrophotometric methods. Quantitative data were submitted to statistical analysis (p <0.05). The absolute and relative weights of the submandibular glands are reduced in relation to the controls (p <0.05). In the histomorphometric analysis, we observed that there was a significant reduction of the acini area (p <0.05) and a non-significant reduction of the striated ducts (p> 0.05). In the granular ducts a not significant increase of the area occurred (p> 0.05). The TAP and TRAP activities were significantly decreased in the experimental groups (p <0.05), while the ALP functional activity decreased moderately (p> 0.05). There was a significant reduction of mucin levels by the effect of chronic alcoholism (p <0.05). From these data it was possible to conclude that chronic alcoholism due to the use of cane brandy affects the biochemical functionality and morphology of the submandibular gland.

Le sujet de cette thèse porte sur la synthèse d'inhibiteurs spécifique de la Sérine-Thréonine phosphatase I (PP1). Un criblage de la chimiothèque de l'institut Curie, réalisé par l'équipe du Dr. Popov a permis d'identifier une 3-aryl-1(2H)isoquinolone, qui perturbe la dynamique des microtubules et qui s’est ensuite avéré être un inhibiteur sélectif de PP1. Dans une première partie, nous avons mis au point une nouvelle méthodologie de synthèse de ces composés hétérocycliques par une réaction tandem d’aminocarbonylation-cyclisation pallado catalysée. L’étude d’une seconde voie de synthèse de ces composés a été étudié par réaction d'arylation direct d'une 1(2H)isoquinolone. Dans le but de trouver d’autres hit, ligand de cette phosphatase, nous avons tenté de développer un test de triple hybride chimique, en collaboration avec la société Hybrigenics. Ce test est basé sur l’interaction de notre inhibiteur hit avec la phosphatase PP1. Pour cela, nous avons synthétisé une sonde à partir de la molécule hit initiale. La deuxième partie a trait à un développement de chimie médicinal pour optimiser le hit initial. Des dérivés de très bonne sélectivité pour l’enzyme cible ont été préparés. / This PhD thesis deals with the synthesis of serine threonine phosphatase I (PPI) inhibitors. This project started with the screening of the Institut Curie’s Library carried out by Dr. Popov team. They identified a 3-aryl-1(2H)isoquinolone (hit molecule) which strongly disturbs the microtubules dynamics. In the first part, we designed an original methodology to prepare those heterocycles, though a tandem palladium catalyzed aminocarbonylation/cyclization reaction. Then, we studied the direct arylation reaction to obtain the desired scaffold. In collaboration with Hybrigenics, we synthesize a probe for a triple hybrid system, based on the specific interaction of the hit molecule with its target PPI. Thanks to this system, one could identify new inhibitors of the targeted phosphatase protein. Eventually, a library of isoquinolones derivatives was synthesized. During the invitro tests, some of those molecules proved to be very specific for the serine threonine phosphatase I.

La fonction des protéines de signalisation est déterminée par la nature des domaines qui les composent. Une meilleure compréhension des voies de signalisation passe par l'étude de ces domaines et de leur régulation. PTPN4 est une tyrosine phosphatase qui joue un rôle anti-apoptotique. Lors de l'infection par une souche atténuée du virus de la rage, sa fonction est perturbée, conduisant à la mort des cellules. Cette perturbation est due à l'interaction du motif de reconnaissance au domaine PDZ (PBM) de la glycoprotéine virale avec le domaine PDZ de PTPN4. Nous avons montré que ce domaine PDZ a un rôle d'inhibiteur allostérique de l'activité catalytique de la phosphatase de PTPN4. Ceci représente la première description de la régulation d'une phosphatase par un domaine PDZ. Cette inhibition est levée lors de la fixation d'un ligand au domaine PDZ, tel que le PBM de la glycoprotéine virale. Notre étude structurale révèle que la fixation d'un PBM perturbe les interactions transitoires entre les deux domaines et rétablit ainsi les propriétés catalytiques de la phosphatase. Nous avons par ailleurs identifié un ligand endogène de PTPN4, la MAP Kinase p38 qui, à travers son interaction avec PTPN4, participerait à la régulation de l'homéostasie cellulaire. La formation du complexe implique le recrutement du PBM de p38 par le domaine PDZ de PTPN4. Ainsi, en plus d'avoir une fonction de régulation du domaine phosphatase, le domaine PDZ permet également le recrutement de partenaires et la présentation de substrats au site actif de la phosphatase de PTPN4. Cette étude contribue ainsi à améliorer notre connaissance du rôle des domaines PDZ dans les voies de signalisation cellulaires. / The function of signaling proteins is determined by the nature of the domains from which they are made up. A better understanding of cell signaling pathways will result from the study of these domains and their regulation. PTPN4 is a non-receptor tyrosine phosphatase with an anti-apoptotic function. Upon infection with an attenuated rabies virus, its function is hijacked, which subsequently leads to cell death. This phenotype is arises from the interaction of the PDZ binding motif (PBM) of the viral glycoprotein with the PDZ domain of PTPN4. In this study, we show that this PDZ domain is an allosteric inhibitor of the catalytic activity of the PTPN4 phosphatase domain. This is the first description of the regulation of a phosphatase by a PDZ domain. This inhibition is released by the interaction of a ligand to the PDZ domain, such as the viral glycoprotein PBM. Our structural study revealed that the PBM recognition disrupts the transient inter-domain interactions and restores the complete phosphatase catalytic properties. As well, we identified a PTPN4 endogenous ligand, the MAP Kinase p38, which may participate in the regulation of the cellular homeostatic through its interaction with PTPN4. Thus, in addition to its phosphatase regulatory role, the PDZ domain also allows the recruitment of partners and the introduction of substrates to the PTPN4 phosphatase active site. This study contributes to our understanding of the role played by PDZ domains in cell signaling pathways.